WO2023086379A2 - Compositions et méthodes de reprogrammation de tcr à l'aide de protéines de fusion - Google Patents

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

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WO2023086379A2
WO2023086379A2 PCT/US2022/049396 US2022049396W WO2023086379A2 WO 2023086379 A2 WO2023086379 A2 WO 2023086379A2 US 2022049396 W US2022049396 W US 2022049396W WO 2023086379 A2 WO2023086379 A2 WO 2023086379A2
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sequence
seq
tcr
domain
operatively linked
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WO2023086379A3 (fr
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Huan Yang
Jian Ding
Robert Hofmeister
Robert Tighe
Patrick Baeuerle
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TCR2 Therapeutics Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464436—Cytokines
    • A61K39/464438—Tumor necrosis factors [TNF], CD70
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/464468—Mesothelin [MSLN]
    • 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
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by targeting or presenting multiple antigens
    • A61K2239/28—Expressing multiple CARs, TCRs or antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector

Definitions

  • cancer immunotherapy Most patients with hematological malignancies or with late-stage solid tumors are incurable with standard therapy. In addition, traditional treatment options often have serious side effects. Numerous attempts have been made to engage a patient’s immune system for rejecting cancerous cells, an approach collectively referred to as cancer immunotherapy. However, several obstacles make it rather difficult to achieve clinical effectiveness. Although hundreds of so-called tumor antigens have been identified, these are often derived from self and thus can direct the cancer immunotherapy against healthy tissue, or are poorly immunogenic. Furthermore, cancer cells use multiple mechanisms to render themselves invisible or hostile to the initiation and propagation of an immune attack by cancer immunotherapies.
  • CAR chimeric antigen receptor
  • BCMA B-cell maturation antigen
  • TCR T cell receptor alpha and beta chains selected for a tumor-associated peptide antigen for genetically engineering autologous T cells.
  • TCR chains will form complete TCR complexes and provide the T cells with a TCR for a second defined specificity. Encouraging results were obtained with engineered autologous T cells expressing NY-ESO-1 -specific TCR alpha and beta chains in patients with synovial carcinoma.
  • Described herein are novel fusion proteins of TCR subunits, including CD3 epsilon, CD3 gamma and CD3 delta, and of TCR alpha and TCR beta chains with binding domains specific for cell surface antigens that have the potential to overcome limitations of existing approaches.
  • novel fusion proteins that more efficiently kill target cells than CARs, but release comparable or lower levels of pro-inflammatory cytokines. These fusion proteins and methods of their use represent an advantage for TFPs relative to CARs because elevated levels of these cytokines have been associated with dose-limiting toxicities for adoptive CAR-T therapies.
  • Described herein are recombinant nucleic acids comprising nucleic acids encoding T cell receptor (TCR) fusion proteins (TFPs) specific for CD70 and mesothelin (MSLN), and T cells comprising such recombinant nucleic acids and TFPs.
  • TCR T cell receptor
  • TFPs T cell receptor fusion proteins
  • MSLN mesothelin
  • TFPs that efficiently kill target cells that express CD70 or MSLN or both CD70 and MSLN. Fusion proteins and methods of their use as described herein represent an advantage relative to CARs or other engineered TCR therapies in their ability to efficiently target both CD70 and MSLN.
  • recombinant nucleic acids comprising: a first nucleic acid sequence encoding binding proteins having specificity for more than one target, and antibodies and T cell receptor (TCR) fusion proteins (TFPs) comprising such dual-specificity binding proteins.
  • TCR T cell receptor
  • TFPs T cell receptor fusion proteins
  • T cells engineered to express one or more TFPs, and methods of use thereof for the treatment of diseases.
  • the TFPs may have dual specificity on a single molecule, or in a single engineered TCR; alternatively, the dual specificity may come from mixing two engineered T cell populations comprising the TFPs, or transducing a single population of T cells with two different viruses.
  • composition comprising (I) a first recombinant nucleic acid sequence encoding a first T cell receptor (TCR) fusion protein (TFP) comprising (a) a first TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising an intracellular domain derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain; and (b) a first antibody domain comprising an anti-CD70 binding domain, wherein the first TCR subunit and the anti-CD70 binding domain are operatively linked, wherein the first TFP functionally incorporates into an endogenous T
  • composition comprising (I) a first recombinant nucleic acid sequence encoding a first T cell receptor (TCR) fusion protein (TFP) comprising (a) a first TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising an intracellular domain derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain; and a first antibody domain comprising an anti-CD70 binding domain and a second antibody domain comprising an anti-MSLN binding domain; wherein the first TCR subunit, the first antibody domain, and the second antibody domain are operatively linked, and wherein the TCR subunit, the first antibody domain, and the second
  • composition comprising a recombinant nucleic acid molecule encoding: (a) a first T cell receptor (TCR) fusion protein (TFP) comprising a first TCR subunit, a first antibody domain comprising a first antigen binding domain that is an anti-CD70 binding domain; and (b) a second T cell receptor (TCR) fusion protein (TFP) comprising a second TCR subunit, a second antibody domain comprising a second antigen binding domain that is an anti- MSLN binding domain, wherein the first TCR subunit of the first TFP and the first antibody domain are operatively linked and the second TCR subunit of the second TFP and the second antibody domain are operatively linked.
  • TCR T cell receptor
  • TFP TCR fusion protein
  • composition comprising a recombinant nucleic acid molecule encoding: a first T cell receptor (TCR) fusion protein (TFP) comprising a first TCR subunit, a first antibody domain comprising a first antigen binding domain that is an anti-CD70 binding domain and a second antibody domain comprising a second antigen binding domain that is an anti- MSLN binding domain; and wherein the first TCR subunit of the first TFP, the first antibody domain and the second antibody domain are operatively linked.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • the first TCR subunit or the second TCR subunit comprises at least a portion of a TCR extracellular domain, a transmembrane domain, and a TCR intracellular domain.
  • the TCR intracellular domain is an intracellular domain derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain.
  • the transmembrane domain is a TCR transmembrane domain from a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain.
  • the TCR extracellular domain is a full-length extracellular domain from a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain.
  • the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit or the second TCR subunit are derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain.
  • the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit or the second TCR subunit are derived only from a TCR subunit selected from the group consisting of a CD3 gamma chain and a CD3 epsilon chain.
  • the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit or the second TCR subunit are derived only from a CD3 gamma chain.
  • the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit or the second TCR subunit are derived only from a CD3 epsilon chain.
  • the first TCR subunit and the second TCR subunit are from the same TCR subunit, and wherein the same TCR subunit is a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain or a CD3 epsilon chain.
  • the same TCR subunit is CD3 epsilon chain.
  • the first TCR subunit and the second TCR subunit are different.
  • the first TCR subunit is from a CD3 epsilon chain
  • the second TCR chain is from a CD3 gamma chain
  • the first TCR subunit is from a CD3 gamma chain
  • the second TCR chain is from a CD3 epsilon chain
  • the first TFP and the second TFP incorporate into a same endogenous TCR complex when expressed in a T cell.
  • the first antibody domain or the second antibody domain is a murine, human or humanized antibody domain.
  • the first TCR subunit or the second TCR subunit comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
  • the first recombinant nucleic acid sequence and the second recombinant nucleic acid sequence are on the same nucleic acid molecule.
  • the first nucleic acid sequence and the second recombinant nucleic acid sequence are on different nucleic acid molecule.
  • the first antibody domain is connected to the TCR extracellular domain of the first TFP by a first linker sequence
  • the second antibody domain is connected to the TCR extracellular domain of the second TFP by a second linker sequence
  • both the first antibody domain is connected to the TCR extracellular domain of the first TFP by a first linker sequence and the second antibody domain is connected to the TCR extracellular domain of the second TFP by a second linker sequence.
  • the first antibody domain is connected to the N-terminus of the TCR extracellular domain of the first TFP by the first linker sequence.
  • the second antibody domain is connected to the N-terminus of the TCR extracellular domain of the second TFP by the second linker sequence.
  • the first linker sequence or the second linker sequence comprises the sequence of AAAGGGGSGGGGSGGGGSLE (SEQ ID NO: 387).
  • the first antibody domain and the second antibody domain are linked by a first linker sequence.
  • the first antibody domain or the second antibody domain is linked to the first TCR subunit by a second linker sequence.
  • the first linker sequence comprises the sequence of GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 1020).
  • the first antibody domain is connected to the N-terminus of the second antibody domain, and wherein the second antibody domain is connected to the N-terminus of the first TCR subunit.
  • the second antibody domain is connected to the N-terminus of the first antibody domain, and wherein the first antibody domain is connected to the N-terminus of the first TCR subunit.
  • the first TCR subunit of the first TFP, the second TCR subunit of the second TFP, or both comprise an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of 4- IBB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
  • the first antibody domain, the second antibody domain, or both comprise an antibody fragment.
  • the first antibody domain, the second antibody domain, or both comprise a scFv or a VH domain.
  • the second antibody domain comprises at least two anti-MSLN binding domains.
  • each of the at least two anti-MSLN binding domains is a VH domain.
  • a first anti-MSLN binding domain is connected to a second anti- MSLN binding domain by a linker.
  • the linker comprises the sequence of
  • the first anti-MSLN binding domain and the second anti-MSLN binding domain comprise the same sequence.
  • the anti-CD70 binding domain comprises (i) a light chain binding domain comprising a light chain (LC) CDR1 of SEQ ID NO:365, LC CDR2 of SEQ ID NO:366, and LC CDR3 of SEQ ID NO:367, and/or (ii) a heavy chain binding domain comprising a heavy chain (HC) CDR1 of SEQ ID NO: 361, HC CDR2 of SEQ ID NO: 362, and HC CDR3 of SEQ ID NO: 363.
  • LC light chain
  • HC heavy chain
  • the anti-CD70 binding domain comprises a light chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a light chain variable region amino acid sequence of SEQ ID NO:368, or a sequence with 95-99% identity to a light chain variable region amino acid sequence of SEQ ID NO:368.
  • the anti-CD70 binding domain comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a heavy chain variable region amino acid sequence of SEQ ID NO:364, or a sequence with 95-99% identity to a heavy chain variable region amino acid sequence of SEQ ID NO:364.
  • the anti-CD70 binding domain comprises a heavy chain comprising a heavy chain (HC) CDR1 of SEQ ID NO: 104, HC CDR2 of SEQ ID NO: 105 and HC CDR3 of SEQ ID NO: 106.
  • HC heavy chain
  • the anti-CD70 binding domain comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a heavy chain variable region amino acid sequence of SEQ ID NO: 107, or a sequence with 95-99% identity to a heavy chain variable region amino acid sequence of SEQ ID NO: 107.
  • the anti-MSLN binding domain comprises a heavy chain comprising a heavy chain (HC) CDR1 of SEQ ID NO:60, HC CDR2 of SEQ ID NO:61 and HC CDR3 of SEQ ID NO: 62, or a heavy chain comprising a HC CDR1 of SEQ ID NO: 63, HC CDR2 of SEQ ID NO: 64 and HC CDR3 of SEQ ID NO: 65.
  • HC heavy chain
  • the anti-MSLN binding domain comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a heavy chain variable region amino acid sequence of SEQ ID NO:69 or SEQ ID NO:70, or a sequence with 95-99% identity to a heavy chain variable region amino acid sequence of SEQ ID NO:69 or SEQ ID NO:70.
  • the first TFP and the second TFP 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, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • 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, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a
  • composition as described herein further comprises a sequence encoding a costimulatory domain.
  • the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA- 1 (CDl la/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
  • the recombinant nucleic acid sequence or the recombinant nucleic acid molecule further comprises a sequence encoding an intracellular signaling domain.
  • the recombinant nucleic acid sequence or the recombinant nucleic acid molecule further comprises a leader sequence.
  • the recombinant nucleic acid molecule further comprises a protease cleavage site.
  • the first recombinant nucleic acid sequence and the second recombinant nucleic acid sequence are linked by a sequence encoding a protease cleavage site.
  • the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the first TFP, the second TFP, or both.
  • the recombinant nucleic acid molecule is an mRNA.
  • the first TFP, the second TFP, or both include an immunoreceptor tyrosine-based activation motif (ITAM) of a TCR subunit that comprises an ITAM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, TAM or portion thereof of a
  • the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon.
  • the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
  • the first TFP or the second TFP does not comprise a costimulatory domain or a heterologous stimulatory domain.
  • the first TFP comprises, from the N-terminus to the C-terminus, an anti-CD70 binding domain operatively linked to the first TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit are derived only from the CD3 epsilon chain
  • the second TFP comprises, from the N-terminus to the C- terminus, an anti-MSLN binding domain operatively linked to the second TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the second TCR subunit are derived only from the CD3 gamma chain.
  • the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO: 364 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 733
  • the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 69 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 734.
  • the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1050 operatively linked to the sequence of SEQ ID NO: 1041.
  • the first TFP comprises, from the N-terminus to the C-terminus, an anti-CD70 binding domain operatively linked to the first TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit are derived only from the CD3 gamma chain
  • the second TFP comprises, from the N-terminus to the C- terminus, an anti-MSLN binding domain operatively linked to the second TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the second TCR subunit are derived only from the CD3 epsilon chain.
  • the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO: 364 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 734
  • the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 69 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 733.
  • the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1040 operatively linked to the sequence of SEQ ID NO: 1041
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1085 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1086 operatively linked to the sequence of SEQ ID NO: 1046.
  • the first TFP comprises, from the N-terminus to the C-terminus, an anti-CD70 binding domain operatively linked to the first TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit are derived only from the CD3 epsilon chain
  • the second TFP from the N-terminus to the C-terminus, comprises an anti-MSLN binding domain operatively linked to the second TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the second TCR subunit are derived only from the CD3 epsilon chain.
  • the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO: 364 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 733
  • the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 69 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 1018.
  • the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1050 operatively linked to the sequence of SEQ ID NO: 1051.
  • the first TFP comprises, from the N-terminus to the C-terminus, an anti-CD70 binding domain operatively linked to the first TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit are derived only from the CD3 epsilon chain
  • the second TFP comprises, from the N-terminus to the C- terminus, a first anti-MSLN binding domain operatively linked to a second anti-MSLN binding domain operatively linked to the second TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the second TCR subunit are derived only from the CD3 gamma chain.
  • the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO: 364 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 733
  • the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 69 operatively linked to the sequence of SEQ ID NO: 248 operatively linked to the sequence of SEQ ID NO: 69 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 734.
  • the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1056 operatively linked to the sequence of SEQ ID NO: 1057 operatively linked to the sequence of SEQ ID NO: 1050 operatively linked to the sequence of SEQ ID NO: 1041.
  • the first TFP comprises, from the N-terminus to the C-terminus, an anti-CD70 binding domain operatively linked to the first TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit are derived only from the CD3 gamma chain
  • the second TFP comprises, from the N-terminus to the C- terminus, a first anti-MSLN binding domain operatively linked to a second anti-MSLN binding domain operatively linked to the second TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the second TCR subunit are derived only from the CD3 epsilon chain.
  • the first TFP is operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of a T2A linker operatively linked to the N-terminus of the second TFP.
  • the T2A linker comprises the sequence of SEQ ID NO: 23.
  • the T2A linker is encoded by the nucleotide sequence of SEQ ID NO: 1048.
  • sequence of GSG is encoded by the nucleotide sequence of SEQ ID NO: 1047.
  • the composition comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1003-1009, 1012, 1013, and 1016.
  • the composition comprises any one of the sequences selected from SEQ ID Nos: 1003-1009, 1012, 1013, and 1016.
  • the composition comprises a sequence encoded by a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the nucleotide sequences selected from SEQ ID NOs: 1024-1030, 1033, 1034, and 1037.
  • the composition comprises a sequence encoded by any one of the nucleotide sequences selected from SEQ ID NOs: 1024-1030, 1033, 1034, and 1037.
  • the first TFP comprises, from the N-terminus to the C-terminus, an anti-CD70 binding domain operatively linked to an anti-MSLN binding domain operatively linked to the first TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit are derived only from the CD3 epsilon chain.
  • the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO: 364 operatively linked to the sequence of SEQ ID NO: 1020 operatively linked to the sequence of SEQ ID NO: 69 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 733.
  • the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1084 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046.
  • the first TFP comprises, from the N-terminus to the C-terminus, an anti-MSLN binding domain operatively linked to an anti-CD70 binding domain operatively linked to the first TCR subunit, wherein the TCR extracellular domain, the transmembrane domain, and the intracellular domain of the first TCR subunit are derived only from the CD3 epsilon chain.
  • the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO: 69 operatively linked to the sequence of SEQ ID NO: 1020 operatively linked to the sequence of SEQ ID NO: 368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO: 364 operatively linked to the sequence of SEQ ID NO: 387 operatively linked to the sequence of SEQ ID NO: 733.
  • the TFP is encoded by a nucleotide sequence comprising, from 5 ’-end to 3 ’-end, the sequence of SEQ ID NO: 1055 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1084 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046.
  • the first TFP comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 1014 or SEQ ID NO: 1015.
  • the first TFP comprises the sequence of SEQ ID NO: 1014 or SEQ ID NO: 1015.
  • the first TFP is encoded by a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 1035 or SEQ ID NO: 1036.
  • the first TFP is encoded by the sequence of SEQ ID NO: 1035 or SEQ ID NO: 1036.
  • a composition comprising a polypeptide molecule encoded by the recombinant nucleic acid molecule of the composition as described herein.
  • the polypeptide comprises a first polypeptide encoded by a first nucleic acid molecule and a second polypeptide encoded by a second nucleic acid molecule.
  • composition comprising a recombinant TFP molecule encoded by the recombinant nucleic acid molecule of the composition as described herein.
  • composition comprising a vector comprising a nucleic acid sequence encoding the polypeptide or recombinant TFP molecule as described herein.
  • the vector comprises a) a first vector comprising a first nucleic acid sequence encoding the first TFP; and b) a second vector comprising a second nucleic acid sequence encoding the second TFP.
  • the vector is selected from the group consisting of a DNA, an RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
  • composition as described herein further comprises a promoter.
  • the vector is an in vitro transcribed vector.
  • the nucleic acid molecule in the vector further encodes a poly(A) tail.
  • the nucleic acid molecule in the vector further encodes a 3’UTR.
  • the nucleic acid molecule in the vector further encodes a protease cleavage site.
  • composition comprising a cell comprising the composition as described herein.
  • the cell is a human T cell.
  • the T cell is a CD8+ or CD4+ T cell.
  • the composition as describe herein further comprises a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain.
  • the inhibitory molecule comprises a first polypeptide that comprises at least a portion of PD 1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.
  • a vector comprising the recombinant nucleic acid sequence as described herein.
  • a vector comprising the first recombinant nucleic acid sequence as described herein.
  • a vector comprising the second recombinant nucleic acid sequence as described herein.
  • a cell comprising the composition as described herein or the vector as described herein.
  • the cell is a human T cell.
  • the human T cell is a CD8+ or CD4+ T cell.
  • the cell is an alpha beta T cell.
  • the cell is not a gamma delta (y5) T cell.
  • the cell is not a V 51+ V 52- y5 T cell, not a V 5 1- V 52+ y5 T cell or not a V 51- V 52- yS T cell.
  • the cell as described herein further comprises a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain.
  • the inhibitory molecule comprises a first polypeptide that comprises at least a portion of PD 1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.
  • the cell as described herein further comprises a nucleic acid encoding an IL- 15 polypeptide or a fragment thereof. In some embodiments, the cell as described herein further comprises a nucleic acid encoding an IL-15Ra polypeptide or fragment thereof. In some embodiments, the cell as described herein further comprises a nucleic acid encoding a fusion protein comprising an IL- 15 polypeptide or a fragment thereof linked to an IL-15Ra polypeptide or a fragment thereof. In some embodiments, the IL- 15 polypeptide or the IL- 15 fusion protein is expressed on cell surface (i.e., is membrane bound) when expressed in a cell, e.g., a T cell. In some embodiments, the IL- 15 polypeptide or the IL- 15 fusion protein is secreted when expressed in a cell, e.g., a T cell.
  • a protein complex comprising: (i) a first TFP molecule comprising an anti-CD70 binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; (ii) a second TFP molecule comprising an anti-MSLN binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and (iii) at least one endogenous TCR subunit or endogenous TCR complex.
  • a protein complex comprising: (i) a TFP molecule comprising an anti-CD70 binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and (ii) at least one endogenous TCR subunit or endogenous TCR complex.
  • a protein complex comprising: (i) a TFP molecule comprising an anti-MSLN binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and (ii) at least one endogenous TCR subunit or endogenous TCR complex.
  • the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, and a CD3 delta TCR subunit.
  • the anti-CD70 binding domain, the anti-MSLN binding domain, or both are connected to the TCR extracellular domain by a linker sequence.
  • a human CD8+ or CD4+ T cell comprising at least two different TFP proteins per the protein complex as described herein.
  • a human CD8+ or CD4+ T cell comprising at least two different TFP molecules encoded by the isolated nucleic acid molecule as described herein.
  • a population of human CD8+ or CD4+ T cells wherein the T cells of the population individually or collectively comprise at least two TFP molecules, the TFP molecules comprising an anti-CD70 binding domain or an anti-MSLN binding domain, or both an anti-CD70 and an anti-MSLN binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+ or CD4+ T cell.
  • a population of human CD8+ or CD4+ T cells wherein the T cells of the population individually or collectively comprise at least two TFP molecules encoded by the recombinant nucleic acid molecule as described herein.
  • compositions comprising an effective amount of the composition as described herein, the vector as described herein, the cell as described herein, or the protein complex as described herein, and a pharmaceutically acceptable excipient.
  • composition comprising an effective amount of the cell as described herein, the population as described herein, and a pharmaceutically acceptable excipient.
  • provided herein is a method of treating a mammal having a disease associated with expression of MSLN or CD70 comprising administering to the mammal an effective amount of the composition as described herein.
  • the disease associated with CD70 or MSLN expression is selected from the group consisting of mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer), ovarian cancer, prostate cancer, colon cancer, colorectal cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, renal cancer, thyroid cancer, bladder cancer, ureter cancer, kidney cancer, endometrial cancer, esophageal cancer, gastric cancer, skin cancer, thymic carcinoma and cholangiocarcinoma.
  • lung cancer e.g., non-small cell lung cancer
  • ovarian cancer prostate cancer
  • colon cancer colorectal cancer
  • cervical cancer brain cancer
  • liver cancer pancreatic cancer
  • renal cancer thyroid cancer
  • bladder cancer ureter cancer
  • kidney cancer endometrial cancer
  • esophageal cancer gastric cancer
  • skin cancer thymic carcinoma
  • cholangiocarcinoma cholangiocarcinoma
  • the disease associated with CD70 or MSLN expression is malignant pleural mesothelioma (MPM), ovarian cancer, renal cell carcinoma, or acute myeloid leukemia (AML).
  • MPM malignant pleural mesothelioma
  • AML acute myeloid leukemia
  • the disease 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), acute myeloid leukemia (AML), 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-Hodg
  • the cells expressing a first TFP molecule and a second TFP molecule are administered in combination with an agent that increases the efficacy of a cell expressing the first TFP molecule and the second TFP molecule.
  • less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing: (a) an anti-MSLN chimeric antigen receptor (CAR); (b) an anti-CD70 CAR; (c) an anti-MSLN CAR and an anti-CD70 CAR; or (d) a combination thereof.
  • a mammal administered an effective amount of a T cell expressing: (a) an anti-MSLN chimeric antigen receptor (CAR); (b) an anti-CD70 CAR; (c) an anti-MSLN CAR and an anti-CD70 CAR; or (d) a combination thereof.
  • the cells expressing the first TFP molecule and a second TFP molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing the first TFP molecule and the second TFP molecule.
  • the cells expressing the first TFP molecule and a second TFP molecule are administered in combination with an agent that treats the disease associated with MSLN or CD70.
  • Figure 1 shows schematic illustrations of exemplary embodiments of the dual TRuC and tandem binder constructs described herein.
  • Figure 2 shows flow cytometry data collected from T cells transduced with CD70 and/or MSLN targeting mono or dual TRuCs.
  • Figure 3 shows percent cytotoxicity of target 786-0, MSTO-MSLN, or 786-O:MSTO-MSLN cells after co-culture with T cells transduced with CD70 and/or MSLN targeting mono or dual TRuCs.
  • Figure 4 shows flow cytometry data collected from T cells transduced with CD70 and/or MSLN targeting mono or dual TRuCs.
  • Figure 5 shows percent cytotoxicity of target MSTO-MSLN, 786-0, OVCAR8 and U2OS cells after co-culture with T cells transduced with CD70 and/or MSLN targeting mono or dual TRuCs.
  • Figure 6 shows flow cytometry data collected from CD4+ T cells transduced with CD70 and/or MSLN targeting mono or dual TRuCs and associated memory phenotype quantification.
  • Figure 7 shows flow cytometry data collected from CD8+ T cells transduced with CD70 and/or MSLN targeting mono or dual TRuCs and associated memory phenotype quantification.
  • Figure 8A and Figure 8B show the tumor volume (mm 3 ) over time in MSTO-MSLN ( Figure 8A) or 786-0 (Figure 8B) tumor bearing mice after administration of T cells transduced with CD70 targeting mono TRuCs (ClOe), MSLN targeting mono TRuCs (MHlg, TC-210), or CD10/MSLN targeting dual TRuCs (C10e-T2A-MHlg), or after administration of non-transduced T cells (NT) or vehicle control.
  • CD70 targeting mono TRuCs CD70 targeting mono TRuCs (ClOe), MSLN targeting mono TRuCs (MHlg, TC-210), or CD10/MSLN targeting dual TRuCs (C10e-T2A-MHlg)
  • NT non-transduced T cells
  • Figure 9A shows the fold expansion over time of non-transduced (NT) T cells or T cells transduced with CD70 targeting mono TRuCs (ClOe), MSLN targeting mono TRuCs (MHlg, TC- 210), or CD70/MSLN targeting dual TRuCs (C10e-T2A-MHlg, C10e-T2A-MHle).
  • Figure 9B shows the tumor volume (mm 3 ) in MSTO-MSLN tumor bearing mice after administration of T cells transduced with CD70 targeting mono TRuCs (ClOe), MSLN targeting mono TRuCs (MHlg, TC- 210), or CD70/MSLN targeting dual TRuCs (C10e-T2A-MHlg, C10e-T2A-MHle); or after administration of NT or vehicle control.
  • CD70 targeting mono TRuCs ClOe
  • MSLN targeting mono TRuCs MHlg, TC- 210
  • CD70/MSLN targeting dual TRuCs C10e-T2A-MHlg, C10e-T2A-MHle
  • Figure 10 shows the fold expansion overtime of non-transduced (NT) T cells or T cells transduced with CD70/MLSN dual TRuCs C10e-T2A-MHlg, C10e-T2A-MHlg Opti 1, C10e-T2A- MHlg Opti 2, ClOe-MHl tandem, MH1-C10 tandem, or C10g-T2A-MHle.
  • NT non-transduced
  • Figure 11 shows flow cytometry data collected from T cells from a representative donor, transduced with the indicated CD70/MSLN dual TRuCs.
  • the X axis indicates expression of MH1 (VHH+) and the Y axis indicates expression of CIO.
  • the MFI for MH1 in each group is provided below each flow plot.
  • MFI for C10a-T2A-MHly Optil is not applicable as surface expression as not detected.
  • Figure 12 shows flow cytometry histogram plots from T cells from a representative donor, transduced with the indicated CD70/MSLN dual TRuCs. The table in the figure provides the MFI for CIO expression in each group.
  • Figure 13 shows percent cytotoxicity of target 786-0, MSTO-MSLN, OVCAR8 and U2OS cells after co-culture at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs Opti2 (i.e., C10e-T2A-MHlg Opti2), C10-MH1 tandem, MH1-C10 tandem, ClOg- T2A-MHle, or C10e-T2A-MHlg.
  • TRuCs Opti2 i.e., C10e-T2A-MHlg Opti2
  • C10-MH1 tandem MH1-C10 tandem
  • ClOg- T2A-MHle or C10e-T2A-MHlg.
  • Figure 14A and Figure 14B show the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of MSTO-MSLN tumor cells at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs Opti2 (i.e., C10e-T2A-MHlg Opti2), C10-MH1 tandem, MH1-C10 tandem, C10g-T2A-MHle, or C10e-T2A-MHlg, for two representative donors.
  • TRuCs Opti2 i.e., C10e-T2A-MHlg Opti2
  • C10-MH1 tandem MH1-C10 tandem
  • C10g-T2A-MHle C10g-T2A-MHle
  • C10e-T2A-MHlg for two representative donors.
  • Figure 15A and Figure 15B show the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of 786-0 tumor cells at a 3: l, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs Opti2 (i.e., C10e-T2A-MHlg Opti2), C10-MH1 tandem, MH1-C10 tandem, C10g-T2A-MHle, or C10e-T2A-MHlg, for two representative donors.
  • TRuCs Opti2 i.e., C10e-T2A-MHlg Opti2
  • C10-MH1 tandem MH1-C10 tandem
  • C10g-T2A-MHle C10e-T2A-MHlg
  • Figure 16A and Figure 16B show the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of OVCAR8 tumor cells at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs Opti2 (i.e., C10e-T2A-MHlg Opti2), C10-MH1 tandem, MH1-C10 tandem, C10g-T2A-MHle, or C10e-T2A-MHlg, for two representative donors.
  • TRuCs Opti2 i.e., C10e-T2A-MHlg Opti2
  • C10-MH1 tandem MH1-C10 tandem
  • C10g-T2A-MHle C10g-T2A-MHle
  • C10e-T2A-MHlg for two representative donors.
  • Figure 17A and Figure 17B show the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of U2OS tumor cells at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs Opti2 (i.e., C10e-T2A-MHlg Opti2), C10-MH1 tandem, MH1-C10 tandem, C10g-T2A-MHle, or C10e-T2A-MHlg, for two representative donors.
  • TRuCs Opti2 i.e., C10e-T2A-MHlg Opti2
  • C10-MH1 tandem MH1-C10 tandem
  • C10g-T2A-MHle C10g-T2A-MHle
  • C10e-T2A-MHlg for two representative donors.
  • Figure 18 shows the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of MSTO-MSLN tumor cells at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells from athird donor transduced with CD70/MSLN dual targeting TRuCs Opti2 (i.e., C10e-T2A-MHlg Opti2), C10-MH1 tandem, MH1-C10 tandem, or C10e-T2A-MHlg, or mono targeting TRuCs ClOe or MHlg.
  • TRuCs Opti2 i.e., C10e-T2A-MHlg Opti2
  • C10-MH1 tandem MH1-C10 tandem
  • C10e-T2A-MHlg mono targeting TRuCs ClOe or MHlg.
  • Figure 19 provides flow cytometry data showing VHH (anti-MSLN binding domain) expression and Fab (anti-CD70 binding domain) expression on the surface of T cells transduced with C10e-T2A-MHlg, C10e-P2A-MHlg, C10e-fP2A-MHlg, MHl-MHlg, or C10e-T2A-(MHl_MHl)g.
  • Figure 20 shows percent cytotoxicity of target 786-0, MSTO-MSLN, OVCAR8 and U2OS cells after co-culture at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A-MHlg, C10e-P2A-MHlg, or C10e-T2A-(MHl_MHl)g, or with mono targeting TRuCs MHl-MHlg, ClOe, or MHlg.
  • Figure 21A shows the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of 786-0 tumor cells at a 3: l, 1 : 1, or 1 :3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A-MHlg, C10e-P2A-MHlg, or C10e-T2A-
  • FIG. 21B shows the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of MSTO-MSLN tumor cells at a 3: l, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A-MHlg, C10e-P2A-MHlg, or C10e-T2A-(MHl_MHl)g, or with mono targeting TRuCs MHl-MHlg, ClOe, or MHlg.
  • Figure 21C shows the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of OVCAR8 tumor cells at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A-MHlg, C10e-P2A-MHlg, or C10e-T2A-(MHl_MHl)g, or with mono targeting TRuCs MHl-MHlg, ClOe, or MHlg.
  • Figure 21D shows the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of U2OS tumor cells at a 3: l, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A-MHlg, C10e-P2A-MHlg, or C10e-T2A-(MHl_MHl)g, or with mono targeting TRuCs MHl-MHlg, ClOe, or MHlg.
  • Figure 22 shows schematic illustrations of exemplary dual TRuCs described herein exhibiting favorable in vitro and in vivo properties.
  • Figure 23A and Figure 23B show the IFNy, IL-2, GM-CSF, and TNFa levels in supernatants collected from co-culture of MSTO-MSLN ( Figure 23A) or 786-0 ( Figure 23B) tumor cells at a 3: 1, 1: 1, or 1:3 E:T ratio with T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A- MHlg, Opti2 (i.e., C10e-T2A-MHlg Opti2), MH1-C10 tandem, or C10g-T2A-MHle.
  • Opti2 i.e., C10e-T2A-MHlg Opti2
  • MH1-C10 tandem or C10g-T2A-MHle.
  • Figure 24 shows flow cytometry data collected from CD4+ T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A-MHlg, C10e-T2A-MHlg Opti2, MH1-C10 tandem, or C10g-T2A-MHle.
  • the flow plots show CD45RA (y-axis) and CCR7 (x-axis) staining, and the bar graph provides the memory phenotype quantification for CD4+ cells.
  • Figure 25 shows flow cytometry data collected from CD8+ T cells transduced with CD70/MSLN dual targeting TRuCs C10e-T2A-MHlg, C10e-T2A-MHlg Opti2, MH1-C10 tandem, or C10g-T2A-MHle.
  • the flow plots show CD45RA (y-axis) and CCR7 (x-axis) staining, and the bar graph provides the memory phenotype quantification for CD8+ cells.
  • Figure 26 shows the tumor volume (mm 3 ) over time in 786-0 (left panel) or MSTO-MSLN (right panel) tumor bearing mice after administration on Day 0 of vehicle control, non-transduced (NT) T cells, or T cells transduced with CD10/MSLN targeting dual TRuCs C10e-T2A-MHlg, ClOe- T2A-MHlg Opti2, MH1-C10 tandem, or C10g-T2A-MHle.
  • NT non-transduced
  • CD10/MSLN transduced with CD10/MSLN targeting dual TRuCs C10e-T2A-MHlg, ClOe- T2A-MHlg Opti2, MH1-C10 tandem, or C10g-T2A-MHle.
  • CD70 mono targeting TRuCs CIO were administered as the mono TRuC control
  • MSLN mono targeting TRuCs TC-210 were administered as the mono TRuC control.
  • compositions of matter and methods of use for the treatment of a disease such as cancer using dual specificity T cell receptor fusion proteins having activity against both CD70 and mesothelin (MSLN).
  • MSLN mesothelin
  • compositions comprising a first recombinant nucleic acid sequence encoding a first T cell receptor (TCR) fusion protein (TFP) comprising (a) a first TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising an intracellular domain derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain; and (b) a first antibody domain comprising an anti-CD70 binding domain, wherein the first TCR subunit and the anti-CD70 binding domain are operatively linked, wherein the first TFP functionally incorporates into an endogenous TCR complex when expressed in
  • composition comprising a first recombinant nucleic acid sequence encoding a first T cell receptor (TCR) fusion protein (TFP) comprising a first TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising an intracellular domain derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain; and a first antibody domain comprising an anti-CD70 binding domain and a second antibody domain comprising an anti-MSLN binding domain; wherein the first TCR subunit, the first antibody domain, and the second antibody domain are operatively linked, and wherein the first TFP functionally incorporate
  • composition comprising a recombinant nucleic acid molecule encoding: a first T cell receptor (TCR) fusion protein (TFP) comprising a first TCR subunit, a first antibody domain comprising a first antigen binding domain that is an anti-CD70 binding domain; and a second T cell receptor (TCR) fusion protein (TFP) comprising a second TCR subunit, a second antibody domain comprising a second antigen binding domain that is an anti-MSLN binding domain, wherein the first TCR subunit of the first TFP and the first antibody domain are operatively linked and the second TCR subunit of the second TFP and the second antibody domain are operatively linked.
  • TCR T cell receptor
  • TCR TCR fusion protein
  • composition comprising a recombinant nucleic acid molecule encoding: a first T cell receptor (TCR) fusion protein (TFP) comprising a first TCR subunit, a first antibody domain comprising a first antigen binding domain that is an anti-CD70 binding domain and a second antibody domain comprising a second antigen binding domain that is an anti- MSLN binding domain; and wherein the first TCR subunit of the first TFP, the first antibody domain and the second antibody domain are operatively linked.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • a protein complex comprising: a first TFP molecule comprising an anti-CD70 binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; a second TFP molecule comprising an anti-MSLN binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and at least one endogenous TCR subunit or endogenous TCR complex.
  • a protein complex comprising: a TFP molecule comprising an anti-CD70 binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and at least one endogenous TCR subunit or endogenous TCR complex.
  • a protein complex comprising: a TFP molecule comprising an anti-MSLN binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and at least one endogenous TCR subunit or endogenous TCR complex.
  • the present disclosure further provides a vector comprising the recombinant nucleic acid, a cell comprising the recombinant nucleic acid or the vector described herein, or a pharmaceutical composition comprising the cell (e.g., modified cell).
  • TCR T cell receptor
  • an element means one element or more than one element.
  • “about” can mean plus or minus less than 1 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or greater than 30 percent, depending upon the situation and known or knowable by one skilled in the art.
  • subject or “subjects” or “individuals” may include, but are not limited to, mammals such as humans or non-human mammals, e.g., domesticated, agricultural or wild, animals, as well as birds, and aquatic animals.
  • “Patients” are subjects suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.
  • treating refers to any indicia of success in the treatment or amelioration of the disease or condition. Treating can include, for example, reducing, delaying or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient.
  • treat or prevent is sometimes used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition, and contemplates a range of results directed to that end, including but not restricted to prevention of the condition entirely.
  • preventing refers to the prevention of the disease or condition, e.g. , tumor formation, in the patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present disclosure and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual.
  • the disease or condition e.g. , tumor formation
  • an “effective amount” or 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)).
  • GMCSFRa also known as CSF2RA, CD116, Cluster of Differentiation 116, CDwl l6, CSF2R, CSF2RAX, CSF2RAY, CSF2RX, CSF2RY, GM-CSF-R-alpha, GMCSFR, GMR, SMDP4, colony stimulating factor 2 receptor alpha subunit, alphaGMR, colony stimulating factor 2 receptor subunit alpha, GMR-alpha, GMCSFR-alpha, granulocyte -macrophage colony-stimulating factor receptor, as used herein, refers to a receptor for granulocyte-macrophage colony-stimulating factor, which stimulates the production of white blood cells.
  • GM-CSF and its receptor play a role in earlier stages of development.
  • GMCSFRa is associated with Surfactant metabolism dysfunction type 4.
  • GMCSFRa includes any of the recombinant or naturally-occurring forms of GMCSFRa or variants or homologs thereof that have or maintain GMCSFRa activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring GMCSFRa.
  • GMCSFRa is substantially identical to the protein identified by the UniProt reference number P 15509 or a variant or homolog having substantial identity thereto.
  • CD28 also known as Cluster of Differentiation 28, CD28, Tp44, and CD28 molecule, as used herein, refers to a protein expressed on T cells that provides co-stimulatory signals required for T cell activation and survival.
  • CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins.
  • CD28 includes any of the recombinant or naturally-occurring forms of CD28 or variants or homologs thereof that have or maintain CD28 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD28.
  • CD28 is substantially identical to the protein identified by the UniProt reference number Pl 0747 or a variant or homolog having substantial identity thereto.
  • 2A 2A self-cleaving peptide
  • 2A peptide refers to a class of peptides, which can induce ribosomal skipping during translation of a protein in a cell. These peptides share a core sequence motif of DxExNPGP, and are found in a wide range of viral families. Exemplary members of 2A include, but are not limited to, P2A, E2A, F2A, and T2A. “T2A” refers to the 2A derived from thosea asigna virus, and the sequence is EGRGSLLTCGDVEENPGP (SEQ ID NO:23).
  • P2A refers to the 2A derived from porcine teschovirus-1 2A, and the sequence is ATNFSLLKQAGDVEENPGP (SEQ ID NO: 1652).
  • E2A refers to the 2A derived from quine rhinitis A virus, and the sequence is QCTNYALLKLAGDVESNPGP (SEQ ID NO: 1653).
  • F2A is derived from foot-and-mouth disease virus 18, and the sequence is VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 1654).
  • adding the 1 linker “GSG” (Gly-Ser-Gly) on the N-terminal of a 2A peptide helps with efficiency.
  • furin cleavage site refers to a cleavage site recognized by protease enzyme furin, also known as FUR, PACE, PCSK3, SPC1, and paired basic amino acid cleaving enzyme.
  • furin is a subtilisin-like proprotein convertase family.
  • furin cleaves proteins just downstream of a basic amino acid target sequence (canonically, Arg-X-(ArgZLys) -Arg').
  • cleave or “cleavage” refer to the hydrolysis of phosphodiester bonds within the backbone of a recognition sequence within a target sequence that results in a doublestranded break within the target sequence, referred to herein as a “cleavage site”.
  • antibody refers to a protein, or polypeptide sequences derived from an immunoglobulin molecule, which specifically binds to an antigen.
  • Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.
  • antibody fragment refers to at least one portion of an antibody, or recombinant variants thereof, that contains the antigen binding domain, i.e., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope.
  • antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, single-chain (sc)Fv (“scFv”) antibody fragments, linear antibodies, single domain antibodies (abbreviated “sdAb”) (either VL or VH), camelid 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” 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.
  • an scFv may have the VL and VH 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 Vn-linker-VL.
  • the term “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 (“K”) and lambda ( ⁇ '/. ”) light chains refer to the two major antibody light chain isotypes.
  • 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 or “Ag” refers to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA.
  • any DNA which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full-length nucleotide sequence of a gene.
  • an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide.
  • a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.
  • anti-tumor effect refers to a biological effect which can be manifested by various means, including but not limited to, e.g. , a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumor cell proliferation, decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition.
  • An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the present disclosure in prevention of the occurrence of tumor in the first place.
  • autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • allogeneic 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.
  • 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, esophageal cancer, gastric cancer, unresectable ovarian cancer with relapsed or refractory disease, and the like.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • one or more amino acid residues within a TFP of the present disclosure can be replaced with other amino acid residues from the same side chain family and the altered TFP can be tested using the functional assays described herein.
  • stimulation refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex.
  • a stimulatory domain or stimulatory molecule e.g., a TCR/CD3 complex
  • signal transduction event such as, but not limited to, signal transduction via the TCR/CD3 complex.
  • Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.
  • the term “stimulatory molecule” or “stimulatory domain,” as used herein, refers to a molecule or portion thereof expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway.
  • the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • a primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or “ITAM”.
  • ITAM immunoreceptor tyrosine-based activation motif
  • T cells may recognize these complexes using their T cell receptors (TCRs).
  • TCRs T cell receptors
  • the portion of the TFP composition of the present disclosure comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), or a single chain antibody (scFv) derived from a murine, humanized or human antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci.
  • sdAb single domain antibody fragment
  • scFv single chain antibody
  • the antigen binding domain of a TFP composition of the present disclosure comprises an antibody fragment.
  • the TFP comprises an antibody fragment that comprises a scFv or a sdAb.
  • MHC molecules are typically bound by TCRs as part of peptide: MHC complex.
  • the MHC molecule may be an MHC class I or II molecule.
  • the complex may be on the surface of an antigen presenting cell, such as a dendritic cell or a B cell, or any other cell, including cancer cells, or it may be immobilized by, for example, coating on to a bead or plate.
  • HLA human leukocyte antigen system
  • MHC major histocompatibility complex
  • HLA class I antigens A, B & C
  • HLA class II antigens DP, DQ, & DR
  • HLA alleles A, B and C present peptides derived mainly from intracellular proteins, e.g., proteins expressed within the cell.
  • T cells undergo a positive selection step to ensure recognition of self MHCs followed by a negative step to remove T cells that bind too strongly to MHC which present self-antigens.
  • a positive selection step to ensure recognition of self MHCs
  • a negative step to remove T cells that bind too strongly to MHC which present self-antigens.
  • certain T cells and the TCRs they express will only recognize peptides presented by certain types of MHC molecules - i.e., those encoded by particular HLA alleles. This is known as HLA restriction.
  • one HLA allele of interest is HLA-A*0201, which is expressed in the vast majority (>50%) of the Caucasian population. Accordingly, TCRs which bind WT1 peptides presented by MHC encoded by HLA-A*0201 (i.e. are HLA-A*0201 restricted) are advantageous since an immunotherapy making use of such TCRs will be suitable for treating a large proportion of the Caucasian population.
  • HLA-A alleles of interest are HLA-A*0101, HLA-A*2402, and HLA-A*0301.
  • HLA-B alleles of interest are HLA-B*3501, HLA- B*0702 and HLA-B*3502.
  • intracellular signaling domain refers to an intracellular portion of a molecule.
  • the intracellular signaling domain generates a signal that promotes an immune effector function of the TFP containing cell, e.g., a TFP -expressing T cell.
  • immune effector function e.g., in a TFP -expressing T cell
  • the intracellular signaling domain comprises a primary intracellular signaling domain.
  • Exemplary primary intracellular signaling domains include, but are not limited to, those derived from the molecules responsible for primary stimulation, or antigen dependent simulation.
  • the intracellular signaling domain comprises a costimulatory intracellular domain.
  • Exemplary costimulatory intracellular signaling domains include, but are not limited to, those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.
  • a primary intracellular signaling domain comprises an 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, and CD66d DAP 10 and DAP 12.
  • costimulatory molecule refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
  • costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response.
  • Exemplary costimulatory molecules include, but are not limited to, an MHC class 1 molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD2, CD27, CD28, CD5, ICAM-1, LFA-1 (CDIIa/CDI8 4-1BB (CD137), IL-15Ra, IL12R, IL18R, IL21R, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, and FcyRIII.
  • MHC class 1 molecule BTLA and a Toll ligand receptor
  • CD2 CD27, CD28, CD5, ICAM-1, LFA-1 (CDIIa/CDI8 4-1BB (CD137), IL-15Ra, IL12R, IL18R, IL21R, ICOS (CD278),
  • a costimulatory intracellular signaling domain is the intracellular portion of a costimulatory molecule.
  • a costimulatory molecule is represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
  • the intracellular signaling domain comprises the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.
  • 4-1BB refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g, mouse, rodent, monkey, ape and the like; and a “4-1BB costimulatory domain,” as used herein, refers to amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g. , mouse, rodent, monkey, ape and the like. .
  • 4-1BB also known as TNFRSF9, 4-1BB, CD137, Cluster of Differentiation 137, CDwl37, ILA, tumor necrosis factor receptor superfamily member 9, and TNF receptor superfamily member 9, as used herein, includes any of the recombinant or naturally-occurring forms of 4- IBB or variants or homologs thereof that have or maintain 4-1BB activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring 4-1BB.
  • 4- IBB is substantially identical to the protein identified by the UniProt reference number Q07011 or a variant or homolog having substantial identity thereto.
  • 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.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain one or more introns.
  • CD3 or “Cluster of Differentiation 3,” as used herein, refers to a protein complex and T cell co-receptor that is involved in activating both the cytotoxic T cell and T helper cells. In some embodiments, it is composed of four distinct chains. For example, in some embodiments, the complex contains a CD3y chain, a CD35 chain, and two CD3a chains in mammals.
  • CD3s.' “CD3a chain,” or “T-cell surface glycoprotein CD3 epsilon chain,” as used herein, includes any of the recombinant or naturally-occurring forms of CD3s or variants or homologs thereof that have or maintain CD3a activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD3s.
  • CD3s is substantially identical to the protein identified by the UniProt reference number P07766 or a variant or homolog having substantial identity thereto.
  • “e” is used interchangeably with “a” for example to refer to the CD3 epsilon, e.g.
  • CD3 epsilon subunit to which a binding domain provided herein is linked may be used interchangeably to refer to an MH1 (anti-MSLN) binding domain linked to a CD3a chain.
  • MHle and MH Is may be used interchangeably to refer to an MH1 (anti-MSLN) binding domain linked to a CD3a chain.
  • ClOe and CIOs may be used interchangeably to refer to a CIO (anti-CD70) binding domain linked to a CD3a chain.
  • ‘CD35,” “CD35 chain,” or “T-cell surface glycoprotein CD3 delta chain,” as used herein, includes any of the recombinant or naturally-occurring forms of CD35 or variants or homologs thereof that have or maintain CD35 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 CD35.
  • CD35 is substantially identical to the protein identified by the UniProt reference number P04234 or a variant or homolog having substantial identity thereto.
  • CD3y includes any of the recombinant or naturally-occurring forms of CD3y or variants or homologs thereof that have or maintain CD3y activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD3y.
  • CD3y is substantially identical to the protein identified by the UniProt reference number P09693 or a variant or homolog having substantial identity thereto.
  • “g” is used interchangeably with “y” for example to refer to the CD3 gamma subunit, e.g., the CD3 gamma subunit to which a binding domain provided herein is linked.
  • “MHlg” and “MHly” may be used interchangeably to refer to an MH1 (anti-MSUN) binding domain linked to a CD3y chain.
  • “ClOg” and “ClOy” may be used interchangeably to refer to a CIO (anti-CD70) binding domain linked to a CD3y chain.
  • mesothelin also known as MPF and SMRP, refers to a tumor differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. In some embodiments, mesothelin is over-expressed in several human tumors, including mesothelioma and ovarian and pancreatic adenocarcinoma.
  • MSLN includes any of the recombinant or naturally-occurring forms of MSLN or variants or homologs thereof that have or maintain MSLN activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring MSLN.
  • MSLN is substantially identical to the protein identified by the UniProt reference number QI 3421 or a variant or homolog having substantial identity thereto.
  • 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.
  • CD70 also known as CD27LG and TNFSF7, as referred herein, refers to a cytokine that is the ligand for CD27.
  • the CD70-CD27 pathway plays an important role in the generation and maintenance of T cell immunity, in particular, during antiviral responses.
  • CD70 induces the proliferation of co-stimulated T-cells and enhances the generation of cytolytic T-cells.
  • CD70 includes any of the recombinant or naturally-occurring forms of CD70 or variants or homologs thereof that have or maintain CD70 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD70.
  • CD70 is substantially identical to the protein identified by the UniProt reference number P32970 or a variant or homolog having substantial identity thereto.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • expression refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
  • transfer vector refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “transfer vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like.
  • Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.
  • lentivirus refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).
  • lentivirus vectors that may be used in the clinic include, but are not limited to, e.g., the LENTIVECTORTM gene delivery technology from Oxford BioMedica, the LENTIMAXTM vector system from Lentigen, and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
  • homologous refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules.
  • two nucleic acid molecules such as, two DNA molecules or two RNA molecules
  • polypeptide molecules between two polypeptide molecules.
  • a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non- human immunoglobulin.
  • humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • a humanized antibody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance.
  • the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Human or “fully human” immunoglobulin refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • A refers to adenosine
  • C refers to cytosine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • 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.
  • nucleotide refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, intratumoral, or infusion techniques.
  • peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • promoter refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
  • promoter/regulatory sequence refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. 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.
  • 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.
  • 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 (Gly ⁇ Scr).
  • linkers described in WO2012/138475 are linkers described in WO2012/138475 (incorporated herein by reference).
  • a 5’ cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the “front” or 5’ end of a eukaryotic messenger RNA shortly after the start of transcription.
  • the 5 ’ cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co- transcriptionally, such that each influences the other.
  • RNA polymerase Shortly after the start of transcription, the 5 ’ end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction.
  • the capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.
  • vitro transcribed RNA refers to RNA, preferably mRNA, which has been synthesized in vitro.
  • the in vitro transcribed RNA is generated from an in vitro transcription vector.
  • the in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.
  • a “poly(A)” refers to a series of adenosines attached by polyadenylation to the mRNA.
  • the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400.
  • Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.
  • polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
  • mRNA messenger RNA
  • the 3’ poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase.
  • poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal.
  • Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm.
  • the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase.
  • the cleavage site is usually characterized by the presence of the base sequence AAUAAA (SEQ ID NO: 1655) near the cleavage site.
  • adenosine residues are added to the free 3’ end at the cleavage site.
  • transient refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.
  • signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • cell surface receptor includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.
  • a “substantially purified” cell refers to a cell that is essentially free of other cell types.
  • a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
  • a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state.
  • the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.
  • terapéutica as used herein means a treatment.
  • a therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.
  • prophylaxis means the prevention of or protective treatment for a disease or disease state.
  • tumor antigen or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” refers to antigens that are common to specific hyperproliferative disorders.
  • the hyperproliferative disorder antigens of the present disclosure are derived from, cancers including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, NHL, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, esophageal cancer, gastric cancer, unresectable ovarian cancer with relapsed or refractory disease.
  • cancers including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, NHL, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ova
  • transfected or “transformed” or “transduced,” as used herein, refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • the term “specifically binds,” as used herein, refers to an antibody, an antibody fragment or a specific ligand, which recognizes and binds a cognate binding partner (e.g., CD70 or mesothelin (MSLN)) present in a sample, but which does not necessarily and substantially recognize or bind other molecules in the sample.
  • a cognate binding partner e.g., CD70 or mesothelin (MSLN)
  • a functional disruption refers to a physical or biochemical change to a specific (e.g. , target) nucleic acid (e.g. , gene, RNA transcript, of protein encoded thereby) that prevents its normal expression and/or behavior in the cell.
  • a functional disruption refers to a modification of the gene via a gene editing method.
  • a functional disruption prevents expression of a target gene (e.g. , an endogenous gene).
  • the term “meganuclease” refers to an endonuclease that binds double -stranded DNA at a recognition sequence that is greater than 12 base pairs.
  • the recognition sequence for a meganuclease of the present disclosure is 22 base pairs.
  • a meganuclease may be an endonuclease that is derived from I-Crel and may refer to an engineered variant of I-Crel that has been modified relative to natural I-Crel with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties.
  • a meganuclease binds to double -stranded DNA as a heterodimer or as a “single-chain meganuclease” in which a pair of DNA-binding domains are joined into a single polypeptide using a peptide linker.
  • meganucleases are substantially non-toxic when expressed in cells, particularly in human T cells, such that cells may be transfected and maintained at 37°C without observing deleterious effects on cell viability or significant reductions in meganuclease cleavage activity when measured using the methods described herein.
  • single-chain meganuclease refers to a polypeptide comprising a pair of nuclease subunits joined by a linker.
  • a single-chain meganuclease has the organization: N- terminal subunit - Linker - C-terminal subunit.
  • the two meganuclease subunits may generally be non-identical in amino acid sequence and may recognize non-identical DNA sequences.
  • single-chain meganucleases typically cleave pseudo- palindromic or non-palindromic recognition sequences.
  • a single-chain meganuclease may be referred to as a “single-chain heterodimer” or “single-chain heterodimeric meganuclease” although it is not, in fact, dimeric.
  • the term “meganuclease” can refer to a dimeric or single-chain meganuclease.
  • TALEN refers to an endonuclease comprising a DNA-binding domain comprising 16-22 TAL domain repeats fused to any portion of the Fokl nuclease domain.
  • Compact TALEN refers to an endonuclease comprising a DNA- binding domain with 16-22 TAL domain repeats fused in any orientation to any catalytically active portion of nuclease domain of the I-Tevl homing endonuclease.
  • CRISPR refers to a caspase-based endonuclease comprising a caspase, such as Cas9, and a guide RNA that directs DNA cleavage of the caspase by hybridizing to a recognition site in the genomic DNA.
  • megaTAL refers to a single-chain nuclease comprising a transcription activator-like effector (TALE) DNA binding domain with an engineered, sequencespecific homing endonuclease.
  • TALE transcription activator-like effector
  • T cell receptor and “T cell receptor complex” are used interchangeably to refer to a molecule found on the surface of T cells that is, in general, responsible for recognizing antigens.
  • the TCR comprises a heterodimer consisting of a TCR alpha and TCR beta chain in 95% of T cells, whereas 5% of T cells have TCRs consisting of TCR gamma and TCR delta chains.
  • the TCR further comprises one or more of CD3a, CD3y, and CD38.
  • the TCR comprises CD3a.
  • the TCR comprises CD3y.
  • the TCR comprises CD35.
  • the TCR comprises CD3 ⁇ .
  • the constant domain of TCR alpha has a sequence of SEQ ID NO:711.
  • the constant domain of TCR alpha has an IgC domain having a sequence of SEQ ID NO:712, a transmembrane domain having a sequence of SEQ ID NO:713, and an intracellular domain having a sequence of SS.
  • the constant domain of TCR beta has a sequence of SEQ ID NO:715.
  • the constant domain of TCR beta has an IgC domain having a sequence of SEQ ID NO:716, a transmembrane domain having a sequence of SEQ ID NO:717, and an intracellular domain having a sequence of SEQ ID NO:719.
  • the constant domain of TCR delta has a sequence of SEQ ID NO:725.
  • the constant domain of TCR delta has an IgC domain having a sequence of SEQ ID NO:726, a transmembrane domain having a sequence of SEQ ID NO:727, and an intracellular domain having a sequence of L.
  • the constant domain of TCR gamma has a sequence of SEQ ID NO:721 .
  • the constant domain of TCR gamma has an IgC domain having a sequence of SEQ ID NO:722, a transmembrane domain having a sequence of SEQ ID NO:723, and an intracellular domain having a sequence of SEQ ID NO:724.
  • CD3 epsilon has a sequence of SEQ ID NO:694.
  • CD3 epsilon has an extracellular domain having a sequence of SEQ ID NO:696, a transmembrane domain having a sequence of SEQ ID NO:697, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO:698.
  • CD3 delta has a sequence of SEQ ID NO:704.
  • CD3 delta has an extracellular domain having a sequence of SEQ ID NO:706, a transmembrane domain having a sequence of SEQ ID NO:707, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO:708.
  • CD3 gamma has a sequence of SEQ ID NO:699.
  • CD3 gamma has an extracellular domain having a sequence of SEQ ID NO:701, a transmembrane domain having a sequence of SEQ ID NO:702, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO:703.
  • Ranges throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6.
  • a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98- 99% identity. This applies regardless of the breadth of the range.
  • compositions of matter and methods of use for the treatment of a disease such as cancer using the composition as provided herein.
  • 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
  • TFPs provide substantial benefits as compared to Chimeric Antigen Receptors.
  • the term “Chimeric Antigen Receptor” or alternatively 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.
  • 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.
  • T cell receptor (TCR) fusion proteins (TFP) T cell receptor (TCR) fusion proteins (TFP)
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD70, , e.g., human CD70, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the present disclosure also encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to mesothelin (MSLN), e.g., human MSLN, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • MSLN mesothelin
  • the present disclosure further encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises an antibody fragment that binds specifically to CD70, , e.g., human CD70, and/or mesothelin (MSLN), e.g., human MSLN, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • MSLN mesothelin
  • the TFPs provided herein are able to associate with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR subunits in order to form a functional TCR complex.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 gamma chain, a CD3 delta chain and a CD3 epsilon chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a TCR subunit selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain and a TCR epsilon chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a TCR alpha chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a TCR beta chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a TCR gamma chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a TCR delta chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a CD3 gamma chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a CD3 delta chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the first TFP are derived only from a CD3 epsilon chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a TCR alpha chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a TCR beta chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a TCR gamma chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a TCR delta chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a CD3 gamma chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a CD3 delta chain.
  • the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit of the second TFP are derived only from a CD3 epsilon chain.
  • the first TFP, the second TFP, or both incorporate into a TCR or functionally interact with a TCR when expressed in a T cell.
  • the first TFP, the second TFP, or both incorporate into a TCR or functionally interact with a TCR when expressed in a T cell.
  • the encoded first antigen binding domain is connected to the TCR extracellular domain of the first TFP by a first linker sequence
  • the encoded second antigen binding domain is connected to the TCR extracellular domain of the second TFP by a second linker sequence
  • both the first antigen binding domain is connected to the TCR extracellular domain of the first TFP by the first linker sequence
  • the encoded second antigen binding domain is connected to the TCR extracellular domain of the second TFP by the second linker sequence.
  • the TCR subunit of the first TFP, the TCR subunit of the second TFP, or both comprise a TCR extracellular domain.
  • the TCR subunit of the first TFP, the TCR subunit of the second TFP, or both comprise a TCR transmembrane domain.
  • the TCR subunit of the first TFP, the TCR subunit of the second TFP, or both comprise a TCR intracellular domain.
  • the TCR subunit of the first TFP, the TCR subunit of the second TFP, or both comprise (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
  • the TCR subunit of the first TFP, the TCR subunit of the second TFP, or both comprise a TCR intracellular domain comprising a stimulatory domain selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one modification thereto.
  • the TCR subunit of the first TFP, the TCR subunit of the second TFP, or both comprise an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of 4- IBB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
  • the first human or humanized antibody domain, the second human or humanized antibody domain, or both comprise an antibody fragment.
  • the first human or humanized antibody domain, the second human or humanized antibody domain, or both comprise a scFv or a VH domain.
  • the composition encodes (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 of a light chain binding domain amino acid sequence with 70-100% sequence identity to a light chain sequence of Table 1, and/or (ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of a heavy chain sequence of Table 1.
  • LC light chain
  • HC heavy chain
  • the composition encodes a light chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a light chain variable region amino acid sequence of Table 1, or a sequence with 95- 99% identity to a light chain variable region amino acid sequence of Table 1.
  • the composition encodes a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a heavy chain variable region amino acid sequence of Table 1, or a sequence with 95-99% identity to a heavy chain variable region amino acid sequence of Table 1.
  • the encoded first TFP, the encoded second TFP, or both 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, 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 encoded first TFP and the encoded second TFP 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, 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 encoded first TFP and the encoded second TFP 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, 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 composition further comprises a sequence encoding a costimulatory domain.
  • the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA- 1 (CDl la/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
  • composition further comprises comprising a sequence encoding an intracellular signaling domain
  • the composition further comprises a leader sequence.
  • the composition further comprises a protease cleavage site.
  • the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the first TFP, the second TFP, or both.
  • the isolated nucleic acid molecule is an mRNA.
  • the first TFP, the second TFP, or both include an immunoreceptor tyrosine-based activation motif (ITAM) of a TCR subunit that comprises an ITAM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, TAM or portion thereof of a
  • the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon.
  • the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
  • the composition further comprises a leader sequence.
  • composition comprising a polypeptide molecule encoded by the nucleic acid molecule of a composition described herein.
  • the polypeptide comprises a first polypeptide encoded by a first nucleic acid molecule and a second polypeptide encoded by a second nucleic acid molecule.
  • composition comprising a recombinant TFP molecule encoded by the nucleic acid molecule of a composition described herein.
  • composition comprising a vector comprising a nucleic acid molecule encoding a polypeptide or recombinant TFP molecule described herein.
  • the vector comprises a) a first vector comprising a first nucleic acid molecule encoding the first TFP; and b) a second vector comprising a second nucleic acid molecule encoding the second TFP.
  • the vector is selected from the group consisting of a DNA, an RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
  • the vector further comprises a promoter.
  • the vector is an in vitro transcribed vector.
  • the nucleic acid molecule in the vector further encodes a poly(A) tail.
  • the nucleic acid molecule in the vector further encodes a 3’UTR.
  • the nucleic acid molecule in the vector further encodes a protease cleavage site.
  • composition comprising a cell comprising a composition described herein.
  • the cell is a human T cell.
  • the T cell is a CD8+ or CD4+ T cell.
  • the composition further comprises a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain.
  • the inhibitory molecule comprises a first polypeptide that comprises at least a portion of PD 1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.
  • provided herein is a method of treating a mammal having a disease associated with expression of MSLN or CD70 comprising administering to the mammal an effective amount of a composition described herein.
  • the disease associated with CD70 or MSLN, expression is selected from the group consisting of a proliferative disease, a cancer, a malignancy, myelodysplasia, a myelodysplastic syndrome, a preleukemia, a non-cancer related indication associated with expression of CD70, a non-cancer related indication associated with expression of MSLN, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, esophageal cancer, gastric cancer and unresectable ovarian cancer with relapsed or refractory disease.
  • the disease 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 cells expressing a first TFP molecule and a second TFP molecule are administered in combination with an agent that increases the efficacy of a cell expressing the first TFP molecule and the second TFP molecule.
  • cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing: an anti-MSLN chimeric antigen receptor (CAR); an anti-CD70 CAR; an anti-MSLN CAR and an anti-CD70 CAR; or a combination thereof.
  • the cells expressing the first TFP molecule and a second TFP molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing the first TFP molecule and the second TFP molecule.
  • the cells expressing the first TFP molecule and a second TFP molecule are administered in combination with an agent that treats the disease associated with MSLN or CD70.
  • an agent that treats the disease associated with MSLN or CD70 is administered in combination with an agent that treats the disease associated with MSLN or CD70.
  • the TCR subunit comprises a TCR extracellular domain.
  • the TCR subunit comprises a TCR transmembrane domain. In yet other embodiments, the TCR subunit comprises a TCR intracellular domain. In further embodiments, the TCR subunit comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
  • the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one, two or three modifications thereto.
  • the TCR subunit comprises an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of 4- IBB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one, two or three modifications thereto.
  • the human or humanized antibody domain comprises an antibody fragment. In some embodiments, the human or humanized antibody domain comprises a scFv or a VH domain.
  • the isolated nucleic acid molecules comprise (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 of any anti -tumor-associated antigen light chain binding domain amino acid sequence provided herein, and/or (ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of any anti-tumor-associated antigen heavy chain binding domain amino acid sequence provided herein.
  • LC light chain
  • HC CDR1 heavy chain binding domain amino acid sequence provided herein
  • the light chain variable region comprises an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein.
  • the heavy chain variable region comprises an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications 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.
  • the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma, or a functional fragment thereof, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto.
  • the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta chain of the TCR or TCR subunits CD3 epsilon, CD3 gamma and CD3 delta, or a functional fragment thereof, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto.
  • the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the TCR or CD3 epsilon, CD3 gamma and CD3 delta CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a functional fragment thereof, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto.
  • a protein selected from the group consisting of the alpha, beta or zeta chain of the TCR or CD3 epsilon, CD3 gamma and CD3 delta CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a functional fragment thereof, or an amino acid sequence having at
  • the encoded anti-tumor-associated antigen binding domain is connected to the TCR extracellular domain by a linker sequence.
  • the isolated nucleic acid molecules further comprise a sequence encoding a costimulatory domain.
  • the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la/CD18), ICOS (CD278), and 4-1BB (CD137), or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto.
  • the isolated nucleic acid molecules further comprise a leader sequence.
  • isolated polypeptide molecules encoded by any of the previously described nucleic acid molecules.
  • isolated T cell receptor fusion protein (TFP) molecules that comprise a human or humanized anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain.
  • the isolated TFP molecules comprises an antibody or antibody fragment comprising a human or humanized anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain.
  • the anti-tumor-associated antigen binding domain is a scFv or a VH domain.
  • the anti-tumor-associated antigen binding domain comprises a light chain and a heavy chain of an amino acid sequence provided herein, or a functional fragment thereof, or an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications 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.
  • the isolated TFP molecules comprise a TCR extracellular domain that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto.
  • the anti-tumor-associated antigen binding domain is connected to the TCR extracellular domain by a linker sequence.
  • the isolated TFP molecules further comprise a sequence encoding a costimulatory domain. In other embodiments, the isolated TFP molecules further comprise a sequence encoding an intracellular signaling domain. In yet other embodiments, the isolated TFP molecules further comprise a leader sequence.
  • vectors that comprise a nucleic acid molecule encoding any of the previously described TFP molecules.
  • the vector is selected from the group consisting of a DNA, an RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector.
  • the vector 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.
  • cells that comprise any of the described vectors.
  • the cell is a human T cell. In some embodiments, the cell is a CD8+ or CD4+ T cell. In other embodiments, the cells further comprise a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain. In some instances, the inhibitory molecule comprises a first polypeptide that comprises at least a portion of PD1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.
  • TFP molecules that comprise a human or humanized anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
  • TFP molecules that comprise a human or humanized anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally integrating into an endogenous TCR complex.
  • human CD8+ or CD4+ T cells that comprise at least two TFP molecules, the TFP molecules comprising a human or humanized anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+ or CD4+ T cell.
  • protein complexes that comprise i) a TFP molecule comprising a human or humanized anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and ii) at least one endogenous TCR complex.
  • the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma.
  • the anti-tumor-associated antigen binding domain is connected to the TCR extracellular domain by a linker sequence.
  • Also provided herein are human CD8+ or CD4+ T cells that comprise at least two different TFP proteins per any of the described protein complexes.
  • a population of human CD8+ or CD4+ T cells wherein the T cells of the population individually or collectively comprise at least two TFP molecules, the TFP molecules comprising a human or humanized anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+ or CD4+ T cell.
  • a population of human CD8+ or CD4+ T cells wherein the T cells of the population individually or collectively comprise at least two TFP molecules encoded by an isolated nucleic acid molecule provided herein.
  • RNA-engineered cells comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, where the RNA comprises a nucleic acid encoding any of the described TFP molecules.
  • kits for providing an anti-tumor immunity in a mammal that comprise administering to the mammal an effective amount of a cell expressing any of the described TFP molecules.
  • the cell is an autologous T cell.
  • the cell is an allogeneic T cell.
  • the mammal is a human.
  • the disease associated with tumor-associated antigen expression is selected from a proliferative disease such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia, or is a non-cancer related indication associated with expression of tumor-associated antigen.
  • the disease is a hematologic cancer selected from the group consisting of one or more acute leukemias including but not limited to B-cell acute lymphoid leukemia (“B-ALL”), T cell acute lymphoid leukemia (“T-ALL”), acute lymphoblastic leukemia (ALL); one or more chronic leukemias including but not limited to chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematologic cancers or hematologic conditions including, but not limited to 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 myel
  • the cells expressing any of the described TFP molecules are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule. In some embodiments, the cells expressing any of the described TFP molecules are administered in combination with an agent that treats the disease associated with tumor-associated antigen.
  • 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 portion of the TFP comprising the antigen binding domain comprises an antigen binding domain that targets CD70.
  • the antigen binding domain targets human CD70.
  • the portion of the TFP comprising the antigen binding domain comprises an antigen binding domain that targets MSLN.
  • the antigen binding domain targets human MSLN.
  • the portion of the TFP comprising the antigen binding domain comprises an antigen binding domain that targets CD70 and an antigen binding domain that targets MSLN.
  • the antigen binding domains target human CD70 and human MSLN.
  • 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 (VHH) of a camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen binding domain, such as a recombinant fibronectin domain, anticalin, DARPIN and the like.
  • VH heavy chain variable domain
  • V L light chain variable domain
  • VHH variable domain
  • a natural or synthetic ligand specifically recognizing and binding the target antigen can be used as antigen binding domain for the TFP.
  • the antigen-binding domain comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment.
  • the murine, humanized or human anti-TAA binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a murine, humanized or human anti-TAA binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a murine, humanized or human anti-CD70 binding domain and/or anti- MSLN binding domain as described herein, e.g.
  • a murine, humanized or human anti-CD70 binding domain and/or anti- MSLN binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs.
  • the murine, humanized or human anti-CD70 binding domain and/or anti- MSLN binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a murine, humanized or human anti- TAA binding domain described herein, e.g., the murine, humanized or human anti-TAA binding domain has two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein.
  • HC CDR1 heavy chain complementary determining region 1
  • HC CDR2 heavy chain complementary determining region 2
  • HC CDR3 heavy chain complementary determining region 3
  • the murine, humanized or human anti-TAA binding domain comprises a humanized or human light chain variable region described herein and/or a murine, humanized or human heavy chain variable region described herein.
  • the murine, humanized or human anti-TAA binding domain comprises a murine, humanized or human heavy chain variable region described herein, e.g., at least two murine, humanized or human heavy chain variable regions described herein.
  • the anti-TAA binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence provided herein.
  • the anti-TAA binding domain (e.g., an scFv or VHH nb) comprises: a light chain variable region as provided herein or 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 as provided herein or 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.
  • the murine, humanized or human anti-TAA binding domain is a scFv, and a light chain variable region comprising an amino acid sequence described herein, is attached to a heavy chain variable region comprising an amino acid sequence described herein, via a linker, e.g., a linker described herein.
  • the murine, humanized, or human anti-TAA binding domain includes a (Gly4-Ser) n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4.
  • the light chain variable region and heavy chain variable region of a scFv can be, e.g., in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.
  • the linker sequence comprises a long linker (LL) sequence.
  • the linker sequence comprises a short linker (SL) sequence.
  • the antigen binding domain is an antibody or a fragment thereof. In some embodiments, the antigen binding domain is a camelid antibody or a binding fragment thereof. In some embodiments, the antigen binding domain is a murine antibody or a binding fragment thereof. In some embodiments, the antigen binding domain is a human or humanized antibody or a binding fragment thereof. In some embodiments, the antigen binding domain is a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain. In some embodiments, the sdAb is a VHH.
  • scFv single-chain variable fragment
  • sdAb single domain antibody
  • the antigen-binding domain comprises an anti-MSLN 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, 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 antigen-binding domain comprises an anti-MSLN single domain antibody or an antibody fragment comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71.
  • the antigen-binding domain comprises an anti-MSLN single domain antibody or an antibody fragment having a CDR1, a CDR2, and a CDR3 of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71.
  • the antigen-binding domain comprises an anti- MSLN single domain antibody or an antibody fragment comprising the sequence of SEQ ID NO:69, SEQ ID NO: 70, or SEQ ID NO:71.
  • the antigen-binding domain comprises an anti-MSLN single domain antibody or an antibody fragment having any one of a CDR1, a CDR2, and a CDR3 sequences listed in Table 1. In some embodiments, the antigen-binding domain comprises an anti-MSLN single domain antibody or an antibody fragment having a CDR1, a CDR2, and a CDR3 of any one of the anti-MSLN single domain antibodies listed in Table 1.
  • the antigen-binding domain comprises an anti-MSLN single domain antibody or an antibody fragment comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the anti-MSLN single domain antibodies listed in Table 1.
  • the antigen-binding domain comprises an anti-MSLN single domain antibody or an antibody fragment comprising any one of the anti-MSLN single domain antibodies listed in Table 1.
  • the antigen-binding domain comprises an anti- MSLN single chain Fv (scFv) or an antibody fragment thereof.
  • the anti-MSLN scFv or antibody fragment thereof can comprise a heavy chain complementary determining region 1 (CDRH1), a CDRH2, and a CDRH3 sequences listed in Table 1.
  • the anti-MSLN scFv or antibody fragment thereof can comprise a light chain complementary determining region 1 (CDRL1), a CDRL2, and a CDRL3 sequences listed in Table 1.
  • the antigen-binding domain comprises an anti- MSLN single chain Fv (scFv) or an antibody fragment thereof.
  • the anti-MSLN scFv or antibody fragment thereof can comprise a CDRH1, a CDRH2, and a CDRH3 of any one of the anti-MSLN single domain antibodies listed in Table 1.
  • the anti-MSLN scFv or antibody fragment thereof can comprise a CDRL1, a CDRL2, and a CDRL3 sequences listed in of any one of the anti-MSLN single domain antibodies listed in Table 1.
  • the anti-MSLN scFv or antibody fragment thereof can comprise a heavy chain variable (VH) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the anti-MSLN scFv VH sequences listed in Table 1.
  • VH heavy chain variable
  • the anti-MSLN scFv or antibody fragment thereof can comprise a light chain variable (VL) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the anti-MSLN scFv VL sequences listed in Table 1.
  • the anti-MSLN scFv or antibody fragment thereof can comprise a heavy chain variable (VH) domain having any one of the anti-MSLN scFv VH sequences listed in Table 1.
  • the anti-MSLN scFv or antibody fragment thereof can comprise a light chain variable (VL) domain having any one of the anti-MSLN scFv VL sequences listed in Table 1.
  • the antigen-binding domain comprises an anti-CD70 humanized or human single domain antibody or an antibody fragment having a CDR1 of SEQ ID NO: 88, a CDR2 of SEQ ID NO:89, and a CDR3 of SEQ ID NOVO, or a CDR1 of SEQ ID NO:92, a CDR2 of SEQ ID NO : 93 , and a CDR3 of SEQ ID NO : 94, or a CDR1 of SEQ ID NO : 96, a CDR2 of SEQ ID NO : 97, and a CDR3 of SEQ ID NO:98, or a CDR1 of SEQ ID NO: 100, a CDR2 of SEQ ID NO: 101, and a CDR3 of SEQ ID NO: 102, or a CDR1 of SEQ ID NO: 104, a CDR2 of SEQ ID NO: 105, and a CDR3 of SEQ ID NO: 106, or a CDR1 of SEQ ID NO:
  • the anti-CD70 single domain antibody or an antibody fragment comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NOVI, 95, 99, 103, 107, 111, 115, 119, or 123.
  • the anti-CD70 single domain antibody or an antibody fragment comprises the sequence of SEQ ID NOVI, 95, 99, 103, 107, 111, 115, 119, or 123.
  • the antigen-binding domain comprises an anti-CD70 single chain Fv (scFv) or an antibody fragment thereof.
  • the anti-CD70 scFv or antibody fragment thereof can comprise a heavy chain complementary determining region 1 (CDRH1) having a sequence of SEQ ID NO:361, a CDRH2 having a sequence of SEQ ID NO:362, and a CDRH3 having a sequence of SEQ ID NOs: 363.
  • the anti-CD70 scFv or antibody fragment thereof can comprise a light chain complementary determining region 1 (CDRL1) having a sequence of SEQ ID NO:365 , a CDRL2 having a sequence of SEQ ID NO:366, and a CDRL3 having a sequence of SEQ ID NO:367.
  • the anti-CD70 scFv or antibody fragment thereof can comprise a heavy chain variable (VH) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:364.
  • the anti-CD70 scFv or antibody fragment thereof can comprise a light chain variable (VL) domain having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:368.
  • the antigen-binding domain comprises an anti-CD70 single domain antibody or an antibody fragment having any one of a CDR1, a CDR2, and a CDR3 sequences listed in Table 1. In some embodiments, the antigen-binding domain comprises an anti-CD70 single domain antibody or an antibody fragment having a CDR1, a CDR2, and a CDR3 of any one of the anti-CD70 single domain antibodies listed in Table 1.
  • the antigen-binding domain comprises an anti-CD70 single domain antibody or an antibody fragment comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the anti-CD70 single domain antibodies listed in Table 1.
  • the antigen-binding domain comprises an anti-CD70 single domain antibody or an antibody fragment comprising any one of the anti-CD70 single domain antibodies listed in Table 1.
  • the antigen-binding domain comprises an anti- MSLN 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), a CDRH2, and a CDRH3 sequences listed in Table 1.
  • the anti-CD70 scFv or antibody fragment thereof can comprise a light chain complementary determining region 1 (CDRL1), a CDRL2, and a CDRL3 sequences listed in Table 1.
  • the antigen-binding domain comprises an anti- MSLN single chain Fv (scFv) or an antibody fragment thereof.
  • the anti-CD70 scFv or antibody fragment thereof can comprise a CDRH1, a CDRH2, and a CDRH3 of any one of the anti-CD70 single domain antibodies listed in Table 1.
  • the anti-CD70 scFv or antibody fragment thereof can comprise a CDRL1, a CDRL2, and a CDRL3 sequences listed in of any one of the anti-CD70 single domain antibodies listed in Table 1.
  • 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%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the anti-CD70 scFv VH sequences listed in Table 1.
  • 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%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the anti-CD70 scFv VL sequences listed in Table 1.
  • the anti-CD70 scFv or antibody fragment thereof can comprise a heavy chain variable (VH) domain having any one of the anti-CD70 scFv VH sequences listed in Table 1.
  • the anti-CD70 scFv or antibody fragment thereof can comprise a light chain variable (VL) domain having any one of the anti-CD70 scFv VL sequences listed in Table 1.
  • a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof.
  • the antigen binding domain is humanized.
  • a humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g., European Patent Nos.
  • framework substitutions are identified by methods well-known in the art, e.g. , by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.)
  • a humanized antibody or antibody fragment has one or more amino acid residues remaining in it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • humanized antibodies or antibody fragments comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions wherein the amino acid residues comprising the framework are derived completely or mostly from human germline.
  • variable domains both light and heavy
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity.
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety).
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • the same framework may be used for several different humanized antibodies (see, e.g., Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference herein in their entirety).
  • the framework region e.g., all four framework regions, of the heavy chain variable region are derived from a VH4-4- 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.
  • a humanized antibody or antibody fragment may retain a similar antigenic specificity as the original antibody, e.g. , in the present disclosure, the ability to bind human a tumor associated antigen (TAA).
  • TAA tumor associated antigen
  • a humanized antibody or antibody fragment may have improved affinity and/or specificity of binding to, e.g., human CD 19, human BCMA, or another tumor associated antigen.
  • the anti -tumor-associated antigen binding domain is a fragment, e.g., a single chain variable fragment (scFv) or a camelid heavy chain (VHH).
  • the anti -TAA binding domain is a Fv, a Fab, a (Fab’)2, or a bi-fimctional (e.g. bi-specific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)).
  • the antibodies and fragments thereof of the present disclosure binds a tumor-associated antigen protein with wild-type or enhanced affinity.
  • a target antigen e.g., MSLN, CD70, or any target antigen described elsewhere herein for targets of fusion moiety binding domains
  • VH domains and scFvs can be prepared according to method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • scFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers.
  • the scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The linker length can greatly affect how the variable regions of a scFv fold and interact.
  • linker orientation and size see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT publication Nos. W02006/020258 and W02007/024715, each of which is incorporated herein by reference.
  • An scFv can comprise a linker of about 10, 11, 12, 13, 14, 15 or greater than 15 residues between its VL and VH regions.
  • the linker sequence may comprise any naturally occurring amino acid.
  • the linker sequence comprises amino acids glycine and serine.
  • the linker sequence comprises sets of glycine and serine repeats such as (Gly4Ser) n , where n is a positive integer equal to or greater than 1.
  • the linker can be (Gly4Ser)4 or (Gly4Ser)3. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.
  • the antigen binding domain described herein can be a camelid antibody or binding fragment thereof.
  • the antigen binding domain can be a murine antibody or binding fragment thereof.
  • the antigen binding domain can be a human or humanized antibody or binding fragment thereof.
  • the antigen binding domain can be a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain.
  • the antigen binding domain is an scFv as listed in Table 1.
  • the antigen binding domain can be a single domain antibody (sdAb).
  • the sdAb can be a VHH.
  • the antigen binding domain is a VHH listed in Table 1.
  • an anti-tumor-associated antigen binding domain e.g., scFv molecules (e.g., soluble scFv)
  • scFv molecules e.g., soluble scFv
  • biophysical properties e.g., thermal stability
  • the humanized or human scFv 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 in the described assays.
  • the improved thermal stability of the anti-TAA (tumor-associated antigen) binding domain e.g., scFv 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 anti-tumor-associated antigen binding domain, e.g., scFv or sdAb can be improved by at least about 2 °C or 3 °C as compared to a conventional antibody.
  • the anti-tumor-associated antigen binding domain, e.g., scFv has a 1 °C improved thermal stability as compared to a conventional antibody.
  • the anti-tumor-associated antigen binding domain e.g., scFv
  • the scFv 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 molecules as described herein and scFv molecules or Fab fragments of an antibody from which the scFv VH and VL were derived.
  • Thermal stability can be measured using methods known in the art. For example, in one embodiment, TM can be measured. Methods for measuring TM and other methods of determining protein stability are described in more detail below.
  • Mutations in antibody sequences alter the stability of the antibody or fragment thereof and improve the overall stability of the antibody and the anti-tumor-associated antigen TFP construct. Stability of the humanized antibody or fragment thereof is compared against the murine antibody or fragment thereof using measurements such as TM, temperature denaturation and temperature aggregation.
  • the anti-tumor-associated antigen binding domain e.g., a scFv or sdAb, comprises at least one mutation arising from the humanization process such that the mutated scFv confers improved stability to the anti-TAA TFP construct.
  • the anti-TAA binding domain e.g., scFv or sdAb
  • the anti-TAA binding domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations arising from the humanization process such that the mutated scFv or sdAb confers improved stability to the TAA-TFP construct.
  • the antigen binding domain of the TFP comprises an amino acid sequence that is homologous to an antigen binding domain amino acid sequence described herein, and the antigen binding domain retains the desired functional properties of the anti-tumor-associated antigen antibody fragments described herein.
  • the TFP composition of the present disclosure comprises an antibody fragment.
  • that antibody fragment comprises a scFv or sdAb.
  • the antigen binding domain of the TFP is engineered by modifying one or more amino acids within one or both variable regions (e.g., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions.
  • the TFP composition of the present disclosure comprises an antibody fragment.
  • that antibody fragment comprises a scFv or sdAb.
  • the antibody or antibody fragment of the present disclosure may further be modified such that they vary in amino acid sequence (e.g., from wild-type), but not in desired activity.
  • additional nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues may be made to the protein.
  • a nonessential amino acid residue in a molecule may be replaced with another amino acid residue from the same side chain family.
  • a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members, e.g., a conservative substitution, in which an amino acid residue is replaced with an amino acid residue having a similar side chain, may be made.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid
  • Percent identity in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences that are the same. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 70%, 71% , 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math.
  • BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • the present disclosure contemplates modifications of the starting antibody or fragment (e.g., scFv or sdAb) amino acid sequence that generate functionally equivalent molecules.
  • the VH or VL of an anti-tumor-associated antigen binding domain, e.g., scFv or sdAb, comprised in the TFP can be modified to retain at least about 70%, 71%. 72%.
  • 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% or 99% identity of the starting TFP construct.
  • the extracellular domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any protein, but in particular a membrane -bound or transmembrane protein. In one aspect, the extracellular domain is capable of associating with the transmembrane domain.
  • An extracellular domain of particular use in this present disclosure may include at least the extracellular region(s) of e.g.
  • the extracellular domain is a TCR extracellular domain.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain.
  • the TCR extracellular domain comprises the extracellular portion of a constant (an IgC) domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain.
  • the extracellular domain comprises, or comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
  • the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% 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,
  • the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular portion of a constant (an IgC) domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma.
  • the extracellular domain comprises a sequence encoding the extracellular portion of a constant (an IgC) domain of TCR alpha, TCR beta, TCR delta, or TCR gamma having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C- terminus.
  • a constant (an IgC) domain of TCR alpha, TCR beta, TCR delta, or TCR gamma having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C- terminus.
  • the extracellular domain comprises, or comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
  • the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% 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 bland 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.
  • the extracellular domain can comprise a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the extracellular domain can comprise a full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the extracellular domain can comprise a fragment (e.g, functional fragment) of the full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the 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 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.
  • 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.
  • the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the extracellular region.
  • the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the intracellular region.
  • the transmembrane domain is one that is associated with one of the other domains of the TFP is used.
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.
  • the transmembrane domain is capable of homodimerization with another TFP on the TFP T cell surface.
  • the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same TFP.
  • the transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane -bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TFP has bound to a target.
  • the TCR- integrating subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a TCR zeta chain, a CD3 epsilon, 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% or more sequence identity to a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the transmembrane domain comprises a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
  • the transmembrane domain can be attached to the extracellular region of the TFP, e.g. , the antigen binding domain of the TFP, via a hinge, e.g. , a hinge from a human protein.
  • a hinge e.g. , a hinge from a human protein.
  • the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge.
  • a short oligo- or polypeptide linker may form the linkage between the binding element and the TCR extracellular domain of the TFP.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the linker may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more in length.
  • the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:690) or a sequence (GGGGS)x wherein X is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more (SEQ ID NO:732).
  • X is 2.
  • X is 4.
  • the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO:691).
  • the cytoplasmic domain of the TFP can include an intracellular domain.
  • the intracellular domain is from CD3 gamma, CD3 delta, CD3 epsilon, TCR alpha, TCR beta, TCR gamma, or TCR delta.
  • the intracellular domain comprises a signaling domain, if the TFP contains CD3 gamma, delta or epsilon polypeptides; TCR alpha, TCR beta, TCR gamma, and TCR delta subunits generally have short (e.g., 1-19 amino acids in length) intracellular domains and are generally lacking in a signaling domain.
  • An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the TFP has been introduced. While the intracellular domains of TCR alpha, TCR beta, TCR gamma, and TCR delta do not have signaling domains, the TCR that forms with any TCR fusion protein is able to recruit proteins having a primary intracellular signaling domain described herein, e.g., CD3 zeta, which functions as an intracellular signaling domain.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
  • intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • intracellular domains for use in the TFP of the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that are able to act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.
  • TCR T cell receptor
  • 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% 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% or more sequence identity to a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the intracellular domain comprises a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
  • naive T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic domain, e.g., a costimulatory domain).
  • a primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • ITAMs containing primary intracellular signaling domains that are of particular use in the present disclosure 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), 0X40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like.
  • CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human TFP-T cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al., Blood. 2012; 119(3):696-706).
  • the intracellular signaling sequences within the cytoplasmic portion of the TFP of the present disclosure may be linked to each other in a random or specified order.
  • a short oligo- or polypeptide linker for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences.
  • a glycine-serine doublet can be used as a suitable linker.
  • a single amino acid e.g., an alanine, a glycine, can be used as a suitable linker.
  • the 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., MSLN, or CD70) or a different target (e.g., Nectin-4, CD19, or MUC16).
  • a second TFP e.g., a second TFP that includes a different antigen binding domain, e.g., to the same target (e.g., MSLN, or CD70) or a different target (e.g., Nectin-4, 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., PD1
  • PD1 can, in some embodiments, decrease the ability of a modified T cell to mount an immune effector response.
  • inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD 160, 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 PD1, 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 PD1, 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 signal
  • the agent comprises a first polypeptide of PD 1 or a fragment thereof (e.g., at least a portion of an extracellular domain of PD1), 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).
  • PD1 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 PD1, PD-L1 and PD-L2, have been shown to downregulate T cell activation upon binding to PD1 (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 (PD1) can be fused to a transmembrane domain and optionally an intracellular signaling domain such as 4 IBB and CD3 zeta (also referred to herein as a PD1 TFP).
  • the PD 1 TFP when used in combinations with the TFP as described herein, improves the persistence of the T cell.
  • the TFP is a PD1 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.
  • 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 an anti-CD70 binding domain and/or an anti- MSLN binding domain described herein, and a second cell expressing a TFP having a binding domain specifically targeting a different antigen, e.g., a binding domain described herein that differs from the anti-CD70 binding domain and/or the anti- MSLN 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 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 e.g., can, in some embodiments, decrease the ability of a modified T cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD 160, 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.
  • TFP constructs can be generated as previously described.
  • An anti -MSLN or CD 19 binder can be linked to a CD3 or TCR DNA fragment by either a DNA sequence encoding a short linker (SL): AAAGGGGSGGGGSGGGGSLE (SEQ ID NO:387) or a long linker (LL): AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE (SEQ ID NO:388) into pLRPO or p510 vector.
  • the TFP used is TC-210 (e.g., an anti-MSLN MHle VHH antibody linked to CD3 epsilon) having the sequence of SEQ ID NO: 195.
  • the TFP used is TC-110 (e.g., an anti-CD19 FMC63 scFv antibody linked to CD3 epsilon) having the sequence of SEQ ID NO: 196.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • the first TFP comprises a GM-CSF signal peptide, an anti-CD70 light chain variable region, a whitlow linker, an anti-CD70 heavy chain variable region, an A3(G4S)3LE Linker, and CD3-epsilon polypeptide sequence.
  • the first TFP comprises, from the N-terminus to the C-terminus, a GM-CSF signal peptide operatively linked to an anti-CD70 light chain variable region operatively linked to a whitlow linker operatively linked to an anti-CD70 heavy chain variable region operatively linked to an A3(G4S)3LE Linker operatively linked to CD3-epsilon polypeptide sequence.
  • the first TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:368, the sequence of SEQ ID NO: 1021, the sequence of SEQ ID NO:364, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:733.
  • the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO:364 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:733.
  • the first TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1038, the sequence of SEQ ID NO: 1042, the sequence of SEQ ID NO: 1043, the sequence of SEQ ID NO: 1044, the sequence of SEQ ID NO: 1045, and the sequence of SEQ ID NO: 1046.
  • the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046.
  • the first TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1049, the sequence of SEQ ID NO: 1042, the sequence of SEQ ID NO: 1043, the sequence of SEQ ID NO: 1044, the sequence of SEQ ID NO: 1045, and the sequence of SEQ ID NO: 1046.
  • the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046.
  • the first TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1060, the sequence of SEQ ID NO: 1061, the sequence of SEQ ID NO: 1062, the sequence of SEQ ID NO: 1063, the sequence of SEQ ID NO: 1064, and the sequence of SEQ ID NO: 1065.
  • the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1060 operatively linked to the sequence of SEQ ID NO: 1061 operatively linked to the sequence of SEQ ID NO: 1062 operatively linked to the sequence of SEQ ID NO: 1063 operatively linked to the sequence of SEQ ID NO: 1064 operatively linked to the sequence of SEQ ID NO: 1065.
  • the first TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1072, the sequence of SEQ ID NO: 1073, the sequence of SEQ ID NO: 1074, the sequence of SEQ ID NO: 1075, the sequence of SEQ ID NO: 1076, and the sequence of SEQ ID NO: 1077.
  • the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1072 operatively linked to the sequence of SEQ ID NO: 1073 operatively linked to the sequence of SEQ ID NO: 1074 operatively linked to the sequence of SEQ ID NO: 1075 operatively linked to the sequence of SEQ ID NO: 1076 operatively linked to the sequence of SEQ ID NO: 1077.
  • the first TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1038, the sequence of SEQ ID NO: 1042, the sequence of SEQ ID NO: 1043, the sequence of SEQ ID NO: 1044, the sequence of SEQ ID NO: 1040, and the sequence of SEQ ID NO: 1041.
  • the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1040 operatively linked to the sequence of SEQ ID NO: 1041.
  • the first TFP comprises a GM-CSF signal peptide, an anti-CD70 light chain variable region, a whitlow linker, an anti-CD70 heavy chain variable region, an A3(G4S)3LE Linker, and CD3-gamma polypeptide sequence.
  • the first TFP comprises, from the N-terminus to the C-terminus, a GM-CSF signal peptide operatively linked to an anti-CD70 light chain variable region operatively linked to a whitlow linker operatively linked to an anti-CD70 heavy chain variable region operatively linked to an A3(G4S)3LE Linker operatively linked to CD3-gamma polypeptide sequence.
  • the first TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:368, the sequence of SEQ ID NO: 1021, the sequence of SEQ ID NO:364, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:734.
  • the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO:364 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:734.
  • the second TFP comprises a GM-CSF signal peptide, an anti-MSLN sdAb, an A3(G4S)3LE Linker, and CD3-gamma polypeptide.
  • the second TFP comprises, from the N-terminus to the C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN sdAb operatively linked to an A3(G4S)3LE Linker operatively linked to CD3-gamma polypeptide.
  • the second TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:69, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:734.
  • the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:69 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:734.
  • the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1038, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1040, and the sequence of SEQ ID NO: 1041.
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1038 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1040 operatively linked to the sequence of SEQ ID NO: 1041.
  • the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1049, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1050, and the sequence of SEQ ID NO: 1041.
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1050 operatively linked to the sequence of SEQ ID NO: 1041.
  • the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1049, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1045, and the sequence of SEQ ID NO: 1041.
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1041.
  • the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1055, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1050, and the sequence of SEQ ID NO: 1041.
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1055 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1050 operatively linked to the sequence of SEQ ID NO: 1041.
  • the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1068, the sequence of SEQ ID NO: 1069, the sequence of SEQ ID NO: 1070, and the sequence of SEQ ID NO: 1071.
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1068 operatively linked to the sequence of SEQ ID NO: 1069 operatively linked to the sequence of SEQ ID NO: 1070 operatively linked to the sequence of SEQ ID NO: 1071.
  • the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1080, the sequence of SEQ ID NO: 1081, the sequence of SEQ ID NO: 1082, and the sequence of SEQ ID NO: 1083.
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1080 operatively linked to the sequence of SEQ ID NO: 1081 operatively linked to the sequence of SEQ ID NO: 1082 operatively linked to the sequence of SEQ ID NO: 1083.
  • the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1085, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1086, and the sequence of SEQ ID NO: 1046.
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1085 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1086 operatively linked to the sequence of SEQ ID NO: 1046.
  • the second TFP comprises a GM-CSF signal peptide, an anti-MSLN sdAb, an A3(G4S)3LE Linker, and CD3-epsilon polypeptide.
  • the second TFP comprises, from the N-terminus to the C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN sdAb operatively linked to an A3(G4S)3LE Linker operatively linked to CD3-epsilon polypeptide.
  • the second TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:69, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO: 1018.
  • the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:69 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO: 1018.
  • the second TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:70, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:733.
  • the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:70 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:733.
  • the second TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:69, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:733.
  • the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:69 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:733.
  • the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1049, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1050, and the sequence of SEQ ID NO: 1051.
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1050 operatively linked to the sequence of SEQ ID NO: 1051.
  • the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1055, the sequence of SEQ ID NO: 1059, the sequence of SEQ ID NO: 1045, and the sequence of SEQ ID NO: 1046.
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1055 operatively linked to the sequence of SEQ ID NO: 1059 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046.
  • the second TFP comprises a GM-CSF signal peptide, a first anti- MSLN sdAb, a TLGM Linker, a first anti-MSLN sdAb, an A3(G4S)3LE Linker, and CD3-gamma polypeptide.
  • the second TFP comprises, from the N-terminus to the C-terminus, a GM-CSF signal peptide operatively linked to a first anti-MSLN sdAb operatively linked to a TLGM Linker operatively linked to a first anti-MSLN sdAb operatively linked to an A3(G4S)3LE Linker operatively linked to CD3 -gamma polypeptide.
  • the second TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:69, the sequence of SEQ ID NO:248, the sequence of SEQ ID NO:69, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:734.
  • the second TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:69 operatively linked to the sequence of SEQ ID NO:248 operatively linked to the sequence of SEQ ID NO:69 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:734.
  • the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1049, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1056, the sequence of SEQ ID NO: 1057, the sequence of SEQ ID NO: 1050, and the sequence of SEQ ID NO: 1041.
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1049 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1056 operatively linked to the sequence of SEQ ID NO: 1057 operatively linked to the sequence of SEQ ID NO: 1050 operatively linked to the sequence of SEQ ID NO: 1041.
  • the second TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1055, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1058, the sequence of SEQ ID NO: 1057, the sequence of SEQ ID NO: 1045, and the sequence of SEQ ID NO: 1041.
  • the second TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1055 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1058 operatively linked to the sequence of SEQ ID NO: 1057 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1041.
  • the first TFP is operatively linked to the second TFP by the sequence of GSG operatively linked to a T2A linker.
  • the first TFP is operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the T2A linker operatively linked to the N-terminus of the second TFP.
  • the second TFP is operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the T2A linker operatively linked to the N-terminus of the first TFP.
  • the T2A linker comprises the sequence of SEQ ID NO:23.
  • the T2A linker is encoded by the nucleotide sequence of SEQ ID NO: 1048.
  • the T2A linker is encoded by the nucleotide sequence of SEQ ID NO: 1067.
  • the T2A linker is encoded by the nucleotide sequence of SEQ ID NO: 1079.
  • sequence of GSG is encoded by the nucleotide sequence of SEQ ID NO: 1047.
  • sequence of GSG is encoded by the nucleotide sequence of SEQ ID NO: 1066.
  • sequence of GSG is encoded by the nucleotide sequence of SEQ ID NO: 1078.
  • the first TFP is operatively linked to the second TFP by a furin cleavage site operatively linked to the sequence of GSG operatively linked to a T2A linker.
  • the first TFP is operatively linked to the N-terminus of the furin cleavage site operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the T2A linker operatively linked to the N-terminus of the second TFP.
  • the second TFP is operatively linked to the N-terminus of the furin cleavage site operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the T2A linker operatively linked to the N-terminus of the first TFP.
  • the furin cleavage site comprises the sequence of SEQ ID NO: 1019.
  • the furin cleavage site is encoded by the nucleotide sequence of SEQ ID NO: 1052.
  • the T2A linker comprises the sequence of SEQ ID NO:23.
  • the T2A linker is encoded by the nucleotide sequence of SEQ ID NO: 1048.
  • sequence of GSG is encoded by the nucleotide sequence of SEQ ID NO: 1053.
  • the first TFP is operatively linked to the second TFP by the sequence of GSG operatively linked to a P2A linker.
  • the first TFP is operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the P2A linker operatively linked to the N-terminus of the second TFP.
  • the second TFP is operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the P2A linker operatively linked to the N-terminus of the first TFP.
  • the P2A linker comprises the sequence of SEQ ID NO: 1652.
  • the P2A linker is encoded by the nucleotide sequence of SEQ ID NO: 1054.
  • sequence of GSG is encoded by the nucleotide sequence of SEQ ID NO: 1053.
  • the first TFP is operatively linked to the second TFP by a furin cleavage site operatively linked to the sequence of GSG operatively linked to a P2A linker.
  • the first TFP is operatively linked to the N-terminus of the furin cleavage site operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the P2A linker operatively linked to the N-terminus of the second TFP.
  • the second TFP is operatively linked to the N-terminus of the furin cleavage site operatively linked to the N-terminus of the sequence of GSG operatively linked to the N-terminus of the P2A linker operatively linked to the N-terminus of the first TFP.
  • the furin cleavage site comprises the sequence of SEQ ID NO: 1019.
  • the furin cleavage site is encoded by the nucleotide sequence of SEQ ID NO: 1052.
  • the P2A linker comprises the sequence of SEQ ID NO: 1652.
  • the P2A linker is encoded by the nucleotide sequence of SEQ ID NO: 1054.
  • sequence of GSG is encoded by the nucleotide sequence of SEQ ID NO: 1053.
  • the first TFP comprises having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 1002.
  • the first TFP comprises the sequence of SEQ ID NO: 1002.
  • the first TFP is encoded by a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 1023.
  • the first TFP is encoded by a nucleotide sequence having the sequence of SEQ ID NO: 1023.
  • the second TFP comprises having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1001, 1010, and 1011.
  • the second TFP comprises any one of the sequences selected from SEQ ID NOs: 1001, 1010, and 1011.
  • the second TFP is encoded by a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1022, 1031, and 1032.
  • the second TFP is encoded by a nucleotide sequence having any one of the sequences selected from SEQ ID NOs: 1022, 1031, and 1032.
  • the composition comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1003-1009, 1012, 1013, and 1016.
  • the composition comprises any one of the sequences selected from SEQ ID Nos: 1003-1009, 1012, 1013, and 1016.
  • the composition comprises a sequence encoded by a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the nucleotide sequences selected from SEQ ID NOs: 1024-1030, 1033, 1034, and 1037.
  • the composition comprises a sequence encoded by any one of the nucleotide sequences selected from SEQ ID NOs: 1024-1030, 1033, 1034, and 1037.
  • the TFP comprises a GM-CSF signal peptide, an anti-CD70 light chain variable region, a whitlow linker, an anti-CD70 heavy chain variable region, a G4S5 Linker, an anti- MSLN sdAb, an A3(G4S)3LE Linker, and CD3-epsilon polypeptide.
  • the TFP comprises, from the N-terminus to the C-terminus, a GM-CSF signal peptide operatively linked to an anti-CD70 light chain variable region operatively linked to a whitlow linker operatively linked to an anti-CD70 heavy chain variable region operatively linked to a G4S5 Linker operatively linked to an anti-MSLN sdAb operatively linked to an A3(G4S)3LE Linker operatively linked to CD3 -epsilon polypeptide.
  • the TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:368, the sequence of SEQ ID NO: 1021, the sequence of SEQ ID NO:364, the sequence of SEQ ID NO: 1020, the sequence of SEQ ID NO:69, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO:733.
  • the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:69 operatively linked to the sequence of SEQ ID NO: 1020 operatively linked to the sequence of SEQ ID NO:368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO:364 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:733.
  • the composition comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1003-1009, 1012, 1013, and 1016.
  • the composition comprises any one of the sequences selected from SEQ ID NOs: 1003-1009, 1012, 1013, and 1016.
  • the composition comprises a sequence encoded by a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the nucleotide sequences selected from SEQ ID NOs: 1024-1030, 1033, 1034, and 1037.
  • the composition comprises a sequence encoded by any one of the nucleotide sequences selected from SEQ ID NOs: 1024-1030, 1033, 1034, and 1037.
  • the first TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1038, the sequence of SEQ ID NO: 1042, the sequence of SEQ ID NO: 1043, the sequence of SEQ ID NO: 1044, the sequence of SEQ ID NO: 1084, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1045, and the sequence of SEQ ID NO: 1046.
  • the first TFP is encoded by a nucleotide sequence comprising, from 5’-end to 3’-end, the sequence of SEQ ID NO: 1055 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1084 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046.
  • the first TFP comprises a GM-CSF signal peptide, an anti-MSLN sdAb, a G4S5 Linker, an anti-CD70 light chain variable region, a whitlow linker, an anti-CD70 heavy chain variable region, an A3(G4S)3LE Linker, and CD3-epsilon polypeptide.
  • the first TFP comprises, from the N-terminus to the C-terminus, a GM-CSF signal peptide operatively linked to an anti-MSLN sdAb operatively linked to a G4S5 Linker operatively linked to an anti-CD70 light chain variable region operatively linked to a whitlow linker operatively linked to an anti-CD70 heavy chain variable region operatively linked to an A3(G4S)3LE Linker operatively linked to CD3 -epsilon polypeptide.
  • the first TFP comprises the sequence of SEQ ID NO: 1017, the sequence of SEQ ID NO:69, the sequence of SEQ ID NO: 1020, the sequence of SEQ ID NO:368, the sequence of SEQ ID NO: 1021, the sequence of SEQ ID NO:364, the sequence of SEQ ID NO:387, and the sequence of SEQ ID NO: 733.
  • the first TFP comprises, from the N-terminus to the C-terminus, the sequence of SEQ ID NO: 1017 operatively linked to the sequence of SEQ ID NO:69 operatively linked to the sequence of SEQ ID NO: 1020 operatively linked to the sequence of SEQ ID NO:368 operatively linked to the sequence of SEQ ID NO: 1021 operatively linked to the sequence of SEQ ID NO:364 operatively linked to the sequence of SEQ ID NO:387 operatively linked to the sequence of SEQ ID NO:733.
  • the first TFP is encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1055, the sequence of SEQ ID NO: 1039, the sequence of SEQ ID NO: 1084, the sequence of SEQ ID NO: 1042, the sequence of SEQ ID NO: 1043, the sequence of SEQ ID NO: 1044, the sequence of SEQ ID NO: 1045, and the sequence of SEQ ID NO: 1046.
  • the TFP is encoded by a nucleotide sequence comprising, from 5 ’-end to 3 ’-end, the sequence of SEQ ID NO: 1055 operatively linked to the sequence of SEQ ID NO: 1039 operatively linked to the sequence of SEQ ID NO: 1084 operatively linked to the sequence of SEQ ID NO: 1042 operatively linked to the sequence of SEQ ID NO: 1043 operatively linked to the sequence of SEQ ID NO: 1044 operatively linked to the sequence of SEQ ID NO: 1045 operatively linked to the sequence of SEQ ID NO: 1046.
  • the first TFP comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 1014 or SEQ ID NO: 1015.
  • the first TFP comprises the sequence of SEQ ID NO: 1014 or SEQ ID NO: 1015.
  • the first TFP is encoded by a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 1035 or SEQ ID NO: 1036.
  • the first TFP is encoded by the sequence of SEQ ID NO: 1035 or SEQ ID NO: 1036.
  • the present disclosure also provides nucleic acid molecules encoding one or more TFP constructs described herein.
  • the nucleic acid molecule is provided as a messenger RNA transcript.
  • the nucleic acid molecule is provided as a DNA construct.
  • 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 TFPs include methods for producing in vitro transcribed RNA encoding TFPs.
  • the present disclosure also includes a TFP encoding RNA construct that can be directly transfected into a cell.
  • 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.
  • RNA so produced can efficiently transfect different kinds of cells.
  • the template includes sequences for the TFP.
  • the TFP is encoded by a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the mRNA encoding the TFP is introduced into a T cell for production of a TFP-T cell.
  • the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection.
  • the RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template.
  • PCR polymerase chain reaction
  • 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.
  • 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.
  • the RNA preferably has 5’ and 3’ UTRs.
  • 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.
  • 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.
  • the use of 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. For example, it is known that AU-rich elements in 3’UTR sequences can decrease the stability of mRNA. Therefore, 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.
  • a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
  • 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 (Schenbom and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003)).
  • 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)).
  • RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence.
  • 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. Hum Gene Ther., 12(8): 861 -70 (2001)).
  • the recombinant nucleic acid molecule described herein can further comprise a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain, a TCR alpha constant domain and a TCR beta constant domain, a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain.
  • the TCR subunit and the antigen binding domain e.g., an anti-CD70 binding domain or antibody domain, and/or an anti- MSLN binding domain or antibody domain
  • the TFP can functionally incorporate into a TCR complex (e.g., an endogenous TCR complex) when expressed in a T cell.
  • the sequence encoding the TFP and the sequence encoding the TCR constant domain can be contained within a same nucleic acid molecule.
  • the sequence encoding the TFP and the sequence encoding the TCR constant domain can be contained within different nucleic acid molecules.
  • the sequence can further encode a cleavage site (e.g., a protease cleavage site) between the encoded TFP and the TCR constant domain.
  • the cleavage site can be a protease cleavage site.
  • the cleavage site can be a self-cleaving peptide such as a T2A, P2A, E2A or F2A cleavage site.
  • the constant domain can comprise a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant domain can comprise a full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant domain can comprise a fragment (e.g., functional fragment) of the full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant domain can comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the sequence encoding the TCR constant domain can further encode the transmembrane domain and/or intracellular region of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the sequence encoding the TCR constant domain can encode a full-length constant region of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant region of a TCR chain can comprise a constant domain, a transmembrane domain, and an intracellular region.
  • the constant region of a TCR chain can also exclude the transmembrane domain and the intracellular region of the TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain described herein can be derived from various species.
  • the TCR chain can be a murine or human TCR chain.
  • the constant domain can comprise a constant domain of a murine or human TCR alpha chain, TCR beta chain, TCR gamma chain or TCR delta chain.
  • the constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.
  • the constant domain can comprise a sequence or fragment thereof of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO : 711 , SEQ ID NO : 715 , SEQ ID NO : 211 , SEQ ID NO : 721 , SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.
  • the constant domain described herein can be a human TCR alpha constant domain.
  • the human TCR alpha constant domain can comprise a sequence of SEQ ID NO:711 .
  • the human TCR alpha constant domain can comprise truncations, additions, or substitutions of a sequence of SEQ ID NO:711 .
  • the human TCR alpha constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:711.
  • the human TCR alpha constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:711.
  • the human TCR alpha constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:711.
  • the human TCR alpha constant domain can comprise a sequence or fragment thereof of SEQ ID NO:711.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:711.
  • the human TCR alpha constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:711.
  • the human TCR alpha constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:711.
  • the constant domain described herein can be a human TCR beta constant domain.
  • the human TCR beta constant domain can comprise a sequence of SEQ ID NO:715 or SEQ ID NO:211.
  • the human TCR beta constant domain can comprise truncations, additions, or substitutions of a sequence of SEQ ID NO:715 or SEQ ID NO:211.
  • the human TCR beta constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:715 or SEQ ID NO:211.
  • the human TCR beta constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:715 or SEQ ID NO:211 .
  • the human TCR beta constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:715 or SEQ ID NO:211.
  • the human TCR beta constant domain can comprise a sequence or fragment thereof of SEQ ID NO:715 or SEQ ID NO:211.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:715 or SEQ ID NO:211.
  • the human TCR beta constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:715 or SEQ ID NO:211.
  • the human TCR beta constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO : 715 or SEQ ID NO : 211.
  • the constant domain described herein can be a human TCR gamma constant domain.
  • the human TCR gamma constant domain can comprise a sequence of SEQ ID NO:721.
  • the human TCR gamma constant domain can comprise truncations, additions, or substitutions of a sequence of SEQ ID NO:721.
  • the human TCR gamma constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:721.
  • the human TCR gamma constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:721.
  • the human TCR gamma constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:721.
  • the human TCR gamma constant domain can comprise a sequence or fragment thereof of SEQ ID NO:721.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:721.
  • the human TCR gamma constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:721.
  • the human TCR gamma constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:721.
  • the constant domain described herein can be a human TCR delta constant domain.
  • the human TCR delta constant domain can comprise a sequence of SEQ ID NO:725.
  • the human TCR delta constant domain can comprise truncations, additions, or substitutions of a sequence of SEQ ID NO:725.
  • the human TCR delta constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:725.
  • the human TCR delta constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:725.
  • the human TCR delta constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:725.
  • the human TCR delta constant domain can comprise a sequence or fragment thereof of SEQ ID NO:725.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:725.
  • the human TCR delta constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:725.
  • the human TCR delta constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO: 725.
  • the constant domain described herein can be a murine TCR alpha constant domain.
  • the murine TCR alpha constant domain can comprise a sequence of SEQ ID NO:212 or SEQ ID NO:213.
  • 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 SEQ ID NO:212 or SEQ ID NO:213.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:212 or SEQ ID NO:213.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:212 or SEQ ID NO:213.
  • the constant domain can comprise a sequence or fragment thereof of SEQ ID NO:212 or SEQ ID NO:213.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:212 or SEQ ID NO:213.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:212 or SEQ ID NO:213.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:212 or SEQ ID NO:213.
  • the constant domain described herein can be a murine TCR beta constant domain.
  • the murine TCR beta constant domain can comprise a sequence of SEQ ID NO:214 or SEQ ID NO:215.
  • 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 SEQ ID NO:214 or SEQ ID NO:215.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:214 or SEQ ID NO:215.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:214 or SEQ ID NO:215.
  • the constant domain can comprise a sequence or fragment thereof of SEQ ID NO:214 or SEQ ID NO:215.
  • the constant domain can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:214 or SEQ ID NO:215.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:214 or SEQ ID NO:215.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:214 or SEQ ID NO:215.
  • 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. Patent No. 9,393,257), meganucleases (endodeoxyribonucleases having large recognition sites comprising double -stranded DNA sequences of 12 to 40 base pairs), zinc finger nuclease (ZFN, see, e.g., Umov et al., Nat. Rev.
  • CRISPR® clustered regularly interspaced short palindromic repeats
  • TALE transcription activator-like effector
  • TALENs transcription activator-like effector
  • meganucleases endodeoxyribonucleases having large recognition sites comprising double -stranded DNA sequences of 12 to 40 base pairs
  • a chimeric construct may be engineered to combine desirable characteristics of each subunit, such as conformation or signaling capabilities. See also Sander & Joung, Nat. Biotech. (2014) v32, 347-55; and June et al., 2009 Nature Review s Immunol . 9.10: 704-716, each incorporated herein by reference.
  • 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).
  • gene editing techniques are employed to disrupt an endogenous TCR gene.
  • mentioned endogenous TCR gene encodes a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
  • mentioned endogenous TCR gene encodes a TCR gamma chain, a TCR delta chain, or a TCR gamma chain and a TCR delta chain.
  • 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 human leukocyte antigens (HLAs), and/or B2M, and/or programmed cell death protein 1 (PD1), and/or other genes.
  • 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.
  • Geno modification of genomic 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 basepairs 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), NatProtoc. 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 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).
  • Casl2a (Cpfl) proteins from Acid- aminococcus sp (AsCpfl) and Lachnospiraceae bacterium (LbCpfl) 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). Specific amino acid substations could reprogram DNA cleavage specificity of homing nucleases (Niyonzima (2017), Protein Eng Des Sei.
  • 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-Crel homing endonuclease. In other embodiments, meganuclease is engineered I-Scel 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 I. 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.
  • the instant invention 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 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.
  • the vector further comprises a promoter.
  • the vector is an in vitro transcribed vector.
  • nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, 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.
  • 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 longterm 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 invention 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.
  • IRES internal ribosomal entry sites
  • self-cleaving peptides may be used instead.
  • 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 Picomaviridae 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-clea
  • the expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art (see, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, each of which is incorporated by reference herein in their entireties).
  • 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).
  • 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.
  • 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.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • a promoter that is capable of expressing a TFP transgene in a mammalian T cell is the EFla promoter.
  • the native EFla 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 EFla 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. Ther. 17(8): 1453-1464 (2009)).
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • other 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- la promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • 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. In general, 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.
  • 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
  • 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.
  • DMPC dimyristyl phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 °C.
  • 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.
  • 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.
  • 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.
  • moleukin 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.
  • the TFP-encoding nucleic acid construct as described herein can be cloned into a lentiviral expression vector and expression validated based on the quantity and quality of the effector T cell response of transduced T cells in response to MSLN+ target cells.
  • Effector T cell responses include, but are not limited to, cellular expansion, proliferation, doubling, cytokine production and target cell lysis or cytolytic activity (i.e., degranulation).
  • 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 (IRES) 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. 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.
  • 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.
  • IVT 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). When the 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. 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' to 3' linked circles.
  • a sequence containing a full-length encephalomyocarditis virus such as EMCV) IRES, a gene encoding a TFP, a CAR, a TCR or combination thereof, two short regions corresponding to exon fragments (El and E2), and of the PIE construct between the 3' and 5' introns of the permuted group I catalytic intron in the thymidylate synthase (Td) gene of the T4 phage or the permuted group I catalytic intron in the pre-tRNA gene of Anabaena.
  • EMCV encephalomyocarditis virus
  • 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 TFP as described herein is encoded by a circular RNA.
  • the circular RNA encoding the TFP as described herein is introduced into a T cell for production of a TFP-T cell.
  • the in vitro transcribed RNA TFP 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 comprising the nucleic acid encoding the TFP of the nucleic acid disclosed herein or a TFP encoded by the nucleic acid disclosed herein. Further disclosed herein, in some embodiments, are modified allogenic T cells comprising the nucleic acid TFP disclosed herein or a TFP encoded by nucleic acid disclosed herein.
  • 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 nucleic acid encoding the TFP disclosed herein or a TFP encoded by the nucleic acid disclosed herein.
  • 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.
  • one or more of TCR alpha, TCR beta, TCR gamma, and TCR delta have been modified to produce an allogeneic T cell. See, e.g., copending PCT Publication No. WO2019173693, which is herein incorporated by reference.
  • the modified T cells are y5 T cells and do not comprise a functional disruption of an endogenous TCR.
  • the y5 T cells are V51+ V52- yd T cells.
  • the y5 T cells are V51- V 52+ yd T cells.
  • the y5 T cells are V51- N 62- y5 T cells.
  • the cell is a human NKT cell.
  • the cell is an allogeneic cell or an autologous cell.
  • the T cell is modified to comprise a functional disruption of the TCR.
  • the modified T cells are T cells and do not comprise a functional disruption of an endogenous TCR.
  • 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 CD8+ T cell, a CD4+ T cell, a naive T cell, a memory stem T cell, a central memory T cell, a double negative T cell, an effector memory T cell, an effector T cell, a ThO cell, a TcO cell, a Thl cell, a Tel cell, a Th2 cell, a Tc2 cell, a Th 17 cell, a Th22 cell, a gamma delta T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a hematopoietic stem cell, or a pluripotent stem cell.
  • NK natural killer
  • NKT natural killer T
  • the present disclosure provides genetically-modified immune cells and populations thereof and methods for producing the same.
  • the genetically-modified immune cells of the presently disclosed compositions and methods are human immune cells.
  • the immune cells are T cells, or cells derived therefrom.
  • the immune cells are natural killer (NK) cells, or cells derived therefrom.
  • the immune cells are B cells, or cells derived therefrom.
  • the immune cells are monocyte or macrophage cells or cells derived therefrom.
  • detectable cell-surface expression of an endogenous TCR refers to the ability to detect one or more components of the TCR complex (e.g., an alpha/beta TCR complex) on the cell surface of an immune cell using standard experimental methods. Such methods can include, for example, immunostaining and/or flow cytometry specific for components of the TCR itself, such as a TCR alpha or TCR beta chain, or for components of the assembled cell-surface TCR complex, such as CD3.
  • Methods for detecting cell-surface expression of an endogenous TCR (e.g., an alpha/beta TCR) on an immune cell include those described in the examples herein, and, for example, those described in MacLeod et al. (2017) Molecular Therapy 25(4): 949-961.
  • 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.
  • the modified T cells can further comprise an enhancing agent or a nucleic acid sequence encoding an enhancing agent.
  • the modified T cells can comprise a nucleic acid sequence encoding a TFP described herein and an additional nucleic acid sequence encoding the enhancing agent.
  • the nucleic acid sequence encoding the TFP and the additional nucleic acid sequence encoding the enhancing agent can be on the same nucleic acid molecule or be on different nucleic acid molecules.
  • the nucleic acid sequence encoding the TFP and the additional nucleic acid sequence encoding the enhancing agent 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 enhancing agent can be a PD-1 switch molecule comprising a PD-1 polypeptide and an intracellular domain of a costimulatory polypeptide, an anti -PD-1 antibody, or a fusion molecule comprising an anti -PD-1 antibody or fragment thereof and a transmembrane domain.
  • the modified cells can comprise 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 modified cells can comprise a 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.
  • the inhibitory molecule can be a PD-1 switch 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.
  • a T cell expressing the TFP as described herein, and a PD-1 switch molecule as descried herein can inhibit tumor growth when expressed in a T cell.
  • the PD-1 switch molecule can enhance the activity of a modified T cell.
  • the nucleic acid sequence can be on the same nucleic acid molecule encoding the TFP described herein or be on a different nucleic acid molecule than the one encoding the TFP described herein.
  • the modified T cells can comprise a first nucleic acid sequence encoding a TFP comprising an anti-CD70 antigen binding domain and/or an anti-MSLN antigen binding domain and a second nucleic acid sequence encoding a PD-1 switch molecule.
  • the first nucleic acid sequence and the second nucleic acid sequence can be linked by a linker.
  • the linker comprises a protease cleavage site.
  • the protease cleavage site is a 2A cleavage site.
  • the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.
  • the PD-1 switch molecule can comprise 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 costimulatory polypeptide is selected from the group consisting of 0X40, 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, CD 160, CD226, FcyRI, FcyRII, and FcyRIII.
  • the costimulatory peptide is CD28.
  • an extracellular domain and a transmembrane domain of PD-1 are linked to an intracellular domain of CD28.
  • the nucleic acid sequence encodes a PD-1 switch molecule comprising the extracellular domain and the transmembrane domain of PD- 1 linked to the intracellular domain of CD28.
  • the fusion protein comprises a sequence with at least 30%, 35%, 40%, 45%, 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:232 or SEQ ID NO:233.
  • the fusion protein comprises the sequence of SEQ ID NO:232 or SEQ ID NO:233.
  • the nucleic acid sequence encodes a PD-1 switch molecule comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15Ra.
  • the fusion protein comprises a sequence with at least 30%, 35%, 40%, 45%, 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:234 or SEQ ID NO:235.
  • the PD-1 switch molecule comprises the sequence of SEQ ID NO:234 or SEQ ID NO:235.
  • the modified T cells described herein may comprise a nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof.
  • the modified T cells described herein may comprise a nucleic acid sequence encoding an Interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof.
  • the modified T cells described herein may comprise a nucleic acid sequence encoding a fusion protein comprising an IL- 15 polypeptide or a fragment thereof linked to an IL- 15Ra polypeptide or a fragment thereof.
  • the modified T cells described herein may comprise a nucleic acid sequence encoding a fusion protein comprising an IL-15Ra polypeptide or a fragment thereof linked to PD-1 or a fragment thereof and/or CD28 or a fragment thereof.
  • 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:256. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:257. 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:256.
  • the IL- 15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:254. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise amino acids 1-162 of SEQ ID NO: 256. In some embodiments, the IL- 15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:257 and a sequence of SEQ ID NO:254. In some embodiments, IL- 15 polypeptide is secreted when expressed in a cell, such as a T cell.
  • the IL-15Ra polypeptide or a fragment thereof may comprise IL-15Ra signal peptide. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 1-30 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 1-30 of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof does not comprise IL-15Ra signal peptide. In some embodiments, the IL-15Ra polypeptide or a fragment thereof does not comprise amino acids 1-30 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof does not comprise amino acids 1-30 of SEQ ID NO:258.
  • the IL-15Ra polypeptide or a fragment thereof may comprise IL-15Ra Sushi domain. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 31-95 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 31-95 of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:260.
  • the IL-15Ra polypeptide or a fragment thereof may comprise an intracellular domain of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 229-267 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 229-267 of a sequence of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:228.
  • the IL-15Ra polypeptide or a fragment thereof may comprise IL-15Ra Sushi domain, transmembrane domain, and intracellular domain. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 31-267 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 31-267 of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:260. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:261 .
  • the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 96-267 of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:260 and a sequence of SEQ ID NO:261.
  • the IL-15Ra polypeptide or a fragment thereof may be a soluble IL- 15Ra (sIL-15Ra).
  • the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 21-205 of IL-15Ra.
  • the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 21-205 of a sequence of SEQ ID NO:258.
  • the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:259.
  • 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-15Ra).
  • IL-15 polypeptide may be linked to N-terminus of IL-15Ra subunit.
  • IL- 15 polypeptide may be linked to C-terminus of IL-15Ra subunit.
  • IL-15 and IL-15Ra are operatively linked by a linker.
  • the linker is not a cleavable linker.
  • the 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 3.
  • the linker 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.
  • the nucleic acid sequence can be on the same nucleic acid molecule encoding the TFP described herein or be on a different nucleic acid molecule than the one encoding the TFP described herein.
  • the modified T cells can comprise a first nucleic acid sequence encoding a TFP comprising an anti-CD70 antigen binding domain and/or an anti-MSLN antigen binding domain and a second nucleic acid sequence encoding an IL-15 polypeptide or a fragment thereof and/or an IL-15R subunit (e.g., an IL-15 polypeptide linked to an IL-15R subunit, e.g., IL-15Ra).
  • the first nucleic acid sequence and the second nucleic acid sequence can be linked by a linker.
  • the linker comprises a protease cleavage site.
  • the protease cleavage site is a 2A cleavage site.
  • the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.
  • the fusion protein may comprise amino acids 30-162 of IL-15. In some embodiments, the fusion protein may comprise amino acids 30-162 of a sequence of SEQ ID NO:256. In some embodiments, the fusion protein may comprise any one of the sequence listed in Table 6 or a fragment thereof. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:254. 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:256. In some embodiments, the fusion protein does not comprise a sequence of SEQ ID NO:257.
  • the fusion protein may comprise a Sushi domain. In some embodiments, the fusion protein may comprise amino acids 31-95 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 31-95 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:260.
  • the fusion protein may comprise the intracellular domain of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 229-267 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 229-267 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:228. [0660] In some embodiments, the fusion protein may comprise a soluble IL-15Ra (sIL-15Ra). In some embodiments, the fusion protein may comprise amino acids 21-205 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 21-205 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:259.
  • the fusion protein may comprise the transmembrane domain and the intracellular domain of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 96-267 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 96-267 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:261.
  • the fusion protein may comprise the Sushi domain, the transmembrane domain, and the intracellular domain of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 31-267 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 31-267 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:260 and a sequence of SEQ ID NO:261.
  • the fusion protein comprising an IL-15 polypeptide or a fragment thereof and an IL-15Ra subunit or a fragment thereof comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NO:263.
  • the fusion protein comprising an IL- 15 polypeptide or a fragment thereof and an IL-15Ra subunit or a fragment thereof comprises the sequence of SEQ ID NO:263.
  • the enhancing agent described herein can be an anti-PD-1 antibody or fragment thereof (e.g., antigen binding fragment).
  • the modified T cells described herein may comprise a nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof that specifically binds programmed cell death protein 1 (PD-1).
  • the anti-PD-1 antibody or fragment thereof can inhibit an interaction of PD-1 with PD-L1 or PD-L2.
  • the anti-PD-1 antibody or fragment thereof can be secreted by the T cell.
  • the modified T cell described herein can comprise a first nucleic acid sequence encoding the TFP described herein and a second nucleic acid sequence encoding the anti-PD-1 antibody or fragment thereof.
  • 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:238.
  • the CDR1 comprises the amino acid sequence of SEQ ID NO:236.
  • the CDR2 comprises the amino acid sequence of SEQ ID NO:237.
  • the variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:239.
  • the variable domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:239.
  • variable domain comprises the amino acid sequence of SEQ ID NO:239. In some embodiments, 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: 240-243. In some embodiments, 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: 240-243. In some embodiments, the 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: 240-243.
  • the second nucleic acid encodes a fusion protein comprising an anti-PD-1 antibody or fragment thereof that specifically binds PD-1.
  • 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 anti-PD-1 antibody is linked to the intracellular domain of the costimulatory polypeptide via the transmembrane domain of PD-1.
  • 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:244 or SEQ ID NO:245.
  • the signal sequence comprises the amino acid sequence of SEQ ID NO:244 or SEQ ID NO:245.
  • 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, CD3a, CD3 ⁇ , CD45, CD4, CD5, CD7, CD8, CD9, CD 16, CD22, CD33, CD37, CD41, CD64, CD68, CD80, CD86, CD134, CD137, CD154, ICOS, 4-1BB, 0X40, PD- I, 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. In some embodiments, 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:246. In some embodiments, the transmembrane domain of the fusion protein comprises the amino acid sequence of SEQ ID NO:246. In some embodiments, the fusion protein further comprises a PD-1 stalk domain. In some embodiments, the PD-1 stalk domain is operatively linked to the transmembrane do-main. In some embodiments, 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:247. In some embodiments, the PD-1 stalk domain comprises the amino acid sequence of SEQ ID NO:247. 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:247 operatively linked to an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:246.
  • the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:247 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:246. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:247 operatively linked to the amino acid sequence of SEQ ID NO:246. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:247 operatively linked to the N-terminus of the amino acid sequence of SEQ ID NO:246. 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), 0X40, 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:224. In some embodiments, the co- stimulatory domain comprises the amino acid sequence of SEQ ID NO:224. In some embodiments, the co-stimulatory domain comprises a CD28 co-stimulatory domain. 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:223. In some embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:223. In some embodiments, the fusion protein comprises two or more anti -PD-1 antibodies or fragments thereof.
  • the two or more anti -PD-1 antibodies or fragments thereof are operatively linked tandemly. In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are identical. In some embodiments, the two or more anti-PD- 1 antibodies or fragments thereof are different. In some embodiments, the two 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 80% sequence identity to the amino acid sequence of SEQ ID NO:248. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:248.
  • 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 do-main.
  • 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 (CD 137) 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 (CD 137) co-stimulatory domain.
  • the fusion protein comprises SEQ ID NO:244 or SEQ ID NO:245, SEQ ID NO:239 or SEQ ID NO:241, SEQ ID NO:247, SEQ ID NO:246, and SEQ ID NO:223 or SEQ ID NO:224.
  • the fusion protein comprises, from the N-terminus to the C- terminus, SEQ ID NO:244 or SEQ ID NO:245 operatively linked to SEQ ID NO:239 or SEQ ID NO:241 operatively linked to SEQ ID NO:247 operatively linked to SEQ ID NO:246 operatively linked to SEQ ID NO:223 or SEQ ID NO:224.
  • the fusion protein comprises, from the N-terminus to the C-terminus, SEQ ID NO:244 or SEQ ID NO:245 operatively linked to SEQ ID NO:239 or SEQ ID NO:241 operatively linked to SEQ ID NO:248 operatively linked to SEQ ID NO:239 or SEQ ID NO:241 operatively linked to SEQ ID NO:247 operatively linked to SEQ ID NO:246 operatively linked to SEQ ID NO:223 or SEQ ID NO:224.
  • the fusion protein comprises an amino acid sequence having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to the amino acid sequence of any one of SEQ ID NOs:249-253. In some embodiments, the fusion protein comprises an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs:249-253.
  • T cells Prior to expansion and genetic modification, a source of T cells is obtained from a subject.
  • subject is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • 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 FicollTM 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).
  • PBS phosphate buffered saline
  • 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.
  • 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 ⁇ x
  • the T cells are y5 T cells.
  • y5 T cells are obtained from a bank of umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cells, for example.
  • 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.
  • a specific subpopulation of T cells such as CD3+, CD28+, CD4+, CD8+, CD45RA+, CD45RO+, alpha-beta, or gamma-delta T cells, can be further isolated by positive or negative selection techniques.
  • CD4+ and CD8+ T cells are isolated with anti-CD4 and anti-CD8 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. In certain aspects, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “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, CD1 lb, 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-y 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 can be varied.
  • it may be desirable to significantly decrease the volume in which beads and cells are mixed together e.g., increase the concentration of cells, to ensure maximum contact of cells and beads.
  • 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.
  • 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.
  • using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
  • the concentration of cells used is 5xlO 6 /mL. In other aspects, the concentration used can be from about IxlO mL to lxlO 6 /mL, and any integer value in between. In other aspects, 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.
  • 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.
  • a blood sample or an apheresis product 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, mycophenolate, and tacrolimus (FK506), antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporin, rapamycin, mycophenolic acid, steroids, romidepsin (formerly FR901228), and irradiation.
  • agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and tacrolimus (FK50
  • 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 fortheir 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.
  • T cells may be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631.
  • the T cells of the present disclosure may be expanded by contact with a surface having atached 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 antigenbinding 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 ligand that binds the accessory molecule is used for costimulation 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 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.
  • T cells may additionally be activated and expanded in the presence of a cytokine with or without an anti-CD3 and/or CD28 antibody.
  • cytokines include IL-2, IL-7, IL-15, and IL-21.
  • 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 24 hours, e.g., 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 gammachain (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.
  • the cells are activated for 24 hours.
  • the cells after transduction, 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, the cells 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. In some embodiments, 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 pM 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 pM 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-l -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. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.
  • an anti-tumor-associated antigen TFP is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following antigen stimulation, sustain T cell expansion in the absence of re-stimulation, and anti-cancer activities in appropriate in vitro and animal models. Assays to evaluate the effects of an anti -tumor- associated antigen TFP are described in further detail below.
  • TFP expression in primary T cells can be used to detect the presence of monomers and dimers (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).
  • T cells (1: 1 mixture of CD4 + and CD8 + T cells) expressing the TFPs are expanded in vitro for more than 10 days followed by lysis and SDS-PAGE under reducing conditions.
  • TFPs are detected by western blotting using an antibody to a TCR chain. The same T cell subsets are used for SDS-PAGE analysis under non-reducing conditions to permit evaluation of covalent dimer formation.
  • TFP + T cells following antigen stimulation can be measured by flow cytometry.
  • a mixture of CD4 + and CD8 + T cells are stimulated with alphaCD3/alphaCD28 and APCs followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed.
  • exemplary promoters include the CMV IE gene, EF-lalpha, ubiquitin C, or phosphoglycerokinase (PGK) promoters.
  • GFP fluorescence is evaluated on day 6 of culture in the CD4+ and/or CD8+ T cell subsets by flow cytometry (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).
  • a mixture of CD4+ and CD8+ T cells are stimulated with alphaCD3/alphaCD28 coated magnetic beads on day 0, and transduced with TFP on day 1 using a bicistronic lentiviral vector expressing TFP as described herein along with eGFP using a 2A ribosomal skipping sequence.
  • Cultures are re-stimulated with either TAA+ K562 cells (K562-TAA), wild-type K562 cells (K562 wild type) or K562 cells expressing hCD32 and 4-1BBL in the presence of anti-CD3 and anti-CD28 antibody (K562 -BBL-3/28) following washing.
  • Exogenous IL-2 is added to the cultures every other day at 100 lU/mL.
  • GFP+ T cells are enumerated by flow cytometry using bead-based counting (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).
  • Sustained TFP+ T cell expansion in the absence of re -stimulation can also be measured (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer III particle counter following stimulation with alphaCD3/alphaCD28 coated magnetic beads on day 0, and transduction with the indicated TFP as described herein on day 1.
  • Animal models can also be used to measure an activity of T cells expressing TFT as described herein.
  • xenograft model using, e.g., human CD70 and/or MSLN-specific TFP+ T cells to treat a cancer in immunodeficient mice (see, e.g., Milone et al., Molecular Therapy 17(8): 1453- 1464 (2009)).
  • mice are randomized as to treatment groups. Different numbers of engineered T cells are coinjected at a 1: 1 ratio into NOD/SCID/y-/- mice bearing cancer. The number of copies of each vector in spleen DNA from mice is evaluated at various times following T cell injection. Animals are assessed for cancer at weekly intervals.
  • Peripheral blood tumor-associated antigen+ cancer cell counts are measured in mice that are injected with anti -tumor- associated antigen-zeta TFP+ T cells or mock-transduced T cells. Survival curves for the groups are compared using the log-rank test.
  • absolute peripheral blood CD4+ and CD8+ T cell counts 4 weeks following T cell injection in NOD/SCID/y-/- mice can also be analyzed. Mice are injected with cancer cells and 3 weeks later are injected with T cells engineered to express TFP as described herein by a bicistronic lentiviral vector that encodes the TFP linked to eGFP.
  • T cells are normalized to 45-50% input GFP+ T cells by mixing with mock-transduced cells prior to injection, and confirmed by flow cytometry. Animals are assessed for cancer at 1-week intervals. Survival curves for the TFP+ T cell groups are compared using the log-rank test.
  • Dose dependent TFP treatment response can be evaluated (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).
  • peripheral blood is obtained 35-70 days after establishing cancer in mice injected on day 21 with TFP T cells, an equivalent number of mock- transduced T cells, or no T cells. Mice from each group are randomly bled for determination of peripheral blood + cancer cell counts and then killed on days 35 and 49. The remaining animals are evaluated on days 57 and 70.
  • Assessment of cell proliferation and cytokine production has been previously described, e.g., at Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).
  • TFP-mediated proliferation is performed in microtiter plates by mixing washed T cells with cells expressing the tumor associated antigen (TAA, e.g., CD70 and/or MSLN) for a final T cell: cell expressing TAA ratio of 2: 1.
  • TAA tumor associated antigen
  • Cells expressing TAA are irradiated with gamma-radiation prior to use.
  • Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultures with KT32-BBL cells to serve as a positive control for stimulating T cell proliferation since these signals support long-term CD8+ T cell expansion ex vivo.
  • T cells are enumerated in cultures using CountB rightTM fluorescent beads (Invitrogen) and flow cytometry as described by the manufacturer.
  • TFP+ T cells are identified by GFP expression using T cells that are engineered with eGFP-2A linked TFP-expressing lentiviral vectors.
  • the TFP+ T cells are detected with biotinylated recombinant TAA protein and a secondary avidin-PE conjugate.
  • CD4+ and CD8+ expression on T cells are also simultaneously detected with specific monoclonal antibodies (BD Biosciences).
  • Cytokine measurements are performed on supernatants collected 24 hours following re-stimulation using the human TH1/TH2 cytokine cytometric bead array kit (BD Biosciences) according the manufacturer’s instructions. Fluorescence is assessed using a FACScaliburTM flow cytometer (BD Biosciences), and data are analyzed according to the manufacturer’s instructions.
  • Cytotoxicity can be assessed by a standard 51 Cr-release assay (see, e.g. , Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Briefly, target cells are loaded with 51 Cr (as NaCrCft, New England Nuclear) at 37 °C for 2 hours with frequent agitation, washed twice in complete RPMI and plated into microtiter plates. Effector T cells are mixed with target cells in the wells in complete RPMI at varying ratios of effector celktarget cell (E:T). Additional wells containing media only (spontaneous release, SR) or a 1% solution of Triton-X 100 detergent (total release, TR) are also prepared.
  • 51 Cr as NaCrCft, New England Nuclear
  • E:T effector celktarget cell
  • % Lysis (ER-SR)/(TR-SR), where ER represents the average 51 Cr released for each experimental condition.
  • Imaging technologies can be used to evaluate specific trafficking and proliferation of TFPs in tumor-bearing animal models. Such assays have been described, e.g., in Barrett et al., Human Gene Therapy 22: 1575-1586 (2011). Briefly, NOD/SCID/yc-/- (NSG) mice are injected IV with cancer cells followed 7 days later with T cells 4 hour after electroporation with the TFP as described herein constructs. The T cells are stably transfected with a lentiviral construct to express firefly luciferase, and mice are imaged for bioluminescence.
  • therapeutic efficacy and specificity of a single injection of TFP+ T cells in a cancer xenograft model can be measured as follows: NSG mice are injected with cancer cells transduced to stably express firefly luciferase, followed by a single tailvein injection of T cells electroporated with a TAA-TFP as described herein 7 days later. Animals are imaged at various time points post injection. For example, photon-density heat maps of firefly luciferase positive cancer in representative mice at day 5 (2 days before treatment) and day 8 (24 hours post TFP+ PBLs) can be generated.
  • the TFP T cells provided herein may be useful for the treatment of any disease or condition involving CD70 and/or MSLN (e.g., Nectin-4-expressing cancers).
  • 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.
  • the disease or condition is a viral infection.
  • provided herein is a method of treating a disease or condition in a subject in need thereof by administering an effective amount of a TFP T cell provided herein to the subject.
  • the disease or condition is a cancer.
  • the disease or condition is a viral infection.
  • the dual specificity TFP T cells are administered with an additional anti-cancer agent; in some embodiments, the anticancer agent is an antibody or fragment thereof, another TFP T cell, a CAR T cell, or a small molecule.
  • tumor-associated antigens include, but are not limited to, oncofetal antigens (e.g., those expressed in fetal tissues and in cancerous somatic cells), oncoviral antigens (e.g., those encoded by tumorigenic transforming viruses), overexpressed/ accumulated antigens (e.g., those expressed by both normal and neoplastic tissue, with the level of expression highly elevated in neoplasia), cancer-testis antigens (e.g., those expressed only by cancer cells and adult reproductive tissues such as testis and placenta), lineage-restricted antigens (e.g., those expressed largely by a single cancer histotype), mutated antigens (e.g., those expressed by cancer as a result of genetic mutation or alteration in transcription), posttranslationally altered antigens (e.g., those tumor- associated alterations in glycosylation, etc.), and idiotypic antigens (e.g., those from highly polymorphic genes where a
  • tumor-associated antigens include, but are not limited to, antigens of alpha-actinin-4, ARTCI, alphafetoprotein (AFP), BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDK12, CDKN2A, CLPP, COA-1, CSNK1A1, CD79, CD79B, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-
  • the present disclosure provides methods for treating a disease associated with at least one tumor-associated antigen expression.
  • the present 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 present 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.
  • provided herein is a method of treating a disease or condition in a subject in need thereof by administering an effective amount of a TFP T cell provided herein to the subject.
  • the disease or condition is a cancer.
  • the disease or condition is a viral infection.
  • any suitable cancer may be treated with the 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 cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma,
  • ALL acute lymph
  • the cancer can be acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia (CLL), chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastric cancer, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), glioblastoma, Hodgkin's lymphoma, hypopharynx cancer,
  • bladder cancer e.
  • the cancer can be characterized by the expression CD70 and/or MSLN.
  • CD70 and/or -MSLN can be highly expressed in breast cancer, lung cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, esophageal cancer, head and neck cancer, bladder cancer, and gastric cancer.
  • the disease or the condition is a cancer or a disease or a condition associated with expression of mesothelin (MSLN) and/or CD70.
  • the cancer is a hematologic cancer.
  • a hematologic cancer include, but are not limited to, 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 pro- lymphocytic 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
  • the present disclosure pertains to a vector comprising an anti-tumor-associated antigen antibody or TFP operably linked to promoter for expression in mammalian T cells.
  • the present 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.
  • the tumor-associated antigen TFP-T of the present disclosure is capable of contacting a tumor cell with at least one tumor-associated antigen TFP of the present disclosure expressed on its surface such that the TFP-T targets the tumor cell and growth of the tumor is inhibited.
  • the present 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 is activated in response to the antigen and targets the cancer cell, wherein the growth of the tumor is inhibited.
  • the present 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.
  • An example of a cancer that is treatable by the tumor-associated antigen TFP T cell of the present disclosure is a cancer associated with expression of tumor-associated antigen.
  • the cancer is a myeloma.
  • the cancer is a lymphoma.
  • the cancer is colon cancer.
  • tumor-associated antigen antibodies or TFP therapy can be used in combination with one or more additional therapies.
  • additional therapies comprise a chemotherapeutic agent, e.g., cyclophosphamide.
  • additional therapies comprise surgical resection or radiation treatment.
  • 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.
  • 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 -modified T cells of the present disclosure may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal.
  • the mammal is a human.
  • cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a TFP disclosed herein.
  • the TFP -modified cell can be administered to a mammalian recipient to provide a therapeutic benefit.
  • the mammalian recipient may be a human and the TFP -modified cell can be autologous with respect to the recipient.
  • the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.
  • ex vivo culture and expansion of T cells comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo.
  • other factors such as flt3-U, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.
  • compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient In addition to using a cell-based vaccine in terms of ex vivo immunization, 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 present disclosure are used in the treatment of diseases, disorders and conditions associated with expression of tumor-associated antigens.
  • the cells of the present 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 present disclosure.
  • the antibodies or TFP-T cells of the present disclosures may be used to treat a proliferative disease such as a cancer or malignancy or is a precancerous condition.
  • a proliferative disease such as a cancer or malignancy or is a precancerous condition.
  • the cancer is a myeloma.
  • the cancer is a lymphoma.
  • the cancer is a colon cancer.
  • a disease associated with tumor-associated antigen expression includes, but is not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing tumor-associated antigens.
  • Non-cancer related indications associated with expression of tumor-associated antigens vary depending on the antigen, but are not limited to, e.g., infectious disease, autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma) and transplantation.
  • the antibodies or TFP-modified 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 such as IL-2 or IL- 12 or other cytokines or cell populations.
  • 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 present 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 present 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 present disclosure that binds to the tumor-associated antigen-expressing cell.
  • the anti- tumor-associated antigen antibody or TFP-T cell of the present 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 present 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).
  • the present disclosure also provides methods for preventing, treating and/or managing a disease associated with tumor-associated antigen-expressing cells, the methods comprising administering to a subject in need an anti- tumor-associated antigen antibody or TFP-T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell.
  • the subject is a human.
  • the present disclosure provides methods for preventing relapse of cancer associated with tumor-associated antigen-expressing cells, the methods comprising administering to a subject in need thereof an anti- tumor-associated antigen antibody or TFP-T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell.
  • the methods comprise administering to the subject in need thereof an effective amount of an anti -tumor-associated antigen antibody or TFP-T cell described herein that binds to the tumor-associated antigen-expressing cell in combination with an effective amount of another therapy.
  • Suitable doses of the 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 reservoiraccess 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 e.g., the amount per each cell to be contacted or the amount per certain body weight
  • an antibody or TFP -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.
  • 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 delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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-y, TNFa, 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 TNFa, and an inhibitor of IL-6.
  • a TNFa 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 (PD1)
  • PD1 can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response.
  • Examples of inhibitory molecules include PD1, 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-L 1 , 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).
  • TIM3 T cell immunoglobulin and mucin-domain containing-3
  • LAG3 Lymphocyte-activation gene 3
  • 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 antitumor-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[3), 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[3 programmed cell death protein 1
  • LAG-3 lymphocyte-activation gene 3
  • Tim-3 hepatitis A virus cellular receptor 2 or
  • the agent is selected from an anti -PD-1 antibody (e.g., pembrolizumab or nivolumab), and anti-PD-Ll 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), CD 160, natural killer cell receptor 2B4 (also CD244 or SLAMF4), and combinations thereof.
  • the agent is pembrolizumab.
  • the agent is nivolumab.
  • the agent is atezolizumab.
  • the additional therapeutic agent is an agent that inhibits the interaction between PD-1 and PD-L1.
  • 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., ChemMed 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.
  • the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in a different pharmaceutical composition from an antibody provided herein.
  • 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, 0X40, 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-2 3 -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.
  • the additional therapeutic agent may be administered by any suitable means.
  • a medicament provided herein, and the additional therapeutic agent are included in the same pharmaceutical composition.
  • 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.
  • the agent is an agent that inhibits DNA methylation. In some embodiments, the agent is an agent that inhibits DNA methyltransferease. In some embodiments, the agent is a hypomethylating agent. Examples of the hypomethylating agent includes, but are not limited to 5 -azacitidine and decitabine and also includes any hypomethylating agent known in the art. In some embodiments, the hypomethylating agent is 5 -azacitidine. In some embodiments, the hypomethylating agent is decitabine. In some embodiments, the hypomethylating agent is a derivative of decitabine or a derivative of 5 -azacitidine. In some embodiments, the hypomethylating agent is an esterificated azacytidine, an acetylated azacitidine, an esterificated decitabine. or an acetylated decitabine.
  • compositions of the present disclosure may comprise a TFP -expressing cell, e.g., a plurality of TFP -expressing cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such 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.
  • Compositions of the present disclosure are in one aspect formulated for intravenous administration.
  • compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented).
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.
  • 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 tumorinhibiting 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 IO 4 to IO 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.
  • 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.
  • 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 by methods known in the art and treated such that one or more TFP constructs of the present disclosure may be introduced, thereby creating a TFP-expressing T cell of the present 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 (CAMPATH®) 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 in U.S. Pat. No. 6,120,766).
  • the TFP as described herein is introduced into T cells, e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of TFP T cells of the present disclosure, and one or more subsequent administrations of the TFP T cells of the present disclosure, 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 TFP T cells of the present disclosure are administered to the subject (e.g., human) per week, e.g., 1, 3, or 4 administrations of the TFP T cells of the present disclosure 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 present disclosure are administered for at least two, three, four, five, six, seven, eight or more weeks.
  • tumor-associated antigen TFP T cells are generated using lentiviral viral vectors, such as lentivirus. TFP-T cells generated that way will have stable TFP expression.
  • TFP T cells transiently express TFP vectors 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 TFP vector delivery. In one aspect, the TFP RNA 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. It is thought that a patient’s antibody producing cells undergo a class switch from IgG isotype (that does not cause anaphylaxis) to IgE isotype when there is a ten- to fourteen -day break in exposure to antigen.
  • TFP T cell infusion breaks should not last more than ten to fourteen days.
  • TFP constructs are made having both binding domains (e.g., an scFv, a sdAb, etc.) in tandem on a single TCR subunit.
  • TFP constructs are made having both binding domains in a single TCR with one binding domain on each of two TCR subunits, e.g., both epsilon subunits, an epsilon and the gamma subunit, etc.
  • TFP constructs are made individually in separate lentiviral vectors, and the target T cell population is transduced with both viruses.
  • the Examples disclose a combination of anti-MSLN TFPs and anti-CD70 TFPs and/or a TFP having specificity to both anti-MSLN and CD70, and/or a mixed T cell population wherein the T cells are a mix of T cells transduced with an anti-MSLN TFP and T cells transduced with an anti-CD70 TFP.
  • the anti-MSLN and anti-CD70 constructs disclosed herein are exemplary only and not meant to be construed as limiting, as noted above. Constructs with a variety of combinations of anti-tumor antigen antibodies are contemplated in the methods of the present disclosure.
  • Subunits of the human T Cell Receptor (TCR) complex all contain an extracellular domain and a transmembrane domain.
  • the CD3 epsilon, CD3 delta, and CD3 gamma subunits have an intracellular domain.
  • 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. Q6ISU 1.
  • 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: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQH NDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCM EMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVP NPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:694).
  • the mature human CD3 -epsilon polypeptide sequence is: DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLK EFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLL VYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:733).
  • the signal peptide of human CD3a is:
  • the transmembrane domain of human CD3a is:
  • the intracellular domain of human CD3s is:
  • the human CD3 -gamma polypeptide canonical sequence is:
  • the mature human CD3 -gamma polypeptide sequence is:
  • the signal peptide of human CD3y is:
  • the transmembrane domain of human CD3y is:
  • GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO:703).
  • the human CD3 -delta polypeptide canonical sequence is:
  • the mature human CD3 -delta polypeptide sequence is:
  • the signal peptide of human CD35 is: MEHSTFLSGLVLATLLSQVSP (SEQ ID NO:705).
  • the extracellular domain of human CD35 is:
  • the transmembrane domain of human CD35 is:
  • GIIVTDVIATLLLALGVFCFA SEQ ID NO:707.

Abstract

L'invention concerne des acides nucléiques recombinants comprenant une séquence codant pour des protéines de fusion (TFP) du récepteur des lymphocytes T (TCR) ayant une spécificité pour plus d'un antigène associé à une cellule tumorale, des lymphocytes T modifiés pour exprimer une ou plusieurs TFP, ainsi que des méthodes d'utilisation de ceux-ci pour le traitement de maladies, notamment du cancer.
PCT/US2022/049396 2021-11-10 2022-11-09 Compositions et méthodes de reprogrammation de tcr à l'aide de protéines de fusion WO2023086379A2 (fr)

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AU2019372673A1 (en) * 2018-11-01 2021-05-27 Gracell Biotechnologies (Shanghai) Co., Ltd. Compositions and methods for T cell engineering
WO2021133959A2 (fr) * 2019-12-24 2021-07-01 TCR2 Therapeutics Inc. Compositions et procédés de reprogrammation de tcr gamma delta à l'aide de protéines de fusion
EP4121079A1 (fr) * 2020-03-16 2023-01-25 University Of Southern California Nouveaux domaines de liaison à l'antigène et récepteurs d'antigènes synthétiques les incorporant

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