WO2023034220A2 - Compositions et procédés de reprogrammation de tcr à l'aide de protéines de fusion et de cxcr6 - Google Patents

Compositions et procédés de reprogrammation de tcr à l'aide de protéines de fusion et de cxcr6 Download PDF

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WO2023034220A2
WO2023034220A2 PCT/US2022/041904 US2022041904W WO2023034220A2 WO 2023034220 A2 WO2023034220 A2 WO 2023034220A2 US 2022041904 W US2022041904 W US 2022041904W WO 2023034220 A2 WO2023034220 A2 WO 2023034220A2
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nucleic acid
sequence
recombinant nucleic
seq
domain
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PCT/US2022/041904
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WO2023034220A3 (fr
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Dana GILMORE
Guangwu GUO
Dario Gutierrez
Robert Hofmeister
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TCR2 Therapeutics Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • cancer immunotherapy Most patients with hematological malignancies or with late-stage solid tumors are incurable with standard therapy. In addition, traditional treatment options often have serious side effects. Numerous attempts have been made to engage a patient’s immune system for rejecting cancerous cells, an approach collectively referred to as cancer immunotherapy. However, several obstacles make it rather difficult to achieve clinical effectiveness. Although hundreds of so-called tumor antigens have been identified, these are often derived from self and thus can direct the cancer immunotherapy against healthy tissue, or are poorly immunogenic. Furthermore, cancer cells use multiple mechanisms to render themselves invisible or hostile to the initiation and propagation of an immune attack by cancer immunotherapies.
  • CAR chimeric antigen receptor
  • CTL019 The clinical results with CD19-specific CAR T cells (called CTL019) have shown complete remissions in patients suffering from chronic lymphocytic leukemia (CLL) as well as in childhood acute lymphoblastic leukemia (ALL) (see, e.g., Kalos et al., Sci Transl Med 3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368: 1509-1518 (2013)).
  • An alternative approach is the use of T cell receptor (TCR) alpha and beta chains selected for a tumor-associated peptide antigen for genetically engineering autologous T cells.
  • TCR T cell receptor
  • TCR chains will form complete TCR complexes and provide the T cells with a TCR for a second defined specificity. Encouraging results were obtained with engineered autologous T cells expressing NY-ESO-1 -specific TCR alpha and beta chains in patients with synovial carcinoma. [0004] Besides the ability for genetically modified T cells expressing a CAR or a second TCR to recognize and destroy respective target cells in vitro/ex vivo, successful patient therapy with engineered T cells may require the T cells to be capable of strong activation, expansion, persistence over time, effective tumor targeting, and, in case of relapsing disease, enabling a ‘memory’ response.
  • recombinant nucleic acids comprising: a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked, and a second nucleic acid sequence encoding C-X-C chemokine receptor type 6 (CXCR6) or a functional fragment thereof.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • CXCR6 C-X-C chemokine receptor type 6
  • the TCR subunit further comprises a TCR intracellular domain.
  • the first and the second nucleic acid molecules are expressed in the same operon.
  • the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a sequence encoding a linker.
  • the linker comprises a protease cleavage site.
  • the protease cleavage site is a 2A cleavage site.
  • the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.
  • the first nucleic acid sequence and the second nucleic acid sequence are present on different nucleic acid molecules.
  • the CXCR6 or functional fragment thereof comprises a sequence with at least 80% sequence identity to any one selected from SEQ ID NOs: 400-402.
  • the CXCR6 or functional fragment thereof comprises a sequence of any one selected from SEQ ID NOs: 400-402.
  • the sequence of the recombinant nucleic acid is codon optimized.
  • the CXCR6 or functional fragment thereof is encoded by a nucleic acid with at least 60% sequence identity to SEQ ID NO: 427.
  • the CXCR6 or functional fragment thereof is encoded by the nucleic acid of SEQ ID NO: 427. [0019] In some embodiments, the CXCR6 or functional fragment thereof comprises at least one, two, three, or four extracellular domains.
  • the CXCR6 or functional fragment thereof comprises four extracellular domains.
  • the CXCR6 or functional fragment thereof comprises an N- terminal extracellular region comprising a sequence with at least 80% sequence identity to SEQ ID NO: 403.
  • the CXCR6 or functional fragment thereof comprises an N- terminal extracellular region comprising the sequence of SEQ ID NO: 403.
  • the CXCR6 or functional fragment thereof comprises a CXCL16- binding domain.
  • the CXCR6 or functional fragment thereof is associated with the cell membrane when expressed in a T cell.
  • the CXCR6 or functional fragment thereof comprises a transmembrane region comprising at least one, two, three, four, five, six, or seven transmembrane domains.
  • the transmembrane region comprises the sequence of any one of SEQ ID NOs 409-415, or any combination thereof.
  • the CXCR6 or functional fragment thereof comprises a transmembrane region comprising seven transmembrane domains.
  • the CXCR6 or functional fragment thereof further comprises the sequence of SEQ ID NO 406, the sequence of SEQ ID NO 407, the sequence of SEQ ID NO 408, the sequence of SEQ ID NO 416, the sequence of SEQ ID NO 417, the sequence of SEQ ID NO 418, or any combination thereof.
  • the CXCR6 or functional fragment thereof further comprises the sequence of SEQ ID NO 406, the sequence of SEQ ID NO 407, the sequence of SEQ ID NO 408, or a combination thereof; and the sequence of the sequence of SEQ ID NO 416, the sequence of SEQ ID NO 417, the sequence of SEQ ID NO 418, or any combination thereof.
  • the CXCR6 or functional fragment thereof comprises a transmembrane region comprising a sequence with at least 80% sequence identity to SEQ ID NO: 428.
  • the CXCR6 or functional fragment thereof comprises a transmembrane region comprising the sequence of SEQ ID NO: 428. [0032] In some embodiments, the CXCR6 or functional fragment thereof comprises at least one, two, three, or four cytoplasmic domains.
  • the CXCR6 or functional fragment thereof comprises four cytoplasmic domains.
  • the CXCR6 or functional fragment thereof comprises a C- terminal cytoplasmic domain comprising a sequence with at least 80% sequence identity to SEQ ID NO: 419.
  • the CXCR6 or functional fragment thereof comprises a cytoplasmic domain comprising the sequence of SEQ ID NO: 419.
  • migration of a cell expressing the CXCR6 or functional fragment thereof increases in response to CXCL16.
  • a migration rate of a cell expressing the CXCR6 or functional fragment thereof increases in response to CXCL16
  • the number of cells expressing the CXCR6 or functional fragment thereof that migrate to a tumor site increases in response to CXCL16
  • a combination thereof a migration rate of a cell expressing the CXCR6 or functional fragment thereof.
  • the recombinant nucleic acid as provided herein comprises a sequence encoding an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 420, SEQ ID NO: 424, SEQ ID NO: 426, or SEQ ID NO: 435.
  • the recombinant nucleic acid as provided herein comprises a sequence encoding the sequence of SEQ ID NO: 420, SEQ ID NO: 424, SEQ ID NO: 426, or SEQ ID NO: 435.
  • the TFP functionally interacts with an endogenous TCR complex when expressed in a T cell.
  • the TCR intracellular domain comprises a stimulatory domain from an intracellular signaling domain of CD3 gamma, CD3 delta, or CD3 epsilon.
  • the TCR intracellular domain comprises an intracellular domain from TCR alpha, TCR beta, TCR gamma, or TCR delta.
  • the antigen binding domain is connected to the TCR extracellular domain by a linker sequence.
  • the linker sequence is 120 amino acids in length or less.
  • the linker sequence comprises (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 10.
  • n is an integer from 1 to 4.
  • at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit.
  • at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR alpha.
  • At least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR beta.
  • At least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma.
  • At least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta.
  • At least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon.
  • At least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta.
  • At least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma.
  • all three of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit.
  • the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon.
  • the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta.
  • the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma.
  • the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR alpha.
  • the constant domain of TCR alpha is murine.
  • the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR beta.
  • the constant domain of TCR beta is murine.
  • the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR gamma.
  • the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain comprise the constant domain of TCR delta.
  • the antigen binding domain is a camelid antibody or binding fragment thereof.
  • the antigen binding domain is a murine antibody or binding fragment thereof.
  • the antigen binding domain is a human or humanized antibody or binding fragment thereof.
  • the antigen binding domain is a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain.
  • the antigen binding domain is a single domain antibody (sdAb).
  • the sdAb is a VH or VHH.
  • the antigen binding domain is selected from the group consisting of an anti-CD19 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, and an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, anti-MUC16 binding domain, an anti-Nectin-4 binding domain, an anti-GPC3 binding domain, and an anti-TROP-2 binding domain.
  • BCMA anti-B-cell maturation antigen
  • MSLN anti-mesothelin
  • a T cell expressing the TFP inhibits tumor growth.
  • the recombinant nucleic acid as provided herein further comprises a leader sequence.
  • the recombinant nucleic acid comprises a sequence encoding (i) a TFP comprising a GM-CSFRa signal peptide, an anti-MSLN scFv or VHH antibody or a fragment thereof, a linker, a CD3 epsilon intracellular signaling domain, and (ii) the CXCR6 or fragment thereof.
  • the recombinant nucleic acid comprises (i) a sequence encoding a TFP comprising, from the N-terminus to the C-terminus, the GM-CSFRa signal peptide operatively linked to the anti-MSLN scFv or VHH antibody or fragment thereof operatively linked to the linker operatively linked to the CD3 epsilon intracellular signaling domain and (ii) a sequence encoding CXCR6 or fragment thereof, or (i) the sequence encoding CXCR6 or fragment thereof and (ii) the sequence encoding the TFP comprising the GM-CSFRa signal peptide operatively linked to the anti-MSLN scFv or VHH antibody or fragment thereof operatively linked to the linker operatively linked to the CD3 epsilon intracellular signaling domain, wherein CXCR6 and the TFP are expressed in the same operon and are separated by a cleavable linker.
  • the linker is a A3(G4S)3LE linker.
  • the CD3 epsilon intracellular signaling domain is operatively linked to the CXCR6 or fragment thereof via a cleavable linker or the CXCR6 or fragment thereof is operatively linked to the GM-CSFRa signal peptide via the cleavable linker.
  • the cleavable linker is a 2A cleavage site or a furin cleavage site.
  • the 2A cleavage site is a P2A cleavage site or a T2A cleavage site.
  • the recombinant nucleic acid encodes a sequence comprising the sequences of SEQ ID NOs: 421, 422, 423, and 400.
  • the recombinant nucleic acid encodes a sequence comprising, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 421 operatively linked to the sequence of SEQ ID NO: 422 operatively linked to the sequence of SEQ ID NO: 423 operatively linked to the sequence of SEQ ID NO: 400, or the sequence of SEQ ID NO: 400 operatively linked to the sequence of SEQ ID NO: 421 operatively linked to the sequence of SEQ ID NO: 422 operatively linked to the linker operatively linked to the sequence of SEQ ID NO: 423.
  • the recombinant nucleic acid comprises a sequence encoding, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 422 is operatively linked to the sequence of SEQ ID NO: 423 via the sequence of SEQ ID NO: 387.
  • the recombinant nucleic acid comprises a sequence encoding, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 423 is operatively linked to the sequence of SEQ ID NO: 400 via the sequence of SEQ ID NO: 23 or the sequence of SEQ ID NO 425 operatively linked to the sequence of SEQ ID NO: 23, or the sequence of SEQ ID NO: 400 is operatively linked to the sequence of SEQ ID NO: 421 via the sequence of SEQ ID NO: 425 linked to the sequence of SEQ ID NO: 23.
  • the recombinant nucleic acid as provided herein further comprises a third nucleic acid sequence.
  • the third nucleic acid sequence encodes a switch polypeptide comprising a transforming growth factor beta receptor II (TGFBr2) extracellular domain or a functional fragment thereof.
  • TGFBr2 transforming growth factor beta receptor II
  • the TGFBr2 extracellular domain or functional fragment thereof comprises a sequence with at least 80% sequence identity to SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437. [0087] In some embodiments, the TGFBr2 extracellular domain or functional fragment thereof comprises the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437.
  • the switch polypeptide further comprises a switch intracellular domain.
  • the TGFBr2 extracellular domain or functional fragment thereof is operably linked to the switch intracellular domain.
  • the switch intracellular domain comprises an intracellular domain of a costimulatory polypeptide.
  • the costimulatory polypeptide is selected from the group consisting of CD28, 4-1BB, IL-15Ra, 0X40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, and FcyRIII.
  • the costimulatory polypeptide is CD28.
  • the costimulatory polypeptide is 4- IBB.
  • the costimulatory polypeptide is IL-15Ra.
  • the switch intracellular domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:273 or SEQ ID NO:277.
  • the switch intracellular domain comprises the sequence of SEQ ID NO:273 or SEQ ID NO:277.
  • the switch polypeptide further comprises a switch transmembrane domain.
  • the TGFBr2 extracellular domain or functional fragment thereof is operably linked to the switch intracellular domain via the switch transmembrane domain.
  • the switch transmembrane domain is a TGFBr2 transmembrane domain.
  • the switch transmembrane domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:272.
  • the switch transmembrane domain comprises the sequence of SEQ ID NO:272.
  • the switch transmembrane domain is a transmembrane domain of the costimulatory polypeptide.
  • the switch transmembrane domain is a transmembrane domain of CD28. [0104] In some embodiments, the switch transmembrane domain is a transmembrane domain of 4-1BB.
  • the switch transmembrane domain is a transmembrane domain of IL-15Ra.
  • the switch transmembrane domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:275 or SEQ ID NO:279.
  • the switch transmembrane domain comprises the sequence of SEQ ID NO:275 or SEQ ID NO:279.
  • the switch polypeptide further comprises an additional intracellular domain.
  • the additional intracellular domain is operably linked to the C- terminus of the switch intracellular domain.
  • the additional intracellular domain comprises an intracellular domain of IL-15Ra or signaling domain thereof.
  • the additional intracellular domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:372 or SEQ ID NO:383.
  • the additional intracellular domain comprises the sequence of SEQ ID NO:372 or SEQ ID NO:383.
  • the switch polypeptide comprises a TGFBr2 transmembrane domain and an intracellular signaling domain of 4-1BB.
  • the switch polypeptide comprises a 4-1BB transmembrane domain and an intracellular signaling domain of 4-1BB.
  • the switch polypeptide comprises a TGFBr2 transmembrane domain and an intracellular signaling domain of CD28.
  • the switch polypeptide comprises a CD28 transmembrane domain and an intracellular signaling domain of CD28.
  • the switch polypeptide comprises a sequence with at least 80% sequence identity to any one selected from SEQ ID NOs: 283, 284, 285, and 286.
  • the switch polypeptide comprises the sequence of SEQ ID Nos: 283, 284, 285, or 286.
  • the third nucleic acid sequence encodes a dominant negative TGFBR2 receptor or a fragment thereof.
  • the dominant negative TGFBR2 receptor or a fragment thereof comprises a sequence with at least 80% sequence identity to SEQ ID NO: 433 or SEQ ID NO: 434.
  • the dominant negative TGFBR2 receptor or a fragment thereof comprises the sequence of SEQ ID NO: 433 or SEQ ID NO: 434.
  • the third nucleic acid sequence encodes an interleukin- 15 (IL- 15) polypeptide or a fragment thereof.
  • IL- 15 interleukin- 15
  • expression of the IL- 15 polypeptide or fragment thereof increases persistence of a cell expressing the IL-15 polypeptide or fragment thereof.
  • the IL- 15 polypeptide or fragment thereof is secreted when expressed in a cell.
  • the IL- 15 polypeptide or fragment thereof comprises a sequence with at least 80% sequence identity to SEQ ID NO: 1242 or SEQ ID NO: 1245.
  • the IL- 15 polypeptide or fragment thereof comprises the sequence of SEQ ID NO: 1242 or SEQ ID NO: 1245.
  • the third nucleic acid sequence further encodes an IL-15 receptor (IL-15R) subunit or a fragment thereof.
  • IL-15R IL-15 receptor
  • the IL-15R subunit is IL-15R alpha (IL-15Ra).
  • the IL-15 polypeptide or fragment thereof and the IL-15Ra are operatively linked by a second linker.
  • the second linker is not a cleavable linker.
  • the second linker comprises a sequence comprising (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 10.
  • n is an integer from 1 to 4.
  • n 3.
  • the second linker comprises the sequence of SEQ ID NO: 1243.
  • the third nucleic acid sequence encodes a fusion protein comprising the IL-15 polypeptide or fragment thereof linked to the IL-15Ra subunit.
  • the IL- 15 polypeptide or fragment thereof is linked to N-terminus of the IL-15Ra subunit.
  • the fusion protein comprises amino acids 30 - 162 of IL-15. [0138] In some embodiments, the fusion protein comprises amino acids 31 - 267 of IL-15Ra. [0139] In some embodiments, the fusion protein further comprises a sushi domain. [0140] In some embodiments, the fusion protein comprises a sequence with at least 80% sequence identity to SEQ ID NO: 1253.
  • the fusion protein comprises the sequence of SEQ ID NO: 1253. [0142] In some embodiments, the fusion protein is expressed on cell surface when expressed in a cell.
  • the fusion protein is secreted when expressed in a cell.
  • the third nucleic acid sequence encodes a PD-1 polypeptide or a fragment thereof.
  • the PD-1 polypeptide or fragment thereof is operably linked via its C-terminus to the N-terminus of an intracellular domain of a costimulatory polypeptide.
  • the PD-1 polypeptide or fragment thereof is linked to the intracellular domain of the costimulatory polypeptide via a transmembrane domain of PD-1.
  • the costimulatory polypeptide is chosen from a group comprising 0X40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, and FcyRIII.
  • the intracellular domain of the costimulatory polypeptide comprises at least a portion of CD28.
  • an extracellular domain and a transmembrane domain of PD-1 are linked to an intracellular domain of CD28.
  • the third nucleic acid sequence encodes a fusion protein comprising the extracellular domain and the transmembrane domain of PD-1 are linked to the intracellular domain of CD28.
  • the fusion protein comprises a sequence with at least 80% sequence identity to SEQ ID NO: 1239 or SEQ ID NO: 1244.
  • the fusion protein comprises the sequence of SEQ ID NO: 1239 or SEQ ID NO: 1244.
  • the third nucleic acid sequence encodes a fusion protein comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15Ra.
  • the fusion protein comprises a sequence with at least 80% sequence identity to SEQ ID NO: 1254 or SEQ ID NO: 1262.
  • the fusion protein comprises the sequence of SEQ ID NO: 1254 or SEQ ID NO: 1262.
  • the third nucleic acid sequence is operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof.
  • the third nucleic acid sequence is operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof by a sequence encoding a third linker.
  • the third linker comprises a protease cleavage site.
  • the protease cleavage site is a 2A cleavage site.
  • the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.
  • the third nucleic acid sequence and the first nucleic acid sequence, the third nucleic acid sequence and the second nucleic acid sequence, or the third nucleic acid sequence, the first nucleic acid sequence, and the second nucleic acid sequence are present on different nucleic acid molecules.
  • the recombinant nucleic acid as provided herein further comprises a fourth nucleic acid sequence.
  • the fourth nucleic acid sequence encodes a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof, a dominant negative TGFBR2 receptor or a fragment thereof, an IL- 15 polypeptide or a fragment thereof, an IL- 15 polypeptide or a fragment thereof operatively linked to an IL-15R subunit or a fragment thereof, a PD-1 polypeptide or a fragment thereof, or a fusion protein comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15Ra.
  • the fourth nucleic acid sequence encodes a polypeptide having a sequence with at least 80% sequence identity to any one selected from SEQ ID NOs: 283, 284, 285, 286, 433, 434, 1242, 1245, 1253, 1239, 1244, 1254, and 1262.
  • the fourth nucleic acid sequence encodes a polypeptide having the sequence of SEQ ID NOs: 283, 284, 285, 286, 433, 434, 1242, 1245, 1253, 1239, 1244, 1254, or 1262.
  • the fourth nucleic acid sequence encodes a polypeptide different from a polypeptide encoded by the third nucleic acid sequence.
  • the fourth nucleic acid sequence is operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, or a combination thereof.
  • the third nucleic acid sequence is operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence by a sequence encoding a fourth linker.
  • the fourth linker comprises a protease cleavage site.
  • the protease cleavage site is a 2A cleavage site.
  • the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.
  • the fourth nucleic acid sequence and the first nucleic acid sequence, the fourth nucleic acid sequence and the second nucleic acid sequence, the fourth nucleic acid sequence and the third nucleic acid sequence, or the fourth nucleic acid sequence, the first nucleic acid sequence, and the second nucleic acid sequence, and the third nucleic acid sequence are present on different nucleic acid molecules.
  • the recombinant nucleic acid is selected from the group consisting of a DNA and an RNA.
  • the recombinant nucleic acid is an mRNA.
  • the recombinant nucleic acid is a circRNA.
  • the recombinant nucleic acid comprises a nucleotide analog.
  • the nucleotide analog is selected from the group consisting of 2’- O-methyl, 2’-O-methoxyethyl (2’-0-M0E), 2’-O-aminopropyl, 2’-deoxy, 2'-deoxy -2’ -fluoro, 2’-O-aminopropyl (2’-O-AP), 2'-O-dimethylaminoethyl (2’-O-DMAOE), 2’-O- dimethylaminopropyl (2’-O-DMAP), 2’-O-dimethylaminoethyloxyethyl (2’-O-DMAEOE), 2’- O-N-methylacetamido (2’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (H
  • LNA locked nucleic
  • the recombinant nucleic acid as provided herein further comprises a promoter.
  • the recombinant nucleic acid is an in vitro transcribed nucleic acid.
  • the recombinant nucleic acid as provided herein further comprises a sequence encoding a poly (A) tail.
  • the recombinant nucleic acid as provided herein further comprises a 3’UTR sequence.
  • polypeptides encoded by the recombinant nucleic acid as provided herein are provided herein.
  • vectors comprising a recombinant nucleic acid as provided herein.
  • the vector is a lentiviral vector.
  • cells comprising the recombinant nucleic acid as provided herein, the polypeptide as provided herein, or the vector as provided herein.
  • the cell is a T cell.
  • the T cell is a human T cell.
  • the T cell is a CD8+ or CD4+ T cell.
  • the T cell is a human aP T cell.
  • the T cell is a human y6 T cell.
  • the cell is a human NKT cell.
  • the cell is an allogeneic cell or an autologous cell.
  • the cell has increased anti-tumor efficacy compared to the antitumor efficacy of a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence.
  • the cell has enhanced migration compared to migration of a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence.
  • the cell in response to CXCL16 (i) the cell has a higher migration rate compared to a migration rate of a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence, (ii) more of the cells migrate to a tumor in response to CXCL16 compared to the number of cells comprising the first nucleic acid sequence and not comprising the second nucleic acid sequence that migrate to a tumor, or (iii) a combination thereof.
  • the cell has enhanced tumor lysis activity compared to a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence.
  • the cell has increased cytokine production compared to a cell that comprises the first nucleic acid sequence and does not comprise the second nucleic acid sequence.
  • the cell comprises a population of cells.
  • the population of cells comprises at least lxl0 A 5 cells or at least lxlO A 6 cells.
  • compositions comprising the cell as provided herein and a pharmaceutically acceptable carrier.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • the method comprising expressing CXCR6 or a functional fragment thereof in the cell: wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked.
  • the cell is the cell as provided herein.
  • the cell has enhanced migration compared to migration of a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof.
  • the migration rate of the cell in response to CXCL16 is faster compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof, (ii) more number of the cell migrates in response to CXCL16 compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof, or (iii) a combination thereof.
  • the cell has enhanced tumor lysis activity compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof.
  • the cell has increased cytokine production compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • the method comprising expressing CXCR6 or a functional fragment thereof in the cell: wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked.
  • the cell is the cell as provided herein. [0209] In some embodiments, (i) the migration rate of the cell in response to CXCL16 is faster compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof, (ii) more number of the cell migrates in response to CXCL16 compared to a cell that comprises the sequence encoding the TFP and does not express the CXCR6 or functional fragment thereof, or (iii) a combination thereof.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • the method comprising expressing CXCR6 or a functional fragment thereof in the cell: wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked.
  • the cell is the cell as provided herein.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • the method comprising expressing CXCR6 or a functional fragment thereof in the cell: wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked.
  • the cell is the cell as provided herein.
  • kits for treating a disease or a condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition as described herein.
  • the disease or the condition is a cancer or a disease or a condition associated with expression of CD 19, B-cell maturation antigen (BCMA), mesothelin (MSLN), CD20, CD70, MUC16, Trop-2, Nectin-4, or GPC3.
  • BCMA B-cell maturation antigen
  • MSLN mesothelin
  • CD20 CD70
  • MUC16 Trop-2
  • Nectin-4 GPC3.
  • the cancer is a hematologic cancer selected from the group consisting of B-cell acute lymphoid leukemia (B-ALL), T cell acute lymphoid leukemia (T- ALL), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell-follicular lymphoma, large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin’s lymphoma, plasmablastic
  • the cancer is mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, thyroid cancer, bladder cancer, ureter cancer, kidney cancer, endometrial cancer, esophageal cancer, gastric cancer, thymic carcinoma or cholangiocarcinoma.
  • the subject is a human.
  • FIG 1 is a graph showing the results of RNA-seq analysis on tumor samples from 33 different tumor types showing CXCL16 expression levels.
  • FIG. 2 is a series of plots showing cell surface expression of MH1 TFPs and cell surface and intracellular expression of CXCR6, as determined by flow cytometry.
  • FIG. 3 is a series of plots showing the memory phenotype (top) and CD4:CD8 T cell distribution (bottom) and of cells transduced with the TFPs as determined by flow cytometry. All plots are gated on CD3+ cells.
  • FIG. 4 is a schematic illustration of the transwell migration assay described in Example 3.
  • FIG. 5 is a series of graphs showing the proportion of cells expressing MH1 TFPs with or without CXCR6 that have migrated to the lower well of the transwell plate containing the concentrations of CXCL16 shown in the transwell migration assay described in Example 3 after 4 or 15 hours of incubation.
  • CXCR6 when CXCR6 is present, it is positioned upstream of the MH1 TFP on the lentiviral vector.
  • FIG 6 is a series of graphs showing the proportion of cells expressing MH1 TFPs with or without CXCR6 that have migrated to the lower well of the transwell plate containing the concentrations of CXCL16 shown in the transwell migration assay described in Example 3 after 4 hours of incubation.
  • FIG. 7 is a series of graphs the proportion of cells expressing MH1 TFPs with or without CXCR6 that have migrated to the lower well of the transwell plate containing supernatant of the cancer cell lines shown in the transwell migration assay described in Example 3 after 4 hours of incubation.
  • CXCR6 when CXCR6 is present, it is positioned upstream of the MH1 TFP on the lentiviral vector. Also shown in the level of soluble CXCL16 expression produced by each of the cancer cell lines.
  • FIG. 8 is a series of graphs showing cytotoxicity of T cells expressing the TFP constructs shown (with or without CXCR6) when contacted with MSTO-msln, OVCAR3, Suit- 2, or Panc-1 target cells, as described in Example 4.
  • FIGs. 9A-9D is a series of graphs showing cytokine expression (fFNy, GM-CSF, IL-2, and TNF-a) by T cells expressing the TFP constructs shown (with or without CXCR6) when contacted with MSTO-msln, OVCAR3, Suit-2, or Panc-1 target cells, as described in Example 5.
  • FIG. 9A shows GM-CSF expression.
  • FIG. 9B shows ZFNy expression.
  • FIG. 9C shows IL-2 expression.
  • FIG. 9D shows TNF-a expression.
  • FIG. 10 is a series of flow cytometry plots showing cell surface expression of CXCR6 on VHH+ (top row) and VHH- (bottom row) cells in each of the indicated groups.
  • FIG. 11 is a series of graphs showing the total CD3+ count; total VHH+ count; and %VHH+ cells that migrated to the lower well of the transwell plate containing the indicated concentration of CXCL16.
  • FIG. 12 is a series of graphs showing the total CD3+ count; total VHH+ count; and %VHH+ of cells that migrated to the lower well of the transwell plate containing 25 or 50 ng/mL CXCL16, in the presence or absence of CXCL16 blocking antibody.
  • FIG. 13 is a series of graphs showing the total CD3+ count; VHH MFI; and % VHH+ of cells that migrated to the lower well of the transwell plate containing MSTO-CXCL16 cells, at the indicated tumor cell:T cell ratio.
  • FIGs. 14A-14D is a series of graphs showing the % tumor lysis in the transwell plate migration and killing assay, at the indicated E:T, when MSTO-msln (FIG. 14A), Suit-2 (FIG. 14B), MSTO-CXCL16 (FIG. 14C), and MSTO-msln-CXCL16 (FIG. 14D) cells were used as the target cells.
  • FIGs. 15A-15D is a series of graphs showing the % of mouse CD45+ cells in the tumor (FIG. 15A) and of human CD45+ cells in the tumor (FIG. 15B), and the % of human CD45+ cells found in the spleen (FIG. 15C) and liver (FIG. 15D), as determined on Day 4 and Day 7 after administration of the CXCR6 TFP or control cells.
  • FIG. 16 is a series of graphs showing the total VHH+ cell count/mg tissue in the tumor, spleen, and liver of mice at Day 4 and Day 7 after administration of CXCR6 TFP or control cells. All groups except the NT group were gated on VHH+ (NT gated on total hCD45+).
  • FIG. 17 is a set of graphs showing the total number of VHH+ cells per tumor at Day 4 and Day 7 after administration of CXCR6 TFP or control cells. All groups except the NT group were gated on VHH+ (NT gated on total hCD45+).
  • FIG. 18 is a series of graphs showing the %VHH+ cells in the tumor, spleen, and liver at Day 4 and Day 7.
  • FIG. 19 is a series of graphs showing the % of VHH+ cells that are CXCR6+ in each group at Days 0, 4, and 7. All groups except the NT group were gated on VHH+ (NT gated on total hCD45+).
  • FIG. 20 is a series of graphs showing the % of VHH+ cells that are Ki67+ in each group at Days 0, 4, and 7. All groups except the NT group were gated on VHH+ (NT gated on total hCD45+).
  • FIG. 21 is a series of graphs showing the CD4:CD8 ratio of VHH+ cells in the tumor, spleen, and liver at Days 0, 4, and 7.
  • FIG. 22A and FIG. 22B provide a series of graphs showing the memory phenotype of VHH+ CD4+ (FIG. 22A) and VHH+ CD8+ (FIG. 22B) cells in the tumors of mice at Days 0, 4, and 7.
  • the present disclosure provides T cells expressing a T cell receptor (TCR) fusion protein (TFP) and CXCR6 or a functional fragment thereof.
  • TCR T cell receptor
  • CXCR6 CXCR6
  • the TFP and CXCR6 can be expressed from the same or different nucleic acid molecules.
  • the disclosure also provides a recombinant nucleic acid comprising a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) and a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof.
  • the TFP can comprise (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain.
  • the TCR subunit and the antigen binding domain can be operatively linked.
  • the first nucleic acid sequence and the second nucleic acid sequence can be present on the same or different nu
  • the present disclosure further provides a vector comprising the recombinant nucleic acid, a cell comprising the recombinant nucleic acid or the vector described herein, or a pharmaceutical composition comprising the cell (e.g., modified cell).
  • the present disclosure also provides a method of increasing migration of a cell expressing a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP).
  • the method can comprise expressing CXCR6 or a functional fragment thereof in the cell.
  • the present disclosure also provides a method of treating a disease such as cancer using the cell described herein.
  • the present disclosure further provides a method of enhancing tumor lysis activity of a cell expressing a recombinant nucleic acid comprising a sequence encoding a TFP, or a method of increasing cytokine production by a cell expressing a recombinant nucleic acid comprising a sequence encoding a TFP.
  • CXCR6 binds the ligand CXCL16.
  • CXCL16 has previously been shown to be highly expressed by pancreatic cancer cells.
  • CXCL16 is highly expressed on a wide variety of different tumor types in addition to pancreatic tumors.
  • CXCL16 can be present as a surface bound molecule or secreted into the surrounding tissue.
  • the secreted form of CXCL16 acts as a chemoattractant and induces proliferation and migration of cancer cells. It has surprisingly been shown herein that expression of CXCR6 in TFP expressing T cells allows migration of the T cells towards a CXCL16 chemokine gradient and towards the supernatant of a variety of types of tumor cells secreting CXCL16.
  • CXCL16 is the only ligand for CXCR6, which is unusual in the chemokine/receptor family and ensures that CXCR6 expressing TFP T cells will only be targeted to CXCL16 expressing cells, and not to cells expressing other cytokines.
  • the discoveries presented herein show that that expression of CXCR6 in TFP expressing T cells facilitates migration of the T cells into the tumor microenvironment where CXCL16 is present, thereby increasing the efficacy of TFP-expressing T cells in treating cancer and solid tumors, in particular.
  • an element means one element or more than one element.
  • the term “comprise” or variations thereof such as “comprises” or “comprising” are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers.
  • the term “comprising,” is inclusive and does not exclude additional, unrecited integers or method/process steps.
  • compositions and methods comprising or may be replaced with “consisting essentially of’ or “consisting of’.
  • the phrase “consisting essentially of’ is used herein to require the specified integer(s) or steps as well as those which do not materially affect the character or function of the claimed invention.
  • the term “consisting” is used to indicate the presence of the recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) alone.
  • “about” can mean plus or minus less than 1 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or greater than 30 percent, depending upon the situation and known or knowable by one skilled in the art.
  • subject or “subjects” or “individuals” may include, but are not limited to, mammals such as humans or non-human mammals, e.g., domesticated, agricultural or wild, animals, as well as birds, and aquatic animals.
  • “Patients” are subjects suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.
  • the term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human).
  • treating or “treatment” refers to any indicia of success in the treatment or amelioration of the disease or condition.
  • Treating can include, for example, reducing, delaying or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient.
  • “treat or prevent” is sometimes used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition, and contemplates a range of results directed to that end, including but not restricted to prevention of the condition entirely.
  • preventing refers to the prevention of the disease or condition, e.g., tumor formation, in the patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present disclosure and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual.
  • the disease or condition e.g., tumor formation
  • modulate and “modulation” refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.
  • a “therapeutically effective amount” is the amount of a composition or an active component thereof sufficient to provide a beneficial effect or to otherwise reduce a detrimental non-beneficial event to the individual to whom the composition is administered.
  • therapeutically effective dose herein is meant a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g. Lieberman, Pharmaceutical Dosage Forms (vols.
  • chemokines or “chemotactic cytokines” refer to a family of small cytokines or signaling proteins secreted by cells, which induce directed chemotaxis in nearby responsive cells.
  • the chemokine is approximately 8-10 kilodaltons in mass.
  • the chemokine has four cysteine residues in conserved locations that are key to forming their 3-dimensional shape.
  • the chemokine is classified into one of the four main subfamilies: CXC, CC, CX3C and XC.
  • the chemokine acts as a chemoattractant to guide the migration of cells.
  • the chemokines exert their biological effects by interacting with G protein-linked transmembrane receptors called chemokine receptors, which are selectively found on the surfaces of their target cells.
  • the chemokine is considered homeostatic and involved in controlling the migration of cells during normal processes of tissue maintenance or development.
  • the homeostatic chemokine controls cells of the immune system during processes of immune surveillance, for example, directing lymphocytes to the lymph nodes so they can screen for invasion of pathogens by interacting with antigen-presenting cells residing in these tissues.
  • the homeostatic chemokine is produced and secreted without any need to stimulate their source cells.
  • the homeostatic chemokine is constitutively produced in certain tissues.
  • the homeostatic chemokine is responsible for basal leukocyte migration.
  • the chemokine has roles in development.
  • the chemokine promotes angiogenesis, i.e., the growth of new blood vessels.
  • the chemokine guides cells to tissues that provide specific signals critical for cellular maturation.
  • Exemplary homeostatic chemokines include, but are not limited to, CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12 and CXCL13.
  • the chemokine is pro-inflammatory and induced during an immune response to recruit cells of the immune system to a site of infection.
  • the inflammatory chemokine is formed under pathological conditions, for example, on pro-inflammatory stimuli, such as TNF-alpha, LPS, and viruses.
  • release of the inflammatory chemokine is stimulated by pro-inflammatory cytokines, for example, interleukin 1.
  • the inflammatory chemokine functions as a chemoattractant for leukocytes and recruits monocytes, neutrophils and other effector cells from the blood to sites of infection or tissue damage.
  • the inflammatory chemokine actively participates in the inflammatory response attracting immune cells to the site of inflammation. In some embodiments, the inflammatory chemokine activates cells to initiate an immune response or promote wound healing. In some embodiments, the inflammatory chemokine is released by many different cell types and serves to guide cells of both innate immune system and adaptive immune system. Exemplary inflammatory chemokines include, but are not limited to CXCL8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10.
  • CXC chemokines also known as “a-chemokines,” refer to the chemokines that contain the two N-terminal cysteines separated by one amino acid, as represented in the name with an “X.”
  • the CXC chemokine is one of 17 different CXC chemokines described in mammals.
  • the CXC chemokine belongs to the category with a specific amino acid sequence (or motif) of glutamic acid-leucine- arginine (or ELR for short) immediately before the first cysteine of the CXC motif (ELR- positive).
  • ELR-negative the CXC chemokine belongs to the category without an ELR motif (ELR-negative).
  • the ELR-positive CXC chemokine specifically induces the migration of neutrophils, and interacts with chemokine receptors CXCR1 and CXCR2.
  • the examples of the ELR-positive CXC chemokine include, but are not limited to, interleukin-8 (IL-8), which, for example, induces neutrophils to leave the bloodstream and enter into the surrounding tissue.
  • the CXC chemokine that lacks the ELR motif tends to be chemoattractant for lymphocytes.
  • the examples of the ELR-negative CXC chemokine include, but are not limited to, CXCL13.
  • CXC chemokines bind to CXC chemokine receptors, of which seven have been discovered to date, designated CXCR1-7.
  • chemokine receptors refer to cytokine receptors found on the surface of certain cells that interact with a chemokine.
  • the chemokine receptors are G protein-coupled receptors containing 7 transmembrane domains that are found predominantly on the surface of leukocytes, making it one of the rhodopsin-like receptors.
  • the chemokine receptors are one of 19 different chemokine receptors that have been characterized and share many common structural features.
  • the chemokine receptors are composed of about 350 amino acids that are divided into a short and acidic N-terminal end, seven helical transmembrane domains with three intracellular and three extracellular hydrophilic loops, and an intracellular C-terminus containing serine and threonine residues that act as phosphorylation sites during receptor regulation.
  • the chemokine receptors comprise a short and acidic N-terminal end.
  • the chemokine receptors comprise seven helical transmembrane domains.
  • the chemokine receptors comprise seven helical transmembrane domains with three intracellular and three extracellular hydrophilic loops.
  • the chemokine receptors comprise seven helical transmembrane domains with three intracellular hydrophilic loops. In some embodiments, the chemokine receptors comprise seven helical transmembrane domains with three extracellular hydrophilic loops. In some embodiments, the chemokine receptors comprise an intracellular C-terminus. In some embodiments, the chemokine receptors comprise an intracellular C-terminus containing serine and threonine residues that act as phosphorylation sites during receptor regulation. In some embodiments, the chemokine receptors comprise the first two extracellular loops that are linked together by disulfide bonding between two conserved cysteine residues.
  • the chemokine receptors comprises the N-terminal end that binds to chemokines and is important for ligand specificity.
  • the G- proteins couple to the C-terminal end of the chemokine receptor, which is important for receptor signaling following ligand binding.
  • the chemokine receptors that share high amino acid identity in their primary sequences bind different ligands. In some embodiments, more than one chemokine bind to a single receptor and the chemokine receptors are redundant in their function.
  • the chemokine receptor is one of 20 distinct chemokine receptors discovered in humans.
  • the chemokine receptor has a rhodopsin-like 7- transmembrane structure and couples to G-protein for signal transduction within a cell.
  • the chemokine receptor is a member of a large protein family of G protein- coupled receptors.
  • the chemokine receptors following interaction with their specific chemokine ligands, the chemokine receptors trigger a flux in intracellular calcium ions (i.e., calcium signaling), which causes cell responses, including the onset of chemotaxis that traffics the cell to a desired location within the organism.
  • the chemokine receptor belongs to one of the following families, CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors and XC chemokine receptors that correspond to the 4 distinct subfamilies of chemokines that they bind.
  • four families of chemokine receptors differ in spacing of cysteine residues near N-terminal of the receptor.
  • CXC chemokine receptors are integral membrane proteins that specifically bind and respond to cytokines of the CXC chemokine family.
  • CXC chemokine receptors represent one subfamily of chemokine receptors, a large family of G protein-linked receptors that are known as seven transmembrane (7-TM) proteins, as they span the cell membrane seven times.
  • the CXC chemokine receptors is one of six known CXC chemokine receptors in mammals, named CXCR1 through CXCR6.
  • CXCR6 also known as C-X-C chemokine receptor type 6, BONZO, CD 186, STRL33, TYMSTR, and C-X-C motif chemokine receptor 6, refers to a chemokine receptor that is named based on its chromosomal location (within the chemokine receptor cluster on human chromosome 3p21) and its similarity to other chemokine receptors in its gene sequence.
  • CXCR6 binds the ligand CXCL16.
  • CXCR6 is more closely related in structure to CC chemokine receptors than to other CXC chemokine receptors.
  • CXCR6 includes any of the recombinant or naturally- occurring forms of CXCR6 or variants or homologs thereof that have or maintain CXCR6 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CXCR6.
  • CXCR6 is substantially identical to the protein identified by the UniProt reference number 000574 or a variant or homolog having substantial identity thereto.
  • CXCL16 also known as Chemokine (C-X-C motif) ligand 16, SCYB16, SR-PSOX, and CXCLG16, refers to a small cytokine that belongs to the CXC chemokine family.
  • CXCL16 is composed of a CXC chemokine domain, a mucin-like stalk, a transmembrane domain and a cytoplasmic tail containing a potential tyrosine phosphorylation site that may bind SH2.
  • CXCL16 includes any of the recombinant or naturally-occurring forms of CXCL16 or variants or homologs thereof that have or maintain CXCL16 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CXCL16.
  • CXCL16 is substantially identical to the protein identified by the UniProt reference number Q9H2A7 or a variant or homolog having substantial identity thereto.
  • fusion protein relates to a protein which is made of polypeptide parts from different sources. Accordingly, in some embodiments, it may be also understood as a chimeric protein.
  • fusion protein is used interchangeably with the term “switch polypeptide” or “switch-receptor.”
  • fusion proteins are proteins created through the joining of two or more genes (or, for example, cDNAs) that originally coded for separate proteins. Translation of this fusion gene (or, for example, fusion cDNA) results in a single polypeptide, for example, with functional properties derived from each of the original proteins.
  • recombinant fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics. Further details to the exemplary production of the fusion protein of the present invention are described herein.
  • TGFBr2 switch polypeptide refers to the TGFBr2 fusion proteins as described herein that receive an inhibitory signal by binding to, e.g., TGF-beta, and transform (e.g., “switch”) the signal via the co-stimulatory domain of the fusion protein into an activating signal.
  • the fusion protein further comprises an epitope tag.
  • An epitope tag as described herein can be a peptide epitope tag or a protein epitope tag. Examples of a peptide epitope tag includes, but are not limited to, 6X His (also known as His-tag or hexahistidine tag), FLAG (e.g., 3X FLAG), HA, Myc, and V5.
  • a protein epitope tag examples include, but are not limited to, green fluorescent protein (GFP), glutathione-S-transferase (GST), P-galactosidase (P-GAL), Luciferase, Maltose Binding Protein (MBP), Red Fluorescence Protein (RFP), and Vesicular Stomatitis Virus Glycoprotein (VSV-G).
  • GFP green fluorescent protein
  • GST glutathione-S-transferase
  • P-GAL P-galactosidase
  • Luciferase Maltose Binding Protein
  • MBP Maltose Binding Protein
  • RFP Red Fluorescence Protein
  • VSV-G Vesicular Stomatitis Virus Glycoprotein
  • the fusion protein further comprises a FLAG tag.
  • the fusion protein further comprises a 3X FLAG tag.
  • TGFBr2 or “transforming growth factor beta receptor II,” also known as transforming growth factor, beta receptor II, TGF beta receptor 2, TGFBR2, TGFBRII, AAT3, FAA3, LDS1B, LDS2, LDS2B, MFS2, RIIC, TAAD2, TGFR-2, TGFbeta-RII, transforming growth factor beta receptor 2, TBR-ii, TBRII, refers to a protein that is a member of the serine/threonine protein kinase family and the TGFB receptor subfamily.
  • TGFBr2 refers to a transmembrane protein that has a protein kinase domain, forms a heterodimeric complex with another receptor protein, and binds TGF-beta.
  • TGFBr2 includes any of the recombinant or naturally-occurring forms of TGFBr2 or variants or homologs thereof that have or maintain TGFBr2 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring TGFBr2.
  • TGFBr2 is substantially identical to the protein identified by the UniProt reference number P37173 or a variant or homolog having substantial identity thereto.
  • GMCSFRa also known as CSF2RA, CD116, Cluster of Differentiation 116, CDwl l6, CSF2R, CSF2RAX, CSF2RAY, CSF2RX, CSF2RY, GM-CSF-R-alpha, GMCSFR, GMR, SMDP4, colony stimulating factor 2 receptor alpha subunit, alphaGMR, colony stimulating factor 2 receptor subunit alpha, GMR-alpha, GMCSFR-alpha, granulocytemacrophage colony-stimulating factor receptor, as used herein, refers to a receptor for granulocyte-macrophage colony-stimulating factor, which stimulates the production of white blood cells.
  • GM-CSF and its receptor play a role in earlier stages of development.
  • GMCSFRa is associated with Surfactant metabolism dysfunction type 4.
  • GMCSFRa includes any of the recombinant or naturally- occurring forms of GMCSFRa or variants or homologs thereof that have or maintain GMCSFRa activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring GMCSFRa.
  • GMCSFRa is substantially identical to the protein identified by the UniProt reference number P15509 or a variant or homolog having substantial identity thereto.
  • CD28 also known as Cluster of Differentiation 28, CD28, Tp44, and CD28 molecule, as used herein, refers to a protein expressed on T cells that provides co-stimulatory signals required for T cell activation and survival.
  • CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins.
  • CD28 includes any of the recombinant or naturally-occurring forms of CD28 or variants or homologs thereof that have or maintain CD28 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD28.
  • CD28 is substantially identical to the protein identified by the UniProt reference number Pl 0747 or a variant or homolog having substantial identity thereto.
  • 2A 2A self-cleaving peptide
  • 2A peptide refers to a class of peptides, which can induce ribosomal skipping during translation of a protein in a cell. These peptides share a core sequence motif of DxExNPGP, and are found in a wide range of viral families. Exemplary members of 2A include, but are not limited to, P2A, E2A, F2A, and T2A. “T2A” refers to the 2A derived from thosea asigna virus, and the sequence is EGRGSLLTCGDVEENPGP (SEQ ID NO:23).
  • P2A refers to the 2A derived from porcine teschovirus-1 2A, and the sequence is ATNFSLLKQAGDVEENPGP (SEQ ID NO:269).
  • E2A refers to the 2 A derived from quine rhinitis A virus, and the sequence is QCTNYALLKLAGDVESNPGP (SEQ ID NO:280).
  • F2A is derived from foot-and-mouth disease virus 18, and the sequence is VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:281).
  • adding the 1 linker “GSG” (Gly-Ser-Gly) on the N-terminal of a 2A peptide helps with efficiency.
  • furin cleavage site refers to a cleavage site recognized by protease enzyme furin, also known as FUR, PACE, PCSK3, SPC1, and paired basic amino acid cleaving enzyme.
  • furin is a subtili sin-like proprotein convertase family.
  • furin cleaves proteins just downstream of a basic amino acid target sequence (canonically, Arg-X-(Arg/Lys) -Arg').
  • a “T cell receptor (TCR) fusion protein” or “TFP,” as used herein, includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T cell.
  • stimulation refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex.
  • a stimulatory domain or stimulatory molecule e.g., a TCR/CD3 complex
  • signal transduction event such as, but not limited to, signal transduction via the TCR/CD3 complex.
  • Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.
  • the term “stimulatory molecule” or “stimulatory domain,” as used herein, refers to a molecule or portion thereof expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway.
  • the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • a primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or “ITAM”.
  • ITAM immunoreceptor tyrosine-based activation motif
  • Examples of an IT AM containing primary cytoplasmic signaling sequence that is of particular use in the invention includes, but is not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”) and CD66d.
  • T cells may recognize these complexes using their T cell receptors (TCRs).
  • TCRs T cell receptors
  • MHC molecules are typically bound by TCRs as part of peptide:MHC complex.
  • the MHC molecule may be an MHC class I or II molecule.
  • the complex may be on the surface of an antigen presenting cell, such as a dendritic cell or a B cell, or any other cell, including cancer cells, or it may be immobilized by, for example, coating on to a bead or plate.
  • HLA human leukocyte antigen system
  • MHC major histocompatibility complex
  • HLA class I antigens A, B & C
  • HLA class II antigens DP, DQ, & DR
  • HLA alleles A, B and C present peptides derived mainly from intracellular proteins, e.g., proteins expressed within the cell.
  • T cells undergo a positive selection step to ensure recognition of self MHCs followed by a negative step to remove T cells that bind too strongly to MHC which present self-antigens.
  • a positive selection step to ensure recognition of self MHCs
  • a negative step to remove T cells that bind too strongly to MHC which present self-antigens.
  • certain T cells and the TCRs they express will only recognize peptides presented by certain types of MHC molecules - i.e., those encoded by particular HLA alleles. This is known as HLA restriction.
  • intracellular signaling domain refers to an intracellular portion of a molecule.
  • the intracellular signaling domain generates a signal that promotes an immune effector function of the TFP containing cell, e.g., a modified T-T cell.
  • immune effector function e.g., in a modified T-T cell, include, but are not limited to, cytolytic activity and T helper cell activity, including the secretion of cytokines.
  • the intracellular signaling domain comprises a primary intracellular signaling domain.
  • Exemplary primary intracellular signaling domains include, but are not limited to, those derived from the molecules responsible for primary stimulation, or antigen dependent simulation.
  • the intracellular signaling domain comprises a costimulatory intracellular domain.
  • Exemplary costimulatory intracellular signaling domains include, but are not limited to, those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.
  • a primary intracellular signaling domain comprises an IT AM (“immunoreceptor tyrosine-based activation motif’).
  • ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP12.
  • costimulatory molecule refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
  • costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response.
  • Exemplary costimulatory molecules include, but are not limited to, an MHC class 1 molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD2, CD27, CD28, CD5, ICAM-1, LFA-1 (CD1 la/CD18), 4-1BB (CD137), IL-15Ra, IL12R, IL18R, IL21R, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, and FcyRIII.
  • MHC class 1 molecule BTLA and a Toll ligand receptor
  • CD2 CD27, CD28, CD5, ICAM-1, LFA-1 (CD1 la/CD18), 4-1BB (CD137), IL-15Ra, IL12R, IL18R, IL21R, ICOS (CD278), GITR
  • a costimulatory intracellular signaling domain is the intracellular portion of a costimulatory molecule.
  • a costimulatory molecule is represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, IL-15Ra, IL12R, IL18R, IL21R, CD27, CD5, ICAM- 1, CD7, CD226, FcyRI, FcyRII, FcyRIII, and the like.
  • the intracellular signaling domain comprises the entire intracellular portion or the entire native intracellular signaling domain of the molecule from which it is derived, or a functional fragment thereof.
  • 4-1BB refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g, mouse, rodent, monkey, ape and the like; and a “4- IBB costimulatory domain,” as used herein, refers to amino acid residues 214-255 of GenBank Acc. No.
  • 4-1BB also known as TNFRSF9, 4-1BB, CD137, Cluster of Differentiation 137, CDwl37, ILA, tumor necrosis factor receptor superfamily member 9, and TNF receptor superfamily member 9, as used herein, includes any of the recombinant or naturally-occurring forms of 4-1BB or variants or homologs thereof that have or maintain 4- 1BB activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring 4-1BB.
  • 4- IBB is substantially identical to the protein identified by the UniProt reference number Q07011 or a variant or homolog having substantial identity thereto.
  • antibody refers to a protein, or polypeptide sequences derived from an immunoglobulin molecule, which specifically binds to an antigen. Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.
  • antibody fragment refers to at least one portion of an antibody, or recombinant variants thereof, that contains the antigen binding domain, i.e., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope.
  • antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, single-chain (sc)Fv (“scFv”) antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • VH heavy chain variable region
  • a camelid “VHH” domain refers to a heavy chain comprising a single variable antibody domain.
  • a scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide.
  • the scFv may comprise -linker-Vu or may comprise Vu-linker- .
  • the portion of the TFP composition of the disclosure comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), or a single chain antibody (scFv) derived from a murine, humanized or human antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci.
  • sdAb single domain antibody fragment
  • scFv single chain antibody
  • the antigen binding domain of a TFP composition of the disclosure comprises an antibody fragment.
  • the TFP comprises an antibody fragment that comprises a scFv or a sdAb.
  • recombinant antibody refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.
  • antigen refers to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response.
  • this immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA.
  • any DNA which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full length nucleotide sequence of a gene.
  • an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide.
  • a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.
  • CD3 or “Cluster of Differentiation 3,” as used herein, refers to a protein complex that is part of the T cell receptor that is involved in activating both the cytotoxic T cell and T helper cells. In some embodiments, it is composed of four distinct chains. For example, in some embodiments, the complex contains a CD3y chain, a CD36 chain, and two CD3s chains in mammals.
  • CD3s includes any of the recombinant or naturally-occurring forms of CD3s or variants or homologs thereof that have or maintain CD3s activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD3s.
  • CD3s is substantially identical to the protein identified by the UniProt reference number P07766 or a variant or homolog having substantial identity thereto.
  • CD36 “CD36 chain,” or “T-cell surface glycoprotein CD3 delta chain,” as used herein, includes any of the recombinant or naturally-occurring forms of CD36 or variants or homologs thereof that have or maintain CD36 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD36.
  • CD36 is substantially identical to the protein identified by the UniProt reference number P04234 or a variant or homolog having substantial identity thereto.
  • CD3y includes any of the recombinant or naturally-occurring forms of CD3y or variants or homologs thereof that have or maintain CD3y activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD3y.
  • CD3y is substantially identical to the protein identified by the UniProt reference number P09693 or a variant or homolog having substantial identity thereto.
  • CD 19 also known as B-lymphocyte antigen CD 19, B4, CVID3, and CD19 molecule, refers to the Cluster of Differentiation 19 protein, which is an antigenic determinant detectable on B cell leukemia precursor cells, other malignant B cells and most cells of the normal B cell lineage.
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD 19.
  • CD 19 is substantially identical to the protein identified by the UniProt reference number P15391 or a variant or homolog having substantial identity thereto.
  • BCMA refers to the B-cell maturation antigen, also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), Cluster of Differentiation 269 protein (CD269), BCM, TNFRSF13A, tumor necrosis factor receptor superfamily member 17, and TNF receptor superfamily member 17, which is a protein that in humans is encoded by the TNFRSF17 gene.
  • TNFRSF17 is a cell surface receptor of the TNF receptor superfamily which recognizes B-cell activating factor (BAFF) (see, e.g., Laabi et al., EMBO 11 (11): 3897- 904 (1992).
  • BCMA includes any of the recombinant or naturally-occurring forms of BCMA or variants or homologs thereof that have or maintain BCMA activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring BCMA.
  • BCMA is substantially identical to the protein identified by the UniProt reference number Q02223 or a variant or homolog having substantial identity thereto.
  • CD 16 also known as FcyRIII, refers to a cluster of differentiation molecule found on the surface of natural killer cells, neutrophil polymorphonuclear leukocytes, monocytes, and macrophages. CD16 has been identified as Fc receptors FcyRIIIa (CD 16a) and FcyRIIIb (CD 16b), which participate in signal transduction. In some embodiments, CD 16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC).
  • IgSF immunoglobulin superfamily
  • CD 16 includes any of the recombinant or naturally-occurring forms of CD 16 or variants or homologs thereof that have or maintain CD 16 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD 16.
  • CD 16 is substantially identical to the protein identified by the UniProt reference number P08637 (CD16a) or a variant or homolog having substantial identity thereto or the protein identified by the UniProt reference number 07501 (CD16b) or a variant or homolog having substantial identity thereto.
  • NKG2D in humans, is expressed by NK cells, y6 T cells and CD8+ aP T cells.
  • NKG2D recognizes induced-self proteins from MIC and RAET1/ULBP families which appear on the surface of stressed, malignant transformed, and infected cells.
  • NKG2D includes any of the recombinant or naturally-occurring forms of NKG2D or variants or homologs thereof that have or maintain NKG2D activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring NKG2D.
  • NKG2D is substantially identical to the protein identified by the UniProt reference number P26718 or a variant or homolog having substantial identity thereto.
  • mesothelin also known as MPF and SMRP, refers to a tumor differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. In some embodiments, mesothelin is over-expressed in several human tumors, including mesothelioma and ovarian and pancreatic adenocarcinoma.
  • MSLN includes any of the recombinant or naturally-occurring forms of MSLN or variants or homologs thereof that have or maintain MSLN activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring MSLN.
  • MSLN is substantially identical to the protein identified by the UniProt reference number QI 3421 or a variant or homolog having substantial identity thereto.
  • tyrosine-protein kinase transmembrane receptor ROR1 also known as ROR1
  • ROR1 neurotrophic tyrosine kinase, receptor-related 1 (NTRKR1), dJ537F10.1, receptor tyrosine kinase-like orphan receptor 1, and receptor tyrosine kinase like orphan receptor 1
  • NTRKR1 receptor-related 1
  • dJ537F10.1 receptor tyrosine kinase-like orphan receptor 1
  • receptor tyrosine kinase like orphan receptor 1 refers to a member of the receptor tyrosine kinase-like orphan receptor (ROR) family. It plays a role in metastasis of cancer.
  • R0R1 includes any of the recombinant or naturally- occurring forms of R0R1 or variants or homologs thereof that have or maintain R0R1 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring ROR1.
  • ROR1 is substantially identical to the protein identified by the UniProt reference number Q01973 or a variant or homolog having substantial identity thereto.
  • MUC16 also known as mucin 16, cell-surface associated, ovarian cancer- related tumor marker CA125, CA-125 (cancer antigen 125, carcinoma antigen 125, or carbohydrate antigen 125), mucin 16, and CA125, refers to a membrane-tethered mucin that contains an extracellular domain at its amino terminus, a large tandem repeat domain, and a transmembrane domain with a short cytoplasmic domain.
  • products of this gene have been used as a marker for different cancers, with higher expression levels associated with poorer outcomes.
  • MUC16 includes any of the recombinant or naturally-occurring forms of MUC16 or variants or homologs thereof that have or maintain MUC16 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring MUC16.
  • MUC16 is substantially identical to the protein identified by the UniProt reference number Q8WXI7 or a variant or homolog having substantial identity thereto.
  • CD22 also known as cluster of differentiation-22, sialic acid binding Ig-like lectin 2, SIGLEC-2, SIGLEC2, CD22 molecule, T cell surface antigen leu- 14, and B cell receptor CD22, refers to a protein that mediates B cell/B cell interactions, and is thought to be involved in, e.g., the localization of B cells in lymphoid tissues.
  • CD22 is associated with diseases including, but not limited to, refractory hematologic cancer and hairy cell leukemia.
  • CD22 includes any of the recombinant or naturally- occurring forms of CD22 or variants or homologs thereof that have or maintain CD22 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD22.
  • CD22 is substantially identical to the protein identified by the UniProt reference number P20273 or a variant or homolog having substantial identity thereto.
  • PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, PD-L1 and PD-L2.
  • PD-1 includes any of the recombinant or naturally-occurring forms of PD-1 or variants or homologs thereof that have or maintain PD-1 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PD-1.
  • PD-1 is substantially identical to the protein identified by the UniProt reference number Q15116 or a variant or homolog having substantial identity thereto.
  • PD- L1 may play a major role in suppressing the adaptive arm of immune system during particular events such as, e.g., pregnancy, tissue allografts, autoimmune disease and other disease states such as, e.g., hepatitis.
  • the adaptive immune system reacts to antigens that are associated with immune system activation by exogenous or endogenous danger signals.
  • clonal expansion of antigen-specific CD8+ T cells and/or CD4+ helper cells is propagated.
  • the binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via Immunoreceptor Tyrosine-Based Switch Motif (ITSM) motif.
  • SHP-1 or SHP-2 phosphatases
  • IMS Immunoreceptor Tyrosine-Based Switch Motif
  • PD-L1 includes any of the recombinant or naturally-occurring forms of PD-L1 or variants or homologs thereof that have or maintain PD-L1 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PD-L1.
  • PD-L1 is substantially identical to the protein identified by the UniProt reference number Q9NZQ7 or a variant or homolog having substantial identity thereto.
  • the term “PD-L2”, also known as B7-DC, is a ligand of PD-1.
  • the amino acid sequence of full length PD-L2 is provided in the Gene Bank under accession number NP 079515.2.
  • the term “PD-L1” also includes protein variants of PD-L1.
  • the term “PD-L2” includes recombinant PD-L2 or fragments thereof.
  • the term also includes, for example, affinity tagged (e.g., histidine tagged) PD-L2 or fragments thereof, mouse or human Fc tagged PD-L2 or fragments thereof, or PD-L2 or fragments thereof coupled to a signal sequence, for example, ROR1.
  • PD- L2 includes any of the recombinant or naturally-occurring forms of PD- L2 or variants or homologs thereof that have or maintain PD-L2 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PD-L2.
  • PD-L2 is substantially identical to the protein identified by the UniProt reference number Q9BQ51 or a variant or homolog having substantial identity thereto.
  • CD79a (Cluster of Differentiation 79a) and CD79P (Cluster of Differentiation 79P)” genes encode proteins that make up the B lymphocyte antigen receptor, a multimeric complex that includes the antigen-specific component, surface immunoglobulin (Ig).
  • Ig surface immunoglobulin
  • Surface Ig non-covalently associates with two other proteins, Ig-alpha and Ig-beta (encoded by CD79a and its paralog CD79P, respectively) which are necessary for expression and function of the B-cell antigen receptor. Functional disruption of this complex can lead to, e.g., human B-cell chronic lymphocytic leukemias.
  • CD79a protein includes any of the recombinant or naturally-occurring forms of CD79a protein or variants or homologs thereof that have or maintain CD79a protein activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD79a protein.
  • CD79a protein is substantially identical to the protein identified by the UniProt reference number Pl 1912 or a variant or homolog having substantial identity thereto.
  • CD79P protein includes any of the recombinant or naturally-occurring forms of CD79P protein or variants or homologs thereof that have or maintain CD79P protein activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD79P protein.
  • CD79P protein is substantially identical to the protein identified by the UniProt reference number P40259 or a variant or homolog having substantial identity thereto.
  • CD70 also known as CD27LG and TNFSF7, as referred herein, refers to a cytokine that is the ligand for CD27.
  • the CD70-CD27 pathway plays an important role in the generation and maintenance of T cell immunity, in particular, during antiviral responses.
  • CD70 induces the proliferation of co-stimulated T-cells and enhances the generation of cytolytic T-cells.
  • CD70 includes any of the recombinant or naturally- occurring forms of CD70 or variants or homologs thereof that have or maintain CD70 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD70.
  • CD70 is substantially identical to the protein identified by the UniProt reference number P32970 or a variant or homolog having substantial identity thereto.
  • Prostate-specific membrane antigen also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I), NAAG peptidase, FOLH1, FGCP, FOLH, GCP2, GCPII, NAALAD1, NAALAdase, PSM, mGCP, folate hydrolase (prostate-specific membrane antigen) 1, or folate hydrolase 1 is a type II membrane protein expressed in all forms of prostate tissue, including carcinoma.
  • the PSMA protein has a unique 3-part structure: a 19-amino-acid internal portion, a 24-amino-acid transmembrane portion, and a 707-amino-acid external portion.
  • PSMA acts as a glutamate- preferring carboxypeptidase.
  • PMSA expression is increased in cancer tissue in the prostate.
  • PSMA includes any of the recombinant or naturally-occurring forms of PSMA or variants or homologs thereof that have or maintain PSMA activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PSMA.
  • PSMA is substantially identical to the protein identified by the UniProt reference number Q04609 or a variant or homolog having substantial identity thereto.
  • HER2 also known as receptor tyrosine-protein kinase erbB-2, CD340 (cluster of differentiation 340), proto-oncogene Neu, ERBB2, human epidermal growth factor receptor 2, HER2/neu, HER-2, HER-2/neu, HER2, MLN 19, NEU, NGL, TKR1, erb-b2 receptor tyrosine kinase 2, encodes a member of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases. This protein has no ligand binding domain of its own and therefore cannot bind growth factors.
  • EGF epidermal growth factor
  • HER2 is amplified and/or overexpressed in 20-30% of invasive breast carcinomas.
  • HER2 -positive breast cancer is treated in a separate manner from other subtypes of breast cancer and commonly presents as more aggressive disease.
  • HER2 includes any of the recombinant or naturally-occurring forms of HER2 or variants or homologs thereof that have or maintain HER2 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HER2.
  • HER2 is substantially identical to the protein identified by the UniProt reference number P04626 or a variant or homolog having substantial identity thereto.
  • CD22 also known as cluster of differentiation-22, sialic acid binding Ig-like lectin 2, SIGLEC-2, SIGLEC2, CD22 molecule, T cell surface antigen leu- 14, and B cell receptor CD22, refers to a protein that mediates B cell/B cell interactions, and is thought to be involved in, e.g., the localization of B cells in lymphoid tissues.
  • CD22 is associated with diseases including, but not limited to, refractory hematologic cancer and hairy cell leukemia.
  • CD22 includes any of the recombinant or naturally- occurring forms of CD22 or variants or homologs thereof that have or maintain CD22 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD22.
  • CD22 is substantially identical to the protein identified by the UniProt reference number P20273 or a variant or homolog having substantial identity thereto.
  • IL-15R refers to a type I cytokine receptor that IL-15 binds to and signals through.
  • IL-15R is composed of three subunits: IL- 15 receptor alpha chain (“IL-15Ra” or CD215), IL-2 receptor beta chain (“IL-2RP” or CD122) and IL-2 receptor gamma/the common gamma chain (“IL-2RY/YC” or CD132).
  • human IL-15Ra precursor protein has a 30 amino acid signal peptide, a 175 amino acid extracellular domain, a 23 amino acid single membrane-spanning transmembrane stretch, and a 39 amino acid cytoplasmic (or intracellular) domain and contains N- and O-linked glycosylation sites.
  • IL-15Ra contains a Sushi domain (amino acid 31-95), which is essential for IL-15 binding.
  • IL-15Ra exists as a soluble form (sIL-15Ra).
  • sIL-15Ra is constitutively generated from the transmembrane receptor through a defined proteolytic cleavage, and this process can be enhanced by certain chemical agents, such as PMA.
  • the human sIL- 15Ra about 42 kDa in size, may prolong the half-life of IL-15 or potentiate IL-15 signaling through IL-15 binding and IL-2RP/YC heterodimer.
  • IL-15R shares subunits with IL-2R that contain the cytoplasmic motifs required for signal transduction
  • IL- 15 signaling has separate biological effects in vivo apart from many biological activities overlapping with IL-2 signaling due to IL-15Ra subunit that is unique to IL-15R, availability and concentration of IL- 15, the kinetics and affinity of IL-15-IL-15Ra binding.
  • IL-15 binds to IL-15Ra specifically with high affinity, which then associates with a complex composed of IL-2RP and fL-2Ry/yc subunits, expressed on the same cell (“cis- presentation”) or on a different cell (“trans-presentation”).
  • the interaction between IL-15 and IL-15Ra is independent of the complex composed of IL-2RP and fL-2Ry/yc subunits.
  • IL- 15 binding to the IL-2Rp/yc heterodimeric receptor induces JAK1 activation that phosphorylates STAT3 via the beta chain, and JAK3 activation that phosphorylates STAT5 via the gamma chain.
  • the IL-15/IL-15R interaction modulates T-cell development and homeostasis in memory CD8+ T-cell.
  • the IL-15/IL-15R interaction also modulates NK cell development, maintenance, expansion and activities.
  • IL-15Ra cytoplasmic (or intracellular) domain comprises amino acids 229-267 of IL-15Ra protein. In some embodiments, IL-15Ra cytoplasmic (or intracellular) domain comprises a sequence of SEQ ID NO:372. In some embodiments, IL-15Ra Sushi domain comprises amino acids 31-95 of IL-15Ra protein. In some embodiments, IL-15Ra Sushi domain comprises a sequence of SEQ ID NO:382. In some embodiments, IL-15Ra comprises the transmembrane domain and the cytoplasmic (intracellular) domain of IL-15Ra protein. In some embodiments, IL-15Ra comprises amino acids 96-267 of IL-15Ra protein.
  • IL-15Ra comprises a sequence of SEQ ID NO:383. In some embodiments, sIL-15Ra comprises amino acids 21-205 of IL-15Ra protein. In some embodiments, sIL-15Ra comprises a sequence of SEQ ID NO:379.
  • SEQ ID NO:372 (IL-15Ra intracellular domain) KSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL
  • SEQ ID NO:379 (Soluble IL-15Ra (sIL-15Ra)) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP
  • SEQ ID NO:382 (IL-15Ra Sushi domain) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIR
  • SEQ ID NO:383 (IL-15Ra region downstream of Sushi domain) DPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPS KSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLL CGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL
  • SEQ ID NO:386 (IL-15Ra full-length protein sequence)
  • SEQ ID NO:300 (IL-15Ra transmembrane domain) VAISTSTVLLCGLSAVSLLACYL
  • IL-15Ra also known as CD215, IL-15 receptor subunit alpha, IL-15R-alpha, IL- 15RA, and Interleukin- 15 receptor subunit alpha, as used herein, includes any of the recombinant or naturally-occurring forms of IL-15Ra or variants or homologs thereof that have or maintain IL-15Ra activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL-15Ra.
  • IL-15Ra is substantially identical to the protein identified by the UniProt reference number QI 3261 or a variant or homolog having substantial identity thereto.
  • IL-2RP also known as CD 122, IL-2 receptor subunit beta, IL-2R subunit beta, IL- 2RB, P70-75, IMD63, and Interleukin-2 receptor subunit beta, as used herein, includes any of the recombinant or naturally-occurring forms of IL-2RP or variants or homologs thereof that have or maintain IL-2RP activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL-2Rp.
  • IL-2RP is substantially identical to the protein identified by the UniProt reference number P14784 or a variant or homolog having substantial identity thereto.
  • IL-2 receptor gamma/the common gamma chain also known as IL-2RY/YC, IL2RG, CIDX, IL-2RG, IMD4, P64, SCIDX, SCIDX1, interleukin 2 receptor subunit gamma, or CD 132, as used herein, includes any of the recombinant or naturally-occurring forms of IL- 2Ry/yc or variants or homologs thereof that have or maintain IL-2RY/YC activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL-2Ry/yc.
  • IL-2Ry/yc is substantially identical to the protein identified by the UniProt reference number P31785 or a variant or homolog having substantial identity thereto.
  • the terms “cleave” or “cleavage” refer to the hydrolysis of phosphodiester bonds within the backbone of a recognition sequence within a target sequence that results in a double-stranded break within the target sequence, referred to herein as a “cleavage site”.
  • the cleavage site can be a self-cleaving peptide such as a T2A, P2A, E2A or F2A cleavage site.
  • the cleavage site can comprise a sequence of SEQ ID NO: 1261 (P2A: GSGATNFSLLKQAGDVEENPG).
  • transfer vector refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “transfer vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like.
  • Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • lentivirus refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).
  • Other examples of lentivirus vectors that may be used in the clinic include but are not limited to, e.g., the LENTIVECTORTM gene delivery technology from Oxford BioMedica, the LENTIMAXTM vector system from Lentigen, and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
  • homologous refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules.
  • two nucleic acid molecules such as, two DNA molecules or two RNA molecules
  • polypeptide molecules between two polypeptide molecules.
  • a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g, 9 of 10), are matched or homologous, the two sequences are 90% homologous.
  • “Humanized” forms of non-human (e.g, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • a humanized antib ody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance.
  • the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Human or “fully human,” as used herein, refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • A refers to adenosine
  • C refers to cytosine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • one or more amino acid residues within a TFP of the present disclosure can be replaced with other amino acid residues from the same side chain family and the altered TFP can be tested using the functional assays described herein.
  • the term “operably linked” or “transcriptional control,” as used herein, refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • operably linked refers to functional linkage between two heterologous nucleic acid sequence resulting in expression of both.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
  • nucleotide refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);
  • peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • promoter refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
  • promoter/regulatory sequence refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • the term “constitutive” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • the term “inducible” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • tissue-specific promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • linker and “flexible polypeptide linker” as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together.
  • the flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3.
  • the linkers include multiple repeats of (Gly2Ser), (GlySer) or (GlysSer). Also included within the scope of the present disclosure are linkers described in WO2012/138475 (incorporated herein by reference).
  • a 5’ cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the “front” or 5’ end of a eukaryotic messenger RNA shortly after the start of transcription.
  • the 5’ cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other.
  • RNA polymerase Shortly after the start of transcription, the 5’ end of the mRNA being synthesized is bound by a capsynthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction.
  • the capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.
  • /// vitro transcribed RNA refers to RNA, preferably mRNA, which has been synthesized in vitro.
  • the in vitro transcribed RNA is generated from an in vitro transcription vector.
  • the in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.
  • a “poly(A)” refers to a series of adenosines attached by polyadenylation to the mRNA.
  • the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400.
  • Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.
  • polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
  • mRNA messenger RNA
  • the 3’ poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase.
  • poly(A) tail is added onto transcripts that contain a specific sequence, the poly adenylation signal.
  • Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm.
  • the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase.
  • the cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site.
  • adenosine residues are added to the free 3’ end at the cleavage site.
  • transient refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.
  • signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • cell surface receptor includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.
  • a “substantially purified” cell refers to a cell that is essentially free of other cell types.
  • a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
  • a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state.
  • the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • transfected or “transformed” or “transduced,” as used herein, refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • the term “specifically binds,” as used herein, refers to an antibody, an antibody fragment or a specific ligand, which recognizes and binds a cognate binding partner (e.g., CD 19) present in a sample, but which does not necessarily and substantially recognize or bind other molecules in the sample.
  • a cognate binding partner e.g., CD 19
  • tumor antigen or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” refers to antigens that are common to specific hyperproliferative disorders.
  • the hyperproliferative disorder antigens of the present disclosure are derived from, cancers including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, NHL, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.
  • anti-tumor effect refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumor cell proliferation, decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition.
  • an “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the present disclosure in prevention of the occurrence of tumor in the first place.
  • autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • allogeneic or, alternatively, “allogenic,” as used herein, refers to any material derived from a different animal of the same species or different patient as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenicallys.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • tumor is a solid tumor.
  • tumor is a hematologic malignancy.
  • cancer refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • a “chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer.
  • Chemotherapeutic agents include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer.
  • encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the noncoding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain one or more introns.
  • effective amount and therapeutically effective amount are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological or therapeutic result.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • expression refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
  • parenteral administration of an immunogenic composition includes, e.g, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, intratumoral, or infusion techniques.
  • terapéutica means a treatment.
  • a therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.
  • prophylaxis means the prevention of or protective treatment for a disease or disease state.
  • a functional disruption refers to a physical or biochemical change to a specific (e.g., target) nucleic acid (e.g., gene, RNA transcript, of protein encoded thereby) that prevents its normal expression and/or behavior in the cell.
  • a functional disruption refers to a modification of the gene via a gene editing method.
  • a functional disruption prevents expression of a target gene (e.g, an endogenous gene).
  • the term “meganuclease” refers to an endonuclease that binds doublestranded DNA at a recognition sequence that is greater than 12 base pairs.
  • the recognition sequence for a meganuclease of the present disclosure is 22 base pairs.
  • a meganuclease may be an endonuclease that is derived from I- Crel and may refer to an engineered variant of I-Crel that has been modified relative to natural I- Crel with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA- binding affinity, or dimerization properties.
  • a meganuclease binds to double-stranded DNA as a heterodimer or as a “single-chain meganuclease” in which a pair of DNA-binding domains are joined into a single polypeptide using a peptide linker.
  • meganucleases are substantially non-toxic when expressed in cells, particularly in human T cells, such that cells may be transfected and maintained at 37°C without observing deleterious effects on cell viability or significant reductions in meganuclease cleavage activity when measured using the methods described herein.
  • single-chain meganuclease refers to a polypeptide comprising a pair of nuclease subunits joined by a linker.
  • a single-chain meganuclease has the organization: N-terminal subunit - Linker - C-terminal subunit.
  • the two meganuclease subunits may generally be non-identical in amino acid sequence and may recognize nonidentical DNA sequences.
  • single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences.
  • a single-chain meganuclease may be referred to as a “single-chain heterodimer” or “single-chain heterodimeric meganuclease” although it is not, in fact, dimeric.
  • the term “meganuclease” can refer to a dimeric or single-chain meganuclease.
  • TALEN refers to an endonuclease comprising a DNA-binding domain comprising 16-22 TAL domain repeats fused to any portion of the Fokl nuclease domain.
  • Compact TALEN refers to an endonuclease comprising a DNA-binding domain with 16-22 TAL domain repeats fused in any orientation to any catalytically active portion of nuclease domain of the I-Tevl homing endonuclease.
  • CRISPR refers to a caspase-based endonuclease comprising a caspase, such as Cas9, and a guide RNA that directs DNA cleavage of the caspase by hybridizing to a recognition site in the genomic DNA.
  • megaTAL refers to a single-chain nuclease comprising a transcription activator-like effector (TALE) DNA binding domain with an engineered, sequencespecific homing endonuclease.
  • TALE transcription activator-like effector
  • T cell receptor and “T cell receptor complex” are used interchangeably to refer to a molecule found on the surface of T cells that is, in general, responsible for recognizing antigens.
  • the TCR comprises a heterodimer consisting of a TCR alpha and TCR beta chain in 95% of T cells, whereas 5% of T cells have TCRs consisting of TCR gamma and TCR delta chains.
  • the TCR further comprises one or more of CD3s, CD3y, and CD36.
  • the TCR comprises CD3s.
  • the TCR comprises CD3y.
  • the TCR comprises CD35.
  • the TCR comprises CD3( ⁇ .
  • the constant domain of human TCR alpha has a sequence of SEQ ID NO: 142.
  • the constant domain of human TCR alpha has an IgC domain having a sequence of SEQ ID NO: 143, a transmembrane domain having a sequence of SEQ ID NO: 144, and an intracellular domain having a sequence of SS.
  • the constant domain of murine TCR alpha has a sequence of SEQ ID NO: 147.
  • the constant domain of murine TCR alpha has a transmembrane domain having a sequence of SEQ ID NO: 144, and an intracellular domain having a sequence of SS.
  • the constant domain of human TCR beta has a sequence of SEQ ID NO: 148.
  • the constant domain of human TCR beta has an IgC domain having a sequence of SEQ ID NO: 149, a transmembrane domain having a sequence of SEQ ID NO: 150, and an intracellular domain having a sequence of SEQ ID NO: 151.
  • the constant domain of murine TCR beta has a sequence of SEQ ID NO: 152.
  • the constant domain of murine TCR beta has a transmembrane domain having a sequence of SEQ ID NO: 152, and an intracellular domain having a sequence of SEQ ID NO: 153.
  • the constant domain of human TCR delta has a sequence of SEQ ID NO:243.
  • the constant domain of human TCR delta has an IgC domain having a sequence of SEQ ID NO:265, a transmembrane domain having a sequence of SEQ ID NO: 158, and an intracellular domain having a sequence of L.
  • the constant domain of human TCR gamma has a sequence of SEQ ID NO:21.
  • the constant domain of human TCR gamma has an IgC domain having a sequence of SEQ ID NO: 155, a transmembrane domain having a sequence of SEQ ID NO: 156, and an intracellular domain having a sequence of SEQ ID NO: 157.
  • human CD3 epsilon has a sequence of SEQ ID NO:258.
  • human CD3 epsilon has an extracellular domain having a sequence of SEQ ID NO: 126, a transmembrane domain having a sequence of SEQ ID NO: 127, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO: 128.
  • human CD3 delta has a sequence of SEQ ID NO: 136.
  • human CD3 delta has an extracellular domain having a sequence of SEQ ID NO: 138, a transmembrane domain having a sequence of SEQ ID NO: 139, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO: 140.
  • human CD3 gamma has a sequence of SEQ ID NO: 130.
  • human CD3 gamma has an extracellular domain having a sequence of SEQ ID NO: 132, a transmembrane domain having a sequence of SEQ ID NO: 133, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO: 134.
  • Ranges throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6.
  • a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.
  • compositions of matter and methods of use for the treatment of a disease such as cancer using recombinant nucleic acids comprising: a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP), wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked, and a second nucleic acid sequence encoding CXCR6 or a functional fragment there.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • a “T cell receptor (TCR) fusion protein” or “TFP” includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when colocated in or on the surface of a T cell.
  • TFPs provide substantial benefits as compared to Chimeric Antigen Receptors.
  • CAR Chimeric Antigen Receptor
  • a CAR refers to a recombinant polypeptide comprising an extracellular antigen binding domain in the form of, e.g., a single domain antibody or scFv, a transmembrane domain, and cytoplasmic signaling domains (also referred to herein as “intracellular signaling domains”) comprising a functional signaling domain derived from a stimulatory molecule as defined below.
  • intracellular signaling domain of a CAR is derived from the CD3 zeta chain that is normally found associated with the TCR complex.
  • the CD3 zeta signaling domain can be fused with one or more functional signaling domains derived from at least one costimulatory molecule such as 4-1BB (i.e., CD137), CD27 and/or CD28.
  • C-X-C chemokine receptor type 6 CXCR6
  • TCR T cell receptor
  • TFP T cell receptor
  • TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked, and a second nucleic acid sequence encoding C-X-C chemokine receptor type 6 (CXCR6) or a functional fragment thereof.
  • the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a linker.
  • the linker comprises a protease cleavage site.
  • the protease cleavage site is a 2A cleavage site.
  • the cleavage site can be a self-cleaving peptide such as a T2A, P2A, E2A or F2A cleavage site.
  • the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.
  • CXCR6 or a functional fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence listed in Table 4 or a fragment thereof. In some embodiments, CXCR6 or a functional fragment thereof comprises any one sequence listed in Table 4 or a fragment thereof.
  • sequence of CXCR6 or a functional fragment thereof is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to any one sequence listed in Table 4 or a fragment thereof. In some embodiments, the sequence of CXCR6 or a functional fragment thereof is any one sequence listed in Table 4 or a fragment thereof.
  • CXCR6 or a functional fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID N0s:400-402. In some embodiments, CXCR6 or a functional fragment thereof comprises any one sequence selected from SEQ ID N0s:400-402.
  • sequence of CXCR6 or a functional fragment thereof is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to any one sequence selected from SEQ ID N0s:400-402. In some embodiments, the sequence of CXCR6 or a functional fragment thereof is any one sequence selected from SEQ ID N0s:400-402.
  • CXCR6 or a functional fragment thereof comprises an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
  • CXCR6 or a functional fragment thereof comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
  • CXCR6 or a functional fragment thereof comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of any one sequence selected from SEQ ID NO:400 - SEQ ID NO:402.
  • CXCR6 or a functional fragment thereof comprises amino acid residue deletions from the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C-terminal end of a sequence of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C-terminal end of a sequence of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C-terminal end of any one sequence selected from SEQ ID NO:400 - SEQ ID NO:402.
  • CXCR6 or a functional fragment thereof comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
  • CXCR6 or a functional fragment thereof comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of a sequence of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of a sequence of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
  • CXCR6 or a functional fragment thereof comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of any one sequence selected from SEQ ID NO:400 - SEQ ID NO:402.
  • the sequence of the recombinant nucleic acid encoding CXCR6 or a functional fragment thereof is codon optimized.
  • the CXCR6 or functional fragment thereof is encoded by a nucleic acid with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 76%, 96%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO:427.
  • the CXCR6 or functional fragment thereof is encoded by a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:427.
  • the CXCR6 or functional fragment thereof is encoded by the nucleic acid sequence of SEQ ID NO:427.
  • CXCR6 or a functional fragment thereof comprises at least one extracellular domain. In some embodiments, CXCR6 or a functional fragment thereof comprises at least two extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at least three extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at least four extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at most one extracellular domain. In some embodiments, CXCR6 or a functional fragment thereof comprises at most two extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at most three extracellular domains.
  • CXCR6 or a functional fragment thereof comprises at most four extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises one extracellular domain. In some embodiments, CXCR6 or a functional fragment thereof comprises two extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises three extracellular domains. In some embodiments, CXCR6 or a functional fragment thereof comprises four extracellular domains.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:403.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region comprising the sequence of SEQ ID NO:403.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:403.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region of which sequence is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to SEQ ID NO:403.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region of which sequence is the sequence of SEQ ID NO:403.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the N-terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises an N- terminal extracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the N-terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:403.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:403.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the N-terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises an N- terminal extracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids deleted from the N-terminal or C-terminal end of the sequence of the N-terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids deleted from the N-terminal or C-terminal end of the sequence of the N-terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids deleted from the N- terminal or C-terminal end of the sequence of SEQ ID NO:403.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids deleted from the N- terminal or C-terminal end of the sequence of SEQ ID NO:403.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of the N-terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of the N- terminal extracellular domain of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:403.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:403.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:404 or SEQ ID NO:405.
  • CXCR6 or a functional fragment thereof comprises an N- terminal extracellular region comprising the sequence of SEQ ID NO:404 or SEQ ID NO:405.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:404 or SEQ ID NO:405.
  • CXCR6 or a functional fragment thereof comprises an N- terminal extracellular region of which sequence is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to SEQ ID NO:404 or SEQ ID NO:405.
  • CXCR6 or a functional fragment thereof comprises an N-terminal extracellular region of which sequence is the sequence of SEQ ID NO:404 or SEQ ID NO:405.
  • CXCR6 or a functional fragment thereof comprises one or more extracellular domains selected from SEQ ID NO:403-SEQ ID NO:405. In some embodiments, CXCR6 or a functional fragment thereof further comprises one or more extracellular domains selected from SEQ ID NO:406-SEQ ID NO:408. [0407] In some embodiments, CXCR6 or a functional fragment thereof comprises a CXCL16- binding domain.
  • CXCR6 or a functional fragment thereof is associated with the cell membrane when expressed in a T cell. In some embodiments, CXCR6 or a functional fragment thereof is a membrane-bound protein.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at least one transmembrane domain. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at least two transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at least three transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at least four transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at least five transmembrane domains.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at least six transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at least seven transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at most one transmembrane domain. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at most two transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at most three transmembrane domains.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at most four transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at most five transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at most six transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising at most seven transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising one transmembrane domain.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region comprising two transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising three transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising four transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising five transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising six transmembrane domains. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region comprising seven transmembrane domains.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:428.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region comprising the sequence of SEQ ID NO:428.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region of which sequence is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to SEQ ID NO:428. In some embodiments, CXCR6 or a functional fragment thereof comprises a transmembrane region of which sequence is the sequence of SEQ ID NO:428.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:428.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the sequence of SEQ ID NO:428.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having a deletion of amino acid residue(s) from the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids deleted from the N-terminal or C-terminal end of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids deleted from the N-terminal or C-terminal end of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:428.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:428.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids independently deleted from both N- terminal and C-terminal ends of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of the transmembrane region of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:428.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids independently deleted from both N-terminal and C- terminal ends of the sequence of SEQ ID NO:428.
  • CXCR6 fragment comprising seven transmembrane domains (amino acid residues 33- 293):
  • CXCR6 transmembrane domain 1 (amino acid residues 33-59): FLPCMYLVVFVCGLVGNSLVLVISIFY (SEQ ID NO:409)
  • CXCR6 transmembrane domain 2 (amino acid residues 69-89): FLVNLPLADLVFVCTLPFWAY (SEQ ID NO:410)
  • CXCR6 transmembrane domain 3 (amino acid residues 104-125): SLLGIYTINFYTSMLILTCITV (SEQ ID NO:411)
  • CXCR6 transmembrane domain 4 (amino acid residues 144-164): RMTWGKVTSLLIWVISLLVSL (SEQ ID NO:412)
  • CXCR6 transmembrane domain 5 (amino acid residues 188-215): STWLATQMTLGFFLPLLTMIVCYSVII (SEQ ID NO:413)
  • CXCR6 transmembrane domain 6 (amino acid residues 232-259): IIFLVMAVFLLTQMPFNLMKFIRSTHWE (SEQ ID NOAM)
  • CXCR6 transmembrane domain 7 (amino acid residues 276-293): IAYLRACLNPVLYAFVSL (SEQ ID NO:415)
  • CXCR6 or a functional fragment thereof comprises a transmembrane region comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to one or more transmembrane domains selected from SEQ ID NO:409-SEQ ID NO:415.
  • CXCR6 or a functional fragment thereof comprises a transmembrane region comprising one or more transmembrane domains selected from SEQ ID NO:409- SEQ ID NO:415.
  • CXCR6 or a functional fragment thereof further comprises (i) one, two, or three cytoplasmic domains, (ii) one, two, or three extracellular domains of CXCR6, or (iii) a combination thereof.
  • CXCR6 or a functional fragment thereof further comprises (i) a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:406, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:407, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:408, or a combination thereof;
  • CXCR6 or a functional fragment thereof further comprises (i) the sequence of SEQ ID NO:406, the sequence of SEQ ID NO:407, the sequence of SEQ ID NO:408, or a combination thereof; (ii) the sequence of SEQ ID NO:416, the sequence of SEQ ID NO:417, the sequence of SEQ ID NO:418, or any combination thereof; or (iii) a combination thereof.
  • CXCR6 or a functional fragment thereof further comprises (i) a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:406, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:407, a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:408, or a combination thereof; and (ii) a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ
  • CXCR6 or a functional fragment thereof further comprises (i) the sequence of SEQ ID NO4:06, the sequence of SEQ ID NO:407, the sequence of SEQ ID NO:408, or a combination thereof; and (ii) the sequence of SEQ ID NO:416, the sequence of SEQ ID NO:417, the sequence of SEQ ID NO:418, or any combination thereof.
  • CXCR6 or a functional fragment thereof comprises at least one cytoplasmic domain. In some embodiments, CXCR6 or a functional fragment thereof comprises at least two cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at least three cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at least four cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at most one cytoplasmic domain. In some embodiments, CXCR6 or a functional fragment thereof comprises at most two cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises at most three cytoplasmic domains.
  • CXCR6 or a functional fragment thereof comprises at most four cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises one cytoplasmic domain. In some embodiments, CXCR6 or a functional fragment thereof comprises two cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises three cytoplasmic domains. In some embodiments, CXCR6 or a functional fragment thereof comprises four cytoplasmic domains.
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:419.
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain comprising the sequence of SEQ ID NO:419.
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain of which sequence is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to SEQ ID NO:419.
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain of which sequence is the sequence of SEQ ID NO:419.
  • the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:419.
  • the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof comprises the sequence of SEQ ID NO:419.
  • sequence of the C- terminal cytoplasmic domain of CXCR6 or a functional fragment thereof is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to SEQ ID NO:419.
  • sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof is the sequence of SEQ ID NO:419.
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a C- terminal cytoplasmic domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasm
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a C- terminal cytoplasmic domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, or 45 amino acids deleted from the N-terminal or C-terminal end of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, or 45 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:419.
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, or 45 amino acids independently deleted from both N-terminal and C- terminal ends of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, or 45 amino acids independently deleted from both N- terminal and C-terminal ends of the sequence of the C-terminal cytoplasmic domain of CXCR6 or a functional fragment thereof as described herein.
  • CXCR6 or a functional fragment thereof comprises a C-terminal cytoplasmic domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, or 45 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:419.
  • CXCR6 or a functional fragment thereof comprises a C- terminal cytoplasmic domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, or 45 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:419.
  • CXCR6 cytoplasmic domain 1 amino acid residues 60-68:
  • CXCR6 cytoplasmic domain 2 (amino acid residues 126-143): DRFIVVVKATKAYNQQAK (SEQ ID NO:417)
  • CXCR6 cytoplasmic domain 3 (amino acid residues 216-231): KTLLHAGGFQKHRSLK (SEQ ID NO:418)
  • CXCR6 C-terminal cytoplasmic domain (amino acid residues 294-342): KFRKNFWKLVKDIGCLPYLGVSHQWKSSEDNSKTFSASHNVEATSMFQL (SEQ ID NO:419)
  • CXCR6 or a functional fragment thereof comprises a cytoplasmic domain comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to one or more cytoplasmic domain selected from SEQ ID NO:416 SEQ ID NO:419.
  • CXCR6 or a functional fragment thereof comprises a cytoplasmic domain comprising one or more cytoplasmic domain selected from SEQ ID NO:416 SEQ ID NO:419.
  • the recombinant nucleic acid comprising CXCR6 or a functional fragment thereof as described herein comprises a sequence encoding an amino acid sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:420, SEQ ID NO:424, SEQ ID NO:426, or SEQ ID NO:435.
  • the recombinant nucleic acid comprising CXCR6 or a functional fragment thereof as described herein comprises a sequence encoding the sequence of SEQ ID NO:420, SEQ ID NO:424, SEQ ID NO:426, or SEQ ID NO:435. In some embodiments, the recombinant nucleic acid comprising CXCR6 or a functional fragment thereof as described herein comprises the sequence of SEQ ID NO:426.
  • the sequence of the recombinant nucleic acid comprising CXCR6 or a functional fragment thereof as described herein is a sequence encoding an amino acid sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:420, SEQ ID NO:424, SEQ ID NO:426, or SEQ ID NO:435.
  • sequence of the recombinant nucleic acid comprising CXCR6 or a functional fragment thereof as described herein is a sequence encoding the sequence of SEQ ID NO:420, SEQ ID NO:424, SEQ ID NO:426, or SEQ ID NO:435.
  • the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein further comprises a sequence encoding an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof.
  • the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein further comprises a sequence encoding the amino acid sequence of SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof.
  • the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein further encodes an amino acid sequence having at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof.
  • the recombinant nucleic acid as described herein or recombinant nucleic acid molecule as described herein further encodes the amino acid sequence of SEQ ID NO:438 or SEQ ID NO:439 or a fragment thereof.
  • TCR T-cell Receptor
  • TFPs Fusion Proteins
  • the present disclosure encompasses recombinant nucleic acid constructs encoding TFPs, wherein the TFP comprises a binding domain, e.g., an antibody or antibody fragment, a ligand, or a ligand binding protein, wherein the sequence of the binding domain is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • a binding domain e.g., an antibody or antibody fragment, a ligand, or a ligand binding protein
  • the antibody or antibody fragment can comprise an antigen binding domain selected from a group consisting of an anti-CD19 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, an anti-CD79b binding domain, , an anti-PMSA binding domain, an anti-MUC16 binding domain, an anti-CD22 binding domain, an anti-PD-Ll binding domain, an anti B AFF receptor binding domain, an anti-Nectin-4 binding domain, an anti-TROP-2 binding domain, an anti-GPC3 binding domain, and anti-ROR-1 binding domain.
  • an antigen binding domain selected from a group consisting of an anti-CD19 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, an anti-CD79b binding domain, ,
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to a tumor associated antigen (a TAA) wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • TAA tumor associated antigen
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD 19, e.g., human CD 19, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant nucleic acid, e.g., DNA, constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to mesothelin, e.g., human mesothelin, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • TFP comprises an antibody fragment that binds specifically to mesothelin, e.g., human mesothelin, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to MUC16, e.g., human MUC16, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD20, e.g., human CD20, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD70, e.g., human CD70, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD79B, e.g., human CD79B, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to HER2, e.g., human HER2, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to PSMA, e.g., human PSMA, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to BCMA, e.g., human BCMA, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to R0R1, e.g., human R0R1, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD22, e.g., human CD22, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to GPC3, e.g., human GPC3, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to Nectin-4, e.g., human Nectin-4, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to Trop-2, e.g., human Trop-2, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the TFPs provided herein are able to associate with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR subunits in order to form a functional TCR complex.
  • the TFP of the present disclosure comprises a target-specific binding element otherwise referred to as an antigen binding domain.
  • the choice of moiety depends upon the type and number of target antigen that define the surface of a target cell.
  • the antigen binding domain may be chosen to recognize a target antigen that acts as a cell surface marker on target cells associated with a particular disease state.
  • examples of cell surface markers that may act as target antigens for the antigen binding domain in a TFP of the present disclosure include those associated with viral, bacterial and parasitic infections; autoimmune diseases; and cancerous diseases (e.g., malignant diseases).
  • the TFP-mediated T cell response can be directed to an antigen of interest by way of engineering an antigen-binding domain into the TFP that specifically binds a desired antigen.
  • the antigen binding domain can be any domain that binds to the antigen including but not limited to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of a camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen binding domain, such as a recombinant fibronectin domain, anticalin, DARPIN and the like.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VHH variable domain of a camelid derived nanobody
  • a natural or synthetic ligand specifically recognizing and binding the target antigen can be used as antigen binding domain for the TFP.
  • the antigen-binding domain comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment.
  • the murine, humanized or human anti-TAA binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a murine, humanized or human anti-TAA binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a murine, humanized or human anti-TAA binding domain described herein, e.g., a murine, humanized or human anti-TAA binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g.
  • the murine, humanized or human anti-TAA binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a murine, humanized or human anti-TAA binding domain described herein, e.g., the murine, humanized or human anti-TAA binding domain has two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein.
  • HC CDR1 heavy chain complementary determining region 1
  • HC CDR2 heavy chain complementary determining region 2
  • HC CDR3 heavy chain complementary determining region 3
  • the murine, humanized or human anti-TAA binding domain comprises a humanized or human light chain variable region described herein and/or a murine, humanized or human heavy chain variable region described herein.
  • the murine, humanized or human anti-TAA binding domain comprises a murine, humanized or human heavy chain variable region described herein, e.g., at least two murine, humanized or human heavy chain variable regions described herein.
  • the anti-TAA binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence provided herein.
  • the anti-TAA binding domain (e.g., a scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity with an amino acid sequence provided herein; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein.
  • a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided here
  • the murine, humanized or human anti-TAA binding domain is a scFv, and a light chain variable region comprising an amino acid sequence described herein, is attached to a heavy chain variable region comprising an amino acid sequence described herein, via a linker, e.g., a linker described herein.
  • the murine, humanized, or human anti-TAA binding domain includes a (Gly4-Ser) n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4.
  • the light chain variable region and heavy chain variable region of a scFv can be, e.g., in any of the following orientations: light chain variable region- linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.
  • the linker sequence comprises a long linker (LL) sequence.
  • the linker sequence comprises a short linker (SL) sequence.
  • the antigen binding domain is an antibody or a fragment thereof. In some embodiments, the antigen binding domain is a camelid antibody or a binding fragment thereof. In some embodiments, the antigen binding domain is a murine antibody or a binding fragment thereof. In some embodiments, the antigen binding domain is a human or humanized antibody or a binding fragment thereof. In some embodiments, the antigen binding domain is a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain. In some embodiments, the sdAb is a VHH.
  • scFv single-chain variable fragment
  • sdAb single domain antibody
  • the antigen binding domain is selected from the group consisting of an anti-CD19 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, anti-MUC16 binding domain, an anti-Nectin-4 binding domain, an anti-GPC3 binding domain, and an anti-TROP-2 binding domain.
  • BCMA anti-B-cell maturation antigen
  • MSLN anti-mesothelin
  • the binding domain is characterized by particular functional features or properties of an antibody or antibody fragment.
  • the portion of a TFP composition of the present disclosure that comprises an antigen binding domain specifically binds human CD 19.
  • the antigen binding domain has the same or a similar binding specificity to human CD19 as the FMC63 scFv described in Nicholson et al., Mol. Immun. 34 (16-17): 1157-1165 (1997).
  • the present disclosure relates to an antigen binding domain comprising an antibody or antibody fragment, wherein the antibody binding domain specifically binds to a CD 19 protein or fragment thereof, wherein the antibody or antibody fragment comprises a variable light chain and/or a variable heavy chain that includes an amino acid sequence provided herein.
  • the scFv is contiguous with and in the same reading frame as a leader sequence.
  • the anti-CD19 antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences listed in Table 5.
  • the anti-CD19 antibody or fragment thereof as described herein comprises any one of the CDR sequences listed in Table 5.
  • the anti-CD19 antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences of the anti-CD19 antibody or fragment thereof sequences listed in Table 5.
  • the anti-CD19 antibody or fragment thereof as described herein comprises a CDR sequence of any one of the CDR sequences of the anti-CD19 antibody or fragment thereof sequences listed in Table 5.
  • the anti-CD19 antibody or fragment thereof as described herein comprises an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the anti-CD19 antibody or fragment thereof sequences listed in Table 5.
  • the anti-CD19 antibody or fragment thereof as described herein comprises any one of the anti-CD19 antibody or fragment thereof sequences listed in Table 5.
  • the antigen-binding domain comprises an anti-CD19 humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment having a light chain CDR1 of SEQ ID NO:73, a CDR2 of SEQ ID NO:75, and a CDR3 of SEQ ID NO: 77 and a heavy chain CDR1 of SEQ ID NO: 79, a CDR2 of SEQ ID NO: 81, and a CDR3 of SEQ ID NO:83.
  • the anti-CD19 antibody is a murine scFv.
  • the anti-CD-19 antibody comprises a VL of SEQ ID NO:85 and a VH of SEQ ID NO:87.
  • the antibody has the antigen binding domain of an anti-mesothelin antibody.
  • exemplary antibodies that bind mesothelin include, but are not limited to, amatuximab and those described in W02006099141, WO2006124641, W02009120769, WO2010111282, W02014004549, WO2014031476, W02014052064, WO2017032293, and WO2017052241, the contents of each of which are incorporated by reference herein in their entirety.
  • the anti-mesothelin antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences listed in Table 5.
  • the anti-mesothelin antibody or fragment thereof as described herein comprises any one of the CDR sequences listed in Table 5.
  • the anti-mesothelin antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences of the anti-mesothelin antibody or fragment thereof sequences listed in Table 5.
  • the anti-mesothelin antibody or fragment thereof as described herein comprises a CDR sequence of any one of the CDR sequences of the anti-mesothelin antibody or fragment thereof sequences listed in Table 5.
  • the anti-mesothelin antibody or fragment thereof as described herein comprises an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the anti-mesothelin antibody or fragment thereof sequences listed in Table 5.
  • the anti-mesothelin antibody or fragment thereof as described herein comprises any one of the anti-mesothelin antibody or fragment thereof sequences listed in Table 5.
  • the antigen-binding domain comprises an anti-mesothelin humanized or human single domain antibody or an antibody fragment having a CDR1 of SEQ ID NO:60, a CDR2 of SEQ ID NO:61, and a CDR3 of SEQ ID NO:62 or a CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64, and a CDR3 of SEQ ID NO: 65 or a CDR1 of SEQ ID NO: 66, a CDR2 of SEQ ID NO: 67, and a CDR3 of SEQ ID NO: 68.
  • the anti-mesothelin antibody has a variable domain of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71.
  • the antibody has the antigen binding domain of an anti-CD70 antibody.
  • exemplary antibodies that bind CD70 include, but are not limited to, cusatuzumab, MDX-1411, vorsetuzumab and those described in WO2014158821 and WO2018152181, the contents of each of which are incorporated by reference herein in their entirety.
  • the anti-CD70 antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences listed in Table 5.
  • the anti-CD70 antibody or fragment thereof as described herein comprises any one of the CDR sequences listed in Table 5.
  • the anti-CD70 antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences of the anti-CD70 antibody or fragment thereof sequences listed in Table 5.
  • the anti-CD70 antibody or fragment thereof as described herein comprises a CDR sequence of any one of the CDR sequences of the anti-CD70 antibody or fragment thereof sequences listed in Table 5.
  • the anti-CD70 antibody or fragment thereof as described herein comprises an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the anti-CD70 antibody or fragment thereof sequences listed in Table 5.
  • the anti-CD70 antibody or fragment thereof as described herein comprises any one of the anti-CD70 antibody or fragment thereof sequences listed in Table 5.
  • the antigen-binding domain comprises an anti-CD70 humanized or human single domain antibody or an antibody fragment having a CDR1 of SEQ ID NO:88, a
  • the antigen-binding domain comprises an anti-CD70 single chain Fv (scFv) or an antibody fragment thereof.
  • the anti-CD70 scFv or antibody fragment thereof can comprise a heavy chain complementary determining region 1 (CDRH1) having a sequence of SEQ ID NO:361, a CDRH2 having a sequence of SEQ ID NO: 362, and a CDRH3 having a sequence of SEQ ID NOs:363.
  • the anti-CD70 scFv or antibody fragment thereof can comprise a light chain complementary determining region 1 (CDRL1) having a sequence of SEQ ID NO:365, a CDRL2 having a sequence of SEQ ID NO:366, and a CDRL3 having a sequence of SEQ ID NO:367.
  • the anti-CD70 scFv or antibody fragment thereof can comprise a heavy chain variable (VH) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:364.
  • the anti-CD70 scFv or antibody fragment thereof can comprise a light chain variable (VL) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 368.
  • the antibody has the antigen binding domain of an anti-BCMA antibody.
  • exemplary antibodies that bind BCMA include, but are not limited to, SEA-BCMA (Seattle Genetics) and those described in W02010104949, WO2011108008, WO2014122143, W02016090327, WO2017143069, WO2017211900, WO2018133877, WO2019066435, WO2019149269.
  • SEA-BCMA ttle Genetics
  • the anti-BCMA antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences listed in Table 5.
  • the anti-BCMA antibody or fragment thereof as described herein comprises any one of the CDR sequences listed in Table 5.
  • the anti-BCMA antibody or fragment thereof as described herein comprises a CDR sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences of the anti-BCMA antibody or fragment thereof sequences listed in Table 5.
  • the anti-BCMA antibody or fragment thereof as described herein comprises a CDR sequence of any one of the CDR sequences of the anti-BCMA antibody or fragment thereof sequences listed in Table 5.
  • the anti-BCMA antibody or fragment thereof as described herein comprises an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the anti-BCMA antibody or fragment thereof sequences listed in Table 5.
  • the anti-BCMA antibody or fragment thereof as described herein comprises any one of the anti-BCMA antibody or fragment thereof sequences listed in Table 5.
  • the antibody has the antigen binding domain of an anti- MUC16 antibody.
  • exemplary antibodies that bind MUC16 include, but are not limited to, oregovomab, 4H11 (Memorial Sloan Kettering Cancer Center), sofituzumab, and those described in W02018058003, the contents of which is incorporated by reference herein in its entirety.
  • the antibody has the antigen binding domain of an anti- CD79B antibody.
  • Exemplary antibodies that bind CD79B include, but are not limited to, those described in W02017009474 and W02016021621, the contents of each of which are incorporated by reference herein in their entirety.
  • the antibody has the antigen binding domain of an anti-HER2 antibody.
  • Exemplary antibodies that bind HER2 include, but are not limited to, trastuzumab, pertuzumab, margetuximab, trastuzumab-pkrb, ertumaxomab, SB3, PF-05280014, CMAB302, trastuzumab-dkst, HD201, GB221, BCD-022, trastuzumab-anns, HLX02, DMB-3111, timigutuzumab, UB-921, IBB 15, RG6194, HLX22, SIBP-01, TX05, and DXL702.
  • the antibody has the antigen binding domain of an anti-PSMA antibody.
  • exemplary antibodies that bind PSMA include, but are not limited to, MDX1201- A488 and those described in W02001009192, W0200303490, W02007002222, W02009130575, WO2010118522, WO2013185117, WO2013188740, WO2014198223, WO2016145139, W02017121905, W02017180713, WO2017212250, WO2018033749, WO2018129284, WO2018142323, and WO2019191728, the contents of each of which are incorporated by reference herein in their entirety.
  • the antigen-binding domain or fragment thereof as described herein comprises a sequence encoding an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the amino acid sequences listed in Table 5.
  • the antigen-binding domain or fragment thereof as described herein comprises a sequence encoding any one of the amino acid sequences listed in Table 5.
  • the antigen-binding domain or fragment thereof as described herein comprises a nucleic acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the nucleic acid sequences listed in Table 5.
  • the antigen-binding domain or fragment thereof as described herein comprises a nucleic acid sequence encoding any one of the nucleic acid sequences listed in Table 5.
  • the antigen-binding domain or fragment thereof as described herein comprises a sequences encoding CDR sequences having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to any one of the CDR sequences listed in Table 5.
  • the antigen-binding domain or fragment thereof as described herein comprises a sequence encoding any one of the CDR sequences listed in Table 5.
  • the antigen-binding domain or fragment thereof as described herein comprises a sequence encoding CDR sequences having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to the CDR sequences of any one of the sequences listed in Table 5.
  • the antigen-binding domain or fragment thereof as described herein comprises a sequence encoding CDR sequences of any one of the sequences listed in Table 5.
  • the present disclosure relates to an antigen binding domain comprising an antibody or antibody fragment, wherein the antibody binding domain specifically binds to a tumor-associated protein or fragment thereof, wherein the antibody or antibody fragment comprises a variable light chain and/or a variable heavy chain that includes an amino acid sequence provided herein.
  • the binding domain is contiguous with and in the same reading frame as a leader sequence.
  • a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof.
  • the antigen binding domain is humanized.
  • a humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g, European Patent No. EP 239,400;
  • framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.)
  • a humanized antibody or antibody fragment has one or more amino acid residues remaining in it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • humanized antibodies or antibody fragments comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions wherein the amino acid residues comprising the framework are derived completely or mostly from human germline.
  • Humanized antibodies are often human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., Proc. Natl. Acad. Sci. USA, 91 :969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.
  • variable domains both light and heavy
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity.
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151 :2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety).
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • the same framework may be used for several different humanized antibodies (see, e.g., Nicholson et al., Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151 :2623 (1993), the contents of which are incorporated herein by reference herein in their entirety).
  • the framework region e.g., all four framework regions, of the heavy chain variable region are derived from a VH -4-59 germline sequence.
  • the framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence.
  • the framework region e.g., all four framework regions of the light chain variable region are derived from a VK3-1.25 germline sequence.
  • the framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence.
  • the portion of a TFP composition of the present disclosure that comprises an antibody fragment is humanized with retention of high affinity for the target antigen and other favorable biological properties.
  • humanized antibodies and antibody fragments are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences.
  • Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody or antibody fragment characteristic, such as increased affinity for the target antigen, is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding.
  • a humanized antibody or antibody fragment may retain a similar antigenic specificity as the original antibody, e.g., in the present disclosure, the ability to bind human a tumor associated antigen (TAA).
  • a humanized antibody or antibody fragment may have improved affinity and/or specificity of binding to, e.g., human CD 19, human BCMA, human MUC16, human mesothelin (MSLN), human CD79B, human HER2, human PSMA, human CD20, human CD70, human Nectin-4, human GPC3, human TROP-2, human PD-1, or another tumor associated antigen.
  • the anti -tumor-associated antigen binding domain is a fragment, e.g., a single chain variable fragment (scFv).
  • the anti-TAA binding domain is a Fv, a Fab, a (Fab’)2, or a bi-functional (e.g., bi-specific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)).
  • the antibodies and fragments thereof of the present disclosure binds a CD 19 protein with wild-type or enhanced affinity.
  • the anti-TAA binding domain comprises a single domain antibody (sdAb or VHH).
  • a target antigen e.g., CD 19, BCMA, MUC16, M
  • VH domains and scFvs can be prepared according to method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • scFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers.
  • the scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The linker length can greatly affect how the variable regions of a scFv fold and interact.
  • the linker sequence comprises a linker sequence.
  • linker orientation and size see, e.g., Hollinger et al., 1993 Proc Natl Acad. Sci. U.S.A. 90:6444- 6448, U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT publication Nos. W02006/020258 and W02007/024715, each of which is incorporated herein by reference.
  • An scFv can comprise a linker of about 10, 11, 12, 13, 14, 15 or greater than 15 residues between its VL and VH regions.
  • the linker sequence may comprise any naturally occurring amino acid.
  • the linker sequence comprises amino acids glycine and serine.
  • the linker sequence comprises sets of glycine and serine repeats such as (Gly4Ser) n , where n is a positive integer equal to or greater than 1.
  • the linker can be (Gly4Ser)4 or (Gly4Ser)3. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.
  • a tumor associated antigen binding domain e.g., scFv or sdAb molecules (e.g., soluble scFv or sdAb)
  • scFv or sdAb molecules e.g., soluble scFv or sdAb
  • biophysical properties e.g., thermal stability
  • the humanized or human scFv or sdAb has a thermal stability that is greater than about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees Celsius than a parent scFv or sdAb in the described assays.
  • the improved thermal stability of the anti-TAA binding domain e.g., scFv or sdAb is subsequently conferred to the entire TAA-TFP construct, leading to improved therapeutic properties of the anti-TAA TFP construct.
  • the thermal stability of the binding domain e.g., scFv or sdAb, can be improved by at least about 2 °C or 3 °C as compared to a conventional antibody.
  • the binding domain has a 1 °C improved thermal stability as compared to a conventional antibody.
  • the binding domain has a 2 °C improved thermal stability as compared to a conventional antibody.
  • the scFv or sdAb has a 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, or 15 °C improved thermal stability as compared to a conventional antibody. Comparisons can be made, for example, between the scFv or sdAb molecules as described herein and scFv or sdAb molecules or Fab fragments of an antibody from which the scFv VH and VL were derived. Thermal stability can be measured using methods known in the art. For example, in one embodiment, TM can be measured. Methods for measuring TM and other methods of determining protein stability are described in more detail below.
  • the binding domain e.g., a scFv or sdAb, comprises at least one mutation arising from the humanization process such that the mutated scFv or sdAb confers improved stability to the anti-TAA TFP construct.
  • the anti-TAA binding domain e.g., scFv or sdAb
  • the anti-TAA binding domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations arising from the humanization process such that the mutated scFv or sdAb confers improved stability to the TAA-TFP construct.
  • the antigen binding domain of the TFP comprises an amino acid sequence that is homologous to an antigen binding domain amino acid sequence described herein, and the antigen binding domain retains the desired functional properties of the anti-tumor-associated antigen antibody fragments described herein.
  • the TFP composition of the present disclosure comprises an antibody fragment.
  • that antibody fragment comprises a scFv or sdAb.
  • the antigen binding domain of the TFP is engineered by modifying one or more amino acids within one or both variable regions (e.g., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions.
  • the TFP composition of the present disclosure comprises an antibody fragment.
  • that antibody fragment comprises a scFv.
  • the antibody or antibody fragment of the present disclosure may further be modified such that they vary in amino acid sequence (e.g., from wild-type), but not in desired activity.
  • additional nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues may be made to the protein.
  • a nonessential amino acid residue in a molecule may be replaced with another amino acid residue from the same side chain family.
  • a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members, e.g., a conservative substitution, in which an amino acid residue is replaced with an amino acid residue having a similar side chain, may be made.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid
  • Percent identity in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences that are the same. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 70%, 71% , 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math.
  • BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • the present disclosure contemplates modifications of the starting antibody or fragment (e.g., scFv or sdAb) amino acid sequence that generate functionally equivalent molecules.
  • the VH or VL of a binding domain, e.g., scFv or sdAb, comprised in the TFP can be modified to retain at least about 70%, 71%. 72%.
  • the present disclosure contemplates modifications of the entire TFP construct, e.g., modifications in one or more amino acid sequences of the various domains of the TFP construct in order to generate functionally equivalent molecules.
  • the TFP construct can be modified to retain at least about 70%, 71%. 72%.
  • the extracellular domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any protein, but in particular a membrane-bound or transmembrane protein. In one aspect the extracellular domain is capable of associating with the transmembrane domain.
  • An extracellular domain of particular use in this present disclosure may include at least the extracellular region(s) of e.g., the alpha, beta or zeta chain of the T cell receptor, or CD3 epsilon, CD3 gamma, or CD3 delta, or in alternative embodiments, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.
  • the extracellular domain is a TCR extracellular domain.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain. In some embodiments, the TCR extracellular domain comprises the extracellular portion of a constant (an IgC) domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain.
  • the extracellular domain comprises, or comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150 or more consecutive amino acid residues of the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a
  • the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to a sequence encoding the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain.
  • the extracellular domain comprises a sequence encoding the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
  • the extracellular domain comprises, or comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
  • the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to a sequence encoding the extracellular portion of a constant (an IgC) domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma.
  • the extracellular domain comprises a sequence encoding the extracellular portion of a constant (an IgC) domain of TCR alpha, TCR beta, TCR delta, or TCR gamma having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C- terminus.
  • a constant (an IgC) domain of TCR alpha, TCR beta, TCR delta, or TCR gamma having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C- terminus.
  • the extracellular domain comprises, or comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
  • the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the extracellular domain comprises a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
  • the extracellular domain can comprise a full-length extracellular portion of the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the extracellular domain can comprise a fragment (e.g., functional fragment) of the full-length extracellular portion of the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the extracellular domain can comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of the extracellular portion of the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the extracellular domain can be a TCR extracellular domain.
  • the TCR extracellular domain can be derived from a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit or a CD3 delta TCR subunit.
  • the extracellular domain can be a full-length TCR extracellular domain or fragment (e.g., functional fragment) thereof.
  • the extracellular domain can comprise a variable domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the extracellular domain can comprise a variable domain and a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. In some cases, the extracellular domain may not comprise a variable domain.
  • the TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain described herein can be derived from various species.
  • the TCR chain can be a murine or human TCR chain.
  • the extracellular domain can comprise a constant domain of a murine TCR alpha chain, a murine TCR beta chain, a human TCR gamma chain or a human TCR delta chain.
  • a TFP sequence contains an extracellular domain and a transmembrane domain encoded by a single genomic sequence.
  • a TFP can be designed to comprise a transmembrane domain that is heterologous to the extracellular domain of the TFP.
  • a transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids of the intracellular region).
  • the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the extracellular region. In some cases, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the intracellular region. In one aspect, the transmembrane domain is one that is associated with one of the other domains of the TFP is used. In some embodiments, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.
  • the transmembrane domain is capable of homodimerization with another TFP on the TFP-T cell surface.
  • the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same TFP.
  • the transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TFP has bound to a target.
  • the TCR-integrating subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the transmembrane domain comprises, or comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more consecutive amino acid residues of the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the transmembrane domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the transmembrane domain comprises a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
  • the transmembrane domain can be attached to the extracellular region of the TFP, e.g., the antigen binding domain of the TFP, via a hinge, e.g., a hinge from a human protein.
  • a hinge e.g., a hinge from a human protein.
  • the hinge can be a human immunoglobulin (Ig) hinge, e.g, an IgG4 hinge, or a CD8a hinge.
  • a short oligo- or polypeptide linker may form the linkage between the binding element and the TCR extracellular domain of the TFP.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the linker may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more in length.
  • the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:429) or a sequence (GGGGS)x or (G4S) n , wherein X or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more (SEQ ID NO:430).
  • X or n is an integer from 1 to 10. In some embodiments, X or n is an integer from 1 to 4. In some embodiments, X or n is 2. In some embodiments, X or n is 4. In some embodiments, the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO:572).
  • the cytoplasmic domain of the TFP can include an intracellular domain.
  • the intracellular domain is from CD3 gamma, CD3 delta, CD3 epsilon, TCR alpha, TCR beta, TCR gamma, or TCR delta.
  • the intracellular domain comprises a signaling domain, if the TFP contains CD3 gamma, delta or epsilon polypeptides; TCR alpha, TCR beta, TCR gamma, and TCR delta subunits generally have short (e.g., 1-19 amino acids in length) intracellular domains and are generally lacking in a signaling domain.
  • An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the TFP has been introduced. While the intracellular domains of TCR alpha, TCR beta, TCR gamma, and TCR delta do not have signaling domains, they are able to recruit proteins having a primary intracellular signaling domain described herein, e.g., CD3 zeta, which functions as an intracellular signaling domain.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
  • intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • intracellular domains for use in the TFP of the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that are able to act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.
  • the intracellular domain comprises the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the intracellular domain comprises, or comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more consecutive amino acid residues of the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain.
  • the intracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to a sequence encoding the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain.
  • the transmembrane domain comprises a sequence encoding the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C- terminus or at both the N- and C-terminus.
  • the intracellular domain comprises, or comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 or more consecutive amino acid residues of the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the intracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the intracellular domain comprises a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
  • naive T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic domain, e.g., a costimulatory domain).
  • a primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosinebased activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosinebased activation motifs
  • ITAMs containing primary intracellular signaling domains include those of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
  • a TFP of the present disclosure comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-epsilon.
  • a primary signaling domain comprises a modified IT AM domain, e.g., a mutated IT AM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain.
  • a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and/or truncated ITAM-containing primary intracellular signaling domain.
  • a primary signaling domain comprises one, two, three, four or more IT AM motifs.
  • the intracellular signaling domain of the TFP can comprise a CD3 signaling domain, e.g., CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta, by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a TFP of the present disclosure.
  • the intracellular signaling domain of the TFP can comprise a CD3 epsilon chain portion and a costimulatory signaling domain.
  • the costimulatory signaling domain refers to a portion of the TFP comprising the intracellular domain of a costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen.
  • examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like.
  • CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human TFP-T cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al., Blood. 2012; 119(3):696-706).
  • the intracellular signaling sequences within the cytoplasmic portion of the TFP of the present disclosure may be linked to each other in a random or specified order.
  • a short oligo- or polypeptide linker for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences.
  • a glycine-serine doublet can be used as a suitable linker.
  • a single amino acid e.g., an alanine, a glycine, can be used as a suitable linker.
  • the TFPs described herein may comprise a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit.
  • At least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR alpha. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR beta. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR gamma. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR delta.
  • At least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 epsilon. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 delta. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 gamma.
  • the TFPs described herein may comprise a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain, wherein all three of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from the same TCR subunit.
  • the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 epsilon.
  • the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 delta.
  • the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 gamma.
  • the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain may comprise the constant domain of TCR alpha.
  • the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain may comprise the constant domain of TCR beta.
  • the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain may comprise the constant domain of TCR gamma.
  • the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain may comprise the constant domain of TCR delta.
  • the constant domain of TCR alpha or the constant domain of TCR beta may be murine.
  • the TFP-expressing cell that co-expresses CXCR6 or a functional fragment thereof as described herein can further comprise a second TFP, e.g., a second TFP that includes a different antigen binding domain, e.g., to the same target or a different target.
  • a second TFP e.g., a second TFP that includes a different antigen binding domain, e.g., to the same target or a different target.
  • the antigen binding domains of the different TFPs can be such that the antigen binding domains do not interact with one another.
  • a cell expressing a first and second TFP can have an antigen binding domain of the first TFP, e.g., as a fragment, e.g., a scFv, that does not form an association with the antigen binding domain of the second TFP, e.g., the antigen binding domain of the second TFP is a VHH.
  • TFP constructs can be generated as previously described.
  • An anti-MSLN or CD 19 binder can be linked to a CD3 or TCR DNA fragment by either a DNA sequence encoding a short linker (SL): AAAGGGGSGGGGSGGGGSLE (SEQ ID NO:387) or a long linker (LL): AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE (SEQ ID NO:388) into pLRPO or p510 vector.
  • the TFP used is TC-110 (e.g., an anti-CD19 FMC63 scFv antibody linked to CD3 epsilon) having the sequence of SEQ ID NO: 196.
  • the TFP used comprises an anti-MSLN antibody and CD3. In some embodiments, the TFP used comprises an anti-MSLN antibody linked to CD3. In some embodiments, the TFP used comprises an anti-MSLN antibody operatively linked to CD3. In some embodiments, the TFP used comprises an anti-MSLN MHle VHH antibody and CD3. In some embodiments, the TFP used comprises an anti-MSLN MHle VHH antibody linked to CD3. In some embodiments, the TFP used comprises an anti-MSLN MHle VHH antibody operatively linked to CD3. In some embodiments, the TFP used comprises an anti-MSLN MHle VHH antibody and CD3 epsilon.
  • the TFP used comprises an anti-MSLN MHle VHH antibody linked to CD3 epsilon. In some embodiments, the TFP used comprises an anti-MSLN MHle VHH antibody operatively linked to CD3 epsilon.
  • the TFP used is TC-210 comprising an anti-MSLN antibody and CD3. In some embodiments, the TFP used is TC-210 comprising an anti-MSLN antibody linked to CD3. In some embodiments, the TFP used is TC-210 comprising an anti-MSLN antibody operatively linked to CD3. In some embodiments, the TFP used is TC-210 comprising an anti- MSLN MHle VHH antibody and CD3. In some embodiments, the TFP used is TC-210 comprising an anti-MSLN MHle VHH antibody linked to CD3. In some embodiments, the TFP used is TC-210 comprising an anti-MSLN MHle VHH antibody operatively linked to CD3.
  • the TFP used is TC-210 comprising an anti-MSLN MHle VHH antibody and CD3 epsilon. In some embodiments, the TFP used is TC-210 comprising an anti-MSLN MHle VHH antibody linked to CD3 epsilon. In some embodiments, the TFP used is TC-210 comprising an anti-MSLN MHle VHH antibody operatively linked to CD3 epsilon.
  • the TFP used comprises a GM-CSFRa Signal Peptide, an anti- MSLN MHle VHH antibody, a A3(G4S)3LE Linker, and CD3 epsilon.
  • the TFP used comprises a GM-CSFRa Signal Peptide operatively linked to an anti-MSLN MHle VHH antibody operatively linked to a A3(G4S)3LE Linker operatively linked to CD3 epsilon.
  • the TFP used comprises a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to SEQ ID NO:421, a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to SEQ ID NO:422, a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to SEQ ID NO:387, and a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to SEQ ID NO:423.
  • the TFP used comprises a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to SEQ ID NO:421 operatively linked to a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to SEQ ID NO:422 operatively linked to a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to SEQ ID NO: 387 operatively linked to a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to SEQ ID NO:423.
  • the TFP used comprises the sequence of SEQ ID NO:421, the sequence of SEQ ID NO:422, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:423.
  • the TFP used comprises the sequence of SEQ ID NO:421 operatively linked to the sequence of SEQ ID NO:422 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:423.
  • the TFP used comprises a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO: 195.
  • the TFP used comprises the sequence of SEQ ID NO: 195.
  • the TFP used is TC-210 comprising a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO: 195.
  • the TFP used is TC-210 comprising the sequence of SEQ ID NO: 195.
  • the TFP used is TC-210 (e.g., an anti-MSLN MHle VHH antibody linked to CD3 epsilon) having the sequence of SEQ ID NO: 195.
  • nucleic acid molecules comprising a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) as described herein and a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • recombinant nucleic acid molecules comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP) and a sequence encoding CXCR6 or a fragment thereof, wherein the recombinant nucleic acid further expresses a TCR constant domain .
  • TCR T cell receptor
  • TFP can comprise a TCR subunit comprising at least a portion of a TCR extracellular domain.
  • the TCR subunit can further comprise a transmembrane domain.
  • the TCR subunit can further comprise an intracellular domain of TCR gamma, TCR delta, TCR alpha or TCR beta or an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, CD3 delta.
  • the TFP can further comprise an antibody (e.g., a human, humanized, or murine antibody) comprising an antigen binding domain.
  • the recombinant nucleic acid molecule can further comprise a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain, a TCR alpha constant domain and a TCR beta constant domain, a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain.
  • the TCR subunit and the antibody can be operatively linked.
  • the TFP can functionally incorporate into a TCR complex (e.g., an endogenous TCR complex) when expressed in a T cell.
  • the constant domain can comprise a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant domain can comprise a full- length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant domain can comprise a fragment (e.g., functional fragment) of the full- length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant domain can comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the sequence encoding the TCR constant domain can further encode the transmembrane domain and/or intracellular region of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the sequence encoding the TCR constant domain can encode a full-length constant region of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant region of a TCR chain can comprise a constant domain, a transmembrane domain, and an intracellular region.
  • the constant region of a TCR chain can also exclude the transmembrane domain and the intracellular region of the TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain described herein can be derived from various species.
  • the TCR chain can be a murine or human TCR chain.
  • the constant domain can comprise a constant domain of a murine or human TCR alpha chain, TCR beta chain, TCR gamma chain or TCR delta chain.
  • the constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO: 16, SEQ ID NO:21, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO:207, SEQ ID NO:209, or SEQ ID NO:243.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO: 16, SEQ ID NO:21, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO:207, SEQ ID NO:209, or SEQ ID NO:243.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO: 16, SEQ ID NO:21, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO:207, SEQ ID NO:209, or SEQ ID NO:243.
  • the constant domain can comprise a sequence or fragment thereof of SEQ ID NO: 16, SEQ ID NO:21, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO:207, SEQ ID NO:209, or SEQ ID NO:243.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO: 16, SEQ ID NO:21, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO:207, SEQ ID NO:209, or SEQ ID NO:243.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO: 16, SEQ ID NO:21, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO:207, SEQ ID NO:209, or SEQ ID NO:243.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO: 16, SEQ ID NO:21, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO:207, SEQ ID NO:209, or SEQ ID NO:243.
  • the murine TCR alpha constant domain can comprise positions 2-137 of SEQ ID NO: 146.
  • the murine TCR alpha constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of positions 2-137 of SEQ ID NO: 146.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of positions 2-137 of SEQ ID NO: 146.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of positions 2-137 of SEQ ID NO: 146.
  • the constant domain can comprise a sequence or fragment thereof of positions 2-137 of SEQ ID NO: 146.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of positions 2-137 of SEQ ID NO: 146.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of positions 2-137 of SEQ ID NO: 146.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of positions 2-137 of SEQ ID NO: 146.
  • the murine TCR beta constant domain can comprise positions 2-173 of SEQ ID NO: 152.
  • the murine TCR beta constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of positions 2-173 of SEQ ID NO: 152.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of positions 2-173 of SEQ ID NO: 152.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of positions 2-173 of SEQ ID NO: 152.
  • the constant domain can comprise a sequence or fragment thereof of positions 22-173 of SEQ ID NO: 152.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of positions 2-173 of SEQ ID NO: 152.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of positions 2-173 of SEQ ID NO: 152.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of positions 2-173 of SEQ ID NO: 152.
  • the TCR constant domain is a TCR delta constant domain.
  • the TCR delta constant domain can comprise SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243 or SEQ ID NO:265, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modification.
  • the TCR delta constant domain can comprise SEQ ID NO:243.
  • the TCR delta constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:243.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:243.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:243.
  • the constant domain can comprise a sequence or fragment thereof of SEQ ID NO:243.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:243.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:243.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:243.
  • the TCR delta constant domain can comprise SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243 or SEQ ID NO:265, functional fragments thereof, or amino acid sequences thereof having at least one but not more than 20 modifications.
  • the sequence encoding a TCR delta constant domain further encodes a TCR delta variable domain, thereby encoding a full TCR delta domain.
  • the full TCR delta domain can be delta 2 or delta 1.
  • the full TCR delta constant domain can comprise SEQ ID NO:256, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the full TCR delta domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the delta domain can comprise a truncated version of a delta domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:256.
  • the delta domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:256.
  • the delta domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:256.
  • the delta domain can comprise a sequence or fragment thereof of SEQ ID NO:256.
  • the delta domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:256.
  • the delta domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:256.
  • the delta domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:256.
  • the TCR gamma constant domain can comprise SEQ ID NO:21.
  • the TCR gamma constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:21.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:21.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:21.
  • the constant domain can comprise a sequence or fragment thereof of SEQ ID NO:21.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:21.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:21.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:243.
  • the TCR gamma constant domain can comprise SEQ ID NO:21 or SEQ ID NO: 155, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the sequence encoding the TCR gamma constant domain further encodes a TCR gamma variable domain, thereby encoding a full TCR gamma domain.
  • the full TCR gamma domain can be gamma 9 or gamma 4.
  • the full TCR gamma domain can comprise SEQ ID NO:255, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the full TCR gamma domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the gamma domain can comprise a truncated version of a gamma domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:255.
  • the gamma domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:255.
  • the gamma domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:255.
  • the gamma domain can comprise a sequence or fragment thereof of SEQ ID NO:255.
  • the gamma domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or gamma of the sequence of SEQ ID NO:255.
  • the gamma domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:255.
  • the gamma domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:255.
  • the TCR constant domain is a TCR delta constant domain.
  • the sequence encoding the TCR delta constant domain can further encode a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR delta constant domain.
  • the second antigen binding domain or ligand binding domain can be the same or different as the antigen binding domain or ligand binding domain of the TFP.
  • the TCR constant domain is a TCR gamma constant domain.
  • the sequence encoding the TCR gamma constant domain can further encode a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR gamma constant domain.
  • the second antigen binding domain or ligand binding domain can be the same or different as the antigen binding domain or ligand binding domain of the TFP.
  • the recombinant nucleic acid comprises a sequence encoding a TCR gamma constant domain and a TCR delta constant domain.
  • the TCR gamma constant domain can comprise SEQ ID NO:21 or SEQ ID NO: 155, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the sequence encoding the TCR gamma constant domain can further encode a TCR gamma variable domain, thereby encoding a full TCR gamma domain.
  • the TCR gamma domain can be gamma 9 or gamma 4.
  • the full TCR gamma domain comprises SEQ ID NO:255, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR delta constant domain can comprise SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243 or SEQ ID NO:265, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the sequence encoding the TCR delta constant domain can further encode a TCR delta variable domain, thereby encoding a full TCR delta domain.
  • the TCR delta domain can be delta 2 or delta 1.
  • the full TCR delta domain can comprise SEQ ID NO:256, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR constant domain incorporates into a functional TCR complex when expressed in a T cell. In some embodiments, the TCR constant domain incorporates into a same functional TCR complex as the functional TCR complex that incorporates the TFP when expressed in a T cell. In some embodiments, the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained within a same nucleic acid molecule. In some embodiments, the sequence encoding the TFP and the sequence encoding the TCR constant domain are contained within different nucleic acid molecules. The sequence can further encode a cleavage site (e.g., a protease cleavage site) between the encoded TFP and the TCR constant domain.
  • a cleavage site e.g., a protease cleavage site
  • the cleavage site can be a protease cleavage site.
  • the cleavage site can be a self-cleaving peptide such as a T2A, P2A, E2A or F2A cleavage site.
  • the cleavage site can comprise a sequence of SEQ ID NO:23.
  • T2A cleavage site EGRGSLLTCGDVEENPGP (SEQ ID NO:23).
  • the TCR subunit of the TFP and the constant domain can comprise a sequence derived from a same TCR chain or a different TCR chain.
  • the TCR subunit of the TFP and the constant domain are derived from different TCR chains.
  • the TCR subunit can comprise (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, where the TCR extracellular domain, the transmembrane domain and the intracellular domain are derived from a TCR alpha chain, and the constant domain can comprise a constant domain of a TCR beta chain.
  • the TCR subunit can comprise (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, where the TCR extracellular domain, the transmembrane domain and the intracellular domain are derived from a TCR beta chain, and the constant domain can comprise a constant domain of a TCR alpha chain.
  • the TCR subunit can comprise (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, where the TCR extracellular domain, the transmembrane domain and the intracellular domain are derived from a TCR gamma chain, and the constant domain can comprise a constant domain of a TCR delta chain.
  • the TCR subunit can comprise (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, where the TCR extracellular domain, the transmembrane domain and the intracellular domain are derived from a TCR delta chain, and the constant domain can comprise a constant domain of a TCR gamma chain.
  • the TCR subunit and the antibody domain, the antigen domain or the binding ligand or fragment thereof are operatively linked by a linker sequence.
  • the TCR subunit of the TFP can comprise the extracellular, transmembrane and intracellular domain of CD3 epsilon, CD3 gamma, or CD3 delta.
  • recombinant nucleic acid comprises a TFP comprising the extracellular, transmembrane and intracellular domain of CD3 epsilon, CD3 gamma, or CD3 delta and the constant domains of TCR beta and TCR alpha.
  • recombinant nucleic acid comprises a TFP comprising the extracellular, transmembrane and intracellular domain of CD3 epsilon and the constant domains of TCR gamma and TCR delta.
  • recombinant nucleic acid comprises a TFP comprising the extracellular, transmembrane and intracellular domain of CD3 epsilon and full length TCF gamma and full length TCR delta.
  • the TCR subunit of the TFP comprises CD3 epsilon.
  • the TCR subunit of CD3 epsilon can comprise the sequence of SEQ ID NO:258 functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprising at least a portion of a murine TCR alpha or murine TCR beta extracellular domain and a murine TCR alpha or murine TCR beta transmembrane domain is or comprises a TCR alpha constant domain or a TCR beta constant domain.
  • the TCR subunit can comprise an intracellular domain of murine TCR alpha or murine TCR beta.
  • the TCR constant domain can be a TCR alpha constant domain, e.g., a TCR alpha constant domain described herein.
  • the TCR alpha constant domain can comprise SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the sequence encoding the TCR alpha constant domain can further encode a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR alpha constant domain.
  • the second antigen binding domain or ligand binding domain can be the same or different as the antigen binding domain or ligand binding domain of the TFP.
  • the TCR alpha constant domain can comprise a murine TCR alpha constant domain.
  • the murine TCR alpha constant domain can comprise amino acids 2-137 of the murine TCR alpha constant domain.
  • the murine TCR alpha constant domain can comprise amino acids 2-137 of SEQ ID NO: 146.
  • the murine TCR alpha constant domain can comprise a sequence of SEQ ID NO:207.
  • the murine TCR alpha constant domain can comprise amino acids 82-137 of SEQ ID NO: 146.
  • the TCR constant domain can be a TCR beta constant domain, e.g., a TCR beta constant domain described herein.
  • the TCR beta constant domain can comprise SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO:209, or functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the sequence encoding the TCR beta constant domain can further encode a second antigen binding domain or ligand binding domain that is operatively linked to the sequence encoding the TCR beta constant domain.
  • the second antigen binding domain or ligand binding domain can be the same or different as the antigen binding domain or ligand binding domain of the TFP.
  • TCR beta constant domain can comprise a murine TCR beta constant domain.
  • the murine TCR beta constant domain can comprise amino acids 2-173 of the murine TCR beta constant domain.
  • the murine TCR beta constant domain can comprise amino acids 2-173 of SEQ ID NO: 152.
  • the murine TCR beta constant domain can comprise SEQ ID NO:209.
  • the TCR beta constant domain can comprise amino acids 123-173 of SEQ ID NO: 152.
  • the recombinant nucleic acid can comprise sequence encoding a TCR alpha constant domain and a TCR beta constant domain.
  • the TCR alpha constant domain can comprise SEQ ID NO: 142, SEQ ID NO: 143, Or SEQ ID NO: 146, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR beta constant domain can comprise SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO:209, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the intracellular signaling domain can be CD3 epsilon, CD3 gamma, or CD3 delta.
  • the intracellular signaling domain can be CD3 epsilon.
  • the sequence encoding the TCR constant domain can comprise from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene sequence.
  • the sequence encoding the TCR constant domain can comprise, from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene sequence.
  • the sequence encoding the TCR constant domain can comprise, from 5’ to 3’, a first leader sequence, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker, and a TRBC gene sequence.
  • the sequence encoding the TCR constant domain can comprise, from 5’ to 3’, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker, and a TRBC gene sequence.
  • the sequence encoding the TCR constant domain can comprise, from 5’-3’, a first leader sequence, a TRAC gene sequence, a first cleavable linker sequence, a second leader sequence, a TRBC gene sequence, a second cleavable linker sequence, a third leader sequence, an antigen binding domain sequence, a linker sequence, and a CD3 epsilon gene sequence.
  • the at least one but not more than 20 modifications thereto of a sequence described herein can comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.
  • the TFP, the TCR gamma constant domain, the TCR delta constant domain, and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
  • the TCR constant domain is a TCR gamma constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR delta, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of TCR gamma, CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof;
  • the TCR constant domain is a TCR gamma constant domain and a TCR delta constant domain and the TFP functionally integrates into a TCR complex comprising an endogenous subunit of CD3 epsilon, CD3 gamma, CD3 delta, or a combination thereof.
  • the antibody or antigen binding domain can be an antibody fragment.
  • the antibody or antigen binding domain can be murine, human or humanized.
  • the murine, human or humanized antibody is an antibody fragment.
  • the antibody fragment is a scFv, a single domain antibody domain, a VH domain or a VL domain.
  • An antigen binding domain described herein can be selected from a group consisting of an anti-CD19 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, an anti-CD79b binding domain, an anti-PMS A binding domain, an anti-MUC16 binding domain, an anti-CD22 binding domain, an anti-PD-Ll binding domain, an anti BAFF receptor binding domain, an anti-Nectin-4 binding domain, an anti-TROP-2 binding domain, an anti-GPC3 binding domain, and anti-ROR-1 binding domain.
  • BCMA anti-B-cell maturation antigen
  • MSLN anti-mesothelin
  • the nucleic acid is selected from the group consisting of a DNA and an RNA. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the recombinant nucleic acid comprises a nucleic acid analog, wherein the nucleic acid analog is not in an encoding sequence of the recombinant nucleic acid.
  • the nucleic analog is selected from the group consisting of 2’-O-methyl, 2’-O-methoxyethyl (2’-O-MOE), 2’-O-aminopropyl, 2’-deoxy, T-deoxy -2 ’-fluoro, 2’-O-aminopropyl (2’-O-AP), 2'-O- dimethylaminoethyl (2’-O-DMAOE), 2’-O-dimethylaminopropyl (2’-O-DMAP), T-O- dimethylaminoethyloxyethyl (2’-O-DMAEOE), 2’-O-N-methylacetamido (2’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino,
  • LNA
  • the recombinant nucleic acid further comprises a leader sequence. In some embodiments, the recombinant nucleic acid further comprises a promoter sequence. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some embodiments, the recombinant nucleic acid further comprises a 3’UTR sequence. In some embodiments, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. In some embodiments, the nucleic acid is an in vitro transcribed nucleic acid.
  • the present disclosure provides a recombinant nucleic acid comprising: a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked, and a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein.
  • TCR T cell receptor
  • TFP TFP fusion protein
  • the recombinant nucleic acid further comprises a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain.
  • a modified T cell further comprises a functional disruption of an endogenous TCR.
  • the present disclosure in some cases, provides a recombinant nucleic acid comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain of TCR alpha, TCR beta, TCR gamma, or TCR delta or an intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta, and; and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked, and a second nucleic acid sequence encoding CXCR6 or a functional fragment thereof as described herein.
  • TCR T cell receptor
  • TCP T cell receptor fusion protein
  • the recombinant nucleic acid further comprises a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain.
  • a modified T cell further comprises a functional disruption of an endogenous TCR.
  • recombinant nucleic acid molecules described herein further comprise a leader sequence.
  • the recombinant nucleic acid molecule is selected from the group consisting of a DNA and an RNA.
  • the recombinant nucleic acid molecule is an mRNA.
  • the recombinant nucleic acid molecule is a circRNA.
  • the recombinant nucleic acid molecule comprises a nucleic acid analog.
  • the nucleic acid analog is not in an encoding sequence of the recombinant nucleic acid.
  • the nucleic analog is selected from the group consisting of 2’-O-methyl, 2’-O-methoxyethyl (2’-0-M0E), 2’-O- aminopropyl, 2’-deoxy, T-deoxy -2 ’-fluoro, 2’-O-aminopropyl (2’-O-AP), 2'-O- dimethylaminoethyl (2’-O-DMAOE), 2’-O-dimethylaminopropyl (2’-O-DMAP), T-O- dimethylaminoethyloxyethyl (2’-O-DMAEOE), 2’-O-N-methylacetamido (2’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a
  • the recombinant nucleic acid molecule further comprises a leader sequence. In some embodiments, the recombinant nucleic acid molecule further comprises a promoter sequence. In some embodiments, the recombinant nucleic acid molecule further comprises a sequence encoding a poly(A) tail. In some embodiments, the recombinant nucleic acid molecule further comprises a 3’UTR sequence. In some embodiments, the recombinant nucleic acid molecule is an isolated nucleic acid or a non-naturally occurring nucleic acid. In some embodiments, the nucleic acid is an in vitro transcribed nucleic acid.
  • recombinant nucleic acids comprising: a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) wherein the TFP comprises: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR transmembrane domain, and (b) an antigen binding domain; and wherein the TCR subunit and the antigen binding domain are operatively linked, and a second nucleic acid sequence encoding C-X-C chemokine receptor type 6 (CXCR6) or a functional fragment thereof.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • CXCR6 C-X-C chemokine receptor type 6
  • the recombinant nucleic acids as described herein further comprises a third nucleic acid sequence encoding a TGFBr2 switch polypeptide comprising a transforming growth factor beta receptor II (TGFBr2) extracellular domain or a functional fragment thereof.
  • TGFBr2 transforming growth factor beta receptor II
  • the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a linker.
  • the linker comprises a protease cleavage site.
  • the protease cleavage site is a 2A cleavage site.
  • the cleavage site can be a self-cleaving peptide such as a T2A, P2A, E2A or F2A cleavage site.
  • the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.
  • the third nucleic acid sequence are operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof. In some embodiments, the third nucleic acid sequence are operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof by a linker.
  • the third nucleic acid sequence are operatively linked to the first nucleic acid sequence, and/or the second nucleic acid sequence, independently. In some embodiments, the third nucleic acid sequence are operatively linked to the first nucleic acid sequence, and/or the second nucleic acid sequence, independently, by a linker. In some embodiments, the linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A cleavage site. In some embodiments, the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.
  • the third nucleic acid sequence and the first nucleic acid sequence, the third nucleic acid sequence and the second nucleic acid sequence, or the third nucleic acid sequence, the first nucleic acid sequence, and the second nucleic acid sequence are present on different nucleic acid molecules.
  • the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs:283, 284, 285, and 286. In some embodiments, the TGFBr2 switch polypeptide comprises any one sequence selected from SEQ ID NOs:283, 284, 285, and 286.
  • sequence of the TGFBr2 switch polypeptide is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to any one sequence selected from SEQ ID NOs:283, 284, 285, and 286. In some embodiments, the sequence of the TGFBr2 switch polypeptide is any one sequence selected from SEQ ID NOs:283, 284, 285, and 286.
  • the TGFBr2 switch polypeptide comprises an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of a TGFBr2 switch polypeptide as described herein.
  • the TGFBr2 switch polypeptide comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of a TGFBr2 switch polypeptide as described herein.
  • the TGFBr2 switch polypeptide comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of a TGFBr2 switch polypeptide as described herein.
  • the TGFBr2 switch polypeptide comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of any one sequence selected from SEQ ID NOs:283, 284, 285, and 286.
  • the TGFBr2 switch polypeptide comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of any one sequence selected from SEQ ID NOs:283, 284, 285, and 286.
  • the TGFBr2 switch polypeptide comprises amino acid residue deletions from the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of a sequence of a TGFBr2 switch polypeptide as described herein.
  • the TGFBr2 switch polypeptide comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C-terminal end of a sequence of a TGFBr2 switch polypeptide as described herein.
  • the TGFBr2 switch polypeptide comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C-terminal end of a sequence of a TGFBr2 switch polypeptide as described herein.
  • the TGFBr2 switch polypeptide comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C- terminal end of any one sequence selected from SEQ ID NOs:283, 284, 285, and 286.
  • the TGFBr2 switch polypeptide comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids deleted from the N-terminal or C-terminal end of any one sequence selected from SEQ ID NOs:283, 284, 285, and 286.
  • the TGFBr2 switch polypeptide comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of a sequence of a TGFBr2 switch polypeptide as described herein.
  • the TGFBr2 switch polypeptide comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of a sequence of a TGFBr2 switch polypeptide as described herein.
  • the TGFBr2 switch polypeptide comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of any one sequence selected from SEQ ID NOs:283, 284, 285, and 286.
  • the TGFBr2 switch polypeptide comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids independently deleted from both N-terminal and C-terminal ends of any one sequence selected from SEQ ID NOs:283, 284, 285, and 286.
  • the TGFBr2 extracellular domain comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437.
  • the TGFBr2 extracellular domain comprises the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437.
  • the sequence of the TGFBr2 extracellular domain is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, or 99.9% sequence identity to SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437. In some embodiments, the sequence of the TGFBr2 extracellular domain is the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437.
  • the TGFBr2 switch polypeptide comprises an extracellular domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the TGFBr2 extracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an extracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the sequence of the TGFBr2 extracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an extracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of the TGFBr2 extracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an extracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437.
  • the TGFBr2 switch polypeptide comprises an extracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437.
  • the TGFBr2 switch polypeptide comprises an extracellular domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of the TGFBr2 extracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an extracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids deleted from the N-terminal or C- terminal end of the sequence of the TGFBr2 extracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an extracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids deleted from the N-terminal or C-terminal end of the sequence of the TGFBr2 extracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an extracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437.
  • the TGFBr2 switch polypeptide comprises an extracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437.
  • the TGFBr2 switch polypeptide comprises an extracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of the TGFBr2 extracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an extracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of the TGFBr2 extracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an extracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437.
  • the TGFBr2 switch polypeptide comprises an extracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:271, SEQ ID NO:432, or SEQ ID NO:437.
  • TGFBr2 extracellular domain TGFBR2 ecto: TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQE VCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPDLLLVIFQ (SEQ ID NO:271)
  • TGFBr2 extracellular domain with a signal peptide: MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAAS PKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ (SEQ ID NO:432) [0556] TGFBr2 signal peptide: MGRGLLRGLWPLHIVLWTRIAS (SEQ ID NO:431)
  • the TGFBr2 switch polypeptide further comprises a switch intracellular domain.
  • the TGFBr2 extracellular domain or functional fragment thereof is operably linked to the switch intracellular domain.
  • the switch intracellular domain comprises an intracellular domain of a costimulatory polypeptide.
  • the costimulatory polypeptide is selected from the group consisting of CD28, 4-lBB(CD137), IL-15Ra, IL12R, IL18R, IL21R, 0X40, CD2, CD27, CD5, ICAM-1, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, CD226, FcyRI, FcyRII, and FcyRIII.
  • the costimulatory polypeptide is CD28.
  • the costimulatory polypeptide is 4- 1BB.
  • the costimulatory polypeptide is IL-15Ra.
  • the switch intracellular domain comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to or SEQ ID NO:273 or SEQ ID NO:277. In some embodiments, the switch intracellular domain comprises the sequence of SEQ ID NO:273 or SEQ ID NO:277.
  • the sequence of the switch intracellular domain is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:273 or SEQ ID NO:277.
  • the sequence of the switch intracellular domain is the sequence of SEQ ID NO:273 or SEQ ID NO:277.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the intracellular domain sequence as described herein.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the intracellular domain sequence as described herein.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the intracellular domain sequence as described herein.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N- terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:273 or SEQ ID NO:277.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C- terminal ends of the sequence of SEQ ID NO:273 or SEQ ID NO:277.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the intracellular domain sequence as described herein.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids deleted from the N-terminal or C-terminal end of the intracellular domain sequence as described herein.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids deleted from the N-terminal or C-terminal end of the intracellular domain sequence as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids deleted from the N-terminal or C- terminal end of the sequence of SEQ ID NO:273 or SEQ ID NO:277.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:273 or SEQ ID NO:277.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids independently deleted from both N-terminal and C-terminal ends of the intracellular domain sequence as described herein.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:273 or SEQ ID NO:277.
  • the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one selected from SEQ ID NOs:287, 277, 288, 289, 273, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 313, 314, 315, 316, and a combination thereof.
  • the TGFBr2 switch polypeptide comprises any one sequence selected from the group consisting of SEQ ID NOs:287, 277, 288, 289, 273, 290, 291, 292, 293, 294, 295, 296,
  • sequence of the switch intracellular domain is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one selected from SEQ ID NOs:287, 277, 288, 289, 273, 290, 291, 292, 293, 294, 295, 296, 297,
  • sequence of the switch intracellular domain is any one sequence selected from the group consisting of SEQ ID NOs:287, 277, 288, 289, 273, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 313, 314, 315, 316, and a combination thereof.
  • PD -1 Intracellular domain CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATI VFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO:287) [0563] 4-1BB Intracellular domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:277) [0564] ICOS Intracellular domain CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO:288) [0565] CTLA4 Intracellular domain AVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO:289) [0566] CD28 Intracellular domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:273) [0567] CD200R Intracellular domain
  • TIM-3 Intracellular domain FKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYV SSRQQPSQPLGCRFAMP (SEQ ID NO:292)
  • TIGIT Intracellular domain LTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCA ELHDYFNVLSYRSLGNCSFFTETG (SEQ ID NO:293)
  • TGFPR2 Intracellular domain
  • IL12R IL-12 receptor subunit beta-2 intracellular domain HYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPE PLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQ LVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEE LEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML (SEQ ID NO:314) [0579] IL18R1 (Interleukin- 18 receptor 1) intracellular domain YRVDLVLFYRHLTRRDETLTDGKTYDAFVSY
  • the costimulatory polypeptide is selected from the group consisting of 0X40, CD2, CD27, CD5, IL12R, IL18R, IL21R, ICAM-1, ICOS (CD278), 4-1BB (CD 137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, FcyRIII, CD3 zeta, CD28, CD27, ICOS, DAP 10, DAP12, LFA-1 (CD1 la/CD18) an MHC class 1 molecule, BTLA and a Toll ligand receptor, lymphocyte function-associated antigen-1 (LFA-1, also known as CDl la/CD18), CD276 (B7- H3), IL-15Ra, and a ligand that specifically binds with CD83.
  • LFA-1 lymphocyte function-associated antigen-1
  • the costimulatory polypeptide is IL-15Ra.
  • the sequence of the switch intracellular domain is from the intracellular domain of IL-15Ra.
  • the sequence of the switch intracellular domain comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:372.
  • the sequence of the switch intracellular domain comprises a sequence or portion thereof of SEQ ID NO:372.
  • the sequence of the switch intracellular domain comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO:383. In some embodiments, the sequence of the switch intracellular domain comprises a sequence or portion thereof of SEQ ID NO: 383.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:372 or SEQ ID NO:383.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids deleted from the N-terminal or C-terminal end of the sequence of an IL-15Ra intracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:372. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:372.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:372. In some embodiments, the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:372.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:383.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:383.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids independently deleted from both N- terminal and C-terminal ends of the sequence of SEQ ID NO: 383.
  • the TGFBr2 switch polypeptide comprises an intracellular domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:383.
  • the TGFBr2 switch polypeptide further comprises a switch transmembrane domain.
  • the TGFBr2 extracellular domain or functional fragment thereof is operably linked to the switch intracellular domain via the switch transmembrane domain.
  • the switch transmembrane domain is derived from a TGFBr2 transmembrane domain. In some embodiments, the switch transmembrane domain is a TGFBr2 transmembrane domain. In some embodiments, the switch transmembrane domain comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272. In some embodiments, the switch transmembrane domain comprises the sequence of SEQ ID NO:272.
  • the sequence of the switch transmembrane domain is a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:272. In some embodiments, the sequence of the switch transmembrane domain is the sequence of SEQ ID NO:272.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of a TGFBr2 transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the sequence of a TGFBr2 transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of a TGFBr2 transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the sequence of SEQ ID NO:272.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:272.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of a TGFBr2 transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids deleted from the N-terminal or C- terminal end of the sequence of a TGFBr2 transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids deleted from the N-terminal or C-terminal end of the sequence of a TGFBr2 transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:272.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:272. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of a TGFBr2 transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids independently deleted from both N- terminal and C-terminal ends of the sequence of a TGFBr2 transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:272.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:272.
  • the switch transmembrane domain is derived from a transmembrane domain of the costimulatory polypeptide. In some embodiments, the switch transmembrane domain is a transmembrane domain of the costimulatory polypeptide. In some embodiments, the switch transmembrane domain is derived from a transmembrane domain of CD28. In some embodiments, the switch transmembrane domain is derived from a transmembrane domain of 4-1BB. In some embodiments, the switch transmembrane domain is a transmembrane domain of CD28. In some embodiments, the switch transmembrane domain is a transmembrane domain of 4-1BB.
  • the switch transmembrane domain comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence to SEQ ID NO:275 or SEQ ID NO:279. In some embodiments, the switch transmembrane domain comprises the sequence of SEQ ID NO:275 or SEQ ID NO:279.
  • the sequence of the switch transmembrane domain is a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence to SEQ ID NO:275 or SEQ ID NO:279. In some embodiments, the sequence of the switch transmembrane domain is the sequence of SEQ ID NO:275 or SEQ ID NO:279.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of a CD28 transmembrane domain or a 4- IBB transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of a CD28 transmembrane domain or a 4- IBB transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the sequence of a CD28 transmembrane domain or a 4- IBB transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the sequence of SEQ ID NO:275 or SEQ ID NO:279.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:275 or SEQ ID NO:279.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of a CD28 transmembrane domain or a 4-1BB transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids deleted from the N-terminal or C-terminal end of the sequence of a CD28 transmembrane domain or a 4-1BB transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids deleted from the N-terminal or C-terminal end of the sequence of a CD28 transmembrane domain or a 4-1BB transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:275 or SEQ ID NO:279.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids deleted from the N-terminal or C-terminal end of the sequence of SEQ ID NO:275 or SEQ ID NO:279.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of a CD28 transmembrane domain or a 4-1BB transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of a CD28 transmembrane domain or a 4-1BB transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:275 or SEQ ID NO:279.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:275 or SEQ ID NO:279.
  • the switch transmembrane domain is derived from a transmembrane domain of IL-15Ra. In some embodiments, the switch transmembrane domain is a transmembrane domain of IL-15Ra. In some embodiments, the switch transmembrane domain comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence to SEQ ID NO:300. In some embodiments, the switch transmembrane domain comprises the sequence of SEQ ID NO:300.
  • the sequence of the switch transmembrane domain is a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence to SEQ ID NO:300.
  • the sequence of the switch transmembrane domain is the sequence of SEQ ID NO:300.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N- terminal and C-terminal ends of the sequence of an IL-15Ra transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:300.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N- terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:300.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having a deletion of amino acid residue(s) from the N-terminal end, C- terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids deleted from the N-terminal or C-terminal end of the sequence of an IL-15Ra transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids deleted from the N-terminal or C-terminal end of the sequence of an IL-15Ra transmembrane domain as described herein. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids deleted from the N-terminal or C- terminal end of the sequence of SEQ ID NO:300. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9,
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of an IL- 15Ra transmembrane domain as described herein.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids independently deleted from both N- terminal and C-terminal ends of the sequence of SEQ ID NO:300. In some embodiments, the TGFBr2 switch polypeptide comprises a transmembrane domain sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of SEQ ID NO:300.
  • the switch transmembrane domain is derived from any one selected from the group consisting of an ICOS transmembrane domain or a fragment thereof, a PD-1 transmembrane domain or a fragment thereof, a CTLA4 transmembrane domain or a fragment thereof, a CD200R transmembrane domain or a fragment thereof, a BTLA transmembrane domain or a fragment thereof, a TIM-3 transmembrane domain or a fragment thereof, a TIGIT transmembrane domain or a fragment thereof, a CD28 transmembrane domain or a fragment thereof, a TGFPR2 transmembrane domain or a fragment thereof, a 4-IBB transmembrane domain or a fragment thereof, an IL- 1 ORA transmembrane domain or a fragment thereof, an IL-7RA transmembrane domain or a fragment thereof, an IL-4RA transmembrane domain or a fragment thereof, a Fa
  • an IL12R transmembrane domain or a fragment thereof is an IL- 12 receptor subunit beta-1 transmembrane domain or a fragment thereof. In some embodiments, an IL12R transmembrane domain or a fragment thereof is an IL-12 receptor subunit beta-2 transmembrane domain or a fragment thereof. In some embodiments, an IL18R transmembrane domain or a fragment thereof is an interleukin- 18 receptor 1 transmembrane domain or a fragment thereof.
  • the TGFBr2 switch polypeptide comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one selected from SEQ ID NOs:301, 279, 302, 303, 275, 304, 305, 306, 307, 272, 308, 309, 310, 311, 312, 317, 318, 319, 320, and a combination thereof.
  • the TGFBr2 switch polypeptide comprises any one sequence selected from the group consisting of SEQ ID NOs:301, 279, 302, 303, 275, 304, 305, 306, 307, 272, 308, 309, 310, 311, 312, 317, 318, 319, 320, and a combination thereof.
  • the sequence of the switch intracellular domain is a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one selected from SEQ ID NOs:301, 279, 302, 303, 275, 304, 305, 306, 307, 272, 308, 309, 310, 311, 312, 317, 318, 319, 320, and a combination thereof.
  • the sequence of the switch intracellular domain is any one sequence selected from the group consisting of SEQ ID NOs:301, 279, 302, 303, 275, 304, 305, 306, 307, 272, 308, 309, 310, 311, 312, 317, 318, 319, 320, and a combination thereof.
  • FWLPIGCAAFVVVCILGCILI (SEQ ID NO: 302) [0601] CTLA4 Transmembrane domain FLLWILAAVSSGLFFYSFLLT (SEQ ID NO:303) [0602] CD28 Transmembrane domain
  • VTGISLLPPLGVAISVIIIFY SEQ ID NO:272
  • IL18R1 Interleukin- 18 receptor 1 transmembrane domain
  • GWNPHLLLLLLLVIVFIPAFW (SEQ ID NO: 320)
  • the TGFBr2 switch polypeptide further comprises an additional intracellular domain.
  • the additional intracellular domain is operably linked to the C-terminus of the switch intracellular domain.
  • the additional intracellular domain is operably linked to the N-terminus of the switch intracellular domain.
  • the additional intracellular domain is operably linked to the C-terminus or the N-terminus of the switch intracellular domain.
  • the additional intracellular domain is operably linked to the C-terminus or the N-terminus of the switch intracellular domain via a linker.
  • the TGFBr2 switch polypeptide may comprise two or more additional intracellular domains.
  • the additional intracellular domains are operably linked to the C-terminus and the N-terminus of the switch intracellular domain, independently. In some embodiments, the additional intracellular domains are operably linked to the C-terminus, to the N-terminus, or independently to the C-terminus and the N-terminus of the switch intracellular domain via a linker.
  • the TGFBr2 switch polypeptide further comprises one or more additional intracellular domains.
  • one or more additional intracellular domains are the same intracellular domain.
  • one or more additional intracellular domains are different intracellular domains.
  • the additional intracellular domain is derived from an intracellular domain of IL-15Ra. In some embodiments, the additional intracellular domain comprises an intracellular domain of IL-15Ra or a fragment thereof.
  • IL-15Ra or a fragment thereof comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
  • IL-15Ra or a fragment thereof comprises a sequence having at least about 50%, 55%, 60%, 6%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to a sequence encoding IL-15Ra.
  • IL-15Ra or a fragment thereof comprises a sequence encoding IL-15Ra having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
  • the additional intracellular domain comprises a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:372 or SEQ ID NO:383. In some embodiments, the additional intracellular domain comprises the sequence of SEQ ID NO:372 or SEQ ID NO:383.
  • sequence of the additional intracellular domain is a sequence with at least 50%, 55%, 60%, 65%, 90%, 75%, 80%, 85%, 90%, 95%, 97%, 99.0%, 99.5%, 99.7%, or 99.9% sequence identity to SEQ ID NO:372 or SEQ ID NO:383.
  • sequence of the additional intracellular domain is the sequence of SEQ ID NO:372 or SEQ ID NO:383.
  • the additional intracellular domain comprises a sequence having an addition of amino acid residue(s) to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein.
  • the additional intracellular domain comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N- terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL- 15Ra intracellular domain as described herein.
  • the additional intracellular domain comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein.
  • the additional intracellular domain comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:372 or SEQ ID NO:383.
  • the additional intracellular domain comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more amino acid residues added to the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of SEQ ID NO:372 or SEQ ID NO:383.
  • the additional intracellular domain comprises a sequence having a deletion of amino acid residue(s) from the N-terminal end, C-terminal end, or both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein.
  • the additional intracellular domain comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids deleted from the N-terminal or C-terminal end of the sequence of an IL- 15Ra intracellular domain as described herein.
  • the additional intracellular domain comprises a sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids deleted from the N-terminal or C-terminal end of the sequence of an IL-15Ra intracellular domain as described herein.
  • the additional intracellular domain comprises a sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, or 165 amino acids independently deleted from both N-terminal and C-terminal ends of the sequence of an IL-15Ra intracellular domain as described herein.

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Abstract

Sont divulgués ici des acides nucléiques recombinants comprenant une séquence codant pour des protéines de fusion (TFP) contenant le récepteur des lymphocytes T (TCR) et une séquence codant pour CXCR6 ou un fragment fonctionnel associé, des cellules comprenant les acides nucléiques recombinants, des procédés d'utilisation des acides nucléiques recombinants et/ou des cellules, notamment des méthodes de traitement de maladies ou d'états, y compris le cancer.
PCT/US2022/041904 2021-08-30 2022-08-29 Compositions et procédés de reprogrammation de tcr à l'aide de protéines de fusion et de cxcr6 WO2023034220A2 (fr)

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