WO2022006451A2 - Compositions et procédés de reprogrammation de tcr faisant intervenir des protéines de fusion et des anticorps anti-pd1 - Google Patents

Compositions et procédés de reprogrammation de tcr faisant intervenir des protéines de fusion et des anticorps anti-pd1 Download PDF

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WO2022006451A2
WO2022006451A2 PCT/US2021/040177 US2021040177W WO2022006451A2 WO 2022006451 A2 WO2022006451 A2 WO 2022006451A2 US 2021040177 W US2021040177 W US 2021040177W WO 2022006451 A2 WO2022006451 A2 WO 2022006451A2
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seq
domain
cell
amino acid
nucleic acid
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PCT/US2021/040177
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WO2022006451A3 (fr
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Derrick Mccarthy
Vania E. Ashminova
Michael Lofgren
Robert Hofmeister
Patrick Baeuerle
Dario Gutierrez
Robert Tighe
Reshma Singh
Courtney Kay ANDERSON
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TCR2 Therapeutics Inc.
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Publication of WO2022006451A2 publication Critical patent/WO2022006451A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • PD-1 and/or PD- L1 have been found in a number of primary tumor biopsies assessed by immunohistochemistry.
  • Such tissues include cancers of the lung, liver, ovary, cervix, skin, colon, glioma, bladder, breast, kidney, esophagus, stomach, oral squamous cell, urothelial cell, and pancreas as well as tumors of the head and neck.
  • PD-ligand expression on tumor cells has been correlated to poor prognosis of cancer patients across multiple tumor types.
  • PD-1 antibody when said PD-1 antibody is expressed in, and secreted from, the same cell as the TFP-expressing T-cell, such PD-1 antibodies can serve to antagonize PD-1 both in the TFP T-cells and also in nearby cytotoxic T-cells, thereby inducing PD-1 blockade in PD-1 expressing cytotoxic T-cells local to the cancer cells having the tumor antigen targeted by the TFP T-cells.
  • the antibodies or other antigen-binding proteins that target PD-1 can be secreted from the cell or membrane-associated.
  • the present disclosure provides a T cell comprising (I) a first recombinant nucleic acid comprising a first 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, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, and (b) a binding domain; and (II) a second sequence encoding an anti-PD-1 antibody or fragment thereof that specifically binds programmed cell death protein 1 (PD-1), wherein the anti-PD-1 antibody or fragment thereof inhibits an interaction of PD-1 with PD-L1 or PD-L2, wherein the anti-PD-1 antibody or fragment thereof is secreted by the T cell, wherein the TCR subunit and the binding domain are operatively linked, and wherein the TFP functionally interacts with an endogenous TCR complex
  • a T cell comprising a first recombinant nucleic acid comprising a first 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, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, and (b) a binding domain; and (II) a second sequence encoding a fusion protein comprising an anti-PD-1 antibody or fragment thereof that specifically binds PD-1, wherein the anti-PD-1 antibody or fragment thereof inhibits an interaction of PD-1 with PD-L1 or PD-L2, and a transmembrane domain operatively linked to the anti-PD-1 antibody or fragment thereof, wherein the TCR subunit and the binding domain are operatively linked, and wherein the TFP functionally interacts with an endogenous TCR
  • a T cell comprising (I) a first recombinant nucleic acid comprising a first 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, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, and (b) a binding domain; and (II) a second sequence encoding a fusion protein comprising an anti-PD-1 antibody or fragment thereof that specifically binds PD-1 wherein the anti-PD-1 antibody or fragment thereof inhibits an interaction of PD-1 with PD-L1 or PD-L2, and an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof, wherein the TCR subunit and the binding domain are operatively linked, and wherein the TFP functionally interacts with an endogenous TCR complex in
  • the first recombinant nucleic acid comprises the second sequence.
  • the first sequence and the second sequence are contained in a single operon.
  • the first sequence and the second sequence are operatively linked by a linker sequence.
  • the linker sequence encodes 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 second sequence is present on a second recombinant nucleic acid.
  • the anti-PD-1 antibody or fragment thereof comprises a variable domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:3.
  • the CDR1 comprises the amino acid sequence of SEQ ID NO:1.
  • the CDR2 comprises the amino acid sequence of SEQ ID NO:2.
  • the variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4.
  • variable domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4.
  • variable domain comprises the amino acid sequence of SEQ ID NO:4.
  • the variable domain is encoded by the nucleic acid sequence of SEQ ID NO:20.
  • the variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8.
  • the variable domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8.
  • variable domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8. [0025] In some embodiments, the variable domain comprises the amino acid sequence of SEQ ID NO:5. [0026] In some embodiments, the variable domain is encoded by the nucleic acid sequence of SEQ ID NO:21. [0027] In some embodiments, the variable domain comprises the amino acid sequence of SEQ ID NO:6. [0028] In some embodiments, the variable domain is encoded by the nucleic acid sequence of SEQ ID NO:22. [0029] In some embodiments, the variable domain comprises the amino acid sequence of SEQ ID NO:7.
  • variable domain is encoded by the nucleic acid sequence of SEQ ID NO:23.
  • variable domain comprises the amino acid sequence of SEQ ID NO:8.
  • variable domain is encoded by the nucleic acid sequence of SEQ ID NO:24.
  • the anti-PD-1 antibody or fragment thereof binds to human PD-1.
  • the anti-PD-1 antibody or fragment thereof binds to human PD-1 with a K D value of about 1 to about 60 nM.
  • the anti-PD-1 antibody or fragment thereof binds to human PD-1 with a KD value of at most 55 nM.
  • the anti-PD-1 antibody or fragment thereof is an antagonist.
  • the anti-PD-1 antibody or fragment thereof is a PD-1 inhibitor.
  • the anti-PD-1 antibody or fragment thereof competes with PD-L1 for binding to PD-1, inhibits PD-L1 from interacting with PD-1, and/or binds to the same epitope of PD-1 to which PD-L1 binds.
  • the anti-PD-1 antibody or fragment thereof blocks an immune checkpoint or blocks immune checkpoint signaling.
  • the anti-PD-1 antibody or fragment thereof is an immune checkpoint inhibitor or inhibits immune checkpoint signaling.
  • the anti-PD-1 antibody or fragment thereof is a human or humanized antibody or fragment thereof.
  • the anti-PD-1 antibody or fragment thereof is a single domain antibody (sdAb).
  • the sdAb is a V HH .
  • the V HH is Fc conjugated.
  • the Fc is an effector-function silent IgG4.
  • the fusion protein further comprises a signal sequence.
  • the signal sequence is a PD-1 signal peptide.
  • the signal sequence comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:256 or SEQ ID NO:335.
  • the signal sequence comprises the amino acid sequence of SEQ ID NO:256 or SEQ ID NO:335.
  • the anti-PD-1 antibody or fragment thereof binds to PD-1 on the surface of the T cell, PD-1 on the surface of a bystander T cell, or a combination thereof.
  • the fusion protein further comprises an intracellular domain operatively linked to the transmembrane domain.
  • the fusion protein further comprises a transmembrane domain operatively linked to the intracellular domain and the anti-PD-1 antibody or fragment thereof.
  • the transmembrane domain of the fusion protein comprises a transmembrane domain of a protein selected from the group consisting of CD28, CD3 ⁇ , CD3 ⁇ , CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD41, CD64, CD68, CD80, CD86, CD134, CD137, CD154, ICOS, 4-1BB, OX40, PD-1, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the transmembrane domain of the fusion protein comprises a PD-1 transmembrane domain.
  • the transmembrane domain of the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:301.
  • the transmembrane domain of the fusion protein comprises the amino acid sequence of SEQ ID NO:301.
  • the fusion protein further comprises a PD-1 stalk domain.
  • the PD-1 stalk domain is operatively linked to the transmembrane domain.
  • the PD-1 stalk domain is operatively linked to the N-terminus of the transmembrane domain.
  • the PD-1 stalk domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:259.
  • the PD-1 stalk domain comprises the amino acid sequence of SEQ ID NO:259.
  • the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:259 operatively linked to an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:301.
  • the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:259 operatively linked to the N terminus of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:301. [0064] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:259 operatively linked to the amino acid sequence of SEQ ID NO:301. [0065] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:259 operatively linked to the N-terminus of the amino acid sequence of SEQ ID NO:301. [0066] In some embodiments, the intracellular domain of the fusion protein comprises a co- stimulatory domain.
  • the co-stimulatory domain comprises a co-stimulatory domain of a protein selected from the group consisting of a CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, PD-1, CD258, ICAM-1, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the co-stimulatory domain comprises a 4-1BB (CD137) co- stimulatory domain.
  • the co-stimulatory domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:277. [0070] In some embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:277. [0071] In some embodiments, the co-stimulatory domain comprises a CD28 co-stimulatory domain. [0072] In some embodiments, the co-stimulatory domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:273. [0073] In some embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:273.
  • the fusion protein comprises two or more anti-PD-1 antibodies or fragments thereof. [0075] In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are operatively linked tandemly. [0076] In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are identical. [0077] In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are different. [0078] In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are operatively linked by a linker. [0079] In some embodiments, the linker comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:371.
  • the linker comprises the amino acid sequence of SEQ ID NO:371.
  • the fusion protein further comprises a PD-1 signal peptide, a PD-1 stalk domain, a PD-1 transmembrane domain, and a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • the fusion protein comprises, from the N-terminus to the C- terminus, a PD-1 signal peptide operatively linked to the anti-PD-1 antibody or fragment thereof operatively linked to a PD-1 stalk domain operatively linked to a PD-1 transmembrane domain operatively linked to a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • the fusion protein comprises, from the N-terminus to the C- terminus, a PD-1 signal peptide operatively linked to a first anti-PD-1 antibody or fragment thereof operatively linked to a linker operatively linked to a second anti-PD-1 antibody or fragment thereof operatively linked to a PD-1 stalk domain operatively linked to a PD-1 transmembrane domain operatively linked to a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • a PD-1 signal peptide operatively linked to a first anti-PD-1 antibody or fragment thereof operatively linked to a linker operatively linked to a second anti-PD-1 antibody or fragment thereof operatively linked to a PD-1 stalk domain operatively linked to a PD-1 transmembrane domain operatively linked to a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • the fusion protein comprises SEQ ID NO:256 or SEQ ID NO:335, SEQ ID NO:4 or SEQ ID NO:6, SEQ ID NO:259, SEQ ID NO:301, and SEQ ID NO:273 or SEQ ID NO:277.
  • the fusion protein comprises, from the N-terminus to the C- terminus, SEQ ID NO:256 or SEQ ID NO:335 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:259 operatively linked to SEQ ID NO:301 operatively linked to SEQ ID NO:273 or SEQ ID NO:277.
  • the fusion protein comprises, from the N-terminus to the C- terminus, SEQ ID NO:256 or SEQ ID NO:335 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:371 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:259 operatively linked to SEQ ID NO:301 operatively linked to SEQ ID NO:273 or SEQ ID NO:277.
  • the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 373-377.
  • the fusion protein comprises an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 373-377. [0089] In some embodiments, the fusion protein is encoded by a nucleic acid sequence having at least 60% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 378-382. [0090] In some embodiments, the fusion protein is encoded by any one of the nucleic acid sequences of SEQ ID NOs: 378-382.
  • the at least a portion of a TCR extracellular domain of the TFP comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, 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 transmembrane domain of the TFP comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, 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 intracellular domain of the TFP comprises an intracellular domain of a protein selected from the group consisting of TCR alpha, TCR beta, TCR delta, TCR gamma, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR intracellular domain of the TFP comprises a stimulatory domain from an intracellular signaling domain of CD3 gamma, CD3 delta, or CD3 epsilon, or an amino acid sequence having at least one modification thereto.
  • the TCR intracellular domain of the TFP does not comprise CD3 zeta.
  • the TFP does not comprise a costimulatory domain.
  • the binding domain is connected to the TCR extracellular domain by a linker sequence.
  • the linker is 120 amino acids in length or less.
  • the linker sequence comprises (G 4 S) n , wherein G is glycine, S is serine, and n is an integer from 1 to 10, e.g., 1 to 4.
  • at least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit.
  • At least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR alpha. [0102] In some embodiments, at least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR beta. [0103] In some embodiments, at least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma. [0104] In some embodiments, at least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta.
  • At least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon. [0106] In some embodiments, at least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta. [0107] In some embodiments, at least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma. [0108] In some embodiments, 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 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 TCR subunit comprises the amino acid sequence of SEQ ID NO:57.
  • the TCR subunit comprises the amino acid sequence of SEQ ID NO:58.
  • the TCR subunit comprises the amino acid sequence of SEQ ID NO:59.
  • 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. [0120] In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma. [0121] In some embodiments, the T cell is a CD8+ or CD4+ T cell. [0122] In some embodiments, the T cell is a human T cell. [0123] In some embodiments, the T cell is a human ⁇ T cell. [0124] In some embodiments, the T cell is a human ⁇ T cell. [0125] In some embodiments, the T cell is an autologous T cell.
  • the T cell is an allogeneic T cell.
  • the T cell exhibits increased cytotoxicity to a human cell expressing an antigen that specifically interacts with the binding domain compared to a T cell not containing the TFP.
  • the binding domain comprises an antibody or a fragment thereof, a ligand, or a ligand binding protein.
  • the ligand or the antibody fragment comprises a scFv or a single domain antibody (sdAb) domain.
  • the sdAb is a VHH.
  • the antibody or fragment thereof comprises an antigen binding domain 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, an anti-Nectin4 binding domain, an anti-GPC3 binding domain, an anti-Trop2 binding domain, and anti-MUC16 binding domain.
  • BCMA anti-B-cell maturation antigen
  • MSLN anti-mesothelin
  • the binding domain comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 comprises an amino acid sequence of SEQ ID NO:60, SEQ ID NO:63, or SEQ ID NO:66; wherein the CDR2 comprises an amino acid sequence of SEQ ID NO:61, SEQ ID NO:64, or SEQ ID NO:67; and wherein the CDR3 comprises an amino acid sequence of SEQ ID NO:62, SEQ ID NO:65, or SEQ ID NO:68. [0133] In some embodiments, the binding domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 69-71.
  • the binding domain comprises an amino acid sequence of any one of the amino acid sequences of ID NOs: 69-71. [0135] In some embodiments, the binding domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:69. [0136] In some embodiments, the binding domain comprises the amino acid sequence of SEQ ID NO:69.
  • the binding domain comprises: (i) a light chain (LC) CDR1, LC CDR2, and LC CDR3 amino acid sequence of SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30, respectively; (ii) a heavy chain (HC) CDR1, HC CDR2, and HC CDR3 amino acid sequence of SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:36, respectively; or (iii) a combination thereof.
  • LC light chain
  • HC CDR3 amino acid sequence of SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30 respectively
  • HC heavy chain
  • the binding domain comprises: (i) a light chain variable region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:38; (ii) a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:40 or (iii) a combination thereof. [0139] In some embodiments, the binding domain comprises: (i) a light chain variable region comprising the amino acid sequence of SEQ ID NO:38; (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:40 or (iii) a combination thereof.
  • the binding domain comprises a CDR1 having an amino acid sequence of SEQ ID NO:204, a CDR2 having an amino acid sequence of SEQ ID NO:205; and a CDR3 having an amino acid sequence of SEQ ID NO:206. [0141] In some embodiments, the binding domain comprises a variable region having an amino acid sequence of SEQ ID NO:207.
  • the binding domain comprises: (i) a light chain (LC) CDR1, LC CDR2, and LC CDR3 amino acid sequence of SEQ ID NO:228, SEQ ID NO:229, and SEQ ID NO:230, respectively; (ii) a heavy chain (HC) CDR1, HC CDR2, and HC CDR3 amino acid sequence of SEQ ID NO:224, SEQ ID NO:225, and SEQ ID NO:226.
  • LC light chain
  • HC CDR3 amino acid sequence of SEQ ID NO:224 amino acid sequence of SEQ ID NO:224, SEQ ID NO:225, and SEQ ID NO:226.
  • the binding domain comprises: (i) a light chain variable region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:231; (ii) a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:227 or (iii) a combination thereof.
  • the TFP further comprises a leader sequence.
  • the leader sequence is a GM-CSFR leader sequence or a CSF2RA leader sequence.
  • the leader sequence comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:359.
  • the leader sequence comprises the amino acid sequence of SEQ ID NO:359.
  • the first recombinant nucleic acid comprises a sequence encoding a GM-CSFR or CSF2RA leader sequence, an anti-mesothelin (MSLN) binding domain or a fragment thereof, a first linker, a CD3 epsilon intracellular domain, a second linker, and an anti- PD-1 antibody or fragment thereof.
  • the first recombinant nucleic acid further comprises a sequence encoding a His tag, a HA tag, a hFc tag or a combination thereof.
  • the first recombinant nucleic acid comprises a sequence encoding, from the N-terminus to the C-terminus, a GM-CSFR or CSF2RA leader sequence operatively linked to an anti-mesothelin (MSLN) binding domain or a fragment thereof operatively linked to a first linker operatively linked to a CD3 epsilon intracellular domain operatively linked to a second linker operatively linked to an anti-PD-1 antibody or fragment thereof operatively linked to a tag.
  • the tag is a His tag, a HA tag, a hFc tag or a combination thereof.
  • the first recombinant nucleic acid comprises a sequence encoding a GM-CSFR or CSF2RA leader sequence, an anti-mesothelin (MSLN) binding domain or a fragment thereof, a first linker, a CD3 epsilon intracellular domain, a second linker, a PD-1 signal peptide, an anti-PD-1 antibody or fragment thereof, a PD-1 stalk domain, a PD-1 transmembrane domain, and a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • MSLN anti-mesothelin
  • the first recombinant nucleic acid comprises a sequence encoding, from the N-terminus to the C-terminus, a GM-CSFR or CSF2RA leader sequence operatively linked to an anti-mesothelin (MSLN) binding domain or a fragment thereof operatively linked to a first linker operatively linked to a CD3 epsilon intracellular domain operatively linked to a second linker operatively linked to a PD-1 signal peptide operatively linked to an anti-PD-1 antibody or fragment thereof operatively linked to a PD-1 stalk domain operatively linked to a PD-1 transmembrane domain operatively linked to a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • MSLN anti-mesothelin
  • the first recombinant nucleic acid comprises a sequence encoding, from the N-terminus to the C-terminus, a GM-CSFR or CSF2RA leader sequence operatively linked to an anti-mesothelin (MSLN) binding domain or a fragment thereof operatively linked to a first linker operatively linked to a CD3 epsilon intracellular domain operatively linked to a second linker operatively linked to a PD-1 signal peptide operatively linked to a first anti-PD-1 antibody or fragment thereof operatively linked to a third linker operatively linked to a second anti-PD-1 antibody or fragment thereof operatively linked to a PD-1 stalk domain operatively linked to a PD-1 transmembrane domain operatively linked to a CD28 or 4-1BB (CD137) co- stimulatory domain.
  • MSLN anti-mesothelin
  • the first recombinant nucleic acid comprises a sequence encoding SEQ ID NO:359, SEQ ID NO:69, SEQ ID NO:118, SEQ ID NO:360, SEQ ID NO:362, SEQ ID NO:256 or SEQ ID NO:355, SEQ ID NO:4 or SEQ ID NO:6, SEQ ID NO:259, SEQ ID NO:301, and SEQ ID NO:273 or SEQ ID NO:277.
  • the first recombinant nucleic acid comprises a sequence encoding, from the N-terminus to the C-terminus, SEQ ID NO:359 operatively linked to SEQ ID NO:69 operatively linked to SEQ ID NO:118 operatively linked to SEQ ID NO:360 operatively linked to SEQ ID NO:362 operatively linked to SEQ ID NO:256 or SEQ ID NO:355 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:259 operatively linked to SEQ ID NO:301 operatively linked to SEQ ID NO:273 or SEQ ID NO:277.
  • the first recombinant nucleic acid comprises a sequence encoding, from the N-terminus to the C-terminus, SEQ ID NO:359 operatively linked to SEQ ID NO:69 operatively linked to SEQ ID NO:118 operatively linked to SEQ ID NO:360 operatively linked to SEQ ID NO:362 operatively linked to SEQ ID NO:256 or SEQ ID NO:355 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:371 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:259 operatively linked to SEQ ID NO:301 operatively linked to SEQ ID NO:273 or SEQ ID NO:277.
  • the first recombinant nucleic acid further comprises a sequence encoding SEQ ID NO:361 operatively linked to the C-terminus of SEQ ID NO:360 and the N- terminus of SEQ ID NO:362.
  • the first recombinant nucleic acid comprises a sequence encoding an amino acid sequence with at least 80% sequence identity to the amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 358, 364, 366, 368, and 370.
  • the first recombinant nucleic acid comprises a sequence encoding any one of the amino acid sequences of SEQ ID NOs: 358, 364, 366, 368, and 370. [0161] In some embodiments, the first recombinant nucleic acid comprises a sequence with at least 60% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 357, 363, 365, 367, and 369. [0162] In some embodiments, the first recombinant nucleic acid comprises any one of the nucleic sequences of SEQ ID NOs: 357, 363, 365, 367, and 369.
  • the T cell as provided herein further comprises one or more nucleic acid sequences selected from the group consisting of a sequence encoding a switch polypeptide comprising a transforming growth factor beta receptor II (TGFBr2) extracellular domain or a functional fragment thereof, an exogenous sequence encoding an IL-15 polypeptide or a fragment thereof, a sequence encoding an IL-15 polypeptide or a fragment thereof operatively linked to an IL-15R subunit or a fragment thereof, a sequence encoding a PD-1 polypeptide or a fragment thereof, and a sequence encoding a fusion protein comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15R ⁇ .
  • TGFBr2 transforming growth factor beta receptor II
  • the one or more nucleic acid sequences encodes a polypeptide having a sequence with at least 80% sequence identity to any one selected from SEQ ID NOs: 283, 284, 285, 286, 242, 245, 253, 239, 244, 254, and 262. [0165] In some embodiments, the one or more nucleic acid sequences encodes a polypeptide having the sequence of SEQ ID NOs: 283, 284, 285, 286, 242, 245, 253, 239, 244, 254, or 262.
  • the switch polypeptide comprising the TGFBr2 extracellular domain or functional fragment thereof further comprises a switch intracellular domain operably linked to the TGFBr2 extracellular domain or functional fragment thereof, wherein 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, OX40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, CD226, Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • the costimulatory polypeptide is selected from the group consisting of CD28, 4-1BB, and 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 polypeptide comprising the TGFBr2 extracellular domain or functional fragment thereof further comprises a switch transmembrane domain, wherein 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, a transmembrane domain of CD28, a transmembrane domain of 4-1BB, or a transmembrane domain of IL-15Ra.
  • the switch transmembrane domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:272, SEQ ID NO:275 or SEQ ID NO:279.
  • the switch polypeptide comprising the TGFBr2 extracellular domain or functional fragment thereof further comprises an additional intracellular domain 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:248 or SEQ ID NO:251.
  • the IL-15R subunit is IL-15R alpha (IL-15R ⁇ ) subunit.
  • 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 costimulatory polypeptide is selected from the group consisting of OX40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, CD226, Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • the PD-1 polypeptide or fragment thereof is operatively linked to the intracellular domain of the costimulatory polypeptide via a transmembrane domain of PD-1.
  • the PD-1 polypeptide or fragment thereof is operatively linked to the intracellular domain of CD28 via a transmembrane domain of PD-1.
  • the extracellular domain and the transmembrane domain of PD-1 is operatively linked to the intracellular domain of CD28.
  • the one or more nucleic acid sequences encode a fusion protein comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15R ⁇ .
  • the one or more nucleic acid sequences are operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof.
  • the one or more nucleic acid sequences are operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof by a linker sequence.
  • the linker sequence 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 one or more nucleic acid sequences and the first nucleic acid sequence, the one or more nucleic acid sequences and the second nucleic acid sequence, or the one or more nucleic acid sequences, the first nucleic acid sequence, and the second nucleic acid sequence are present on different nucleic acid molecules.
  • the first recombinant nucleic acid comprises a DNA or an RNA.
  • the second recombinant nucleic acid comprises a DNA or an RNA.
  • the first and/or second recombinant nucleic acid comprises a nucleotide analog.
  • the nucleotide 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’-O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide
  • LNA locked nucleic acid
  • ENA ethylene
  • the T cell promotes anti-tumor activity or inhibits inhibition of anti-tumor activity.
  • the T cell thereof promotes T cell mediated tumor cell killing or inhibits inhibition of T cell mediated tumor cell killing.
  • the T cell inhibits tumor growth.
  • the present disclosure an anti-PD-1 antibody or a fragment thereof that specifically binds programmed cell death protein 1 (PD-1) comprising a variable domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, and wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:3.
  • the CDR1 comprises the amino acid sequence of SEQ ID NO:1.
  • the CDR2 comprises the amino acid sequence of SEQ ID NO:2.
  • the variable domain further comprises a framework region 1 (FR1) comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.
  • the variable domain further comprises a framework region 2 (FR2) comprising the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.
  • variable domain further comprises a framework region 3 (FR3) comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.
  • variable domain further comprises a framework region 4 (FR4) comprising the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:19.
  • variable domain comprises one or more mutations in one or more of framework region 1 (FR1), FR2, FR3, or FR4 relative to FR1, FR2, FR3, and/or FR4 of the amino acid sequence of SEQ ID NO.4.
  • variable domain has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4. [0205] In some embodiments, the variable domain has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4. [0206] In some embodiments, the variable domain comprises the amino acid sequence of SEQ ID NO:4. [0207] In some embodiments, the variable domain is encoded by the nucleic acid sequence of SEQ ID NO:20. [0208] In some embodiments, the variable domain comprises one or more mutations at amino acid position selected from the group consisting of amino acid positions 1, 5, 14, 34, 35, 49, 75, 76, 78, 79, 87, 88, 93, and 115 of the amino acid sequence of SEQ ID NO:4.
  • variable domain comprises a mutation at amino acid positions 1, 5, 14, 34, 49, 76, 78, 79, 87, 93, and 115 of the amino acid sequence of SEQ ID NO:4.
  • variable domain comprises a mutation at amino acid positions 1, 5, 14, 34, 49, 75, 76, 78, 79, 87, 93, and 115 of the amino acid sequence of SEQ ID NO:4.
  • variable domain comprises a mutation at amino acid positions 1, 5, 14, 34, 35, 49, 75, 76, 78, 79, 87, 93, and 115 of the amino acid sequence of SEQ ID NO:4.
  • variable domain comprises a mutation at amino acid positions 1, 5, 14, 34, 35, 49, 75, 76, 78, 79, 87, 88, 93, and 115 of the amino acid sequence of SEQ ID NO:4.
  • the variable domain comprises one or more mutations selected from the group consisting of Q1E, Q5V, A14P, I34M, G35S, A49S, A75S, N76K, A78T, V79L, K87R, P88A, I93V and Q115L relative to the amino acid sequence of SEQ ID NO:4.
  • variable domain comprises Q1E, Q5V, A14P, I34M, A49S, N76K, A78T, V79L, K87R, I93V and Q115L mutations relative to the amino acid sequence of SEQ ID NO:4.
  • variable domain comprises Q1E, Q5V, A14P, I34M, A49S, A75S, N76K, A78T, V79L, K87R, I93V and Q115L mutations relative to the amino acid sequence of SEQ ID NO:4.
  • variable domain comprises Q1E, Q5V, A14P, I34M, G35S, A49S, A75S, N76K, A78T, V79L, K87R, I93V and Q115L mutations relative to the amino acid sequence of SEQ ID NO:4.
  • the variable domain comprises Q1E, Q5V, A14P, I34M, G35S, A49S, A75S, N76K, A78T, V79L, K87R, P88A, I93V and Q115L mutations relative to the amino acid sequence of SEQ ID NO:4.
  • the variable domain has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4.
  • variable domain has at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8. [0220] In some embodiments, the variable domain has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8. [0221] In some embodiments, the variable domain has at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8. [0222] In some embodiments, the variable domain comprises the amino acid sequence of SEQ ID NO:5. [0223] In some embodiments, the variable domain is encoded by the nucleic acid sequence of SEQ ID NO:21. [0224] In some embodiments, the variable domain comprises the amino acid sequence of SEQ ID NO:6.
  • variable domain is encoded by the nucleic acid sequence of SEQ ID NO:22.
  • variable domain comprises the amino acid sequence of SEQ ID NO:7.
  • variable domain is encoded by the nucleic acid sequence of SEQ ID NO:23.
  • variable domain comprises the amino acid sequence of SEQ ID NO:8.
  • variable domain is encoded by the nucleic acid sequence of SEQ ID NO:24.
  • the anti-PD-1 antibody or fragment thereof binds to human PD-1.
  • the anti-PD-1 antibody or fragment thereof binds to human PD-1 with a K D value of about 1 to about 60 nM. [0232] In some embodiments, the anti-PD-1 antibody or fragment thereof binds to human PD-1 with a KD value of at most 55 nM. [0233] In some embodiments, the anti-PD-1 antibody or fragment thereof is secreted when expressed in a cell. [0234] In some embodiments, the anti-PD-1 antibody or fragment thereof is an antagonist. [0235] In some embodiments, the anti-PD-1 antibody or fragment thereof is a PD-1 inhibitor.
  • the anti-PD-1 antibody or fragment thereof competes with PD-L1 for binding to PD-1, inhibits PD-L1 from interacting with PD-1, and/or binds to the same epitope of PD-1 to which PD-L1 binds.
  • the anti-PD-1 antibody or fragment thereof blocks an immune checkpoint or blocks immune checkpoint signaling.
  • the anti-PD-1 antibody or fragment thereof is an immune checkpoint inhibitor or inhibits immune checkpoint signaling.
  • the anti-PD-1 antibody or fragment thereof promotes anti-tumor activity or inhibits inhibition of anti-tumor activity.
  • the anti-PD-1 antibody or fragment thereof promotes antibody or antibody fragment mediated tumor cell killing or inhibits inhibition of antibody or antibody fragment mediated tumor cell killing. [0241] In some embodiments, the anti-PD-1 antibody or fragment thereof inhibits tumor growth. [0242] In some embodiments, anti-PD-1 antibody or fragment thereof is a human or humanized antibody or fragment thereof. [0243] In some embodiments, the anti-PD-1 antibody or fragment thereof is a single domain antibody (sdAb). [0244] In some embodiments, the sdAb is a V HH . [0245] In some embodiments, the VHH is Fc conjugated. [0246] In some embodiments, the Fc is an effector-function silent IgG4.
  • the present disclosure a recombinant nucleic acid comprising (I) a first 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, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, and (b) a binding domain; and (II) a second sequence encoding an anti-PD-1 antibody or fragment thereof that specifically binds programmed cell death protein 1 (PD-1), wherein the anti-PD-1 antibody or fragment thereof inhibits an interaction of PD-1 with PD-L1 or PD-L2, wherein the anti-PD-1 antibody or fragment thereof is secreted when expressed in a T cell, wherein the TCR subunit and the binding domain are operatively linked, and wherein the TFP functionally interacts with an endogenous TCR complex when expressed in the T
  • the present disclosure a recombinant nucleic acid comprising (I) a first 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, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, and (b) a binding domain; and (ii) a second sequence encoding a fusion protein comprising an anti-PD-1 antibody or fragment thereof that specifically binds PD-1, wherein the anti-PD-1 antibody or fragment thereof inhibits an interaction of PD-1 with PD-L1 or PD-L2, and a transmembrane domain operatively linked to the anti-PD-1 antibody or fragment thereof, wherein the TCR subunit and the binding domain are operatively linked, and wherein the TFP functionally interacts with an endogenous TCR complex when expressed in
  • the present disclosure a recombinant nucleic acid comprising (I) a first 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, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, and (b) a binding domain; and (ii) a second sequence encoding a fusion protein comprising an anti-PD-1 antibody or fragment thereof that specifically binds PD-1, wherein the anti-PD-1 antibody or fragment thereof inhibits an interaction of PD-1 with PD-L1 or PD-L2, and an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof, wherein the TCR subunit and the binding domain are operatively linked, and wherein the TFP functionally interacts with an endogenous TCR complex when expressed in the T cell.
  • TCR T-cell
  • the first sequence and the second sequence are contained in a single operon.
  • the first sequence and the second sequence are encoded in frame and the first sequence and the second sequence are operatively linked by a linker sequence.
  • the linker sequence encodes 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 sequence and the second sequence are present on different nucleic acid molecules.
  • the anti-PD-1 antibody or fragment thereof comprises a variable domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:3.
  • CDR1 comprises the amino acid sequence of SEQ ID NO:1.
  • the CDR2 comprises the amino acid sequence of SEQ ID NO:2.
  • the variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4.
  • the variable domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4.
  • variable domain comprises the amino acid sequence of SEQ ID NO:4.
  • variable domain is encoded by the nucleic acid sequence of SEQ ID NO:20.
  • variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8.
  • variable domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8.
  • variable domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8.
  • variable domain comprises the amino acid sequence of SEQ ID NO:5.
  • variable domain is encoded by the nucleic acid sequence of SEQ ID NO:21.
  • variable domain comprises the amino acid sequence of SEQ ID NO:6.
  • variable domain is encoded by the nucleic acid sequence of SEQ ID NO:22.
  • variable domain comprises the amino acid sequence of SEQ ID NO:7.
  • the variable domain is encoded by the nucleic acid sequence of SEQ ID NO:23.
  • the variable domain comprises the amino acid sequence of SEQ ID NO:8.
  • variable domain is encoded by the nucleic acid sequence of SEQ ID NO:24.
  • the anti-PD-1 antibody or fragment thereof binds to human PD-1.
  • the anti-PD-1 antibody or fragment thereof binds to human PD-1 with a KD value of about 1 to about 60 nM.
  • the anti-PD-1 antibody or fragment thereof binds to human PD-1 with a K D value of at most 55 nM.
  • the anti-PD-1 antibody or fragment thereof is an antagonist.
  • the anti-PD-1 antibody or fragment thereof is a PD-1 inhibitor.
  • the anti-PD-1 antibody or fragment thereof competes with PD-L1 for binding to PD-1, inhibits PD-L1 from interacting with PD-1, and/or binds to the same epitope of PD-1 to which PD-L1 binds.
  • the anti-PD-1 antibody or fragment thereof blocks an immune checkpoint or blocks immune checkpoint signaling.
  • the anti-PD-1 antibody or fragment thereof is an immune checkpoint inhibitor or inhibits immune checkpoint signaling.
  • the recombinant nucleic acid promotes anti-tumor activity or inhibits inhibition of anti-tumor activity when expressed in a T cell.
  • the recombinant nucleic acid promotes T cell mediated tumor cell killing or inhibits inhibition of T cell mediated tumor cell killing when expressed in a T cell.
  • the recombinant nucleic acid inhibits tumor growth when expressed in a T cell.
  • the anti-PD-1 antibody or fragment thereof is a human or humanized antibody or fragment thereof.
  • the anti-PD-1 antibody or fragment thereof is a single domain antibody (sdAb).
  • the sdAb is a V HH .
  • the V HH is Fc conjugated.
  • the Fc is an effector-function silent IgG4.
  • the fusion protein further comprises a signal sequence.
  • the signal sequence is a PD-1 signal peptide.
  • the signal sequence comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:256 or SEQ ID NO:335.
  • the signal sequence comprises the amino acid sequence of SEQ ID NO:256 or SEQ ID NO:335.
  • the anti-PD-1 antibody or fragment thereof binds to PD-1 on the surface of the T cell, PD-1 on the surface of a bystander T cell, or a combination thereof.
  • the fusion protein further comprises an intracellular domain operatively linked to the transmembrane domain.
  • the fusion protein further comprises a transmembrane domain operatively linked to the intracellular domain and the anti-PD-1 antibody or fragment thereof.
  • the transmembrane domain of the fusion protein comprises a transmembrane domain of a protein selected from the group consisting of CD28, CD3 ⁇ , CD3 ⁇ , CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD41, CD64, CD68, CD80, CD86, CD134, CD137, CD154, ICOS, 4-1BB, OX40, PD-1, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the transmembrane domain of the fusion protein comprises a PD-1 transmembrane domain.
  • the transmembrane domain of the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:301.
  • the transmembrane domain of the fusion protein comprises the amino acid sequence of SEQ ID NO:301.
  • the fusion protein further comprises a PD-1 stalk domain.
  • the PD-1 stalk domain is operatively linked to the transmembrane domain.
  • the PD-1 stalk domain is operatively linked to the N-terminus of the transmembrane domain.
  • the PD-1 stalk domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:259. [0305] In some embodiments, the PD-1 stalk domain comprises the amino acid sequence of SEQ ID NO:259. [0306] In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:259 operatively linked to an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:301.
  • the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:259 operatively linked to the N terminus of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:301. [0308] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:259 operatively linked to the amino acid sequence of SEQ ID NO:301. [0309] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:259 operatively linked to the N-terminus of the amino acid sequence of SEQ ID NO:301. [0310] In some embodiments, the intracellular domain of the fusion protein comprises a co- stimulatory domain.
  • the co-stimulatory domain comprises a co-stimulatory domain of a protein selected from the group consisting of a CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, PD-1, CD258, ICAM-1, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the co-stimulatory domain comprises a 4-1BB (CD137) co- stimulatory domain.
  • the co-stimulatory domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:277. [0314] In some embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:277. [0315] In some embodiments, the co-stimulatory domain comprises a CD28 co-stimulatory domain. [0316] In some embodiments, the co-stimulatory domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:273. [0317] In some embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:273.
  • the fusion protein comprises two or more anti-PD-1 antibodies or fragments thereof. [0319] In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are operatively linked tandemly. [0320] In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are identical. [0321] In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are different. [0322] In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are operatively linked by a linker. [0323] In some embodiments, the linker comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:371.
  • the linker comprises the amino acid sequence of SEQ ID NO:371.
  • the fusion protein further comprises a PD-1 signal peptide, a PD-1 stalk domain, a PD-1 transmembrane domain, and a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • the fusion protein comprises, from the N-terminus to the C- terminus, a PD-1 signal peptide operatively linked to the anti-PD-1 antibody or fragment thereof operatively linked to a PD-1 stalk domain operatively linked to a PD-1 transmembrane domain operatively linked to a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • the fusion protein comprises, from the N-terminus to the C- terminus, a PD-1 signal peptide operatively linked to a first anti-PD-1 antibody or fragment thereof operatively linked to a linker operatively linked to a second anti-PD-1 antibody or fragment thereof operatively linked to a PD-1 stalk domain operatively linked to a PD-1 transmembrane domain operatively linked to a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • a PD-1 signal peptide operatively linked to a first anti-PD-1 antibody or fragment thereof operatively linked to a linker operatively linked to a second anti-PD-1 antibody or fragment thereof operatively linked to a PD-1 stalk domain operatively linked to a PD-1 transmembrane domain operatively linked to a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • the fusion protein comprises SEQ ID NO:256 or SEQ ID NO:335, SEQ ID NO:4 or SEQ ID NO:6, SEQ ID NO:259 , SEQ ID NO:301 , and SEQ ID NO:273 or SEQ ID NO:277.
  • the fusion protein comprises, from the N-terminus to the C- terminus, SEQ ID NO:256 or SEQ ID NO:335 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:259 operatively linked to SEQ ID NO:301 operatively linked to SEQ ID NO:273 or SEQ ID NO:277.
  • the fusion protein comprises, from the N-terminus to the C- terminus, SEQ ID NO:256 or SEQ ID NO:335 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:371 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:259 operatively linked to SEQ ID NO:301 operatively linked to SEQ ID NO:273 or SEQ ID NO:277.
  • the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 373-377.
  • the fusion protein comprises an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 373-377
  • the recombinant nucleic acid as provided herein comprises a nucleic acid sequence having at least 60% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 378-382.
  • the recombinant nucleic acid as provided herein comprises any one of the nucleic acid sequences of SEQ ID NOs: 378-382.
  • the at least a portion of a TCR extracellular domain of the TFP comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, 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 transmembrane domain of the TFP comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, 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 intracellular domain of the TFP comprises an intracellular domain of a protein selected from the group consisting of TCR alpha, TCR beta, TCR delta, TCR gamma, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR intracellular domain of the TFP comprises a stimulatory domain from an intracellular signaling domain of CD3 gamma, CD3 delta, or CD3 epsilon, or an amino acid sequence having at least one modification thereto.
  • the TCR intracellular domain of the TFP does not comprise CD3 zeta.
  • the TFP does not comprise a costimulatory domain.
  • the binding domain is connected to the TCR extracellular domain by a linker sequence.
  • the linker 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, e.g., 1 to 4.
  • at least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit.
  • At least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR alpha. [0346] In some embodiments, at least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR beta. [0347] In some embodiments, at least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma. [0348] In some embodiments, at least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta.
  • At least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon. [0350] In some embodiments, at least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta [0351] In some embodiments, at least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma. [0352] In some embodiments, 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 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 TCR subunit comprises the amino acid sequence of SEQ ID NO:57.
  • the TCR subunit comprises the amino acid sequence of SEQ ID NO:58.
  • the TCR subunit comprises the amino acid sequence of SEQ ID NO:59.
  • 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. [0364] In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma. [0365] In some embodiments, the binding domain comprises an antibody or a fragment thereof, a ligand, or a ligand binding protein. [0366] In some embodiments, the ligand or the antibody fragment comprises a scFv or a single domain antibody (sdAb) domain. [0367] In some embodiments, the sdAb is a V HH .
  • the antibody or fragment thereof is camelid, murine, human or humanized.
  • the antibody or fragment thereof comprises an antigen binding domain 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, an anti-Nectin4 binding domain, an anti-GPC3 binding domain, an anti-Trop2 binding domain, and anti-MUC16 binding domain.
  • BCMA anti-B-cell maturation antigen
  • MSLN anti-mesothelin
  • the binding domain comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 comprises an amino acid sequence of SEQ ID NO:60, SEQ ID NO:63, or SEQ ID NO:66; wherein the CDR2 comprises an amino acid sequence of SEQ ID NO:61, SEQ ID NO:64, or SEQ ID NO:67; and wherein the CDR3 comprises an amino acid sequence of SEQ ID NO:62, SEQ ID NO:65, or SEQ ID NO:68.
  • the binding domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 69-71.
  • the binding domain comprises an amino acid sequence of any one of the amino acid sequences of ID NOs: 69-71.
  • the antigen binding domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:69.
  • the binding domain comprises the amino acid sequence of SEQ ID NO:69.
  • the binding domain comprises: (i) a light chain (LC) CDR1, LC CDR2, and LC CDR3 amino acid sequence of SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30, respectively; (ii) a heavy chain (HC) CDR1, HC CDR2, and HC CDR3 amino acid sequence of SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:36, respectively; or (iii) a combination thereof.
  • LC light chain
  • HC CDR3 amino acid sequence of SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30 respectively
  • HC heavy chain
  • the binding domain comprises: (i) a light chain variable region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:38; (ii) a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:40 or (iii) a combination thereof. [0377] In some embodiments, the binding domain comprises: (i) a light chain variable region comprising the amino acid sequence of SEQ ID NO:38; (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:40 or (iii) a combination thereof.
  • the binding domain comprises a CDR1 having an amino acid sequence of SEQ ID NO:204, a CDR2 having an amino acid sequence of SEQ ID NO:205; and a CDR3 having an amino acid sequence of SEQ ID NO:206. [0379] In some embodiments, the binding domain comprises a variable region having an amino acid sequence of SEQ ID NO:207.
  • the binding domain comprises: (i) a light chain (LC) CDR1, LC CDR2, and LC CDR3 amino acid sequence of SEQ ID NO:228, SEQ ID NO:229, and SEQ ID NO:230, respectively; (ii) a heavy chain (HC) CDR1, HC CDR2, and HC CDR3 amino acid sequence of SEQ ID NO:224, SEQ ID NO:225, and SEQ ID NO:226.
  • LC light chain
  • HC CDR3 amino acid sequence of SEQ ID NO:224 amino acid sequence of SEQ ID NO:224, SEQ ID NO:225, and SEQ ID NO:226.
  • the binding domain comprises: (i) a light chain variable region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:231; (ii) a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:227 or (iii) a combination thereof.
  • the TFP further comprises a leader sequence.
  • the leader sequence is a GM-CSFR leader sequence or a CSF2RA leader sequence.
  • the leader sequence comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:359.
  • the leader sequence comprises the amino acid sequence of SEQ ID NO:359
  • the recombinant nucleic acid as provided herein encodes a GM- CSFR or CSF2RA leader sequence, an anti-mesothelin (MSLN) binding domain or a fragment thereof, a first linker, a CD3 epsilon intracellular domain, a second linker, and an anti-PD-1 antibody or fragment thereof.
  • the first recombinant nucleic acid further comprises a sequence encoding a His tag, a HA tag, a hFc tag or a combination thereof.
  • the recombinant nucleic acid as provided herein encodes, from the N-terminus to the C-terminus, a GM-CSFR or CSF2RA leader sequence operatively linked to an anti-mesothelin (MSLN) binding domain or a fragment thereof operatively linked to a first linker operatively linked to a CD3 epsilon intracellular domain operatively linked to a second linker operatively linked to an anti-PD-1 antibody or fragment thereof operatively linked to a tag.
  • the tag is a His tag, a HA tag, a hFc tag or a combination thereof.
  • the recombinant nucleic acid as provided herein encodes a GM- CSFR or CSF2RA leader sequence, an anti-mesothelin (MSLN) binding domain or a fragment thereof, a first linker, a CD3 epsilon intracellular domain, a second linker, a PD-1 signal peptide, an anti-PD-1 antibody or fragment thereof, a PD-1 stalk domain, a PD-1 transmembrane domain, and a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • MSLN anti-mesothelin
  • the recombinant nucleic acid as provided herein encodes, from the N-terminus to the C-terminus, a GM-CSFR or CSF2RA leader sequence operatively linked to an anti-mesothelin (MSLN) binding domain or a fragment thereof operatively linked to a first linker operatively linked to a CD3 epsilon intracellular domain operatively linked to a second linker operatively linked to a PD-1 signal peptide operatively linked to an anti-PD-1 antibody or fragment thereof operatively linked to a PD-1 stalk domain operatively linked to a PD-1 transmembrane domain operatively linked to a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • MSLN anti-mesothelin
  • the recombinant nucleic acid as provided herein encodes, from the N-terminus to the C-terminus, a GM-CSFR or CSF2RA leader sequence operatively linked to an anti-mesothelin (MSLN) binding domain or a fragment thereof operatively linked to a first linker operatively linked to a CD3 epsilon intracellular domain operatively linked to a second linker operatively linked to a PD-1 signal peptide operatively linked to a first anti-PD-1 antibody or fragment thereof operatively linked to a third linker operatively linked to a second anti-PD-1 antibody or fragment thereof operatively linked to a PD-1 stalk domain operatively linked to a PD-1 transmembrane domain operatively linked to a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • MSLN anti-mesothelin
  • the recombinant nucleic acid as provided herein encodes SEQ ID NO:359, SEQ ID NO:69, SEQ ID NO:118, SEQ ID NO:360, SEQ ID NO:362, SEQ ID NO:256 or SEQ ID NO:355, SEQ ID NO:4 or SEQ ID NO:6, SEQ ID NO:259, SEQ ID NO:301, and SEQ ID NO:273 or SEQ ID NO:277.
  • the recombinant nucleic acid as provided herein encodes, from the N-terminus to the C-terminus, SEQ ID NO:359 operatively linked to SEQ ID NO:69 operatively linked to SEQ ID NO:118 operatively linked to SEQ ID NO:360 operatively linked to SEQ ID NO:362 operatively linked to SEQ ID NO:256 or SEQ ID NO:355 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:259 operatively linked to SEQ ID NO:301 operatively linked to SEQ ID NO:273 or SEQ ID NO:277.
  • the recombinant nucleic acid as provided herein encodes, from the N-terminus to the C-terminus, SEQ ID NO:359 operatively linked to SEQ ID NO:69 operatively linked to SEQ ID NO:118 operatively linked to SEQ ID NO:360 operatively linked to SEQ ID NO:362 operatively linked to SEQ ID NO:256 or SEQ ID NO:355 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:371 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:259 operatively linked to SEQ ID NO:301 operatively linked to SEQ ID NO:273 or SEQ ID NO:277.
  • the recombinant nucleic acid as provided herein further encodes SEQ ID NO:361 operatively linked to the C-terminus of SEQ ID NO:360 and the N-terminus of SEQ ID NO:362.
  • the recombinant nucleic acid as provided herein encodes an amino acid sequence with at least 80% sequence identity to the amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 358, 364, 366, 368, and 370.
  • the recombinant nucleic acid as provided herein encodes any one of the amino acid sequences of SEQ ID NOs: 358, 364, 366, 368, and 370.
  • the recombinant nucleic acid as provided herein comprises a sequence with at least 60% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 357, 363, 365, 367, and 369. [0400] In some embodiments, the recombinant nucleic acid as provided herein comprises any one of the nucleic acid sequences of SEQ ID NOs: 357, 363, 365, 367, and 369.
  • the recombinant nucleic acid as provided herein further comprises one or more nucleic acid sequences selected from the group consisting of a sequence encoding a switch polypeptide comprising a transforming growth factor beta receptor II (TGFBr2) extracellular domain or a functional fragment thereof, an exogenous sequence encoding an IL-15 polypeptide or a fragment thereof, a sequence encoding an IL-15 polypeptide or a fragment thereof operatively linked to an IL-15R subunit or a fragment thereof, a sequence encoding a PD- 1 polypeptide or a fragment thereof, and a sequence encoding a fusion protein comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15R ⁇ .
  • TGFBr2 transforming growth factor beta receptor II
  • the one or more nucleic acid sequences encodes a polypeptide having a sequence with at least 80% sequence identity to any one selected from SEQ ID NOs: 283, 284, 285, 286, 242, 245, 253, 239, 244, 254, and 262. [0403] In some embodiments, the one or more nucleic acid sequences encodes a polypeptide having the sequence of SEQ ID NOs: 283, 284, 285, 286, 242, 245, 253, 239, 244, 254, and 262.
  • the switch polypeptide comprising the TGFBr2 extracellular domain or functional fragment thereof further comprises a switch intracellular domain operably linked to the TGFBr2 extracellular domain or functional fragment thereof, wherein 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, OX40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, CD226, Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • the costimulatory polypeptide is selected from the group consisting of CD28, 4-1BB, and 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 polypeptide comprising the TGFBr2 extracellular domain or functional fragment thereof further comprises a switch transmembrane domain, wherein 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, a transmembrane domain of CD28, a transmembrane domain of 4-1BB, or a transmembrane domain of IL-15Ra.
  • the switch transmembrane domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:272, SEQ ID NO:275 or SEQ ID NO:279.
  • the switch polypeptide comprising the TGFBr2 extracellular domain or functional fragment thereof further comprises an additional intracellular domain 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:248 or SEQ ID NO:251.
  • the IL-15R subunit is IL-15R alpha (IL-15R ⁇ ) subunit.
  • 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 costimulatory polypeptide is selected from the group consisting of OX40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, CD226, Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • the PD-1 polypeptide or fragment thereof is operatively linked to the intracellular domain of the costimulatory polypeptide via a transmembrane domain of PD-1.
  • PD-1 polypeptide or fragment thereof is operatively linked to the intracellular domain of CD28 via a transmembrane domain of PD-1.
  • the extracellular domain and the transmembrane domain of PD-1 is operatively linked to the intracellular domain of CD28.
  • the one or more nucleic acid sequences encode a fusion protein comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15R ⁇ .
  • the one or more nucleic acid sequences are operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof.
  • the one or more nucleic acid sequences are operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof by a linker sequence.
  • the linker sequence 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 one or more nucleic acid sequences and the first nucleic acid sequence, the one or more nucleic acid sequences and the second nucleic acid sequence, or the one or more nucleic acid sequences, the first nucleic acid sequence, and the second nucleic acid sequence are present on different nucleic acid molecules.
  • the recombinant nucleic acid comprises a DNA or an RNA.
  • 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’-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’-O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1’,5’- anhydrohexitol nucleic acid (HNA), a
  • LNA locked nucleic
  • the present disclosure a fusion protein comprising an anti-PD-1 antibody or fragment thereof that specifically binds PD-1, wherein the anti-PD-1 antibody or fragment thereof inhibits an interaction of PD-1 with PD-L1 or PD-L2, and a transmembrane domain.
  • the present disclosure a fusion protein comprising an anti-PD-1 antibody or fragment thereof that specifically binds PD-1, wherein the anti-PD-1 antibody or fragment thereof inhibits an interaction of PD-1 with PD-L1 or PD-L2, and an intracellular domain.
  • the anti-PD-1 antibody or fragment thereof is stably linked to the transmembrane domain.
  • the anti-PD-1 antibody or fragment thereof is stably linked to the intracellular domain.
  • the anti-PD-1 antibody or fragment thereof comprises a variable domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:3.
  • the CDR1 comprises the amino acid sequence of SEQ ID NO:1.
  • the CDR2 comprises the amino acid sequence of SEQ ID NO:2.
  • the variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4.
  • variable domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4.
  • variable domain comprises the amino acid sequence of SEQ ID NO:4.
  • the variable domain is encoded by the nucleic acid sequence of SEQ ID NO:20.
  • the variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8.
  • the variable domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8.
  • variable domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8. [0444] In some embodiments, the variable domain comprises the amino acid sequence of SEQ ID NO:5. [0445] In some embodiments, the variable domain is encoded by the nucleic acid sequence of SEQ ID NO:21. [0446] In some embodiments, the variable domain comprises the amino acid sequence of SEQ ID NO:6. [0447] In some embodiments, the variable domain is encoded by the nucleic acid sequence of SEQ ID NO:22. [0448] In some embodiments, the variable domain comprises the amino acid sequence of SEQ ID NO:7.
  • variable domain is encoded by the nucleic acid sequence of SEQ ID NO:23. [0450] In some embodiments, the variable domain comprises the amino acid sequence of SEQ ID NO:8. [0451] In some embodiments, the variable domain is encoded by the nucleic acid sequence of SEQ ID NO:24. [0452] In some embodiments, the anti-PD-1 antibody or fragment thereof binds to human PD-1. [0453] In some embodiments, the anti-PD-1 antibody or fragment thereof is a single domain antibody (sdAb). [0454] In some embodiments, the sdAb is a V HH . [0455] In some embodiments, the VHH is Fc conjugated.
  • sdAb single domain antibody
  • the Fc is an effector-function silent IgG4.
  • the fusion protein further comprises a signal sequence.
  • the signal sequence is a PD-1 signal peptide.
  • the signal sequence comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence SEQ ID NO:256 or SEQ ID NO:335.
  • the signal sequence comprises the amino acid sequence of SEQ ID NO:256 or SEQ ID NO:335
  • the fusion protein further comprises an intracellular domain.
  • the fusion protein further comprises a transmembrane domain.
  • the transmembrane domain of the fusion protein comprises a transmembrane domain of a protein selected from the group consisting of CD28, CD3 ⁇ , CD3 ⁇ , CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD41, CD64, CD68, CD80, CD86, CD134, CD137, CD154, ICOS, 4-1BB, OX40, PD-1, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the transmembrane domain of the fusion protein comprises a PD-1 transmembrane domain.
  • the transmembrane domain of the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:301.
  • the transmembrane domain of the fusion protein comprises the amino acid sequence of SEQ ID NO:301.
  • the fusion protein further comprises a PD-1 stalk domain.
  • the PD-1 stalk domain is operatively linked to the transmembrane domain.
  • the PD-1 stalk domain is operatively linked to the N-terminus of the transmembrane domain.
  • the PD-1 stalk domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:259.
  • the PD-1 stalk domain comprises the amino acid sequence of SEQ ID NO:259.
  • the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:259 operatively linked to an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:301.
  • the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:259 operatively linked to the N terminus of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:301.
  • the fusion protein comprises the amino acid sequence of SEQ ID NO:259 operatively linked to the amino acid sequence of SEQ ID NO:301.
  • the fusion protein comprises the amino acid sequence of SEQ ID NO:259 operatively linked to the N-terminus of the amino acid sequence of SEQ ID NO:301.
  • the intracellular domain of the fusion protein comprises a co- stimulatory domain.
  • the co-stimulatory domain comprises a co-stimulatory domain of a protein selected from the group consisting of a CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, PD-1, CD258, ICAM-1, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the co-stimulatory domain comprises a 4-1BB (CD137) co- stimulatory domain.
  • the co-stimulatory domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:277. [0480] In some embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:277. [0481] In some embodiments, the co-stimulatory domain comprises a CD28 co-stimulatory domain. [0482] In some embodiments, the co-stimulatory domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:273. [0483] In some embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:273.
  • the fusion protein comprises two or more anti-PD-1 antibodies or fragments thereof. [0485] In some embodiments, the fusion protein comprises the two or more anti-PD-1 antibodies or fragments thereof operatively linked tandemly [0486] In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are identical. [0487] In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are different. [0488] In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are operatively linked by a linker. [0489] In some embodiments, the linker comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:371.
  • the linker comprises the amino acid sequence of SEQ ID NO:371.
  • the fusion protein as provided herein comprises a PD-1 signal peptide, an anti-PD-1 antibody or fragment thereof, a PD-1 stalk domain, a PD-1 transmembrane domain, and a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • the fusion protein as provided herein comprises, from the N- terminus to the C-terminus, a PD-1 signal peptide operatively linked to an anti-PD-1 antibody or fragment thereof operatively linked to a PD-1 stalk domain operatively linked to a PD-1 transmembrane domain operatively linked to a CD28 or 4-1BB (CD137) co-stimulatory domain.
  • the fusion protein as provided herein comprises, from the N- terminus to the C-terminus, the PD-1 signal peptide operatively linked to a first anti-PD-1 antibody or fragment thereof operatively linked to a linker operatively linked to a second anti- PD-1 antibody or fragment thereof operatively linked to the PD-1 stalk domain operatively linked to the PD-1 transmembrane domain operatively linked to the CD28 or 4-1BB (CD137) co- stimulatory domain.
  • the fusion protein as provided herein comprises SEQ ID NO:256 or SEQ ID NO:335, SEQ ID NO:4 or SEQ ID NO:6, SEQ ID NO:259 , SEQ ID NO:301 , and SEQ ID NO:273 or SEQ ID NO:277.
  • the fusion protein as provided herein comprises, from the N- terminus to the C-terminus, SEQ ID NO:256 or SEQ ID NO:335 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:259 operatively linked to SEQ ID NO:301 operatively linked to SEQ ID NO:273 or SEQ ID NO:277.
  • the fusion protein as provided herein comprises, from the N- terminus to the C-terminus, SEQ ID NO:256 or SEQ ID NO:335 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:371 operatively linked to SEQ ID NO:4 or SEQ ID NO:6 operatively linked to SEQ ID NO:259 operatively linked to SEQ ID NO:301 operatively linked to SEQ ID NO:273 or SEQ ID NO:277.
  • the fusion protein as provided herein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 373-377.
  • the fusion protein as provided herein comprises an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 373-377 [0499] In some embodiments, the fusion protein is encoded by a nucleic acid sequence having at least 60% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 378-382. [0500] In some embodiments, the fusion protein is encoded by any one of the nucleic acid sequences of SEQ ID NOs: 378-382.
  • the fusion protein is encoded by a recombinant nucleic acid further comprising a 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, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, and (b) a binding domain; and wherein the TCR subunit and the binding domain are operatively linked, and wherein the TFP functionally interacts with an endogenous TCR complex in the T cell.
  • TCR T-cell receptor
  • the present disclosure a recombinant nucleic acid comprising a sequence encoding an anti-PD-1 antibody or fragment thereof that specifically binds PD-1, wherein the anti-PD-1 antibody or fragment thereof inhibits an interaction of PD-1 with PD-L1 or PD-L2, operatively or stably linked to a transmembrane domain.
  • the present disclosure a recombinant nucleic acid comprising a sequence encoding an anti-PD-1 antibody or fragment thereof that specifically binds PD-1, wherein the anti-PD-1 antibody or fragment thereof inhibits an interaction of PD-1 with PD-L1 or PD-L2, operatively or stably linked to an intracellular domain.
  • the present disclosure the first recombinant nucleic acid as provided herein.
  • the present disclosure a recombinant nucleic acid comprising a sequence encoding the anti-PD-1 antibody or fragment thereof as provided herein or the fusion protein as provided herein.
  • the recombinant nucleic acid as provided herein further comprises a promoter.
  • the nucleic acid is an in vitro transcribed nucleic acid.
  • the nucleic acid further comprises a sequence encoding a poly(A) tail.
  • the nucleic acid further comprises a 3’UTR sequence.
  • the present disclosure a polypeptide encoded by the first recombinant nucleic acid as provided herein or the recombinant nucleic acid as provided herein.
  • the present disclosure a vector comprising the first recombinant nucleic acid as provided herein or the recombinant nucleic acid as provided herein.
  • the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
  • the vector as provided herein further comprises a promoter.
  • the vector is an in vitro transcribed vector.
  • a nucleic acid sequence in the vector further comprises a poly(A) tail.
  • a nucleic acid sequence in the vector further comprises a 3’UTR.
  • the present disclosure a cell 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, e.g., a human T cell.
  • the T cell is a CD8+ or CD4+ T cell.
  • the T cell is a human alpha beta T cell.
  • the T cell is a human gamma delta T cell.
  • the cell is a human NKT cell.
  • the present disclosure a pharmaceutical composition comprising the cell as provided herein and a pharmaceutically acceptable carrier.
  • the present disclosure a method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition as provided herein.
  • the disease or the condition is a cancer or a disease or a condition associated with expression of CD19, B-cell maturation antigen (BCMA), mesothelin (MSLN), CD20, CD70, Nectin-4, GPC3, Trop-2, or MUC16.
  • BCMA B-cell maturation antigen
  • MSLN mesothelin
  • CD20 CD70
  • Nectin-4 GPC3, Trop-2
  • MUC16 MUC16
  • 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.
  • the present disclosure a method of treating a subject in need thereo comprising administering to the subject (A) a T cell comprising 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, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, and (b) a binding domain; wherein the TCR subunit and the binding domain are operatively linked, and wherein the TFP functionally interacts with an endogenous TCR complex in the T cell; and (B) the anti-PD1 antibody as provided herein.
  • TCR T-cell receptor
  • the T cell comprises the TFP as provided herein.
  • the anti-PD1 antibody is administered before administering the T cell, concurrently with the T cell, or after administering the T cell.
  • the T cell, the anti-PD1 antibody, or a combination thereof is administered in one, two, three or more doses. INCORPORATION BY REFERENCE [0533] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
  • FIG.1 is a schematic illustration of the PD-1/PD-L1 inhibitor assay described in Example 2.
  • FIGs.2A and 2B are graphs showing the ability of the anti-PD-1 VHH antibodies shown to inhibit the interaction of PD-1 and PD-L1 as determined by the PD-1/PD-L1 inhibitor assay described in Example 2.
  • FIG.3 is a series of graphs showing the results of the epitope binning experiment described in Example 2.
  • FIG.4 is a series of graphs showing the results of the octet assay measuring the KD of clone 51 (2PDE172) having a His or Fc tag, or Nivolomab, as is described in Example 3.
  • FIG.5 is a schematic illustration and a series of graphs showing staining of T cells induced to express PD-1 with dynabeads and IL-2 or dynabeads, IL-2 and TGF-beta by the clone 51 VHH antibody or by a commercial anti-PD-1 antibody, as is described in Example 3.
  • FIG.6 is a series of graphs showing the results of an SEB assay described in Example 3.
  • FIG.7 is a graph showing the fold inhibition of PD-1/PD-L1 binding achieved by clone 51 VHH, clone 51 VHH-Fc tag, control anti-PD-1 antibodies, or an hIgG1 control as determined by the PD-1/PD-L1 blockade assay described in Example 3.
  • FIGs.8A-8D are a series of graphs showing binding of clone 51 VHH (CDS parental and Hz and MD codon optimized versions) secreted from T cells binding to antigen presenting cells, as is described in Example 4.
  • FIG.9 is a series of graphs showing binding affinity of non-humanized and four humanized variants of clone 51 VHH as determined by octet assay as described in Example 5.
  • FIGs.10A and 10B show the characterization of humanized clone 51 h12 variants.
  • FIG. 10A is an SDS-page gel of the clone 51 h12 variants described in Example 6.
  • FIG.10B is a graph showing the results of an octet assay to determine the affinity of each of the variants for PD-1 as described in Example 6.
  • Samples shown are (i) clone 51 h12 VHH-hIgG4 tag; (ii) clone 51 h12 VHH- clone 51 h12 VHH – anti-HSA - SrtA tag-His tag; (iii) clone 51 h12 VHH-anti-HSA-SrtA tag-His tag; (iv) clone 51 h12 VHH- clone 51 h12 VHH - SrtA tag-His tag; and (v) clone 51 h12 VHH-SrtA tag-His tag, from top to bottom, at 200 nM PD-1 binder.
  • FIG.11 is a graph showing the mean expansion of three donors of T cells transduced with the constructs shown, as is described in Example 8.
  • FIGs.12A and 12B are a series of graphs showing transduction efficiency as determined by staining for VHH positive T cells transduced with the TFPs shown with an anti-VHH antibody, as is described in Example 8.
  • FIGs.13A-13E are a series of graphs showing phenotyping of T cells transduced with the TFPs shown by flow cytometry, as is described in Example 9.
  • FIGs.14A and 14B are a series of graphs showing VHH and PD-1 expression in T cells transduced with the TFPs shown by flow cytometry, as is described in Example 9.
  • FIG.15 is a series of graphs showing detection of anti-MSLN TFP by anti-VHH and an anti-idiotype antibody to anti-MSLN, as is described in Example 10.
  • FIG.16 is a graph showing transduction efficiency as determined by staining T cells transduced with the TFPs shown with an anti-anti-MSLN antibody by flow cytometry, as is described in Example 10.
  • FIGs.17A-17C are a series of graphs showing detection of anti-PD-1 binding to T-cells transduced with the TFP shown as determined by staining with an anti-HA or anti-Fc antibody by flow cytometry.
  • FIG.17A shows TFP+ T-cells.
  • FIG.17B shows TFP- T cells.
  • FIG.17C shows staining of untransduced T-cells when cultured with supernatants from T cells transduced with the TFPs shown.
  • FIG.18 is a graph showing cytotoxicity of TFP T-cells having the TFP shown when cultured with C30, msto-MSLN, or msto-MLSN-PD-L1 cells, as is described in Example 11.
  • FIGs.19A-19C are a series of graphs showing cytokine secretion of TFP T-cells having the TFP shown when cultured with C30, msto-MSLN, or msto-MLSN-PD-L1 cells, as is described in Example 12.
  • FIG.20 is a series of graphs showing expansion of three donors of T cells transduced with the constructs shown in Table 9.
  • FIG.21 is a series of plots and a graph showing transduction efficiency as determined by staining for VHH positive T cells transduced with the constructs shown with an anti-VHH antibody.
  • FIG.22 is a series of plots and a graph showing the CD4:CD8 T cell ratio of T cells transduced with the constructs shown as determined by flow cytometry. In the graph on the right, the proportion of CD8 T+ cells is shown at the top and the proportion of CD4+ T cells is shown at the bottom.
  • FIG.23 is a series of plots and a graph showing the memory phenotype of T cells transduced with the constructs shown as determined by detection of CD45RA and CCR7 by flow cytometry. In the graphs on the right, the proportion of TEMRA, TEM, TCM, and TNAIVE cells are shown from top to bottom.
  • FIG.24 is a series of plots and a graph showing detection of PD-1 expression in T cells transduced with the constructs shown with an anti-PD-1 antibody by flow cytometry.
  • FIG.25 is a series of plots and a graph showing detection of CD5 and CD25 expression in T cells transduced with the constructs shown by flow cytometry.
  • FIG.26 is a series of graph showing cytotoxicity of TFP T-cells having the constructs shown when cultured with C30, msto-MSLN, or msto-MLSN-PD-L1 cells.
  • FIG.27A and 27B are a series of graphs showing cytokine secretion of TFP T-cells having the constructs shown when cultured with msto-MSLN or msto-MLSN-PD-L1 cells.
  • FIG.28 is a schematic diagram illustrating cis and trans signaling through the anti-PD-1 switch constructs described herein.
  • FIG.29 is a schematic diagram illustrating the experimental protocol described in Example 16 to measure signaling in cis by the anti-PD-1 switch constructs described herein.
  • FIG.30 is a schematic diagram illustrating the experimental protocol described in Example 16 to measure signaling in cis and trans by the anti-PD-1 switch constructs described herein.
  • FIG.31 is a series of plots and a graph showing PD-1 expression in TFP T-cells having the constructs shown after incubation with or without plate bound MSLN and PD-1 in the assay described in Example 16. The graphs on the right show the proportion of PD-1+ CD4+ (in the top graph) TFP T cells and PD-1+ CD8+ TFP T cells (in the bottom graph).
  • FIG.32 is a series of graphs showing cytokine expression of TFP T-cells having the constructs shown after incubation with or without plate bound MSLN and PD-1 in the assay described in Example 16.
  • FIG.33 is a series of graphs showing the proportion of CD4+ TFP T cells having the constructs shown that are CD25+ or CD69+ after incubation with or without plate bound MSLN and PD-1 in the assay described in Example 16.
  • FIGs.34A and 34B are a series of plots and a graph showing PD-1 expression in TFP T- cells having the constructs shown after incubation with or without plate bound MSLN in the assay described in Example 17.
  • the graphs on the right show the proportion of PD- 1+ CD4+ (in the top graph) TFP T cells and PD-1+ CD8+ TFP T cells (in the bottom graph).
  • the graphs on the right show the MFI for PD-1+ CD4+ (in the top graph) TFP T cells and PD-1+ CD8+ TFP T cells (in the bottom graph).
  • FIG.34A and 34B For the graphs shown in FIG.34A and 34B, for each MSLN level, non-transduced cells, TC-210, TC-510, PD-1-41BB(IC), PDS-CD28(IC), hPDS-CD28(IC), hPDS-CD28(IC) #2, hPDS-41BB(IC), and 2X(hPDS)-CD28(IC) are shown from left to right.
  • FIGs.35A and 35B are a series of graphs showing cytokine expression of TFP T-cells having the constructs shown after incubation with or without plate bound MSLN, PD-1, and/or PD-L1 in the assay described in Example 17.
  • IFN- ⁇ and IL-2 levels are shown in FIG.35A and TNF- ⁇ and GM-CSF levels are shown in FIG.35B.
  • non-transduced cells TC-210, TC-510, PD-1-41BB(IC), PDS-CD28(IC), hPDS-CD28(IC), hPDS-CD28(IC) #2, hPDS-41BB(IC), and 2X(hPDS)-CD28(IC) are shown from left to right.
  • FIG.36 is a series of graphs showing the proportion of CD4+ TFP T-cells having the constructs shown that are CD25+ or CD69+ after incubation with or without plate bound MSLN, PD-1, and/or PD-L1 in the assay described in Example 17.
  • MSLN plate bound MSLN
  • PD-1 plate bound MSLN
  • PD-L1 plate bound MSLN
  • PD-L1 plate bound MSLN, PD-1, and/or PD-L1 in the assay described in Example 17.
  • non-transduced cells TC-210, TC-510, PD-1-41BB(IC), PDS-CD28(IC), hPDS-CD28(IC), hPDS-CD28(IC) #2, hPDS-41BB(IC), and 2X(hPDS)-CD28(IC) are shown from left to right.
  • FIG.37 is a series of graphs showing cytokine expression of TFP T-cells from donor R020 having the constructs shown after incubation with or without plate bound MSLN and/or PD-1 in the assay described in Example 18.
  • MSLN level non-transduced cells, TC-210, TC-510, PD-1-41BB(IC), hPDS-CD28(IC), hPDS-CD28(IC) #2, hPDS-41BB(IC), and 2X(hPDS)-CD28(IC) are shown from left to right.
  • FIG.38 is a series of graphs showing cytokine expression of TFP T-cells from donor R024 having the constructs shown after incubation with or without plate bound MSLN and/or PD-1 in the assay described in Example 18.
  • MSLN level non-transduced cells, TC-210, TC-510, PD-1-41BB(IC), hPDS-CD28(IC), hPDS-CD28(IC) #2, hPDS-41BB(IC), and 2X(hPDS)-CD28(IC) are shown from left to right.
  • the present disclosure provides antigen-binding proteins such as antibodies that can specifically bind a programmed cell death protein 1 (PD-1) on T cells and blocking the interaction between PD-1 and PD-L1, thereby modulating immune responses by the T cells.
  • PD-1 programmed cell death protein 1
  • blockade of the PD-1 signaling pathway can inhibit apoptosis/anergy of the T cells.
  • the present disclosure also provides T cells expressing a T-cell receptor fusion protein (TFP) comprising a binding domain, e.g., an antibody or antibody fragment, a ligand, or a ligand binding protein, that binds specifically to an antigen such as a tumor-associated antigen (TAA) and further expressing the PD-1 binding antibody.
  • TFP T-cell receptor fusion protein
  • the invention described herein is particularly advantageous because the anti-PD-1 antibody is delivered to the site of the cancer cells having the tumor-associated antigen by the TFP T cells, thereby allowing for targeted co-administration of the anti-PD-1 antibodies and TFP cells at the site of the cancer.
  • the secreted PD-1 antibody advantageously, can then block PD-1/PD-L1 and/or PD-1/PD-L2 signaling not only in the TFP T-cells, but also in nearby cytotoxic T cells.
  • the PD-1 antibody is expressed as a fusion protein linked to the intracellular domain of a co-stimulatory molecule such as 4-1BB or CD28, optionally by the stalk and transmembrane domain of PD-1.
  • the present disclosure provides a modified T cell for T cell therapy.
  • the modified T cell can comprise a nucleic acid sequence encoding the TFP disclosed herein or a TFP encoded by the nucleic acid sequence disclosed herein.
  • the modified T cell can further comprise an anti-PD-1 antibody or a fusion protein comprising the PD-1 antibody or a nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising the PD-1 antibody or fragment thereof.
  • the anti-PD-1 antibody blocks the interaction between PD-1 and PD-L1.
  • the anti-PD-1 antibody blocks the interaction between PD-1 and PD-L2.
  • the nucleic acid sequence encoding an anti-PD-1 antibody or fusion protein comprising the PD-1 antibody and the nucleic acid sequence encoding the TFP can be on a same nucleic acid molecule or two separate nucleic acid molecules.
  • the anti-PD-1 antibody or fragment thereof may not be membrane bound or may be fused to a transmembrane domain.
  • the modified T cell may secrete the anti-PD-1 antibody or fragment thereof.
  • the anti- PD-1 antibody or fragment thereof can be an scFv or sdAb.
  • are modified allogenic T cells comprising (i) the TFP or the anti-PD-1 antibody or (ii) a sequence encoding the TFP or the anti- PD-1 antibody.
  • the present disclosure provides pharmaceutical compositions comprising a modified T cell.
  • the modified T cell can comprise a TFP or a sequence encoding a TFP.
  • the modified T cell can further comprise an anti-PD-1 antibody or a sequence encoding the anti-PD-1 antibody, such as PD-1 antibody that can be secreted by the modified T cell.
  • the modified T cells e.g., a TFP-T cell
  • the modified T cells may be useful for the treatment of any disease or condition involving the TFP. Methods of treating a subject in need thereof are also provided. Definitions [0578] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains.
  • the term “comprising,” is inclusive and does not exclude additional, unrecited integers or method/process steps.
  • “comprising” 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.
  • 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.
  • the term “antigen-binding domain” means the portion of an antibody that is capable of specifically binding to an antigen or epitope.
  • an antigen-binding domain is an antigen-binding domain formed by a VH -VL dimer of an antibody.
  • Another example of an antigen-binding domain is an antigen-binding domain formed by diversification of certain loops from the tenth fibronectin type III domain of an AdnectinTM.
  • antibody fragment or “antibody binding domain” refer 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 (abbreviated “sdAb”) (either VL or VH, or VL or VH), camelid VHH or VHH domains, and multi-specific 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.
  • “Heavy chain variable region” or “VH” or “V H ” refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.
  • 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 VL-linker-VH or may comprise VH-linker-VL.
  • 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 heavy chain antibodies HCAb, 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
  • HCAb heavy chain antibodies
  • scFv single chain antibody
  • the antigen binding domain of a TFP composition of the present disclosure comprises an antibody fragment.
  • the TFP comprises an antibody fragment that comprises a scFv or a sdAb.
  • antibody heavy chain refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations.
  • Kappa (“ ⁇ ”) and lambda (“ ⁇ ”) light chains refer to the two major antibody light chain isotypes.
  • the term “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. A skilled artisan will understand that 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. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that 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.
  • Such 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.
  • CD70 is a is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This cytokine is a ligand for TNFRSF27/CD27. It is a surface antigen on activated, but not on resting, T and B lymphocytes. CD70 induces proliferation of costimulated T cells, enhances the generation of cytolytic T cells, and contributes to T cell activation. CD70 is also reported to play a role in regulating B-cell activation, cytotoxic function of natural killer cells, and immunoglobulin synthesis.
  • TNF tumor necrosis factor
  • CD19 also known as B-lymphocyte antigen CD19, 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.
  • CD19 includes any of the recombinant or naturally-occurring forms of CD19 or variants or homologs thereof that have or maintain CD19 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 CD19.
  • CD19 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.
  • CD16 also known as Fc ⁇ RIII, 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 Fc ⁇ RIIIa (CD16a) and Fc ⁇ RIIIb (CD16b), which participate in signal transduction. In some embodiments, CD16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC).
  • IgSF immunoglobulin superfamily
  • CD16 includes any of the recombinant or naturally-occurring forms of CD16 or variants or homologs thereof that have or maintain CD16 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 CD16.
  • CD16 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 O7501 (CD16b) or a variant or homolog having substantial identity thereto.
  • the term “NKG2D,” also known as KLRK1, CD314, D12S2489E, KLR, NKG2-D, NKG2D, natural killer group 2D, killer cell lectin-like receptor K1, and killer cell lectin like receptor K1 refers to a transmembrane protein belonging to the CD94/NKG2 family of C-type lectin-like receptors.
  • NKG2D is expressed by NK cells, ⁇ T cells and CD8+ ⁇ 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 Q13421 or a variant or homolog having substantial identity thereto.
  • ROR1 includes any of the recombinant or naturally- occurring forms of ROR1 or variants or homologs thereof that have or maintain ROR1 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.
  • the “CD79 ⁇ (Cluster of Differentiation 79 ⁇ )” and “CD79 ⁇ (Cluster of Differentiation 79 ⁇ )” genes encode proteins that make up the B lymphocyte antigen receptor, a multimeric complex that includes the antigen-specific component, surface immunoglobulin (Ig). Surface Ig non-covalently associates with two other proteins, Ig-alpha and Ig-beta (encoded by CD79 ⁇ and its paralog CD79 ⁇ , respectively) which are necessary for expression and function of the B-cell antigen receptor.
  • CD79 ⁇ protein includes any of the recombinant or naturally-occurring forms of CD79 ⁇ protein or variants or homologs thereof that have or maintain CD79 ⁇ 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 CD79 ⁇ protein.
  • CD79 ⁇ protein is substantially identical to the protein identified by the UniProt reference number P11912 or a variant or homolog having substantial identity thereto.
  • CD79 ⁇ protein includes any of the recombinant or naturally-occurring forms of CD79 ⁇ protein or variants or homologs thereof that have or maintain CD79 ⁇ 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 CD79 ⁇ protein.
  • CD79 ⁇ protein is substantially identical to the protein identified by the UniProt reference number P40259 or a variant or homolog having substantial identity thereto.
  • B cell activating factor or “BAFF,” also known as tumor necrosis factor ligand superfamily member 13B, TNFSF13B, BLYS, CD257, DTL, TALL-1, TALL1, THANK, TNFSF20, ZTNF4, TNLG7A, tumor necrosis factor superfamily member 13b, and TNF superfamily member 13b, refers to a cytokine that belongs to the tumor necrosis factor (TNF) ligand family.
  • TNF tumor necrosis factor
  • This cytokine is a ligand for receptors TNFRSF13B/TACI, TNFRSF17/BCMA, and TNFRSF13C/BAFF-R. This cytokine is expressed in B cell lineage cells, and acts as a potent B cell activator.
  • BAFF plays an important role in the proliferation and differentiation of B cells.
  • BAFF includes any of the recombinant or naturally- occurring forms of BAFF or variants or homologs thereof that have or maintain BAFF 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 BAFF.
  • BAFF is substantially identical to the protein identified by the UniProt reference number Q9Y275 or a variant or homolog having substantial identity thereto.
  • PSMA Prostate-specific membrane antigen
  • GCPII glutamate carboxypeptidase II
  • NAALADase I N-acetyl-L-aspartyl-L-glutamate peptidase I
  • NAAG peptidase FOLH1, FGCP, FOLH, GCP2, GCPII, NAALAD1, NAALAdase, PSM, mGCP
  • folate hydrolase prostate-specific membrane antigen
  • 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.
  • 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 disclosure in prevention of the occurrence of tumor in the first place.
  • 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
  • IRS 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.
  • “secreted” or “secreted protein” refers to a molecule or protein produced or expressed intracellularly and secreted from the cell to an extracellular environment.
  • “Humanized” forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.
  • a humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody).
  • the donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect.
  • selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody.
  • Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.
  • a “human antibody” is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.
  • “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope).
  • affinity refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen or epitope).
  • the affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD).
  • KD dissociation equilibrium constant
  • the kinetic components that contribute to the dissociation equilibrium constant are described in more detail below.
  • Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE ® ) or biolayer interferometry (e.g., FORTEBIO ® ).
  • SPR surface plasmon resonance
  • BIACORE ® BIACORE ®
  • biolayer interferometry e.g., FORTEBIO ®
  • the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule).
  • Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule.
  • Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule.
  • autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • allogeneic 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 antigenically.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • treating refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • 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.1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).
  • 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 co-located in or on the surface of a T cell.
  • TCR T cell receptor
  • TFP T cell receptor
  • the T cell is a CD4+ T cell, a CD8+ T cell, or a CD4+ / CD8+ T cell.
  • the TFP-T cell is an NK cell or a regulatory T cell.
  • 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 CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ .
  • the TCR comprises CD3 ⁇ .
  • the TCR comprises CD3 ⁇ .
  • the TCR comprises CD3 ⁇ .
  • the TCR comprises CD3 ⁇ .
  • the TCR comprises CD3 ⁇ .
  • the TCR comprises CD3 ⁇ .
  • the constant domain of human TCR alpha has a sequence of SEQ ID NO:101.
  • the constant domain of human TCR alpha has an IgC domain having a sequence of SEQ ID NO:102, a transmembrane domain having a sequence of SEQ ID NO:103, and an intracellular domain having a sequence of SS.
  • the constant domain of human TCR beta has a sequence of SEQ ID NO:105.
  • the constant domain of human TCR beta has an IgC domain having a sequence of SEQ ID NO:106, a transmembrane domain having a sequence of SEQ ID NO:107, and an intracellular domain having a sequence of SEQ ID NO:109.
  • the constant domain of murine TCR alpha has a sequence of SEQ ID NO:386.
  • the constant domain of murine TCR beta has a sequence of SEQ ID NO:395.
  • the constant domain of TCR delta has a sequence of SEQ ID NO:115.
  • the constant domain of TCR delta has an IgC domain having a sequence of SEQ ID NO:116, a transmembrane domain having a sequence of SEQ ID NO:117, and an intracellular domain having a sequence of L.
  • the constant domain of TCR gamma has a sequence of SEQ ID NO:111.
  • the constant domain of TCR gamma has an IgC domain having a sequence of SEQ ID NO:112, a transmembrane domain having a sequence of SEQ ID NO:113, and an intracellular domain having a sequence of SEQ ID NO:114.
  • CD3 epsilon has a sequence of SEQ ID NO:57.
  • CD3 epsilon has an extracellular domain having a sequence of SEQ ID NO:89, a transmembrane domain having a sequence of SEQ ID NO:89, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO:90.
  • CD3 delta has a sequence of SEQ ID NO:59.
  • CD3 delta has an extracellular domain having a sequence of SEQ ID NO:96, a transmembrane domain having a sequence of SEQ ID NO:97, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO:98.
  • CD3 gamma has a sequence of SEQ ID NO:58. In some embodiments, CD3 gamma has an extracellular domain having a sequence of SEQ ID NO:92, a transmembrane domain having a sequence of SEQ ID NO:93, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO:94.
  • the term “subject” means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human.
  • a “patient” is a subject suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.
  • the subject has cancer, e.g., a cancer described herein.
  • “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.
  • kits are used to refer to instructions customarily included in commercial packages of therapeutic or diagnostic products (e.g., kits) that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic or diagnostic products.
  • 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.
  • 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, primary or metastatic melanoma, thymoma, Non-Hodgkin lymphoma (NHL), solid cancer, sarcoma, colon cancer, kidney cancer, stomach cancer, bladder cancer, uterine cancer and the like.
  • NDL Non-Hodgkin lymphoma
  • composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.
  • modulate and “modulation” refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.
  • the terms “increase” and “activate” refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.
  • the terms “reduce” and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100- fold, or greater in a recited variable.
  • the term “agonize” refers to the activation of receptor signaling to induce a biological response associated with activation of the receptor.
  • An “agonist” is an entity that binds to and agonizes a receptor.
  • the term “antagonize” refers to the inhibition of receptor signaling to inhibit a biological response associated with activation of the receptor.
  • An “antagonist” is an entity that binds to and antagonizes a receptor.
  • effector T cell includes T helper (i.e., CD4+) cells and cytotoxic (i.e., CD8+) T cells.
  • CD4+ effector T cells contribute to the development of several immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages.
  • CD8+ effector T cells destroy virus-infected cells and tumor cells. See Seder and Ahmed, Nature Immunol., 2003, 4:835-842, incorporated by reference in its entirety, for additional information on effector T cells.
  • the term “regulatory T cell” includes cells that regulate immunological tolerance, for example, by suppressing effector T cells.
  • the regulatory T cell has a CD4+CD25+Foxp3+ phenotype.
  • the regulatory T cell has a CD8+CD25+ phenotype.
  • the term “dendritic cell” refers to a professional antigen-presenting cell capable of activating a na ⁇ ve T cell and stimulating growth and differentiation of a B cell.
  • the phrase “disease associated with expression of a tumor-associated antigen (TAA)” includes, but is not limited to, a disease associated with expression of any TAA described herein or condition associated with cells which express a TAA including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition.
  • the disease is a disease associated with expression of PD-1, PD-L1, or PD-L2.
  • the disease is a cancer.
  • the cancer is a solid tumor.
  • the cancer is a hematological malignancy.
  • the cancer is a leukemia.
  • the cancer is a lymphoma.
  • the cancer is a mesothelioma.
  • the cancer is a pancreatic cancer.
  • the cancer is an ovarian cancer.
  • the cancer is a stomach cancer.
  • the cancer is a lung cancer.
  • the cancer is an endometrial cancer.
  • cancers include but are not limited to, breast cancer, prostate cancer, cervical cancer, skin cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lung cancer, primary or metastatic melanoma, thymoma, Non-Hodgkin lymphoma (NHL), solid cancer, sarcoma, colon cancer, kidney cancer, stomach cancer, bladder cancer, uterine cancer, and the like.
  • NDL Non-Hodgkin lymphoma
  • Non-cancer related indications associated with expression of a target described herein such as PD-1, PD-L1, or PD-L2 include, but are not limited to, e.g., autoimmune disease, (e.g., lupus, rheumatoid arthritis, colitis), inflammatory disorders (allergy and asthma), and transplantation.
  • autoimmune disease e.g., lupus, rheumatoid arthritis, colitis
  • inflammatory disorders allergy and asthma
  • transplantation e.g., autoimmune disease, (e.g., lupus, rheumatoid arthritis, colitis), inflammatory disorders (allergy and asthma), and transplantation.
  • 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 disclosure by standard techniques known in the art, such
  • 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. These families include 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) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains
  • 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. Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.
  • a stimulatory domain or stimulatory molecule e.g., a TCR/CD3 complex
  • the term “stimulatory molecule” or “stimulatory domain” 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 ITAM containing primary cytoplasmic signaling sequence that is of particular use in the disclosure includes, but is not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”) and CD66d.
  • the term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC’s) on its surface. T cells may recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.
  • the intracellular signaling domain generates a signal that promotes an immune effector function of the TFP containing cell, e.g., a TFP-expressing T cell.
  • immune effector function e.g., in a TFP-expressing T cell
  • examples of immune effector function, e.g., in a TFP-expressing T cell include cytolytic activity and T helper cell activity, including the secretion of cytokines.
  • the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation.
  • the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.
  • a primary intracellular signaling domain can comprise an ITAM (“immunoreceptor tyrosine-based activation motif”).
  • ITAM immunoglobulin-based activation motif
  • Examples of 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, 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.
  • Costimulatory molecules include, but are not limited to, an MHC class 1 molecule, BTLA and a Toll ligand receptor, as well as DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137).
  • a costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule.
  • a costimulatory molecule can be 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), OX40, 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, and the like.
  • the intracellular signaling domain can comprise 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 “4-1BB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species, e.g., mouse, rodent, monkey, ape and the like.
  • the term “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.
  • 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 version contain one or more introns.
  • the term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.
  • the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • the term “expression” refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
  • the term “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).
  • a target gene e.g., an endogenous gene.
  • the term “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. Thus, 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.
  • 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.
  • viruses e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses
  • 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.
  • the term “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, e.g., in Milone et al., Mol. Ther.17(8): 1453-1464 (2009).
  • lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR TM gene delivery technology from Oxford BioMedica, the LENTIMAX TM vector system from Lentigen Technology, and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
  • the term “circularized RNA” or “circRNA” refers to a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. CircRNAs are 3-5’ covalently closed RNA rings, and circRNAs do not display Cap or poly(A) tails.
  • CircRNAs lack the free ends necessary for exonuclease-mediated degradation, rendering them resistant to several mechanisms of RNA turnover and granting them extended lifespans as compared to their linear mRNA counterparts. For this reason, circularization may allow for the stabilization of mRNAs that generally suffer from short half-lives and may therefore improve the overall efficacy of mRNA in a variety of applications. CircRNAs are produced by the process of splicing, and circularization occurs using conventional splice sites mostly at annotated exon boundaries (Starke et al., 2015; Szabo et al., 2015).
  • RNA circularization For circularization, splice sites are used in reverse: downstream splice donors are “backspliced” to upstream splice acceptors (see Jeck and Sharpless, 2014; Barrett and Salzman, 2016; Szabo and Salzman, 2016; Holdt et al., 2018 for review).
  • Three general strategies have been reported so far for RNA circularization: chemical methods using cyanogen bromide or a similar condensing agent, enzymatic methods using RNA or DNA ligases, and ribozymatic methods using self-splicing introns.
  • precursor RNA is synthesized by run-off transcription and then heated in the presence of magnesium ions and GTP to promote circularization. RNA so produced can efficiently transfect different kinds of cells.
  • the template includes sequences for the TFP, CAR, and TCR, or combination thereof.
  • a ribozymatic method utilizing a permuted group I catalytic intron is used. This method is more applicable to long RNA circularization and requires only the addition of GTP and Mg2+ as cofactors.
  • This permuted intron-exon (PIE) splicing strategy consists of fused partial exons flanked by half-intron sequences. In vitro, these constructs undergo the double transesterification reactions characteristic of group I catalytic introns, but because the exons are fused, they are excised as covalently 5’ and 3’linked circles.
  • 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.
  • isolated means altered or removed from the natural state.
  • 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.
  • nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.
  • operably linked 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.
  • 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.
  • nucleic acid or “polynucleotide” 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); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al., Mol. Cell.
  • 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.
  • 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, that can initiate the specific transcription of a polynucleotide sequence.
  • promoter/regulatory sequence refers to a nucleic acid sequence which can be used for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, 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 (Gly3Ser).
  • 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.
  • in 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)” is 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. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal.
  • the poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases.
  • 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. After transcription has been terminated, 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 (SEQ ID NO:119) near 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.
  • terapéutica means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.
  • proliferative means the prevention of or protective treatment for a disease or disease state.
  • 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, mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, gastric cancer, ovarian cancer, Non- Hodgkin lymphoma (NHL), leukemias, uterine cancer, prostate cancer, colon cancer, cervical cancer, bladder cancer, kidney cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, brain cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, endometrial cancer, primary or metastatic melanoma, thymoma, solid cancer, sarcoma, uterine cancer, and stomach cancer.
  • NDL Non- Hodgkin lymphoma
  • leukemias uterine cancer
  • prostate cancer colon cancer
  • cervical cancer bladder cancer
  • kidney cancer prostate
  • the disease is a cancer selected from the group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, endometroid mucinous ovarian carcinoma, malignant pleural disease, pancreatic adenocarcinoma, ductal pancreatic adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, breast adenocarcinoma, a disease associated with a TAA expression, relapsed or refractory neoplastic disease, and any combination thereof.
  • the TFP targets CD70 and the disease is a cancer selected from the group consisting of T cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), an Epstein-Barr virus (EBV) + cancer, a human papilloma virus (HPV) + cancer, kidney cancer, renal cell carcinoma, lung cancer, pancreatic cancer, ovarian cancer, esophageal cancer, nasopharyngeal carcinoma, mesothelioma, glioblastoma, thymic carcinoma, breast cancer, head and neck cancer, gastric cancer, and any combinations thereof.
  • DLBCL diffuse large B-cell lymphoma
  • MCL mantle cell lymphoma
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • EBV Epstein-Barr virus
  • HPV human papilloma
  • the TFP targets mesothelin (MSLN) and the disease is a cancer selected from 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, cholangiocarcinoma, stomach cancer, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, endometroid mucinous ovarian carcinoma, pancreatic adenocarcinoma, ductal pancreatic adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorec
  • the TFP targets CD19 and the disease is a cancer selected from the group consisting of B-cell acute lymphoid leukemia (“BALL”), T-cell acute lymphoid leukemia (“TALL”), acute lymphoid 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- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin’s lymphoma, plasmablastic lymphoma, plasmacyto
  • the TFP targets MUC16 and the disease is a cancer selected from the group consisting of pancreatic cancer, ovarian cancer, stomach cancer, lung cancer, endometrial cancer, atypical cancers expressing MUC16, or any combinations thereof.
  • the TFP targets BCMA and the disease is a cancer selected from the group consisting of B-cell acute lymphoid leukemia (“BALL”), T-cell acute lymphoid leukemia (“TALL”), acute lymphoid 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- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mant
  • the TFP targets CD79B and the disease is a cancer selected from the group consisting of lymphoma, myeloma, non-Hodgkin lymphoma (NHL), diffuse large-cell B-cell lymphoma, aggressive NHL, asymptomatic NHL, follicular lymphoma, recurrent aggressive NHL, recurrent non-chronic non- treatable NHL, non-treatable asymptomatic NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, reticuloendotheliosis (RE), acute lymphocytic leukemia (ALL), lymphoma of the head cortex brain, and any combinations thereof.
  • NHL non-Hodgkin lymphoma
  • NHL non-Hodgkin lymphoma
  • NHL non-Hodgkin lymphoma
  • NHL non-Hodgkin lymphoma
  • NHL non-Hodgkin lymphoma
  • NHL non-Hodgkin lymphom
  • the TFP targets HER2 and the disease is a cancer selected from breast cancer, ovarian cancer, endometrial cancer, gastric cancer, pancreatic cancer, prostate cancer, salivary gland cancer, and any combinations thereof.
  • the TFP targets PSMA and the disease is a cancer selected from the group consisting of prostate cancer, endometrial cancer, breast cancer, kidney cancer, colon cancer, and any combinations thereof.
  • the TFP targets CD20 and the disease is a cancer selected from the group consisting of non-Hodgkin lymphomas, Hodgkin's disease, acute lymphoblastic leukemias, myelomas, chronic lymphocytic leukemias, myeloblastic leukemias, and any combinations thereof.
  • transfected or “transformed” or “transduced” 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,” refers to an antibody, an antibody fragment or a specific ligand, which recognizes and binds a cognate binding partner present in a sample, but which does not necessarily and substantially recognize or bind other molecules in the sample.
  • stably linked“ or “stable linkage,” as used herein, refers to non-cleavably linked or non-cleavable linkage. In some embodiment, the term “stably linked“ or “stable linkage” refers to operatively linked or operative linkage.
  • 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.
  • 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.
  • Co-receptor signaling may be a mechanism for coordinating and tightly regulating immune responses.
  • the usual scheme of activation of alpha beta ( ⁇ ) T cells can rely on positive signals given by peptide antigens presented by HLA class I or II. Co-receptor signals can either increase or prevent this activation.
  • the negative signaling molecules can include those belonging to CD28/B7 families. Three members of this family have been described: CTL-associated antigen-4 (CTLA-4), programmed death-1 (PD-1) and B and T lymphocyte attenuator (BTLA). They can play a role in the control of tolerance. They can provide negative signals that limit, terminate and/or attenuate immune responses.
  • PD-1 (or CD279) can be expressed on activated T and B cells as well as on activated myeloid cells. Upon colligation with the T-cell receptor (TCR), PD-1 can elicit inhibitory signals.
  • TCR T-cell receptor
  • the PD-1 cytoplasmic domain can contain two tyrosines, one that constitutes an immunoreceptor tyrosine inhibitory receptor (ITIM) and the other one an immunoreceptor tyrosine based switch motif (ITSM).
  • ITIM immunoreceptor tyrosine inhibitory receptor
  • ITSM immunoreceptor tyrosine based switch motif
  • the phosphorylation of the second tyrosine can lead to the recruitment of the tyrosine phosphatases SHP2 and to some extent SHP1.
  • phosphatases can dephosphorylate ZAP70, CD3 ⁇ and PKC ⁇ and consequently can attenuate T cell signals.
  • PD-1 may inhibit T and B cell proliferation by causing cell arrest in G0/G1 and inhibiting cytokine production in T cells.
  • PD-1 can bind to its ligand(s) including but not limited to PD-L1/B7H1/CD274 and PD- L2/B7-DC/CD273.
  • PD-L1 can be expressed at low levels on immune cells such as B cells, dendritic cells, macrophages and T cells and is up regulated following activation.
  • PD-L1 can also be expressed on non-lymphoid organs such as endothelial cells, heart, lung, pancreas, muscle, keratinocytes and placenta.
  • PD-L1 may regulate the function of self-reactive T and B cells as well as myeloid cells in peripheral tissues or may regulate inflammatory responses in the target organs.
  • PD-L1 expression can be regulated by type 1 and 2 interferon on endothelial and epithelial cells.
  • PD-L1 can be expressed in tumor samples and may be associated to poor prognosis.
  • PD-L2/B7-DC cell surface expression can be restricted to macrophages and dendritic cells, though PD-L2 transcript may be found in non hematopoietic tissues such as heart, liver and pancreas. Its surface expression can depend on the production of IFN ⁇ and Th2 cytokines. Both PD-L1 and PD-L2 may inhibit T cell proliferation, cytokine production and ⁇ 1 and ⁇ 2 integrins mediated adhesion. The expression patterns of PD-L1 and PD-L2 suggest both overlapping and differential roles in immune regulation. PD-L1 may be abundant in a variety of human cancers.
  • the interaction between PD-1 and PD-L1 can result in a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and immune evasion by the cancerous cells. Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect can be additive when the interaction of PD-1 with PD-L2 is blocked as well.
  • Blocking the interaction of PD-1 with its ligand(s) may increase effector functions of T cells and lead to improved therapeutic effects of cell therapy in treating a disease such as cancer.
  • the present disclosure provides a nucleic acid sequence encoding an antibody or fragment thereof that specifically binds programmed cell death protein 1 (PD-1).
  • the antibody or fragment thereof that specifically binds PD-1 can be referred to as anti-PD-1 antibody.
  • the anti- PD-1 antibody can block the interaction between PD-1 and PD-L1 and/or PD-L2.
  • the anti-PD-1 antibody can be a fragment or a binding domain of an antibody.
  • the nucleic acid sequence can be a DNA or a RNA.
  • the nucleic acid sequence can be a sequence within a vector.
  • the present disclosure also provides a polypeptide encoded by the nucleic acid sequence.
  • the polypeptide can be the antibody or fragment thereof that specifically binds PD-1.
  • the antibody or fragment thereof provided herein can be any domain that binds to PD-1 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 (V L ) and a variable domain (V HH ) of a camelid derived nanobody, and to an alternative scaffold to function as antigen binding domain, such as a recombinant fibronectin domain, anticalin, DARPIN and the like.
  • VH heavy chain variable domain
  • V L light chain variable domain
  • V HH variable domain of a camelid derived nanobody
  • the antibody or fragment thereof that specifically binds PD-1 can be a humanized or non-humanized antibody or fragment thereof.
  • the antigen binding domain is a fragment, e.g., a single chain variable fragment (scFv).
  • the antigen binding domain is a Fv, a Fab, a (Fab’)2, or a bi- functional (e.g., bi-specific) hybrid antibody.
  • the antibodies and fragments thereof disclosed herein bind a PD-1 protein with wild-type or enhanced affinity.
  • 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 PD-1.
  • a humanized antibody or antibody fragment may have improved affinity and/or specificity of binding to PD-1.
  • Various methods can be used to humanize an antibody, including but not limited to the methods described here in “T cell receptor (TCR) fusion proteins (TFPs)” section below.
  • TCR T cell receptor
  • TFPs T cell receptor fusion proteins
  • the present disclosure provides an antibody or fragment thereof that specifically binds PD-1.
  • the anti-PD-1 antibody or antibody fragment can comprise a variable domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3.
  • the CDR3 can comprise the amino acid sequence of SEQ ID NO:3.
  • the CDR1 can comprise the amino acid sequence of SEQ ID NO:1.
  • the CDR2 can comprise the amino acid sequence of SEQ ID NO:2.
  • variable domain can further comprise a framework region 1 (FR1) comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.
  • the variable domain can further comprise a framework region 2 (FR2) comprising the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.
  • the variable domain can further comprise a framework region 3 (FR3) comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.
  • FR4 framework region 4 comprising the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:19.
  • variable domain can comprise one or more mutations in one or more of framework region 1 (FR1), FR2, FR3, or FR4 relative to FR1, FR2, FR3, and/or FR4 of the amino acid sequence of SEQ ID NO. 4.
  • the variable domain can have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4.
  • the variable domain can have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to the amino acid sequence of SEQ ID NO:4.
  • the variable domain can comprise the amino acid sequence of SEQ ID NO:4.
  • the variable domain can be encoded by the nucleic acid sequence of SEQ ID NO:20.
  • the variable domain can comprise one or more mutations at amino acid position selected from the group consisting of amino acid positions 1, 5, 14, 34, 35, 49, 75, 76, 78, 79, 87, 88, 93, and 115 of the amino acid sequence of SEQ ID NO:4.
  • the variable domain can comprise a mutation at amino acid positions 1, 5, 14, 34, 49, 76, 78, 79, 87, 93, and 115 of the amino acid sequence of SEQ ID NO:4.
  • the variable domain can comprise a mutation at amino acid positions 1, 5, 14, 34, 49, 75, 76, 78, 79, 87, 93, and 115 of the amino acid sequence of SEQ ID NO:4.
  • the variable domain can comprise a mutation at amino acid positions 1, 5, 14, 34, 35, 49, 75, 76, 78, 79, 87, 93, and 115 of the amino acid sequence of SEQ ID NO:4.
  • the variable domain can comprise a mutation at amino acid positions 1, 5, 14, 34, 35, 49, 75, 76, 78, 79, 87, 88, 93, and 115 of the amino acid sequence of SEQ ID NO:4.
  • the variable domain can comprise one or more mutations selected from the group consisting of Q1E, Q5V, A14P, I34M, G35S, A49S, A75S, N76K, A78T, V79L, K87R, P88A, I93V and Q115L relative to the amino acid sequence of SEQ ID NO:4.
  • the variable domain can comprise Q1E, Q5V, A14P, I34M, A49S, N76K, A78T, V79L, K87R, I93V and Q115L mutations relative to the amino acid sequence of SEQ ID NO:4.
  • the variable domain can comprise Q1E, Q5V, A14P, I34M, A49S, A75S, N76K, A78T, V79L, K87R, I93V and Q115L mutations relative to the amino acid sequence of SEQ ID NO:4. [0703]
  • the variable domain can comprise Q1E, Q5V, A14P, I34M, G35S, A49S, A75S, N76K, A78T, V79L, K87R, I93V and Q115L mutations relative to the amino acid sequence of SEQ ID NO:4.
  • variable domain can comprise Q1E, Q5V, A14P, I34M, G35S, A49S, A75S, N76K, A78T, V79L, K87R, P88A, I93V and Q115L mutations relative to the amino acid sequence of SEQ ID NO:4.
  • the variable domain can have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to the amino acid sequence of SEQ ID NO:4.
  • variable domain can have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8.
  • the variable domain can have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8.
  • the variable domain can have at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9% or more sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8.
  • the variable domain can comprise the amino acid sequence of SEQ ID NO:5.
  • the variable domain can be encoded by the nucleic acid sequence of SEQ ID NO:21.
  • the variable domain can comprise the amino acid sequence of SEQ ID NO:6.
  • the variable domain can be encoded by the nucleic acid sequence of SEQ ID NO:22.
  • the variable domain can comprise the amino acid sequence of SEQ ID NO:7.
  • the variable domain can be encoded by the nucleic acid sequence of SEQ ID NO:23.
  • the variable domain can comprise the amino acid sequence of SEQ ID NO:8.
  • the variable domain can be encoded by the nucleic acid sequence of SEQ ID NO:24.
  • the anti-PD-1 antibody or fragment thereof can bind to human PD-1 with a KD value of about 1 to about 60 nM. In some cases, the K D value is at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more nM.
  • the anti-PD-1 antibody or fragment thereof can bind to human PD-1 with a K D value of at most 55 nM. In some cases, the KD value is at most about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or less nM.
  • the anti-PD-1 antibody or fragment thereof can be secreted when expressed in a cell.
  • the anti-PD-1 antibody or fragment thereof can be fused to a transmembrane domain and may not be secreted in some cases.
  • the transmembrane domain may be further linked to a co-stimulatory domain.
  • the co-stimulatory domain can be any co-stimulatory domain disclosed herein.
  • the anti-PD-1 antibody or fragment thereof can be an antagonist.
  • the anti-PD-1 antibody or fragment thereof can be a PD-1 inhibitor.
  • the anti-PD-1 antibody or fragment thereof can compete with PD-L1 and/or PD-L2 for binding to PD-1, inhibit PD-L1 and/or PD-L2 from interacting with PD-1, and/or bind to the same epitope of PD-1 to which PD-L1 and/or PD-L2 binds.
  • the anti-PD-1 antibody or fragment thereof can block an immune checkpoint or block immune checkpoint signaling.
  • the anti-PD-1 antibody or fragment thereof can be an immune checkpoint inhibitor or can inhibit immune checkpoint signaling.
  • the anti-PD-1 antibody or fragment thereof can promote anti-tumor activity or inhibit inhibition of anti-tumor activity.
  • the anti-PD-1 antibody or fragment thereof can promote antibody or antibody fragment mediated tumor cell killing or inhibit inhibition of antibody or antibody fragment mediated tumor cell killing.
  • the anti-PD-1 antibody or fragment thereof can inhibit tumor growth.
  • the anti-PD-1 antibody or fragment thereof can be a human or humanized antibody or antibody fragment.
  • the anti-PD-1 antibody or fragment thereof can be a single domain antibody (sdAb).
  • the sdAb can be a VHH.
  • the VHH can be Fc conjugated.
  • the Fc can be a variant of IgG.
  • the Fc can be from an effector-function silent IgG.
  • the effector-function silent IgG can contain point mutations that abrogate binding of Fc receptors (Fc ⁇ R, FcR), abolishing antibody directed cytotoxicity (ADCC) effector function.
  • the effector-function silent IgG Fc can comprise one or more mutations. The one or more mutations can be in the hinge and/or CH 2 regions.
  • the Fc can be from an effector-function silent IgG4.
  • the Fc can be from an effector-function silent IgG2, which can be engineered with V234A/G237A /P238S/H268A/V309L/A330S/P331S substitutions to eliminate affinity for Fc ⁇ R and C1q complement protein and consequently, immune effector functions.
  • the Fc can be from an effector-function silent IgG1 (e.g., aglycosylated IgG1).
  • the Fc can be from an effector-function silent IgG2m4 (H268Q/V309L/A330S/P331S, changes to IgG4).
  • the Fc can be from an effector-function silent IgG4 ProAlaAla (S228P/L234A/L235A).
  • the anti-PD-1 antibody or fragment thereof as described herein comprises a sequence encoded by 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 10.
  • the anti-PD-1 antibody or fragment thereof as described herein comprises a sequence encoded by any one of the nucleotide sequences listed in Table 10.
  • the anti-PD-1 antibody or fragment thereof as described herein is encoded by 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 10.
  • the anti-PD-1 antibody or fragment thereof as described herein comprises a sequence encoded by any one of the nucleic acid sequence listed in Table 10.
  • the anti-PD-1 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 10. In some embodiments, the anti-PD-1 antibody or fragment thereof as described herein comprises any one of the CDR sequences listed in Table 10.
  • the anti-PD-1 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-PD-1 antibody or fragment thereof sequences listed in Table 10.
  • the anti-PD-1 antibody or fragment thereof as described herein comprises a CDR sequence of any one of the CDR sequences of the anti-PD-1 antibody or fragment thereof sequences listed in Table 10.
  • the anti-PD-1 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-PD-1 antibody or fragment thereof sequences listed in Table 10.
  • the anti-PD-1 antibody or fragment thereof as described herein comprises any one of the anti-PD-1 antibody or fragment thereof sequences listed in Table 10.
  • the anti-PD-1 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 sequences of SEQ ID NOs: 1-3.
  • the anti-PD-1 antibody or fragment thereof as described herein comprises a CDR sequence of SEQ ID NOs: 1-3.
  • the anti-PD-1 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 SEQ ID NOs: 4-8.
  • the anti-PD-1 antibody or fragment thereof as described herein comprises the CDR sequence of SEQ ID NOs: 4-8.
  • the anti-PD-1 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 amino acid sequences selected from SEQ ID NOs: 1-24, 75, 77, 79, 81, 83, and 85.
  • the anti-PD-1 antibody or fragment thereof as described herein comprises the amino acid sequences selected from SEQ ID NOs : 1-24, 75, 77, 79, 81, 83, and 85.
  • the anti-PD-1 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 the amino acid sequence of SEQ ID NO:4 or 6.
  • the anti-PD-1 antibody or fragment thereof as described herein comprises the amino acid sequence of SEQ ID NOs : 4 or 6.
  • sequence of the anti-PD-1 antibody or fragment thereof as described herein is 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 the amino acid sequence of SEQ ID NO:4 or 6.
  • sequence of the anti-PD-1 antibody or fragment thereof as described herein is the amino acid sequence of SEQ ID NOs : 4 or 6.
  • the recombinant nucleic acid molecule 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 selected from SEQ ID NOs: 1-24, 75, 77, 79, 81, 83, and 85.
  • the recombinant nucleic acid molecule as described herein comprises a sequence encoding any one of the amino acid sequences selected from SEQ ID NOs: 1-24, 75, 77, 79, 81, 83, and 85.
  • the recombinant nucleic acid molecule 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 the amino acid sequence of SEQ ID NO:4 or 6.
  • the recombinant nucleic acid molecule as described herein comprises the amino acid sequence of SEQ ID NOs : 4 or 6.
  • the anti-PD-1 antibody or fragment thereof that specifically binds programmed cell death protein 1 (PD-1) inhibits an interaction of PD-1 with PD-L1 or PD-L2.
  • the anti-PD-1 antibody or fragment thereof that specifically binds programmed cell death protein 1 (PD-1) is secreted by the T cell.
  • a fusion protein comprises an anti-PD-1 antibody or fragment thereof that specifically binds PD-1 and a transmembrane domain operatively linked to the anti-PD-1 antibody or fragment thereof, wherein the anti-PD-1 antibody or fragment thereof inhibits an interaction of PD-1 with PD-L1 or PD-L2.
  • the anti-PD-1 antibody or fragment thereof is stably linked to the transmembrane domain.
  • the fusion protein further comprises an intracellular domain operatively linked to the transmembrane domain.
  • the anti-PD-1 antibody is any of the PD-1 antibodies described above and in Table 10.
  • the PD-1 antibody of the fusion protein is selected from any of those disclosed in Table 15.
  • the PD-1 antibody of the fusion protein comprises a HC CDR1, CDR2, CDR3 and/or a LC CDR1, CDR2, and CDR3 of any of the PD-1 antibodies disclosed in Table 15.
  • the anti-PD-1 antibody or fragment thereof as described herein comprises any one of the anti-PD- 1 antibody or fragment thereof sequences listed in Table 15.
  • a fusion protein comprising an anti-PD-1 antibody or fragment thereof that specifically binds PD-1 and an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof, wherein the anti-PD-1 antibody or fragment thereof inhibits an interaction of PD-1 with PD-L1 or PD-L2.
  • the anti-PD- 1 antibody or fragment thereof is stably linked to the intracellular domain.
  • the fusion protein further comprises an intracellular domain operatively linked to the transmembrane domain.
  • the fusion protein as described herein further comprises a signal sequence.
  • the signal sequence comprises a PD-1 signal peptide.
  • the signal sequence is a PD-1 signal peptide.
  • the signal sequence 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 the amino acid sequence of SEQ ID NO:256 or SEQ ID NO:335.
  • the signal sequence comprises the amino acid sequence of SEQ ID NO:256 or SEQ ID NO:335.
  • the sequence of the signal sequence is 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 the amino acid sequence of SEQ ID NO:256 or SEQ ID NO:335. In some embodiments, the sequence of the signal sequence is the amino acid sequence of SEQ ID NO:256 or SEQ ID NO:335.
  • the transmembrane domain of the fusion protein as describe herein comprises a transmembrane domain of a protein selected from the group consisting of CD28, CD3 ⁇ , CD3 ⁇ , CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD41, CD64, CD68, CD80, CD86, CD134, CD137, CD154, ICOS, 4-1BB, OX40, PD-1, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the transmembrane domain of the fusion protein as describe herein comprises a PD-1 transmembrane domain.
  • the transmembrane domain of the fusion protein as describe herein comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more consecutive amino acid residues of the PD-1 transmembrane domain.
  • the transmembrane domain of the fusion protein as describe 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 the amino acid sequence of the PD-1 transmembrane domain.
  • the transmembrane domain of the fusion protein as describe herein comprises the amino acid sequence of the PD-1 transmembrane domain. In some embodiments, the transmembrane domain of the fusion protein as describe herein comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more consecutive amino acid residues of SEQ ID NO:301. In some embodiments, the transmembrane domain of the fusion protein as describe 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 the amino acid sequence of SEQ ID NO:301.
  • the transmembrane domain of the fusion protein as describe herein comprises the amino acid sequence of SEQ ID NO:301.
  • the sequence of the transmembrane domain of the fusion protein as describe herein is 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 the amino acid sequence of SEQ ID NO:301.
  • the sequence of the transmembrane domain of the fusion protein as describe herein is the amino acid sequence of SEQ ID NO:301.
  • the fusion protein further comprises a PD-1 stalk domain.
  • the PD-1 stalk domain is operatively linked to the transmembrane domain. In some embodiments, the PD-1 stalk domain is operatively linked to the N-terminus of the transmembrane domain. [0721] In some embodiments, the transmembrane domain of the fusion protein as describe herein further comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,, 23, 24, 25 or more consecutive amino acid residues of the PD-1 stalk domain.
  • the transmembrane domain of the fusion protein as describe herein further 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 the amino acid sequence of the PD-1 stalk domain.
  • the transmembrane domain of the fusion protein as describe herein further comprises the amino acid sequence of the PD-1 stalk domain.
  • the transmembrane domain of the fusion protein as describe herein further comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,, 23, 24, 25 or more consecutive amino acid residues of SEQ ID NO:259.
  • the transmembrane domain of the fusion protein as describe herein further 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 the amino acid sequence of SEQ ID NO:259.
  • the transmembrane domain of the fusion protein as describe herein further comprises the amino acid sequence of SEQ ID NO:259.
  • the fusion protein as describe 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 the amino acid sequence of SEQ ID NO:259 operatively linked to 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 the amino acid sequence of SEQ ID NO:301.
  • the fusion protein as describe 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 the amino acid sequence of SEQ ID NO:259 operatively linked to the N terminus of 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 the amino acid sequence of SEQ ID NO:301.
  • the fusion protein as describe herein comprises the amino acid sequence of SEQ ID NO:259 operatively linked to the amino acid sequence of SEQ ID NO:301. In some embodiments, the fusion protein as describe herein comprises the amino acid sequence of SEQ ID NO:259 operatively linked to the N-terminus of the amino acid sequence of SEQ ID NO:301. [0723] In some embodiments, the intracellular domain of the fusion protein as describe herein comprises a co-stimulatory domain.
  • the co-stimulatory domain comprises a co-stimulatory domain of a protein selected from the group consisting of a CD27, CD28, 4- 1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, PD- 1, CD258, ICAM-1, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the co-stimulatory domain comprises a 4-1BB (CD137) co-stimulatory domain.
  • the co-stimulatory domain of the fusion protein as describe herein 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, 39, 40, 41, 42 or more consecutive amino acid residues of the 4-1BB intracellular domain or 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 or more consecutive amino acid residues of the CD28 co-stimulatory domain.
  • the co-stimulatory domain of the fusion protein as describe 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 the amino acid sequence of the 4-1BB intracellular domain or the CD28 co-stimulatory domain.
  • the co-stimulatory domain of the fusion protein as describe herein comprises the amino acid sequence of the 4-1BB intracellular domain or the CD28 co-stimulatory domain.
  • the co-stimulatory domain of the fusion protein as describe herein is the amino acid sequence of the 4-1BB intracellular domain or the CD28 co-stimulatory domain.
  • the co-stimulatory domain of the fusion protein as describe herein 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, 39, 40, 41, 42 or more consecutive amino acid residues of SEQ ID NO:277 or 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 or more consecutive amino acid residues of SEQ ID NO:273.
  • the co- stimulatory domain of the fusion protein as describe 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 the amino acid sequence of SEQ ID NO:277 or SEQ ID NO:273.
  • the co-stimulatory domain of the fusion protein as describe herein comprises the amino acid sequence of SEQ ID NO:277 or SEQ ID NO:273.
  • the sequence of the co-stimulatory domain of the fusion protein as describe herein is 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 the amino acid sequence of SEQ ID NO:277 or SEQ ID NO:273.
  • the sequence of the co- stimulatory domain of the fusion protein as describe herein is the amino acid sequence of SEQ ID NO:277 or SEQ ID NO:273.
  • the fusion protein comprises at least two, three, four, five, six, seven, eight, nine, ten or more anti-PD-1 antibodies or fragments thereof.
  • the at least two, three, four, five, six, seven, eight, nine, ten or more anti-PD-1 antibodies or fragments thereof are operatively linked tandemly. In some embodiments, the at least two, three, four, five, six, seven, eight, nine, ten or more anti-PD-1 antibodies or fragments thereof are identical. In some embodiments, the at least two, three, four, five, six, seven, eight, nine, ten or more anti-PD-1 antibodies or fragments thereof are different. In some embodiments, the at least two, three, four, five, six, seven, eight, nine, ten or more anti-PD-1 antibodies or fragments thereof are operatively linked by a linker.
  • the linker 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 the amino acid sequence of SEQ ID NO:371.
  • the linker comprises the amino acid sequence of SEQ ID NO:371.
  • the sequence of the linker is 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 the amino acid sequence of SEQ ID NO:371.
  • the sequence of the linker is the amino acid sequence of SEQ ID NO:371.
  • the fusion protein as describe 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 amino acid sequences listed in Table 16. In some embodiments, the fusion protein as describe herein comprises any one of the amino acid sequences listed in Table 16.
  • the fusion protein as describe 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 amino acid sequences of SEQ ID NOs: 373-377. In some embodiments, the fusion protein as describe herein comprises any one of the amino acid sequences of SEQ ID NOs: 373- 377.
  • the sequence of the fusion protein as describe herein is 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 of SEQ ID NO:373-377. In some embodiments, the sequence of the fusion protein as describe herein is any one of the amino acid sequences of SEQ ID NOs: 373-377.
  • the fusion protein as describe herein is encoded by a nucleic acid sequence having at least 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 of SEQ ID NOs: 378-382. In some embodiments, the fusion protein as describe herein is encoded by any one of the nucleic acid sequences of SEQ ID NOs: 378-382.
  • T cell receptor (TCR) fusion proteins T cell receptor (TCR) [0730]
  • TFP T cell receptor
  • TFP T cell receptor
  • the present disclosure encompasses recombinant nucleic acid constructs encoding TFPs and variants thereof, wherein the TFP comprises a binding domain, e.g., an antibody or antibody fragment, a ligand, or a ligand binding protein, that binds specifically to a tumor-associated antigen (TAA), e.g., human TAA, 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.
  • TAA tumor-associated antigen
  • TFPs 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.
  • 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 domains also referred to herein as “intracellular signaling domains”
  • the central 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 co-stimulatory molecule such as 4-1BB (i.e., CD137), CD27 and/or CD28.
  • 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 (V L ) and a variable domain (V HH ) 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
  • V L light chain variable domain
  • V HH variable domain of a camelid derived nanobody
  • the TFP comprises an antigen-binding domain comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment.
  • the murine, human, or humanized antigen binding domain is, e.g., an antibody or antibody fragment.
  • the antibody or antibody fragment is an scFv or single domain antibody (sdAb).
  • the antibody is an scFv and 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 humanized or human anti-tumor- associated antigen 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 humanized or human anti-tumor-associated antigen binding domain described herein, e.g., a humanized or human anti-CD19, anti-BCMA, anti-MUC16, anti-mesothelin (anti-MSLN), anti-CD79B, anti-HER2, anti-PSMA, anti-CD20, anti-CD70
  • the humanized or human anti-tumor-associated antigen (anti-TAA) binding domain comprises a humanized heavy chain variable region described herein, e.g., at least two 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 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 humanized 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.
  • 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 including those of the antigen binding domains and the anti-PD-1 antibodies described herein, 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; International Publication No.
  • 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.
  • Multiple techniques for humanization of antibodies or antibody fragments are well-known in the art and can essentially be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239,400; PCT Publication No.
  • WO 91/09967 and U.S. Pat. Nos.4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference in their entirety).
  • Humanized antibodies and antibody fragments substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species.
  • 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.
  • the 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 V H 4-4-59 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.
  • 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 a human TAA or PD-1.
  • a humanized antibody or antibody fragment may have improved affinity and/or specificity of binding to human CD19, 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, or human PD-1.
  • 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 CD19, human BCMA, human MUC16, human mesothelin, human CD79B, human HER2, human PSMA, human CD70, human CD20, human Nectin-4, human GPC3, human TROP-2, or human PD-1.
  • 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 antibody has the antigen binding domain of FMC63 or another anti-CD19 antibody.
  • exemplary antibodies that bind CD19 include, but are not limited to, inebilizumab, MDX-1342, tafasitamab, obexelimab, B4 (Merck), immunomedics hA19, and those described in WO2019112347, WO2010142952, WO2018108106, WO2009086514, WO2006121852, WO2010095031, WO2016059253, WO2009102473, WO2001058916, WO2012010561, WO2005012493, WO2008031056, WO2019214332, WO2017126587, WO2000035409, WO2019137518, WO2007002223, WO2012067981, WO2007076950, WO2002020615, WO2001070266, WO2005035582, WO2015179236, WO2013184218, WO2018101448, WO2016112855, WO2010021697, WO2001057226, WO2017165
  • 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:26, a CDR2 of SEQ ID NO:28, and a CDR3 of SEQ ID NO:30 and a heavy chain CDR1 of SEQ ID NO:32, a CDR2 of SEQ ID NO:34, and a CDR3 of SEQ ID NO:36.
  • the anti-CD19 antibody is a murine scFv.
  • the anti-CD-19 antibody comprises a VL of SEQ ID NO:38 and a VH of SEQ ID NO:40.
  • 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 11.
  • the anti-CD19 antibody or fragment thereof as described herein comprises any one of the CDR sequences listed in Table 11.
  • 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 11.
  • 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 11.
  • 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 11.
  • 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 11. [0747]
  • 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 WO2010104949, WO2011108008, WO2014122143, WO2016090327, WO2017143069, WO2017211900, WO2018133877, WO2019066435, WO2019149269.
  • SEA-BCMA ttle Genetics
  • WO2010104949 WO2011108008, WO2014122143
  • WO2016090327 WO2017143069
  • WO2017211900 WO2018133877
  • WO2019066435 WO2019149269.
  • WO2019190969, and WO2019195017 the contents of each of which are incorporated by reference herein in their entirety.
  • 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 11.
  • the anti-BCMA antibody or fragment thereof as described herein comprises any one of the CDR sequences listed in Table 11.
  • 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 11.
  • 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 11.
  • 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 11.
  • 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 11. [0749]
  • 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 WO2006099141, WO2006124641, WO2009120769, WO2010111282, WO2014004549, WO2014031476, WO2014052064, WO2017032293, and WO2017052241, the contents of each of which are incorporated by reference herein in their entirety.
  • 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 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 11.
  • the anti-mesothelin antibody or fragment thereof as described herein comprises any one of the CDR sequences listed in Table 11.
  • 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 11.
  • 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 11.
  • 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 11.
  • 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 11. [0752]
  • 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 WO2018058003, 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 WO2017009474 and WO2016021621, 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, IBI315, 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 WO2001009192, WO200303490, WO2007002222, WO2009130575, WO2010118522, WO2013185117, WO2013188740, WO2014198223, WO2016145139, WO2017121905, WO2017180713, WO2017212250, WO2018033749, WO2018129284, WO2018142323, and WO2019191728, the contents of each of which are incorporated by reference herein in their entirety.
  • 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 antigen-binding domain comprises an anti-CD70 humanized or human single domain antibody or an antibody fragment having a CDR1 of SEQ ID NO:188, a CDR2 of SEQ ID NO:189, and a CDR3 of SEQ ID NO:190, or a CDR1 of SEQ ID NO:192, a CDR2 of SEQ ID NO:193, and a CDR3 of SEQ ID NO:194, or a CDR1 of SEQ ID NO:196, a CDR2 of SEQ ID NO:197, and a CDR3 of SEQ ID NO:198, or a CDR1 of SEQ ID NO:200, a CDR2 of SEQ ID NO:201, and a CDR3 of SEQ ID NO:202, or a CDR1 of SEQ ID NO:204, a CDR2 of SEQ ID NO:205, and a CDR3 of SEQ ID NO:206, or a CDR1 of SEQ ID NO:208, a CDR1 of SEQ ID NO:
  • the anti-CD70 antibody fragment thereof can comprise a variable (VL) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:207.
  • 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:224, a CDRH2 having a sequence of SEQ ID NO:225, and a CDRH3 having a sequence of SEQ ID NOs: 226.
  • 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:228, a CDRL2 having a sequence of SEQ ID NO:229, and a CDRL3 having a sequence of SEQ ID NO:230.
  • 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:227.
  • 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:231.
  • 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 11.
  • the anti-CD70 antibody or fragment thereof as described herein comprises any one of the CDR sequences listed in Table 11.
  • 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 11.
  • 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 11.
  • 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 11.
  • 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 11.
  • a TFP that specifically binds human CD19 comprises a heavy chain variable region having the sequence of SEQ ID NO:40 and a light chain variable region having the sequence of SEQ ID NO:38.
  • a TFP that specifically binds human MSLN comprises or consists of a VHH having a sequence of SEQ ID NO:69, SEQ ID NO:70 or SEQ ID NO:71.
  • 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 11.
  • the antigen-binding domain or fragment thereof as described herein comprises a sequence encoding any one of the amino acid sequences listed in Table 11.
  • 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 11.
  • 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 11.
  • 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 11.
  • the antigen-binding domain or fragment thereof as described herein comprises a sequence encoding any one of the CDR sequences listed in Table 11.
  • 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 11.
  • 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 11.
  • 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.
  • 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.
  • the antibodies and fragments thereof of the present disclosure binds a tumor-associated protein with wild-type or enhanced affinity.
  • the anti-TAA binding domain comprises a single domain antibody (sdAb or V HH ).
  • a target antigen e.g., CD19, BCMA, MUC16, MSLN, CD20, CD70, CD79B, HER
  • V H 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.
  • linker orientation and size see, e.g., Hollinger et al., 1993, Proc Natl Acad. Sci. U.S.A.90:6444-6448, U.S.
  • An scFv can comprise a linker of about 10, 11, 12, 13, 14, 15 or greater than 15 residues between its V L and V H 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 (Gly 4 Ser) 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.
  • Stability and Mutations The stability of a tumor associated antigen binding domain or an anti-PD-1 antibody described herein, e.g., scFv or sdAb molecules (e.g., soluble scFv or sdAb) can be evaluated in reference to the biophysical properties (e.g., thermal stability) of a conventional control scFv or sdAb molecule or a full-length antibody.
  • scFv or sdAb molecules e.g., soluble scFv or sdAb
  • 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 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 or anti- PD-1 antibody, 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 disclosed herein and scFv or sdAb molecules or Fab fragments of an antibody from which the scFv VH and VL or sdAb VH were derived. Thermal stability can be measured using methods known in the art. For example, in one embodiment, T M can be measured.
  • the binding domain or anti-PD-1 antibody comprises at least one mutation arising from the humanization process such that the mutated scFv or sdAb confers improved stability to the anti-PD-1 antibody or anti-TAA TFP construct.
  • the anti-TAA binding domain or anti-PD-1 antibody e.g., scFv or sdAb, 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 or anti-PD-1 antibody.
  • the antigen binding domain of the TFP or anti-PD-1 antibody 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 or anti-PD-1 antibody 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 or anti-PD-1 antibody is engineered by modifying one or more amino acids within one or both variable regions (e.g., VH and/or V L ), 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.
  • 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 algorithm 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.
  • the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math.2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat’l. Acad. Sci.
  • 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 V H or V L of a binding domain or anti-PD-1 antibody, e.g., scFv or sdAb, comprised in the TFP can be modified to retain at least about 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% identity of the starting VH or VL framework region of the starting antibody fragment, e.g., scFv or sdAb.
  • 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%.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% identity of the starting TFP construct.
  • the TFP comprises an antigen-binding domain that comprises a ligand.
  • the ligand or fragment thereof is capable of binding to an antibody or fragment thereof. In some instances, the ligand binds to the polypeptide expressed on a surface of a cell. In some instances, the receptor or polypeptide expressed on a surface of a cell comprises a stress response receptor or polypeptide. In some instances, the receptor or polypeptide expressed on a surface of a cell is an MHC class I-related glycoprotein.
  • the MHC class I- related glycoprotein is selected from the group consisting of MICA (MHC class I polypeptide- related sequence A; UniProt ID: Q29983), MICB (MHC class I polypeptide-related sequence B; UniProt ID: Q29980), RAET1E (Retinoic acid early transcript 1E; UniProt ID: Q8TD07), RAET1G (UL-16 binding protein 5; UniProt ID: Q6H3X3), ULBP1 (UL16-binding protein 1; UniProt ID: Q9BZM6), ULBP2 (UL16-binding protein 2; UniProt ID: Q9BZM5), ULBP3 (UL16-binding protein 3; UniProt ID: Q9BZM4), ULBP4 (Retinoic acid early transcript 1E; UniProt ID: Q8TD07) and combinations thereof.
  • MICA MHC class I polypeptide- related sequence A
  • MICB MHC class I polypeptide-related sequence
  • the antigen binding domain comprises a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the antigen binding domain comprises a monomer or a dimer of the ligand or fragment thereof.
  • the ligand or fragment thereof is a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nonamer, or a decamer.
  • the ligand or fragment thereof is a monomer or a dimer.
  • the antigen binding domain does not comprise an antibody or fragment thereof.
  • the antigen binding domain does not comprise a variable region.
  • the antigen binding domain does not comprise a CDR.
  • the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or a fragment thereof.
  • 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.
  • 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, gamma, or delta 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, CD154.
  • 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.
  • the TCR extracellular domain comprises the extracellular region of a constant domain or 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,
  • 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, 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 or more consecutive amino acid residues of the extracellular region of a constant domain or IgC domain of TCR alpha, a TCR beta, a TCR delta,
  • 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 region of a constant domain or IgC domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma.
  • the extracellular domain comprises a sequence encoding the extracellular region of a constant domain or 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, 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 or more consecutive amino acid residues of the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit
  • 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 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. [0784]
  • the extracellular domain can comprise an extracellular portion of a 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 the extracellular portion of a full-length 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 extracellular portion 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 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 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.
  • Transmembrane Domain [0786]
  • 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 instances, 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.
  • 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, CD16, 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.
  • the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge.
  • Ig immunoglobulin
  • Linkers [0790]
  • a short oligo- or polypeptide linker between 2 and 10 amino acids in length 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:120) or a sequence (GGGGS)x wherein X is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more (SEQ ID NO:122).
  • X is 2.
  • X is 4.
  • the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO:121).
  • Cytoplasmic Domain [0791] 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.
  • 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.
  • intracellular signaling domain 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.
  • the term 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.
  • 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 TCR subunit can comprise (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain of a TCR gamma chain or a TCR delta chain.
  • the TCR extracellular domain can comprise the extracellular portion of a constant domain of a TCR gamma chain or a TCR delta chain, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain is or comprises a delta constant domain, or a fragment thereof, e.g., a delta constant domain described herein, or a gamma constant domain, e.g., a gamma constant domain described herein.
  • the extracellular domain of the TFP may not comprise the variable domain of a gamma chain or a delta chain.
  • 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 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 TCR constant domain can be a TCR beta constant domain, e.g., a TCR beta constant domain described herein.
  • 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.
  • 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 alpha constant domain can comprise amino acids 2-137 of SEQ ID NO:386.
  • 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:386.
  • 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:386.
  • 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:386.
  • the constant domain can comprise a sequence or fragment thereof of positions 2-137 of SEQ ID NO:386.
  • 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:386.
  • 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:386.
  • 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:386.
  • the murine TCR beta constant domain can comprise amino acids 2-173 of SEQ ID NO:395.
  • 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:395.
  • 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:395.
  • 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:395.
  • the constant domain can comprise a sequence or fragment thereof of positions 22-173 of SEQ ID NO:395.
  • 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:395.
  • 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:395.
  • 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:395.
  • 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.
  • na ⁇ ve 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).
  • 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 tyrosine- based activation motifs (ITAMs).
  • 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, CD3 delta, or CD3 gamma.
  • a primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM 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 ITAM 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), OX40, 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.
  • 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 TFP-expressing cell 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 (e.g., CD70) or a different target (e.g., MSLN, CD19, or MUC16).
  • 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.
  • the TFP-expressing cell described herein can further express another agent, e.g., an agent which enhances the activity of a modified T cell.
  • the agent can be an agent which inhibits an inhibitory molecule.
  • Inhibitory molecules e.g., PD-1
  • inhibitory molecules include PD-1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.
  • the agent which inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
  • the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD-1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4 and TIGIT, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4- 1BB, CD27 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein).
  • an inhibitory molecule such as PD-1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4 and TIGIT
  • a fragment of any of these e.g., at least a portion of an extracellular domain of any of these
  • a second polypeptide which is an intra
  • the agent comprises a first polypeptide of PD-1 or a fragment thereof (e.g., at least a portion of an extracellular domain of PD-1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein).
  • PD-1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA.
  • PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al., 1996, Int. Immunol 8:765-75).
  • PD-L1 Two ligands for PD-1, PD-L1 and PD-L2, have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al., 2000 J. Exp. Med. 192:1027-34; Latchman et al., 2001 Nat. Immunol.2:261-8; Carter et al., 2002 Eur. J. Immunol. 32:634-43).
  • PD-L1 is abundant in human cancers (Dong et al., 2003 J. Mol. Med.81:281-7; Blank et al., 2005 Cancer Immunol. Immunother.54:307-314; Konishi et al., 2004 Clin. Cancer Res.10:5094).
  • the agent comprises the extracellular domain (ECD) of an inhibitory molecule, e.g., Programmed Death 1 (PD-1) can be fused to a transmembrane domain and optionally an intracellular signaling domain such as 41BB and CD3 zeta (also referred to herein as a PD-1 TFP).
  • ECD extracellular domain
  • PD-1 TFP when used in combinations with an anti- TAA TFP described herein, improves the persistence of the T cell.
  • the TFP is a PD-1 TFP comprising the extracellular domain of PD 1.
  • TFPs containing an antibody or antibody fragment such as a scFv that specifically binds to the Programmed Death-Ligand 1 (PD-L1) or Programmed Death-Ligand 2 (PD-L2).
  • the agent that inhibits an inhibitory molecule is a fusion protein that comprises an extracellular domain having an antibody or antibody fragment that specifically binds PD-1, e.g., an anti-PD-1 antibody described herein, and further comprises a transmembrane domain.
  • transmembrane domains for use with the present disclosure include, but are not limited to, transmembrane domains derived from CD28, CD3 ⁇ , CD3 ⁇ , CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD41, CD64, CD68, CD80, CD86, CD134, CD137, CD154, ICOS, 4-1BB, OX40, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the fusion protein further comprises a co-stimulatory domain.
  • Suitable co-stimulatory domains for use with the present disclosure include, but are not limited to, co-stimulatory domains derived from CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, PD-1, CD258, ICAM-1, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the present disclosure provides a population of TFP-expressing T cells, e.g., TFP-T cells.
  • the population of TFP-expressing T cells comprises a mixture of cells expressing different TFPs.
  • the population of TFP-T cells can include a first cell expressing a TFP having a binding domain described herein, and a second cell expressing a TFP having a different anti-TAA binding domain, e.g., a binding domain described herein that differs from the binding domain in the TFP expressed by the first cell.
  • the population of TFP-expressing cells can include a first cell expressing a TFP that includes an a first binding domain binding domain, e.g., as described herein, and a second cell expressing a TFP that includes an antigen binding domain to a target other than the binding domain of the first cell (e.g., another tumor-associated antigen).
  • the present disclosure provides a population of cells wherein at least one cell in the population expresses a TFP having a domain described herein, and a second cell expressing another agent, e.g., an agent which enhances the activity of a modified T cell.
  • the agent can be an agent which inhibits an inhibitory molecule.
  • Inhibitory molecules can, in some embodiments, decrease the ability of a modified T cell to mount an immune effector response.
  • inhibitory molecules include PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.
  • the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
  • the agent is a cytokine.
  • the cytokine is IL-15.
  • IL-15 increases the persistence of the T cells described herein.
  • Recombinant Nucleic Acids [0814] Disclosed herein, in some embodiments, is a recombinant nucleic acid encoding a TFP disclosed herein. Also provided herein is a recombinant nucleic acid encoding an antibody or antibody fragment that specifically binds PD-1. The recombinant nucleic acid provided herein can comprise a first sequence encoding the TFP and a second sequence encoding the antibody or antibody fragment that specifically binds PD-1.
  • a recombinant nucleic acid comprising (I) a first sequence encoding a T-cell receptor (TCR) fusion protein (TFP); and (II) a second sequence encoding an antibody or fragment thereof that specifically binds programmed cell death protein 1 (PD-1).
  • the TFP can comprise (i) a TCR subunit comprising: at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain, and a binding domain.
  • the anti-PD-1 antibody or fragment thereof can be secreted when expressed in a T cell.
  • the anti-PD-1 antibody can inhibit the interaction between PD-1 and PD-L1 and/or PD- L2.
  • TCR subunit and the binding domain can be operatively linked.
  • the TFP can functionally interact with an endogenous TCR complex when expressed in the T cell.
  • a recombinant nucleic acid comprising (i) a first sequence encoding a T-cell receptor (TCR) fusion protein (TFP); and (ii) a second sequence encoding a fusion protein comprising an antibody or fragment thereof that specifically binds programmed cell death protein 1 (PD-1) and a transmembrane domain.
  • TCR T-cell receptor
  • PD-1 programmed cell death protein 1
  • the TFP can comprise (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, and (b) a binding domain.
  • the TCR subunit and the binding domain can be operatively linked.
  • the TFP can functionally interact with an endogenous TCR complex when expressed in the T cell.
  • the first sequence encoding the TFP and the second sequence encoding the anti-PD-1 antibody or fragment thereof can be contained in a single operon.
  • the first sequence and the second sequence can be encoded in frame.
  • the recombinant nucleic acid can further comprise a third sequence in between the first and second sequences encoding a self-cleaving peptide.
  • the self-cleaving peptide can be a T2A peptide.
  • the anti-PD-1 antibody or fragment thereof can comprise a variable domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3.
  • the CDR3 can comprise the encoded amino acid sequence of SEQ ID NO:3.
  • the CDR1 can comprise the encoded amino acid sequence of SEQ ID NO:1.
  • the CDR2 can comprise the encoded amino acid sequence of SEQ ID NO:2.
  • the variable domain of the anti-PD-1 antibody or fragment thereof can comprise a sequence of the variable domain described herein.
  • the binding domain of the TFP encoded by the recombinant nucleic acid can specifically bind to a TAA described herein.
  • the binding domain can specifically bind to CD19, BCMA, MUC16, MSLN, CD79B, HER2, PSMA, CD20, CD70, Nectin-4, GPC3, TROP-2, or PD-1.
  • the binding domain can comprise an antibody domain of an anti-CD19, anti-BCMA, anti- MUC16, anti-mesothelin (anti-MSLN), anti-CD79B, anti-HER2, anti-PSMA, anti-CD20, anti- CD70, anti-Nectin-4, anti-GPC3, anti-TROP-2, or anti-PD-1 antibody.
  • the recombinant nucleic acid further comprises a leader sequence. In some instances, the recombinant nucleic acid further comprises a promoter sequence. In some instances, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some instances, the recombinant nucleic acid further comprises a 3’UTR sequence. In some instances, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. Non-naturally occurring nucleic acids are well known to those of skill in the art. In some instances, the nucleic acid is an in vitro transcribed nucleic acid.
  • RNA encoding TFP further encodes the anti-PD-1 antibody.
  • a method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3’ and 5’ untranslated sequence (“UTR”), a 5’ cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length.
  • IVTT in vitro transcription
  • UTR untranslated sequence
  • IRS Internal Ribosome Entry Site
  • the template includes sequences for the TFP.
  • the anti-TAA TFP e.g., anti-TAA TFP or anti-TFP and anti-PD-1 antibody
  • mRNA messenger RNA
  • the mRNA encoding the anti-TAA TFP e.g., anti-TAA TFP or anti-TFP and anti-PD-1 antibody, e.g., in the same coding sequence
  • the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection.
  • RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template.
  • DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase.
  • the source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.
  • the desired template for in vitro transcription is a TFP of the present disclosure.
  • the DNA to be used for PCR contains an open reading frame.
  • the DNA can be from a naturally occurring DNA sequence from the genome of an organism.
  • the nucleic acid can include some or all of the 5’ and/or 3’ untranslated regions (UTRs).
  • the nucleic acid can include exons and introns.
  • the DNA to be used for PCR is a human nucleic acid sequence.
  • the DNA to be used for PCR is a human nucleic acid sequence including the 5’ and 3’ UTRs.
  • the DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism.
  • An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.
  • PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection. Methods for performing PCR are well known in the art.
  • Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR.
  • “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR.
  • the primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5’ and 3’ UTRs.
  • the primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest.
  • the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5’ and 3’ UTRs.
  • Primers useful for PCR can be generated by synthetic methods that are well known in the art.
  • “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified.
  • Upstream is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand.
  • “Reverse primers” are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified. “Downstream” is used herein to refer to a location 3’ to the DNA sequence to be amplified relative to the coding strand.
  • Any DNA polymerase useful for PCR can be used in the methods disclosed herein. The reagents and polymerase are commercially available from a number of sources.
  • Chemical structures with the ability to promote stability and/or translation efficiency may also be used.
  • the RNA preferably has 5’ and 3’ UTRs. In one embodiment, the 5’ UTR is between one and 3,000 nucleotides in length.
  • the length of 5’ and 3’ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5’ and 3’ UTR lengths that can be used to achieve optimal translation efficiency following transfection of the transcribed RNA.
  • the 5’ and 3’ UTRs can be the naturally occurring, endogenous 5’ and 3’ UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
  • UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and/or translation efficiency of the RNA.
  • 3’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • the 5’ UTR can contain the Kozak sequence of the endogenous nucleic acid.
  • a consensus Kozak sequence can be redesigned by adding the 5’ UTR sequence.
  • Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation.
  • the 5’ UTR can be 5’UTR of an RNA virus whose RNA genome is stable in cells.
  • various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.
  • the RNA polymerase promoter When a sequence that functions as a promoter for an RNA polymerase is added to the 5’ end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
  • the promoter is a T7 polymerase promoter, as described elsewhere herein.
  • Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
  • the mRNA has both a cap on the 5’ end and a 3’ poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
  • a circular DNA template for instance, plasmid DNA
  • RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells.
  • the transcription of plasmid DNA linearized at the end of the 3’ UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.
  • phage T7 RNA polymerase can extend the 3’ end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).
  • the conventional method of integration of polyA/T stretches into a DNA template is molecular cloning.
  • polyA/T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable.
  • the polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100 T tail (size can be 50-5000 Ts), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination.
  • Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.
  • Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP).
  • E-PAP E. coli polyA polymerase
  • increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA.
  • the attachment of different chemical groups to the 3’ end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds.
  • ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
  • RNAs produced by the methods disclosed herein include a 5’ cap.
  • the 5’ cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).
  • the RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence.
  • IRS internal ribosome entry site
  • the IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
  • RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al.
  • the modified T cells disclosed herein are engineered using a gene editing technique such as clustered regularly interspaced short palindromic repeats (CRISPR®, see, e.g., U.S. Patent No.8,697,359), transcription activator-like effector (TALE) nucleases (TALENs, see, e.g., U.S.
  • CRISPR® clustered regularly interspaced short palindromic repeats
  • TALE transcription activator-like effector
  • Patent No.9,393,257 discloses, meganucleases (endodeoxyribonucleases having large recognition sites comprising double-stranded DNA sequences of 12 to 40 base pairs), zinc finger nuclease (ZFN, see, e.g., Urnov et al., Nat. Rev. Genetics (2010) v11, 636-646), or megaTAL nucleases (a fusion protein of a meganuclease to TAL repeats) methods.
  • ZFN zinc finger nuclease
  • megaTAL nucleases a fusion protein of a meganuclease to TAL repeats
  • one or more of the extracellular domain, the transmembrane domain, or the cytoplasmic domain of a TFP subunit are engineered to have aspects of more than one natural TCR subunit domain (i.e., are chimeric).
  • TCR subunit domain i.e., are chimeric.
  • mentioned endogenous TCR gene encodes a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain. In some embodiments, mentioned endogenous TCR gene encodes a TCR gamma chain, a TCR delta chain, or a TCR gamma chain and a TCR delta chain. In some embodiments, gene editing techniques pave the way for multiplex genomic editing, which allows simultaneous disruption of multiple genomic loci in endogenous TCR gene.
  • multiplex genomic editing techniques are applied to generate gene-disrupted T cells that are deficient in the expression of endogenous TCR, and/or B2M, and/or human leukocyte antigens (HLAs), and/or programmed cell death protein 1 (PD-1), and/or other genes. Techniques to reduce expression of these genes, such as RNAi, can also be used.
  • Current gene editing technologies comprise meganucleases, zinc-finger nucleases (ZFN), TAL effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system.
  • DSB double-stranded DNA break
  • NHEJ non-homologous end joining
  • HR homologous recombination
  • DSBs may be repaired by single strand DNA incorporation (ssDI) or single strand template repair (ssTR), an event that introduces the homologous sequence from a donor DNA.
  • Genome DNA can be performed using site-specific, rare-cutting endonucleases that are engineered to recognize DNA sequences in the locus of interest.
  • Methods for producing engineered, site-specific endonucleases are known in the art.
  • ZFNs zinc- finger nucleases
  • ZFNs are chimeric proteins comprising a zinc finger DNA-binding domain fused to the nuclease domain of the Fokl restriction enzyme.
  • the zinc finger domain can be redesigned through rational or experimental means to produce a protein that binds to a pre-determined DNA sequence -18 base pairs in length.
  • TAL-effector nucleases can be generated to cleave specific sites in genomic DNA.
  • a TALEN comprises an engineered, site-specific DNA-binding domain fused to the Fokl nuclease domain (reviewed in Mak et al. (2013), Curr Opin Struct Biol.23:93-9).
  • the DNA binding domain comprises a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA basepair.
  • Compact TALENs have an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley et al. (2013), Nat Commun.4: 1762).
  • a Compact TALEN comprises an engineered, site-specific TAL-effector DNA-binding domain fused to the nuclease domain from the I-TevI homing endonuclease. Unlike Fokl, I-TevI does not need to dimerize to produce a double-strand DNA break so a Compact TALEN is functional as a monomer.
  • Engineered endonucleases based on the CRISPR/Cas9 system are also known in the art (Ran et al. (2013), Nat Protoc.8:2281-2308; Mali et al. (2013), Nat Methods 10:957-63).
  • the CRISPR gene-editing technology is composed of an endonuclease protein whose DNA-targeting specificity and cutting activity can be programmed by a short guide RNA or a duplex crRNA/TracrRNA.
  • a CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically microbial Cas9; and (2) a short "guide RNA” or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome.
  • a caspase effector nuclease typically microbial Cas9
  • a short "guide RNA” or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome.
  • Class 1 contains type I and type III CRISPR systems that are commonly found in Archaea.
  • Class II contains type II, IV, V, and VI CRISPR systems.
  • CRISPR/Cas system is the type II CRISPR- Cas9 system
  • CRISPR/Cas systems have been repurposed by researchers for genome editing. More than 10 different CRISPR/Cas proteins have been remodeled within last few years (Adli (2016) Nat. Commun.9:1911).
  • Cas12a (Cpf1) proteins from Acid- aminococcus sp (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) are particularly interesting.
  • Homing endonucleases are a group of naturally occurring nucleases that recognize 15-40 base-pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as group 1 self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by producing a double -stranded break in the chromosome, which recruits the cellular DNA-repair machinery (Stoddard (2006), Q. Rev. Biophys.38: 49-95).
  • meganucleases are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases and engineered for a unique target site (Gersbach (2016), Molecular Therapy.24: 430–446).
  • meganuclease is engineered I-CreI homing endonuclease. In other embodiments, meganuclease is engineered I-SceI homing endonuclease.
  • chimeric proteins comprising fusions of meganucleases, ZFNs, and TALENs have been engineered to generate novel monomeric enzymes that take advantage of the binding affinity of ZFNs and TALENs and the cleavage specificity of meganucleases (Gersbach (2016), Molecular Therapy.24: 430–446).
  • a megaTAL is a single chimeric protein, which is the combination of the easy-to- tailor DNA binding domains from TALENs with the high cleavage efficiency of meganucleases.
  • nucleases In order to perform the gene editing technique, the nucleases, and in the case of the CRISPR/ Cas9 system, a gRNA, must be efficiently delivered to the cells of interest. Delivery methods such as physical, chemical, and viral methods are also know in the art (Mali (2013). Indian J. Hum. Genet.19: 3-8.). In some instances, physical delivery methods can be selected from the methods but not limited to electroporation, microinjection, or use of ballistic particles. On the other hand, chemical delivery methods require use of complex molecules such calcium phosphate, lipid, or protein. In some embodiments, viral delivery methods are applied for gene editing techniques using viruses such as but not limited to adenovirus, lentivirus, and retrovirus.
  • viruses such as but not limited to adenovirus, lentivirus, and retrovirus.
  • TGFBr2 Switch Polypeptides 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) a binding domain; wherein the TCR subunit and the binding domain are operatively linked, and wherein the TFP functionally interacts with an endogenous TCR complex when expressed in the T cell; and a second sequence encoding an anti-PD-1 antibody or fragment thereof that specifically binds programmed cell death protein 1 (PD-1), wherein the anti-PD-1 antibody or fragment thereof is secreted when expressed in a T cell, or a second sequence encoding a fusion protein comprising an anti-PD-1 antibody or fragment thereof that specifically binds PD-1, wherein the anti-PD
  • the recombinant nucleic acids as described 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 third nucleic acid sequence are operatively linked to the first nucleic acid sequence, the second nucleic acid sequence, or a combination thereof.
  • 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.
  • the TGFBr2 switch polypeptide comprises any one sequence selected from SEQ ID NOs: 283, 284, 285, and 286.
  • the 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.
  • 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 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 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 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 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 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 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 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 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.
  • the TGFBr2 extracellular domain comprises the sequence of SEQ ID NO:271.
  • 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.
  • the sequence of the TGFBr2 extracellular domain is the sequence of SEQ ID NO:271.
  • TGFBr2 extracellular domain TGFBR2 ecto: TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQE VCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPDLLLVIFQ (SEQ ID NO:271) Switch Intracellular Domain
  • 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, 4-1BB(CD137), IL-15Ra, IL12R, IL18R, IL21R, OX40, CD2, CD27, CD5, ICAM-1, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, CD226, Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • the costimulatory polypeptide is CD28. In some embodiments, the costimulatory polypeptide is 4-1BB. In some embodiments, 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. In some embodiments, 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 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, 297, 298, 299, 313, 314, 315, 316, 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:287, 277, 288, 289, 273, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 313, 314, 315, 316, and a combination thereof.
  • the 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.
  • 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:248.
  • the sequence of the switch intracellular domain comprises a sequence or portion thereof of SEQ ID NO:248.
  • 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:251.
  • the sequence of the switch intracellular domain comprises a sequence or portion thereof of SEQ ID NO:251.
  • Switch Transmembrane Domain [0879]
  • 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. [0881] In some embodiments, 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.
  • 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. [0882] In some embodiments, 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.
  • 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:252. In some embodiments, the switch transmembrane domain comprises the sequence of SEQ ID NO:252. In some embodiments, 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:252.
  • the sequence of the switch transmembrane domain is the sequence of SEQ ID NO:252.
  • 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 TGF ⁇ R2 transmembrane domain or a fragment thereof, a 4-IBB transmembrane domain or a fragment thereof, an IL-10RA transmembrane domain or a fragment thereof, an IL-7RA transmembrane domain
  • 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.
  • an IL18R transmembrane domain or a fragment thereof is an interleukin-18 receptor 1 transmembrane domain or a fragment [0885]
  • 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.
  • the additional intracellular domain is operably linked to the C-terminus of the switch intracellular domain. In some embodiments, the additional intracellular domain is operably linked to the N-terminus of the switch intracellular domain. In some embodiments, the additional intracellular domain is operably linked to the C-terminus or the N-terminus of the switch intracellular domain. In some embodiments, the additional intracellular domain is operably linked to the C-terminus or the N-terminus of the switch intracellular domain via a linker. In some embodiments, the TGFBr2 switch polypeptide may comprise two or more additional intracellular domains. In some embodiments, the additional intracellular domains are operably linked to the C-terminus and the N-terminus of the switch intracellular domain, independently.
  • 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.
  • 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, 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114
  • 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-15R ⁇ 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, 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, or more amino acids at the N- or C- terminus or at both
  • 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:248 or SEQ ID NO:251. In some embodiments, the additional intracellular domain comprises the sequence of SEQ ID NO:248 or SEQ ID NO:251.
  • the 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:248 or SEQ ID NO:251.
  • the sequence of the additional intracellular domain is the sequence of SEQ ID NO:248 or SEQ ID NO:251.
  • Switch Polypeptides [0909]
  • the TGFBr2 switch polypeptide comprises a transmembrane domain derived from a TGFBr2 transmembrane domain and an intracellular signaling domain of 4-1BB.
  • the TGFBr2 switch polypeptide 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 and 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:277.
  • the TGFBr2 switch polypeptide 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 operatively linked 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:277.
  • the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:272 and the sequence of SEQ ID NO:277.
  • the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:272 operatively linked to the sequence of SEQ ID NO:277. [0911] In some embodiments, the TGFBr2 switch polypeptide 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:285. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:285.
  • the sequence of the TGFBr2 switch polypeptide 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:285.
  • the sequence of the TGFBr2 switch polypeptide is the sequence of SEQ ID NO:285.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain derived from a 4-1BB transmembrane domain and an intracellular signaling domain of 4- 1BB.
  • the TGFBr2 switch polypeptide 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:279 and 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:277.
  • the TGFBr2 switch polypeptide 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:279 operatively linked 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:277.
  • the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:279 and the sequence of SEQ ID NO:277.
  • the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:279 operatively linked to the sequence of SEQ ID NO:277. [0914] In some embodiments, the TGFBr2 switch polypeptide 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:286. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:286.
  • the sequence of the TGFBr2 switch polypeptide 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:286.
  • the sequence of the TGFBr2 switch polypeptide is the sequence of SEQ ID NO:286.
  • the TGFBr2 switch polypeptide comprises a transmembrane domain derived from a TGFBr2 transmembrane domain and an intracellular signaling domain of CD28.
  • the TGFBr2 switch polypeptide 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 and 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:273.
  • the TGFBr2 switch polypeptide 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 operatively linked 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:273.
  • the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:272 and the sequence of SEQ ID NO:273.
  • the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:272 operatively linked to the sequence of SEQ ID NO:273. [0917] In some embodiments, the TGFBr2 switch polypeptide 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:283. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:283.
  • the sequence of the TGFBr2 switch polypeptide 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:283.
  • the sequence of the TGFBr2 switch polypeptide is the sequence of SEQ ID NO:283. [0918]
  • the TGFBr2 switch polypeptide comprises a transmembrane domain derived from a CD28 transmembrane domain and an intracellular signaling domain of CD28.
  • the TGFBr2 switch polypeptide 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:275 and 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:273.
  • the TGFBr2 switch polypeptide 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:275 operatively linked 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:273.
  • the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:275 and the sequence of SEQ ID NO:273.
  • the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:275 operatively linked to the sequence of SEQ ID NO:273. [0920] In some embodiments, the TGFBr2 switch polypeptide 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:284. In some embodiments, the TGFBr2 switch polypeptide comprises the sequence of SEQ ID NO:284.
  • the sequence of the TGFBr2 switch polypeptide 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:284.
  • the sequence of the TGFBr2 switch polypeptide is the sequence of SEQ ID NO:284.
  • PD-1 Switch Molecule [0921]
  • the third nucleic acid sequence encodes an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain.
  • the inhibitory molecule comprises the first polypeptide comprising at least a portion of PD-1 and the second polypeptide comprising a costimulatory domain and primary signaling domain.
  • a cell e.g., a T cell, expressing the recombinant nucleic acids comprising a third nucleic acid sequence that encodes a PD-1 switch molecule as descried herein can inhibit tumor growth when expressed in a T cell.
  • nucleic acid molecules comprising a first sequence encoding a TFP as described herein, a second sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding an agent that can enhance the activity of a modified cell, e.g., T cell, expressing the TFP as described herein and an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein.
  • a modified cell e.g., T cell
  • the third nucleic acid sequence is included in a separate nucleic acid sequence from the recombinant nucleic acid molecules. In some embodiments, the third nucleic acid sequence is included in the same nucleic acid molecule as the first sequence, the second sequence, or a combination thereof.
  • the agent that can enhance the activity of a modified cell e.g., T cell
  • the PD-1 polypeptide may be operably linked to the N-terminus of an intracellular domain of a costimulatory polypeptide via the C-terminus of the PD-1 polypeptide.
  • the PD-1 polypeptide is linked to the intracellular domain of the costimulatory polypeptide via the transmembrane domain of PD-1.
  • the costimulatory polypeptide is selected from the group consisting of OX40, CD2, CD27, CD5, ICAM-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, IL-15Ra, IL12R, IL18R, IL21R, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, CD226, Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • the costimulatory peptide is CD28.
  • recombinant nucleic acid molecules comprising a first sequence encoding a TFP as described herein and a second sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, wherein the recombinant nucleic acid molecules further comprising an agent that can enhance the activity of a modified cell, e.g., T cell, expressing the TFP as described herein and an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein.
  • the cells expressing TFP as described herein and an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein can further express another agent, e.g., an agent which enhances the activity of a modified cell, e.g., T cell.
  • the agent can be an agent which inhibits an inhibitory molecule.
  • Inhibitory molecules, e.g., PD-1 can, in some embodiments, decrease the ability of a modified cell, e.g., T cell, to mount an immune effector response.
  • inhibitory molecules examples include PD-1, PD- L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, and 2B4.
  • the agent which inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain as described herein.
  • the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD-1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4, and TIGIT, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4- 1BB, CD27 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein).
  • an inhibitory molecule such as PD-1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4, and TIGIT
  • a fragment of any of these e.g., at least a portion of an extracellular domain of any of these
  • a second polypeptide which is an intra
  • the agent comprises a first polypeptide of PD-1 or a fragment thereof (e.g., at least a portion of an extracellular domain of PD-1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein).
  • the recombinant nucleic acid molecules as described herein further comprises a sequence encoding PD-1 or a fragment thereof.
  • the recombinant nucleic acid molecules as described herein further comprises a sequence encoding the extracellular domain of PD-1.
  • the recombinant nucleic acid molecules as described herein comprises a sequence encoding the extracellular domain and transmembrane domain of PD-1. In some embodiments, the recombinant nucleic acid molecules as described herein may further comprise a sequence encoding CD28 or a fragment thereof. In some embodiments, the recombinant nucleic acid molecules as described herein comprises a sequence encoding the intracellular domain of CD28. In some embodiments, the recombinant nucleic acid molecules as described herein comprises a sequence encoding a fusion protein comprising the PD-1 extracellular domain and transmembrane domain linked to the CD28 intracellular domain linked to intracellular domain.
  • the agent comprises the extracellular and transmembrane domain of PD-1 fused to the intracellular signaling domain of CD28.
  • the agent comprises SEQ ID NO:239.
  • PD-1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA.
  • PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al., 1996, Int. Immunol 8:765-75). Two ligands for PD-1, PD-L1 and PD-L2, have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al., 2000 J. Exp.
  • PD-L1 is abundant in human cancers (Dong et al., 2003 J. Mol. Med.81:281-7; Blank et al., 2005 Cancer Immunol. Immunother.54:307-314; Konishi et al., 2004 Clin. Cancer Res.10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1.
  • the agent comprises the extracellular domain (ECD) of an inhibitory molecule, e.g., PD-1 can be fused to a transmembrane domain and optionally an intracellular signaling domain such as 41BB and CD3 zeta (also referred to herein as a PD-1 TFP).
  • the PD-1 TFP when used in combinations with an anti-TAA TFP as described herein and an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, improves the persistence of the T cell.
  • the TFP is a PD-1 TFP comprising the extracellular domain of PD-1.
  • TFPs containing an antibody or antibody fragment such as a scFv that specifically binds to the Programmed Death-Ligand 1 (PD-L1) or Programmed Death- Ligand 2 (PD-L2).
  • the present disclosure provides a population of TFP-expressing T cells, e.g., TFP-T cells co-expressing an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein.
  • the population of TFP-expressing T cells co-expressing an anti- PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein comprises a mixture of cells expressing different TFPs.
  • the population of TFP-T cells co- expressing an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD- 1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein can include a first cell expressing a TFP having a binding domain described herein, and a second cell expressing a TFP having a different anti-TAA binding domain, e.g., a binding domain described herein that differs from the binding domain in the TFP expressed by the first cell.
  • the population of TFP-expressing cells co-expressing an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein can include a first cell expressing a TFP that includes a first binding domain binding domain, e.g., as described herein, and a second cell expressing a TFP that includes an antigen binding domain to a target other than the binding domain of the first cell (e.g., another tumor-associated antigen).
  • the present disclosure provides a population of cells wherein at least one cell in the population expresses a TFP having a domain as described herein and an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti- PD-1 antibody or fragment thereof as descried herein, and a second cell expressing another agent, e.g., an agent which enhances the activity of a modified cell, e.g., T cell.
  • the agent can be an agent which inhibits an inhibitory molecule.
  • Inhibitory molecules can, in some embodiments, decrease the ability of a modified cell, e.g., T cell, to mount an immune effector response.
  • inhibitory molecules include PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, and 2B4.
  • the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
  • recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding a PD-1 switch molecule as described herein.
  • TCR T cell receptor
  • TFP T cell receptor
  • second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein
  • a third nucleic acid sequence encoding a PD-1 switch molecule as described herein.
  • recombinant nucleic acid molecules comprise a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • recombinant nucleic acid molecules comprise a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding a inhibitory molecule comprising the first polypeptide comprising at least a portion of PD-1 and the second polypeptide comprising a costimulatory domain and primary signaling domain.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • a cell e.g., T cell, expressing the TFP as descried herein, an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a PD-1 switch molecule as descried herein can inhibit tumor growth when expressed in a T cell.
  • the third nucleic acid sequence encodes another agent, for example, an agent that can enhance longevity or activity of TFP-expressing cells that co-express an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti- PD-1 antibody or fragment thereof as descried herein.
  • another agent for example, an agent that can enhance longevity or activity of TFP-expressing cells that co-express an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti- PD-1 antibody or fragment thereof as descried herein.
  • the TFP-expressing cells that coexpress an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein can further express another agent, for example, an agent that can enhance longevity or activity of TFP- expressing cells that co-express an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein.
  • another agent for example, an agent that can enhance longevity or activity of TFP- expressing cells that co-express an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein.
  • the agent is a cytokine such as a pleiotropic cytokine that plays important roles in maintenance and homeostatic expansion of immune cells.
  • TEE tumor microenvironment
  • the agent activates a cytokine signaling.
  • the agent activates interleukin-15 (IL-15) signaling.
  • the agent comprises interleukin-15 (IL-15) and/or interleukin-15 receptor (IL-15R).
  • the IL-15R is an IL-15R alpha (IL-15R ⁇ ) subunit.
  • the present disclosure encompasses recombinant nucleic acid molecules encoding an interleukin-15 (IL-15) polypeptide or a fragment thereof.
  • the IL-15 polypeptide 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, 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,
  • the IL-15 polypeptide or a fragment thereof 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 IL-15.
  • the IL-15 polypeptide or a fragment thereof comprises a sequence encoding IL-15 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 IL-15 polypeptide or a fragment thereof may comprise an IL- 15 signal peptide. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise amino acids 1-29 of IL-15. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise amino acids 1-29 of SEQ ID NO:245. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:246. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise amino acids 30-162 of IL-15. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise amino acids 30-162 of SEQ ID NO:245.
  • the IL-15 polypeptide or a fragment thereof may comprise any one of the sequence listed in Table 14 or a fragment thereof. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:242. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise amino acids 1-162 of SEQ ID NO:245. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:246 and a sequence of SEQ ID NO:242. In some embodiments, IL-15 polypeptide is secreted when expressed in a cell, such as a T cell.
  • the present disclosure further encompasses recombinant nucleic acid molecules encoding an interleukin-15 receptor (IL-15R) subunit polypeptide or a fragment thereof.
  • IL-15R subunit may be IL-15 receptor alpha chain (“IL-15R ⁇ ” or CD215), IL-2 receptor beta chain (“IL-2R ⁇ ” or CD122) and IL-2 receptor gamma/the common gamma chain (“IL-2R ⁇ / ⁇ c” or CD132).
  • the IL-15R subunit is an IL-15R ⁇ or a fragment thereof.
  • the IL-15R ⁇ polypeptide 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, 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114
  • the IL-15R ⁇ polypeptide or a fragment thereof 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 IL-15R ⁇ .
  • the IL- 15R ⁇ polypeptide or a fragment thereof comprises a sequence encoding IL-15R ⁇ 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, 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, or more amino acids at the N- or C-
  • the IL-15R ⁇ polypeptide or a fragment thereof may comprise IL- 15R ⁇ signal peptide. In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof may comprise amino acids 1-30 of IL-15R ⁇ . In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof may comprise amino acids 1-30 of SEQ ID NO:247. In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof does not comprise IL-15R ⁇ signal peptide. In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof does not comprise amino acids 1- 30 of IL-15R ⁇ .
  • the IL-15R ⁇ polypeptide or a fragment thereof does not comprise amino acids 1-30 of SEQ ID NO:247. [0933] In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof may comprise IL- 15R ⁇ Sushi domain. In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof may comprise amino acids 31-95 of IL-15R ⁇ . In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof may comprise amino acids 31-95 of SEQ ID NO:247. In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:250.
  • the IL-15R ⁇ polypeptide or a fragment thereof may comprise an intracellular domain of IL-15R ⁇ . In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof may comprise amino acids 229-267 of IL-15R ⁇ . In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof may comprise amino acids 229-267 of a sequence of SEQ ID NO:247. In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:248. [0935] In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof may comprise IL- 15R ⁇ Sushi domain, transmembrane domain, and intracellular domain.
  • the IL-15R ⁇ polypeptide or a fragment thereof may comprise amino acids 31-267 of IL-15R ⁇ . In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof may comprise amino acids 31-267 of SEQ ID NO:247. In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:250. In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:251. In some embodiments, the IL-15R ⁇ polypeptide or a fragment thereof may comprise amino acids 96-267 of SEQ ID NO:247.
  • the IL-15R ⁇ polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:250 and a sequence of SEQ ID NO:251.
  • the IL-15R ⁇ polypeptide or a fragment thereof may be a soluble IL-15R ⁇ (sIL-15R ⁇ ).
  • the IL-15R ⁇ polypeptide or a fragment thereof may comprise amino acids 21-205 of IL-15R ⁇ .
  • the IL-15R ⁇ polypeptide or a fragment thereof may comprise amino acids 21-205 of a sequence of SEQ ID NO:247.
  • the IL-15R ⁇ polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:249.
  • the present disclosure encompasses recombinant nucleic acid molecules encoding a fusion protein comprising an IL-15 polypeptide linked to an IL-15R subunit.
  • IL-15 and IL-15R subunit are operatively linked by a linker.
  • the IL-15R subunit is IL-15R alpha (IL-15R ⁇ ).
  • IL-15 polypeptide may be linked to N-terminus of IL-15R ⁇ subunit.
  • IL-15 polypeptide may be linked to C-terminus of IL-15R ⁇ subunit.
  • IL-15 and IL-15R ⁇ are operatively linked by a linker.
  • the linker is not a cleavable linker.
  • the linker may comprise a sequence comprising (G 4 S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 10. In some embodiments, n is an integer from 1 to 4. In some embodiments, n is 3.
  • the linker comprises a sequence of SEQ ID NO:243. [0938] SG3(SG4)3SG3SLQ Linker SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO:243) [0939]
  • the fusion protein may comprise amino acids 30-162 of IL-15.
  • the fusion protein may comprise amino acids 30-162 of a sequence of SEQ ID NO:245. In some embodiments, the fusion protein may comprise any one of the sequence listed in Table 14 or a fragment thereof. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:242. In some embodiments, the fusion protein does not comprise IL- 15 signal peptide. In some embodiments, the fusion protein does not comprise amino acids 1-29 of IL-15. In some embodiments, the fusion protein does not comprise amino acids 1-29 of a sequence of SEQ ID NO:245. In some embodiments, the fusion protein does not comprise a sequence of SEQ ID NO:246. [0940] In some embodiments, the fusion protein may comprise a Sushi domain.
  • the fusion protein may comprise amino acids 31-95 of IL-15R ⁇ . In some embodiments, the fusion protein may comprise amino acids 31-95 of a sequence of SEQ ID NO:247. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:250. [0941] In some embodiments, the fusion protein may comprise the intracellular domain of IL- 15R ⁇ . In some embodiments, the fusion protein may comprise amino acids 229-267 of IL-15R ⁇ . In some embodiments, the fusion protein may comprise amino acids 229-267 of a sequence of SEQ ID NO:247. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:248.
  • the fusion protein may comprise a soluble IL-15R ⁇ (sIL-15R ⁇ ). In some embodiments, the fusion protein may comprise amino acids 21-205 of IL-15R ⁇ . In some embodiments, the fusion protein may comprise amino acids 21-205 of a sequence of SEQ ID NO:247. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:249. [0943] In some embodiments, the fusion protein may comprise the transmembrane domain and the intracellular domain of IL-15R ⁇ . In some embodiments, the fusion protein may comprise amino acids 96-267 of IL-15R ⁇ . In some embodiments, the fusion protein may comprise amino acids 96-267 of a sequence of SEQ ID NO:247.
  • the fusion protein may comprise a sequence of SEQ ID NO:251.
  • the fusion protein may comprise the Sushi domain, the transmembrane domain, and the intracellular domain of IL-15R ⁇ .
  • the fusion protein may comprise amino acids 31-267 of IL-15R ⁇ .
  • the fusion protein may comprise amino acids 31-267 of a sequence of SEQ ID NO:247.
  • the fusion protein may comprise a sequence of SEQ ID NO:250 and a sequence of SEQ ID NO:251.
  • 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.
  • 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), ⁇ -galactosidase ( ⁇ -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
  • ⁇ -GAL ⁇ -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.
  • the fusion protein further comprises a sequence of SEQ ID NO:255.
  • the fusion protein is expressed on cell surface when expressed in a cell, e.g., a T cell. In some embodiments, the fusion protein is secreted when expressed in a cell, e.g., a T cell.
  • cells expressing TFPs, an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, an IL-15 polypeptide or a fragment thereof, an IL-15R ⁇ polypeptide or a fragment thereof, and/or a fusion protein comprising an IL-15 polypeptide and an IL-15R ⁇ polypeptide described herein can yet further express another agent that can enhance the activity of a modified T cell expressing TFPs and an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein.
  • the agent that can enhance the activity of a modified T cell can be a PD-1 polypeptide.
  • the PD-1 polypeptide may be operably linked to the N-terminus of an intracellular domain of a costimulatory polypeptide via the C- terminus of the PD-1 polypeptide.
  • the agent that can enhance the activity of a modified T cell expressing TFPs can be an anti-PD-1 antibody, or antigen binding fragment thereof.
  • the anti-PD-1 antibody or antigen binding fragment thereof may be operably linked to the N-terminus of an intracellular domain of a costimulatory polypeptide via the C-terminus of the anti-PD-1 antibody, or antigen binding fragment thereof.
  • the PD-1 polypeptide or anti-PD-1 antibody is linked to the intracellular domain of the costimulatory polypeptide via the transmembrane domain of PD- 1.
  • the costimulatory polypeptide is selected from the group consisting of OX40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, CD226, Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • the costimulatory polypeptide is CD28.
  • the agent that can enhance the activity of a modified T cell expressing TFPs and an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein can be linked to an IL- 15R ⁇ polypeptide or a fragment thereof.
  • the agent can be an agent that can inhibit an inhibitory molecule that can decrease the ability of a T cell expressing a TFP and an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti- PD-1 antibody or fragment thereof as descried herein to mount an immune effector response.
  • the agent which inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
  • the agent may comprise a first polypeptide, e.g., of an inhibitory molecule such as PD-1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4, and TIGIT, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain as described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27, or CD28, as described herein)) and/or a primary signaling domain (e.g., IL-15R ⁇ described herein).
  • the agent may be PD-1 or a fragment thereof.
  • the agent may comprise the extracellular domain of PD-1.
  • the agent may comprise the extracellular domain and transmembrane domain of PD-1.
  • the agent may further comprise CD28 or a fragment thereof.
  • the agent may comprise the intracellular domain of CD28.
  • the agent may comprise a fusion protein comprising the PD-1 extracellular domain and transmembrane domain linked to the CD28 intracellular domain linked to IL-15R ⁇ .
  • the CD28 intracellular domain is linked to the intracellular domain of IL-15R ⁇ .
  • the PD-1 or a fragment thereof may comprise any one of the sequence listed in Table 13 or a fragment thereof.
  • the PD-1 or a fragment thereof may comprise a sequence of SEQ ID NO:256. In some embodiments, the PD-1 or a fragment thereof may comprise a sequence of SEQ ID NO:257. In some embodiments, the PD-1 or a fragment thereof may comprise a sequence of SEQ ID NO:258. In some embodiments, the PD-1 or a fragment thereof may comprise a sequence of SEQ ID NO:259. In some embodiments, the transmembrane domain of PD-1 may comprise a sequence of SEQ ID NO:239. In some embodiments, the intracellular domain of CD28 may comprise a sequence of SEQ ID NO:273.
  • the intracellular domain of IL-15R ⁇ comprises amino acids 229-267 of IL- 15R ⁇ . In some embodiments, the intracellular domain of IL-15R ⁇ comprises amino acids 229- 267 of a sequence of SEQ ID NO:247. In some embodiments, the fusion protein comprises a sequence of SEQ ID NO:248.
  • the agent that can enhance the activity of a modified T cell expressing TFPs and an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein can be linked to a fusion protein comprising an IL-15 polypeptide and an IL-15R ⁇ polypeptide.
  • the agent may be PD-1 or a fragment thereof.
  • the agent may comprise the extracellular domain of PD-1.
  • the agent may comprise the extracellular domain and transmembrane domain of PD-1.
  • the agent may further comprise CD28 or a fragment thereof.
  • the agent may comprise the intracellular domain of CD28.
  • the agent may comprise a fusion protein comprising the PD-1 extracellular domain and transmembrane domain linked to the CD28 intracellular domain linked to the fusion protein comprising an IL-15 polypeptide and an IL-15R ⁇ polypeptide.
  • the CD28 intracellular domain is linked to the intracellular domain of IL-15R ⁇ .
  • the intracellular domain of IL-15R ⁇ is linked to the IL-15 polypeptide by a linker described herein.
  • the linker comprises 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:261 (P2A: GSGATNFSLLKQAGDVEENPG).
  • the fusion protein may comprise a PD-1 or a fragment thereof comprising any one of the sequence listed in Table 13 or a fragment thereof.
  • the fusion protein may comprise a PD-1 or a fragment thereof comprising a sequence of SEQ ID NO:256.
  • the fusion protein may comprise a PD-1 or a fragment thereof comprising a sequence of SEQ ID NO:257. In some embodiments, the fusion protein may comprise a PD-1 or a fragment thereof comprising a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a PD-1 or a fragment thereof comprising a sequence of SEQ ID NO:259. In some embodiments, the fusion protein may comprise a PD-1 or a fragment thereof comprising a transmembrane domain of PD-1 comprising a sequence of SEQ ID NO:239.
  • the fusion protein may comprise a CD28 or a fragment comprising the intracellular domain of CD28 comprising a sequence of SEQ ID NO:273.
  • the intracellular domain of IL-15R ⁇ comprises amino acids 229-267 of IL-15R ⁇ .
  • the intracellular domain of IL-15R ⁇ comprises amino acids 229-267 of a sequence of SEQ ID NO:247.
  • the fusion protein comprises a sequence of SEQ ID NO:248.
  • the IL-15 polypeptide comprises IL-15 signal peptide.
  • the IL-15 polypeptide comprises amino acids 1-29 of IL-15.
  • the IL-15 polypeptide comprises amino acids 1-29 of a sequence of SEQ ID NO:245. In some embodiments, the IL-15 polypeptide comprises a sequence of SEQ ID NO:246. In some embodiments, the IL-15 polypeptide comprises amino acids 30-162 of IL-15. In some embodiments, the IL-15 polypeptide comprises amino acids 30-162 of a sequence of SEQ ID NO:245. In some embodiments, the IL-15 polypeptide comprises a sequence of SEQ ID NO:242. [0953] Disclosed herein, in some embodiments, are polypeptides encoded by any of recombinant nucleic acid molecules described herein.
  • recombinant nucleic acid molecules comprising a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding an Interleukin-15 (IL-15) polypeptide or a fragment thereof as described herein.
  • TCR T cell receptor
  • TFP T cell receptor
  • a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein
  • a third nucleic acid sequence encoding an Interleukin
  • nucleic acid molecules comprising a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding an Interleukin-15 receptor alpha (IL-15R ⁇ ) polypeptide or a fragment thereof as described herein.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • nucleic acid molecules comprising a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding a fusion protein comprising an IL-15 polypeptide or a fragment thereof linked to an IL-15R ⁇ polypeptide or a fragment thereof as described herein.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • nucleic acid molecules comprising a first nucleic acid sequence encoding a T cell receptor (TCR) fusion protein (TFP) as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding a fusion protein comprising a fusion protein comprising an IL-15R ⁇ polypeptide or a fragment thereof linked to PD-1 or a fragment thereof and/or CD28 or a fragment thereof as described herein.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof as described herein, wherein the third nucleic acid sequence are included in a separate nucleic acid molecule from the first nucleic acid sequence and/or the second nucleic acid sequence.
  • nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding an anti- PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof as described herein, wherein the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are included in a single nucleic acid molecule.
  • first nucleic acid sequence and the second nucleic acid sequence, the first nucleic acid sequence and the third nucleic acid sequence, and/or the second nucleic acid sequence and the third nucleic acid sequence are operatively linked by a linker.
  • nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding an IL-15R ⁇ polypeptide or a fragment thereof as described herein, wherein the third nucleic acid sequence is included in a separate nucleic acid molecule from the first nucleic acid sequence and the second nucleic acid sequence.
  • nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding an IL-15R ⁇ polypeptide or a fragment thereof as described herein, wherein the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are included in a single nucleic acid molecule.
  • the first nucleic acid sequence and the second nucleic acid sequence, the first nucleic acid sequence and the third nucleic acid sequence, and/or the second nucleic acid sequence and the third nucleic acid sequence are operatively linked by a linker.
  • the linker may be a cleavable linker.
  • the linker may comprise a protease cleavage site.
  • the cleavage site can be a self-cleaving peptide, for example, a 2A cleavage site such as a T2A, P2A, E2A or F2A cleavage site.
  • the protease cleavage site is a T2A cleavage site.
  • the cleavage site can comprise a sequence of SEQ ID NO:362, when expressed.
  • the linker comprises a sequence of SEQ ID NO:362, when expressed.
  • nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof and an IL-15R subunit or a fragment thereof as described herein, wherein the first nucleic acid sequence, the second nucleic acid sequence, and third nucleic acid sequence are included in a single nucleic acid molecule.
  • the first nucleic acid sequence and the second nucleic acid sequence, the first nucleic acid sequence and the third nucleic acid sequence, and/or the second nucleic acid sequence and the third nucleic acid sequence are operatively linked by a linker as described herein.
  • An IL-15R subunit may be an IL-15R alpha (IL-15R ⁇ ), an IL-2R beta (IL-2 ⁇ ), or an IL-2R gamma/the common gamma chain (IL-2R ⁇ / ⁇ c).
  • the IL-15R subunit is IL-15R alpha (IL-15R ⁇ ).
  • IL-15 and IL-15R subunit are operatively linked by a second linker.
  • IL-15 and IL-15R ⁇ are operatively linked by a second linker.
  • the second linker is not a cleavable linker.
  • the second linker may comprise a sequence comprising (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 10. In some embodiments, n is an integer from 1 to 4. In some embodiments, n is 3. In some embodiments, the second linker comprises a sequence of SEQ ID NO:243.
  • nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding a fusion protein comprising an IL-15 polypeptide linked to an IL-15R ⁇ subunit as described herein, wherein the third nucleic acid sequence is included in a separate nucleic acid molecule from the first nucleic acid sequence and the second nucleic acid sequence.
  • nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding a fusion protein comprising an IL-15 polypeptide linked to an IL-15R ⁇ subunit as described herein, wherein the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are included in a single nucleic acid molecule.
  • first nucleic acid sequence and the second nucleic acid sequence, the first nucleic acid sequence and the third nucleic acid sequence, and/or the first nucleic acid sequence and the third nucleic acid sequence are operatively linked by a first linker as described herein.
  • IL-15 polypeptide may be linked to N-terminus of IL-15R ⁇ subunit.
  • IL-15 polypeptide may be linked to C-terminus of IL-15R ⁇ subunit.
  • nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, a third nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof as described herein, and a fourth nucleic acid sequence encoding an agent that can enhance the activity of a modified T cell expressing the TFP and an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein.
  • the fourth nucleic acid sequence is included in a separate nucleic acid sequence. In some embodiments, the fourth nucleic acid sequence is included in the same nucleic acid molecule as the first nucleic acid sequence, the second nucleic acid sequence, or the third nucleic acid sequence, or the first, the second, and the third nucleic acid sequences.
  • the agent that can enhance the activity of a modified T cell can be a PD-1 polypeptide. In these embodiments, the PD-1 polypeptide may be operably linked to the N- terminus of an intracellular domain of a costimulatory polypeptide via the C-terminus of the PD- 1 polypeptide.
  • the agent that can enhance the activity of a modified T cell can be an anti-PD-1 antibody, or antigen binding fragment thereof.
  • the anti-PD-1 antibody or antigen binding fragment thereof may be operably linked to the N-terminus of an intracellular domain of a costimulatory polypeptide via the C-terminus of the anti-PD-1 antibody, or antigen binding fragment thereof.
  • the PD-1 polypeptide or anti-PD-1 antibody is linked to the intracellular domain of the costimulatory polypeptide via the transmembrane domain of PD-1.
  • the costimulatory polypeptide is selected from the group consisting of OX40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, CD226, Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a third nucleic acid sequence encoding an IL-15R ⁇ polypeptide or a fragment thereof as described herein, wherein the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are operatively linked by a first linker as described herein, and wherein the third nucleic acid sequence further encodes an agent that can enhance the activity of a modified T cell expressing the TFP and an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transme
  • the agent can be an agent that can inhibit an inhibitory molecule that can decrease the ability of a T cell expressing a TFP and an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein to mount an immune effector response.
  • the agent which inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
  • the agent may comprise a first polypeptide, e.g., of an inhibitory molecule such as PD-1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4, and TIGIT, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27, or CD28, as described herein)) and/or a primary signaling domain (e.g., IL-15R ⁇ described herein).
  • an inhibitory molecule such as PD-1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4, and TIGIT
  • a fragment of any of these e.g., at least a portion of an extracellular domain of any of these
  • a second polypeptide which is an intracellular signaling domain described herein (e
  • the third nucleic acid sequence further comprises a sequence encoding PD-1 or a fragment thereof. In some embodiments, the third nucleic acid sequence comprises a sequence encoding the extracellular domain of PD-1. In some embodiments, the third nucleic acid sequence comprises a sequence encoding the extracellular domain and transmembrane domain of PD-1. In some embodiments, the third nucleic acid sequence may further comprise a sequence encoding CD28 or a fragment thereof. In some embodiments, the third nucleic acid sequence comprises a sequence encoding the intracellular domain of CD28.
  • the third nucleic acid sequence comprises a sequence encoding a fusion protein comprising the PD-1 extracellular domain and transmembrane domain linked to the CD28 intracellular domain linked to IL-15R ⁇ .
  • the CD28 intracellular domain is linked to the intracellular domain of IL-15R ⁇ .
  • the intracellular domain of IL-15R ⁇ comprises amino acids 229-267 of IL-15R ⁇ .
  • the intracellular domain of IL-15R ⁇ comprises amino acids 229-267 of SEQ ID NO:247.
  • the intracellular domain of IL-15R ⁇ comprises a sequence of SEQ ID NO:248.
  • the third nucleic acid sequence encoding PD-1, or a fragment thereof may comprise a nucleic acid sequence encoding any one of the sequence listed in Table 13 or a fragment thereof. In some embodiments, the third nucleic acid sequence encoding PD-1, or a fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:256. In some embodiments, the third nucleic acid sequence encoding PD-1, or a fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:257.
  • the third nucleic acid sequence encoding PD-1, or a fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:258. In some embodiments, the third nucleic acid sequence encoding PD-1, or a fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:259. In some embodiments, the nucleic acid sequence encoding the transmembrane domain of PD-1 may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:239. In some embodiments, the nucleic acid sequence encoding the intracellular domain of CD28 may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:273.
  • the intracellular domain of IL-15R ⁇ comprises amino acids 229-267 of IL-15R ⁇ .
  • the nucleic acid encoding the intracellular domain of IL-15R ⁇ comprises a nucleic acid encoding amino acids 229-267 of SEQ ID NO:247.
  • the nucleic acid encoding the intracellular domain of IL-15R ⁇ comprises a nucleic acid encoding a sequence of SEQ ID NO:248.
  • nucleic acid molecules comprising a first nucleic acid sequence encoding a TFP as described herein, a second nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, a third nucleic acid sequence encoding an IL-15R ⁇ polypeptide or a fragment thereof as described herein and an agent that can enhance the activity of a modified T cell expressing the TFP and an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a fourth nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof as described here
  • the third nucleic sequence is included in a separate nucleic acid sequence from the first nucleic acid sequence and the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence, the first nucleic acid sequence and the third nucleic acid sequence, the second nucleic acid sequence and the second nucleic acid sequence, or the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are included in a single nucleic acid sequence.
  • first nucleic acid sequence and the second nucleic acid sequence, the first nucleic acid sequence and the third nucleic acid sequence, and/or the second nucleic acid sequence and the third nucleic acid sequence are operatively linked by a first linker as described herein.
  • the fourth nucleic acid sequence is included in a separate nucleic acid sequence.
  • the fourth nucleic acid sequence is included in the same nucleic acid molecule as the first nucleic acid sequence, the second nucleic acid sequence, or the third nucleic acid sequence, or the first and the second nucleic acid sequences, the first and the third nucleic acid sequences, the second and the third nucleic acid sequences, or the first, the second, and the third nucleic acid sequences.
  • the fourth nucleic acid sequence encoding the IL- 15 polypeptide may comprise a sequence encoding any one of the sequence listed in Table 14 or a fragment thereof.
  • the fourth nucleic acid sequence encoding the IL-15 polypeptide may comprise a sequence encoding a sequence of SEQ ID NO:242.
  • the IL-15 polypeptide is secreted when expressed in a T cell.
  • recombinant nucleic acid molecules comprising a nucleic acid sequence encoding a TFP as described herein, a nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, a nucleic acid sequence encoding a PD-1 polypeptide or a fragment thereof as described herein, a nucleic acid sequence encoding CD28 polypeptide or a fragment thereof as described herein, a nucleic acid sequence encoding an IL-15R ⁇ or a fragment thereof as described herein, and/or a nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof as described herein.
  • the nucleic acid sequence encoding a TFP may comprise a sequence encoding CSF2RA signal peptide. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:359. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a sequence encoding CD3 ⁇ . In some embodiments, the nucleic acid sequence encoding a TFP may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:360.
  • the anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein comprises a sequence encoding PD-1 signal peptide.
  • a nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:256 or SEQ ID NO:335.
  • the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a sequence encoding PD-1 signal peptide. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding any one of the sequence listed in Table 13 or a fragment thereof. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding a sequence of SEQ ID NO:256. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a sequence encoding PD-1 N-Loop.
  • the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding a sequence of SEQ ID NO:257. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a sequence encoding PD-1 IgV. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding a sequence of SEQ ID NO:258. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a sequence encoding PD-1 Stalk.
  • the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding a sequence of SEQ ID NO:259. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a sequence encoding PD-1 transmembrane domain. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding a sequence of SEQ ID NO:239. In some embodiments, the nucleic acid sequence encoding the CD28 polypeptide or a fragment thereof comprises a sequence encoding CD28 intracellular domain.
  • the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding a sequence of SEQ ID NO:273.
  • the nucleic acid sequence encoding IL-15R ⁇ polypeptide or fragment thereof comprise a sequence encoding amino acids 229-267 of IL-15R ⁇ .
  • the nucleic acid sequence encoding IL-15R ⁇ polypeptide or fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:248.
  • the nucleic acid sequence encoding IL-15 polypeptide or fragment thereof comprise a sequence encoding amino acids 1-29 of IL-15.
  • the nucleic acid sequence encoding IL-15 polypeptide or fragment thereof comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:246. In some embodiments, the nucleic acid sequence encoding IL-15 polypeptide or fragment thereof may comprise a sequence encoding amino acids 30-162 of IL-15. In some embodiments, the nucleic acid sequence encoding IL-15 polypeptide or fragment thereof may comprise a nucleic acid sequence encoding any one of the sequence listed in Table 14 or a fragment thereof. In some embodiments, the nucleic acid sequence encoding IL- 15 polypeptide or fragment thereof may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:242.
  • the nucleic acid sequence encoding the TFP and the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof are operatively linked by a T2A linker.
  • the T2A linker may comprise a sequence of SEQ ID NO:362.
  • the nucleic acid sequence encoding the IL-15R ⁇ or a fragment thereof and the nucleic acid sequence encoding the IL-15 polypeptide or a fragment thereof are operatively linked by a P2A linker.
  • the P2A linker may comprise a sequence of SEQ ID NO:261.
  • nucleic acid molecules comprising a nucleic acid sequence encoding a TFP as described herein, a nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a nucleic acid sequence encoding a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof as described herein, a PD-1 polypeptide or a fragment thereof as described herein, a CD28 polypeptide or a fragment thereof as described herein, an IL-15 polypeptide, or a fragment thereof described herein, an IL-15R ⁇ or a fragment thereof as described herein, or a combination thereof.
  • the nucleic acid sequence encoding a TFP may comprise a sequence encoding CSF2RA signal peptide. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:359. In some embodiments, the nucleic acid sequence encoding a TFP may comprise a sequence encoding CD3 ⁇ . In some embodiments, the nucleic acid sequence encoding a TFP may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:360.
  • the anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein comprises a sequence encoding PD-1 signal peptide.
  • a nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein may comprise a nucleic acid sequence encoding a sequence of SEQ ID NO:256 or SEQ ID NO:335.
  • the nucleic acid sequence encoding the switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof comprises a nucleic acid sequence encoding any one of the sequence listed in Table 12 or a fragment thereof. In some embodiments, the nucleic acid sequence encoding the switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof comprises a nucleic acid sequence encoding a sequence of SEQ ID NO:283, 284, 285 or 286. In some embodiments, the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a sequence encoding PD-1 signal peptide.
  • the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding any one of the sequence listed in Table 13 or a fragment thereof.
  • the nucleic acid sequence encoding the PD-1 polypeptide or a fragment thereof comprises a nucleic acid sequence encoding a sequence selected from the group consisting of SEQ ID NOs: 256, 257, 258.259, 273, and 239.
  • the nucleic acid sequence encoding the CD28 polypeptide or a fragment thereof comprises a sequence encoding CD28 intracellular domain.
  • the nucleic acid sequence encoding IL-15 polypeptide or fragment thereof may comprise a nucleic acid sequence encoding any one of the sequence listed in Table 14 or a fragment thereof.
  • the nucleic acid sequence encoding IL-15 polypeptide or fragment thereof comprise a nucleic acid sequence encoding a sequence selected from the group consisting of SEQ ID NOs: 242 and 246.
  • the nucleic acid sequence encoding IL-15R ⁇ polypeptide or fragment thereof may comprise a nucleic acid sequence encoding a sequence selected from the group consisting of SEQ ID NO:248, 249, 250, and 251.
  • the nucleic acid sequence encoding the IL-15 polypeptide or a fragment thereof and the nucleic acid sequence encoding the IL-15R ⁇ or a fragment thereof are operatively linked by a non- cleavable linker.
  • the non-cleavable linker may comprise a sequence of SEQ ID NO:243.
  • the nucleic acid sequences are operatively linked by a T2A linker.
  • the T2A linker may comprise a sequence of SEQ ID NO:362.
  • 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. In some embodiments, the recombinant nucleic acid molecule is an mRNA. In some embodiments, the recombinant nucleic acid molecule is a circRNA. In some embodiments, the recombinant nucleic acid molecule comprises a nucleic acid analog. In some embodiments, 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’-O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methyl, 2’-
  • 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.
  • the recombinant nucleic acid molecule 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 16. In some embodiments, the recombinant nucleic acid molecule as described herein comprises a sequence encoding any one of the amino acid sequences listed in Table 16.
  • the recombinant nucleic acid molecule 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 selected from SEQ ID NOs:358, 364, 366, 368, and 370.
  • the recombinant nucleic acid molecule as described herein comprises a sequence encoding any one of the amino acid sequences selected from SEQ ID NOs: 358, 364, 366, 368, and 370.
  • the recombinant nucleic acid molecule as described herein comprises a sequence having at least about 40%, 45%, 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 16.
  • the recombinant nucleic acid molecule as described herein comprises any one of the nucleic acid sequences listed in Table 16.
  • the recombinant nucleic acid molecule as described herein comprises a sequence having at least about 40%, 45%, 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 selected from SEQ ID NOs: 357, 363, 365, 367, and 369.
  • the recombinant nucleic acid molecule as described herein comprises any one of the nucleic acid sequences selected from SEQ ID NOs: 357, 363, 365, 367, and 369.
  • the sequence of the recombinant nucleic acid molecule as described herein is a sequence having at least about 40%, 45%, 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 selected from SEQ ID NOs: 357, 363, 365, 367, and 369.
  • the sequence of the recombinant nucleic acid molecule as described herein is the any one of the nucleic acid sequences selected from SEQ ID NOs: 357, 363, 365, 367, and 369.
  • the instant disclosure provides vectors comprising the recombinant nucleic acid(s) encoding the TFP and/or additional molecules of interest (e.g., a protein or proteins to be secreted by the TFP-T cell).
  • the vector can comprise a recombinant nucleic acid encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein.
  • the vector can comprise a recombinant nucleic acid comprising a first sequence encoding a TFP and a second sequence encoding an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein.
  • the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, an adeno-associated viral vector (AAV), a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
  • the vector is an AAV6 vector. In some instances, the vector further comprises a promoter. In some instances, the vector is an in vitro transcribed vector. [0967] The present disclosure further provides a vector comprising a nucleic acid molecule encoding a TFP as described herein, an anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein, and a sequence encoding a switch polypeptide comprising a TGFBr2 extracellular domain or a functional fragment thereof as described herein, a PD-1 polypeptide or a fragment thereof as described herein, a CD28 polypeptide or a fragment thereof as described herein, an IL-15 polypeptide, or a fragment thereof described herein, an IL-15R ⁇ or a fragment thereof as described herein, or a combination thereof.
  • a vector encoding the nucleic acid molecules as described herein can be directly transduced into a cell, e.g., a T cell.
  • the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs.
  • the vector is capable of expressing the TFP construct, the anti-PD-1 antibody or fragment thereof or a fusion protein comprising an anti-PD-1 antibody or fragment thereof and a transmembrane domain or an intracellular domain operatively linked to the anti-PD-1 antibody or fragment thereof as descried herein construct, an IL-15 construct, and/or an IL-15R ⁇ construct in mammalian T cells.
  • the mammalian T cell is a human T cell.
  • the nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.
  • the present disclosure also provides vectors in which a DNA of the present disclosure is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non- proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • the vector comprising the nucleic acid encoding the desired TFP of the present disclosure is an adenoviral vector (A5/35).
  • the expression of nucleic acids encoding TFPs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See, e.g., June et al., 2009 Nature Reviews Immunology 9.10: 704-716, which is incorporated herein by reference.
  • the TFP of the present disclosure may be used in multicistronic vectors or vectors expressing several proteins in the same transcriptional unit.
  • Such vectors may use internal ribosomal entry sites (IRES). Since IRES are not functional in all hosts and do not allow for the stoichiometric expression of multiple protein, self-cleaving peptides may be used instead. For example, several viral peptides are cleaved during translation and allow for the expression of multiple proteins form a single transcriptional unit.
  • Such peptides include 2A-peptides, or 2A- like sequences, from members of the Picornaviridae virus family. See for example Szymczak et al., 2004, Nature Biotechnology; 22:589-594.
  • the recombinant nucleic acid described herein encodes the TFP in frame with the agent, with the two sequences separated by a self-cleaving peptide, such as a 2A sequence, or a T2A sequence.
  • the recombinant nucleic acid described herein can encode a TFP in frame with a secreted PD-1 antibody binding fragment, such as an scFv or sdAb, with the two sequences separated by a self- cleaving peptide, such as a 2A sequence, or a T2A sequence.
  • the expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols.
  • the present disclosure provides a gene therapy vector.
  • the nucleic acid can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193).
  • selectable markers e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • tk thymidine kinase
  • a promoter that is capable of expressing a TFP transgene in a mammalian T cell is the EF1a promoter.
  • the native EF1a promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome.
  • the EF1a promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving TFP expression from transgenes cloned into a lentiviral vector (see, e.g., Milone et al., Mol.
  • CMV immediate early cytomegalovirus
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor-1a promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the present disclosure should not be limited to the use of constitutive promoters.
  • inducible promoters are also contemplated as part of the present disclosure.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline-regulated promoter.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic- resistance genes, such as neo and the like.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • Methods of introducing and expressing genes into a cell are known in the art.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection [0982] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like (see, e.g., U.S. Pat. Nos.5,350,674 and 5,585,362.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.
  • an exemplary delivery vehicle is a liposome.
  • lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St.
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 °C. Chloroform is used as the only solvent since it is more readily evaporated than methanol.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10).
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine- nucleic acid complexes are also contemplated.
  • Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and western blots) or by assays described herein to identify agents falling within the scope of the present disclosure.
  • the present disclosure further provides a vector comprising a TFP encoding nucleic acid molecule.
  • a TFP vector can be directly transduced into a cell, e.g., a T cell.
  • the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs.
  • the vector is capable of expressing the TFP construct in mammalian T cells.
  • the mammalian T cell is a human T cell.
  • Circular RNA [0988]
  • TFP-T cells are transduced with an RNA molecule.
  • the RNA is circular RNA.
  • the circular RNA is exogenous.
  • circular RNA is endogenous.
  • circular RNAs with an internal ribosomal entry site can be translated in vitro or in vivo or ex vivo.
  • Circular RNAs are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Circular RNAs are 3-5’ covalently closed RNA rings, and circular RNAs do not display Cap or poly(A) tails. Since circular RNAs lack the free ends necessary for exonuclease-mediated degradation, rendering them resistant to several mechanisms of RNA turnover and granting them extended lifespans as compared to their linear mRNA counterparts.
  • Circular RNAs are produced by the process of splicing, and circularization occurs using conventional splice sites mostly at annotated exon boundaries (Starke et al., 2015; Szabo et al., 2015).
  • splice sites are used in reverse: downstream splice donors are “backspliced” to upstream splice acceptors (see Jeck and Sharpless, 2014; Barrett and Salzman, 2016; Szabo and Salzman, 2016; Holdt et al., 2018 for review).
  • RNA circularization To generate circular RNAs that we could subsequently transfer into cells, in vitro production of circular RNAs with autocatalytic-splicing introns can be programmed.
  • IVTT in vitro transcription
  • Three general strategies have been reported so far for RNA circularization: chemical methods using cyanogen bromide or a similar condensing agent, enzymatic methods using RNA or DNA ligases, and ribozymatic methods using self-splicing introns.
  • precursor RNA was synthesized by run-off transcription and then heated in the presence of magnesium ions and GTP to promote circularization. RNA so produced can efficiently transfect different kinds of cells.
  • the template includes sequences for the TFP, CAR, and TCR, or combination thereof.
  • the group I intron of phage T4 thymidylate synthase (td) gene is well characterized to circularize while the exons linearly splice together (Chandry and Bel- fort, 1987; Ford and Ares, 1994; Perriman and Ares, 1998).
  • td intron order is permuted flanking any exon sequence, the exon is circularized via two autocatalytic transesterification reactions (Ford and Ares, 1994; Puttaraju and Been, 1995).
  • the group I intron of phage T4 thymidylate synthase (td) gene is used to generate exogenous circular RNA.
  • a ribozymatic method utilizing a permuted group I catalytic intron has been used since it is more applicable to long RNA circularization and requires only the addition of GTP and Mg 2+ as cofactors.
  • This permuted intron-exon (PIE) splicing strategy consists of fused partial exons flanked by half-intron sequences.
  • a full-length encephalomyocarditis virus such as EMCV
  • IRES full-length encephalomyocarditis virus
  • TFP thymidylate synthase
  • T4 thymidylate synthase
  • the mentioned sequence further comprises complementary ‘homology arms’ placed at the 5′ and 3′ ends of the precursor RNA with the aim of bringing the 5′ and 3′ splice sites into proximity of one another.
  • the splicing reaction can be treated with RNase R.
  • the anti-TAA TFP and/or the anti-PD-1 antibody is encoded by a circular RNA.
  • the circular RNA encoding the anti-TAA TFP and/or the anti-PD-1 antibody is introduced into a T cell for production of a TFP-T cell.
  • the in vitro transcribed RNA TFP and/or anti-PD-1 antibody can be introduced to a cell as a form of transient transfection.
  • linear precursor RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template as is described herein.
  • PCR polymerase chain reaction
  • Modified T cells Disclosed herein are modified T cells comprising the sequence encoding the TFP disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein.
  • modified allogenic T cells comprising the sequence encoding the TFP disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein.
  • the TFP T cells disclosed herein further comprises an anti-PD-1 antibody or a nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof unless otherwise specified.
  • the nucleic acid sequence encoding an anti-PD-1 antibody and the nucleic acid sequence encoding the TFP can be on a same nucleic acid molecule.
  • the modified T cell may secrete the anti-PD-1 antibody or fragment thereof.
  • the anti-PD-1 antibody or fragment thereof can be an scFv or sdAb.
  • the modified T cells comprising the recombinant nucleic acid disclosed herein, or the vectors disclosed herein comprises a functional disruption of an endogenous TCR.
  • modified allogenic T cells comprising the sequence encoding the TFP disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein.
  • a T cell comprising (i) a first recombinant nucleic acid comprising a sequence encoding a T-cell receptor (TCR) fusion protein (TFP); and (ii) a sequence encoding an antibody or fragment thereof that specifically binds programmed cell death protein 1 (PD-1).
  • TCR T-cell receptor
  • PD-1 programmed cell death protein 1
  • the TFP can comprise: (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, and (b) a binding domain.
  • the anti-PD-1 antibody or fragment thereof can be secreted by the T cell.
  • the TCR subunit and the binding domain can be operatively linked.
  • the TFP can functionally interact with an endogenous TCR complex in the T cell.
  • a T cell comprising (i) a first recombinant nucleic acid comprising a sequence encoding a T-cell receptor (TCR) fusion protein (TFP); and (ii) a sequence encoding a fusion protein comprising an antibody or fragment thereof that specifically binds programmed cell death protein 1 (PD-1) and a transmembrane domain.
  • the TFP can comprise (a) a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, and (b) a binding domain.
  • the TCR subunit and the binding domain can be operatively linked.
  • the TFP can functionally interact with an endogenous TCR complex in the T cell.
  • the first recombinant nucleic acid can comprise the sequence encoding the anti-PD-1 antibody or fragment thereof.
  • the sequence encoding the TFP and the sequence encoding the anti-PD-1 antibody or fragment thereof can be contained in a single operon.
  • a second recombinant nucleic acid can comprise the sequence encoding the anti-PD-1 antibody or fragment thereof.
  • the anti-PD-1 antibody or fragment thereof can comprise a variable domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:3.
  • CDR1 complementarity determining region 1
  • CDR2 complementarity determining region 1
  • CDR3 comprises the amino acid sequence of SEQ ID NO:3.
  • the CDR1 can comprise the amino acid sequence of SEQ ID NO:1.
  • the CDR2 can comprise the amino acid sequence of SEQ ID NO:2.
  • the anti-PD-1 antibody or fragment thereof can comprise a variable domain described herein (see, for example, “PD-1” section).
  • the T cell can promote anti-tumor activity or inhibits inhibition of anti-tumor activity.
  • the T cell can promote T cell mediated tumor cell killing or inhibit inhibition of T cell mediated tumor cell killing.
  • the secreted anti-PD-1 antibody can bind to PD-1 on the surface of the T cell and/or to PD-1 on the surface of a bystander T cell.
  • the T cell can inhibit tumor growth.
  • the TFP can include an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, 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 TFP can include a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, 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 intracellular domain can comprise an intracellular domain of TCR alpha, TCR beta, TCR delta, or TCR gamma, or an amino acid sequence having at least one modification thereto.
  • the TCR intracellular domain can comprise a stimulatory domain from an intracellular signaling domain of CD3 gamma, CD3 delta, or CD3 epsilon, or an amino acid sequence having at least one modification thereto.
  • the TCR intracellular domain may not comprise CD3 zeta.
  • the TFP may not comprise a costimulatory domain.
  • the binding domain can be connected to the TCR extracellular domain by a linker sequence.
  • the linker can be 120 amino acids in length or less.
  • the linker sequence can comprise (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 10, e.g., 1 to 4.
  • the at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from the same TCR subunit.
  • the at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR alpha.
  • the at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR beta.
  • the at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR gamma.
  • the at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from TCR delta.
  • the at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 epsilon.
  • the at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 delta
  • the at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain can be from CD3 gamma.
  • 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 subunit can comprise the amino acid sequence of SEQ ID NO:57.
  • the TCR subunit can comprise the amino acid sequence of SEQ ID NO:58.
  • the TCR subunit can comprise the amino acid sequence of SEQ ID NO:59.
  • the T cell can be a CD8+ or CD4+ T cell.
  • the T cell can be a human T cell.
  • the T cell can be an autologous T cell.
  • the T cell can be an allogeneic T cell.
  • the T cell can exhibit increased cytotoxicity to a human cell expressing an antigen that specifically interacts with the binding domain compared to a T cell not containing the TFP.
  • the binding domain can comprise an antibody or antibody fragment, a ligand, or a ligand binding protein.
  • the ligand or the antibody fragment can comprise a scFv or a single domain antibody (sdAb) domain.
  • the sdAb can be a VHH.
  • the T cell expressing the recombinant nucleic acid exhibits increased cytotoxicity to a human cell expressing an antigen that specifically interacts with the binding domain compared to a T cell not containing the TFP.
  • the antibody fragment comprises an antigen binding domain 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, anti-Nectin-4 binding domain, anti-GPC3 binding domain, and an anti-TROP-2 binding domain.
  • BCMA anti-B-cell maturation antigen
  • MSLN anti- mesothelin
  • the antigen binding domain can comprise: (i) a light chain (LC) CDR1, LC CDR2, and LC CDR3 amino acid sequence of SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30, respectively; (ii) a heavy chain (HC) CDR1, HC CDR2, and HC CDR3 amino acid sequence of SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:36, respectively; or (iii) a combination thereof.
  • the antigen binding domain can comprise a CDR1, a CDR2, and a CDR3.
  • the CDR1 can comprise an amino acid sequence of SEQ ID NO:60, SEQ ID NO:63, or SEQ ID NO:66.
  • the CDR2 can comprise an amino acid sequence of SEQ ID NO:61, SEQ ID NO:64, or SEQ ID NO:67.
  • the CDR3 can comprise an amino acid sequence of SEQ ID NO:62, SEQ ID NO:65, or SEQ ID NO:68.
  • the first recombinant nucleic acid can comprise a DNA or an RNA.
  • the second recombinant nucleic acid can comprise a DNA or an RNA.
  • the recombinant nucleic acid can comprise a nucleotide analog.
  • the nucleotide analog can be 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’-O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a
  • the transmembrane domain of the fusion protein can comprise a transmembrane domain of a protein selected from the group consisting of CD28, CD3 ⁇ , CD3 ⁇ , CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD41, CD64, CD68, CD80, CD86, CD134, CD137, CD154, ICOS, 4-1BB, OX40, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the fusion protein can further comprise a co-stimulatory domain.
  • the co-stimulatory domain of the fusion protein can comprise a co-stimulatory domain of a protein selected from the group consisting of a CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, PD-1, CD258, ICAM-1, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • a protein selected from the group consisting of a CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, PD-1, CD258, ICAM-1, functional fragments thereof, and amino
  • the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain.
  • the endogenous TCR that is functionally disrupted is an endogenous TCR alpha chain, an endogenous TCR beta chain, or an endogenous TCR alpha chain and an endogenous TCR beta chain.
  • the T cell further comprises a heterologous 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.
  • the endogenous TCR that is functionally disrupted is an endogenous TCR gamma chain, an endogenous TCR delta chain, or an endogenous TCR gamma chain and an endogenous TCR delta chain.
  • the endogenous TCR that is functionally disrupted has reduced binding to MHC-peptide complex compared to that of an unmodified control T cell.
  • the functional disruption is a disruption of a gene encoding the endogenous TCR.
  • the disruption of a gene encoding the endogenous TCR is a removal of a sequence of the gene encoding the endogenous TCR from the genome of 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 an allogenic T cell.
  • the T cell is a TCR alpha-beta T cell.
  • the T cell is a TCR gamma-delta T cell.
  • the modified T cells are ⁇ T cells and do not comprise a functional disruption of an endogenous TCR.
  • the ⁇ T cells are V ⁇ 1+ V ⁇ 2- ⁇ ⁇ T cells.
  • the ⁇ T cells are V ⁇ 1- V ⁇ 2+ ⁇ ⁇ T cells.
  • the ⁇ T cells are V ⁇ 1- V ⁇ 2- ⁇ ⁇ T cells.
  • the modified T cells further comprise a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain.
  • the inhibitory molecule comprises the first polypeptide comprising at least a portion of PD-1 and the second polypeptide comprising a costimulatory domain and primary signaling domain.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • PBMCs peripheral blood mononuclear cells
  • T cells can be obtained from a leukopak.
  • any number of T cell lines available in the art may be used.
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll TM separation.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation.
  • a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe® 2991 cell processor, the Baxter OncologyCytoMate, or the Haemonetics® Cell Saver® 5) according to the manufacturer’s instructions.
  • a semi-automated “flow-through” centrifuge for example, the Cobe® 2991 cell processor, the Baxter OncologyCytoMate, or the Haemonetics® Cell Saver® 5
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture media.
  • the T cells are ⁇ T cells. In some embodiments, the T cells are ⁇ T cells. ⁇ T cells are obtained from a bank of umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cells, for example. [1021] In one aspect, T cells are isolated from peripheral blood lymphocytes by positive selection. In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL ® gradient or by counterflow centrifugal elutriation.
  • T cells such as CD3+, CD28+, CD4+, CD8+, CD45RA+, CD45RO+, alpha-beta, or gamma-delta T cells
  • CD4+ and CD8+ T cells are isolated by positive selection with anti-CD4 and anti- CD8 antibodies.
  • the anti-CD4 and anti-CD8 antibodies are conjugated to microbeads.
  • T cells are isolated by incubation with anti-CD3/anti-CD28 (e.g., 3x28)-conjugated beads, such as DYNABEADS ® M-450 CD3/CD28 T or Trans-Act ® beads, for a time period sufficient for positive selection of the desired T cells.
  • the time period is about 30 minutes.
  • the time period ranges from 30 minutes to 36 hours or longer and all integer values there between.
  • the time period is at least 1, 2, 3, 4, 5, or 6 hours.
  • the time period is 10 to 24 hours.
  • the incubation time period is 24 hours.
  • TIL tumor infiltrating lymphocytes
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.
  • multiple rounds of selection can also be used in the context of this present disclosure.
  • Unselected” cells can also be subjected to further rounds of selection.
  • Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.
  • T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.
  • a T cell population can be selected that expresses one or more of IFN- ⁇ TNF-alpha, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g., other cytokines.
  • Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No. WO2013126712, which is herein incorporated by reference.
  • the concentration of cells and surface e.g., particles such as beads
  • the concentration of cells and surface can be varied.
  • a concentration of 2 billion cells/mL is used.
  • a concentration of 1 billion cells/mL is used.
  • greater than 100 million cells/mL is used.
  • a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mL is used.
  • concentrations of 125 or 150 million cells/mL can be used.
  • Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression. [1025] In a related aspect, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized.
  • target antigens of interest such as CD28-negative T cells
  • CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations.
  • the concentration of cells used is 5x10 6 /mL. In other aspects, the concentration used can be from about 1x10 5 /mL to 1x10 6 /mL, and any integer value in between.
  • the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10 °C or at room temperature. [1026] T cells for stimulation can also be frozen after a washing step.
  • the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
  • the cells may be suspended in a freezing solution.
  • one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80 °C at a rate of 1 per minute and stored in the vapor phase of a liquid nitrogen storage tank.
  • cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure.
  • the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed.
  • the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein.
  • a blood sample or an apheresis is taken from a generally healthy subject.
  • a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use.
  • the T cells may be expanded, frozen, and used at a later time.
  • samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments.
  • the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, and mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, and irradiation.
  • agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, and mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3
  • T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells.
  • the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo.
  • these cells may be in a preferred state for enhanced engraftment and in vivo expansion.
  • mobilization for example, mobilization with GM-CSF
  • conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy.
  • Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
  • Activation and Expansion of T Cells [1029] T cells may be activated and expanded generally using methods as described, for example, in U.S. Pat.
  • the T cells of the present disclosure may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells.
  • T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
  • a protein kinase C activator e.g., bryostatin
  • a ligand that binds the accessory molecule is used for co-stimulation of an accessory molecule on the surface of the T cells.
  • a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
  • an anti-CD3 antibody and an anti-CD28 antibody To stimulate proliferation of either CD4+ T cells, CD8+ T cells, or CD4+CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody.
  • an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med.190(9):13191328, 1999; Garland et al., J. Immunol. Meth.227(1-2):53-63, 1999).
  • T cells are activated by incubation with anti-CD3/anti-CD28-conjugated beads, such as DYNABEADS ® or Trans-Act ® beads, for a time period sufficient for activation of the T cells.
  • the time period is at least 1, 2, 3, 4, 5, or 6 hours.
  • the time period is 10 to 36 hours, e.g., about 24 hours.
  • T cells are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with cytokines that bind the common gamma-chain (e.g., IL-2, IL-7, IL-12, IL-15, IL-21, and others).
  • T cells are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 100 U/mL of IL-2, IL-7, and/or IL-15. In some embodiments, the cells are activated for 24 hours. In some embodiments, after transduction, the cells are expanded in the presence of anti- CD3 antibody, anti-CD28 antibody in combination with the same cytokines.
  • cells activated in the presence of an anti-CD3 antibody and an anti-CD28 antibody in combination with cytokines that bind the common gamma-chain are expanded in the presence of the same cytokines in the absence of the anti-CD3 antibody and anti-CD28 antibody after transduction.
  • the cells after transduction, are expanded in the presence of anti-CD3 antibody, anti-CD28 antibody in combination with the same cytokines up to a first washing step, when the cells are sub-cultured in media that includes the cytokines but does not include the anti-CD3 antibody and anti-CD28 antibody.
  • the cells are subcultured every 1, 2, 3, 4, 5, or 6 days.
  • cells are expanded for 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 days.
  • the expansion of T cells may be stimulated with zoledronic acid (Zometa), alendronic acid (Fosamax) or other related bisphosphonate drugs at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10, or 100 ⁇ M in the presence of feeder cells (irradiated cancer cells, PBMCs, artificial antigen presenting cells).
  • T cells may be stimulated with isopentyl pyrophosphate (IPP), (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP or HMB-PP) or other structurally related compounds at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10, or 100 ⁇ M in the presence of feeder cells (irradiated cancer cells, PBMCs, artificial antigen presenting cells).
  • IPP isopentyl pyrophosphate
  • HMBPP or HMB-PP HMB-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate
  • feeder cells irradiated cancer cells, PBMCs, artificial antigen presenting cells.
  • the expansion of T cells may be stimulated with synthetic phosphoantigens (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1 PP, or 2-methyl- 3-butenyl-1 -pyrophosphate in the presence of IL-2 for one-to-two weeks.
  • the expansion of T cells may be stimulated with immobilized anti-TCRyd (e.g., pan TCRY6) in the presence of IL-2, e.g., for approximately 14 days.
  • the expansion of T cells may be stimulated with culture of immobilized anti-CD3 antibodies (e.g., OKT3) in the presence of IL-2.
  • the aforementioned culture is maintained for about seven days prior to subculture in soluble anti-CD3, and IL-2.
  • T cells that have been exposed to varied stimulation times may exhibit different characteristics.
  • typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+).
  • TH, CD4+ helper T cell population
  • TC cytotoxic or suppressor T cell population
  • Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells.
  • infusing a subject with a T cell population comprising predominately of TH cells may be advantageous.
  • an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.
  • other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.
  • the modified T cells (e.g., a TFP-T cell) provided herein may be useful for the treatment of any disease or condition involving the TFP.
  • the disease or condition is a disease or condition that can benefit from treatment with adoptive cell therapy.
  • the disease or condition is a cell proliferative disorder.
  • the disease or condition is a cancer.
  • the disease or condition is a blood cancer.
  • the disease or condition is a tumor. In some embodiments, the disease or condition is a viral infection.
  • the disease or condition is a cancer.
  • the disease or condition is a viral infection.
  • the modified T cell e.g., TFP-T cell
  • the TFP-T cell can further comprise a nucleic acid sequence encoding an antibody or fragment thereof targeting PD-1 (or anti-PD-1 antibody).
  • the TFP-T cell can comprise a nucleic acid molecule comprising a first sequence encoding a TFP and a second sequence encoding an antibody or fragment thereof targeting PD-1.
  • the modified T cell described herein can express the TFP or the anti-PD-1 antibody before administering the modified T cell into the subject.
  • the modified T cell described herein can secrete the anti-PD-1 antibody before administering the modified T cell into the subject.
  • the secreted anti-PD-1 antibody can be administered into the subject in the medium together with the modified T cell.
  • the modified T cell described herein can express the TFP or the anti-PD-1 antibody after administering the modified T cell into the subject.
  • the modified T cell described herein can secrete anti-PD-1 antibody after administering the modified T cell into the subject.
  • Any suitable cancer may be treated with the modified T cells (e.g., TFP-T cells) provided herein.
  • suitable cancers include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder
  • the present disclosure also provides methods of treating PD-1-mediated diseases or disorders in a subject, e.g., a human patient, comprising administering a therapeutically effective amount of an antibody (e.g., anti-PD-1 antibody or fragment thereof) of the present disclosure to a subject in need thereof.
  • the method is a method of treating cancer.
  • the method is a method of treating inflammation.
  • the method is a method of treating an autoimmune disease, e.g., Crohn's disease.
  • the cancer is selected from the group consisting of: melanoma, renal cancer, prostate cancer, pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer, esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, and lymphoma.
  • the therapeutically effective amount can be the amount of anti-PD-1 antibody already secreted out of the modified T cells.
  • the effective amount can be the amount of anti-PD-1 antibody secreted after the modified T cells have been administered into a subject.
  • the disease may be associated with at least one tumor-associated antigen expression.
  • the method provided herein can comprise administering a modified T cell in a subject with a disease.
  • the disclosure provides methods for treating a disease wherein part of the tumor is negative for the tumor associated antigen and part of the tumor is positive for the tumor associated antigen.
  • the antibody or TFP of the disclosure is useful for treating subjects that have undergone treatment for a disease associated with elevated expression of said tumor antigen, wherein the subject that has undergone treatment for elevated levels of the tumor associated antigen exhibits a disease associated with elevated levels of the tumor associated antigen.
  • the modified T cell being administered in a subject is a T cell.
  • the T cell can have an increased effector function or proliferation rate.
  • the modified T cell can comprise a TFP or a sequence encoding a TFP described herein.
  • the modified T cell can further comprise an anti-PD-1 antibody or a sequence encoding the anti-PD-1 antibody.
  • the modified T cell can secrete the anti-PD-1 antibody.
  • the secreted PD-1 antibody can block the interaction between PD-1 and PD-L1 and/or PD-L2.
  • the present disclosure provides a method for increasing T cell effector function, where the T cell comprises an anti-PD-1 antibody (e.g., a secreted PD-1 antibody or a PD-1 antibody fusion) or a sequence encoding the anti-PD-1 antibody.
  • the present disclosure provides a method for increasing T cell effector function in a subject, comprising administering in the subject a modified T cell, where the modified T cell comprises an anti-PD-1 antibody (e.g., a secreted PD-1 antibody or a PD-1 antibody fusion) or a sequence encoding the anti-PD-1 antibody.
  • the modified T cell can further comprise a TFP or a sequence encoding the TFP.
  • the present disclosure provides a method for increasing lymphocyte secretion of a cytokine selected from the group consisting of IL-6, IL-12, IL-18, TNF- ⁇ , IL-1 ⁇ and GM-CSF in a subject in need of increased T cell effector function, comprising administering in the subject a modified T cell, where the modified T cell can comprise anti-PD-1 antibody or a sequence encoding the anti-PD-1 antibody.
  • the modified T cell can further comprise a TFP or a sequence encoding the TFP.
  • the present disclosure provides a method for increasing lymphocyte secretion of a cytokine selected from the group consisting of IL-6, IL-12, IL-18, TNF- ⁇ , IL-1 ⁇ and GM-CSF in a subject in need of increased T cell effector function, comprising administering in the subject a modified T cell, where the modified T cell can comprise a TFP and an anti-PD-1 antibody or a sequence encoding the TFP and the anti-PD-1 antibody.
  • the disclosure pertains to a vector comprising a sequence encoding an anti- PD-1 antibody (e.g., a secreted PD-1 antibody or a PD-1 antibody fusion).
  • the vector can further comprise an additional sequence encoding a TFP described herein.
  • the modified T cell can comprise the vector for expression of the TFP and/or the anti-PD-1 antibody (e.g., a secreted PD- 1 antibody or a PD-1 antibody fusion).
  • the disclosure pertains to a vector comprising a sequence encoding an anti- tumor-associated antigen antibody or TFP operably linked to promoter for expression in mammalian T cells.
  • the disclosure provides a recombinant T cell expressing a tumor-associated antigen TFP for use in treating tumor-associated antigen-expressing tumors, wherein the recombinant T cell expressing the tumor-associated antigen TFP is termed a tumor- associated antigen TFP-T cell.
  • the tumor-associated antigen TFP-T cell of the disclosure is capable of contacting a tumor cell with at least one tumor-associated antigen TFP of the disclosure expressed on its surface such that the TFP-T targets the tumor cell and growth of the tumor is inhibited.
  • the disclosure pertains to a method of inhibiting growth of a tumor- associated antigen-expressing tumor cell, comprising contacting the tumor cell with a tumor- associated antigen antibody or TFP-T cell of the present disclosure such that the TFP-T cell is activated in response to the antigen and targets the cancer cell, wherein the growth of the tumor is inhibited.
  • the disclosure pertains to a method of treating cancer in a subject. The method comprises administering to the subject a tumor-associated antigen antibody, bispecific antibody, or TFP-T cell of the present disclosure such that the cancer is treated in the subject.
  • tumor-associated antigen antibodies or TFP therapy can be used in combination with one or more additional therapies described herein.
  • T cells are genetically modified to express a TFP and the TFP-expressing T cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Unlike antibody therapies, TFP-expressing T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.
  • the T cells administered to the patient, or their progeny persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of the T cell to the patient.
  • T cells are modified, e.g., by in vitro transcribed RNA, to transiently express a TFP and the TFP-expressing T cell is infused to a recipient in need thereof.
  • the infused cell is able to kill tumor cells in the recipient.
  • the T cells administered to the patient is present for less than one month, e.g., three weeks, two weeks, or one week, after administration of the T cell to the patient.
  • the anti-tumor immunity response elicited by the TFP-expressing T cells may be an active or a passive immune response, or alternatively may be due to a direct vs indirect immune response.
  • the TFP transduced T cells exhibit specific proinflammatory cytokine secretion and potent cytolytic activity in response to human cancer cells expressing the tumor-associated antigen, resist soluble tumor-associated antigen inhibition, mediate bystander killing and/or mediate regression of an established human tumor.
  • antigen-less tumor cells within a heterogeneous field of tumor-associated antigen-expressing tumor may be susceptible to indirect destruction by tumor- associated antigen-redirected T cells that has previously reacted against adjacent antigen-positive cancer cells.
  • the human TFP-T cells of the disclosure may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal.
  • the mammal is a human.
  • ex vivo immunization at least one of the following occurs in vitro prior to administering the cell into a mammal: i) expansion of the cells, ii) introducing a nucleic acid encoding a TFP to the cells or iii) cryopreservation of the cells, as is described herein.
  • the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.
  • the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised.
  • the TFP-modified T cells of the disclosure are used in the treatment of diseases, disorders and conditions associated with expression of tumor-associated antigens.
  • the cells of the disclosure are used in the treatment of patients at risk for developing diseases, disorders and conditions associated with expression of tumor-associated antigens.
  • the present disclosure provides methods for the treatment or prevention of diseases, disorders and conditions associated with expression of tumor-associated antigens comprising administering to a subject in need thereof, a therapeutically effective amount of the TFP-modified T cells of the disclosure.
  • the antibodies or TFP-T cells of the present disclosure may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components as is described in further detail below.
  • the present disclosure also provides methods for inhibiting the proliferation or reducing a tumor-associated antigen-expressing cell population, the methods comprising contacting a population of cells comprising a tumor-associated antigen-expressing cell with an anti-tumor- associated antigen TFP-T cell of the disclosure that binds to the tumor-associated antigen- expressing cell.
  • the present disclosure provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing tumor-associated antigen, the methods comprising contacting the tumor-associated antigen-expressing cancer cell population with an anti-tumor-associated antigen antibody or TFP-T cell of the disclosure that binds to the tumor-associated antigen-expressing cell.
  • the present disclosure provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing tumor- associated antigen, the methods comprising contacting the tumor-associated antigen-expressing cancer cell population with an anti-tumor-associated antigen antibody or TFP-T cell of the disclosure that binds to the tumor-associated antigen-expressing cell.
  • the anti- tumor-associated antigen antibody or TFP-T cell of the disclosure reduces the quantity, number, amount or percentage of cells and/or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a subject with or animal model for multiple myeloma or another cancer associated with tumor-associated antigen-expressing cells relative to a negative control.
  • the subject is a human.
  • the present disclosure also provides methods for preventing, treating and/or managing a disease associated with tumor-associated antigen-expressing cells (e.g., a cancer expressing tumor-associated antigen), the methods comprising administering to a subject in need an anti- tumor-associated antigen antibody or TFP-T cell of the disclosure that binds to the tumor- associated antigen-expressing cell.
  • the subject is a human.
  • disorders associated with tumor-associated antigen-expressing cells include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma) and cancers (such as hematological cancers or atypical cancers expressing tumor-associated antigen).
  • Suitable doses of the modified T cells (e.g., TFP-T cells) described herein for a therapeutic effect would be at least 10 5 or between about 10 5 and about 10 10 cells per dose, for example, preferably in a series of dosing cycles.
  • An exemplary dosing regimen consists of four one-week dosing cycles of escalating doses, starting at least at about 10 5 cells on Day 0, for example increasing incrementally up to a target dose of about 10 10 cells within several weeks of initiating an intra-patient dose escalation scheme.
  • Suitable modes of administration include intravenous, subcutaneous, intracavitary (for example by reservoir-access device), intraperitoneal, and direct injection into a tumor mass.
  • an effective amount or sufficient number of the isolated, T cells is present in the composition and introduced into the subject such that long-term, specific, anti-cancer and/or anti- tumor responses are established to reduce the size of a tumor or eliminate tumor growth or regrowth than would otherwise result in the absence of such treatment.
  • the amount of T cells introduced into the subject causes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in tumor size when compared to otherwise same conditions wherein the T cells are not present.
  • the amount of T cells administered should take into account the route of administration and should be such that a sufficient number of the T cells will be introduced so as to achieve the desired therapeutic response.
  • the amounts of each active agent included in the compositions described herein can vary in different applications.
  • the TFP-T cell described herein can further comprise an anti- PD-1 antibody or a sequence encoding the anti-PD-1 antibody.
  • the methods provided herein can comprise administering a pharmaceutical composition comprising a modified T cell.
  • the modified T cell can be a TFP-T cell.
  • the TFP-T cell can comprise a TFP or a sequence encoding a TFP.
  • the modified T cell can further comprise an anti- PD-1 antibody (e.g., a secreted PD-1 antibody or a PD-1 antibody fusion) or a sequence encoding the anti-PD-1 antibody.
  • the composition administered into a subject can comprise a therapeutically effective amount of modified T cells and a therapeutically effective amount of anti-PD-1 antibody already secreted out of the modified T cells.
  • the composition administered into a subject can comprise a therapeutically effective amount of modified T cells, where the modified T cells can express or secrete therapeutically effective amount of anti-PD-1 antibody after being administered into a subject.
  • the present disclosure also provides the method of treating a subject in need thereof comprising administering to the subject a T cell comprising TFP as described herein and an anti-PD-1 antibody as described herein.
  • the anti-PD-1 antibody is administered before administering the T cell, concurrently with the T cell, or after administering the T cell. In some embodiments, the T cell, the anti-PD-1 antibody, or a combination thereof is administered in one, two, three or more doses.
  • the anti-PD-1 antibody can be an anti-PD-1 antibody described herein, for example, an anti-PD-1 antibody in Table 10.
  • the anti-PD-1 antibody may be an off-the-shelf anti-PD-1 antibody.
  • the anti-PD-1 antibody is administered every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.
  • the anti-PD-1 antibody is administered for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, r45 weeks, 46 weeks, 47 weeks, or 48 weeks.
  • the anti-PD-1 antibody is administered with the dose of 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, 38, 39, 40, 45, 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, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 500, 550, 600, 650,
  • the anti-PD-1 antibody 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 amino acid sequences selected from SEQ ID NOs: 1-19, 75, 77, 79, 81, 83, 85, 388, and 391.
  • the anti-PD-1 antibody as described herein comprises any one of the amino acid sequences selected from SEQ ID NOs: 1-19, 75, 77, 79, 81, 83, 85, 388, and 391.
  • the anti-PD-1 antibody as described herein is encoded by a nucleic acid sequence having at least about 40%, 45%, 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 selected from SEQ ID NOs: 20-24, 72, 73, 74, 76, 78, 80, 82, 84, 86, 389, 390, 392, and 393.
  • the anti-PD-1 antibody as described herein is encoded by any one of the nucleic acid sequences selected from SEQ ID NOs: 20-24, 72, 73, 74, 76, 78, 80, 82, 84, 86, 389, 390, 392, and 393.
  • the anti-PD-1 antibody as described herein comprises 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 of SEQ ID NOs: 1-19, 75, 77, 79, 81, 83, 85, 388, and 391.
  • the anti-PD-1 antibody as described herein comprises any one of the CDR sequences of SEQ ID NOs: 1-19, 75, 77, 79, 81, 83, 85, 388, and 391.
  • the anti-PD-1 antibody as described herein comprises CDR sequences encoded by a nucleic acid sequence having at least about 40%, 45%, 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 selected from SEQ ID NOs: 20-24, 72, 73, 74, 76, 78, 80, 82, 84, 86, 389, 390, 392, and 393.
  • the anti- PD-1 antibody as described herein comprises CDR sequences encoded by any one of the nucleic acid sequences selected from SEQ ID NOs: 20-24, 72, 73, 74, 76, 78, 80, 82, 84, 86, 389, 390, 392, and 393.
  • the anti-PD-1 antibody 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-PD-1 antibody amino acid sequences listed in Table 10.
  • the anti-PD-1 antibody as described herein comprises any one of the anti-PD-1 antibody amino acid sequences listed in Table 10.
  • the anti-PD-1 antibody as described herein is encoded by a nucleic acid sequence having at least about 40%, 45%, 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-PD-1 antibody nucleic acid sequences listed in Table 10.
  • the anti-PD- 1 antibody as described herein is encoded by any one of the anti-PD-1 antibody nucleic acid sequences listed in Table 10.
  • the anti-PD-1 antibody as described herein comprises 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 of the anti-PD-1 antibody amino acid sequences listed in Table 10.
  • the anti-PD-1 antibody as described herein comprises any one of the CDR sequences of the anti-PD-1 antibody amino acid sequences listed in Table 10.
  • the anti-PD-1 antibody as described herein comprises CDR sequences encoded by a nucleic acid sequence having at least about 40%, 45%, 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-PD-1 antibody nucleic acid sequences listed in Table 10.
  • the anti-PD- 1 antibody as described herein comprises CDR sequences encoded by any one of the anti-PD-1 antibody nucleic acid sequences listed in Table 10.
  • an antibody or a modified T cell (e.g., aTFP and anti-PD-1-expressing cell) described herein may be used in combination with other known agents and therapies.
  • Administered “in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”.
  • the delivery of one treatment ends before the delivery of the other treatment begins.
  • the treatment is more effective because of combined administration.
  • the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment or the analogous situation is seen with the first treatment.
  • delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other.
  • the effect of the two treatments can be partially additive, wholly additive, or greater than additive.
  • the “at least one additional therapeutic agent” includes a TFP- expressing cell.
  • T cells that express multiple TFPs, which bind to the same or different target antigens, or same or different epitopes on the same target antigen.
  • populations of T cells in which a first subset of T cells expresses a first TFP and a second subset of T cells expresses a second TFP.
  • the second subset of TFP- expressing T cells does not comprise an anti-PD-1 antibody.
  • a TFP-expressing cell described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the TFP-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.
  • the TFP-T cells provided herein are administered with at least one additional therapeutic agent. Any suitable additional therapeutic agent may be administered with a TFP-T cell provided herein.
  • the additional therapeutic agent is selected from radiation, a cytotoxic agent, a chemotherapeutic agent, a cytostatic agent, an anti-hormonal agent, an EGFR inhibitor, an immunostimulatory agent, an anti-angiogenic agent, and combinations thereof.
  • a TFP-expressing cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and tacrolimus, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cyclophosphamide, fludarabine, cyclosporin, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, cytokines, and irradiation.
  • peptide vaccine such as that described in Izumoto et al. 2008 J Neurosurg 108:963-971.
  • the subject can be administered an agent which reduces or ameliorates a side effect associated with the administration of a TFP-expressing cell.
  • Side effects associated with the administration of a TFP-expressing cell include, but are not limited to, cytokine release syndrome (CRS), and hemophagocytic lymphohistiocytosis (HLH), also termed Macrophage Activation Syndrome (MAS).
  • CRS cytokine release syndrome
  • HHL hemophagocytic lymphohistiocytosis
  • MAS Macrophage Activation Syndrome
  • Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like.
  • the methods described herein can comprise administering a TFP-expressing cell described herein to a subject and further administering an agent to manage elevated levels of a soluble factor resulting from treatment with a TFP-expressing cell.
  • the soluble factor elevated in the subject is one or more of IFN- ⁇ , TNF ⁇ , IL-2 and IL-6. Therefore, an agent administered to treat this side effect can be an agent that neutralizes one or more of these soluble factors.
  • agents include, but are not limited to a steroid, an inhibitor of TNF ⁇ , and an inhibitor of IL-6.
  • An example of a TNF ⁇ inhibitor is etanercept (marketed under the name ENBREL®).
  • An example of an IL-6 inhibitor is tocilizumab (marketed under the name ACTEMRA®).
  • the subject can be administered an agent which enhances the activity of a TFP-expressing cell.
  • the agent can be an agent which inhibits an inhibitory molecule.
  • Inhibitory molecules e.g., Programmed Death 1 (PD-1)
  • PD-1 can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response.
  • inhibitory molecules include PD-1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.
  • Inhibition of an inhibitory molecule e.g., by inhibition at the DNA, RNA or protein level, can optimize a TFP-expressing cell performance.
  • an inhibitory nucleic acid e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA
  • an inhibitory nucleic acid e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA
  • the inhibitor is a shRNA.
  • the inhibitory molecule is inhibited within a TFP-expressing cell.
  • a dsRNA molecule that inhibits expression of the inhibitory molecule is linked to the nucleic acid that encodes a component, e.g., all of the components, of the TFP.
  • the inhibitor of an inhibitory signal can be, e.g., an antibody or antibody fragment that binds to an inhibitory molecule.
  • the agent can be an antibody or antibody fragment that binds to PD-1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX-101, and marketed as YERVOY®; Bristol-Myers Squibb; tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP- 675,206)).
  • ipilimumab also referred to as MDX-010 and MDX-101, and marketed as YERVOY®
  • tremelimumab IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP- 675,206
  • the agent is an antibody or antibody fragment that binds to T cell immunoglobulin and mucin-domain containing-3 (TIM3). In an embodiment, the agent is an antibody or antibody fragment that binds to Lymphocyte-activation gene 3 (LAG3).
  • the agent which enhances the activity of a TFP-expressing cell can be, e.g., a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule, or fragment thereof, and the second domain is a polypeptide that is associated with a positive signal, e.g., a polypeptide comprising an intracellular signaling domain as described herein.
  • the polypeptide that is associated with a positive signal can include a costimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27 and/or ICOS, and/or a primary signaling domain, e.g., of CD3 zeta, e.g., described herein.
  • the fusion protein is expressed by the same cell that expressed the TFP.
  • the fusion protein is expressed by a cell, e.g., a T cell that does not express an anti-tumor-associated antigen TFP.
  • the additional therapeutic agent comprises an immunostimulatory agent.
  • the immunostimulatory agent is an agent that blocks signaling of an inhibitory receptor of an immune cell, or a ligand thereof.
  • the inhibitory receptor or ligand is selected from cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), programmed cell death protein 1 (also PD-1 or CD279), programmed death ligand 1 (also PD-L1 or CD274), transforming growth factor beta (TGF ⁇ ), lymphocyte-activation gene 3 (LAG-3, also CD223), Tim-3 (hepatitis A virus cellular receptor 2 or HAVCR2 or CD366), neuritin, B- and T-lymphocyte attenuator (also BTLA or CD272), killer cell immunoglobulin-like receptors (KIRs), and combinations thereof.
  • CTL-4 cytotoxic T-lymphocyte-associated protein 4
  • TGF ⁇ programmed cell death protein 1
  • LAG-3 lymphocyte-activation gene 3
  • Tim-3 hepatitis A virus cellular receptor 2 or H
  • the agent is selected from an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), and anti-PD-L1 antibody (e.g., atezolizumab), an anti-CTLA-4 antibody (e.g., ipilimumab), an anti-TIM3 antibody, carcinoembryonic antigen-related cell adhesion molecule 1 (CECAM-1, also CD66a) and 5 (CEACAM-5, also CD66e), vset immunoregulatory receptor (also VISR or VISTA), leukocyte-associated immunoglobulin-like receptor 1 (also LAIR1 or CD305), CD160, natural killer cell receptor 2B4 (also CD244 or SLAMF4), and combinations thereof.
  • an anti-PD-1 antibody e.g., pembrolizumab or nivolumab
  • anti-PD-L1 antibody e.g., atezolizumab
  • an anti-CTLA-4 antibody e.g., ipi
  • the agent is pembrolizumab. In some aspects, the agent is nivolumab. In some aspects, the agent is atezolizumab. [1082] In some embodiments, the additional therapeutic agent is an agent that inhibits the interaction between PD-1 and PD-L1. In some aspects, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is selected from an antibody, a peptidomimetic and a small molecule.
  • the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is selected from pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), atezolizumab, avelumab, pidilizumab, durvalumab, sulfamonomethoxine 1, and sulfamethizole 2.
  • the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is any therapeutic known in the art to have such activity, for example as described in Weinmann et al., Chem Med Chem, 2016, 14:1576 (DOI: 10.1002/cmdc.201500566), incorporated by reference in its entirety.
  • the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in the same pharmaceutical composition an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in a different pharmaceutical composition from an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is administered prior to administration of an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is administered after administration of an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is administered contemporaneously with an antibody provided herein, but the agent and antibody are administered in separate pharmaceutical compositions.
  • the immunostimulatory agent is an agonist of a co-stimulatory receptor of an immune cell.
  • the co-stimulatory receptor is selected from GITR, OX40, ICOS, LAG-2, CD27, CD28, 4-1BB, CD40, STING, a toll-like receptor, RIG-1, and a NOD-like receptor.
  • the agonist is an antibody.
  • the immunostimulatory agent modulates the activity of arginase, indoleamine-23-dioxygenase, or the adenosine A2A receptor.
  • the immunostimulatory agent is a cytokine.
  • the cytokine is selected from IL-2, IL-5, IL-7, IL-12, IL-15, IL-21, and combinations thereof.
  • the immunostimulatory agent is an oncolytic virus.
  • the oncolytic virus is selected from a herpes simplex virus, a vesicular stomatitis virus, an adenovirus, a Newcastle disease virus, a vaccinia virus, and a maraba virus.
  • additional therapeutic agents include a taxane (e.g., paclitaxel or docetaxel); a platinum agent (e.g., carboplatin, oxaliplatin, and/or cisplatin); a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, and/or mitoxantrone); folinic acid (e.g., leucovorin); or a nucleoside metabolic inhibitor (e.g., fluorouracil, capecitabine, and/or gemcitabine).
  • the additional therapeutic agent is folinic acid, 5- fluorouracil, and/or oxaliplatin.
  • the additional therapeutic agent is 5- fluorouracil and irinotecan. In some embodiments, the additional therapeutic agent is a taxane and a platinum agent. In some embodiments, the additional therapeutic agent is paclitaxel and carboplatin. In some embodiments, the additional therapeutic agent is pemetrexate. In some embodiments, the additional therapeutic agent is a targeted therapeutic such as an EGFR, RAF or MEK-targeted agent. [1088] The additional therapeutic agent may be administered by any suitable means. In some embodiments, a medicament provided herein, and the additional therapeutic agent are included in the same pharmaceutical composition. In some embodiments, an antibody provided herein, and the additional therapeutic agent are included in different pharmaceutical compositions.
  • administration of the antibody can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about one month of each other.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about one week of each other.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about one day of each other.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about twelve hours of each other.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about one hour of each other.
  • compositions of the present disclosure may comprise a modified T cell.
  • the modified T cell can comprise a TFP or a sequence encoding a TFP.
  • the modified T cell can further comprise an anti-PD-1 antibody or a sequence encoding the anti-PD-1 antibody.
  • Pharmaceutical compositions of the present disclosure may comprise a TFP-expressing cell, e.g., a plurality of TFP-expressing cells, as described herein.
  • the pharmaceutical compositions of the present disclosure may comprise a modified T cell such as the TFP-expressing T cell, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins polypeptides or amino acids
  • antioxidants e.g., antioxidants
  • chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.
  • a contaminant e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.
  • the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.
  • an immunologically effective amount When “an immunologically effective amount,” “an anti-tumor effective amount,” “a tumor-inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 10 4 to 10 9 cells/kg body weight, in some instances 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988).
  • T cells can be activated from blood draws of from 10 cc to 400 cc.
  • T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.
  • the administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
  • the compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection. In one aspect, the T cell compositions of the present disclosure are administered by i.v. injection.
  • the compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.
  • subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g., T cells.
  • T cell isolates may be expanded and treated such that one or more TFP constructs (e.g., constructs encoding a TFP and/or a PD-1 antibody) of the disclosure may be introduced, thereby creating a TFP-expressing T cell of the disclosure.
  • Subjects in need thereof may subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • subjects receive an infusion of the expanded TFP-T cells of the present disclosure.
  • expanded cells are administered before or following surgery.
  • the dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices.
  • the dose for alemtuzumab will generally be in the range 1 to about 100 mg for an adult patient, usually administered daily for a period between 1 and 30 days.
  • the preferred daily dose is 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used (described, e.g., in U.S. Pat. No.6,120,766).
  • the TFP and/or the anti-PD-1 antibody is introduced into T cells, e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of the modified T cells (e.g., TFP-T cells) of the disclosure, and one or more subsequent administrations of the modified T cells, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration.
  • more than one administration of the modified T cells are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the modified T cells are administered per week.
  • the subject receives more than one administration of the TFP-T cells per week (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no TFP-T cells administrations, and then one or more additional administration of the TFP-T cells (e.g., more than one administration of the TFP-T cells per week) is administered to the subject.
  • the subject receives more than one cycle of TFP-T cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days.
  • the TFP-T cells are administered every other day for 3 administrations per week.
  • the TFP-T cells of the disclosure are administered for at least two, three, four, five, six, seven, eight or more weeks.
  • the TFP-T cells are generated using lentiviral vectors, such as lentivirus. TFP-T cells generated that way can have stable TFP expression.
  • the lentiviral vector may comprise a sequence encoding an anti-PD-1 antibody.
  • the TFP-T cells transiently express TFPs for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. Transient expression of TFPs can be effected by RNA vector delivery.
  • the RNA vector comprising a sequence encoding a TFP is transduced into the T cell by electroporation.
  • a potential issue that can arise in patients being treated using transiently expressing TFP- T cells (particularly with murine scFv bearing TFP-T cells) is anaphylaxis after multiple treatments.
  • an anaphylactic response might be caused by a patient developing humoral anti-TFP response, i.e., anti-TFP antibodies having an anti-IgE isotype.
  • Cytokine Release syndrome is a form of systemic inflammatory response syndrome that arises as a complication of some diseases or infections, and is also an adverse effect of some monoclonal antibody drugs, as well as adoptive T cell therapies.
  • TFP-T cells can exhibit better killing activity than CAR-T cells.
  • TFP-T cells administered to a subject can exhibit better killing activity than CAR-T cells administered to a subject.
  • This can be one of the advantages of TFP-T cells over CAR-T cells.
  • TFP-T cells can exhibit less cytokine release CAR-T cells.
  • a subject administered TFP-T cells can exhibit less cytokine release than a subject administered CAR-T cells.
  • This can be one of the advantages of TFP-T cell therapies over CAR-T cell therapies.
  • TFP- T cells can exhibit similar or better killing activity than CAR-T cells and the TFP-T cells can exhibit less cytokine release than the CAR-T cells.
  • TFP-T cells administered to a subject can exhibit similar or better killing activity than CAR-T cells administered to a subject and the subject can exhibit less cytokine release than a subject administered CAR-T cells. This can be one of the advantages of TFP-T cell therapies over CAR-T cell therapies. [1107] In some cases, the cytokine release of a treatment with TFP-T cells is less than the cytokine release of a treatment with CAR-T cells.
  • the cytokine release of a treatment with TFP-T cells is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% less than the cytokine release of a treatment with CAR-T cells.
  • Various cytokines can be released less in the T cell treatment with TFP-T cells than CAR-T cells.
  • the cytokine is IL-2, IFN- ⁇ , IL-4, TNF- ⁇ , IL-6, IL-13, IL-5, IL-10, sCD137, GM-CSF, MIP-1 ⁇ , MIP-1 ⁇ , or a combination thereof.
  • the treatment with TFP-T cells release less perforin, granzyme A, granzyme B, or a combination thereof, than the treatment with CAR-T cells.
  • the perforin, granzyme A, or granzyme B released in a treatment with TFP-T cells is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% less than a treatment with CAR-T cells.
  • at least 10% less amount of the given cytokine is released following treatment compared to an amount of the given cytokine of a mammal treated with a CAR-T cell comprising the same binding domain.
  • the given cytokine comprises one or more cytokines selected from the group consisting of IL-2, IFN- ⁇ , IL-4, TNF- ⁇ , IL-6, IL-13, IL-5, IL-10, sCD137, GM-CSF, MIP-1 ⁇ , MIP-1 ⁇ , and any combination thereof.
  • the TFP-T cells may exhibit similar or better activity in killing tumor cells than CAR-T cells.
  • a tumor growth in the mammal is inhibited such that a size of the tumor is at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, or at most 60% of a size of a tumor in a mammal treated with T cells that do not express the TFP after at least 8 days of treatment, wherein the mammal treated with T cells expressing TFP and the mammal treated with T cells that do not express the TFP have the same tumor size before the treatment.
  • the tumor growth in the mammal is completely inhibited.
  • the tumor growth in the mammal is completely inhibited for at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least 100 days, or more.
  • the population of T cells transduced with TFP kill similar amount of tumor cells compared to the CAR-T cells comprising the same binding domain.
  • the TFP-T cells can exhibit different gene expression profile than cells that do not express TFP. In some cases, the TFP-T cells may exhibit similar gene expression profiles than CAR-T cells. In some other cases, the TFP-T cells may exhibit different gene expression profiles than CAR-T cells.
  • the population of T cells transduced with TFP have a different gene expression profile than the CAR-T cells comprising the same binding domain.
  • an expression level of a gene is different in the T cells transduced with the TFP than an expression level of the gene in the CAR-T cells comprising the same binding domain.
  • the gene has a function in antigen presentation, TCR signaling, homeostasis, metabolism, chemokine signaling, cytokine signaling, toll like receptor signaling, MMP and adhesion molecule signaling, or TNFR related signaling.
  • Example 1 Production of anti-PD-1 Antibodies
  • a llama was immunized subcutaneously on days 0,7, 14, 21, and 25 with 100 ⁇ g of human PD-1 protein (amino acids 25-167) C-terminally fused to a hexahistidine tag. Gerbu P was used as an adjuvant.100 mL of non-coagulated blood was collected prepare lymphocytes for RNA extraction. RNA was used as template for first strand cDNA synthesis with oligo dT primer.
  • VHH encoding sequences were amplified by PCR, digested with PstI and NotI, and cloned into the phagemid pMECS-GG vector.
  • the phagemid vectors were transformed into E. coli, generating a phage-displayed VHH library having a 6xHis tag and consisting of approximately 10 8 transformants.
  • VHH-his for secretion into the E. coli periplasm was induced by the addition of IPTG to a final concentration of 1mM. Cultures were then grown overnight at 30 ⁇ C in a shaking incubator.
  • VHH-his proteins were purified from the periplasmic extract using Ni-NTA agarose using a stepwise gradient comprised 10 column- volume equivalents 50 mM Tris at pH 8.0 containing 150 mM NaCl and 10 mM imidazole, followed by elution with 2.5 column volume equivalents with the same buffer and containing 0.5 M imidazole.
  • hPD-L1 2 ug/mL hPD-L1 was fixed on a high- binding plate and 500 nM/0.006 mg/mL of VHH antibody was added followed by 1.25 ug/mL PD-1-Fc biotin (66 nM) (Acro Cat. PD1-H82F1).
  • PD-1-Fc biotin binding was measured using high-sensitivity streptavidin HRP (ThermoFisher catalog 21130) and antibodies observed to diminish PD-1-biotin binding signal were identified as PD-1/PD-L1 blocking antibodies.
  • the optimal concentration range of VHH antibody to be used in the assay was determined by performing a titration with 18-0.01 ug/mL of nivolumab scFv and identifying the IC50. As is shown in FIG.2A and FIG.2B, only clone 51 showed inhibition of the interaction between PD-1 and PD-L1.
  • Octet assay [1120] Bio-layer interferometry was used to measure the affinity of the VHH antibodies for PD- 1.
  • Each of the 53 VHH antibodies was diluted in Octet Buffer [PBS containing 0.01% Tween20 (vol./vol.) and 0.1% fetal bovine serum (wt./vol.)] and immobilized on Ni-NTA biosensors (ForteBio cat.18-5101) to a final biolayer thickness of 0.5-1.0 nm. Following VHH immobilization, biosensors were immersed in Octet Buffer to remove unbound VHH antibodies and to establish a flat baseline sensor signal. VHH-loaded Biosensors were then transferred to Octet buffer containing 100 ⁇ M human PD-1-Fc Biotin (Acro Cat.
  • Table 1 shows that 28 of the 29 binders belong to clonotype 1 based on their CDR3 sequence, whereas clone 51 belongs to clonotype 3.
  • Table 1 Exemplary Anti-PD-1 VHH [1121] Epitope Binning [1122] In-tandem epitope binning of nivolumab scFv, clone 2PDE172 (clone 51), and clone 2PDE145 (clone 35) was performed by biolayer interferometry. Clone 2PDE145 was selected because of its ability to bind PD-1 without blocking binding to PD-L1.
  • Nivolumab scFv and clone 2PDE172 were selected based on their ability to bind PD-1 and blocking its association with PD-L1. Briefly, 2 ⁇ g/mL of PD-1 (Acro catalog PD1-H82F1) was immobilized on streptavidin (SA) biosensor (ForteBio cat.18-5019) to a final thickness of approximately 1 nm followed by baseline establishment. Immobilized PD-1 was titrated to full saturation for 5 minutes with antibody solution (Ab1) comprised of 400 nM of nivolumab scFv-6His, 2PDE145, 2PDE172 or buffer lacking antibody.
  • SA streptavidin
  • Binding of a second antibody (Ab2) was screened by transferring biosensors to a solution containing 400 nM of the already-associated antibody (Ab1) and 400 nM of the competing antibody (Ab2) or buffer for 5 minutes.
  • the exclusion of a competing antibody Ab2 in this step establishes self-blocking signal and establishes the threshold to identify competitive Ab2 binding.
  • Clones 35 and 51 were chosen for further characterization by epitope binning.
  • Epitope binning pairs were identified based on a PD-1 binding threshold for the competition step established by the self-blocking signal of Nivolumab scFv, 2PDE145, and 2PDE172.
  • the signal threshold was defined as the largest self-blocking PD-1 binding signal observed when the same binder is used for saturation and competition steps.
  • Non-competitive Ab1/Ab2 pairs were sorted into unique epitope bins when neither Ab1 nor Ab2 blocked PD-1 binding during the competition step and produced a signal > self-blocking threshold.
  • Competitive Ab1/Ab2 pairs enforced mutual blockade of PD-1 binding signal by generating values ⁇ the self- blocking threshold.
  • Data were analyzed by Pall ForteBio Data Analysis 9.0 software to generate an epitope binning matrix and identify binning, non-binning, and unidirectional pairs.
  • hPD-1-hFc-biotin was diluted in Octet Buffer [PBS containing 0.01% Tween20 (vol./vol.) and 0.1% fetal bovine serum (wt./vol.)] and immobilized on SA (streptavidin) biosensors to a final biolayer thickness of approximately 1.0 nm. Following immobilization, biosensors were immersed in Octet Buffer to remove unbound PD-1 and to establish a flat baseline sensor signal.
  • Octet Buffer PBS containing 0.01% Tween20 (vol./vol.) and 0.1% fetal bovine serum (wt./vol.)
  • SA streptavidin
  • PD-1-loaded Biosensors were then transferred to Octet buffer containing 6 – 800 nM anti-PD-1 antibody (Nivolumab, Fc-tagged Clone 51 VHH, or His- tagged Clone 51 VHH and association was monitored for 20 minutes at 30°C whilst agitating at 1,000 RPM. VHH antibody dissociation was initiated by transferring sensors to Octet buffer and monitored for 5 minutes. Data were analyzed using ForteBio Data Analysis Suite 9.0. The observed antibody binding response values were plotted as a function of antibody concentration and the data were analyzed and fitted is Prism 7 using a one-site specific binding model to determine values of K D for PD-1 binding.
  • Staphyloccocal aureus enterotoxin B is a super-antigen that induces peptide-independent activation of T cells by crosslinking specific TCR V ⁇ chains (V ⁇ 3, V ⁇ 5, V ⁇ 12, V ⁇ 14, V ⁇ 17) with MHC class II.
  • SEB is a potent inducer of toxic shock syndrome through production of IFN- ⁇ , IL-2, and TNF- ⁇ by T cells, but is costimulation dependent and therefore requires antigen-presenting cells.
  • SEB has been commonly used as a positive control for T cell activation, and also as a tool to examine mechanisms of T cell anergy and exhaustion.
  • a titration curve of human PBMCs cultured with SEB and Pembrolizumab was first generated to determine the optimal concentration of SEB in the assay.200,000 PBMCs were plated with 10-fold dilutions of SEB ranging from 0.1 ng/mL to 100.0 ⁇ g/mL of SEB. The highest level of IL-2 production was seen with 1.0 ⁇ g/ml of SEB, regardless of concentration of Pembrolizumab.
  • PBMCs were then cultured with 1.0 ⁇ g/ml or 3.0 ⁇ g/ml of SEB in the presence of no antibody, hIgG4, hIgG1, Pembrolizumab, clone 51 VHH with an hFc fusion, clone 51 VHH or clone 51 VHH at a 10x increase in concentration.
  • Control antibodies, Pembrolizumab, clone 51 IgG1 hFc and clone 51 VHH were added at a concentration of 6.7114 nM and 67.14 nM, respectively.
  • This experiment was performed in two separate experiments with the same donor PBMCs. All conditions were plated in triplicate wells in a flat bottom 96 well plate.
  • Error bars in the first panel represent the SEM between two independent experiments using the same donor. This experiment was repeated a third time with a new donor (Donor 2) after determining that its best response to SEB was observed at 3.0 ⁇ g/mL in an SEB titration curve. Identical concentrations of antibodies were used. Error bars represent the standard deviation between technical replicates. As is shown in FIG.6, clone 51 VHH or clone 51 VHH hFc fusion blocked PD-1 signaling as evidenced by an increase in IL-2 levels.
  • PD-1/PDL-1 Blockade BioAssay A PD-1/PDL-1 blockade bioassay (Promega) was performed according to the manufacturer’s instructions to further assess the ability of the clone 51 anti-PD-1 VHH antibody to block PD-1/PDL-1 signaling.
  • the assay consists of two cell lines: (i) PD-1 effector cells that are Jurkat T cells stably expressing human PD-1 and NFAT-induced luciferase, and (ii) PD-L1 aAPC/CHO-K1 cells that are CHO-K1 cells stably expressing human PD-L1 and a cell surface protein designed to activate cognate TCRs in an antigen-independent manner.
  • the PD-1/PD-L1 interaction inhibits TCR signaling and NFAT-mediated luciferase activity.
  • Addition of an antibody that blocks either PD-1 or PD-L1 releases the inhibitory signal and results in TCR signaling and NFAT-mediated luciferase activity.
  • the PD- L1 aAPC/CHO-K1 cells were incubated at 37 for 16-20 hours in a 96-well plate and antibody and PD-1 effector cells were then added. The samples were then incubated at 37oC for an additional 6 hours before the addition of Bio-GloTM reagent.
  • Supernatants were collected from the T cells and were used to stain T cells that had been stimulated to express PD-1 treatment with IL-2 with or without (1) dynabeads with anti-CD3 and anti-CD28 antibodies, and/or (2) 500 ng/ml TGF- ⁇ for 72 hours.
  • a standard curve was generated by treating the stimulated T cells with known amounts of the CDS clone 51 VHH. The MFI was measured by FITC.
  • FIGs.8A-8D show that the clone 51 VHH antibody secreted from T cells was able to detect cell surface expression of PD-1 induced by IL-2 with dynabeads or IL-2 with dynabeads and TGF- ⁇ even at a 1:20 dilution of supernatant, with the highest signal seen with clone 51 VHH Hz.
  • Example 5 Humanized Clone 51 VHH [1137] Clone 51 Hz framework sequence was humanized based on comparison to thirteen highly homologous human VH sequences using Standard Protein Blast. To achieve full clone 51 framework humanization, eighteen amino acids needed to be mutated to their human counterparts.
  • biosensors were immersed in Octet Buffer to remove unbound PD-1 and to establish a flat baseline sensor signal.
  • PD-1-loaded Biosensors were then transferred to Octet buffer containing 6–400 nM anti-PD-1 antibody (parental or humanized clone 51 VHH) and association was monitored for 20 minutes at 30 °C whilst agitating at 1,000 RPM.
  • VHH antibody dissociation was initiated by transferring sensors to Octet buffer and monitored for 5 minutes. Data were analyzed using ForteBio Data Analysis Suite 9.0. The observed antibody binding response values were plotted as a function of antibody concentration and the data were analyzed and fitted is Prism 7 using a one-site specific binding model to determine values of KD for PD-1 binding.
  • Variants of clone 51 h12 were generated in several different configurations as shown in FIG.10A including (i) clone 51 h12 VHH-SrtA tag-His tag; (ii) clone 51 h12 VHH-hIgG4 tag; (iii) clone 51 h12 VHH- clone 51 h12 VHH - SrtA tag-His tag; (iv) clone 51 h12 VHH-anti- HSA-SrtA tag-His tag; and (v) clone 51 h12 VHH- clone 51 h12 VHH – anti-HSA - SrtA tag-His tag.
  • FIG.10A shows each of the different antibodies run on an SDS-page gel.
  • Bio-layer interferometry was used to measure the steady state affinity of each of the configurations of the humanized versions of clone 51 for PD-1.2.5 ug/mL of biotinylated human PD-1-Fc fusion protein (Acro catalog PD1-H82F1) was immobilized on streptavidin (SA) biosensors (ForteBio cat.18-5019) to a final thickness of approximately 1 nm followed by the establishment of a baseline and measurement of the association and dissociation rates constants, ka and kd, for binding of 400 nM of the indicated anti-PD-1 fusion protein.
  • SA streptavidin
  • the dissociation constant, KD was determined from the ratio of kd to ka. [1142] As is shown in Table 6 below, multimerization of clone 51 and addition of an hIgG4 tag positively improve clone 51 apparent affinity.
  • the PD-1 V HH DNA fragment from clone 51 (2PDE172) (non-humanized, codon optimized (CDS), or humanized, codon-optimized (Hz)) having HA and 6xHis tags or hIgG Fc tags were cloned into the pLRPC vector downstream of an anti-MSLN TFP.
  • the VHH antibody and the TFP were separated by a 2A self-cleaving peptide.
  • the anti-MSLN TFP comprised the MH1 anti-MSLN VHH linked to a CD3 epsilon TCR DNA fragment by a DNA sequence encoding a short linker (SL): AAAGGGGSGGGGSGGGGSLE (SEQ ID NO:118).
  • TFP human T Cell Receptor
  • TCR human T Cell Receptor
  • a human TCR complex contains the CD3-epsilon polypeptide, the CD3-gamma poly peptide, the CD3-delta polypeptide, and the TCR alpha chain polypeptide and the TCR beta chain polypeptide or the TCR delta chain polypeptide and the TCR gamma chain polypeptide.
  • TCR alpha, TCR beta, TCR gamma, and TCR delta recruit the CD3 zeta polypeptide.
  • the human CD3-epsilon polypeptide canonical sequence is Uniprot Accession No. P07766.
  • the human CD3-gamma polypeptide canonical sequence is Uniprot Accession No. P09693.
  • the human CD3-delta polypeptide canonical sequence is Uniprot Accession No. P043234.
  • the human CD3-zeta polypeptide canonical sequence is Uniprot Accession No. P20963.
  • the human TCR alpha chain canonical sequence is Uniprot Accession No. Q6ISU1.
  • the human TCR beta chain C region canonical sequence is Uniprot Accession No. P01850, a human TCR beta chain V region sequence is P04435.
  • the human CD3-epsilon polypeptide canonical sequence is: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILW QHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARV CENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQ NKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:57).
  • the mature human CD3-epsilon polypeptide sequence is: DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHL SLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITG GLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYS GLNQRRI (SEQ ID NO:383).
  • the signal peptide of human CD3 ⁇ is: MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO:87).
  • the extracellular domain of human CD3 ⁇ is: DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHL SLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO:88).
  • the transmembrane domain of human CD3 ⁇ is: VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID NO:89).
  • the intracellular domain of human CD3 ⁇ is: KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:90).
  • the human CD3-gamma polypeptide canonical sequence is: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDG KMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS GFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQ GNQLRRN (SEQ ID NO:58).
  • the mature human CD3-gamma polypeptide sequence is: QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAK DPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQ DGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO:384).
  • the signal peptide of human CD3 ⁇ is: MEQGKGLAVLILAIILLQGTLA (SEQ ID NO:91).
  • the extracellular domain of human CD3 ⁇ is: QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAK DPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS (SEQ ID NO:92).
  • the transmembrane domain of human CD3 ⁇ is: GFLFAEIVSIFVLAVGVYFIA (SEQ ID NO:93).
  • the intracellular domain of human CD3 ⁇ is: GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO:94).
  • the human CD3-delta polypeptide canonical sequence is: MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLG KRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALG VFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKS (SEQ ID NO:59).
  • the mature human CD3-delta polypeptide sequence is: FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKE STVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQAL LRNDQVYQPLRDRDDAQYSHLGGNWARNKS (SEQ ID NO:385).
  • the signal peptide of human CD3 ⁇ is: MEHSTFLSGLVLATLLSQVSP (SEQ ID NO:95).
  • the extracellular domain of human CD3 ⁇ is: FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKE STVQVHYRMCQSCVELDPATVA (SEQ ID NO:96).
  • the transmembrane domain of human CD3 ⁇ is: GIIVTDVIATLLLALGVFCFA (SEQ ID NO:97).
  • the intracellular domain of human CD3 ⁇ is: GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO:98).
  • the human CD3-zeta polypeptide canonical sequence is: MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:99).
  • the human TCR alpha chain canonical sequence is: MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLD SPIWFSAGNGSALDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHS RSTQPMHLSGEASTARTCPQEPLRGTPGGALWLGVLRLLLFKLLLFDLLLTCSCLCDPAG PLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVWGEG SYLSSYPTCPAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGAA (SEQ ID NO:100).
  • the human TCR alpha chain constant region canonical sequence is: PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFK SNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIG FRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:101).
  • the human TCR alpha chain IgC sequence is: PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFK SNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS (SEQ ID NO:102).
  • the transmembrane domain of the human TCR alpha chain is: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO:103).
  • the intracellular domain of the human TCR alpha chain is: SS.
  • the human TCR alpha chain V region CTL-L17 canonical sequence is: MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDY FLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAAK GAGTASKLTFGTGTRLQVTL (SEQ ID NO:104).

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Abstract

La présente divulgation concerne des protéines de fusion de lymphocytes T (récepteurs) (TFP) comprenant des domaines de liaison ciblant divers antigènes associés à une maladie ou des molécules d'acides nucléiques codant pour de telles TFP. La présente divulgation concerne également des protéines de liaison à l'antigène anti-PD-1 ou un fragment de celles-ci, ainsi que des molécules d'acides nucléiques codant pour les protéines de liaison à l'antigène anti-PD-1. La présente divulgation concerne en outre des lymphocytes T modifiés exprimant de telles TFP ou une protéine de liaison à l'antigène ou un fragment de celle-ci, et des méthodes de régulation de lymphocytes T ou de traitement de patients faisant intervenir de telles constructions ou de tels lymphocytes T modifiés.
PCT/US2021/040177 2020-07-02 2021-07-01 Compositions et procédés de reprogrammation de tcr faisant intervenir des protéines de fusion et des anticorps anti-pd1 WO2022006451A2 (fr)

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KR20210049816A (ko) * 2018-07-26 2021-05-06 티씨알2 테라퓨틱스 인크. 표적 특이적 융합 단백질을 사용하는 tcr 리프로그래밍을 위한 조성물 및 방법

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EP4146233A4 (fr) * 2020-05-05 2024-05-22 TCR2 Therapeutics Inc. Compositions et procédés pour la reprogrammation de tcr au moyen de protéines de fusion spécifiques cd70
WO2022192439A1 (fr) * 2021-03-11 2022-09-15 Kite Pharma, Inc. Amélioration de la fonction de cellules immunitaires
CN116904514A (zh) * 2022-04-18 2023-10-20 玥特农生物科技河北有限责任公司 用双aav载体同步定点基因编辑制备car-t细胞的方法
WO2023235479A1 (fr) * 2022-06-02 2023-12-07 TCR2 Therapeutics Inc. Compositions et méthodes pour traiter le cancer
WO2024103107A1 (fr) * 2022-11-14 2024-05-23 Peter Maccallum Cancer Institute Protéines de fusion et leurs utilisations

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