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WO2022177966A1 - Compositions et méthodes de traitement du vih - Google Patents

Compositions et méthodes de traitement du vih Download PDF

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Publication number
WO2022177966A1
WO2022177966A1 PCT/US2022/016569 US2022016569W WO2022177966A1 WO 2022177966 A1 WO2022177966 A1 WO 2022177966A1 US 2022016569 W US2022016569 W US 2022016569W WO 2022177966 A1 WO2022177966 A1 WO 2022177966A1
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WIPO (PCT)
Prior art keywords
tcr
cells
domain
cell
pharmaceutical composition
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PCT/US2022/016569
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English (en)
Inventor
Robert Hofmeister
Patrick Baeuerle
Dario Gutierrez
Philippe KIEFFER-KWON
Courtney Kay ANDERSON
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TCR2 Therapeutics Inc.
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Application filed by TCR2 Therapeutics Inc. filed Critical TCR2 Therapeutics Inc.
Publication of WO2022177966A1 publication Critical patent/WO2022177966A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • 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
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • 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
    • A61K39/12—Viral antigens
    • 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
    • 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
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • HIV 1 human immunodeficiency virus type 1
  • T cell therapy has undergone significant advancement over the years, specifically in cancer therapy.
  • One significant advantage is the discovery of chimeric antigen receptors (CAR), that can specifically direct a T cell to a target of choice by expressing a CAR that can bind to the target.
  • CAR chimeric antigen receptors
  • T cells themselves are subject to HIV infection.
  • adoptive T cells can generate a large cytokine response which may be the major cause of non-specific inflammatory response, known as the cytokine storm. Potential avenues are thereby sought after to address these issues and successfully design T cell therapies against HIV.
  • the disclosure provides a pharmaceutical composition comprising a T cell, that comprises a recombinant nucleic acid encoding a T cell receptor (TCR) fusion protein (TFP) and a pharmaceutically acceptable carrier.
  • TCR T cell receptor
  • TFP T cell fusion protein
  • the TFP of the pharmaceutical composition comprises: a TCR-integrating subunit comprising an extracellular domain, and a TCR transmembrane domain; and an antigen binding domain that specifically binds an HIV antigen (e.g., GP120); wherein the TCR-integrating subunit and the first binding domain are operatively linked; and wherein the TFP functionally interacts with an endogenous TCR when expressed in a T cell.
  • the TFP may further comprise a TCR intracellular domain.
  • the TCR intracellular domain may comprise a stimulatory domain from an intracellular signaling domain.
  • the T cell (i) does not express CCR5 at physiological levels or (ii) comprises a genetically modified CCR5 gene and has a reduced expression level of CCR5 as compared with a wild-type counterpart.
  • the T cell further comprises a nuclease (e.g., a nuclease for reducing or eliminating CCR5 expression in the T cell) or a sequence encoding the nuclease, or the genetically disrupted CCR5 gene is genetically disrupted by a nuclease.
  • the nuclease is selected from: (i) a Cas protein or nuclease; (ii) a TAL-effector nuclease (TALEN); (iii) a zinc-finger nuclease (ZFN); and (iv) a meganuclease.
  • the nuclease is a Cas nuclease and the pharmaceutical composition further comprises a guide RNA.
  • the antigen binding domain that specifically binds GP120 specifically binds to the CD4 binding region of GP120; the V1-V2 loop of GP120; the V3 loop of GP120; or the membrane proximal external region of GP120.
  • the antigen binding domain is a CD4 receptor or a fragment thereof.
  • the antigen binding domain is an antibody.
  • the antibody is an scFv or a single domain antibody.
  • the antibody is human or humanized.
  • the T cell exhibits increased cytotoxicity to a gp120 expressing cell compared to a T cell not containing the TFP.
  • the T cell is from a human subject.
  • the T cell is a CD4+ T cell or a CD8+ T cell.
  • the extracellular domain comprises at least a portion of a TCR extracellular domain.
  • the encoded binding domain is connected to the TCR extracellular domain by a linker sequence.
  • the encoded linker sequence comprises (G4S)n, wherein n is 1 to 4 (SEQ ID NO: 85).
  • the encoded linker sequence comprises AAAGGGGSGGGGSGGGGSLE (SEQ ID NO: 2 or 72).
  • the TCR-integrating 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 intracellular domain comprises an intracellular 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 and functional fragments thereof.
  • the intracellular signaling domain comprises a stimulatory domain selected from an intracellular signaling domain of CD3 gamma, CD3 delta, or CD3 epsilon, or an amino acid sequence thereof having at least one modification but not more than 20 modifications.
  • the intracellular signaling domain comprises a stimulatory domain selected from a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
  • 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 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 encoded TFP includes 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 gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, a CD3 zeta TCR subunit, 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 gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, a CD3 zeta TCR subunit,
  • the encoded TFP includes 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.
  • 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
  • the encoded TFP 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 OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TFP includes 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 2b1 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, CD89, CD278, CD66d
  • ITAM immunoreceptor
  • 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.
  • nucleic acid encoding the TFP of any of the paragraphs described above.
  • the nucleic acid is selected from the group consisting of a DNA and an RNA.
  • the nucleic acid is an mRNA.
  • the nucleic acid is circRNA.
  • the T cell further comprises a sequence encoding a broadly neutralizing antibody against HIV.
  • the broadly neutralizing antibody against HIV is secreted.
  • the recombinant nucleic acid molecule comprises the sequence encoding the broadly neutralizing antibody against HIV.
  • the sequence encoding the TFP and the sequence encoding the broadly neutralizing antibody against HIV are contained in the same operon.
  • the recombinant nucleic acid further encodes a self-cleaving peptide between the sequence encoding the TFP and the sequencing encoding the broadly neutralizing antibody against HIV.
  • the broadly neutralizing antibody against HIV is selected from bl2, VRC01, VRC02, VRC03, VRC06, and VRC07.
  • the broadly neutralizing antibody against HIV is selected from 3BNC117, IOMA, N6, PGT121, PGT122, PGT123, PGT127, PGT128, PGT135, 10-1074, BG18, 35022, N123-VRC34.01, 3BC315, PGT151, 10E8, 10E8n4, 10E8n4, S100cF, Dh5l l.2_k3, Z13, 4E10, 2F5, PGT141, PGT142, PGT143, PGT145, or PG9.
  • the broadly neutralizing antibody comprises a heavy chain having a sequence of SEQ ID NO: 82 or SEQ ID NO: 83, or a sequence having at least 80% identity thereto.
  • the broadly neutralizing antibody comprises a light chain having a sequence of SEQ ID NO: 80 or SEQ ID NO: 81, or a sequence having at least 80% identity thereto.
  • the broadly neutralizing antibody is a scFv of 10-1074.
  • the recombinant nucleic acid comprises a nucleic acid analog, wherein the nucleic acid analog is not in an encoding sequence of the recombinant nucleic acid.
  • the recombinant nucleic acid further comprises a leader sequence. [0048] In some embodiments, the recombinant nucleic acid further comprises a promoter sequence. [0049] In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. [0050] In some embodiments, the recombinant nucleic acid further comprises a 3’UTR sequence. [0051] In some embodiments, the recombinant nucleic acid is an isolated nucleic acid or a non- naturally occurring nucleic acid. [0052] In some embodiments, the nucleic acid is an in vitro transcribed nucleic acid.
  • the recombinant nucleic acid comprises a nucleotide analog.
  • the nucleotide analog is selected from the group consisting of 2’- O-methyl, 2’-O-methoxyethyl (2’-O-MOE), 2’-O-aminopropyl, 2’-deoxy, T-deoxy-2’-fluoro, 2’- O-aminopropyl (2’-O-AP), 2'-O-dimethylaminoethyl (2’-O-DMAOE), 2’-O- dimethylaminopropyl (2’-O-DMAP), T-O-dimethylaminoethyloxyethyl (2’-O-DMAEOE), 2’-O- N-methylacetamido (2’-O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (LNA), an ethylene nucleic acid (ENA),
  • a vector comprising the recombinant nucleic acid of any one of the preceding paragraphs.
  • 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 in vitro transcribed vector.
  • a circular RNA may comprise the recombinant nucleic acid of any one of the preceding paragraphs.
  • a method for treating HIV comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of the preceding paragraphs.
  • the method for treating described in any one of the preceding paragraphs is conducted with a subject not receiving anti-retroviral therapy or wherein the subject is receiving anti-retroviral therapy that is disrupted.
  • a composition for use in the manufacture of a medicament for use in treating HIV comprising the composition of any one of the preceding paragraphs.
  • the subject is not receiving anti-retroviral therapy or in cases where the subject is receiving anti-retroviral therapy, the anti-retroviral therapy is disrupted.
  • the present disclosure provides a method of producing the pharmaceutical composition described herein comprising: isolating CD8+ T cells from a population of cells; and transducing the CD8+ T cell with the vector described herein.
  • the present disclosure provides a method of producing the pharmaceutical composition described herein comprising: transducing a population of T cells with the vector described herein; and disrupting an endogenous CCR5 gene in the T cells prior to or after transduction.
  • the endogenous CCR5 gene is disrupted with a nuclease.
  • the nuclease is selected from: a Cas protein or a Cas nuclease; a TAL-effector nuclease (TALEN); a zinc-finger nuclease (ZFN); and a meganuclease.
  • TALEN TAL-effector nuclease
  • ZFN zinc-finger nuclease
  • FIG.1A shows an exemplary TFP comprising a CD4 extracellular region fused to CD3 epsilon transmembrane and intracellular domains.
  • FIG.1B shows an exemplary TFP comprising a CD4 extracellular region fused via a linker (L) to CD3 epsilon transmembrane and intracellular domains.
  • FIG.1C shows an exemplary TFP comprising a CD4 extracellular region fused to CD3 epsilon transmembrane and intracellular domains and comprising an additional intracellular signaling domain (S*) fused to the cytoplasmic end.
  • FIG.2A shows an exemplary TFP comprising a gp120 binding scFV as an extracellular region fused to CD3 epsilon transmembrane and intracellular domains.
  • FIG.2B shows an exemplary TFP comprising a gp120 binding scFV as an extracellular region fused via a linker (L) to CD3 epsilon transmembrane and intracellular domains.
  • FIG.2C shows an exemplary TFP comprising a gp120 binding scFV as an extracellular region fused to CD3 epsilon transmembrane and intracellular domains and comprising an additional intracellular signaling domain (S*) fused at the cytoplasmic end.
  • FIG.3 shows exemplary TRuC T cells expressing CD4 T cell receptor fusion proteins.
  • FIG.4 shows exemplary flow cytometry plots demonstrating successful transduction of CD4 TRuC T cells.
  • FIG.5 shows exemplary flow cytometry plots and graphs demonstrating the differentiation state of the transduced TRuC T cells.
  • FIG.6 shows quantification of % cell lysis in a 24hr co-culture luciferase-based cytotoxicity assay.
  • FIG.7A shows quantification of IFN ⁇ release after 24hr co-culture of CD4 TRuC T cells and HIV-envelope expressing cells.
  • FIG.7B shows quantification of GM-CSF release after 24hr co-culture of CD4 TRuC T cells and HIV-envelope expressing cells.
  • FIG.7C shows quantification of TNF ⁇ release after 24hr co-culture of CD4 TRuC T cells and HIV-envelope expressing cells.
  • FIG.8 shows results of a 4hr flow cytometry-based cytotoxicity assay of CD4 TRuC T cells and HIV-envelope expressing cells.
  • the term “about” indicates the designated value(s) ⁇ one standard deviation of that value(s).
  • 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.
  • antigen-binding domain means the portion of an antibody or any other polypeptide that is capable of specifically binding to an antigen or epitope.
  • an antigen-binding domain is an antigen-binding domain formed by a VH -VL dimer of an antibody.
  • Another example of an antigen-binding domain is an antigen-binding domain formed by diversification of certain loops from the tenth fibronectin type III domain of an Adnectin.
  • Another example of an antigen binding domain is a receptor or fragment thereof, e.g., a CD4 receptor that binds an antigen.
  • Another example of an antigen binding domain is a receptor ligand.
  • antibody fragment or “antibody binding domain” refer to at least one portion of an antibody, or recombinant variants thereof, that contains the 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” (or, in the case of single domain antibodies, e.g., nanobodies, “VHH”) with regard to an antibody 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.
  • Light chain variable region or “VL” with regard to an antibody refers to the fragment of the light chain that contains three CDRs interposed between framework regions.
  • a scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.
  • the portion of the TFP composition of the invention comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb) or heavy chain antibodies HCAb, a single chain antibody (scFv) derived from a murine, humanized or human antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci.
  • sdAb single domain antibody fragment
  • HCAb heavy chain antibodies
  • scFv single chain antibody
  • the antigen binding domain of a TFP composition of the present disclosure comprises an antibody fragment.
  • the TFP comprises an antibody fragment that comprises a scFv or a sdAb.
  • antibody heavy chain refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations.
  • Kappa (“ ⁇ ”) and lambda (“ ⁇ ”) light chains refer to the two major antibody light chain isotypes.
  • the term “recombinant antibody” refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.
  • antigen binding domain of a recombinant protein may be able to bind an antigen or a target, such as an antigen on a different cell, such as a microbial cell or a virus.
  • the antigen may be a viral antigen.
  • the viral antigen may be a full-length antigenic viral protein or a portion of the antigenic viral protein that contains the predominant antigen, neutralizing antigen, or epitope of the viral protein.
  • the viral antigen contains the constant region of glycoproteins of at least two strains of the pathogenic virus.
  • the viral antigen may be a modified antigen that is mutated from a glycoprotein of the pathogenic virus such that the viral antigen is rendered non-functional as a viral component but retains its antigenicity.
  • modification of the viral antigen includes deletions in the proteolytic cleavage site of the glycoprotein, and duplications and rearrangement of immunosuppressive peptide regions of the glycoprotein.
  • the antigen or target antigen is a target glycoprotein on the surface of a pathogen that causes a disease, or on a diseased cell; such that a therapeutic recombinant protein described herein is designed to bind to the target antigen.
  • the disease may be an infection, such as a viral infection.
  • Exemplary viral infection could be any infection caused by a pathogenic virus, such as a retrovirus, such as an HIV virus.
  • acquired immune deficiency syndrome (AIDS) is a disease caused by HIV.
  • HIV-1 and HIV-2 There are at least two distinct types of HIV: HIV-1 and HIV-2.
  • HIV envelope surface glycoproteins are synthesized as a single 160kD precursor protein (gp160) which is cleaved by a cellular protease during viral budding into two glycoproteins, gp4l and gp120.
  • gp4l is a transmembrane protein and gp120 is an extracellular protein which remains noncovalently associated with gp4l, possibly in a trimeric or multimeric form.
  • a target or a target antigen may be a gp120 glycoprotein, an aggregate, a conjugate or a fragment thereof.
  • 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. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response.
  • an antigen need not be encoded by a “gene” at all.
  • 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.
  • affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope).
  • affinity refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen or epitope).
  • the affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD).
  • KD dissociation equilibrium constant
  • the kinetic components that contribute to the dissociation equilibrium constant are described in more detail below.
  • Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE ® ) or biolayer interferometry (e.g., FORTEBIO ® ).
  • SPR surface plasmon resonance
  • BIACORE ® BIACORE ®
  • biolayer interferometry e.g., FORTEBIO ®
  • the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule).
  • Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule.
  • Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule.
  • autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • allogeneic refers to any material derived from a different animal of the same species or different patient as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unalike genetically to interact antigenically.
  • a “gp120 antigen ” refers to an antigen of the gp120 exterior envelope glycoprotein of HIV-1. gp120 binds sequentially to CD4 and chemokine receptors on cells to initiate virus entry.
  • the gp120 core is composed of inner and outer domains and a bridging sheet. Components of both domains and the bridging sheet contribute to CD4 binding.
  • a gp120 antigen targeted by the antigen binding domains described herein comprises the inner and outer domains and bridging sheet of the gp120 core of the gp120 exterior envelope glycoprotein of HIV-1.
  • a gp120 antigen targeted by the antigen binding domains described herein may comprise one or more of: residues 83–118, 204– 256, and 474–492 of the inner domain; residues 257–421 and 436–473 of the outer domain; and residues 119–203 and 422–435 of the bridging sheet of HXBc2 gp120 or YU2 gp120.
  • CD4 refers to an integral membrane protein encoded by the CD4 gene in humans.
  • CD4 can comprise a sequence of MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQ IK ILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQL LVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSG TWTCTVLQNQKKVEFKIDIVVLAFQKASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELW W QAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTL A LEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKREKA VWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIV
  • a CD4 TM domain can comprise a sequence of MALIVLGGVAGLLLFIGLGIFF [SEQ ID NO: 21] [0106]
  • a CD4 intracellular domain (ICD), as used herein, refers to the ICD of the integral membrane protein encoded by the CD4 gene in humans.
  • a CD4 ICD can comprise a sequence of CVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI [SEQ ID NO: 22] [0107]
  • the extracellular domain of CD4 does not comprise a signal peptide.
  • a CD4 ECD or portion thereof that is capable of binding to an HIV antigen, such as gp120 can comprise the sequence, which comprises the extracellular domain of CD4 without the signal peptide: KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRR S LWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLES PPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVK R VTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQL Q
  • a CD4 ECD or portion thereof that is capable of binding to an HIV antigen, such as gp120 can comprise the sequence [0109] KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDR TLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIV VLAF [SEQ ID NO: 28]
  • a CD4 ECD domain or portion thereof that is capable of binding to an HIV antigen, such as gp120 can comprise amino acids at least 8, 9, 10, 11, 12, 13, 15, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more contiguous amino acids of the CD4 full length CD4 ECD.
  • a CD4 ECD domain or portion thereof that is capable of binding to an HIV antigen, such as gp120 can comprise at least 8, 9, 10, 11, 12, 13, 15, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more contiguous amino acids of the sequence KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRR S LWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLES PPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVK R VTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQL QKNLTCEVWGPT
  • treating refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • a “therapeutically effective amount” is the amount of a composition or an active component thereof sufficient to provide a beneficial effect or to otherwise reduce a detrimental non-beneficial event to the individual to whom the composition is administered.
  • therapeutically effective dose herein is meant a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g. Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).
  • the term "broad neutralizing antibody against HIV” refers to an antibody which inhibits HIV-l infection.
  • the antibody inhibits HIV-l infection as defined by at least about 50% inhibition of infection in vitro, in more than about 50%, 60%, 70%, 80%, 90%, 95%, 99% or greater, of a large panel of (greater than 100) HIV-l envelope pseudotyped viruses and/or viral isolates.
  • the broad neutralizing antibody is an antibody that inhibits HIV-l infection as defined by at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% inhibition of infection in vitro in more than about 50%, 60%, 70%, 80%, 90%, 95%, 99% or greater, of a large panel of (greater than 100) HIV-l envelope pseudotyped viruses and/or viral isolates.
  • the disclosure relates to a composition or pharmaceutical composition comprising one or a plurality of broad neutralizing antibodies.
  • the broadly neutralizing antibody is 10-1074.
  • a “T cell receptor (TCR) fusion protein” or “TFP” or “TRuCTM” 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
  • TRuCTM 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.
  • a “TFP T cell” or TRuC T cell is a T cell that has been transduced according to the methods disclosed herein and that expresses a TFP, e.g., incorporated into the natural T
  • the T cell is a CD4+ T cell, a CD8+ T cell, or a CD4+ / CD8+ T cell.
  • the TFP T cell is an NK cell or a regulatory T cell.
  • the term “subject” means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human.
  • a “patient” is a subject suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein. In some embodiments, a subject has HIV. In some embodiments, a subject has AIDS.
  • preventing refers to the prevention of the disease or condition, e.g., infection, in the patient. For example, if an individual at risk of developing an infection is treated with the methods of the present invention and does not later develop the infection, then the disease has been prevented, at least over a period of time, in that individual.
  • packaging insert is used to refer to instructions customarily included in commercial packages of therapeutic or diagnostic products (e.g., kits) that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic or diagnostic products.
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • pharmaceutical composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.
  • modulate refers to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.
  • the terms “increase” and “activate” refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.
  • the terms “reduce” and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100- fold, or greater in a recited variable.
  • the term “agonize” refers to the activation of receptor signaling to induce a biological response associated with activation of the receptor.
  • An “agonist” is an entity that binds to and agonizes a receptor.
  • the term “antagonize” refers to the inhibition of receptor signaling to inhibit a biological response associated with activation of the receptor.
  • An “antagonist” is an entity that binds to and antagonizes a receptor.
  • effector T cell includes T helper (i.e., CD4+) cells and cytotoxic (i.e., CD8+) T cells.
  • CD4+ effector T cells contribute to the development of several immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages.
  • CD8+ effector T cells destroy virus-infected cells and tumor cells. See Seder and Ahmed, Nature Immunol., 2003, 4:835-842, incorporated by reference in its entirety, for additional information on effector T cells.
  • the term “regulatory T cell” includes cells that regulate immunological tolerance, for example, by suppressing effector T cells.
  • the regulatory T cell has a CD4+CD25+Foxp3+ phenotype.
  • the regulatory T cell has a CD8+CD25+ phenotype.
  • the term “dendritic cell” refers to a professional antigen-presenting cell capable of activating a na ⁇ ve T cell and stimulating growth and differentiation of a B cell.
  • the phrase “disease associated with expression of viral antigen such as GP120” includes, but is not limited to, a disease associated with expression of viral antigen such as GP120 or condition associated with cells which express viral antigen such as GP120 including, e.g., HIV or AIDS.
  • RNA viruses of the subfamily Lentivirus include a human immunodeficiency virus type 1 or 2 (e.g., HIV-1 or HIV-2, wherein HIV-1 was formerly called lymphadenopathy associated virus 3 (HTLV-III) and acquired immune deficiency syndrome (AIDS)-related virus (ARV)), or another virus related to HIV-1 or HIV-2 that has been identified and associated with AIDS or AIDS-like disease.
  • HIV human immunodeficiency virus type 1 or 2
  • HIV-1 was formerly called lymphadenopathy associated virus 3 (HTLV-III) and acquired immune deficiency syndrome (AIDS)-related virus (ARV)
  • HIV-1 or HIV-2 formerly called lymphadenopathy associated virus 3 (HTLV-III) and acquired immune deficiency syndrome (AIDS)-related virus (ARV)
  • HIV-1 or HIV-2 formerly called lymphadenopathy associated virus 3 (HTLV-III) and acquired immune deficiency syndrome (AIDS)-related virus (ARV)
  • HIV lymphadenopathy associated virus 3
  • ARV acquired immune
  • 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 invention 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
  • stimulation refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.
  • a stimulatory domain or stimulatory molecule e.g., a TCR/CD3 complex
  • 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” refers to a molecule or portion thereof expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway.
  • the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • a primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or “ITAM”.
  • ITAM immunoreceptor tyrosine-based activation motif
  • Examples of an ITAM containing primary cytoplasmic signaling sequence that is of particular use in the invention includes, but is not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”) and CD66d.
  • the term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC’s) on its surface. T cells may recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.
  • the intracellular signaling domain generates a signal that promotes an immune effector function of the TFP containing cell, e.g., a TFP-expressing T cell.
  • immune effector function e.g., in a TFP-expressing T cell
  • examples of immune effector function, e.g., in a TFP-expressing T cell include cytolytic activity and T helper cell activity, including the secretion of cytokines.
  • the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation.
  • the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.
  • a primary intracellular signaling domain can comprise an ITAM (“immunoreceptor tyrosine-based activation motif”).
  • ITAM immunoglobulin-based activation motif
  • Examples of ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, DAP10 and DAP12.
  • costimulatory molecule refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response.
  • Costimulatory molecules include, but are not limited to, an MHC class 1 molecule, BTLA and a Toll ligand receptor, as well as DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137).
  • a costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule.
  • a costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like.
  • the intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.
  • 4-1BB refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No.
  • AAA62478.2 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like; and a “4-1BB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species, e.g., mouse, rodent, monkey, ape and the like.
  • the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain one or more introns.
  • the term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.
  • the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • the term “expression” refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
  • the term “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.
  • transfer vector includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like.
  • viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.
  • 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, e.g., in Milone et al., Mol. Ther.17(8): 1453-1464 (2009).
  • lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR TM gene delivery technology from Oxford BioMedica, the LENTIMAX TM vector system from Lentigen Technology, and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
  • the term “homologous” or “identity” 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.
  • 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” 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.
  • a “human antibody” is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies. [0154]
  • isolated means altered or removed from the natural state.
  • nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al., Mol. Cell.
  • pathogen can refer to a disease causing entity, such as a virus, or a microorganism.
  • the pathogen may be capable of causing an infection to a host, such as a human host.
  • a host may be a mammal.
  • a host may also be referred to as a subject, such as a human subject or a mammalian subject.
  • the pathogen may be referred to as a disease causing entity that can cause an active virulent infection, or a latent infection, an acute infection or a chronic infection or any combination thereof.
  • a pathogen frequently referred to in the context of the disclosure may be a retrovirus.
  • Retroviruses are small enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase.
  • the pathogenic virus of the instant disclosure may be HIV, including various types (e.g., HIV-1 and HIV-2), strains (e.g, strain BH10 and pNL4-3 of HIV-1), isolates, clades within a group of isolates (e.g., clade A, B, C, D, E, F, and G of group M of HIV-1 isolates) of HIV.
  • the viral antigen may be an HIV glycoprotein (or surface antigen) such as HIV envelope protein Env, either full length wild type (gp160), truncated (e.g, gp120 and gp41), or modified with insertions, deletions or substitutions; or an HIV structural protein Gag, either full length wild type, modified, or protease-processed products or fragments in various forms (e.g., natural, secreted, or membrane bound forms of HIV capsid proteins such as HIV p24 and p17; and 3) HIV regulatory proteins such as Tat, Vif, Nef, and Rev.
  • the polynucleotide may further encode HIV regulatory proteins such as Tat, Vif, Nef, and Rev.
  • HIV is a member of the lentivirus family of retroviruses.
  • retroviruses include, for example, oncogenic viruses such as human T-cell leukemia viruses (HTLV-1,-II,-III), and feline leukemiavirus.
  • the HIV viral particle consists of a viral core, made up of proteins designated p24 and p18.
  • the viral core contains the viral RNA genome and those enzymes required for replicative events.
  • Myristylated gag protein forms an outer viral shell around the viral core, which is, in turn, surrounded by a lipid membrane envelope derived from the infected cell membrane.
  • the HIV viral envelope comprises glycoproteins such as the gp120.
  • the gp120 molecule of HIV-1 is a glycoprotein that is part of the outer layer of the virus. gp120 forms viral membrane spikes consisting of 3 molecules of gp120 linked together and anchored to the membrane by gp41 protein. gp120 is essential for viral infection as it facilitates HIV entry into a cell, that is a CD4+ T cell.
  • “Infection” of a viral pathogen, such as HIV is the process of attaching to and delivering viral nucleic acid into a cell, such that the cell then replicates and produces live viruses. In some instances, infection is successful or envigored by the presence of more than one receptors that the virus can attach to, such receptors may be termed co-receptors for infection.
  • HIV co-receptors are receptors present in a cell that facilitate the infection of a virus.
  • the chemokine receptors CXCR4 and CCR5 function as coreceptors for HIV-1 entry into CD4 + cells.
  • CXCR4 can mediate entry of T cell line-tropic (T-tropic) HIV-1 strains.
  • M-tropic macrophage-tropic
  • the importance of chemokine receptors in HIV-1 pathogenesis is underscored by the observation that individuals deficient in CCR5 and peripheral blood mononuclear cells (PBMC) from these individuals are resistant to infection by HIV-1.
  • PBMC peripheral blood mononuclear cells
  • the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer 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. As used herein, 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, that can initiate the specific transcription of a polynucleotide sequence.
  • promoter/regulatory sequence refers to a nucleic acid sequence which can be used for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • the term “constitutive” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • the term “inducible” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • tissue-specific promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • linker and “flexible polypeptide linker” as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together.
  • the flexible polypeptide linkers include, but are not limited to, (Gly 4 Ser) 4 (SEQ ID NO: 87) or (Gly 4 Ser) 3 (SEQ ID NO: 88).
  • the linkers include multiple repeats of (Gly 2 Ser), (GlySer) or (Gly 3 Ser) (SEQ ID NO: 89).
  • 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.
  • in vitro transcribed RNA refers to RNA, preferably mRNA, which has been synthesized in vitro. Generally, the in vitro transcribed RNA is generated from an in vitro transcription vector.
  • the in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.
  • a “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA.
  • the polyA is between 50 and 5000 (SEQ ID NO: 92), 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
  • 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.
  • polyadenylate polymerase an enzyme that catalyzes the adenylation of adenine nucleotides
  • the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal.
  • the poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation.
  • Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm.
  • the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase.
  • the cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site.
  • adenosine residues are added to the free 3’ end at the cleavage site.
  • transient refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.
  • signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • cell surface receptor includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.
  • a “substantially purified” cell refers to a cell that is essentially free of other cell types.
  • a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
  • a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cells 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.
  • the term “therapeutic” as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.
  • the term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state.
  • the term “transfected” or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.
  • the term “specifically binds,” refers to an antibody, an antibody fragment or a specific ligand, which recognizes and binds a cognate binding partner (e.g., viral antigen such as GP120) 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., viral antigen such as GP120
  • Ranges throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range.
  • a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6.
  • a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.
  • T cell receptor (TCR) fusion proteins T cell receptor (TCR) [0183]
  • TFP T cell receptor
  • TFP T cell receptor
  • the present disclosure encompasses recombinant DNA constructs encoding TFPs and variants thereof, wherein the TFP comprises a binding domain, e.g., an antibody or antibody fragment, a ligand, or a ligand binding protein, that binds specifically to a target, such as a target antigen, for example, gp120 viral antigen, wherein the sequence of the binding domain is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • a binding domain e.g., an antibody or antibody fragment, a ligand, or a ligand binding protein
  • a target antigen for example, gp120 viral antigen
  • 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.
  • TFPs when expressed in a T-cell, can target and attack HIV-infected cells.
  • TFP-expressing cells When administered to a subject, can treat or reduce the disease burden of HIV.
  • the TFPs of the present disclosure comprise 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 invention include those associated with viral, bacterial and parasitic infections; autoimmune diseases; and cancerous diseases (e.g., malignant diseases).
  • the TFP-mediated T cell response can be directed to an antigen of interest by way of engineering an antigen-binding domain into the TFP that specifically binds a desired antigen.
  • the antigen binding domain can be any domain that binds to the antigen including any polypeptide including but not limited to a ligand or fragment thereof, a receptor or fragment thereof, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of a camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen binding domain, such as a recombinant fibronectin domain, anticalin, DARPIN and the like.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VHH variable domain
  • a natural or synthetic ligand specifically recognizing and binding the target antigen can be used as antigen binding domain for the TFP.
  • 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 to a target, such as a target antigen, for example, gp120.
  • a humanized antibody or antibody fragment may have improved affinity and/or specificity of binding to the target antigen.
  • the present disclosure contemplates modifications of the starting antibody or fragment (e.g., scFv) amino acid sequence that generate functionally equivalent molecules.
  • the VH or VL of a binding domain, e.g., scFv, comprised in the TFP can be modified to retain at least about 70%, 71%.72%.73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting VH or VL framework region of the target binding domain, for example, gp120 binding domain, e.g., scFv.
  • the present disclosure contemplates modifications of the entire TFP construct, e.g., modifications in one or more amino acid sequences of the various domains of the TFP construct in order to generate functionally equivalent molecules.
  • the TFP construct can be modified to retain at least about 70%, 71%.72%.73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting TFP construct.
  • the TFP of the instant disclosure binds a component of a pathogen or a pathogen infected cell, such as a virus, e.g., HIV.
  • the antigen binding domain comprises a binding domain for a protein or glycoprotein of a retrovirus.
  • the antigen binding domain comprises a binding domain for a protein or glycoprotein of a lentivirus.
  • the antigen binding domain comprises a binding domain for a protein or glycoprotein of an HIV.
  • the antigen binding domain comprises a binding domain for gp120, or a fragment thereof.
  • the antigen binding domain comprises CD4 or a fragment thereof.
  • the antigen binding domain comprises an extracellular domain of CD4, e.g., an extracellular domain of CD4 that specifically binds GP120.
  • the CD4 extracellular domain may comprise a sequence of SEQ ID NO.23.
  • the antigen binding domain comprises a portion or a fragment of CD4 that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the extracellular domain of CD4, and that is capable of binding to gp120.
  • the antigen binding domain comprises a portion or a fragment of CD4 that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the gp120 binding domain of CD4, and that is capable of binding to gp120.
  • the antigen binding comprises a region that is 100% identical to the gp120 binding domain of CD4.
  • the antigen binding domain comprises a sequence having at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.23.
  • the antigen binding domain comprises a portion (e.g., the first two domains) of CD4 extracellular domain.
  • the antigen binding domain can comprise a sequence having at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.28.
  • the antigen binding domain comprises an antibody or a fragment thereof that can bind to HIV-gp120.
  • the antigen binding domain comprises an antibody or a fragment thereof specifically binds gp120.
  • the antigen binding domain comprises an antibody or a fragment thereof specifically binds the V1- V2 loop of gp120. In some embodiments, the antigen binding domain comprises an antibody or a fragment thereof specifically binds the V3 loop of gp120. In some embodiments, the antigen binding domain comprises an antibody or a fragment thereof specifically binds the membrane proximal external region of gp120. In some embodiments, the antigen binding domain comprises a gp120-binding antibody heavy chain and light chain variable regions, a VH domain and a VL domain respectively. In some embodiments, the gp120-binding antigen binding domain of the TFP comprises a single chain variable fraction antibody (scFv).
  • scFv single chain variable fraction antibody
  • the gp120-binding antigen binding domain of TFP comprises a diabody. In some embodiments, the gp120-binding antigen binding domain of TFP comprises a portion of a bivalent or a multivalent antibody. [0192] In some embodiments, the TFP extracellular domain comprises a gp120-binding scFv, that can specifically bind to the exposed epitope of gp120, with high specificity. [0193] In some embodiments, the TFP extracellular domain binds to gp120 with a Kd of less than 10 ⁇ -7, or less than 10 ⁇ -8, or less than 10 ⁇ -9, or less than 10 ⁇ -10, or less than 10 ⁇ -11.
  • the TFP extracellular domain comprises a humanized antibody or fraction thereof, that can bind to gp120.
  • the TFP comprises two antigen binding domains.
  • the first antigen binding domain comprises CD4 or a fraction thereof
  • the second antigen binding domain comprises a gp120-binding antibody or fragment thereof.
  • Extracellular domain [0196] The extracellular domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any protein, but in particular a membrane-bound or transmembrane protein. In one aspect the extracellular domain is capable of associating with the transmembrane domain.
  • An extracellular domain of particular use in this present disclosure may include at least the extracellular region(s) of e.g., the alpha, beta, gamma, delta, or zeta chain of the T cell receptor, or CD3 epsilon, CD3 gamma, or CD3 delta, or in alternative embodiments, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • the extracellular domain comprises, or comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more consecutive amino acid residues of the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR
  • 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, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more consecutive amino acid residues of an IgC domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma
  • 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 an IgC domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma.
  • the extracellular domain comprises a sequence encoding an IgC domain of TCR alpha, TCR beta, TCR delta, or TCR gamma having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
  • the extracellular domain comprises, or comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more consecutive amino acid residues of the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit
  • the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the extracellular domain comprises a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
  • the extracellular domain can be a TCR extracellular domain.
  • the TCR extracellular domain can be derived from a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit or a CD3 delta TCR subunit.
  • the extracellular domain can be a full-length TCR extracellular domain or fragment (e.g., functional fragment) thereof.
  • the extracellular domain can comprise a variable domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the extracellular domain can comprise a variable domain and the extracellular component of 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. [0201]
  • the extracellular domain can comprise the extracellular component of a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the extracellular domain can comprise the extracellular component of a full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the extracellular domain can comprise a fragment (e.g., functional fragment) of the extracellular component 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 the extracellular component 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.
  • the TFP may comprise a CD4 extracellular domain that is fused to a transmembrane region of a receptor comprising entirely or at least a partial segment of CD3 receptor, resulting in a CD4-CD3 TFP of the disclosure.
  • the TFP may comprise a CD4 extracellular domain fused to the transmembrane and intracellular segments of a CD3 epsilon molecule, resulting in a CD4-CD3 epsilon TFP of the disclosure.
  • the TFP extracellular domain is operably linked with the transmembrane and intracellular regions of the TFP such that binding of the extracellular domain to the target, such as gp120 triggers activation of the intracellular domains, such as one or more intracellular signaling domains.
  • Transmembrane Domain [0205]
  • 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 acids associated with the extracellular region of the protein from which the transmembrane was derived (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids of the intracellular region).
  • the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the extracellular region. In some cases, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the intracellular region. In one aspect, the transmembrane domain is one that is associated with one of the other domains of the TFP. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.
  • the transmembrane domain is capable of homodimerization with another TFP on the TFP-T cell surface.
  • the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same TFP.
  • the transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TFP has bound to a target.
  • a transmembrane domain of particular use in this present disclosure may include at least the transmembrane region(s) of e.g., the alpha, beta, gamma, delta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, 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 region(s) e.g., the alpha, beta, gamma, delta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, 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 comprises a CD3 transmembrane domain.
  • the transmembrane domain comprises a CD3 epsilon transmembrane domain, or a portion thereof.
  • the extracellular region of the TFP can be attached to the antigen binding domain of the TFP, via a hinge, e.g., a hinge from a human protein.
  • the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge.
  • a short oligo- or polypeptide linker between 2 and 10 amino acids in length may form the linkage between binding element and the TCR extracellular domain of the TFP.
  • 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.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 93) or a sequence (GGGGS)x or (G4S)n, wherein X or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more (SEQ ID NO: 94).
  • X or n is an integer from 1 to 10.
  • X or n is an integer from 1 to 4.
  • X or n is 2. In some embodiments, X or n is 4.
  • the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 95).
  • Cytoplasmic Domain [0211]
  • the cytoplasmic domain of the TFP can include an intracellular domain.
  • the intracellular domain is from CD3 gamma, CD3 delta, CD3 epsilon, TCR alpha, TCR beta, TCR gamma, or TCR delta.
  • the intracellular domain comprises a signaling domain, if the TFP contains CD3 gamma, delta or epsilon polypeptides; TCR alpha, TCR beta, TCR gamma, and TCR delta subunits generally have short (e.g., 1-19 amino acids in length) intracellular domains and are generally lacking in a signaling domain.
  • An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the TFP has been introduced.
  • intracellular domains of TCR alpha, TCR beta, TCR gamma, and TCR delta do not have signaling domains, they are able to recruit proteins having a primary intracellular signaling domain described herein, e.g., CD3 zeta, which functions as an intracellular signaling domain.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function.
  • intracellular signaling domain While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
  • the term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • Examples of intracellular signaling domains for use in the TFP of the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that 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.
  • Examples of intracellular domains for use in the TFP of the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that are able to act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.
  • the intracellular domain comprises the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the intracellular domain comprises, or comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more consecutive amino acid residues of the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain.
  • the intracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% 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 intracellular 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.
  • na ⁇ ve T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic domain, e.g., a costimulatory domain).
  • primary 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 include those of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
  • a TFP of the present disclosure comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-epsilon.
  • a primary signaling domain comprises a modified 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 the CD3 zeta signaling domain by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a TFP of the present disclosure.
  • the intracellular signaling domain of the TFP can comprise a CD3 epsilon chain portion and a costimulatory signaling domain.
  • the costimulatory signaling domain refers to a portion of the TFP comprising the intracellular domain of a costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, 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.
  • the intracellular portion of the TFP comprises at least a functional domain comprising at least a portion of CD3 epsilon intracellular signaling domains.
  • the intracellular signaling domains are operably linked with the extracellular domain, such that binding of the extracellular domain to its target activates the intracellular signaling domains.
  • 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 described herein comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO.70.
  • the TFP comprises from N-terminus to C- terminus a GM-CSFRa signal peptide, a CD4 extracellular domain, a linker, and a CD3 ⁇ subunit.
  • the TFP comprises from N-terminus to C-terminus a sequence of SEQ ID NO.71, a sequence of SEQ ID NO.23, a sequence of SEQ ID NO.72, and a sequence of SEQ ID NO.55.
  • the CD4 extracellular domain may comprise a mutation, e.g., a F68A mutation.
  • the CD4 extracellular domain may comprise a sequence of SEQ ID NO. 74.
  • the TFP may comprise a sequence of SEQ ID NO.73.
  • the TFP described herein comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO.84.
  • the TFP comprises from N-terminus to C- terminus a GM-CSFRa signal peptide, a first two domains of CD4 extracellular domain, a linker, and a CD3 ⁇ subunit. In some embodiments, the TFP comprises from N-terminus to C-terminus a sequence of SEQ ID NO.71, a sequence of SEQ ID NO.28, a sequence of SEQ ID NO.72, and a sequence of SEQ ID NO.55.
  • 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., GP120) or a different target.
  • a first TFP has an anti-GP120 antigen binding domain and a second TFP comprising a CD4 antigen binding domain.
  • 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 comprise one or more TCR constant domains, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain, a TCR alpha constant domain and a TCR beta constant domain, a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain.
  • the TCR subunit and the antibody can be operatively linked.
  • the TFP can functionally incorporate into a TCR complex (e.g., an endogenous TCR complex) when expressed in a T cell.
  • the 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 TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain of CD3 epsilon, CD3 gamma, or CD3 delta, and further comprises the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • a TFP-expressing cell comprises a constant domain of a TCR alpha chain and a TCR beta chain.
  • a TFP- expressing cell comprises a constant domain of a TCR gamma chain and a TCR delta chain.
  • the TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) the constant domain of a TCR alpha chain (i.e., comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain), and further comprises the constant domain of a TCR beta chain.
  • the TCR beta constant domain can further comprise second binding domain that is operatively linked to the TCR beta constant domain.
  • the second binding domain can be the same or different as the binding domain of the TFP.
  • the TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) the constant domain of a TCR beta chain (i.e., comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain), and further comprises the constant domain of a TCR alpha chain.
  • the TCR alpha constant domain can further comprise a second binding domain that is operatively linked to the TCR alpha constant domain.
  • the second binding domain can be the same or different as the binding domain of the TFP.
  • the TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) the constant domain of a TCR gamma chain (i.e., comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain), and further comprises the constant domain of a TCR delta chain.
  • the TCR delta constant domain can further comprise a second binding domain that is operatively linked to the TCR delta constant domain.
  • the second binding domain can be the same or different as the binding domain of the TFP.
  • the TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) the constant domain of a TCR delta chain (i.e., comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain), and further comprises the constant domain of a TCR gamma chain.
  • the TCR gamma constant domain can further comprise a second binding domain that is operatively linked to the TCR gamma constant domain.
  • the second binding domain can be the same or different as the binding domain of the TFP.
  • 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, CD160, 2B4 and TGFR beta.
  • the agent which inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
  • the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as 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 PD1 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 41BB and CD3 zeta (also referred to herein as a PD1 TFP).
  • ECD extracellular domain
  • PD1 TFP when used in combinations with a TFP 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).
  • a population of TFP-expressing T cells e.g., TFP-T cells.
  • the population of TFP-expressing T cells comprises a mixture of cells expressing different TFPs.
  • the population of TFP-T cells can include a first cell expressing a TFP having a binding domain described herein, e.g., CD4 or a fragment thereof, and a second cell expressing a TFP having a different binding domain, e.g., a binding domain described herein that differs from the binding domain in the TFP expressed by the first cell, e.g., an anti-GP120 antibody fragment.
  • a first cell expressing a TFP having a binding domain described herein, e.g., CD4 or a fragment thereof
  • a second cell expressing a TFP having a different binding domain, e.g., a binding domain described herein that differs from the binding domain in the TFP expressed by the first cell, e.g., an anti-GP120 antibody fragment.
  • 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 can, in some embodiments, decrease the ability of a modified T cell to mount an immune effector response.
  • inhibitory molecules include PD1, PD-L1, PD- L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.
  • the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
  • the TFP-expressing cell described herein can further express a broadly neutralizing antibody against HIV (or “bNAbs”).
  • a broadly neutralizing antibody is defined as a bNAb that neutralizes HIV- 1 species belonging to two or more different clades.
  • the different clades are selected from the group consisting of clades A, B, C, D, E, AE, AG, G or F.
  • the HIV-l strains from two or more clades comprise virus from non-B clades.
  • bNAbs target conserved sites of vulnerability on the HIV-l envelope (env).
  • the bNAb anti -HIV-l antibody is sufficient to neutralize or bind up to or at least about 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or up to 100% of viral isolates in culture or in a subject.
  • the bNAb is selected based on its neutralization activity.
  • the bNAb is selected based on its ability to bind HIV-l infected cells (predictive of ADCC).
  • Various bNAbs are known in the art and can be used according to this disclosure.
  • the present disclosure comprises a composition or cell comprising bispecific, trispecific or tetraspecific anti-HIV bNAbs.
  • examples include but are not limited to those described in U.S. Patent No.8673307, W02014063059, WO2012158948, WO2015/117008, and PCT/US2015/41272, including antibodies 3BNC117, 3BNC60, 12A12, 12A21, NIH45-46, bANCl3l, 8ANC134, IB2530, INC9, 8ANC195.8ANC196, 10-259, 10- 303, 10-410, 10- 847, 10-996, 10-1074, 10-1121, 10-1130, 10-1146, 10-1341, 10-1369, and 10-1074GM.
  • Certain bNAbs target conserved sites of vulnerability on the HIV-l envelope (ENV) such as the CD4 binding site (CD4bs).
  • ENV HIV-l envelope
  • CD4bs CD4 binding site
  • the bl2 monoclonal antibody was for many years considered the prototype and optimal CD4bs bNAb, although it was only able to neutralize about 40% of HIV-l strains.
  • a new group of CD4bs antibodies named VRC01, VRC02, and VRC03 was disclosed. Of these, VRC01 was the most potent and broad.
  • VRC01 neutralized 91% of viruses with an IC50 less than 50 pg/ml and 72% of viruses with an IC50 less than 1 pg/ml (Wu et al, Science, 329(5993):856-86l, 2010). Structural analyses have explained VRCOl's high potency and breadth: VRC01 partially mimics the CD4 interaction with gpl20. Specifically, the majority of the gpl20 area targeted by VRC01 is the highly conserved site of initial CD4 attachment in the outer domain of gpl20, which allows VRC01 to bypass conformational and glycan masking that impaired previously identified CD4bs bNAbs.
  • VRC01 Both the heavy and light chain of VRC01 contribute to the binding of gpl20, with the CDRH2 providing the primary interaction, and CDRL1, CDRL3, CDRH1, and CDRH3 providing additional contact points. It has been shown that passive transfer of VRC01 protects against intrarectal or intravaginal simian-HIV (SHIV) challenge in non-human primates.
  • VRC01 is a monoclonal antibody that specifically binds to gpl20 and neutralizes a broad range of HIV viruses.
  • variable heavy (VH) chain and variable light (VL) chain of VRC01 have been described in Wu et al, Science, 329(5993):856-86l, 2010, and PCT publication WO2012/154312, incorporated by reference herein in their entireties.
  • VRCOl-like antibodies are described, for example in US20170267748, incorporated by reference herein in its entirety. Generally, these antibodies bind to the CD4 binding surface of gpl20 in substantially the same orientation as VRC01, and are broadly neutralizing VRCOl-like antibodies, with several of the important contacts between CD4 and gpl20 mimicked by the VRCOl-like antibodies.
  • VRCOl-like antibodies are available, including VRCOl-like antibodies, heavy chains and light chains disclosed in PCT International Application No. PCT/US2010/050295, filed Sep.24, 2010, which is incorporated by reference herein and Wu et al, "Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-l," Science, 329(5993):856-86l, 2010, which is incorporated by reference herein.
  • These include heavy and light chains of the VRC01, VRC02, VRC03, VRC06, VRC07, 3BNC117, IOMA and N6.
  • the amino acid sequences of the heavy and light variable regions of VRC03 have been described in Wu et al, (Science.2010 Aug 13; 329(5993):856-6l; PMID 20616233).
  • the amino acid sequences of the heavy and light variable regions of VRC06 have been described in Li et al, (J Virol.2012 Oct; 86(20): 11231-41; PMID 22875963).
  • the amino acid sequences of the heavy and light variable regions of VRC07 have been described in Rudicell et al., (J Virol. 2014 Nov; 88(21): 12669-82; PMID 25142607).
  • amino acid sequences of the heavy and light variable regions of 3BNC117 have been described in Scheid et al, (Science.2011 Sep 16; 333(6049): 1633-7; PMID 21764753).
  • the amino acid sequences of the heavy and light variable regions of IOMA have been described in Gristick et al., (Nat Struct Mol Biol.2016 Oct; 23(10):906-915; PMID 27617431).
  • the amino acid sequences of the heavy and light variable regions of N6 have been described in Huang et al, (Immunity.2016 Nov 15; 45(5): 1108-1121; PMID 27851912). These amino acid sequences are herein incorporated by reference in their entireties.
  • PGT121, PGT122, PGT123, PGT127, PGT128, PGT135, 10-1074 and BG18 are a family of neutralizing monoclonal antibodies that specifically bind to the V1/V2 and V3 regions of HIV- l Env and can inhibit HIV-l infection of target cells.
  • PGT121, PGT122, and PGT123 mAbs and methods of producing them are described in, for example, Walker et al, Nature, 477:466-470, 2011, and Int. Pub. No. WO 2012/030904, each of which is incorporated by reference herein.
  • PGT127 and PGT128 are described in, for example Pejchal et al. (Science, 2011 Nov 25, 334 (6059): 1097-103).
  • PGT135 is described, for example, in Kong et al. (Nature Structural and Molecular Biology, 2013 Jul, 20:796-803).
  • the amino acid sequences of the heavy and light variable regions of 10-1074 have been described in Mouquet et al. ((2012) Proc. Natl. Acad. Sci. USA 109: E3268-E3277).
  • the amino acid sequences of the heavy and light variable regions of BG18 have been described in Freund et al. ((2012) Sci Transl Med.2017 Jan l8;9(373); PMID 28100831).
  • the amino acid sequences of the heavy and light variable regions of PGT135 have been described in Kong et al.
  • PGT151 are broadly neutralizing monoclonal antibodies that specifically bind to the gpl20/gp4l interface of HIV-l Env in its prefusion mature (cleaved) conformation, and which can inhibit HIV-l infection of target cells.
  • PGT151 antibody and methods of producing this antibody are described in, for example, Blattner et al., Immunity, 40, 669-680, 2014, and Falkowska et al, Immunity, 40, 657-668, 2014, each of which is incorporated by reference herein in its entirety).
  • the amino acid sequences of the heavy and light variable regions of the PGT151 mAb are known and have been deposited in GenBank as Nos.
  • KJ700282.1 (PGT151 VH) and KJ700290.1 (PGT151 VL), each of which is incorporated by reference herein in its entirety).
  • the amino acid sequences of the heavy and light variable regions of N123-VRC34.01 have been described in Kong et al, (Science 352 (6287), 828-833 (2016)).
  • the amino acid sequences of the heavy and light variable regions of 3BC315 have been described in Lee et al. (Nat Commun.2015 Sep 25; 6:8167; PMID 26404402).
  • the amino acid sequences of the heavy and light variable regions of PGT151 have been described in Blattner et al. (Immunity.2014 May 15; 40(5):669-80; PMID 24768348).
  • 10E8, 10E8n4, 10E8n4 S100cF, Dh5l l.2_k3, Z13, 4E10, and 2F5 are broadly neutralizing monoclonal antibodies that primarily target a HIV Env membrane proximal external region (MPER) helix spanning residues 671-683.
  • MPER HIV Env membrane proximal external region
  • the amino acid sequences of the heavy and light variable regions of 10E8n4 S100cF have been described in PCT/US2016/060390 and WO2017079479.
  • the amino acid sequences of the heavy and light variable regions of DH5l l.2_k3 have been described in Williams et al. (Sci Immunol.2017 Jan 27; 2(7); PMID 28783671).
  • the amino acid sequences of the heavy and light variable regions of 4E10 have been described in Rujas et al. (J Virol.2015 Dec; 89(23): 11975-89; PMID 26378169).
  • the amino acid sequences of the heavy and light variable regions of 2F5 have been described in Julien et al.
  • PGT141, PGT142, PGT143, and PGT145 are a family of broadly neutralizing monoclonal antibodies that specifically bind to the V1/V2 domain of the HIV-l Env ectodomain trimer in its prefusion mature closed conformation, and which can inhibit HIV-l infection of target cells.
  • PGT141, PGT142, PGT143, and PGT145 mAbs and methods of producing them are described in, for example, Walker et al., Nature, 477:466-470, 2011, and Int. Pub. No.
  • JN201906.1 (PGT141 VH), JN201923.1 (PGT141 VL), JN201907.1 (PGT142 VH), JN201924.1 (PGT142 VL), JN201908.1 (PGT143 VH), JN201925.1 (PGT143 VL), JN201909.1 (PGT144 VH), JN201926.1 (PGT144 VL), JN201910.1 (PGT145 VH), and JN201927.1 (PGT145 VL), each of which is incorporated by reference herein in its entirety).
  • the HIV-l neutralizing single domain antibody JM4 (Matz J, Kessler P, Bouchet J, Combes O, Ramos OH, Barin F, Baty D, Martin L, Benichou S, Chames P. Straightforward selection of broadly neutralizing single-domain antibodies targeting the conserved CD4 and coreceptor binding sites of HIV-l gpl20. J Virol.2013 Jan;87(2): 1137-49. doi: 10.1128/JVI.00461-12. Epub 2012 Nov 14) is antoher neutralizing antibody that can be used in the present disclosure.
  • the bNAb is selected from 10-1074, VRC01, VRC07, 3BNC117, N6, PCT121, 2G12, GDM1400, CAP256, PG16, 10E8, 2F5, 4E10, PG9, JM4, and VRC01.
  • the bNAb is PG9 or a salt thereof.
  • the bNAb is JM4 or a salt thereof.
  • the bNAb is 10-1074 or a salt thereof (Mouquet, et al., Proc. Natl. Acad. Sci.
  • Monoclonal antibody 10-1074 targets the V3 glycan supersite on the HIV-l envelope (Env) protein. It is among the most potent anti -HIV-l neutralizing antibodies isolated to date. [0248] The amino acid sequences of the heavy and light variable regions of 10-1074 have been described in Mouquet et al. ((2012) Proc.Natl. Acad. Sci. USA 109: E3268-E3277). Nucleic Acid Constructs Encoding a TFP [0249] The present disclosure provides nucleic acid molecules encoding one or more TFP constructs described herein.
  • the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct. [0250] In some embodiments, when the cell comprises a first polypeptide, e.g., a TFP, and a second polypeptide, e.g., a second TFP, a constant domain, a PD-1 fusion protein, or a broadly neutralizing antibody against HIV, the polypeptides are expressed from the same recombinant nucleic acid molecule. In some embodiments, the polypeptides are expressed from the same operon.
  • a first polypeptide e.g., a TFP
  • a second polypeptide e.g., a second TFP, a constant domain, a PD-1 fusion protein, or a broadly neutralizing antibody against HIV
  • the polypeptides are expressed from the same recombinant nucleic acid molecule. In some embodiments, the polypeptides are expressed from the same operon.
  • the first and second polypeptide are expressed as a single polypeptide separated by a 2A cleavage site, e.g., a P2A or T2A cleavage site.
  • the 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. Alternatively, the gene of interest can be produced synthetically, rather than cloned.
  • the nucleic acid is selected from the group consisting of a DNA and an RNA.
  • the nucleic acid is an mRNA.
  • the recombinant nucleic acid comprises a nucleic acid analog, wherein the nucleic acid analog is not in an encoding sequence of the recombinant nucleic acid.
  • the nucleic analog is selected from the group consisting of 2’-O-methyl, 2’-O-methoxyethyl (2’-O-MOE), 2’-O- aminopropyl, 2’-deoxy, T-deoxy-2’-fluoro, 2’-O-aminopropyl (2’-O-AP), 2'-O- dimethylaminoethyl (2’-O-DMAOE), 2’-O-dimethylaminopropyl (2’-O-DMAP), T-O- dimethylaminoethyloxyethyl (2’-O-DMAEOE), 2’-O-N-methylacetamido (2’-O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphoric acid
  • 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. In some instances, the nucleic acid is an in vitro transcribed nucleic acid. [0254] Disclosed herein are 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 (SEQ ID NO: 96) 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).
  • 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.
  • 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.
  • Gene Editing of TCR Complex or Endogenous Protein-coding Genes [0257]
  • 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.
  • CRISPR® clustered regularly interspaced short palindromic repeats
  • Patent No.8,697,359 transcription activator-like effector (TALE) nucleases
  • TALE transcription activator-like effector
  • TALENs transcription activator-like effector nucleases
  • meganucleases endodeoxyribonucleases having large recognition sites comprising double-stranded DNA sequences of 12 to 40 base pairs
  • ZFN zinc finger nuclease
  • ZFN zinc finger nuclease
  • 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 Reviews 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 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 an 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 programmed cell death protein 1 (PD1), and/or other genes.
  • HLAs human leukocyte antigens
  • PD1 programmed cell death protein 1
  • DSB may then be repaired by either non-homologous end joining (NHEJ) or –when donor DNA is present- homologous recombination (HR), an event that introduces the homologous sequence from a donor DNA fragment.
  • NHEJ non-homologous end joining
  • HR homologous recombination
  • nickase nucleases generate single-stranded DNA breaks (SSB). 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.
  • ssDI single strand DNA incorporation
  • ssTR single strand template repair
  • Genetic 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. For example, zinc- finger nucleases (ZFNs) can be engineered to recognize and cut predetermined sites in a genome.
  • 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.
  • ZFNs have been used extensively to target gene addition, removal, and substitution in a wide range of eukaryotic organisms (reviewed in Durai et al. (2005) Nucleic Acids Res 33, 5978).
  • TAL-effector nucleases can be generated to cleave specific sites in genomic DNA.
  • a TALEN comprises an engineered, site-specific DNA-binding domain fused to the Fokl nuclease domain (reviewed in Mak et al. (2013), Curr Opin Struct Biol.23:93-9).
  • the DNA binding domain comprises a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA basepair.
  • Compact TALENs have an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley et al. (2013), Nat Commun.4: 1762).
  • a Compact TALEN comprises an engineered, site-specific TAL-effector DNA-binding domain fused to the nuclease domain from the I-TevI homing endonuclease. Unlike Fokl, I-TevI does not need to dimerize to produce a double-strand DNA break so a Compact TALEN is functional as a monomer.
  • Engineered endonucleases based on the CRISPR/Cas9 system are also known in the art (Ran et al. (2013), Nat Protoc.8:2281-2308; Mali et al. (2013), Nat Methods 10:957-63).
  • the CRISPR gene-editing technology is composed of an endonuclease protein whose DNA-targeting specificity and cutting activity can be programmed by a short guide RNA or a duplex crRNA/TracrRNA.
  • a CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically microbial Cas9; and (2) a short "guide RNA” or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome.
  • CRISPR systems There are two classes of CRISPR systems known in the art (Adli (2016) Nat. Commun. 9:1911), each containing multiple CRISPR types. Class 1 contains type I and type III CRISPR systems that are commonly found in Archaea. And, Class II contains type II, IV, V, and VI CRISPR systems. Although the most widely used 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).
  • 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.
  • MN Meganucleases
  • meganuclease is engineered I-CreI homing endonuclease. In other embodiments, meganuclease is engineered I-SceI homing endonuclease.
  • chimeric proteins comprising fusions of meganucleases, ZFNs, and TALENs have been engineered to generate novel monomeric enzymes that take advantage of the binding affinity of ZFNs and TALENs and the cleavage specificity of meganucleases (Gersbach (2016), Molecular Therapy.24: 430–446).
  • a megaTAL is a single chimeric protein, which is the combination of the easy-to- tailor DNA binding domains from TALENs with the high cleavage efficiency of meganucleases.
  • the nucleases and in the case of the CRISPR/ Cas9 system, a gRNA, must be efficiently delivered to the cells of interest. Delivery methods such as physical, chemical, and viral methods are also know in the art (Mali (2013). Indian J. Hum. Genet.19: 3-8.). In some instances, physical delivery methods can be selected from the methods but not limited to electroporation, microinjection, or use of ballistic particles.
  • 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.
  • the 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. Alternatively, the gene of interest can be produced synthetically, rather than cloned.
  • the present disclosure also provides vectors in which a DNA of the present disclosure is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non- proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • the vector comprising the nucleic acid encoding the desired TFP of the present disclosure is an adenoviral vector (A5/35).
  • the expression of nucleic acids encoding TFPs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See, e.g., June et al., 2009 Nature Reviews Immunology 9.10: 704-716, which is incorporated herein by reference.
  • the 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).
  • a number of virally based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • 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.
  • An example of a promoter that is capable of expressing a TFP transgene in a mammalian T cell is the EF1a promoter.
  • the native EF1a promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome.
  • the EF1a promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving TFP expression from transgenes cloned into a lentiviral vector (see, e.g., Milone et al., Mol. Ther.17(8): 1453-1464 (2009)).
  • Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • CMV immediate early cytomegalovirus
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor-1a promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the present disclosure should not be limited to the use of constitutive promoters.
  • inducible promoters are also contemplated as part of the present disclosure.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence to which it is operatively linked when such expression is desired or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline-regulated promoter.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic- resistance genes, such as neo and the like.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • Methods of introducing and expressing genes into a cell are known in the art.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY. A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
  • 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, the contents of which are herein incorporated by reference in their entirety).
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.
  • an exemplary delivery vehicle is a liposome.
  • lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St.
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 °C. Chloroform is used as the only solvent since it is more readily evaporated than methanol.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10).
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine- nucleic acid complexes are also contemplated.
  • the present disclosure further provides a vector comprising a TFP encoding nucleic acid molecule.
  • a TFP vector can be directly transduced into a cell, e.g., a T cell.
  • the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs.
  • the vector is capable of expressing the TFP construct in mammalian T cells.
  • the mammalian T cell is a human T cell.
  • Circular RNA [0287]
  • TFP T cells are transduced with an RNA molecule.
  • the RNA is circular RNA.
  • the circular RNA is exogenous.
  • circular RNA is endogenous.
  • circular RNAs with an internal ribosomal entry site can be translated in vitro or in vivo or ex vivo.
  • Circular RNAs are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Circular RNAs are 3-5’ covalently closed RNA rings, and circular RNAs do not display Cap or poly(A) tails. Since circular RNAs lack the free ends necessary for exonuclease-mediated degradation, rendering them resistant to several mechanisms of RNA turnover and granting them extended lifespans as compared to their linear mRNA counterparts.
  • Circular RNAs are produced by the process of splicing, and circularization occurs using conventional splice sites mostly at annotated exon boundaries (Starke et al., 2015; Szabo et al., 2015).
  • splice sites are used in reverse: downstream splice donors are “backspliced” to upstream splice acceptors (see Jeck and Sharpless, 2014; Barrett and Salzman, 2016; Szabo and Salzman, 2016; Holdt et al., 2018 for review).
  • RNA circularization To generate circular RNAs that we could subsequently transfer into cells, in vitro production of circular RNAs with autocatalytic-splicing introns can be programmed.
  • IVTT in vitro transcription
  • Three general strategies have been reported so far for RNA circularization: chemical methods using cyanogen bromide or a similar condensing agent, enzymatic methods using RNA or DNA ligases, and ribozymatic methods using self-splicing introns.
  • precursor RNA was synthesized by run-off transcription and then heated in the presence of magnesium ions and GTP to promote circularization. RNA so produced can efficiently transfect different kinds of cells.
  • the template includes sequences for the TFP, CAR, and TCR, or combination thereof.
  • the group I intron of phage T4 thymidylate synthase (td) gene is well characterized to circularize while the exons linearly splice together (Chandry and Bel- fort, 1987; Ford and Ares, 1994; Perriman and Ares, 1998).
  • td intron order is permuted flanking any exon sequence, the exon is circularized via two autocatalytic transesterification reactions (Ford and Ares, 1994; Puttaraju and Been, 1995).
  • the group I intron of phage T4 thymidylate synthase (td) gene is used to generate exogenous circular RNA.
  • a ribozymatic method utilizing a permuted group I catalytic intron has been used since it is more applicable to long RNA circularization and requires only the addition of GTP and Mg 2+ as cofactors.
  • This permuted intron-exon (PIE) splicing strategy consists of fused partial exons flanked by half-intron sequences.
  • a full-length encephalomyocarditis virus such as EMCV
  • IRES full-length encephalomyocarditis virus
  • TFP thymidylate synthase
  • T4 thymidylate synthase
  • the mentioned sequence further comprises complementary ‘homology arms’ placed at the 5′ and 3′ ends of the precursor RNA with the aim of bringing the 5′ and 3′ splice sites into proximity of one another.
  • the splicing reaction can be treated with RNase R.
  • the TFP is encoded by a circular RNA.
  • the circular RNA 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.
  • linear precursor RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template.
  • DNA of interest from any source can be directly converted by PCR into a template for in vitro RNA synthesis using appropriate primers and buffer and RNA polymerase and nucleotides modified or not.
  • the source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, digested DNA, 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 linear precursor RNA 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.
  • Chemical structures with the ability to promote stability and/or translation efficiency may also be used.
  • the RNA preferably has 5’ and 3’ UTRs.
  • the 5’ UTR is between one and 3000 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 required to achieve optimal RNA stability or/and 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.
  • AU-rich elements in 3’UTR sequences can decrease the stability of mRNA whereas protein binding motifs can increase the stability of mRNA and circular RNA. 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 do not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art.
  • the 5’ UTR can be the 5’UTR of an RNA virus whose RNA genome is stable in cells.
  • 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.
  • RNA polymerase 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 RNA has both a cap on the 5’ end and a 3’ poly(A) tail which determine ribosome binding, initiation of translation and stability of mRNA in the cell.
  • RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells.
  • phage T7 RNA polymerase can extend the 3’ end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).
  • the conventional method of integration of polyA/T stretches into a DNA template is molecular cloning.
  • 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 T) (SEQ ID NOS 97 and 98, respectively, in order of appearance), 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.
  • the poly(A) tail is between 100 and 5000 adenosines (SEQ ID NO: 99).
  • 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 (SEQ ID NO: 100) 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.
  • RNAs produced by the methods disclosed herein include a 5’ cap.
  • the 5’ cap is provided using techniques known in the art and described herein (Cougot et al., Trends in Biochem. Sci., 29:436- 444 (2001); Stepinski et al., RNA, 7:1468-95 (2001); Elango et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).
  • the RNAs e.g. circular RNA
  • IRISPR internal ribosome entry site
  • the IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
  • RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector®-II (Amaxa Biosystems, Cologne, Germany)), ECM® 830 (BTX) (Harvard Instruments, Boston, Mass.), Neon Transfection System (ThermoFisher), Cell squeezing (SQZ Biotechnologies) or the Gene Pulser® II (BioRad, Denver, Colo.), Multiporator® (Eppendorf, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa et al.
  • the TFP T cells provided herein may be useful for the treatment of any disease or condition involving a viral antigen such as gp120. Without being bound by theory, it is believed that the gp120-targeting domain binds to gp120 on the surface of an HIV-infected cell, thereby activating the TFP-expressing T cell to direct cytotoxic activity towards the infected cell and reduce the viral burden of HIV in the subject.
  • the disease or condition is a disease or condition that can benefit from treatment with adoptive cell therapy.
  • the disease or condition is an infection.
  • the disease or condition is a viral infection, e.g., HIV.
  • 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 viral infection, e.g., HIV.
  • the TFP T cells provided herein can be useful in treating viral diseases, such as AIDS and HIV infection.
  • the TFP T cells provided herein can be useful in preventing viral infection, such as HIV infection.
  • Modified T cells [0312] Disclosed herein, in some embodiments, are modified T cells comprising the recombinant nucleic acid disclosed herein, or the vectors disclosed herein; wherein the modified T cell comprises a functional disruption of an endogenous TCR.
  • modified T cells comprising the sequence encoding the TFP of the nucleic acid disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein, wherein the modified T cell comprises a functional disruption of an endogenous TCR.
  • modified allogenic T cells comprising the sequence encoding the TFP disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein.
  • the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain.
  • the endogenous TCR that is functionally disrupted is an endogenous TCR gamma chain, an endogenous TCR delta chain, or an endogenous TCR gamma chain and an endogenous TCR delta chain.
  • the endogenous TCR that is functionally disrupted has reduced binding to MHC-peptide complex compared to that of an unmodified control T cell.
  • the functional disruption is a disruption of a gene encoding the endogenous TCR.
  • the disruption of a gene encoding the endogenous TCR is a removal of a sequence of the gene encoding the endogenous TCR from the genome of a T cell.
  • the T cell is a human T cell.
  • the T cell is a CD8+ or CD4+ T cell.
  • the T cell is an allogenic T cell.
  • the T cell is a TCR alpha-beta T cell.
  • the T cell is a TCR gamma-delta T cell.
  • the modified T cells 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 PD1 and the second polypeptide comprising a costimulatory domain and primary signaling domain.
  • the modified T cells further comprise one or more genetic modifications, other than expressing a recombinant nucleic acid molecule encoding a TFP.
  • the genetic modification may comprise a deletion of a portion of one or more genes, for example, a gene that encodes for a protein that assists in viral entry or propagation.
  • the modified T cell may comprise a deletion of a gene that assists in the entry or propagation of HIV.
  • the modified T cell comprise a deletion of CCR5 or CXCR4.
  • the gene deletion may be permanent, or transient.
  • CCR5 or CXCR4 deletion may be achieved by CRISPR method, using a Cas nuclease, to delete CCR5 or CXCR4 gene at the chromosomal level.
  • the nuclease is Cas9.
  • a TALEN of ZFP endonuclease may be employed for gene deletion in the modified T cell expressing a recombinant nucleic acid expressing the TFP as described herein.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects of the present disclosure, any number of T cell lines available in the art, may be used. In certain aspects of the present disclosure, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll TM separation. In one preferred aspect, 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.
  • the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture media.
  • T cells such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD95+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.
  • such cells are selected by incubation with one or more antibody or binding partner that specifically binds to such markers.
  • the antibody or binding partner can be conjugated, such as directly or indirectly, to a solid support or matrix to effect selection, such as a magnetic bead or paramagnetic bead.
  • CD3+, CD28+ T cells can be positively selected using anti- CD3/anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander, and/or ExpACT® beads).
  • T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL ® gradient or by counterflow centrifugal elutriation.
  • T cells can be further isolated by positive or negative selection techniques.
  • T cells are isolated by incubation with anti-CD3/anti-CD28 (e.g., 3x28)-conjugated beads, such as DYNABEADS ® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells.
  • the time period is about 30 minutes. In a further aspect, 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. In yet another preferred aspect, the time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells.
  • TIL tumor infiltrating lymphocytes
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process.
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.
  • the skilled artisan would recognize that 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.
  • a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells.
  • Such CD4+ and CD8+ populations can be further sorted into sub- populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
  • Enrichment of a T cell population by positive selection can be accomplished with antibodies directed to surface markers unique to the positively selected cells, e.g., anti-CD4 or anti-CD8 antibodies.
  • Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.
  • T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.
  • a T cell population can be selected that expresses one or more of IFN- ⁇ TNF-alpha, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g., other cytokines.
  • Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No. WO2013126712, which is herein incorporated by reference.
  • the concentration of cells and surface e.g., particles such as beads
  • the concentration of cells and surface can be varied.
  • a concentration of 2 billion cells/mL is used.
  • a concentration of 1 billion cells/mL is used.
  • greater than 100 million cells/mL is used.
  • a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mL is used.
  • concentrations of 125 or 150 million cells/mL can be used.
  • Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression. [0321] In a related aspect, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized.
  • target antigens of interest such as CD28-negative T cells
  • CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations.
  • the concentration of cells used is 5x10 6 /mL. In other aspects, the concentration used can be from about 1x10 5 /mL to 1x10 6 /mL, and any integer value in between.
  • the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10 °C or at room temperature. [0322] T cells for stimulation can also be frozen after a washing step.
  • the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
  • the cells may be suspended in a freezing solution.
  • one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80 °C at a rate of 1 per minute and stored in the vapor phase of a liquid nitrogen storage tank.
  • cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure.
  • the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed.
  • the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein.
  • a blood sample or an apheresis is taken from a generally healthy subject.
  • a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use.
  • the T cells may be expanded, frozen, and used at a later time.
  • samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments.
  • the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, and mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, and irradiation.
  • agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, and mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3
  • T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells.
  • the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo.
  • these cells may be in a preferred state for enhanced engraftment and in vivo expansion.
  • mobilization for example, mobilization with GM-CSF
  • conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy.
  • Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
  • Activation Expansion and Characterization of T Cells [0325] T cells may be activated and expanded generally using methods as described, for example, in U.S. Pat.
  • the T cells of the present disclosure may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells.
  • T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
  • a protein kinase C activator e.g., bryostatin
  • a ligand that binds the accessory molecule is used for co-stimulation of an accessory molecule on the surface of the T cells.
  • a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
  • an anti-CD3 antibody and an anti-CD28 antibody may be used.
  • an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med.190(9):13191328, 1999; Garland et al., J. Immunol. Meth.227(1-2):53-63, 1999).
  • T cells are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with cytokines that bind the common gamma-chain (e.g., IL-2, IL-7, IL-12, IL-15, IL-21, and others).
  • T cells are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 100 U/mL of IL-2, IL-7, and/or IL-15.
  • 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 by stimulation with 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.
  • 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. [0327] 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
  • TH, CD4+ helper T cell population
  • TC cytotoxic or suppressor T cell population
  • a T cell expressing a TFP, that is a functional component of the TCR complex, that has an antigen binding domain fused to T cell receptor subunits and can recognize a target antigen in an HLA-independent manner.
  • the TFP expressing T cell provided herein can target the CD4-binding epitope of gp120 with high specificity.
  • the TFP-expressing T cells also comprise a deletion of a viral coreceptor gene, such as the CCR5 gene, thereby conferring resistance to HIV infection.
  • the TFP T cells provided herein produce significantly lower cytokine release.
  • the TFP T cell that has been engineered by transducing primary T cells with a TFP described herein is able to recognize and kill cells that have been engineered to overexpress gp-120.
  • the TFP expressing cells described herein are able to recognize and kill CD4+ T cells that have been infected with HIV.
  • the TFP expressing cells described herein can kill HIV infected cells in vivo, as examined from peripheral blood samples after administration of the TFP expressing cells.
  • the TFP expressing cells induce about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% less cytokines as compared to conventional CAR-T cells.
  • the TFP T cells provided herein are administered with at least one additional therapeutic agent. Any suitable additional therapeutic agent may be administered with a TFP T cell provided herein.
  • the at least one additional therapeutic agent is an antiviral agent.
  • the antiviral agent may be a nucleoside analog. Nucleoside analogs as a class have a well-established regulatory history, with more than 10 currently approved by the US Food and Drug Administration (US FDA) for treating human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV).
  • US FDA US Food and Drug Administration
  • the challenge in developing antiviral therapies is to inhibit viral replication without injuring the infected cell.
  • the at least one additional therapeutic agent may be a drug that targets reverse transcriptase (HIV-RT), the viral polymerase.
  • HIV-RT reverse transcriptase
  • This enzyme is active early in the viral replication cycle and converts the virus' genetic information from RNA into DNA, a process necessary for continued viral replication.
  • Nucleoside reverse transcriptase inhibitors mimic natural nucleosides. In the triphosphate form, each NRTI competes with one of the four naturally occurring 2'-deoxynucleoside 5'-triphosphate (dNTP), namely, dCTP, dTTP, dATP, or dGTP for binding and DNA chain elongation near the active site of HIV-1 RT.
  • dNTP 2'-deoxynucleoside 5'-triphosphate
  • the at least one additional therapeutic agent may be a synthetic nucleoside 3′-azido-3′-deoxythymidine (AZT).
  • the at least one additional therapeutic agent may be 2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxycytidine (ddC), 2′,3′-dideoxy- 2′,3′-didehydrothymidine (d4T), ( ⁇ )-2′,3′-dideoxy-3′-thiacytidine (3TC), and ( ⁇ )-carbocyclic 2′,3′-didehydro-2′,3′-dideoxyguanosine (carbovir) and its prodrug abacavir.
  • ddI 2′,3′-dideoxyinosine
  • ddC 2′,3′-dideoxycytidine
  • d4T 2′,3′-dideoxy- 2′,3′-didehydrothymidine
  • TC
  • the at least one additional therapeutic agent may be Cis-2- hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC), racemates and enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolane-5-yl]cytosine, 1,3-dioxolane nucleosides, (+/ ⁇ )-1-[(2- ⁇ ,4- ⁇ )-2-(hydroxymethyl)-4-dioxolanyl]thymine (referred to as (+/ ⁇ )- dioxolane-T) and others.
  • FTC Cis-2- hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane
  • FTC Cis-2- hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane
  • the additional therapeutic agent is one or more of: emtricitabine, lamivudine, tenofovir disoproxil fumarate, zidovudine, doravirine, efavirine, nevirapine, rilpivirine, azatanavir, darunavir, ritonavir, saquinavir, tipranavir, enfurvirtide, maraviroc, dolutegravir, rategravir, cobicistat, ibalizumab-iuyk or any combination thereof.
  • the TFP- T cells are administered with ribozyme that are at least partially effective at inhibiting the propagation of HIV.
  • the ribozyme is a hammerhead ribozyme. In some embodiments, the ribozyme may be designed to cleave within the transcribed region of the HIV gag gene.
  • the additional therapeutic agent comprises an immunostimulatory agent. In some embodiments, the additional therapeutic agent may be a cytokine, a chemokine or a mimic thereof. In some embodiments, the additional therapeutic agent may be an interferon. [0340] The additional therapeutic agent may be administered by any suitable means. In some embodiments, a medicament provided herein, and the additional therapeutic agent are included in the same pharmaceutical composition. In some embodiments, an antibody provided herein, and the additional therapeutic agent are included in different pharmaceutical compositions.
  • administration of the antibody can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about one month of each other.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about one week of each other.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about one day of each other.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about twelve hours of each other.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about one hour of each other.
  • the TFP-expressing cell described herein is not administered with an additional therapeutic agent.
  • a subject is receiving an additional therapeutic agent, e.g., an antiviral agent, and administration of the additional therapeutic agent is discontinued before, e.g., 1 days, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more before treatment with the TFP-expressing T cell.
  • Immunodeficiency, HIV and HIV targeted TFP [0343]
  • the invention provides methods for treating a disease associated a viral infection.
  • the invention provides methods for treating a disease that is caused by a viral infection, such as HIV.
  • the disease is immunodeficiency or AIDS.
  • the invention pertains to a vector comprising TFP operably linked to promoter for expression in mammalian T cells.
  • the invention provides a recombinant T cell expressing a HIV antigen-binding TFP for use in treating HIV infection.
  • the HIV antigen-binding TFP-T cell of the invention is capable of contacting an HIV- infected T cell with at least one antigen binding domain of the receptor (TFP) of the invention expressed on its surface such that the TFP-T cell targets the HIV infected cell.
  • TFP antigen binding domain of the receptor
  • the invention pertains to a method of inhibiting growth of a HIV infected cell, comprising contacting the cell with a HIV antigen-binding TFP T cell of the present invention such that the TFP-expressing T cell is activated in response to the antigen and targets the virus or the infected cells, wherein the growth of the infected cell is inhibited.
  • a method of cellular therapy wherein 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 HIV infected cells in the recipient, or prevent an infection by HIV thereof.
  • TFP-expressing T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained infection 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 infected 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-HIV 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 cells expressing the anti-HIV-associated antigen antibody or a binding domain, resist soluble HIV-associated antigen inhibition, mediate bystander killing and/or mediate regression of an established human infection.
  • the human TFP-modified T cells of the invention may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal.
  • the mammal is a human.
  • ex vivo immunization at least one of the following occurs in vitro prior to administering the cell into a mammal: i) expansion of the cells, ii) introducing a nucleic acid encoding a TFP to the cells or iii) cryopreservation of the cells.
  • Ex vivo procedures are well known in the art and are discussed more fully below.
  • 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.
  • the procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described, e.g., in U.S. Pat.
  • 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-L, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.
  • the present invention 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 invention are used in the treatment of diseases, disorders and conditions associated with expression of HIV antigen-binding TFP.
  • the TFP-modified T cells of the present invention 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 invention also provides methods for inhibiting the proliferation or reducing a HIV infection in a cell population, the methods comprising contacting a population of cells comprising a T cell expressing HIV antigen-binding TFP, a TFP-T cell of the invention that binds to the HIV or HIV-infected cell.
  • the present invention provides methods for inhibiting the proliferation or reducing the population of cells expressing HIV antigen-binding TFP, the methods comprising contacting the antigen-expressing infected cell population with an HIV antigen-binding TFP-T cell of the invention.
  • the present invention provides methods for inhibiting the proliferation or reducing the population of cells expressing HIV infected cells, the methods comprising contacting the infected cell population with an HIV antigen-binding TFP-T cell of the invention that binds to the HIV antigen.
  • the TFP-T cell of the invention reduces the quantity, number, amount or percentage of cells and/or HIV-infected 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 HIV infection relative to a negative control.
  • the subject is a human.
  • the present invention also provides methods for preventing, treating and/or managing a disease associated with HIV infection, the methods comprising administering to a subject in need an HIV antigen-binding TFP-T cell of the invention that binds to HIV or the infected cell.
  • the subject is a human.
  • the present invention also provides methods for preventing, treating and/or managing a disease associated with HIV, the methods comprising administering to a subject in need thereof an HIV antigen-binding TFP or TFP-T cell of the invention that binds to the HIV antigen or HIV antigen-expressing cell.
  • the subject is a human.
  • 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. In some embodiments of either case, 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. Also provided are 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. For sequential administration, the TFP-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.
  • a TFP-expressing cell described herein may be used in a treatment regimen in combination with the at least one additional therapeutic agents described herein.
  • 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
  • HHLH hemophagocytic lymphohistiocytosis
  • MAS Macrophage Activation Syndrome
  • Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like.
  • the methods described herein can comprise administering a TFP-expressing cell described herein to a subject and further administering an agent to manage elevated levels of a soluble factor resulting from treatment with a TFP-expressing cell.
  • the soluble factor elevated in the subject is one or more of IFN- ⁇ , TNF ⁇ , IL-2 and IL-6. Therefore, an agent administered to treat this side effect can be an agent that neutralizes one or more of these soluble factors.
  • agents include, but are not limited to a steroid, an inhibitor of TNF ⁇ , and an inhibitor of IL-6.
  • An example of a TNF ⁇ inhibitor is etanercept (marketed under the name ENBREL®).
  • an 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.
  • inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.
  • 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 PD1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX-101, and marketed as YERVOY®; Bristol-Myers Squibb; tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP- 675,206)).
  • the agent is an antibody or antibody fragment that binds to T cell immunoglobulin and mucin-domain containing-3 (TIM3).
  • the agent is an antibody or antibody fragment that binds to Lymphocyte-activation gene 3 (LAG3).
  • 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 anti-HIV antigen TFP.
  • compositions of the present invention 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 invention are in one aspect formulated for intravenous administration.
  • Pharmaceutical compositions of the present invention 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 “or “therapeutic amount”
  • the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, extent of infection or metastasis, and condition of the patient (subject).
  • a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 10 4 to 10 9 cells/kg body weight, in some instances 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • T cells can be activated from blood draws of from 10 cc to 400 cc.
  • T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.
  • 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 invention are administered to a patient by intradermal or subcutaneous injection.
  • the T cell compositions of the present invention are administered by i.v. injection.
  • 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 invention may be introduced, thereby creating a TFP- expressing T cell of the invention.
  • 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 invention.
  • 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, e.g., in U.S. Pat. No.6,120,766).
  • the TFP 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 invention, and one or more subsequent administrations of the TFP T cells of the invention, 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 invention are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the TFP T cells of the invention 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 invention are administered for at least two, three, four, five, six, seven, eight or more weeks.
  • HIV antigen-binding TFP or TFP T cells are generated using lentiviral viral vectors, such as lentivirus. TFP-T cells generated that way can 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 affected by RNA TFP vector delivery.
  • 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.
  • EXAMPLES [0379] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
  • exemplary recombinant proteins of various embodiments comprises a CD4- gp120 binding extracellular domain, fused to a CD3 epsilon TCR, thereby obtaining the fused protein, CD4-TCR.
  • FIG.1A shows an exemplary CD4 extracellular domain gp120 binding region fused to a CD3 epsilon transmembrane and intracellular regions.
  • FIG.1B shows a CD4 extracellular domain gp120 binding region fused via a linker (L) to a CD3 epsilon transmembrane and intracellular regions.
  • FIG.1C shows a CD4 extracellular domain gp120 binding region fused to a CD3 epsilon transmembrane and intracellular regions, with additional intracellular signaling domains (S*) to facilitate T cell activation and killing of virus.
  • FIG.2A-C shows graphical view of exemplary scFv-CD3 epsilon fusion proteins of the disclosure, comprising a gp120 binding scFv, fused to a CD3 epsilon TCR.
  • FIG.2A shows a scFv protein that binds to gp120, fused to a CD3 epsilon transmembrane and intracellular regions.
  • FIG.2B shows a scFv protein that binds to gp120, fused via a linker (L) to a CD3e transmembrane and intracellular regions.
  • FIG.2C shows a scFv protein that binds to gp120, fused to a CD3 epsilon transmembrane and intracellular regions, with additional intracellular signaling domains (S*) to facilitate T cell activation and killing of virus.
  • S* intracellular signaling domains
  • TFP Constructs The extracellular domain of the CD4 receptor is recombinantly linked to CD3-epsilon or other TCR subunits using a linker sequence, such as G4S (SEQ ID NO: 101), (G4S)2 (SEQ ID NO: 93) (G 4 S) 3 (SEQ ID NO: 88) or (G 4 S) 4 (SEQ ID NO: 87).
  • a linker sequence such as G4S (SEQ ID NO: 101), (G4S)2 (SEQ ID NO: 93) (G 4 S) 3 (SEQ ID NO: 88) or (G 4 S) 4 (SEQ ID NO: 87).
  • the linker also has an HA-tag.
  • TCR alpha and TCR beta chains are used for generation of TFPs either as full-length polypeptides or only their constant domains.
  • TCR T Cell Receptor
  • a human TCR complex contains the CD3-epsilon polypeptide, the CD3-gamma polypeptide, the CD3-delta polypeptide, the CD3- zeta polypeptide, the TCR alpha chain polypeptide and the TCR beta chain polypeptide.
  • the human CD3-epsilon polypeptide canonical sequence is Uniprot Accession No. P07766.
  • the human CD3-gamma polypeptide canonical sequence is Uniprot Accession No. P09693.
  • the human CD3-delta polypeptide canonical sequence is Uniprot Accession No. P043234.
  • the human CD3-zeta polypeptide canonical sequence is Uniprot Accession No. P20963.
  • the human TCR alpha chain canonical sequence is Uniprot Accession No. Q6ISU1.
  • the human TCR beta chain C region canonical sequence is Uniprot Accession No. P01850, a human TCR beta chain V region sequence is P04435. Table 1. Examples of linker sequences, TCR subunit sequences and binder sequences
  • Expression vectors include: a promoter (an EF1alpha promoter), a signal sequence to enable secretion, a polyadenylation signal and transcription terminator (Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes (e.g., SV40 origin and ColE1 or others known in the art) and elements to allow selection (ampicillin resistance gene and zeocin marker).
  • a promoter an EF1alpha promoter
  • a signal sequence to enable secretion e.g., a polyadenylation signal and transcription terminator (Bovine Growth Hormone (BGH) gene
  • BGH Bovine Growth Hormone
  • an element allowing episomal replication and replication in prokaryotes e.g., SV40 origin and ColE1 or others known in the art
  • elements to allow selection e.g., SV40 origin and ColE1 or others known in the art
  • TFP-encoding nucleic acid construct having the full or first two domains of extracellular CD4 linked to CD3 epsilon is cloned into a lentiviral expression vector and expression validated based on the quantity and quality of the effector T cell response of TFP.CD4-transduced T cells (“TFP.CD4(ECD FL)” or “TFP.CD4(ECD FL) T cells” or “TFP.CD4(2D)” or “TFP.CD4(2D) T cells” to represent those cells expressing the first two domains of CD4) in response to GP120 expressing cells.
  • TFP.CD4(F68A) A negative control is included (TFP.CD4(F68A)) which comprises the full extracellular domain of CD4 comprising a F68A mutation linked to CD3 epsilon. The position of the mutation is relative to the FL sequence including the signal sequence. This mutation blocks CD4 from interacting with GP120. 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). [0386] The TFP.CD4 (ECD FL, 2D, and F68A) lentiviral transfer vectors are used to produce the genomic material packaged into the VSV-G pseudotyped lentiviral particles.
  • Lentiviral transfer vector DNA is mixed with the three packaging components of VSV-G, gag/pol and rev in combination with Lipofectamine® reagent to transfect them together into HEK-293 (embryonic kidney, ATCC® CRL-1573TM) cells. After 24 and 48 hours, the media is collected, filtered and concentrated. The resulting viral preparation is stored at -80°C. The number of transducing units is determined by titration on Sup-T1 (T cell lymphoblastic lymphoma, ATCC® CRL-1942TM) cells.
  • Redirected TFP.CD4 T cells are produced by activating fresh na ⁇ ve T cells with, e.g., anti- CD3 anti-CD28 beads for 24 hrs and then adding the appropriate number of transducing units to obtain the desired percentage of transduced T cells, as is described in further detail below. These modified T cells are allowed to expand until they become rested and come down in size at which point they are cryopreserved for later analysis. The cell numbers and sizes are measured using a Coulter MultisizerTM III. Before cryopreserving, the percentage of cells transduced (expressing TFP.CD4 on the cell surface) and the relative fluorescence intensity of that expression are determined by flow cytometric analysis.
  • TFP.CD4 T cells for treating HIV, while protecting the TFP.CD4 T cells from infection.
  • the first strategy is to reduce or knock out CCR5 in the TFP.CD4 T cells to prevent HIV infection upon administration to a HIV+ patient. This can be done using CRISPR, for example. This strategy is ideal for an allogeneic approach, in which the cells are not obtained from a subject with HIV.
  • the second strategy involves the use of enriched CD8+ TFP.CD4 T cells.
  • CRISPR-based deletion of CCR5 [0387] CCR5 is disrupted in PBMCs or CD8+ T cells isolated from PBMCs by negative selection to prevent infection of TFP.CD4 T cells with HIV.
  • Exemplary guide DNA target sequences are as follows: TCAGTTTACACCCGATCCAC (SEQ ID NO: 14) TCTGAACTTCTCCCCGACAA (SEQ ID NO: 15) TCATCCTCCTGACAATCGAT (SEQ ID NO: 16) GTAAACTGAGCTTGCTCGCT (SEQ ID NO: 17) ACAATGTGTCAACTCTTGAC (SEQ ID NO: 18) ACAGCATTTGCAGAAGCGTT (SEQ ID NO: 19) [0388] Primary T cells or CD8+ T cells prepared from either whole blood or buffy coat and isolated by negative selection are stimulated with IL-7 and IL-15 (primary T cells) or IL-2 and IL-15 (CD8+ T cells) in the presence of bead-bound anti-CD3 and anti-CD28 antibodies (TransAct beads) in TexMACS media containing 3% human serum for four days prior to electroporation.
  • IL-7 and IL-15 primary T cells
  • IL-2 and IL-15 CD8+ T cells
  • SpCas9 ribonucleoproteins targeting CCR5 are prepared by annealing crRNA targeting CCR5 with tracrRNA at a molecular ratio of 1:1. Annealed duplexes are mixed with SpCas9 protein at a molecular ratio of 1.5:1. RNPs were mixed with T cells and electroporated following the manufacturer’s instructions for the Neon Transfection System. Electroporation is set at 1600 V, 10ms, 3 pulses. Cells are immediately transferred to warm medium and incubated at 37°C to allow expansion of edited T cells with an approximate doubling time of 3 to 5 days. Editing efficacy is assessed by measuring loss of surface expression of CCR5 by flow cytometry.
  • Edited T cells are purified ten days post activation using Magnetic-Activated Cell Sorting (MACS®, Miltenyi Biotec) according to the manufacturer cell separation system and are negatively selected against anti-CCR5 antibody.
  • Cells expressing CCR5 on their surface are immobilized on MACS MS (Cat. #130-041-301) or LS (Cat# 130-041-306) columns, while edited T cells negative for CCR5 are collected in the column flow-through and maintained in culture.
  • MACS MS Cat. #130-041-301
  • LS Cat# 130-041-306
  • Expi293F-cells are suspended in FS media and allowed to incubate at 37 degrees C, 8% CO 2 , 150 rpm for 1-3 hours.
  • the transfer DNA plasmid, Gag/Pol plasmid, Rev plasmid, and VSV-G plasmid are diluted in FS media.
  • PEIpro is then diluted in FS media and added to the mixture of DNA and media.
  • the incubated cells are added to this mixture and are incubated at 37 degrees C, 8% CO 2 , 150 rpm for 18-24 hours. The following day, the supernatant is replaced with fresh media and supplemented with sodium butyrate and incubated at 37°C for an additional 24 hours.
  • the lentivirus containing supernatant is then collected into a 50 mL sterile, capped conical centrifuge tube and put on ice. After centrifugation at 3000 rpm for 30 minutes at 4°C, the cleared supernatant is filtered with a low-protein binding 0.45 ⁇ m sterile filter. The virus is subsequently concentrated by Lenti-X. The virus stock preparation is either used for infection immediately or aliquoted and stored at -80°C for future use.
  • CCR5+ Primary T cells CD4+ and CD8+ T cells
  • CCR5+ CD8+ T cells CCR5- Primary T cells (CD4+ and CD8+ T cells)
  • CCR5- CD8+ T cells prepared from either whole blood or buffy coat are stimulated with IL-7 and IL-15 or IL-2 and IL-15 in the presence of bead- bound anti-CD3 and anti-CD28 antibodies (TransAct beads) for 24 hours prior to viral transduction.
  • CD4+ and CD8+ T cells or CD8+ T cells are first isolated from the PBMCs by selection prior to expansion. The appropriate number of transducing units is then added to obtain the desired percentage of transduced T cells.
  • IL-7 and IL-15 or IL-2 and IL-15 are then grown in the continued presence of IL-7 and IL-15 or IL-2 and IL-15 for a period of 6-14 days (total incubation time is dependent on the final number of TFP-expressing cells required). Cell concentrations are analyzed every 2-3 days, with media being added at that time to maintain the cell suspension at 1x10 6 cells/mL. Verification of TFP expression by cell staining [0391] Following lentiviral transduction, expression of CD4 TFPs is confirmed by flow cytometry in three ways. First, in a population of primary T cells transduced with a CD4.TFP in which the linker has an HA tag, an HA antibody is used.
  • both anti-CD4 and anti-CD8 antibodies are used, such that the transduction efficacy can be measured in the CD8+ T cell population only.
  • an anti-CD4 antibody is used in the population of cells transduced with a CD4.TFP that only comprises CD8+ T cells. T cells are washed three times in 3 mL staining buffer (PBS, 4% BSA) and re-suspended in PBS at 1x10 6 cells per well. For dead cell exclusion, cells are incubated with LIVE/DEAD® Fixable Aqua Dead Cell Stain (Invitrogen) for 30 minutes on ice.
  • TFPs are washed twice with PBS and re-suspended in 50 ⁇ L staining buffer.
  • 1 ⁇ L of 1:100 diluted normal goat lgG (BD Bioscience) is added to each tube and incubated in ice for 10 minutes.
  • 1.0 mL FACS buffer is added to each tube, mixed well, and cells are pelleted by centrifugation at 300g for 5 min.
  • Surface expression of TFPs is detected by anti-CD4 or anti-HA, as described above, or human IgG1 isotype control.1 ⁇ g antibodies are added to the respective samples and incubated for 30 minutes on ice.
  • T Cells are then washed twice, and stained for surface markers using Anti-CD3 APC (clone, UCHT1), anti-CD4-Pacific blue (Clone RPA-T4), anti-CD8 APCCy7(Clone SK1), from BD bioscience.
  • Flow cytometry is performed using LSRFortessaTM X20 (BD Biosciences) and data is acquired using FACS diva software and is analyzed with FlowJo® (Treestar, Inc. Ashland, OR).
  • Example 5 Measurement of Activation of T Cells by FACS [0392] Activation of the T cells expressing TFP Constructs are performed. As described above, activated cells are transduced and expanded.
  • TFP.CD4 T cells are harvested and stimulated with plate-bound anti-human pan ⁇ ⁇ TCR, anti-human CD3 (UCHT1), or anti-CD4 for 24 hours.
  • Expression of CD25 and CD69 are determined by flow cytometry with Ab panel including anti-human CD3, anti-human TCR ⁇ ⁇ , anti-human CD25 (Clone BC96) and anti-human CD69 (clone FN50).
  • Activation of T cells may be similarly assessed by analysis of granzyme B production. T- cells are cultured and expanded as described above, and intracellular staining for granzyme B is done according to the manufacturer’s kit instructions (Gemini Bioproducts; 100-318).
  • Cells are harvested, washed with PBS three times and blocked with human Fc block for 10 min.
  • Cells are stained for surface antigens with anti-CD3 APC (clone, UCHT1), and anti-CD8 APCcy7(Clone SK1) for 30 min at 4 o C.
  • Cells are then fixed with Fixation/Permeabilization solution (BD Cytofix/Cytoperm Fixation/Permeabilization kit cat #554714) for 20 min at 4 C, flowed by washing with BD Perm/Wash buffer.
  • Cells are subsequently stained with anti-Granzyme B Alexafluor700 (Clone GB11), washed with BD Perm/Wash buffer twice and resuspended in FACS buffer.
  • Example 6 Luciferase-based cytotoxicity assay
  • the luciferase-based cytotoxicity assay assesses the cytotoxicity of TFP T cells by indirectly measuring the luciferase enzymatic activity in the residual live target cells after co- culture. Loaded YU2 Env + K562 cells modified to express GP120 and to overexpress firefly luciferase are used as target cells with K562 cells overexpressing firefly luciferase used as a control.
  • the target cells are plated at 10000 cells per well in 96-well plate.
  • the TFP T or control cells are added to the target cells at different effector-to-target ratios.
  • the mixture of cells is then cultured for 24 or 48 hours at 37°C with 5 % CO2 before the luciferase enzymatic activity in the live target cells is measured by the Bright-Glo® Luciferase Assay System (Promega®, Catalogue number E2610).
  • the cells are spun into a pellet and resuspended in medium containing the luciferase substrate.
  • % Cytotoxicity 100% x [1 – RLU (Tumor cells + T cells) / RLU (Tumor cells)].
  • T cells expressing the CD4.TFP ECD FL and 2D but not F68A will demonstrate enhanced cytotoxicity towards loaded YU2 Env + K562 target cells relative to K562 cells having no target.
  • Example 7 Cytokine Secretion measurement by MSD [0396]
  • a measure of effector T-cell activation and proliferation associated with the recognition of cells bearing cognate antigen is the production of effector cytokines such as interleukin-2 (IL- 2) and interferon-gamma (IFN- ⁇ ), granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF- ⁇ ).
  • IL-2 interleukin-2
  • IFN- ⁇ interferon-gamma
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • TNF- ⁇ tumor necrosis factor alpha
  • Target-specific cytokine production including IL-2, IFN- ⁇ , GM-CSF, and TNF- ⁇ by monospecific TFP T cells is measured from supernatants harvested 48 hours after the co-culture of T cells with K562 cells, expressing no target or YU2 Env + K562 target cells using the U- PLEX® Biomarker Group I (hu) Assays (Meso Scale Diagnostics®, LLC, catalog number: K15067L-4) or after culture of TFP containing cells with plate-bound anti-CD3 ⁇ , anti-pan ⁇ ⁇ TCR, anti-CD4 or medium alone.
  • T cells expressing the CD4.TFP will demonstrate enhanced cytokine secretion when contacted with loaded YU2 Env + K562 target cells relative to K562 cells having no target.
  • Example 8 In vitro HIV replication control assay and intracellular Gag stain [0399] CD4+ T cells are infected with the CCR5-tropic HIV strain Bal, and 24 hours later are co- cultured at varying effector to target (E:T) ratios with TFP.CD4 (ECD FL, 2D, and F68A) expressing CD8 T cells.
  • Bal viral stocks (280ng/ml p24) are prepared by harvesting the cell-free supernatant from anti CD3/CD28 activated CD4 T cells and freezing in aliquots. Activated CD4 T cells are infected by adding approximately 1ml of supernatant per 20 million cells. The following day CD4 and CD8 T cells are co-cultured at varying E:T ratios and HIV spread is monitored by intracellular p24 Gag with the KC57 anti-Gag-RD1 antibody (Beckman Coulter) and the Invitrogen Fix and Perm buffers, according to the manufacturers’ instructions, gating on a population of uninfected cells.
  • CD4 T cells contacted with TFP + CD8 T cells will demonstrate reduced HIV replication relative to CD4 T cells contacted with control CD8 T cells.
  • Example 9 HIV prevention humanized mouse model [0400] NSG (NOD-scid IL2Rg null ) mice at 7 weeks of age are treated with 30mg/kg Busulfan mixed 1:1 with PBS.24 hours later are injected via tail vein with TFP.CD4 T cells.24 hours later mice are injected via tail vein with 10x10 6 human lymphocytes, comprised of 8 million CD4 T cells and 2 million TFP.CD4 CD8 T cells or untransduced CD8 control T cells. Three weeks later mice are tail vein injected with 15ng HIV Bal mixed 1:1 with PBS.
  • Peripheral blood is obtained by retro-orbital bleeding, and human CD4 and TFP + CD8 lymphocyte counts are enumerated using BD lysis buffer and BD TruCount tubes, staining with mouse anti human CD45 PerCp Cy5.5 (BD Biosciences), mouse anti human CD4 BV421 (Bio- legend), and mouse anti human CD8 ⁇ BV711 (Biolegend). Viral load is measured in plasma.
  • mice injected with CD4 T cells and TFP.CD4 (TFP.CD4(ECD) and TFP.CD4(2D)) CD8 T cells will maintain higher levels of CD4 T cells and will have a reduced viral load, over time, than mice injected with CD4 T cells and control CD8 T cells or control TFP.CD4(F68A) CD8+ T cells.
  • Example 10 HIV treatment humanized mouse model [0401] NSG (NOD-scid IL2Rg null ) mice at 6 weeks of age are injected with 5 million CD8- depleted human PBMCs, and 12 days later injected with 1 million HIV Bal-infected (or mock- infected) autologous CD4 T cells that have been in vitro infected with HIV Bal and cultured with ART for 2 days prior to freezing. The same day as HIV infection, mice begin receiving 200mg/kg daily intraperitoneal injections of the reverse transcriptase inhibitor nucleotide analog tenofovir disoproxil fumarate (TDF) for 4 days.
  • TDF reverse transcriptase inhibitor nucleotide analog tenofovir disoproxil fumarate
  • TFP.CD4 CD8 T cells On Day 16, 5 million TFP.CD4 CD8 T cells, or untransduced control CD8 T cells are injected. Peripheral blood is obtained by retroorbital bleeding, and human CD4 and TFP + CD8 lymphocyte counts are enumerated using BD lysis buffer and BD TruCount tubes, staining with mouse anti human CD45 PerCP-Cy5.5 (BD Biosciences), mouse anti human CD4 BV421 (Biolegend), and mouse anti human CD8 ⁇ BV711 (Biolegend). Viral load is measured in plasma.
  • mice injected with TFP.CD4 (TFP.CD4(ECD) and TFP.CD4(2D)) CD8 T cells will maintain higher levels of CD4 T cells and will have a reduced viral load, over time, than mice injected with control CD8 T cells or control TFP.CD4(F68A) CD8+ T cells.
  • Example 11 Generation and testing of HIV-envelope targeting TRuC T cells [0402] Though much success has been achieved in the field of HIV/AIDS treatment(s), there remains a need for improved therapeutics and/or a cure. Several HIV-specific CAR T cell therapies have been pursued but have suffered from setbacks such as poor durability and limited viral control (see Maldini et al 2018 Nat. Rev.
  • the first TRuC, CD4(EC)e or CD4e when read N-terminus to C- terminus included a GM-CSF-Ra signal peptide (SEQ ID NO: 22), a full-length CD4 extracellular domain (SEQ ID NO: 23), an A3(G4S)3LE linker (SEQ ID NO: 2 or 72), and a mature human CD3e component (SEQ ID NO: 55).
  • a truncated CD4 sequence was used having only the first two N-terminal domains (SEQ ID NO: 28), while the other sequence components remained the same.
  • CD4(F68A)e or CD4(mut)e SEQ ID NO: 73
  • a mutation phenylalanine (F) to alanine (A)
  • This F68A mutation has been shown to decrease the affinity of CD4 to gp120 by approximately 500-fold (see Moebius et al 1992 JEMA 176:507-517, the contents of which are herein incorporated by reference in their entirety) and was used as a negative control.
  • Transduction of TC-110 was visualized using a FITC-conjugated human CD19 protein, was only capable of interacting with the binder of TC- 110 and not the CD4 TRuCs. Exemplary flow cytometry plots are shown in FIG.4. [0405] The differentiation state of CD3+ TRuC T cells was also assessed using flow cytometry. Detection of CD45RA and CCR7 was used to sort the cells by phenotype (T NAIVE , T EMRA , T EM , T CM ). These data showed robust populations of T NAIVE and T CM cells, substantially similar to those populations seen in the non-transduced control (gated on total CD3+).
  • FIG. 5 Exemplary flow cytometry plots and histograms showing the associated quantification of TRuC T cells having each phenotype are provided in FIG.5.
  • two cell lines expressing an HIV envelope protein gp120
  • Jurkat or Nalm6 cells expressing luciferase were plated at 1x10 5 cells per well in a 24-well plate and grown for 3 days.
  • the media was replaced and cells transduced with lentiviral vectors encoding a group M subtype B (isolate BRU/LAI) gp160-GFP (SEQ ID NO: 75) and Lentiboost; samples were then spun down at 1200 RPM for 30 minutes.
  • group M subtype B isolated BRU/LAI
  • gp160-GFP SEQ ID NO: 75
  • the gp160 was processed into cell surface gp120 and transmembrane gp41 components.
  • Both Jurkat-luc and Nalm6-luc cells transduced with gp160 had a population of GFP positive cells, with Nalm6-luc GFP expression being slightly higher (16.3%) than Jurkat-luc GFP expression (14.3%).
  • Mock transduced cells showed no detectable GFP (0%). Remaining cells were allowed to continue expanding for 5 additional days and on Day 11, GFP positive cells were sorted by FACS/flow cytometry.
  • HIV-envelope expressing cells or mock transduced controls (Jurkat-luc + mock; Jurkat-luc + gp160; Nalm6-luc + mock; Nalm6- luc + gp160) were plated at 1x10 4 per well in 96-well plates.
  • TRuC T cells were added at an effector to target ratio (E:T) of 9:1, 3:1, 1:1 or 1:3. After 24hr, the supernatant was collected for cytokine analysis by MSD and the cells harvested for a luciferase-based cytotoxicity assay. [0409] Due to poor luciferase expression in the Jurkat cells, the cytotoxicity assay was conducted only with the Nalm6 cells.
  • the luciferase expression was quantified and an inverse calculation made to determine the percent cell killing for each condition and type of TRuC T cell tested.
  • the data are shown in FIG.6 and demonstrated that CD4e and CD4(D1-2)e TRuC T cells yielded comparable cell killing ability against HIV-envelope expressing Nalm6 cells as compared to TC- 110 against both the mock and transduced Nalm6 cell lines.
  • the CD4(mut)e meanwhile, like the NT control, was ineffective in killing HIV-envelope expressing cells.
  • the supernatant collected after 24 hours of co-culture of CD4 TRuC T cells and HIV- envelope expressing cells was analyzed by MSD for cytokine release.
  • CD4e and CD4(D1-2)e TRuC T cells generated cytokine responses after co-culture with HIV-envelope expressing cells, but to a lesser degree than the robust response of TC-110 in the Nalm6 cells.
  • a secondary cytotoxicity assay was conducted using flow cytometry methods.
  • TRuC T cells transduced with CD4e, CD4(D1-2)e, CD4(mut)e, TC-110 and non-transduced (NT) control cells were thawed and washed in R10 media, then normalized to 70%.
  • HIV- envelope expressing cells Jurkat-luc + gp160; Nalm6-luc + gp160
  • TRuC T cells were added at an effector to target ratio (E:T) of 10:1, 5:1, 2.5:1, 1.25:1 or 0.625:1. The co-cultures were incubated for 4 hours then stained with Live/Dead Blue for dead cells at 1:1000 and fixed.

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Abstract

L'invention concerne des compositions et des méthodes de traitement d'une infection par le VIH chez un sujet. Les compositions comprennent une cellule T comprenant une molécule d'acide nucléique recombinant codant pour une protéine de fusion (TFP) de récepteur de lymphocyte T (TCR) comprenant un domaine de liaison à l'antigène qui se lie à un antigène du VIH, tel que gp120. Les protéines de fusion de TCR comprennent une sous-unité d'intégration de TCR comprenant un domaine extracellulaire, un domaine transmembranaire de TCR et un domaine intracellulaire de TCR.
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US11965012B2 (en) 2015-05-18 2024-04-23 TCR2 Therapeutics Inc. Compositions and methods for TCR reprogramming using fusion proteins

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US10442849B2 (en) * 2015-05-18 2019-10-15 Tcr2 Therabeutics Inc. Compositions and methods for TCR reprogramming using fusion proteins
US20200392459A1 (en) * 2017-04-13 2020-12-17 Cellectis New sequence specific reagents targeting ccr5 in primary hematopoietic cells

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* Cited by examiner, † Cited by third party
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US10442849B2 (en) * 2015-05-18 2019-10-15 Tcr2 Therabeutics Inc. Compositions and methods for TCR reprogramming using fusion proteins
US20200392459A1 (en) * 2017-04-13 2020-12-17 Cellectis New sequence specific reagents targeting ccr5 in primary hematopoietic cells

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11965012B2 (en) 2015-05-18 2024-04-23 TCR2 Therapeutics Inc. Compositions and methods for TCR reprogramming using fusion proteins

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