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WO2022083667A1 - Polypeptides comprenant un tcr spécifique d'un vaccin et un récepteur antigénique chimérique et leurs utilisations - Google Patents

Polypeptides comprenant un tcr spécifique d'un vaccin et un récepteur antigénique chimérique et leurs utilisations Download PDF

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WO2022083667A1
WO2022083667A1 PCT/CN2021/125142 CN2021125142W WO2022083667A1 WO 2022083667 A1 WO2022083667 A1 WO 2022083667A1 CN 2021125142 W CN2021125142 W CN 2021125142W WO 2022083667 A1 WO2022083667 A1 WO 2022083667A1
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vaccine
domain
cell
virus
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Ming Zeng
Zhigang Li
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Nanjing Legend Biotechnology Co Ltd
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Nanjing Legend Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4264—Cancer antigens from embryonic or fetal origin
    • A61K40/4265—Alpha-feto protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/46—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • polypeptides comprising a chimeric antigen receptor (CAR) fused to at least one additional domain derived from a virus (e.g., a virus vaccine) specific T cell receptor (TCR) , and polynucleotides encoding same.
  • a virus e.g., a virus vaccine
  • TCR T cell receptor
  • a composition comprising CAR-T cells expressing a CAR as well as a virus (e.g., a virus vaccine) specific TCR or a domain derived therefrom, and uses thereof for treating a disease or disorder, such as cancer.
  • T cells engineered with chimeric antigen receptor (CAR) induce potent clinical response in patients with blood cancers, demonstrating promising superior prognosis comparing with conventional therapies. Nonetheless, the translation of CAR-T cell therapy from liquid tumor to solid tumor is clinically challenging. T cell exhaustion and lack of in vivo persistence is the main deficiency for traditional CAR-T cell therapies.
  • CAR chimeric antigen receptor
  • CAR-T cells expressing IL-7 and CCL19 have been shown to improve immune cell infiltration and CAR-T cell survival in the tumor.
  • IL15 and IL18 have been demonstrated to enhance CAR-T cell expansion and persistence, and anti-tumor activity.
  • IL-12 has also been shown to improve activation of T cells and NK cells, and to modulate the immunological and vascular tumor microenvironment, thereby enhancing CAR-T cell anti-tumor efficacy.
  • cytokines and chemokines Despite the potent anti-tumor efficacy by CAR-T cells armored with certain cytokines and chemokines, the cytotoxicity limits its clinical application. Instead of constitutively expressing soluble cytokines, several strategies have been proposed to minimize side effects of cytokines and chemokines, including immobilizing IL-15 on the CAR-T cell surface (membrane bound) to reduce the trans-activity, and expressing IL-12 under control of inducible promoter (e.g., NFAT6 minimal promoter) . However, these strategies are insufficient to eliminate the side effects.
  • inducible promoter e.g., NFAT6 minimal promoter
  • a polypeptide comprising a chimeric antigen receptor (CAR) ; and a domain derived from a virus specific T cell receptor (TCR) , wherein the CAR and the domain are fused by a first peptide linker.
  • CAR chimeric antigen receptor
  • TCR virus specific T cell receptor
  • the CAR comprises (i) an extracellular domain, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • the domain derived from a virus specific TCR is a domain derived from a vaccine specific TCR.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain and/or a ⁇ -chain of the virus specific TCR. In some embodiments, the domain derived from a virus specific TCR comprises a ⁇ -chain and a ⁇ -chain of the virus specific TCR, and wherein the ⁇ -chain and the ⁇ -chain of the virus specific TCR are linked via a second peptide linker.
  • the virus specific TCR is capable of binding to a vaccine selected from a group consisting of yellow fever vaccine, influenza vaccine, hepatitis A vaccine, human papillomavirus vaccine, smallpox vaccine, rotavirus vaccine, adenovirus vaccine, poliovirus vaccine, chickenpox vaccine, typhoid vaccine, and measles, mumps, rubella (MMR) vaccine.
  • a vaccine selected from a group consisting of yellow fever vaccine, influenza vaccine, hepatitis A vaccine, human papillomavirus vaccine, smallpox vaccine, rotavirus vaccine, adenovirus vaccine, poliovirus vaccine, chickenpox vaccine, typhoid vaccine, and measles, mumps, rubella (MMR) vaccine.
  • the virus specific TCR is a yellow fever vaccine specific TCR capable of binding to a yellow fever vaccine antigen.
  • the yellow fever vaccine antigen is selected from a group consisting of HLA-A02 restricted yellow fever virus antigen, HLA-A24 restricted yellow fever virus antigen, and HLA-A11 restricted yellow fever virus antigen.
  • the domain derived from a virus specific TCR is capable of binding a yellow fever vaccine peptide comprising an amino acid sequence of LLWNGPMAV (SEQ ID NO: 70) .
  • the domain derived from a virus specific TCR comprises: (i) a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence selected from a group consisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35; and/or (ii) a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 9
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.
  • the first peptide linker and/or the second peptide linker is a cleavable peptide linker.
  • the peptide linker is a 2A self-cleaving peptide.
  • the 2A self-cleaving peptide is selected from a group consisting of F2A, E2A, P2A, T2A, or variants thereof.
  • the first peptide linker and/or the second peptide linker comprises an amino acid sequence of SEQ ID NO: 55.
  • the first peptide linker and/or the second peptide linker comprises an amino acid sequence of SEQ ID NO: 56.
  • the CAR is at the N-terminus of the domain derived from the virus specific TCR. In other embodiments, the CAR is at the C-terminus of the domain derived from the virus specific TCR.
  • a polynucleotide comprising a nucleic acid sequence encoding the polypeptide provided herein.
  • a polynucleotide comprising a first region encoding a CAR comprising (i) an extracellular domain; (ii) a transmembrane domain, and (iii) an intracellular domain; and a second region encoding a domain derived from a virus specific TCR.
  • the first region and the second region are under control of the same promoter. In other embodiments, the first region and the second region are under control of different promoters.
  • a vector comprising the polynucleotide provided herein.
  • provided herein is a method for making a CAR-T cell, comprising introducing the polynucleotide or the vector provided herein into a T cell.
  • a method for making a CAR-T cell comprising introducing into a T cell a first polynucleotide encoding a CAR comprising (i) an extracellular domain, (ii) a transmembrane domain, and (iii) an intracellular domain; and a second polynucleotide encoding a domain derived from a virus specific TCR.
  • provided herein is a CAR-T cell produced according to the method provided herein.
  • a CAR-T cell comprising the polynucleotide or the vector provided herein.
  • a CAR-T cell expressing a chimeric antigen receptor (CAR) ; and a domain derived from a virus vaccine specific T cell receptor (TCR) .
  • the CAR comprises (i) an extracellular domain, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • the domain derived from a virus vaccine specific TCR comprises a ⁇ -chain and/or a ⁇ -chain of the virus vaccine specific TCR. In some embodiments, the domain derived from a virus vaccine specific TCR comprises a ⁇ -chain and a ⁇ -chain of the virus vaccine specific TCR, and wherein the ⁇ -chain and the ⁇ -chain of the virus vaccine specific TCR are linked via a peptide linker.
  • the virus vaccine is selected from a group consisting of yellow fever vaccine, influenza vaccine, hepatitis A vaccine, human papillomavirus vaccine, smallpox vaccine, rotavirus vaccine, adenovirus vaccine, poliovirus vaccine, chickenpox vaccine, typhoid vaccine, and measles, mumps, rubella (MMR) vaccine.
  • the virus vaccine specific TCR is a yellow fever vaccine specific TCR capable of binding to a yellow fever vaccine antigen.
  • the yellow fever vaccine antigen is selected from a group consisting of HLA-A02 restricted yellow fever virus antigen, HLA-A24 restricted yellow fever virus antigen, and HLA-A11 restricted yellow fever virus antigen.
  • the domain derived from a virus vaccine specific TCR is capable of binding a yellow fever vaccine peptide comprising an amino acid sequence of LLWNGPMAV (SEQ ID NO: 70) .
  • the domain derived from a virus vaccine specific TCR comprises (i) a ⁇ -chain of the virus vaccine specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence selected from a group consisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35; and/or (ii) a ⁇ -chain of the virus vaccine specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%
  • the domain derived from a virus vaccine specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.
  • the peptide linker is a cleavable peptide linker. In some embodiments, the peptide linker is a 2A self-cleaving peptide. In some embodiments, the 2A self-cleaving peptide is selected from a group consisting of F2A, E2A, P2A, T2A, or variants thereof. In some embodiments, the peptide linker comprises an amino acid sequence of SEQ ID NO: 55. In other embodiments, the peptide linker comprises an amino acid sequence of SEQ ID NO: 56.
  • composition comprising the polypeptide, the vector, or the CAR-T cell provided herein, and a pharmaceutically acceptable excipient.
  • provided herein is a method for treating a disease or disorder in a subject comprising administering to the subject a therapeutically effective amount of the CAR-T cell, or the pharmaceutical composition provided herein.
  • the method further comprises administering to the subject the virus vaccine after administering to the subject a therapeutically effective amount of the CAR-T cell or the pharmaceutical composition.
  • a method for treating a disease or disorder in a subject comprising administering to the subject (i) a therapeutically effective amount of the CAR-T cell provided herein, and (ii) a virus vaccine, wherein the CAR-T cell expresses a TCR or domain thereof capable of binding to the virus vaccine.
  • the disease or disorder is cancer.
  • the cancer is blood cancer.
  • the cancer is solid tumor cancer.
  • the subject is a human subject in need thereof.
  • FIG. 1A shows a schematic illustration of Vax-CAR and CAR design.
  • FIG. 1B shows the expression of CAR and GFP in transduced primary T cells.
  • FIG. 2 shows the flow cytometry analysis of surface expression of CD209 and CD86 in monocytes and mDCs.
  • FIG. 3 shows the expression of CD69 upon pYF peptide-pulsed DCs stimulation.
  • FIG. 4A shows the schematic of pYF peptide-pulsed DCs stimulation.
  • FIG. 4B shows the proliferation of T cells upon three times of stimulation by pYF peptide-pulsed DCs or controls.
  • FIG. 4C shows the cell cytotoxicity assay of T cells on HepG2 cells upon three times of stimulation by pYF peptide-pulsed DCs or controls.
  • FIG. 4D shows the flow cytometry analysis of surface expression of CD62L and CD45RO in T cells upon three times of stimulation by pYF peptide-pulsed DCs or controls.
  • the present disclosure is based, in part, on the surprising finding of improved expansion, activation, and function of engineered T cells transfected with a polynucleotide encoding a polypeptide comprising a CAR fused to a virus (e.g., a virus vaccine) specific TCR derived domain.
  • a virus e.g., a virus vaccine
  • T cells e.g., CAR-T cells
  • CAR-T cells adoptive transfer of T cells
  • the proliferative capacity of genetically engineered T cells is generally limited, known as exhaustion, due to lack of full spectrum of stimulations. Consequently, the efficacy of CAR-T cells against tumors is greatly impaired.
  • CAR-T cells engineered with secretion of pro-inflammatory cytokines (see, e.g., Adachi et al., Nature Biotechnology, 36 (4) : 346-51 (2016) ; Hurton et al., Proceedings of the National Academy of Sciences, 113 (48) : E7788-E97 (2016) ; Ma et al., Nature Biotechnology, 38 (4) : 448-59 (2020) ) , armored with dominant negative inhibitory signal receptors (see, e.g., Kloss et al., Molecular Therapy, 26 (7) : 1855-66 (2016) ) , or utilization of multi-co-stimulatory signals (see, e.g., Tokarew et al., British Journal of Cancer, 120 (1) : 26-37 (2019) ) .
  • these approaches do not produce sufficient effects.
  • a virus vaccine or a derivative thereof is used to boost CAR-T therapies by using a novel construct named Vax-CAR.
  • a novel construct named Vax-CAR More specifically, for example, polypeptides comprising domains derived from a vaccine specific TCR (e.g., a domain comprising ⁇ -and ⁇ -chain of a virus vaccine specific TCR) linked to a CAR, e.g., via a self-cleaving peptide linker are provided herein.
  • T cells transfected with a polynucleotide encoding such a polypeptide express vaccine specific TCR in addition to CAR on cell surface (named Vax-CAR-T cells) .
  • Such T cells respond to vaccine antigen, and thus vaccination can be used to induce activation, expansion, proliferation, cytotoxicity efficacy and/or anti-tumor efficacy of Vax-CAR-T cells.
  • vaccine induces the central memory phenotype of Vax-CAR-T cells, and restores the exhausted Vax-CAR-T cells upon repeated tumor antigen stimulation.
  • the compositions and methods provided herein provide the advantage of that the in vivo expansion of CAR-T cells is controllable, by modifying the dose and frequency of vaccine administration.
  • vacuna refers to a biological preparation that provides active acquired immunity, e.g., to a particular infectious disease.
  • a vaccine provided herein contains an agent that resembles a disease-causing microorganism (e.g., a virus) or a portion thereof, often made from weakened or killed forms.
  • antibody immunoglobulin, ” or “Ig” is used interchangeably herein, and is used in the broadest sense and specifically covers, for example, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies) , antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) , formed from at least two intact antibodies, single chain antibodies, and fragments thereof (e.g., domain antibodies) , as described below.
  • an antibody can be human, humanized, chimeric and/or affinity matured, as well as an antibody from other species, for example, mouse, rabbit, llama, etc.
  • the term “antibody” is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa) , each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region.
  • Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies including from Camelidae species (e.g., llama or alpaca) or their humanized variants, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • Camelidae species e.g., llama or alpaca
  • anti-Id anti-idiotypic antibodies
  • functional fragments e.g., antigen-binding fragments
  • Non-limiting examples of functional fragments include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc. ) , Fab fragments, F (ab’) fragments, F (ab) 2 fragments, F (ab’) 2 fragments, disulfide-linked Fvs (dsFv) , Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody.
  • scFv single-chain Fvs
  • Fab fragments fragments
  • F (ab’) fragments fragments
  • F (ab) 2 fragments F (ab’) 2 fragments
  • dsFv disulfide-linked Fvs
  • antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen-binding domains or molecules that contain an antigen-binding site that binds to an antigen (e.g., one or more CDRs of an antibody) .
  • an antigen e.g., one or more CDRs of an antibody
  • Such antibody fragments can be found in, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989) ; Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995) ; Huston et al., 1993, Cell Biophysics 22: 189-224; Plückthun and Skerra, 1989, Meth. Enzymol. 178: 497-515; and Day, Advanced Immunochemistry (2d ed. 1990) .
  • the antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule.
  • Antibodies may be agonistic antibodies or antagonistic antibodies .
  • Antibodies may be neither agonistic nor antagonistic.
  • an “antigen” is a structure to which an antibody can selectively bind.
  • a target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound.
  • the target antigen is a polypeptide.
  • an antigen is associated with a cell, for example, is present on or in a cell.
  • an “intact” antibody is one comprising an antigen-binding site as well as a CL and at least heavy chain constant regions, CH1, CH2 and CH3.
  • the constant regions may include human constant regions or amino acid sequence variants thereof.
  • an intact antibody has one or more effector functions.
  • Single-chain Fv also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding.
  • Single domain antibody refers to a single monomeric variable antibody domain and which is capable of antigen binding.
  • Single domain antibodies include VHH domains as described herein. Examples of single domain antibodies include, but are not limited to, antibodies naturally devoid of light chains such as those from Camelidae species (e.g., llama) , single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.
  • Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, and bovine.
  • a single domain antibody can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco, as described herein. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; VHHs derived from such other species are within the scope of the disclosure.
  • the single domain antibody e.g., VHH
  • the single domain antibody has a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • Single domain antibodies may be genetically fused or chemically conjugated to another molecule (e.g., an agent) as described herein.
  • Single domain antibodies may be part of a bigger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor) .
  • binding refers to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope.
  • the ratio of dissociation rate (koff) to association rate (kon) of a binding molecule (e.g., an antibody) to a monovalent antigen (koff/kon) is the dissociation constant KD, which is inversely related to affinity.
  • KD dissociation constant
  • the value of KD varies for different complexes of antibody and antigen and depends on both kon and koff.
  • the dissociation constant KD for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art.
  • the affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen.
  • binding molecules described herein terms such as “bind to, ” “that specifically bind to, ” and analogous terms are also used interchangeably herein and refer to binding molecules of antigen binding domains that specifically bind to an antigen, such as a polypeptide.
  • a binding molecule or antigen binding domain that binds to or specifically binds to an antigen can be identified, for example, by immunoassays, or other techniques known to those of skill in the art.
  • a binding molecule or antigen binding domain binds to or specifically binds to an antigen when it binds to an antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) .
  • RIA radioimmunoassay
  • ELISA enzyme linked immunosorbent assay
  • a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion regarding binding specificity.
  • the extent of binding of a binding molecule or antigen binding domain to a “non-target” protein is less than about 10%of the binding of the binding molecule or antigen binding domain to its particular target antigen, for example, as determined by FACS analysis or RIA.
  • a binding molecule or antigen binding domain that binds to an antigen includes one that is capable of binding the antigen with sufficient affinity such that the binding molecule is useful, for example, as a therapeutic and/or diagnostic agent in targeting the antigen.
  • a binding molecule or antigen binding domain that binds to an antigen has a dissociation constant (KD) of less than or equal to 1 ⁇ M, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM.
  • KD dissociation constant
  • a binding molecule or antigen binding domain binds to an epitope of an antigen that is conserved among the antigen from different species.
  • the binding molecules or antigen binding domains can comprise “chimeric” sequences in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851-55) .
  • Chimeric sequences may include humanized sequences.
  • the binding molecules or antigen binding domains can comprise portions of “humanized” forms of nonhuman (e.g., camelid, murine, non-human primate) antibodies that include sequences from human immunoglobulins (e.g., recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (e.g., donor antibody) such as camelid, mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
  • a nonhuman species e.g., donor antibody
  • one or more FR region residues of the human immunoglobulin sequences are replaced by corresponding nonhuman residues.
  • humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • a humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the binding molecules or antigen binding domains can comprise portions of a “fully human antibody” or “human antibody, ” wherein the terms are used interchangeably herein and refer to an antibody that comprises a human variable region and, for example, a human constant region.
  • the binding molecules may comprise a single domain antibody sequence.
  • the terms refer to an antibody that comprises a variable region and constant region of human origin.
  • “Fully human” antibodies in certain embodiments, can also encompass antibodies which bind polypeptides and are encoded by nucleic acid sequences which are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequence.
  • the term “fully human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) .
  • a “human antibody” is one that possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries (Hoogenboom and Winter, J. Mol.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., mice (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6 (5) : 561-66 (1995) ; Brüggemann and Taussing, Curr. Opin. Biotechnol. 8 (4) : 455-58 (1997) ; and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology) . See also, for example, Li et al., Proc. Natl. Acad. Sci. USA 103: 3557-62 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
  • the binding molecules or antigen binding domains can comprise portions of a “recombinant human antibody, ” wherein the phrase includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse or cow) that is transgenic and/or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, L. D. et al., Nucl. Acids Res.
  • human antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (See Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) .
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • the binding molecules or antigen binding domains can comprise a portion of a “monoclonal antibody, ” wherein the term as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts or well-known post-translational modifications such as amino acid iomerizatio or deamidation, methionine oxidation or asparagine or glutamine deamidation, each monoclonal antibody will typically recognize a single epitope on the antigen.
  • a “monoclonal antibody, ” as used herein is an antibody produced by a single hybridoma or other cell.
  • the term “monoclonal” is not limited to any particular method for making the antibody.
  • the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., Nature 256: 495 (1975) , or may be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567) .
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352: 624-28 (1991) and Marks et al., J. Mol. Biol. 222: 581-97 (1991) , for example.
  • a typical 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • the 4-chain unit is generally about 150,000 daltons.
  • Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the ⁇ and ⁇ chains and four CH domains for ⁇ and ⁇ isotypes.
  • Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end.
  • the VL is aligned with the VH
  • the CL is aligned with the first constant domain of the heavy chain (CH1) .
  • Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • the pairing of a VH and VL together forms a single antigen-binding site.
  • Fab refers to an antibody region that binds to antigens.
  • a conventional IgG usually comprises two Fab regions, each residing on one of the two arms of the Y-shaped IgG structure.
  • Each Fab region is typically composed of one variable region and one constant region of each of the heavy and the light chain. More specifically, the variable region and the constant region of the heavy chain in a Fab region are VH and CH1 regions, and the variable region and the constant region of the light chain in a Fab region are VL and CL regions.
  • the VH, CH1, VL, and CL in a Fab region can be arranged in various ways to confer an antigen binding capability according to the present disclosure.
  • VH and CH1 regions can be on one polypeptide, and VL and CL regions can be on a separate polypeptide, similarly to a Fab region of a conventional IgG.
  • VH, CH1, VL and CL regions can all be on the same polypeptide and oriented in different orders as described in more detail the sections below.
  • variable region refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen.
  • the variable region of the heavy chain may be referred to as “VH. ”
  • the variable region of the light chain may be referred to as “VL. ”
  • variable refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen.
  • variable regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” that are each about 9-12 amino acids long.
  • FRs framework regions
  • hypervariable regions that are each about 9-12 amino acids long.
  • the variable regions of heavy and light chains each comprise four FRs, largely adopting a ⁇ sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the ⁇ sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991) ) .
  • the constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) .
  • the variable regions differ extensively in sequence between different antibodies.
  • the variable region is a human variable region.
  • variable region residue numbering refers to the numbering system used for heavy chain variable regions or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, an FR or CDR of the variable domain.
  • a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 and three inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra) .
  • the “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra) .
  • the “EU index as in Kabat” refers to the residue numbering of the human IgG 1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.
  • the term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy-terminal portion includes a constant region.
  • the constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha ( ⁇ ) , delta ( ⁇ ) , epsilon ( ⁇ ) , gamma ( ⁇ ) , and mu ( ⁇ ) , based on the amino acid sequence of the heavy chain constant region.
  • the distinct heavy chains differ in size: ⁇ , ⁇ , and ⁇ contain approximately 450 amino acids, while ⁇ and ⁇ contain approximately 550 amino acids.
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin G
  • IgM immunoglobulin M
  • light chain when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy-terminal portion includes a constant region.
  • the approximate length of a light chain is 211 to 217 amino acids.
  • CDR refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH ⁇ -sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL ⁇ -sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences.
  • CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems.
  • the Kabat Complementarity Determining Regions are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra) .
  • Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196: 901-17 (1987) ) .
  • the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34) .
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Dübel eds., 2d ed.
  • IMGT ImMunoGeneTics
  • IG immunoglobulins
  • TCR T-cell receptors
  • MHC major histocompatibility complex
  • CDR complementary determining region
  • individual CDRs e.g., CDR-H1, CDR-H2
  • the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR is given.
  • CDR regions may also be defined by a combination of various numbering systems, e.g., a combination of Kabat and Chothia numbering systems, or a combination of Kabat and IMGT numbering systems. Therefore, the term such as “a CDR as set forth in a specific VH” includes any CDR1 as defined by the exemplary CDR numbering systems described above, but is not limited thereby.
  • a variable region e.g., a VH or VL
  • those skilled in the art would understand that CDRs within the region can be defined by different numbering systems or combinations thereof.
  • Hypervariable regions may comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (L1) , 46-56 or 50-56 (L2) , and 89-97 or 89-96 (L3) in the VL, and 26-35 or 26-35A (H1) , 50-65 or 49-65 (H2) , and 93-102, 94-102, or 95-102 (H3) in the VH.
  • constant region refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor.
  • the term refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding site.
  • the constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.
  • FR refers to those variable region residues flanking the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies (e.g., single domain antibodies) , diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.
  • a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • a “functional Fc region” possesses an “effector function” of a native sequence Fc region.
  • exemplary “effector functions” include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor) , etc.
  • effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays known to those skilled in the art.
  • a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., substituting, addition, or deletion) .
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide.
  • the variant Fc region herein can possess at least about 80%homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, or at least about 90%homology therewith, for example, at least about 95%homology therewith.
  • an “epitope” is a term in the art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody comprising a single domain antibody sequence) can specifically bind.
  • An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope.
  • an epitope can be contiguous amino acids of the polypeptide (a “linear” epitope) or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide (a “conformational, ” “non-linear” or “discontinuous” epitope) .
  • a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure.
  • a binding molecule binds to a group of amino acids regardless of whether they are folded in a natural three dimensional protein structure.
  • a binding molecule requires amino acid residues making up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.
  • Percent (%) amino acid sequence identity and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • CAR Chimeric antigen receptor
  • CAR genetically engineered receptors, which can be used to graft one or more antigen specificity onto immune effector cells, such as T cells.
  • Some CARs are also known as “artificial T-cell receptors, ” “chimeric T cell receptors, ” or “chimeric immune receptors. ”
  • the CAR comprises an extracellular antigen binding domain specific for one or more antigens (such as tumor antigens) , a transmembrane domain, and an intracellular signaling domain of a T cell and/or other receptors.
  • CAR-T cell refers to a T cell that expresses a CAR.
  • the CAR is single CAR.
  • the CAR is tandem CAR.
  • the CAR is dual CAR.
  • polypeptide and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.
  • polypeptides containing one or more analogs of an amino acid including but not limited to, unnatural amino acids, as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a “polypeptide” can occur as a single chain or as two or more associated chains.
  • Polynucleotide or “nucleic acid, ” as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • Oligonucleotide refers to short, generally single-stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length.
  • oligonucleotide and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
  • a cell that produces a binding molecule of the present disclosure may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced.
  • the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction.
  • the direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences” ; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences. ”
  • an “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or a mixed nucleic acids, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence.
  • An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an “isolated” nucleic acid molecule, such as a cDNA molecule can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid molecules encoding a single domain antibody or an antibody as described herein are isolated or purified.
  • the term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a substantially pure molecule may include isolated forms of the molecule.
  • an “isolated” nucleic acid molecule encoding a CAR described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • operatively linked, ” and similar phrases when used in reference to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequence, respectively, placed in functional relationships with each other.
  • an operatively linked promoter, enhancer elements, open reading frame, 5' and 3' UTR, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA) .
  • operatively linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame) .
  • an operatively linked peptide is one in which the functional domains are placed with appropriate distance from each other to impart the intended function of each domain.
  • vector refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, in order to introduce a nucleic acid sequence into a host cell.
  • Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences.
  • Selection control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art.
  • both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.
  • nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA
  • immunoblotting for expression of gene products or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product.
  • host refers to an animal, such as a mammal (e.g., a human) .
  • host cell refers to a particular subject cell that may be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • autologous is meant to refer to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • Allogeneic refers to a graft derived from a different individual of the same species.
  • transfected or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • pharmaceutically acceptable means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
  • Excipient means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material.
  • Excipients include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof.
  • the term “excipient” can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) or vehicle.
  • excipients are pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN TM , polyethylene glycol (PEG) , and PLURONICS TM .
  • buffers such as phosphate,
  • each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable excipients are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • a pharmaceutically acceptable excipient is an aqueous pH buffered solution.
  • excipients are sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • Water is an exemplary excipient when a composition (e.g., a pharmaceutical composition) is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions.
  • An excipient can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
  • Oral compositions, including formulations can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • compositions including pharmaceutical compounds, may contain a binding molecule (e.g., an antibody) , for example, in isolated or purified form, together with a suitable amount of excipients.
  • a binding molecule e.g., an antibody
  • an effective amount or “therapeutically effective amount” as used herein refers to the amount of a single domain antibody or a therapeutic molecule comprising an agent and the single domain antibody or pharmaceutical composition provided herein which is sufficient to result in the desired outcome.
  • a subject is a mammal, such as a non-primate or a primate (e.g., human) .
  • the subject is a human.
  • the subject is a mammal, e.g., a human, diagnosed with a disease or disorder.
  • the subject is a mammal, e.g., a human, at risk of developing a disease or disorder.
  • administer refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art.
  • treat, ” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a disease or condition resulting from the administration of one or more therapies. Treating may be determined by assessing whether there has been a decrease, alleviation and/or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder.
  • Treating includes both managing and ameliorating the disease.
  • the terms “manage, ” “managing, ” and “management” refer to the beneficial effects that a subject derives from a therapy which does not necessarily result in a cure of the disease.
  • prevent, ” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom (s) (e.g., diabetes or a cancer) .
  • “delaying” the development of cancer means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.
  • a method that "delays" development of cancer is a method that reduces probability of disease development in a given time frame and/or reduces the extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of individuals.
  • Cancer development can be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT Scan) , Magnetic Resonance Imaging (MRI) , abdominal ultrasound, clotting tests, arteriography, or biopsy. Development may also refer to cancer progression that may be initially undetectable and includes occurrence, recurrence, and onset.
  • CAT Scan computerized axial tomography
  • MRI Magnetic Resonance Imaging
  • abdominal ultrasound clotting tests
  • arteriography arteriography
  • biopsy biopsy.
  • cancer progression may be initially undetectable and includes occurrence, recurrence, and onset.
  • Polypeptides Comprising a Chimeric Antigen Receptor and a Binding Domain Derived from a Virus Specific T Cell Receptor
  • a polypeptide comprising at least two regions-a chimeric antigen receptor (CAR) and a domain derived from a virus (e.g., a virus vaccine) specific TCR (e.g., a domain comprising ⁇ -and ⁇ -chains of a vaccine specific TCR) , wherein the two regions are linked by a linker.
  • a virus e.g., a virus vaccine
  • TCR e.g., a domain comprising ⁇ -and ⁇ -chains of a vaccine specific TCR
  • the linker is cleavable in cells (e.g., a self-cleaving peptide linker) , so that the two regions are presented as or in two individual domains on the surface of an engineered immune cell (e.g., a T cell) , when the immune cell (e.g., a T cell) is transfected with a polynucleotide encoding such a polypeptide.
  • an engineered immune cell e.g., a T cell
  • the immune cell e.g., a T cell
  • the CAR expressed on the engineered immune cell can bind an antigen (e.g., a tumor antigen) ; and the domain derived from a virus (e.g., a virus vaccine) specific TCR can bind to and respond to an vaccine antigen, thereby inducing activation, expansion, proliferation, cytotoxicity efficacy and/or anti-tumor efficacy of the CAR-T cells upon vaccination.
  • an antigen e.g., a tumor antigen
  • a virus e.g., a virus vaccine
  • a polypeptide comprising (a) a CAR comprising (i) an extracellular domain, (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) a domain derived from a vaccine specific TCR, wherein the CAR and the domain are fused by a peptide linker.
  • the CAR and the domain can be in any order in the polypeptide provided herein.
  • the CAR is at the N-terminus of the domain.
  • the CAR is at the C-terminus of the domain.
  • TCR engineered T cell receptor
  • TAC T cell antigen coupler
  • a polypeptide comprising at least two regions-a TCR (e.g., engineered TCR) or a domain thereof (e.g., a TCR subunit) and a domain derived from a vaccine specific TCR, wherein the TCR subunit and the domain are fused by a peptide linker.
  • a TCR e.g., engineered TCR
  • a domain thereof e.g., a TCR subunit
  • a domain derived from a vaccine specific TCR wherein the TCR subunit and the domain are fused by a peptide linker.
  • the linker is cleavable in cells (e.g., a self-cleaving peptide linker) , so that the two regions are expressed as (or as part of) two individual domains on the surface of the engineered immune cell (e.g., a T cell) , when the immune cell (e.g., a T cell) is transfected with a polynucleotide encoding such a polypeptide.
  • the TCR subunit and the domain can be in any order in the polypeptide provided herein.
  • the TCR subunit is at the N-terminus of the domain. In other embodiments, the TCR subunit is at the C-terminus of the domain.
  • the TCR region (e.g., a TCR subunit) in the present polypeptide comprises (i) at least a portion of a TCR extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain.
  • a polypeptide comprising at least two regions-a TAC (or a domain thereof) and a domain derived from a vaccine specific TCR, wherein the TAC (or a domain thereof) and the domain are fused by a peptide linker.
  • the linker is cleavable in cells (e.g., a self-cleaving peptide linker) , so that the two regions are expressed as (or as part of) two individual domains on the surface of the engineered immune cell (e.g., a T cell) , when the immune cell (e.g., a T cell) is transfected with a polynucleotide encoding such a polypeptide.
  • the TAC (or a domain thereof) and the domain can be in any order in the polypeptide provided herein.
  • the TAC (or a doman thereof) is at the N-terminus of the domain.
  • the TAC (or a domain thereof) is at the C-terminus of the domain.
  • the TAC region in the present polypeptide comprises an antigen-binding domain, a TCR-recruitment domain, and a co-receptor domain (hinge, transmembrane, and cytosolic regions) .
  • the CAR provided herein comprises a polypeptide comprising: (a) an extracellular antigen binding domain; (b) a transmembrane domain; and (c) an intracellular signaling domain, each of which and additional regions are described in more detail below.
  • the extracellular antigen binding domain of the CARs described herein comprises one or more antigen binding domains.
  • the extracellular antigen binding domain of the CAR provided herein is mono-specific.
  • the extracellular antigen binding domain of the CAR provided herein is multispecific.
  • the extracellular antigen binding domain of the CAR provided herein is multivalent.
  • the extracellular antigen binding domain comprises two or more antigen binding domains which are fused to each other directly via peptide bonds, or via peptide linkers.
  • the extracellular antigen binding domain comprises an antibody or a fragment thereof.
  • the binding domain may be derived from monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies) , antibody with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) , formed from at least two intact antibodies, single chain antibodies, and fragments thereof (e.g., domain antibodies) .
  • An antibody can be human, humanized, chimeric and/or affinity matured, as well as an antibody from other species, for example, mouse, rabbit, llama, etc.
  • the antibody include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa) , each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region.
  • each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa)
  • each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids
  • each carboxy-terminal portion of each chain includes a constant region.
  • Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies including from Camelidae species (e.g., llama or alpaca) or their humanized variants, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • functional fragments include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.
  • antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen-binding domains or molecules that contain an antigen-binding site that binds to an antigen (e.g., one or more CDRs of an antibody) .
  • antigen-binding domains or molecules that contain an antigen-binding site that binds to an antigen e.g., one or more CDRs of an antibody
  • the antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule.
  • Antibodies may be agonistic antibodies or antagonistic antibodies .
  • Antibodies may be neither agonistic nor antagonistic.
  • the extracellular antigen binding domain of the present CARs comprise a single-chain Fv (sFv or scFv) .
  • ScFvs are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. See Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994) .
  • the extracellular antigen binding domain of the present CARs comprises one or more single domain antibodies (sdAbs) .
  • the sdAbs may be of the same or different origins, and of the same or different sizes.
  • Exemplary sdAbs include, but are not limited to, heavy chain variable domains from heavy-chain only antibodies (e.g., VHH or V NAR ) , binding molecules naturally devoid of light chains, single domains (such as V H or V L ) derived from conventional 4-chain antibodies, humanized heavy-chain only antibodies, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies.
  • sdAbs known in the art or developed by the present disclosure may be used to construct the CARs described herein.
  • the sdAbs may be derived from any species including, but not limited to mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine.
  • Single domain antibodies contemplated herein also include naturally occurring single domain antibody molecules from species other than Camelidae and sharks.
  • the sdAb is derived from a naturally occurring single domain antigen binding molecule known as heavy chain antibody devoid of light chains (also referred herein as “heavy chain only antibodies” ) .
  • heavy chain antibody devoid of light chains also referred herein as “heavy chain only antibodies”
  • single domain molecules are disclosed in WO 94/04678 and Hamers-Casterman, C. et al., Nature 363: 446-448 (1993) , for example.
  • the variable domain derived from a heavy chain molecule naturally devoid of light chain is known herein as a VHH to distinguish it from the conventional V H of four chain immunoglobulins.
  • VHH molecule can be derived from antibodies raised in Camelidae species, for example, camel, llama, vicuna, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain molecules naturally devoid of light chain, and such VHHs are within the scope of the present disclosure.
  • humanized versions of VHHs as well as other modifications and variants are also contemplated and within the scope of the present disclosure.
  • the sdAb is derived from a variable region of the immunoglobulin found in cartilaginous fish.
  • the sdAb can be derived from the immunoglobulin isotype known as Novel Antigen Receptor (NAR) found in the serum of shark.
  • NAR Novel Antigen Receptor
  • Methods of producing single domain molecules derived from a variable region of NAR are described in WO 03/014161 and Streltsov, Protein Sci. 14: 2901-2909 (2005) .
  • naturally occurring VHH domains against a particular antigen or target can be obtained from ( or immune) libraries of camelid VHH sequences. Such methods may or may not involve screening such a library using said antigen or target, or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known in the field. Such libraries and techniques are for example described in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694.
  • improved synthetic or semi-synthetic libraries derived from ( or immune) VHH libraries may be used, such as VHH libraries obtained from ( or immune) VHH libraries by techniques such as random mutagenesis and/or CDR shuffling, as for example described in WO 00/43507.
  • the sdAb is recombinant, CDR-grafted, humanized, camelized, de-immunized and/or in vitro generated (e.g., selected by phage display) .
  • the amino acid sequence of the framework regions may be altered by “camelization” of specific amino acid residues in the framework regions. Camelization refers to the replacing or substitution of one or more amino acid residues in the amino acid sequence of a (naturally occurring) VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position (s) in a VHH domain of a heavy chain antibody. This can be performed in a manner known in the field, which will be clear to the skilled person.
  • Such “camelizing” substitutions are preferably inserted at amino acid positions that form and/or are present at the VH-VL interface, and/or at the so-called Camelidae hallmark residues, as defined herein (see for example WO 94/04678, Davies and Riechmann FEBS Letters 339: 285-290 (1994) ; Davies and Riechmann, Protein Engineering 9 (6) : 531-537 (1996) ; Riechmann, J. Mol. Biol. 259: 957-969 (1996) ; and Riechmann and Muyldermans, J. Immunol. Meth. 231: 25-38 (1999) ) .
  • the sdAb is a human single domain antibody produced by transgenic mice or rats expressing human heavy chain segments. See, e.g., US20090307787, U.S. Pat. No. 8, 754, 287, US20150289489, US20100122358, and WO2004049794.
  • the single domain antibodies are generated from conventional four-chain antibodies. See, for example, EP 0 368 684; Ward et al., Nature, 341 (6242) : 544-6 (1989) ; Holt et al., Trends Biotechnol., 21 (11) : 484-490 (2003) ; WO 06/030220; and WO 06/003388.
  • the extracellular antigen binding domain comprises humanized antibodies or fragment thereof.
  • a humanized antibody can comprise human framework region and human constant region sequences.
  • Humanized antibodies can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239, 400; International publication No. WO 91/09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089) , veneering or resurfacing (European Patent Nos. EP 592, 106 and EP 519, 596; Padlan, 1991, Molecular Immunology 28 (4/5) : 489-498; Studnicka et al., 1994, Protein Engineering 7 (6) : 805-814; and Roguska et al., 1994, PNAS 91: 969-973) , chain shuffling (U.S. Patent No.
  • a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization may be performed, for example, following the method of Jones et al., 1986, Nature 321: 522-25; Riechmann et al., 1988, Nature 332: 323-27; and Verhoeyen et al., 1988, Science 239: 1534-36) , by substituting hypervariable region sequences for the corresponding sequences of a human antibody.
  • the humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of the parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework.
  • CDR grafting in which the amino acid sequences of the six CDRs of the parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework.
  • the amino acid sequences of the six CDRs of the parent non-human antibody e.g., rodent
  • SDRs the “specificity determining residues, ” or SDRs (Padlan et al., 1995, FASEB J. 9: 133-39) .
  • SDR grafting only the SDR residues are grafted onto the human antibody framework (see, e.g., Kashmiri et al., 2005, Methods 36: 25-34) .
  • variable domains both light and heavy
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity.
  • the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence that is closest to that of the rodent may be selected as the human framework for the humanized antibody (Sims et al., 1993, J. Immunol. 151: 2296-308; and Chothia et al., 1987, J. Mol. Biol. 196: 901-17) .
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • the same framework may be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89: 4285-89; and Presta et al., 1993, J. Immunol. 151: 2623-32) .
  • the framework is derived from the consensus sequences of the most abundant human subclasses, VL6 subgroup I (VL6I) and VH subgroup III (VHIII) .
  • VL6I VL6 subgroup I
  • VHIII VH subgroup III
  • human germline genes are used as the source of the framework regions.
  • FR homology is irrelevant.
  • the method consists of comparison of the non-human sequence with the functional human germline gene repertoire. Those genes encoding the same or closely related canonical structures to the murine sequences are then selected. Next, within the genes sharing the canonical structures with the non-human antibody, those with highest homology within the CDRs are chosen as FR donors. Finally, the non-human CDRs are grafted onto these FRs (see, e.g., Tan et al., 2002, J. Immunol. 169: 1119-25) .
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng. 13: 819-24) , Modeller (Sali and Blundell, 1993, J. Mol. Biol.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen (s) , is achieved.
  • the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
  • HSC Human String Content
  • Antibody variants may be isolated from phage, ribosome, and yeast display libraries as well as by bacterial colony screening (see, e.g., Hoogenboom, 2005, Nat. Biotechnol. 23: 1105-16; Dufner et al., 2006, Trends Biotechnol. 24: 523-29; Feldhaus et al., 2003, Nat. Biotechnol. 21: 163-70; and Schlapschy et al., 2004, Protein Eng. Des. Sel. 17: 847-60) .
  • residues to be substituted may include some or all of the “Vernier” residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, 1992, J. Mol. Biol. 224: 487-99) , or from the more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem. 272: 10678-84) .
  • FR shuffling whole FRs are combined with the non-human CDRs instead of creating combinatorial libraries of selected residue variants (see, e.g., Dall’A cqua et al., 2005, Methods 36: 43-60) .
  • the libraries may be screened for binding in a two-step process, first humanizing VL, followed by VH.
  • a one-step FR shuffling process may be used. Such a process has been shown to be more efficient than the two-step screening, as the resulting antibodies exhibited improved biochemical and physicochemical properties including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., 2007, Mol. Immunol. 44: 3049-60) .
  • the “humaneering” method is based on experimental identification of essential minimum specificity determinants (MSDs) and is based on sequential replacement of non-human fragments into libraries of human FRs and assessment of binding. It begins with regions of the CDR3 of non-human VH and VL chains and progressively replaces other regions of the non-human antibody into the human FRs, including the CDR1 and CDR2 of both VH and VL. This methodology typically results in epitope retention and identification of antibodies from multiple subclasses with distinct human V-segment CDRs. Humaneering allows for isolation of antibodies that are 91-96%homologous to human germline gene antibodies (see, e.g., Alfenito, Cambridge Healthtech Institute’s Third Annual PEGS, The Protein Engineering Summit, 2007) .
  • the “human engineering” method involves altering a non-human antibody or antibody fragment, such as a mouse or chimeric antibody or antibody fragment, by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies.
  • the technique involves classifying amino acid residues of a non-human (e.g., mouse) antibody as “low risk, ” “moderate risk, ” or “high risk” residues. The classification is performed using a global risk/reward calculation that evaluates the predicted benefits of making particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the resulting antibody’s folding.
  • the particular human amino acid residue to be substituted at a given position (e.g., low or moderate risk) of a non-human (e.g., mouse) antibody sequence can be selected by aligning an amino acid sequence from the non-human antibody’s variable regions with the corresponding region of a specific or consensus human antibody sequence.
  • the amino acid residues at low or moderate risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence according to the alignment.
  • a composite human antibody can be generated using, for example, Composite Human Antibody TM technology (Antitope Ltd., Cambridge, United Kingdom) .
  • variable region sequences are designed from fragments of multiple human antibody variable region sequences in a manner that avoids T cell epitopes, thereby minimizing the immunogenicity of the resulting antibody.
  • Such antibodies can comprise human constant region sequences, e.g., human light chain and/or heavy chain constant regions.
  • a deimmunized antibody is an antibody in which T-cell epitopes have been removed. Methods for making deimmunized antibodies have been described. See, e.g., Jones et al., Methods Mol Biol. 2009; 525: 405-23, xiv, and De Groot et al., Cell. Immunol. 244: 148-153 (2006) ) .
  • Deimmunized antibodies comprise T-cell epitope-depleted variable regions and human constant regions. Briefly, VH and VL of an antibody are cloned and T-cell epitopes are subsequently identified by testing overlapping peptides derived from the VH and VL of the antibody in a T cell proliferation assay.
  • T cell epitopes are identified via in silico methods to identify peptide binding to human MHC class II. Mutations are introduced in the VH and VL to abrogate binding to human MHC class II. Mutated VH and VL are then utilized to generate the deimmunized antibody.
  • the extracellular antigen binding domain comprises multiple binding domains.
  • the extracellular antigen binding domain comprises multispecific antibodies or fragments thereof, e.g., an extracellular antigen binding domain comprising multiple binding domains (e.g., multiple VHHs) in tandem.
  • the extracellular antigen binding domain comprises multivalent antibodies or fragments thereof.
  • specificity refers to selective recognition of an antigen binding protein for a particular epitope of an antigen.
  • multispecific denotes that an antigen binding protein has two or more antigen-binding sites of which at least two bind different antigens.
  • the term “valent” as used herein denotes the presence of a specified number of binding sites in an antigen binding protein.
  • a full length antibody has two binding sites and is bivalent.
  • the terms “trivalent” , “tetravalent” , “pentavalent” and “hexavalent” denote the presence of two binding site, three binding sites, four binding sites, five binding sites, and six binding sites, respectively, in an antigen binding protein.
  • Multispecific antibodies such as bispecific antibodies are antibodies that have binding specificities for at least two different antigens.
  • Methods for making multipecific antibodies are known in the art, such as, by co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305: 537-40) .
  • multispecific antibodies e.g., bispecific antibodies
  • Bispecific Antibodies Kontermann ed., 2011
  • the antibodies of the present disclosure can be multivalent antibodies with two or more antigen binding sites (e.g., tetravalent antibodies) , which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody.
  • a multivalent antibody comprises (or consists of) three to about eight antigen binding sites.
  • a multivalent antibody comprises (or consists of) four antigen binding sites.
  • the multivalent antibody comprises at least one polypeptide chain (e.g., two polypeptide chains) , wherein the polypeptide chain (s) comprise two or more variable domains.
  • the polypeptide chain (s) may comprise VD1- (X1) n-VD2-(X2) n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1.
  • the polypeptide chain (s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain.
  • the multivalent antibody herein may further comprise at least two (e.g., four) light chain variable domain polypeptides.
  • the multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides.
  • the light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.
  • the various domains may be fused to each other via peptide linkers. In some embodiments, the domains are directly fused to each other without any peptide linkers.
  • the peptide linkers may be the same or different. Each peptide linker may have the same or different length and/or sequence depending on the structural and/or functional features of the various domains. Each peptide linker may be selected and optimized independently. The length, the degree of flexibility and/or other properties of the peptide linker (s) used in the CARs may have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more particular antigens or epitopes.
  • a peptide linker comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move relative to each other.
  • a glycine-serine doublet can be a suitable peptide linker.
  • the peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence.
  • a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO1996/34103.
  • the peptide linker is a flexible linker.
  • Exemplary flexible linkers include but not limited to glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , (GSGGS) n , (GGGS) n , and (GGGGS) n , where n is an integer of at least one) , glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Exemplary peptide linkers are listed in the table below.
  • the extracellular antigen binding domain provided in the present CARs recognizes an antigen that acts as a cell surface marker on target cells associated with a special disease state.
  • the antigen is a tumor antigen. Tumors express a number of proteins that can serve as a target antigen for an immune response, particularly T cell mediated immune responses.
  • the antigens targeted by the CAR may be antigens on a single diseased cell or antigens that are expressed on different cells that each contribute to the disease.
  • the antigens targeted by the CAR may be directly or indirectly involved in the diseases.
  • Tumor antigens are proteins that are produced by tumor cells that can elicit an immune response, particularly T-cell mediated immune responses.
  • Exemplary tumor antigens include, but not limited to, a glioma-associated antigen, carcinoembryonic antigen (CEA) , ⁇ -human chorionic gonadotropin, alphafetoprotein (AFP) , lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS) , intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA) , PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2/neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1) , MAGE, ELF2M, neutrophil elastase
  • the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor.
  • Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase and gp100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer.
  • Other target molecules belong to the group of transformation-related molecules such as the oncogene HER2/Neu/ErbB-2.
  • Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA) .
  • CEA carcinoembryonic antigen
  • the tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA) .
  • TSA tumor-specific antigen
  • TAA tumor-associated antigen
  • a TSA is unique to tumor cells and does not occur on other cells in the body.
  • a TAA associated antigen is not unique to a tumor cell, and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen.
  • the expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen.
  • TAAs may be antigens that are expressed on normal cells during fetal development, when the immune system is immature, and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells, but which are expressed at much higher levels on tumor cells.
  • TSA or TAA antigens include: differentiation antigens such as MART-1/MelanA (MART-I) , gp 100 (Pmel 17) , tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7.
  • differentiation antigens such as MART-1/MelanA (MART-I) ,
  • Additional non-limiting exemplary targets of the CARs provided herein include GPC2, CD276, Delta-like protein ligand 3 (DLL3) , NY-ESO-1, melanoma associated antigen 4; survivin protein, synovial sarcoma X breakpoint protein 2, CD3, epidermal growth factor receptor (EGFR) , erbb2 tyrosine kinase receptor, HER2, CEA, CD66, CD66e, ROR1, ntrkr1 tyrosine kinase receptor, GPC3, mesothelin, glutamate carboxypeptidase II, PMSA, PD-L1, folate receptor alpha, PSCA, Mucin 1, HLA antigen (such as HLA class I antigen A-2 alpha, HLA class I antigen A-11 alpha, and HLA class II antigen) , c-Met, hepatocyte growth factor receptor, K-Ras GTPase (KRAS) , IL-15 receptor, Kit
  • At least one target antigen of the present CARs is AFP. In some specific embodiments, at least one target antigen of the present CARs is CD19. In other specific embodiments, at least one target antigen of the present CARs is CD20. In yet other specific embodiments, at least one target antigen of the present CARs is CD22. In yet other specific embodiments, at least one target antigen of the present CARs is BCMA. In yet other specific embodiments, at least one target antigen of the present CARs is VEGFR2. In yet other specific embodiments, at least one target antigen of the present CARs is FAP. In yet other specific embodiments, at least one target antigen of the present CARs is EpCam.
  • At least one target antigen of the present CARs is GPC3. In yet other specific embodiments, at least one target antigen of the present CARs is CD133. In yet other specific embodiments, at least one target antigen of the present CARs is IL13Ra. In yet other specific embodiments, at least one target antigen of the present CARs is EGFRIII. In yet other specific embodiments, at least one target antigen of the present CARs is EphA2. In yet other specific embodiments, at least one target antigen of the present CARs is Muc1. In yet other specific embodiments, at least one target antigen of the present CARs is CD70. In yet other specific embodiments, at least one target antigen of the present CARs is CD123.
  • At least one target antigen of the present CARs is ROR1. In yet other specific embodiments, at least one target antigen of the present CARs is PSMA. In yet other specific embodiments, at least one target antigen of the present CARs is CD5. In yet other specific embodiments, at least one target antigen of the present CARs is GD2. In yet other specific embodiments, at least one target antigen of the present CARs is GAP. In yet other specific embodiments, at least one target antigen of the present CARs is CD33. In yet other specific embodiments, at least one target antigen of the present CARs is CEA. In yet other specific embodiments, at least one target antigen of the present CARs is PSCA. In yet other specific embodiments, at least one target antigen of the present CARs is Her2. In yet other specific embodiments, at least one target antigen of the present CARs is Mesothelin.
  • the CARs of the present disclosure comprise a transmembrane domain that can be directly or indirectly fused to the extracellular antigen binding domain.
  • the transmembrane domain may be derived either from a natural or from a synthetic source.
  • a “transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane, preferably an eukaryotic cell membrane.
  • Transmembrane domains compatible for use in the CARs described herein may be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non-naturally occurring protein segment, e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane.
  • Transmembrane domains are classified based on the three dimensional structure of the transmembrane domain.
  • transmembrane domains may form an alpha helix, a complex of more than one alpha helix, a beta-barrel, or any other stable structure capable of spanning the phospholipid bilayer of a cell.
  • transmembrane domains may also or alternatively be classified based on the transmembrane domain topology, including the number of passes that the transmembrane domain makes across the membrane and the orientation of the protein. For example, single-pass membrane proteins cross the cell membrane once, and multi-pass membrane proteins cross the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7 or more times) .
  • Membrane proteins may be defined as Type I, Type II or Type III depending upon the topology of their termini and membrane-passing segment (s) relative to the inside and outside of the cell.
  • Type I membrane proteins have a single membrane-spanning region and are oriented such that the N-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the C-terminus of the protein is present on the cytoplasmic side.
  • Type II membrane proteins also have a single membrane-spanning region but are oriented such that the C-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the N-terminus of the protein is present on the cytoplasmic side.
  • Type III membrane proteins have multiple membrane-spanning segments and may be further sub-classified based on the number of transmembrane segments and the location of N-and C-termini.
  • the transmembrane domain of the CAR described herein is derived from a Type I single-pass membrane protein.
  • transmembrane domains from multi-pass membrane proteins may also be compatible for use in the CARs described herein.
  • Multi-pass membrane proteins may comprise a complex (at least 2, 3, 4, 5, 6, 7 or more) alpha helices or a beta sheet structure.
  • the N-terminus and the C-terminus of a multi-pass membrane protein are present on opposing sides of the lipid bilayer, e.g., the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the extracellular side.
  • Transmembrane domains for use in the CARs described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment.
  • the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet.
  • the protein segment is at least approximately 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example in U.S. Patent No. 7,052,906 and PCT Publication No. WO 2000/032776, the relevant disclosures of which are incorporated by reference herein.
  • the transmembrane domain provided herein may comprise a transmembrane region and a cytoplasmic region located at the C-terminal side of the transmembrane domain.
  • the cytoplasmic region of the transmembrane domain may comprise three or more amino acids and, in some embodiments, helps to orient the transmembrane domain in the lipid bilayer.
  • one or more cysteine residues are present in the transmembrane region of the transmembrane domain.
  • one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain.
  • the cytoplasmic region of the transmembrane domain comprises positively charged amino acids.
  • the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.
  • the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues.
  • the transmembrane domain of the CAR provided herein comprises an artificial hydrophobic sequence.
  • a triplet of phenylalanine, tryptophan and valine may be present at the C terminus of the transmembrane domain.
  • the transmembrane region comprises mostly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine.
  • the transmembrane region is hydrophobic.
  • the transmembrane region comprises a poly-leucine-alanine sequence.
  • the hydropathy, or hydrophobic or hydrophilic characteristics of a protein or protein segment can be assessed by any method known in the art, for example the Kyte and Doolittle hydropathy analysis.
  • the transmembrane domain of the CAR comprises a transmembrane domain chosen from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDl la, CD18) , ICOS (CD278) , 4-1BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRFl) , CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl
  • the transmembrane domain is derived from CD8 ⁇ . In other specific embodiments, the transmembrane domain is derived from CD28 ⁇ .
  • the intracellular signaling domain in the CARs provided herein is responsible for activation of at least one of the normal effector functions of the immune effector cell expressing the CARs.
  • 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.
  • cytoplasmic signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire cytoplasmic signaling domain can be employed, in many cases it is not necessary to use the entire chain.
  • cytoplasmic signaling domain is thus meant to include any truncated portion of the cytoplasmic signaling domain sufficient to transduce the effector function signal.
  • the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.
  • the CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell.
  • Primary intracellular signaling domain refers to cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions.
  • the primary intracellular signaling domain contains a signaling motif known as immunoreceptor tyrosine-based activation motif, or ITAM.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITAM immunoreceptor tyrosine-based activation motif
  • the motif may comprises two repeats of the amino acid sequence YxxL/I separated by 6-8 amino acids, wherein each x is independently any amino acid, producing the conserved motif YxxL/Ix (6-8) YxxL/I.
  • ITAMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule. ITAMs may also function as docking sites for other proteins involved in signaling pathways.
  • ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3z, FcR gamma (FCER1G) , FcR beta (Fc Epsilon Rib) , CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
  • the CAR comprises at least one co-stimulatory signaling domain.
  • co-stimulatory signaling domain refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function.
  • Many immune effector cells require co-stimulation, in addition to stimulation of an antigen-specific signal, to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cell.
  • the co-stimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a co-stimulatory protein, which transduces a signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils.
  • “Co-stimulatory signaling domain” can be the cytoplasmic portion of a co-stimulatory molecule.
  • co-stimulatory molecule refers to a cognate binding partner on an immune cell (such as T cell) that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the immune cell, such as, but not limited to, proliferation and survival.
  • the intracellular signaling domain comprises a single co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (such as about any of 2, 3, 4, or more) co-stimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more of the same co-stimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more co-stimulatory signaling domains from different co-stimulatory proteins, such as any two or more co-stimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3z) and one or more co-stimulatory signaling domains.
  • a primary intracellular signaling domain such as cytoplasmic signaling domain of CD3z
  • the one or more co-stimulatory signaling domains and the primary intracellular signaling domain are fused to each other via optional peptide linkers.
  • the primary intracellular signaling domain, and the one or more co-stimulatory signaling domains may be arranged in any suitable order.
  • the one or more co-stimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3z) . Multiple co-stimulatory signaling domains may provide additive or synergistic stimulatory effects.
  • Activation of a co-stimulatory signaling domain in a host cell may induce the cell to increase or decrease the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival, and/or cytotoxicity.
  • the co-stimulatory signaling domain of any co-stimulatory molecule may be compatible for use in the CARs described herein.
  • the type (s) of co-stimulatory signaling domain is selected based on factors such as the type of the immune effector cells in which the effector molecules would be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effect) .
  • co-stimulatory signaling domains for use in the CARs can be the cytoplasmic signaling domain of co-stimulatory proteins, including, without limitation, members of the B7/CD28 family (e.g., B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, and PDCD6) ; members of the TNF superfamily (e.g., 4-1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TN
  • the one or more co-stimulatory signaling domains are selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands that specially bind to CD83.
  • LFA-1 lymphocyte function-associated antigen-1
  • the co-stimulatory signaling domains are variants of any of the co-stimulatory signaling domains described herein, such that the co-stimulatory signaling domain is capable of modulating the immune response of the immune cell.
  • the co-stimulatory signaling domains comprises up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) as compared to a wild-type counterpart.
  • Such co-stimulatory signaling domains comprising one or more amino acid variations may be referred to as variants. Mutation of amino acid residues of the co-stimulatory signaling domain may result in an increase in signaling transduction and enhanced stimulation of immune responses relative to co-stimulatory signaling domains that do not comprise the mutation. Mutation of amino acid residues of the co-stimulatory signaling domain may result in a decrease in signaling transduction and reduced stimulation of immune responses relative to co-stimulatory signaling domains that do not comprise the mutation.
  • the CARs provided herein may comprise a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide.
  • signal peptides are peptide sequences that target a polypeptide to the desired site in a cell.
  • the signal peptide targets the effector molecule to the secretory pathway of the cell and will allow for integration and anchoring of the effector molecule into the lipid bilayer.
  • Signal peptides including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences, which are compatible for use in the CARs described herein will be evident to one of skill in the art.
  • the signal peptide is derived from a molecule selected from the group consisting of CD8 ⁇ , GM-CSF receptor ⁇ , and IgG1 heavy chain.
  • the CARs provided herein comprise a hinge domain that is located between the extracellular antigen binding domain and the transmembrane domain.
  • a hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen binding domain relative to the transmembrane domain of the effector molecule can be used.
  • Hinge domains of antibodies are also compatible for use in the pH-dependent chimeric receptor systems described herein.
  • the hinge domain is the hinge domain that joins the constant domains CH1 and CH2 of an antibody.
  • the hinge domain is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody.
  • the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody.
  • the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody.
  • the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgG1 antibody.
  • Non-naturally occurring peptides may also be used as hinge domains for the chimeric receptors described herein.
  • the hinge domain between the C-terminus of the extracellular ligand-binding domain of an Fc receptor and the N-terminus of the transmembrane domain is a peptide linker, such as a (GxS) n linker, wherein x and n, independently can be an integer between 3 and 12 or greater than 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.
  • the hinge domain may contain about 10-100 amino acids, e.g., about any one of 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. In some embodiments, the hinge domain may be at least about any one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.
  • the hinge domain is a hinge domain of a naturally occurring protein. Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor.
  • the hinge domain is derived from CD8 ⁇ . In some embodiments, the hinge domain is a portion of the hinge domain of CD8 ⁇ , e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8 ⁇ . In other embodiments, the hinge domain is derived from CD28 ⁇ . In some embodiments, the hinge domain is a portion of the hinge domain of CD28 ⁇ , e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD28 ⁇ .
  • the CAR provided herein comprises amino acid sequences with certain percent identity relative to any one of the CARs exemplified in Section 6 below.
  • a CAR comprising or consisting of an extracellular domain having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of the CARs exemplified in Section 6 below.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 87: 2264 2268 (1990) , modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 90: 5873 5877 (1993) .
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215: 403 (1990) .
  • Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25: 3389 3402 (1997) .
  • PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.
  • amino acid sequence modification (s) of the CARs described herein are contemplated.
  • variants of the domains described herein can be prepared.
  • scFv variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide.
  • amino acid changes may alter post-translational processes of the antibody.
  • Variations may be a substitution, deletion, or insertion of one or more codons encoding the polypeptide that results in a change in the amino acid sequence as compared with the original antibody or polypeptide.
  • Sites of interest for substitutional mutagenesis include the CDRs and FRs.
  • Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements.
  • Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids.
  • the substitution, deletion, or insertion includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule.
  • the substitution is a conservative amino acid substitution made at one or more predicted non-essential amino acid residues. The variation allowed may be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the parental polypeptides.
  • polypeptides generated by conservative amino acid substitutions are included in the present disclosure.
  • a conservative amino acid substitution an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge.
  • families of amino acid residues having side chains with similar charges 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
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed and the activity of the protein can be determined.
  • Conservative (e.g., within an amino acid group with similar properties and/or side chains) substitutions may be made, so as to maintain or not significantly change the properties.
  • Amino acids may be grouped according to similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975) ) : (1) non-polar: Ala (A) , Val (V) , Leu (L) , Ile (I) , Pro (P) , Phe (F) , Trp (W) , Met (M) ; (2) uncharged polar: Gly (G) , Ser (S) , Thr (T) , Cys (C) , Tyr (Y) , Asn (N) , Gln (Q) ; (3) acidic: Asp (D) , Glu (E) ; and (4) basic: Lys (K) , Arg (R) , His (H) .
  • Naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, for example, with another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and to prevent aberrant crosslinking.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody) or fragment thereof in the extraceullar antigen binding domain of the present CARs.
  • a parent antibody e.g., a humanized or human antibody
  • the resulting variant (s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity) .
  • Alterations may be made in CDRs, e.g., to improve antibody affinity. Such alterations may be made in CDR “hotspots, ” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008) ) , and/or SDRs (a-CDRs) , with the resulting variant antibody or fragment thereof being tested for binding affinity.
  • CDR “hotspots i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008) )
  • SDRs a-CDRs
  • affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis) .
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling.
  • substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells, Science, 244: 1081-1085 (1989) .
  • a residue or group of target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen.
  • Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
  • Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino-and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody molecule include the fusion to the N-or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • the variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.
  • Site-directed mutagenesis see, e.g., Carter, Biochem J. 237: 1-7 (1986) ; and Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)
  • cassette mutagenesis see, e.g., Wells et al., Gene 34: 315-23 (1985)
  • other known techniques can be performed on the cloned DNA to produce the antibody variant DNA.
  • the present polypeptides include a TCR derived binding domain in addition to the CAR, more specifically, a virus specific TCR (e.g., a vaccine specific TCR) .
  • a virus specific TCR e.g., a vaccine specific TCR
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR. In other embodiments, the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain and a ⁇ -chain of the virus specific TCR.
  • the ⁇ -chain and the ⁇ -chain of the virus specific TCR can be linked via a peptide linker (e.g., a self-cleaving peptide linker) .
  • the ⁇ -chain and the ⁇ -chain of the virus specific TCR can be in any order in the present polypeptide.
  • the ⁇ -chain is at the N-terminus of the ⁇ -chain.
  • the ⁇ -chain is at the N-terminus of the ⁇ -chain.
  • the ⁇ -chain and the ⁇ -chain are separated by other domains.
  • one of the ⁇ -chain and the ⁇ -chain is at the N-terminus of the CAR and the other one is at the C-terminus of the CAR.
  • the virus specific TCR is a virus vaccine specific TCR.
  • Exemplary vaccines include (but not limited to) yellow fever vaccine, influenza vaccine, hepatitis A vaccine, human papillomavirus vaccine, smallpox vaccine, rotavirus vaccine, adenovirus vaccine, poliovirus vaccine, chickenpox vaccine, typhoid vaccine, and measles, mumps, rubella (MMR) vaccine.
  • the vaccine is yellow fever vaccine.
  • the vaccine is influenza vaccine.
  • the vaccine is hepatitis A vaccine.
  • the vaccine is human papillomavirus vaccine.
  • the vaccine is smallpox vaccine.
  • the vaccine is rotavirus vaccine. In some embodiments, the vaccine is adenovirus vaccine. In some embodiments, the vaccine is poliovirus vaccine. In some embodiments, the vaccine is chickenpox vaccine. In some embodiments, the vaccine is typhoid vaccine. In some embodiments, the vaccine is MMR vaccine.
  • the virus specific TCR is a yellow fever vaccine specific TCR capable of binding to a yellow fever vaccine antigen.
  • the yellow fever vaccine antigen is a HLA-A02 restricted yellow fever virus antigen.
  • the yellow fever vaccine antigen is a HLA-A24 restricted yellow fever virus antigen.
  • the yellow fever vaccine antigen is a HLA-A11 restricted yellow fever virus antigen.
  • the domain derived from a virus specific TCR is capable of binding a yellow fever vaccine peptide comprising an amino acid sequence of LLWNGPMAV (SEQ ID NO: 70) .
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 22.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 23.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 24.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 25.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 26.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 27.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 28.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 29.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 30.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 31.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 32.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 33.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 34.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 35.
  • ⁇ -chain of the virus specific TCR is one of those listed in Table 4.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 36.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 37.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 38.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 39.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 40.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 41.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 42.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 43.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 44.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 45.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 46.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 47.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 48.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 49.
  • the domain derived from a virus specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 50.
  • the ⁇ -chain of the virus specific TCR is one of those listed in Table 5.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 6.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 7.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 8.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 9.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 10.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 11.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 12.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 13.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 14.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 15.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 16.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 17.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 18.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 19.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 20.
  • the domain derived from a virus specific TCR comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 21.
  • the TCR derived domain comprising ⁇ -chain and ⁇ -chain of the virus specific TCR is one of those listed in Table 3.
  • the ⁇ -chain and ⁇ -chain of the virus specific TCR can be linked by a peptide linker.
  • the peptide linker is a 2A self-cleaving peptide, which is described in more detail in the section below.
  • the 2A self-cleaving peptide is selected from a group consisting of F2A, E2A, P2A, T2A, or variants thereof.
  • the peptide linker comprises a P2A peptide, e.g., comprising an amino acid sequence of SEQ ID NO: 55.
  • the peptide linker comprises a F2A peptide, e.g., comprising an amino acid sequence of SEQ ID NO: 56.
  • linkers that are cleavable in cells may be used in the present disclosure to link the CARs and the domains derived from virus (e.g., virus vaccine) specific TCRs.
  • virus e.g., virus vaccine
  • the peptide linker is a 2A self-cleaving peptide.
  • the members of 2A peptides are named after the virus in which they have been first described.
  • F2A the first described 2A peptide
  • the self-cleaving 18-22 amino acids long 2A peptides mediate ‘ribosomal skipping’ between the proline and glycine residues and inhibit peptide bond formation without affecting downstream translation.
  • These peptides allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation.
  • Self-cleaving peptides are found in members of the picornaviridae virus family, including aphthoviruses such as foot-and-mouth disease virus (FMDV) , equine rhinitis A virus (ERAV) , thosea asigna virus (TaV) and porcine teschovirus-1 (PTV-1) (see Donnelly et al., J. Gen. Virol., 82: 1027-101 (2001) ; Ryan et al., J. Gen. Virol., 72: 2727-2732 (2001) ) and cardioviruses such as theilovirus (e.g., theiler's murine encephalomyelitis) and encephalomyocarditis viruses.
  • aphthoviruses such as foot-and-mouth disease virus (FMDV) , equine rhinitis A virus (ERAV) , thosea asigna virus (TaV) and porcine teschovirus-1
  • the 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are sometimes referred to as “F2A, ” “E2A, ” “P2A, ” and “T2A, ” respectively, and are included in the present disclosure, e.g., as described in Donnelly et al., J. Gen. Virol., 78: 13-21 (1997) ; Ryan and Drew, EMBO J., 13: 928-933 (1994) ; Szymczak et al., Nature Biotech., 5: 589-594 (2004) ; Hasegawa et al., Stem Cells, 25 (7) : 1707-12 (2007) .
  • intein mediated protein splicing system is used herein, e.g., as described in Shah and Muir, Chem Sci., 5 (1) : 446–461 (2014) and Topilina and Mills, Mobile DNA, 5 (5) (2014) .
  • Other methods known in the art can also be used in the present constructs.
  • the 2A self-cleaving peptide is selected from a group consisting of F2A, E2A, P2A, T2A, or variants thereof.
  • the peptide linker comprises a P2A peptide, e.g., comprising an amino acid sequence of SEQ ID NO: 55.
  • the peptide linker comprises a F2A peptide, e.g., comprising an amino acid sequence of SEQ ID NO: 56.
  • the disclosure provides polynucleotides that encode the polypeptide provided herein, including those described in Section 5.2 above. More specifically, in some embodiments, provided herein is a polynucleotide encoding a polypeptide comprising (a) a chimeric antigen receptor (CAR) and (b) a domain derived from a virus (e.g., a virus vaccine) specific TCR, wherein the CAR and the domain are fused by a peptide linker.
  • CAR chimeric antigen receptor
  • a polynucleotide comprising a first region encoding a CAR, and a second region encoding a domain derived from a virus (e.g., a virus vaccine) specific TCR.
  • the CAR and the domain derived from a virus (e.g., a virus vaccine) specific TCR are as described in Section 5.2 above.
  • the first region and the second region are controlled by the same promoter.
  • internal ribosomal entry sites are used herein to express multiple genes from one promoter.
  • the first region and the second region are controlled by separate promoters.
  • a first polynucleotide encoding a CAR
  • a second polynucleotide encoding a domain derived from a virus (e.g., a virus vaccine) specific TCR.
  • the CAR and the domain derived from a virus (e.g., a virus vaccine) specific TCR are as described in Section 5.2 above.
  • a polynucleotide encoding a polypeptide comprising a TCR subunit and a domain derived from a vaccine specific TCR, wherein the TCR subunit and the domain are fused by a peptide linker.
  • a polynucleotide comprising a first region encoding a TCR subunit, and a second region encoding a domain derived from a virus (e.g., a virus vaccine) specific TCR.
  • the first region and the second region are controlled by the same promoter.
  • internal ribosomal entry sites IVS are used herein to express multiple genes from one promoter.
  • the first region and the second region are controlled by separate promoters.
  • a first polynucleotide encoding a TCR subunit
  • a second polynucleotide encoding a domain derived from a virus (e.g., a virus vaccine) specific TCR.
  • a polynucleotide encoding a polypeptide comprising a TAC (or a domain thereof) and a domain derived from a vaccine specific TCR, wherein the TAC (or a domain thereof) and the domain are fused by a peptide linker.
  • a polynucleotide comprising a first region encoding a TAC (or a domain thereof) , and a second region encoding a domain derived from a virus (e.g., a virus vaccine) specific TCR.
  • the first region and the second region are controlled by the same promoter.
  • internal ribosomal entry sites IVS are used herein to express multiple genes from one promoter.
  • the first region and the second region are controlled by separate promoters.
  • a first polynucleotide encoding a TAC (or a domain thereof)
  • a second polynucleotide encoding a domain derived from a virus (e.g., a virus vaccine) specific TCR.
  • the polynucleotides of the disclosure can be in the form of RNA or in the form of DNA.
  • DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.
  • the polynucleotide is in the form of cDNA.
  • the polynucleotide is a synthetic polynucleotide.
  • the present disclosure further relates to variants of the polynucleotides described herein, wherein the variant encodes, for example, fragments, analogs, and/or derivatives of the polypeptide of the disclosure.
  • the present disclosure provides a polynucleotide comprising a polynucleotide having a nucleotide sequence at least about 75%identical, at least about 80%identical, at least about 85%identical, at least about 90%identical, at least about 95%identical, and in some embodiments, at least about 96%, 97%, 98%or 99%identical to a polynucleotide encoding the polypeptide of the disclosure.
  • a polynucleotide having a nucleotide sequence at least, for example, 95%“identical” to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence up to 5%of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted into the reference sequence.
  • These mutations of the reference sequence can occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both.
  • a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide.
  • a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code) .
  • Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (i.e., change codons in the human mRNA to those preferred by a bacterial host such as E. coli) .
  • a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.
  • a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.
  • vectors comprising the polynucleotides or nucleic acid molecules described herein.
  • the nucleic acid molecules can be incorporated into a recombinant expression vector.
  • the present disclosure provides vectors for cloning and expressing any one of the polypeptides described herein.
  • the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells.
  • the vector is a viral vector.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, lentiviral vector, retroviral vectors, vaccinia vector, herpes simplex viral vector, and derivatives thereof.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) , and in other virology and molecular biology manuals.
  • retroviruses provide a convenient platform for gene delivery systems.
  • the heterologous nucleic acid 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 the engineered mammalian cell in vitro 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.
  • self-inactivating lentiviral vectors are used.
  • self-inactivating lentiviral vectors carrying the immunomodulator (such as immune checkpoint inhibitor) coding sequence and/or self-inactivating lentiviral vectors carrying chimeric antigen receptors can be packaged with protocols known in the art.
  • the resulting lentiviral vectors can be used to transduce a mammalian cell (such as primary human T cells) using methods known in the art.
  • Vectors derived from retroviruses such as lentivirus are suitable tools to achieve long-term gene transfer, because they allow long-term, stable integration of a transgene and its propagation in progeny cells.
  • Lentiviral vectors also have low immunogenicity, and can transduce non-proliferating cells.
  • the vector comprises any one of the nucleic acids encoding a polypeptide described herein.
  • the nucleic acid can be cloned into the vector using any known molecular cloning methods in the art, including, for example, using restriction endonuclease sites and one or more selectable markers.
  • the nucleic acid is operably linked to a promoter. Varieties of promoters have been explored for gene expression in mammalian cells, and any of the promoters known in the art may be used in the present disclosure. Promoters may be roughly categorized as constitutive promoters or regulated promoters, such as inducible promoters.
  • the nucleic acid encoding the polypeptide is operably linked to a constitutive promoter.
  • Constitutive promoters allow heterologous genes (also referred to as transgenes) to be expressed constitutively in the host cells.
  • Exemplary constitutive promoters contemplated herein include, but are not limited to, murine stem cell virus (MSCV) promoter, cytomegalovirus (CMV) promoters, human elongation factors-1 alpha (hEF1 ⁇ ) , ubiquitin C promoter (UbiC) , phosphoglycerokinase promoter (PGK) , simian virus 40 early promoter (SV40) , and chicken ⁇ -Actin promoter coupled with CMV early enhancer (CAGG) .
  • MSCV murine stem cell virus
  • CMV cytomegalovirus
  • hEF1 ⁇ human elongation factors-1 alpha
  • UbiC ubiquitin C promoter
  • PGK phosphoglycerokina
  • the nucleic acid encoding the CAR is operably linked to a hEF1 ⁇ promoter. In some embodiments, the nucleic acid encoding the CAR is operably linked to a MSCV promoter.
  • the nucleic acid encoding the polypeptide is operably linked to an inducible promoter.
  • Inducible promoters belong to the category of regulated promoters.
  • the inducible promoter can be induced by one or more conditions, such as a physical condition, microenvironment of the engineered immune effector cell, or the physiological state of the engineered immune effector cell, an inducer (i.e., an inducing agent) , or a combination thereof.
  • the inducing condition does not induce the expression of endogenous genes in the engineered mammalian cell, and/or in the subject that receives the pharmaceutical composition.
  • the inducing condition is selected from the group consisting of: inducer, irradiation (such as ionizing radiation, light) , temperature (such as heat) , redox state, tumor environment, and the activation state of the engineered mammalian cell.
  • the vector also contains a selectable marker gene or a reporter gene to select cells expressing the polypeptide from the population of host cells transfected through lentiviral vectors.
  • selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in the host cells.
  • the vector may contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid sequences.
  • host cells comprising any one of the polypeptides, polynucleotides, or vectors described herein.
  • an engineered immune effector cell e.g., a CAR-T cell or a TCR-T cell or a TAC-T cell
  • introducing the polynucleotide or the vector provided herein e.g., as described in Section 5.3 and Section 5.4 above
  • an immune effector cell e.g., a T cell
  • a method for making a CAR-T cell comprising introducing the polynucleotide encoding a polypeptide comprising (a) a chimeric antigen receptor (CAR) comprising (i) an extracellular domain, (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) a domain derived from a vaccine specific TCR, wherein the CAR and the domain are fused by a peptide linker.
  • CAR chimeric antigen receptor
  • a method of making a CAR-T cell comprising introducing into a T cell a polynucleotide comprising a first region encoding a CAR comprising (i) an extracellular domain, (ii) a transmembrane domain, and (iii) an intracellular domain; and a second region encoding a domain derived from a vaccine specific TCR.
  • a method of making a CAR-T cell comprising introducing into a T cell a first polynucleotide encoding a CAR comprising (i) an extracellular domain, (ii) a transmembrane domain, and (iii) an intracellular domain; and a second polypeptide encoding a domain derived from a vaccine specific TCR.
  • the above approaches may also be used for generating a TCR-T cell or a TAC-T cell.
  • an engineered immune effector cell e.g., a CAR-T cell
  • a CAR-T cell e.g., a CAR-T cell
  • Immuno effector cells are immune cells that can perform immune effector functions.
  • the immune effector cells express at least Fc ⁇ RIII and perform ADCC effector function.
  • immune effector cells which mediate ADCC include peripheral blood mononuclear cells (PBMC) , natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • monocytes cytotoxic T cells
  • neutrophils neutrophils
  • eosinophils eosinophils.
  • the immune effector cells are T cells.
  • the T cells are CD4+/CD8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-, or combinations thereof.
  • the T cells produce IL-2, TNF, and/or TNF upon expressing the CAR and binding to the target cells.
  • the CD8+ T cells lyse antigen-specific target cells upon expressing the CAR and binding to the target cells.
  • the immune effector cells are NK cells.
  • the immune effector cells can be established cell lines, for example, NK-92 cells.
  • the immune effector cells are differentiated from a stem cell, such as a hematopoietic stem cell, a pluripotent stem cell, an iPS, or an embryonic stem cell.
  • a stem cell such as a hematopoietic stem cell, a pluripotent stem cell, an iPS, or an embryonic stem cell.
  • the engineered immune effector cells are prepared by introducing the polypeptide provided herein into the immune effector cells, such as T cells.
  • the polypeptide is introduced to the immune effector cells by transfecting any one of the isolated nucleic acids or any one of the vectors described above.
  • vectors or isolated nucleic acids into a mammalian cell are known in the art.
  • the vectors described can be transferred into an immune effector cell by physical, chemical, or biological methods.
  • Physical methods for introducing the vector into an immune effector 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, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, the vector is introduced into the cell by electroporation.
  • Biological methods for introducing the vector into an immune effector cell include the use of DNA and RNA vectors.
  • Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Chemical means for introducing the vector into an immune effector 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.
  • 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 is a liposome (e.g., an artificial membrane vesicle) .
  • RNA molecules encoding any of the polypeptides described herein may be prepared by a conventional method (e.g., in vitro transcription) and then introduced into the immune effector cells via known methods such as mRNA electroporation. See, e.g., Rabinovich et al., Human Gene Therapy 17: 1027-1035 (2006) .
  • the transduced or transfected immune effector cell is propagated ex vivo after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced or transfected immune effector cell is cultured to propagate for at least about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. In some embodiments, the transduced or transfected immune effector cell is further evaluated or screened to select the engineered mammalian cell.
  • Reporter genes may be 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. FEBS Letters 479: 79-82 (2000) ) .
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • nucleic acid encoding the polypeptide in the engineered immune effector cell include, for example, molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological methods (such as ELISAs and Western blots) .
  • molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays such as detecting the presence or absence of a particular peptide, e.g., by immunological methods (such as ELISAs and Western blots) .
  • a source of T cells is obtained from a subject.
  • 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.
  • any number of T cell lines available in the art may be used.
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll TM separation.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS) .
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium may 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 CytoMate, 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 CytoMate, or the Haemonetics Cell Saver 5
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca 2+ -free, Mg 2+ -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 are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL TM gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of T cells such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selection techniques.
  • T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3 ⁇ 28) -conjugated beads, such as 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 embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the time period is 10 to 24 hours. In some embodiments, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. 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 immune-compromised individuals.
  • TIL tumor infiltrating lymphocytes
  • 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.
  • multiple rounds of selection can also be used. In some embodiments, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.
  • Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.
  • T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.
  • the concentration of cells and surface can be varied.
  • it may be desirable to significantly decrease the volume in which beads and cells are mixed together i.e., increase the concentration of cells
  • 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. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations may result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations may allow 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 (i.e., leukemic blood, tumor tissue, etc. ) . Such populations of cells may have therapeutic value and would be desirable to obtain. In some embodiments, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
  • the concentration of cells used is 5 ⁇ 10 6 /ml. In some embodiments, the concentration used can be from about 1 ⁇ 10 5 /ml to 1 ⁇ 10 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.
  • T cells for stimulation can also be frozen after a washing step.
  • the freeze and subsequent thaw step may provide 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.
  • Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.
  • cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation.
  • a blood sample or an apheresis product is taken from a generally healthy subject.
  • a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use.
  • the T cells may be expanded, frozen, and used at a later time.
  • samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments.
  • the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.
  • agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as
  • the cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, simultaneously or following) bone marrow or stem cell transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT) , cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
  • chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT) , cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
  • XRT external-beam radiation therapy
  • cyclophosphamide cyclophosphamide
  • antibodies such as OKT3 or CAMPATH.
  • the cells are isolated prior to and can be frozen for later use for treatment following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.
  • T cells are obtained from a patient directly following treatment.
  • 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.
  • the Vax-CAR-T cells provided herein can be activated in vivo by adminstration of a vaccine after the infusion of the Vax-CAR-T cells to a subject as described in more detail in Section 5.7 below.
  • the T cells prior to the adminstration of the cells to the subject, in some embodiments, can be activated and expanded, prior to or after genetic modification of the T cells with the polypeptides described herein, generally using methods as described, for example, in U.S. Pat. Nos.
  • T cells can 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 co-stimulatory 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 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 can be used as can other methods commonly known in the art (Graves J, et al., J. Immunol. 146: 2102 (1991) ; Li B, et al., Immunology 116: 487 (2005) ; Rivollier A, et al., Blood 104: 4029 (2004) ) .
  • an anti-CD28 antibody examples 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) ) .
  • the primary stimulatory signal and the co-stimulatory signal for the T cell may be provided by different protocols.
  • the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation) .
  • one agent may be coupled to a surface and the other agent in solution.
  • the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution.
  • the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents.
  • a surface such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents.
  • aAPCs artificial antigen presenting cells
  • the T cells are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured.
  • the agent-coated beads and cells prior to culture, are not separated but are cultured together.
  • the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.
  • cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3 ⁇ 28 beads) to contact the T cells.
  • the cells for example, 10 4 to 4 ⁇ 10 8 T cells
  • beads for example, anti-CD3/CD28 MACSiBead particlesa at a recommended titer of 1: 100
  • a buffer preferably PBS (without divalent cations such as, calcium and magnesium)
  • the target cell may be very rare in the sample and comprise only 0.01%of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest.
  • any cell number is within the context of the present disclosure.
  • it may be desirable to significantly decrease the volume in which particles and cells are mixed together i.e., increase the concentration of cells
  • a concentration of about 2 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 may result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations may allow more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain embodiments. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
  • the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment, the beads and the T cells are cultured together for about eight days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more.
  • Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15 (Lonza) ) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum) , interleukin-2 (IL-2) , insulin, IFN- ⁇ , IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF ⁇ , and TNF- ⁇ or any other additives for the growth of cells known to the skilled artisan.
  • Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.
  • Media can include RPMI 1640, AIM-V, DMEM, MEM, ⁇ -MEM, F-12, X-Vivo 15, and X-Vivo 20, optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine (s) sufficient for the growth and expansion of T cells.
  • Antibiotics e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject.
  • the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37 °C) and atmosphere (e.g., air plus 5%CO 2 ) .
  • T cells that have been exposed to varied stimulation times may exhibit different characteristics.
  • typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8) .
  • TH, CD4+ helper T cell population
  • TC, CD8 cytotoxic or suppressor T cell population
  • Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells.
  • infusing a subject with a T cell population comprising predominately of TH cells may be advantageous.
  • an antigen-specific subset of TC cells may be beneficial to expand this subset to a greater degree.
  • CD4 and CD8 markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.
  • an engineered immune effector cell e.g., a CAR-T cell
  • a CAR-T cell expressing (a) a CAR and (b) a virus vaccine specific TCR or a domain derived therefrom.
  • the CAR and the domain derived from the virus vaccine specific TCR are as described in Section 5.2 above and briefly in the following paragraphs.
  • the CAR in the present CAR-T cells comprises an extracellular antigen binding domain; a transmembrane domain; and an intracellular signaling domain.
  • the CAR further comprises one or more additional regions/domains such as a signal peptide, hinge region, co-stimulatory signaling domain, linkers, etc., each of which can be as described in Section 5.2.1 above.
  • the CARs provided herein may comprise a signal peptide at the N-terminus of the polypeptide.
  • the signal peptide targets the effector molecule to the secretory pathway of the cell and will allow for integration and anchoring of the effector molecule into the lipid bilayer.
  • Signal peptides including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences, which are compatible for use in the CARs described herein will be evident to one of skill in the art.
  • the signal peptide is derived from a molecule selected from the group consisting of CD8 ⁇ , GM-CSF receptor ⁇ , and IgG1 heavy chain.
  • the extracellular antigen binding domain of the CARs described herein comprises one or more antigen binding domains.
  • the extracellular antigen binding domain comprises an antibody or a fragment thereof.
  • the extracellular antigen binding domain of the present CARs comprise a single-chain Fv (sFv or scFv) .
  • the extracellular antigen binding domain of the present CARs comprise a single domain antibody (e.g., a VHH domain) .
  • the extracellular antigen binding domain comprises humanized antibodies or fragment thereof.
  • the extracellular antigen binding domain comprises multiple binding domains (e.g., tandem CAR) .
  • the extracellular antigen binding domain comprises multispecific antibodies or fragments thereof.
  • the extracellular antigen binding domain comprises multivalent antibodies or fragments thereof. In case there are multiple binding domains in the extracellular antigen binding domain of the present CARs.
  • the various domains may be fused to each other via peptide linkers. In some embodiments, the domains are directly fused to each other without any peptide linkers.
  • the peptide linkers may be the same or different. Each peptide linker may have the same or different length and/or sequence depending on the structural and/or functional features of the various domains.
  • the CAR-T cell provided herein comprises multiple CARs, e.g., 2 or more different CARs. In some embodiments, the CAR-T cell provided herein is a split CAR-T cell.
  • the extracellular antigen binding domain provided in the present CARs recognizes an antigen that acts as a cell surface marker on target cells associated with a special disease state.
  • the antigen is a tumor antigen. Tumors express a number of proteins that can serve as a target antigen for an immune response, particularly T cell mediated immune responses.
  • the antigens targeted by the CAR may be antigens on a single diseased cell or antigens that are expressed on different cells that each contribute to the disease.
  • the antigens targeted by the CAR may be directly or indirectly involved in the diseases.
  • Tumor antigens are proteins that are produced by tumor cells that can elicit an immune response, particularly T-cell mediated immune responses.
  • Exemplary tumor antigens include, but not limited to, a glioma-associated antigen, carcinoembryonic antigen (CEA) , ⁇ - human chorionic gonadotropin, alphafetoprotein (AFP) , lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS) , intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA) , PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2/neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1) , MAGE, ELF2M, neutrophil elastase
  • the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor.
  • Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase and gp100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer.
  • Other target molecules belong to the group of transformation-related molecules such as the oncogene HER2/Neu/ErbB-2.
  • Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA) .
  • CEA carcinoembryonic antigen
  • the tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA) .
  • TSA tumor-specific antigen
  • TAA tumor-associated antigen
  • a TSA is unique to tumor cells and does not occur on other cells in the body.
  • a TAA associated antigen is not unique to a tumor cell, and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen.
  • the expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen.
  • TAAs may be antigens that are expressed on normal cells during fetal development, when the immune system is immature, and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells, but which are expressed at much higher levels on tumor cells.
  • TSA or TAA antigens include: differentiation antigens such as MART-1/MelanA (MART-I) , gp 100 (Pmel 17) , tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7.
  • differentiation antigens such as MART-1/MelanA (MART-I) ,
  • Additional non-limiting exemplary targets of the CARs provided herein include GPC2, CD276, Delta-like protein ligand 3 (DLL3) , NY-ESO-1, melanoma associated antigen 4; survivin protein, synovial sarcoma X breakpoint protein 2, CD3, epidermal growth factor receptor (EGFR) , erbb2 tyrosine kinase receptor, HER2, CEA, CD66, CD66e, ROR1, ntrkr1 tyrosine kinase receptor, GPC3, mesothelin, glutamate carboxypeptidase II, PMSA, PD-L1, folate receptor alpha, PSCA, Mucin 1, HLA antigen (such as HLA class I antigen A-2 alpha, HLA class I antigen A-11 alpha, and HLA class II antigen) , c-Met, hepatocyte growth factor receptor, K-Ras GTPase (KRAS) , IL-15 receptor, Kit
  • At least one target antigen of the present CARs is AFP. In some specific embodiments, at least one target antigen of the present CARs is CD19. In other specific embodiments, at least one target antigen of the present CARs is CD20. In yet other specific embodiments, at least one target antigen of the present CARs is CD22. In yet other specific embodiments, at least one target antigen of the present CARs is BCMA. In yet other specific embodiments, at least one target antigen of the present CARs is VEGFR2. In yet other specific embodiments, at least one target antigen of the present CARs is FAP. In yet other specific embodiments, at least one target antigen of the present CARs is EpCam.
  • At least one target antigen of the present CARs is GPC3. In yet other specific embodiments, at least one target antigen of the present CARs is CD133. In yet other specific embodiments, at least one target antigen of the present CARs is IL13Ra. In yet other specific embodiments, at least one target antigen of the present CARs is EGFRIII. In yet other specific embodiments, at least one target antigen of the present CARs is EphA2. In yet other specific embodiments, at least one target antigen of the present CARs is Muc1. In yet other specific embodiments, at least one target antigen of the present CARs is CD70. In yet other specific embodiments, at least one target antigen of the present CARs is CD123.
  • At least one target antigen of the present CARs is ROR1. In yet other specific embodiments, at least one target antigen of the present CARs is PSMA. In yet other specific embodiments, at least one target antigen of the present CARs is CD5. In yet other specific embodiments, at least one target antigen of the present CARs is GD2. In yet other specific embodiments, at least one target antigen of the present CARs is GAP. In yet other specific embodiments, at least one target antigen of the present CARs is CD33. In yet other specific embodiments, at least one target antigen of the present CARs is CEA. In yet other specific embodiments, at least one target antigen of the present CARs is PSCA. In yet other specific embodiments, at least one target antigen of the present CARs is Her2. In yet other specific embodiments, at least one target antigen of the present CARs is Mesothelin.
  • the CARs provided herein comprise a hinge domain that is located between the extracellular antigen binding domain and the transmembrane domain.
  • the hinge domain is a hinge domain of a naturally occurring protein. Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the chimeric receptors described herein.
  • the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor.
  • the hinge domain is derived from CD8 ⁇ .
  • the hinge domain is a portion of the hinge domain of CD8 ⁇ , e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8 ⁇ .
  • the CARs of the present disclosure comprise a transmembrane domain that can be directly or indirectly fused to the extracellular antigen binding domain.
  • the transmembrane domain may be derived either from a natural or from a synthetic source.
  • Transmembrane domains compatible for use in the CARs described herein may be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non-naturally occurring protein segment, e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane.
  • the transmembrane domains are derived from membrane proteins of Type I, Type II or Type III.
  • the transmembrane domain of the CAR described herein is derived from a Type I single-pass membrane protein.
  • transmembrane domains from multi-pass membrane proteins may also be compatible for use in the CARs described herein.
  • Transmembrane domains for use in the CARs described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment.
  • the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet.
  • the protein segment is at least approximately 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids.
  • the transmembrane domain provided herein may comprise a transmembrane region and a cytoplasmic region located at the C-terminal side of the transmembrane domain.
  • the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues.
  • the transmembrane domain of the CAR comprises a transmembrane domain chosen from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDl la, CD18) , ICOS (CD278) , 4-1BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRFl) , CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl
  • the intracellular signaling domain in the CARs provided herein is responsible for activation of at least one of the normal effector functions of the immune effector cell expressing the CARs.
  • the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.
  • the CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell.
  • the primary intracellular signaling domain contains a signaling motif known as immunoreceptor tyrosine-based activation motif, or ITAM.
  • ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3z, FcR gamma (FCER1G) , FcR beta (Fc Epsilon Rib) , CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
  • the CAR comprises at least one co-stimulatory signaling domain.
  • the co-stimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a co-stimulatory protein, which transduces a signal and modulates responses mediated by immune cells.
  • the intracellular signaling domain comprises a single co-stimulatory signaling domain.
  • the intracellular signaling domain comprises two or more (such as about any of 2, 3, 4, or more) co-stimulatory signaling domains.
  • the intracellular signaling domain comprises two or more of the same co-stimulatory signaling domains.
  • the intracellular signaling domain comprises two or more co-stimulatory signaling domains from different co-stimulatory proteins, such as any two or more co-stimulatory proteins described herein.
  • the intracellular signaling domain comprises a primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3z) and one or more co-stimulatory signaling domains.
  • the one or more co-stimulatory signaling domains and the primary intracellular signaling domain are fused to each other via optional peptide linkers.
  • the primary intracellular signaling domain, and the one or more co-stimulatory signaling domains may be arranged in any suitable order.
  • the one or more co-stimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3z) .
  • Multiple co-stimulatory signaling domains may provide additive or synergistic stimulatory effects.
  • co-stimulatory signaling domain of any co-stimulatory molecule may be compatible for use in the CARs described herein.
  • co-stimulatory signaling domains for use in the CARs can be the cytoplasmic signaling domain of co-stimulatory proteins, including, without limitation, members of the B7/CD28 family (e.g., B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, and PDCD6) ; members of the TNF superfamily (e.g., 4-1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF
  • the one or more co-stimulatory signaling domains are selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands that specially bind to CD83.
  • LFA-1 lymphocyte function-associated antigen-1
  • the CAR provided herein comprises amino acid sequences with certain percent identity relative to any one of the CARs exemplified in the Section 6 below.
  • a CAR comprising or consisting of an extracellular domain having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of the CARs exemplified in the Section 6 below.
  • the vaccine specific TCR or domain derived therefrom are as described above in Section 5.2.2.
  • the domain derived from a virus vaccine specific TCR comprises a ⁇ -chain of the virus specific TCR. In other embodiments, the domain derived from a virus vaccine specific TCR comprises a ⁇ -chain of the virus vaccine specific TCR. In some embodiments, the domain derived from a virus vaccine specific TCR comprises a ⁇ -chain and a ⁇ -chain of the virus vaccine specific TCR. In some embodiments, the ⁇ -chain of the virus specific TCR and the ⁇ -chain of the virus vaccine specific TCR can be assembled in vivo with other components of a TCR to generate and present an intact TCR complex on the T cell surface.
  • Exemplary vaccines include (but not limited to) yellow fever vaccine, influenza vaccine, hepatitis A vaccine, human papillomavirus vaccine, smallpox vaccine, rotavirus vaccine, adenovirus vaccine, poliovirus vaccine, chickenpox vaccine, typhoid vaccine, and measles, mumps, rubella (MMR) vaccine.
  • the vaccine is yellow fever vaccine.
  • the vaccine is influenza vaccine.
  • the vaccine is hepatitis A vaccine.
  • the vaccine is human papillomavirus vaccine.
  • the vaccine is smallpox vaccine.
  • the vaccine is rotavirus vaccine.
  • the vaccine is adenovirus vaccine. In some embodiments, the vaccine is poliovirus vaccine. In some embodiments, the vaccine is chickenpox vaccine. In some embodiments, the vaccine is typhoid vaccine. In some embodiments, the vaccine is MMR vaccine.
  • the virus vaccine specific TCR is a yellow fever vaccine specific TCR capable of binding to a yellow fever vaccine antigen.
  • the yellow fever vaccine antigen is a HLA-A02 restricted yellow fever virus antigen.
  • the yellow fever vaccine antigen is a HLA-A24 restricted yellow fever virus antigen.
  • the yellow fever vaccine antigen is a HLA-A11 restricted yellow fever virus antigen.
  • the domain derived from a virus specific TCR is capable of binding a yellow fever vaccine peptide comprising an amino acid sequence of LLWNGPMAV (SEQ ID NO: 70) .
  • the domain derived from a virus vaccine specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of any one of SEQ ID NOs: 22-35.
  • ⁇ -chain of the virus specific TCR is one of those listed in Table 4.
  • the domain derived from a virus vaccine specific TCR comprises a ⁇ -chain of the virus specific TCR comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence of any one of SEQ ID NOs: 36-50.
  • ⁇ -chain of the virus specific TCR is one of those listed in Table 5.
  • the present disclosure further provides pharmaceutical compositions comprising an engineered T cell of the present disclosure.
  • a pharmaceutical composition comprises a therapeutically effective amount of the engineered T cell of the present disclosure and a pharmaceutically acceptable excipient.
  • excipient can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) , carrier or vehicle.
  • adjuvant e.g., Freunds’ adjuvant (complete or incomplete)
  • Pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions will contain a prophylactically or therapeutically effective amount of the active ingredient provided herein, such as in purified form, together with a suitable amount of excipient so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the choice of excipient is determined in part by the particular cell, and/or by the method of administration. Accordingly, there are a variety of suitable formulations.
  • acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metal complexes (e.g. Zn-protein complexes) ; chelating agents such as EDTA and/or non-ionic surfactants.
  • Buffers may be used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent.
  • Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and salts thereof.
  • buffers may comprise histidine and trimethylamine salts such as Tris.
  • Preservatives may be added to retard microbial growth.
  • Suitable preservatives for use with the present disclosure include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide) , benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.
  • octadecyldimethylbenzyl ammonium chloride hexamethonium chloride
  • benzalkonium halides e.g., chloride, bromide, iodide
  • benzethonium chloride thimerosal, phenol, butyl or
  • Tonicity agents can be present to adjust or maintain the tonicity of liquid in a composition.
  • stabilizers When used with large, charged biomolecules such as proteins and antibodies, they are often termed “stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter and intra-molecular interactions.
  • exemplary tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
  • excipients include: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) agents preventing denaturation or adherence to the container wall.
  • excipients include: polyhydric sugar alcohols (enumerated above) ; amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol) , polyethylene glycol; sulfur
  • Non-ionic surfactants or detergents may be present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody.
  • Suitable non-ionic surfactants include, e.g., polysorbates (20, 40, 60, 65, 80, etc. ) , polyoxamers (184, 188, etc. ) , polyols, polyoxyethylene sorbitan monoethers ( etc.
  • lauromacrogol 400 lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl celluose and carboxymethyl cellulose.
  • Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents include benzalkonium chloride or benzethonium chloride.
  • the route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means.
  • a pharmaceutical composition can be provided as a controlled release or sustained release system.
  • a pump may be used to achieve controlled or sustained release (see, e.g., Sefton, Crit. Ref. Biomed. Eng. 14: 201-40 (1987) ; Buchwald et al., Surgery 88: 507-16 (1980) ; and Saudek et al., N. Engl. J. Med. 321: 569-74 (1989) ) .
  • polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., a fusion protein as described herein) or a composition provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974) ; Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984) ; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23: 61-126 (1983) ; Levy et al., Science 228: 190-92 (1985) ; During et al., Ann. Neurol.
  • a prophylactic or therapeutic agent e.g., a fusion protein as described herein
  • a composition provided herein see, e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974) ; Controlled Drug Bioavailability, Drug Product Design and Performance (
  • polymers used in sustained release formulations include, but are not limited to, poly (2-hydroxy ethyl methacrylate) , poly (methyl methacrylate) , poly (acrylic acid) , poly (ethylene-co-vinyl acetate) , poly (methacrylic acid) , polyglycolides (PLG) , polyanhydrides, poly (N-vinyl pyrrolidone) , poly (vinyl alcohol) , polyacrylamide, poly (ethylene glycol) , polylactides (PLA) , poly (lactide-co-glycolides) (PLGA) , and polyorthoesters.
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • a controlled or sustained release system can be placed in proximity of a particular target tissue, for example, the nasal passages or lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984) ) . Controlled release systems are discussed, for example, by Langer, Science 249: 1527-33 (1990) . Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more agents as described herein (see, e.g., U.S. Pat.
  • compositions described herein may also contain more than one active compound or agent as necessary for the particular indication being treated.
  • the composition may comprise a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressive agent, or growth inhibitory agent.
  • cytotoxic agent chemotherapeutic agent
  • cytokine cytokine
  • immunosuppressive agent or growth inhibitory agent.
  • growth inhibitory agent Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • compositions and delivery systems are known and can be used with the therapeutic agents provided herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the single domain antibody or therapeutic molecule provided herein, construction of a nucleic acid as part of a retroviral or other vector, etc.
  • the pharmaceutical composition provided herein contains the binding molecules and/or cells in amounts effective to treat or prevent the disease or disorder, such as a therapeutically effective or prophylactically effective amount.
  • Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined.
  • methods for using and uses of the engineered T cells provided herein.
  • Such methods and uses include therapeutic methods and uses, for example, involving administration of the cells, or compositions containing the same, to a subject having a disease or disorder.
  • the cell is administered in an effective amount to effect treatment of the disease or disorder.
  • Uses include uses of the cells in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods.
  • the methods are carried out by administering the cells, or compositions comprising the same, to the subject having or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or disorder in the subject.
  • the treatment provided herein cause complete or partial amelioration or reduction of a disease or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith.
  • Desirable effects of treatment include, but are not limited to, 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.
  • the terms include, but do not imply, complete curing of a disease or complete elimination of any symptom or effect (s) on all symptoms or outcomes.
  • the treatment provided herein delay development of a disease or disorder, e.g., defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer) .
  • This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated.
  • a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease or disorder.
  • a late stage cancer such as development of metastasis, may be delayed.
  • the method or the use provided herein prevents a disease or disorder.
  • the present CAR-T cell therapies are used for treating solid tumor cancer. In other embodiments, the present CAR-T cell therapies are used for treating blood cancer. In other embodiments, the disease or disorder is an autoimmune and inflammatory disease.
  • the disease or disorder is a disease of abnormal cell growth and/or dysregulated apoptosis.
  • diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, bone cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and/or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra,
  • the disease or disorder is selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small-cell lung cancer, prostate cancer, small-cell lung cancer and spleen cancer.
  • the disease or disorder is a hematological cancer, such as leukemia, lymphoma, or myeloma.
  • the cancer is selected from a group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL) , cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL) , mantle cell lymphoma (MCL) , follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL) , diffuse poorly differentiated lymphocytic lymphoma (PDL) , centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL) , peripheral T-cell lymphomas (PTCL) , cutaneous T-Cell lymphoma, mantle zone lymphoma
  • NHL non-Hod
  • the disease or disorder is myelodysplastic syndromes (MDS) .
  • the disease or disorder is acute myeloid leukemia (AML) .
  • the disease or disorder is chronic lymphocytic leukemia (CLL) .
  • the disease or disorder is multiple myeloma (MM) .
  • the disease or disorder is a solid tumor cancer.
  • the solid tumor cancer is selected from a group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, a liver cancer and a lung cancer.
  • the disease or disorder is an immune or autoimmune disorder.
  • disorders include autoimmune bullous disease, abetalipoprotemia, acquired immunodeficiency-related diseases, acute immune disease associated with organ transplantation, acquired acrocyanosis, acute and chronic parasitic or infectious processes, acute pancreatitis, acute renal failure, acute rheumatic fever, acute transverse myelitis, adenocarcinomas, aerial ectopic beats, adult (acute) respiratory distress syndrome, AIDS dementia complex, alcoholic cirrhosis, alcohol-induced liver injury, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allergy and asthma, allograft rejection, alpha-l-antitrypsin deficiency, Alzheimer's disease, amyotrophic lateral sclerosis, anemia, angina pectoris, ankylosing spondylitis-associated lung disease, anterior horn cell degeneration, antibody mediated cytotoxicity, antiphospholipid syndrome,
  • the disease or disorder is an inflammatory disease.
  • Inflammation plays a fundamental role in host defenses and the progression of immune-mediated diseases.
  • the inflammatory response is initiated in response to injury (e.g., trauma, ischemia, and foreign particles) and infection (e.g., bacterial or viral infection) by a complex cascade of events, including chemical mediators (e.g., cytokines and prostaglandins) and inflammatory cells (e.g., leukocytes) .
  • the inflammatory response is characterized by increased blood flow, increased capillary permeability, and the influx of phagocytic cells. These events result in swelling, redness, warmth (altered heat patterns) , and pus formation at the site of injury or infection.
  • Cytokines and prostaglandins control the inflammatory response, and are released in an ordered and self-limiting cascade into the blood or affected tissues. This release of cytokines and prostaglandins increases the blood flow to the area of injury or infection, and may result in redness and warmth. Some of these chemicals cause a leak of fluid into the tissues, resulting in swelling. This protective process may stimulate nerves and cause pain. These changes, when occurring for a limited period in the relevant area, work to the benefit of the body.
  • a delicate well-balanced interplay between the humoral and cellular immune elements in the inflammatory response enables the elimination of harmful agents and the initiation of the repair of damaged tissue.
  • the inflammatory response may result in considerable damage to normal tissue and may be more harmful than the original insult that initiated the reaction.
  • clinical intervention is needed to prevent tissue damage and organ dysfunction.
  • Diseases such as psoriasis, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, Crohn’s disease, asthma, allergies or inflammatory bowel disease, are characterized by chronic inflammation.
  • Inflammatory diseases such as arthritis, related arthritic conditions (e.g., osteoarthritis, rheumatoid arthritis, and psoriatic arthritis) , inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis) , sepsis, psoriasis, atopic dermatitis, contact dermatitis, and chronic obstructive pulmonary disease, chronic inflammatory pulmonary diseases are also prevalent and problematic ailments.
  • inflammatory bowel disease e.g., Crohn's disease and ulcerative colitis
  • the methods include adoptive cell therapy, whereby genetically engineered cells are administered to a subject.
  • Such administration can promote activation of the cells (e.g., T cell activation) , such that the cells of the disease or disorder are targeted for destruction.
  • the methods include administration of the cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having the disease or disorder.
  • the cells, populations, and compositions are administered to a subject having the particular disease or disorder to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy.
  • the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or disorder.
  • the methods thereby treat, e.g., ameliorate one or more symptom of the disease or disorder.
  • the cell therapy (e.g., adoptive T cell therapy) is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject.
  • the cells are derived from a subject in need of a treatment and the cells, following isolation and processing are administered to the same subject.
  • the cell therapy (e.g., adoptive T cell therapy) is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject.
  • the cells then are administered to a different subject, e.g., a second subject, of the same species.
  • a different subject e.g., a second subject
  • the first and second subjects are genetically identical.
  • the first and second subjects are genetically similar.
  • the second subject expresses the same HLA class or supertype as the first subject.
  • the subject, to whom the cells, cell populations, or compositions are administered is a primate, such as a human.
  • the subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
  • the subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes.
  • composition provided herein can be administered by any suitable means, for example, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the amount of a prophylactic or therapeutic agent provided herein that will be effective in the prevention and/or treatment of a disease or condition can be determined by standard clinical techniques. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the appropriate dosage of the binding molecule or cell may depend on the type of disease or disorder to be treated, the type of binding molecule, the severity and course of the disease or disorder, whether the therapeutic agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician.
  • the compositions, molecules and cells are in some embodiments suitably administered to the patient at one time or over a series of treatments. Multiple doses may be administered intermittently. An initial higher loading dose, followed by one or more lower doses may be administered.
  • a subject may be administered the range of about one million to about 100 billion cells and/or that amount of cells per kilogram of body weight.
  • the pharmaceutical composition comprises any one of the engineered immune cells described herein, the pharmaceutical composition is administered at a dosage of at least about any of 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 cells/kg of body weight of the individual. Dosages may vary depending on attributes particular to the disease or disorder and/or patient and/or other treatments.
  • the pharmaceutical composition is administered for a single time. In some embodiments, the pharmaceutical composition is administered for multiple times (such as any of 2, 3, 4, 5, 6, or more times) . In some embodiments, the pharmaceutical composition is administered once or multiple times during a dosing cycle.
  • a dosing cycle can be, e.g., 1, 2, 3, 4, 5 or more week (s) , or 1, 2, 3, 4, 5, or more month (s) .
  • the optimal dosage and treatment regime for a particular patient can be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • a vaccine is administered to the subject to activate Vax-CAR-T cells in vivo.
  • the vaccine can be administered immediately after the infusion of the Vax-CAR-T cells, and/or after 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, 40, 50, 60, 70, 80, 90 or even more days after the infusion of the Vax-CAR-T cells.
  • the frequency and dosages of the vaccination may vary depending on attributes particular to the disease or disorder and/or patient and/or other treatments.
  • yellow fever vaccine is administered (one dose, 4.74 log10 plaque forming units (PFU) per 0.5 mL dose) at 1, 30 and 60 days post Vax-CAR T cell infusion.
  • compositions provided herein are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as another antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
  • another therapeutic intervention such as another antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
  • compositions provided herein are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order.
  • the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa.
  • the compositions provided herein are administered prior to the one or more additional therapeutic agents. In some embodiments, the compositions provided herein are administered after to the one or more additional therapeutic agents.
  • the biological activity of the engineered cell populations is measured by any of a number of known methods.
  • Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry.
  • the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32 (7) : 689-702 (2009) , and Herman et al. J.
  • the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as CD107a, IFN ⁇ , IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
  • kits, unit dosages, and articles of manufacture comprising any of the engineered immune effector cells described herein.
  • a kit is provided which contains any one of the pharmaceutical compositions described herein and preferably provides instructions for its use.
  • kits of the present application are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags) , and the like. Kits may optionally provide additional components such as buffers and interpretative information.
  • the present application thus also provides articles of manufacture, which include vials (such as sealed vials) , bottles, jars, flexible packaging, and the like.
  • the article of manufacture can comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating a disease or disorder (such as cancer) described herein, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) .
  • the label or package insert indicates that the composition is used for treating the particular condition in an individual.
  • the label or package insert will further comprise instructions for administering the composition to the individual.
  • the label may indicate directions for reconstitution and/or use.
  • the container holding the pharmaceutical composition may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the reconstituted formulation.
  • Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • kits or article of manufacture may include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.
  • the disclosure is generally disclosed herein using affirmative language to describe the numerous embodiments.
  • the disclosure also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis.
  • the disclosure is generally not expressed herein in terms of what the disclosure does not include, aspects that are not expressly included in the disclosure are nevertheless disclosed herein.
  • Exemplary CAR and Vax-CAR molecules were constructed as illustrated in FIG. 1A. More specifically, the ⁇ -and ⁇ -chains of yellow fever virus-specific TCR, and CAR are connected with 2A peptide linkers (e.g., a P2A linker) . GFP gene was incorporated as a selection marker. The nucleic acids encoding the CAR and Vax-CAR polypeptides were cloned into a lentiviral vector under the control of an EF1 ⁇ promoter.
  • a lentiviral vector was modified using pLVX-Puro (Clontech#632164) by replacing the original promoter with murine stem cell virus (MSCV) promoter and the puromycin resistance gene was depleted with a EcoRI and BamHI.
  • CARs targeting AFP were used as exemplary CARs in the following examples, and exemplary AFP CAR sequences (SEQ ID NOs: 1-5) are shown in Table 2 below.
  • Exemplary domains comprising ⁇ -and ⁇ -chains of yellow fever virus-specific TCR are shown in Table 3 below.
  • the linker sequence used between ⁇ -and ⁇ -chains of the TCR of these exemplary constructs is SEQ ID NO: 55 as underlined in the sequences in Table 3.
  • Exemplary ⁇ -chains of yellow fever virus-specific TCR are shown in Table 4.
  • Exemplary ⁇ -chains of yellow fever virus-specific TCR are shown in Table 5.
  • Exemplary constructs comprising domains derived from yellow fever specific TCR, AFP targeting CAR, and GFP are shown in Table 6.
  • Exemplary linker sequence between ⁇ -and ⁇ -chains of the TCR SEQ ID NO: 55
  • exemplary linker sequence between the CAR and the domain derived from the TCR SEQ ID NO: 56
  • GFP sequence SEQ ID NO: 57
  • nucleic acid sequences encoding exemplary domains derived from yellow fever virus-specific TCRs are shown in Table 7 below.
  • G was pre-mixed with CAR or Vax-CAR construct at a pre-optimized ratio with polyetherimide (PEI) , then mixed properly and incubated at room temperature for 5 minutes. The transfection mix was then added dropwise to the 293-T cells and mixed gently. Afterwards, cells were incubated overnight in a 37°C and 5%CO 2 cell incubator. The supernatant was collected after centrifugation at 4°C, 500 g for 10 min.
  • PEI polyetherimide
  • the lentiviral supernatant was then filtered through a 0.45 ⁇ m PES filter, and concentrated with ultracentrifugation. After centrifugation, the supernatant was carefully discarded and the virus pellet was rinsed cautiously with pre-chilled DPBS. The concentration of virus was then measured. Virus was aliquoted properly, then stored at -80°C immediately. The virus titer was determined by functional transduction on T cell line.
  • T cells Primary T cells were purified from human PBMCs using Miltenyi Pan T cell isolation kit (Cat#130-096-535) , following the product manual. The T cells were then activated for 48 hours with human T cell Activation/Expansion kit (Milteny#130-091-441) . An optimal activation of T cells was accomplished by using one loaded Anti-Biotin MACSiBead Particle per two cells (bead-to-cell ratio 1: 2) .
  • the pre-activated T cells were harvested and re-suspended in RPMI 1640 medium containing 300 IU/mL of IL-2, and transduced with lentivirus at MOI 5 in the presence of 8 ⁇ g/ml polybrene at 1000 g, 32 °C for 1h.
  • the transduced cells were incubated overnight in a 37°C and 5%CO2 cell incubator before replaced with fresh media. The cell density was monitored every other day, and fresh media were added if necessary.
  • CAR-T cells and Vax-CAR-T cells were harvested and stained with PE-conjugated MHC tetramer. Flow cytometry analysis was then used to confirm the expression of CAR and GFP tag (FIG.
  • DCs dendritic cells
  • rhIL-4 100 ng/ml of rhGM-CSF, 20 ng/ml of rhTNF- ⁇ , and 200 ng/ml of ionomycin for 3-5 days.
  • mDCs Mature DCs
  • monocytes unstimulated THP-1 cells
  • APC-conjugated anti-CD209 antibody and APC-Cy7-conjugated anti-CD86 antibody
  • flow cytometry analysis Mature DCs derived from monocytes were confirmed by the expression of DC markers CD209 and CD86 (FIG. 2) .
  • Untransduced (UNT) , CAR-T and Vax-CAR-T cells were left unstimulated (T) , or stimulated with DCs (T+DC) , or yellow fever virus antigen peptide-pulsed DCs (T+DC+pYF) for 24 hours, and then stained with APC-conjugated anti-CD69 antibody followed by flow cytometry analysis.
  • the sequence of HLA-A02 restricted yellow fever virus antigen peptide is LLWNGPMAV (SEQ ID NO: 70) (pYF) .
  • T cell activation marker CD69 was significantly induced in Vax-CAR-T cells, but not in CAR-T or UNT cells; while the expression in the control groups and unpulsed DC groups was minimal (FIG. 3) .
  • UNT, CAR-T and Vax-CAR-T cells (1E+6/well) were subjected to three rounds of stimulation of pYF peptide-pulsed DCs, and the cell count, T cell phenotyping and killing efficacy was investigated as shown in FIG. 4A.
  • the proliferation of Vax-CAR-T cells, but not CAR-T or UNT cells were enhanced by pYF peptide-pulsed DCs, while the effect of unpulsed DCs was minimal (see FIG. 4B) .
  • T cells were harvested and re-suspended with AIMV medium with 5%FBS (Invitrogen) .
  • HepG2 target cells were pre-stained with CMFDA and the residual dye was washed out with DPBS.
  • T cells were co-cultured with HepG2 cells at 1.25: 1 of effector to target ratios (E: T) at 37°C for 24 hours in 96 well plate in the presence of 8 ⁇ g/ml of propidium iodide.
  • E effector to target ratios
  • the dead target cells showed both green and red fluorescence, while the live ones only showed green.
  • the data was analyzed and exported by IncuCyte S3 software, plotted by GraphPad Prism 6 (FIG. 4C) .
  • the cytotoxicity of Vax-CAR-T cells was significantly enhanced by pYF peptide-pulsed DCs (FIG. 4C) .
  • T cells were harvested stained with Brilliant Violet 605-conjugated anti-CD62L antibody and APC-Cy7-conjugated anti-CD45RO antibody, followed by flow cytometry analysis.
  • the CD62L and CD45RO double positive population was referred as central memory T cells.
  • pYF peptide-pulsed DCs significantly induced accumulation of central memory T cell population (FIG. 4D) .
  • yellow fever vaccine activates Vax-CAR-T cell, induces T cell proliferation and memory T cell accumulation, and enhances T cell cytotoxicity effect against tumor cells.

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Abstract

L'invention concerne un polypeptide comprenant un récepteur antigénique chimérique (CAR) et un domaine dérivé d'un récepteur de lymphocyte T (TCR) spécifique au virus, le CAR et le domaine étant fusionnés par un lieur peptidique.
PCT/CN2021/125142 2020-10-21 2021-10-21 Polypeptides comprenant un tcr spécifique d'un vaccin et un récepteur antigénique chimérique et leurs utilisations Ceased WO2022083667A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013126726A1 (fr) * 2012-02-22 2013-08-29 The Trustees Of The University Of Pennsylvania Lymphocytes t doubles transgéniques comportant un car et un tcr, et leurs procédés d'utilisation
CN106632659A (zh) * 2016-08-05 2017-05-10 武汉赛云博生物科技有限公司 一种eb病毒特异性tcr及其重组慢病毒载体与应用
CN108463548A (zh) * 2015-10-30 2018-08-28 加利福尼亚大学董事会 由干细胞产生t细胞的方法及使用所述t细胞的免疫治疗方法
CN108472314A (zh) * 2015-07-31 2018-08-31 明尼苏达大学董事会 修饰的细胞和治疗方法
CN108603170A (zh) * 2015-06-12 2018-09-28 莱蒂恩技术公司 用工程改造的t细胞治疗癌症的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013126726A1 (fr) * 2012-02-22 2013-08-29 The Trustees Of The University Of Pennsylvania Lymphocytes t doubles transgéniques comportant un car et un tcr, et leurs procédés d'utilisation
CN108603170A (zh) * 2015-06-12 2018-09-28 莱蒂恩技术公司 用工程改造的t细胞治疗癌症的方法
CN108472314A (zh) * 2015-07-31 2018-08-31 明尼苏达大学董事会 修饰的细胞和治疗方法
CN108463548A (zh) * 2015-10-30 2018-08-28 加利福尼亚大学董事会 由干细胞产生t细胞的方法及使用所述t细胞的免疫治疗方法
CN106632659A (zh) * 2016-08-05 2017-05-10 武汉赛云博生物科技有限公司 一种eb病毒特异性tcr及其重组慢病毒载体与应用

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