WO2018236986A1 - Récepteurs de lymphocytes t modifiés et méthodes d'utilisation correspondantes - Google Patents

Récepteurs de lymphocytes t modifiés et méthodes d'utilisation correspondantes Download PDF

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Publication number
WO2018236986A1
WO2018236986A1 PCT/US2018/038478 US2018038478W WO2018236986A1 WO 2018236986 A1 WO2018236986 A1 WO 2018236986A1 US 2018038478 W US2018038478 W US 2018038478W WO 2018236986 A1 WO2018236986 A1 WO 2018236986A1
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Prior art keywords
cell
polynucleotide
antigen
cells
vector
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PCT/US2018/038478
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English (en)
Inventor
David Sulzer
Patrick Ho
Alessandro Sette
Bjoern Peters
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The Trustees Of Columbia University In The City Of New York
La Jolla Institute For Allergy & Immunology
The Research Foundation For Mental Hygiene, Inc.
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Publication of WO2018236986A1 publication Critical patent/WO2018236986A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46432—Nervous system antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • Hits disclosure relates to novel polynucleotides, cells, and compositions, and methods of their use.
  • a polynucleotide encoding an engineered T-cell receptor comprising, consisting of. or alternatively consisting essentially of: (a) an extracellular antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the extracellular antigen binding domain binds an antigen that binds an MHC molecule, wherein the antigen binds to the MHC molecule with high affinity, and/or wherein the antigen induces a specific cytokine response, optionally an 1L-17. IFNy, and/or IL-5 response.
  • the antigen is bound to the MHC molecule.
  • the engineered T-cell receptor is a modified T-cell receptor. In other aspects, the engineered T-cell receptor is a chimeric antigen receptor (CAR). In certain aspects, the extracellular antigen binding domain binds both the antigen and the MHC molecule. Also provided herein are the expression product(s) of the polynucleotide encoding an engineered T-cell receptor.
  • the antigen comprises all or part of an epitope derived from an antigen of the group of: a microbial antigen, a viral antigen, a bacterial antigen, a fungal antigen, a protozoan antigen, an antigen involved in autoimmune disease, an auloanligcn, an allergy antigen, a graft rejection antigen, a neurodegenerative disease or disorder, a tumor antigen, or a cancer antigen.
  • the antigen comprises all or part of a toxin.
  • the antigen is an antigen involved in the etiology of autoimmune disease.
  • the disclosure relates to a vector comprising the polynucleotide according to any of the embodiments disclosed herein, optionally operatively linked to a promoter.
  • the vector further comprises an enhancer, a polynucleotide encoding FoxP3 optionally operatively linked to a promoter, a polynucleotide encoding IL-10 optionally operatively linked to a promoter, a suicide gene optionally operatively linked to a promoter, a ubiquitin binding domain, and/or STUB1 optionally operatively linked to a promoter.
  • the vector is from the group of: a plasmid, a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector. Also provided herein are the expression producl(s) of the vector comprising the polynucleotide encoding an engineered T-cell receptor.
  • the disclosure relates to a cell comprising or expressing the polynucleotide or vector according to any one of the embodiments disclosed herein.
  • the cell comprises two or more distinct polynucleotides or two or more distinct vectors according to the embodiments disclosed herein, and wherein the engineered T-cell receptors encoded by the two or more polynucleotides or two or more vectors bind distinct antigens.
  • the cell is an isolated cell.
  • the cell is a leukocyte, a T-cell, or an NK cell.
  • the cell is a regulatory T-ccll, a regulatory memory T-cell, a central memory T-cell, an effector memory T-cell, a CD4+ T-cell, or a CD8+ T-cell.
  • a cell comprising the expression product(s) of the polynucleotide or vector encoding an engineered T-cell receptor.
  • the engineered T-cell receptor is expressed on the surface of the cell.
  • the disclosure provides a non-human animal comprising one or more of a polynucleotide, and/or vector, and/or cell, and/or a population of cells according to any of the embodiments described herein.
  • the non-human animal comprises two or more polynucleotides, two or more vectors, two or more cells, or two or more populations of cells that encode distinct engineered T-cell receptors that bind distinct antigen targets.
  • composition comprising a carrier and one or more of: a polynucleotide, vector, engineered T cell receptor, cell, modified cell, or population comprising said cells or modified cells according to any of the embodiments described herein.
  • Engineered T- ccll receptors described herein may comprise, consist of, or alternatively consist essentially of: (a) an extracellular antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the extracellular antigen binding domain binds an antigen that binds an MHC molecule, wherein the antigen binds to the MHC molecule with high affinity, and/or wherein the antigen induces a specific cytokine response, optionally an IL-17, IFNy, and/or IL-5 response.
  • the engineered T-cell receptor is a modified T-cell receptor.
  • the engineered T-ccll receptor is a chimeric antigen receptor (CAR).
  • the extracellular antigen binding domain binds both the antigen and the MHC molecule.
  • the polynucleotide or vector encodes the engineered T cell receptor.
  • the cell or modified cell expresses the polynucleotide or vector and/or comprises an engineered T cell receptor on a surface of the cell or modified cell.
  • the disclosure provides a kit comprising a composition according to any of the embodiments described herein and instructions for use.
  • step (i) comprises CRISPR mediated gene editing.
  • the cell is a leukocyte, a T-ccll, or an NK cell.
  • the cell is a regulatory T-cell, a regulatory memory T-cell, a central memory T-cell, an effector memory T-ccll, a CD4+ T-ccll, or a CD8+ T-ccll.
  • the method further comprises inducing a regulatory T-cell phenotype or a memory regulatory T-cell phenotype in the cell or subpopulatiun of modified cells by inducing FoxP3 expression.
  • the method further comprises introducing a polynucleotide encoding IL-10 and/or a suicide gene to the cell or subpopulation of modified cells.
  • the method further comprises contacting the cell or subpopulation of modified cells with activation-induced soluble anti-lFNy antibody and/or anli-IL-5 antibody. Also provided are the cells prepared by these methods, that are optionally isolated.
  • Engineered T-cell receptors described herein may comprise, consist of, or alternatively consist essentially of: (a) an extracellular antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the extracellular antigen binding domain binds an antigen that binds an MHC molecule, wherein the antigen binds to the MHC molecule with high affinity, and/or wherein the antigen induces a specific cytokine response, optionally an IL-17. IFNy, and/or II .-5 response.
  • the engineered T-cell receptor is a modified T-cell receptor.
  • the engineered T-ccll receptor is a chimeric antigen receptor (CAR).
  • the extracellular antigen binding domain binds both the antigen and the MHC molecule.
  • the polynucleotide or vector encodes the engineered T cell receptor.
  • the cell or modified cell expresses the polynucleotide or vector and/or comprises an engineered T cell receptor on a surface of the cell or modified cell.
  • the composition comprises one or more of the polynucleotide, vector, engineered T cell receptor, cell, modified cell, population comprising said cells or modified cells and, optionally, a carrier, such as but not limited to a pharmaceutically acceptable carrier.
  • the response is characterized by suppression of pathogenic T- cells.
  • the response is characterized by decreased expression of one or more proinflammatory cytokines in the cell, tissue, or subject, and optionally wherein the one or more proinflammatory cytokines comprise ⁇ .- ⁇ . TNF- ⁇ . IFN- ⁇ . II.-8. II. -6, II.- 12, II.- 15, II. -16, ⁇ .- ⁇ 7, IL-18, GM-CSF, IL-21. IL-23, IL-27, and/or TGF- ⁇ .
  • the response is characterized by increased expression of one or more anti-inflammatory cytokines in the cell, tissue, or subject, and optionally wherein the one or more anti-inflammatory cytokines comprise TGF-P.
  • IL-IRa 1L-4. IL-6, lL-10, IL-11, IL-13. IL-3S, and/or INF-a.
  • the subject is murine, canine, feline, simian, equine, rat or human.
  • a method of inducing an antiinflammatory response, mediating an immune response, or mediating an inflammatory response in a subject in need thereof comprising administering an effective amount of a polynucleotide, vector, engineered T cell receptor, cell, modified cell, population comprising said cells or modified cells, or composition according to any of the embodiments described herein, linginecrcd 1 -cell receptors described herein may comprise, consist of.
  • the engineered T-ccll receptor is a modified T-ccII receptor.
  • the engineered T-cell receptor is a chimeric antigen receptor (CAR).
  • the extracellular antigen binding domain binds both the antigen and the MHC molecule.
  • the polynucleotide or vector encodes the engineered T cell receptor.
  • the cell or modified cell expresses the polynucleotide or vector and/or comprises an engineered T cell receptor on a surface of the cell or modified cell.
  • the composition comprises one or more of the polynucleotide, vector, engineered T cell receptor, cell, modified cell, population comprising said cells or modified cells and, optionally, a carrier, such as but not limited to a pharmaceutically acceptable carrier.
  • the response is characterized by suppression of pathogenic T-cells.
  • the response is characterized by decreased expression of one or more pro-inflammatory cytokines in the cell, tissue, or subject, and optionally wherein the one or more pro-inflammatory cytokines comprise II.- IB, TNF-o, IFN- ⁇ , IL-8. IL-6, IL-12, IL-15, IL-16. IL-17, IL-18. GM-CSF. IL-21. IL-23, IL-27. and/or TGF- ⁇ .
  • the response is characterized by increased expression of one or more antiinflammatory cytokines in the cell, tissue, or subject, and optionally wherein the one or more antiinflammatory cytokines comprise TGF- ⁇ , II.-I Ra, II.-4. II.-6, 11.
  • the subject is murine, canine, feline, simian, equine, rat or human.
  • Engineered T-cell receptors described herein may comprise, consist of, or alternatively consist essentially of: (a) an extracellular antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the extracellular antigen binding domain binds an antigen that binds an MI IC molecule, wherein the antigen binds to the MI IC molecule with high affinity, and/or wherein the antigen induces a specific cytokine response, optionally an 1L-17, ⁇ , and/or 1L-5 response.
  • the engineered T-cell receptor is a modified T-cell receptor.
  • the engineered T-ccll receptor is a chimeric antigen receptor (CAR).
  • the extracellular antigen binding domain hinds both the antigen and the MHC molecule.
  • the polynucleotide or vector encodes the engineered T cell receptor.
  • the cell or modified cell expresses the polynucleotide or vector and/or comprises an engineered T cell receptor on a surface of the cell or modified cell.
  • the composition comprises one or more of the polynucleotide, vector, engineered T cell receptor, cell, modified cell, population comprising said cells or modified cells and. optionally, a carrier, such as but not limited to a pharmaceutically acceptable carrier.
  • the enhanced activity of the regulatory T-cell or memory regulatory T-cell is characterized by increased expression of IL- 10.
  • the administration is to a cell or tissue.
  • Administration to a cell or tissue may be in vivo, ex vivo, or in vitro.
  • the administration is to a subject.
  • the subject is murine, canine, feline, simian, or human.
  • Another aspect of this disclosure relates to a method of treating a disease or condition involving an inflammatory 1 response or related to inflammation in a subject in need thereof, comprising administering to the subject an clTcclivc amount of a polynucleotide, vector, engineered T-cell receptor, cell, modified cell, population comprising said cells or modified cells, or composition according to any of the embodiments described herein.
  • Engineered T-ccll receptors described herein may comprise, consist of.
  • the engineered T-cell receptor is a modified T-cell receptor. In other aspects, the engineered T-cell receptor is a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the extracellular antigen binding domain binds both the antigen and the MHC molecule.
  • the polynucleotide or vector encodes the engineered T cell receptor.
  • the cell or modified cell expresses the polynucleotide or vector and/or comprises an engineered T cell receptor on a surface of the cell or modified cell.
  • the composition comprises one or more of the polynucleotide, vector, engineered T cell receptor, cell, modified cell, population comprising said cells or modified cells and. optionally, a carrier, such as hut not limited to a pharmaceutically acceptable carrier.
  • the subject is murine, canine, feline, simian, equine, rat or human.
  • a method of treating a neurodegenerative disease or disorder in a subject in need thereof comprising administering to the subject an cITcctivc amount of a polynucleotide, vector, engineered T cell receptor, cell, modified cell, population comprising said cells or modified cells, or composition according to any of the embodiments described herein.
  • Engineered T-cell receptors described herein may comprise, consist of.
  • the engineered T-ccll receptor is a modified T-ccll receptor.
  • the engineered T-cell receptor is a chimeric antigen receptor (CAR).
  • the extracellular antigen binding domain binds both the antigen and the MHC molecule.
  • the polynucleotide or vector encodes the engineered T cell receptor.
  • the cell or modified cell expresses the polynucleotide or vector and/or comprises an engineered T cell receptor on a surface of the cell or modified cell.
  • the composition comprises one or more of the polynucleotide, vector, engineered T cell receptor, cell, modified cell, population comprising said cells or modified cells and. optionally, a carrier, such as but not limited to a pharmaceutically acceptable carrier.
  • the neurodegenerative disease or disorder is a-synucleinopathy. Parkinson's disease.
  • the method further comprises administering to the subject an effective amount of one or more immunosuppressive therapeutic compounds, optionally wherein the compounds are selected from calcineurin inhibitor, chemokine receptor inhibitor, a glucocorticoid, an mTOR inhibitor, an anti-metabolic compound, a phosphodiesterase- 5 inhibitor, an antibody, or a leukocyte function antigen-3/Fc fusion protein.
  • the subject is murine, canine, feline, simian, equine, rat or human.
  • Ihe cell, modified cell, or population comprising said cell or modified cell is autologous to the subject being treated.
  • the cull is allogeneic to the subject.
  • the cell can be from any appropriate species, e.g., mammalian, canine, feline, murine, rat, equine or human.
  • the term "4- IBB costimulatory signaling region" refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%. or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity with the 4- 1 BB costimulatory signaling region sequence as shown herein.
  • the example sequence of the 4- IBB costimulatory signaling region is provided in U.S. Pub. No. US20130266551 ⁇ 1.
  • the sequence of the 4- IBB costimulatory signaling region associated disclosed in the U.S. Pub. No. US20130266551A1 is set forth herein as SEQ ID NO: 1 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.
  • the term "about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.
  • AAV adeno-associaled virus
  • AAV refers to a member of the class of viruses belonging to the genus dependoparvovirus, family Parvoviridae. Multiple serotypes of this virus arc known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 1 1 sequentially numbered, AAV serotypes are known in the art.
  • Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 1 1 serotypes, e.g., AAV2, AAV8, AAV9. or variant serotypes. e.g., AAV-DJ.
  • Nonlimiting exemplary AAV vectors arc disclosed in U.S. Pub. Nos. US20070036760A1, US20120I37379A1, and US20130195801 Al.
  • administer and “administering” are used to mean introducing the therapeutic agent (e.g. polynucleotide, vector, cell, modified cell, population) into a subject.
  • the therapeutic administration of this substance serves to attenuate any symptom, or prevent additional symptoms from arising.
  • administration is for the purposes of preventing or reducing the likelihood of developing an autoimmune disease or disorder, the substance is provided in advance of any visible or detectable symptom.
  • Routes of administration include, but are not limited to, oral (such as a tablet, capsule or suspension), topical, transdermal, intranasal. vaginal, rectal, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal.
  • affinity refers to the strength of the reversible biomolccular interaction between two molecules.
  • affinity refers to the interaction between the MHC molecule (or the antigen binding domain (e.g. peptide binding cleft) of an MHC molecule) and an antigen, antigen fragment, peptide, or epitope.
  • affinity refers to the interaction between the ' ICR's variable domain regions and an antigen or antigen-MHC complex. Affinity is determined by calculating the equilibrium dissociation constant (KD) using a known method (e.g.
  • antigen-MHC affinities reported herein reflect Kn and were calculated according to the method disclosed in Sidney, J. et al. J. Immunol. 185: 4189-4198 (2010). Affinity and KD are inversely related - the smaller the Kn value, the greater the binding affinity.
  • high affinity refers to a KD of less than or equal to 1000 nM. Antigens bound to MHC with intermediate high affinity exhibit a Kn of between 10-100 nM. Antigens bound to MHC with very high affinity exhibit a KD of less than or equal to 10 nM.
  • animal refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • mamal includes both human and non-human mammals.
  • antibody collectively refers to immunoglobulins (or “Ig”) or immunoglobnlin-like molecules including hut not limited to antibodies of the following isolypes: IgM, IgA, IgD, IgE, IgG, and combinations thereof.
  • Immunoglobulin-like molecules include but are not limited to similar molecules produced during an immune response in a vertebrate, for example, in mammals such as humans, rats, goats, rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins (see Feige, M. et al. Proc. Nat. Ac. Sci. 41(22): 8155-60 (2014)).
  • the term “antibody” includes intact immunoglobulins and "antibody fragments” or "antigen binding fragments” that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 10 3 M' 1 greater, at least 10 4 M' 1 greater or at least 10* M " ' greater than a binding constant for other molecules in a biological sample).
  • the term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as. bispecific antibodies). See also. Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, III.); Kuby, J., Immunology, 3 rf Ed., W.H. Freeman & Co., New York, 1997.
  • the general structure of an antibody is comprised of heavy (H) chains and light (I .) chains connected by disulfide bonds.
  • the structure can also comprise glycans attached at conserved amino acid residues.
  • Each heavy and light chain contains a constant region and a variable region (also known as "domains").
  • the constant regions of the heavy chain also contribute to the effector function of the antibody molecule.
  • Antibodies comprising the heavy chains ⁇ , ⁇ , ⁇ 3, ⁇ , ol, ⁇ 2, ⁇ 4.
  • ⁇ , and ⁇ 2 result in the following isotypes: IgM, IgD. Ig03, IgGI, IgAI. lgG2, IgG4, IgF, and lgA2. respectively.
  • An IgY isotype. related to mammalian IgU. is found in reptiles and birds.
  • An IgW isotype. related to mammalian IgD. is found in cartilaginous fish.
  • Class switching is the process by which the constant region of an immunoglobulin heavy chain is replaced with a different immunoglobulin heavy chain through recombination of the heavy chain locus of a B-cell to produce an antibody of a different isotype.
  • Antibodies may exist as monomers (e.g. IgG), dimcrs (e.g. IgA), tetramcrs (e.g. fish IgM), pentamers (e.g. mammalian IgM). and/or in complexes with other molecules.
  • antibodies can be bound to the surface of a cell or secreted by a cell.
  • the variable regions of the immunoglobulin heavy and the light chains specifically bind the antigen.
  • the 'Tramework” region is a portion of the Fab that acts as a scaffold Tor three hypervariable regions called “complementarity-determining regions” (CDRs).
  • CDRs complementarity-determining regions
  • a set of CDRs is known as a paratope.
  • the framework regions ⁇ difTcrcnl light or heavy chains are relatively conserved within a species.
  • the combined framework region of an antibody (comprising regions from both light and heavy chains), largely adopts a ⁇ -shccl conformation and the CDRs form loops which connect, and in some cases form part of. the ⁇ -sheet structure.
  • framework regions act to position the CDRs in correct orientation by inter-chain, non-covalent interactions.
  • the framework region and CDRs for numerous antibodies have been defined and are available in a database maintained online (Rabat et ai. Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991).
  • ITic CDRs of the variable regions of heavy and light chains (Vn and VL) arc responsible for binding to an epitope of an antigen.
  • a limited number of amino acid positions within the CDRs arc directly involved in antigen binding. These positions within the CDRs arc called specificity determining residues (SDRs).
  • the CDRs of a heavy or light chain are numbered sequentially starting from the N-lcrminal end (i.e. CDR1, CDR2, and CDR3). l or example, a Vi.CDR3 is the middle CDR located in the variable domain of the light chain of an antibody.
  • a VH CDR I is the first CDR in the variable domain of a heavy chain of an antibody.
  • An antibody that binds a specific antigen will have specific VH and VL region sequences, and thus specific CDR sequences.
  • Antibodies with different specificities i.e. dilTcrcnt combining sites for different antigens) have different CDRs.
  • an "antigen-binding fragment” refers to the regions of an antibody corresponding to two of the three fragments produced by papain digestion.
  • the Fab fragment comprises the region that binds to an antigen and is composed of one variable region and one constant region from both a heavy chain and a light chain.
  • An F(ab')2 fragment refers to a fragment of an antibody digested by pepsin or the enzyme IdeS (immunoglobulin degrading enzyme from S. pyogenes) comprising two Fab regions connected by disulfide bonds.
  • a single chain variable fragment (“scFv”) refers to a fusion protein comprising at least one Vn and at least one VL region connected by a linker of between 5 to 30 amino acids. Methods and techniques of developing scFv that bind to specific antigens are known in the art (see. e.g. Ahmad, Z. A. et al.. Clinical and Developmental Immunology, 2012: 980250 (2012)).
  • the term "antigen” refers to a compound, composition, or substance that may be specifically bound and/or recognized by the products of specific humoral or cellular immunity and antigen recognition molecules, including but not limited to an antibody molecule, single-chain variable fragment (scFv), cell surface immunoglobulin receptor, B-ccll receptor (DCR), T-cell receptor (TCR), engineered TCR, modified TCR. or CAR.
  • scFv single-chain variable fragment
  • DCR B-ccll receptor
  • TCR T-cell receptor
  • engineered TCR modified TCR.
  • CAR CAR
  • epitopope refers to an antigen or a fragment, region, site, or domain of an antigen that is recognized by an antigen recognition molecule.
  • Antigens can be any type of molecule including but not limited to peptides, proteins, lipids, phospholipids haptens, simple intermediary metabolites, sugars (e.g., monosaccharides or oligosaccharides), hormones, and macromolecules such as complex carbohydrates (e.g., polysaccharides).
  • Common categories of antigens include, but are not limited to microbial antigens such as viral antigens, bacterial antigens, fungal antigens, protozoa, and other parasitic antigens, antigens involved in autoimmune disease (including autoantigens), allergy, and graft rejection, tumor antigens, toxins, and other miscellaneous antigens.
  • antigen binding domain refers to any protein or polypeptide domain that can specifically bind to an antigen target (including target complexes of antigens and MHC molecules).
  • autologous in reference to cells, tissue, and/or grafts refers to cells, tissue, and/or grafts dial arc isolated from and then and administered back into the same subject, patient, recipient, and/or host.
  • Allogeneic refers to non-autologous cells, tissue, and/or grafts.
  • B cell refers to a type of lymphocyte in the humoral immunity of the adaptive immune system. D cells principally function to make antibodies, serve as antigen presenting cells, release cytokines, and develop memory B cells after activation by antigen interaction. B cells are distinguished from other lymphocytes, such as T cells, by the presence of a B-cell receptor on the cell surface. B cells may either be isolated or obtained from a commercially available source. Non-limiting examples of commercially available B cell lines include lines AHH-1 (ATCC® CRL-8146 ,M ).
  • BC-1 (ATCC® CRL-2230TM), BC-2 (A ICC® CRL-2231 '"), BC-3 (ATCCtt CRL-2277TM), CA46 (ATCC® CRL-1648TM), DG-75 [D.G.-75] (ATCC® CRL- 2625 ,M ), DS-I (ATCC® CRL-1 1 102 IM ), EB-3 [BB3] (ATCC® CCL-85 rM ), Z-138 (ATCC #CRL-300I ).
  • DB (ATCC CRL-2289).
  • Toledo (ATCC CRL-2631 ), PfifTer( ATCC CRL-2632), SR (ATCC CRL-2262), JM-I (ATCC CRL-I042 I ).
  • NFS-5 C-l (ATCC CRL-1693); NFS-70 CIO (ATCC CRL-1694), NFS-25 C-3 (ATCC CRL-1695), AND SUP-B15 (ATCC CRL-1929).
  • Further examples include but are not limited to cell lines derived from anaplastic and large cell lymphomas, e.g., DEL, DL-40.
  • FE-PD JB6.
  • ATCC American Type Culture Collection
  • ATCC www.atcc.org/
  • German Collection of Microorganisms and Cell Cultures https://www.dsmz.de/).
  • Cas9 reruns to a CRISPR associated cndonuclease referred to by this name.
  • Non-limiting exemplary Cas9s include Staphylococcus aureus Cas9, nuclease dead C&s9, and orthologs and biological equivalents each thereof.
  • Urthologs include but are not limited to Streptococcus pyogenes Cas9 ("spCas9 ,, ) I Cas 9 from Streptococcus thermophiles. Uglonella pneumophilia. Neisseria lactamica.
  • CD3 zeta signaling domain or ⁇ 03 ⁇ refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% ammo acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity with the CD3 zeta signaling domain sequence as shown herein.
  • Non-limiting exemplary sequences of the CD3 zeta signaling domain arc provided in U.S. Pub. No. US 2013/0266SS 1 A 1.
  • An example oia CD3 zeta signaling domain sequence is set forth herein as SEQ ID NO: 2 RVKFSRSADAPAYQOGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYStlGMKGERRRGKGHDGLYQGLSTA l KDl YDALHMQALPPR.
  • CD8 u hinge domain refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity with the CD8 a hinge domain sequence as shown herein.
  • the example sequences of CD8 a hinge domain for human, mouse, and other species arc provided in Pinto, R.D. el al. (2006) Vet. Immunol. Immunopalhol. 1 10: 169- 177.
  • Non-limiting examples of such sequences include: I luman CD8 alpha hinge domain set forth herein as SEQ ID NO: 3:
  • PAKPTTTPAPRPPTPAPTIASQPLSI.RPEACRPAAGGAVIITRGI.DFACDIY Mouse CDS alpha hinge domain set forth herein as SEQ ID NO: 4: KVNSTTTKPVI.RTPSPVIIPTCiTSQPQRPEDCRPRGSVKGTGLDFACDIY, and Cat CDS alpha hinge domain set forth herein as SEQ ID NO: 5: PVKPTTTPAPRPPTQAPITTSQRVSLRPGTCQPSAGSTVEASGLDLSCDIY.
  • CD8 u transmembrane domain refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity with the CDS a transmembrane domain sequence as shown herein, llic fragment sequences associated with the amino acid positions 183 to 203 of the human T-cell surface glycoprotein CD8 alpha chain (NCRI Reference Sequence: NP_001759.3), or the amino acid positions 197 to 217 of the mouse T-cell surface glycoprotein CD8 alpha chain (NCBI Reference Sequence: NP_00l 074579.1 ), and the amino acid positions 190 to 210 of the rat T-ccll surface glycoprotein CD8 alpha chain(NCBI Reference Sequence: NP_ 1 13726.1 ) provide additional example sequences of the CD8 a transmembrane domain.
  • sequences associated with each of the listed NCBI arc provided as follows: Human CD8 alpha transmembrane domain set forth herein as SF.Q ID NO: 6: lYIWAPLAGTCGVLLLSLVIT; Mouse CDS alpha transmembrane domain set forth herein as SF.Q ID NO: 7: IWAPI.AGICVAI.I.I.SI.IITI.I; Rat CD8 alpha transmembrane domain set forth herein as SEQ ID NO: 8: IWAPLAGICAVLLLSLVITLI.
  • CD28 costimulatory signaling region refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% ammo acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity with the CD28 costimulatory signaling region sequence shown herein.
  • Exemplary CD28 costimulatory signaling domains are provided in U.S. Pat. No. 5,686,281 ; Geiger. T. L. et al.. Blood 98: 2364-2371 (2001 ); Hombach, A. et al.. J Immunol 167: 6123-6131 (2001 ): Maher, J. et al.
  • Non-limiting examples include residues 1 14-220 of the CD28 Sequence set forth herein as SEQ ID NO: 9: MLRLLLALNL FPS1QVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLDSAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPPPYLDNEKSNG TIII1VKGK1IL CPSPLFPGPS KPFVWLVVVG GVLACYSLLVTVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS, and equivalents thereof.
  • CD28 transmembrane domain refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%. or alternatively at least 80% amino acid sequence identity, at least 90% sequence identity, or alternatively at least 95% sequence identity with the CD28 transmembrane domain sequence as shown herein.
  • the fragment sequences associated with the NCDI Accession Nos: XM_006712862.2 and XM_009444056.1 provide additional, non-limiting, example sequences of the CD28 transmembrane domain.
  • the sequences associated with each of the listed accession numbers are incorporated herein as SEQ ID NO: 10 and SEQ ID NO: 1 1, respectively.
  • chimera intends that the sequence contains is comprised of at least one substiiutcnt unit (e.g. fragment, region, portion, domain, polynucleotide, or polypeptide) that is derived from, obtained or isolated from, or based upon other distinct physical or chemical entities.
  • a chimera of two or more dilTcrcnl proteins may comprise the sequence of a variable region domain from an antibody fused to the transmembrane domain of a cell signaling molecule.
  • a chimera intends that the sequence is comprised of sequences from at least two distinct species.
  • chimeric antigen receptor refers to a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from a polypeptide from which the extracellular domain is derived, and at least one intracellular domain.
  • the "chimeric antigen receptor (CAR)” is also known as a “T-body”, “chimeric receptor”, or “chimeric immune receptor (CIR).”
  • the "extracellular domain capable of binding to an antigen” means any oligopeptide or polypeptide that can bind to a certain antigen.
  • the "intracellular domain” means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.
  • the intracellular domain may comprise, alternatively consist essentially of, or yet further comprise one or more costimulatory signaling domains in addition to the primary signaling domain.
  • the "transmembrane domain” means any oligopeptide or polypeptide known to span the cell membrane and that can function to link the extracellular and signaling domains.
  • a chimeric antigen receptor may optionally comprise a "hinge domain" which serves as a linker between the extracellular and transmembrane domains.
  • Non-limiting exemplary polynucleotide sequences that encode for components of each domain are disclosed herein, e.g.: Hinge domain: IgGl heavy chain hinge sequence set forth herein as SEQ ID NO: 12: CTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCG; Transmembrane domain: CD28 transmembrane region set forth herein as SEQ ID NO: 13: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACA GTGGCCTTTATTATTTTCTGGGTG; Intracellular domain: 4- IBB co-stimulatory signaling region set forth herein as SEQ ID NO: 14: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGT ACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAG GAGGATGTGAACTG; Intracellular domain: CD28 co-stimulatory signaling region
  • each exemplary domain component include other proteins that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity with the proteins encoded by the above disclosed nucleic acid sequences. Further, non- limiting examples of such domains are provided herein.
  • composition typically intends a combination of the active agent, e.g., an engineered T-cell receptor, a modified T-cell receptor, a chimeric antigen receptor, a cell comprising an engineered T-cell receptor, a CAR T cell or a CAR NK cell, an antibody, a compound or composition, and a naturally-occurring or non-naturally-occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.
  • the active agent e.g., an engineered T-cell receptor, a modified T-cell receptor, a chimeric antigen receptor, a cell comprising an engineered T-cell receptor, a CAR T cell or a CAR NK cell, an antibody, a compound or composition, and a naturally-occurring or non-naturally-occur
  • Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates ⁇ e.g., sugars, including monosaccharides, di-, tri-, tetra- oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination I -99.99% by weight or volume.
  • Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rllA), gelatin, casein, and the like.
  • amino acid/antibody components which can also function in a buffering capacity, include alanine, arginine, glycine, arginine. betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
  • Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose.
  • disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like: polysaccharides, such as raffinose, melezitose. maltodextrins, dextrans, starches, and the like: and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.
  • disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like
  • polysaccharides such as raffinose, melezitose. maltodextrins, dextrans, starches, and the like
  • alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and my
  • compositions and methods that include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination for the stated purpose.
  • a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed disclosure, such as compositions for treating or preventing an autoimmune disease or disorder, a neurodegerative disease or disorder, such as Parkinson's disease .
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.
  • consensus sequence refers to an amino acid or nucleic acid sequence that is determined by aligning a series of multiple sequences and that defines an idealized sequence that represents the predominant choice of amino acid or base at each corresponding position of the multiple sequences.
  • the consensus sequence for the series can differ from each of the sequences by zero. one. a few. or more substitutions. Also, depending on the sequences of the series of multiple sequences, more than one consensus sequence may be determined for the series. The generation of consensus sequences has been subjected to intensive mathematical analysis. Various software programs can be used to determine a consensus sequence.
  • CKISI'K refers to a technique of sequence specific genetic manipulation relying on the clustered regularly interspaced short palindromic repeats pathway.
  • C'KISI'R can be used to perform gene editing and/or gene regulation, as well as to simply target proteins to a specific genomic location.
  • Gene editing refers to a type of genetic engineering in which the nucleotide sequence of a target polynucleotide is changed through introduction of deletions, insertions, single stranded or double stranded breaks, or base substitutions to the polynucleotide sequence.
  • CRISPR-mcdiatcd gene editing utilizes the pathways of nonhomologous end-joining (NHFJ) or homologous recombination to perform the edits.
  • Gene regulation refers to increasing or decreasing the production of specific gene products such as protein or RNA.
  • cytotoxic cdl intends a cell that is capable of killing other cells or microbes.
  • cytotoxic cells include but are not limited to CD8+ T cells, natural-killer (NK) cells, NKT cells, and neutrophils, which cells are capable of mediating cytotoxicity responses.
  • the term "detectable marker” refers to at least one marker capable of directly or indirectly, producing a detectable signal.
  • a non-exhaustive list of this marker includes enzymes which produce a detectable signal, for example by colorimctry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, ⁇ -galaclosidase, glucose-6- phosphate dehydrogenase, chromophorcs such as fluorescent, luminescent dyes, groups with electron density detected by electron microscopy or by their electrical property such as conductivity, ampcromcU), voltammctry, impedance, detectable groups, for example whose molecules are of sufficient size to induce detectable modifications in their physical and/or chemical properties, such detection may be accomplished by optical methods such as diffraction, surface plasmon resonance, surface variation, the contact angle change or physical methods such as atomic force spectroscopy, tunnel effect, or radioactive molecules such as 32 P, 35 S or
  • disease-relevant antigen refers to an antigen, epitope, or fragment thereof involved in the disease process or mechanism.
  • an inflammation-relevant antigen is an antigen or fragment thereof that, when presented, produces an immune response. An inflammation-relevant antigen producing such an effect is selected to treat the inflammation.
  • an autoimmunity-related antigen is an antigen that is relevant to an autoimmune disease and would not be selected for the treatment of a disorder or disease other than autoimmunity, e.g., cancer.
  • Non-limiting, exemplary disease-relevant antigens are disclosed herein and further, such antigens may be determined for a particular disease based on the epitope screening techniques, mechanisms, and methods described herein.
  • an effective amount or “efficacious amount” is an amount sufficient to achieve the intended purpose, non-limiting examples of such include: initiation of the immune response, modulation of the immune response, suppression of an inflammatory response and modulation of T cell activity or T cell populations.
  • the effective amount is one that functions to achieve a stated therapeutic purpose, e.g., a therapeutically effective amount.
  • the effective amount, or dosage depends on the purpose and the composition, and can be determined according to the present disclosure.
  • nucleic acid sequences refers to a polynucleotide which is said to "encode” an KNA or polypeptide if. in its native state or when manipulated by methods well known to those skilled in the art, the nucleic acid can be transcribed and/or translated to produce a functional KNA (e.g. miRNA, siRNA, RNAi. tRNA. rRNA. snRNA. etc), an mRNA, or a polypeptide and/or a fragment thereof.
  • the anliscnse strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • an engineered T-cell receptor refers to a molecule comprising the elements of (a) an extracellular antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain.
  • an engineered T-cell receptor is a genetically modified I CR, a modified I CR, a recombinant I CR. a transgenic ICR, a partial I CR, a chimeric fusion protein, a CAR, a first generation CAR, a second generation CAR, a third generation CAR, or a fourth generation TRUCK.
  • the engineered T-cell receptor comprises an antibody or a fragment of an antibody.
  • the engineered T-cell receptor is a genetically modified I CR or a CAR.
  • denotes regulatory sequence elements that augment, improve or ameliorate transcription of a nucleic acid sequence irrespective of its location and orientation in relation to the nucleic acid sequence to be expressed.
  • An enhancer may enhance transcription from a single promoter or simultaneously from more than one promoter.
  • any truncated, mutated or otherwise modified variants of a wild- type enhancer sequence are also within the above definition.
  • the term "equivalent” or “biological equivalent” of an antibody means the ability of the antibody to selectively bind its epitope protein or fragment thereof as measured by ELISA or other suitable methods.
  • Biologically equivalent antibodies include, but arc not limited to. those antibodies, peptides, antibody fragments, antibody variant, antibody derivative and antibody mimctics that bind to the same epitope as the reference antibody. It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide or antibody, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure.
  • biological equivalent thereof is intended to be synonymous with "equivalent thereof when referring to a reference protein, antibody, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. For example, an equivalent intends at least about 70% homology or identity, or at least 80 % homology or identity and alternatively, or at least about 85 %, or alternatively at least about 90 %. or alternatively at least about 95 %. or alternatively 98 % percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.
  • an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.
  • the term "equivalent” also includes but is not limited to a sub-sequence, portion, homologuc, variant or derivative thereof.
  • the term "expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • the expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample.
  • the expression level of a gene from one sample may be directly compared to the expression level of that gene from a control or reference sample.
  • the expression level of a gene from one sample may be directly compared to the expression level of that gene from the same sample following administration of a compound.
  • a “first generation CAR” refers to a CAR comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from a polypeptide from which the extracellular domain is derived, and at least one intracellular domain.
  • a “second generation CAR” refers to a first generation CAR further comprising one costimulation domain (e.g. 4-1 BB or CD28).
  • a “third generation CAR” refers to a first generation CAR further comprising two costimulation domains (e.g. CD27, CD28, 1COS, 4-1 BB. or OX40).
  • a "fourth generation CAR” (also known as a “TRUCK”) refers to a CAR T- ccll further engineered to secrete an additional factor (e.g. proinflammatory cytokine 1L-12).
  • gRNA or "guide RNA” as used herein refers to guide RNA sequences used to target specific polynucleotide sequences for gene editing employing the CRISPR technique.
  • Techniques of designing gRNAs and donor therapeutic polynucleotides for target specificity are well known in the art. For example, Doench, J., el al. Nature biotechnology 2014; 32( 12): 1262-7, Mohr, S. et al. (2016) FEBS Journal 283: 3232-38, and Graham, D., etal. Genome Biol. 20 IS; 16: 260.
  • gRNA comprises or alternatively consists essentially of, or yet further consists of a fusion polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA); or a polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA).
  • a gRNA is synthetic (Kelley. M. et al. (2016) J of Biotechnology 233 (2016) 74-83).
  • HLA-A refers to an MHC class I cell surface receptor associated with this name and any other molecules that have analogous biological function that share at least 80% amino acid sequence identity, preferably 90% sequence identity, or alternatively at least 95% sequence identity with any HLA-A variant, including but not limited to any one of its several variants, including but not limited to IILA-A serotypes A I to A69.
  • the gene locus of HLA-A is located at chromosome 6p21.3 (mRNA: NM_001242758, included herein as SEQ ID NO: 18, and NM_0021 16 included herein as SEQ ID NO: 19 ).
  • ⁇ _ ⁇ microglobulin is encoded by the B2M gene located at chromosome 15:44.71-44.72 in humans and chromosome 2: 122.15 in mice (mRNA: NM_004048 included herein as SEQ ID NO: 20).
  • IILA-A sequences are known in the art and a non-limited example is IILA- A*03:01:0:01 precursor set forth herein as SEQ ID NO: 17: MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFV RFDSDAASQRMEPRAPWIEQEGPEYWDQE ' I RNVKAQSQl DRVDLGl LRGY YNQSEAG SIITIQIMYGCDVGSDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRICWE AAHEAEQLRAYLDG TCVEWLRRYLENGKETLQR TDPPK MM THHP1SDHEA I LRCWAL GFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHn GLPKPLI LRWELSSQP riPlVGIIAGLV
  • a I type I diabetes
  • A2 spontaneous abortion
  • A3 hemochromatosis, myasthenia gravis, and multiple sclerosis
  • a 1 1 papilloma virus susceptibility
  • A24 ankylosing spondylitis, type I diabetes, and myasthenia gravis
  • A26 adult T-cell leukemia
  • ⁇ 30 myasthenia gravis
  • ⁇ 68 adult T-cell leukemia
  • ⁇ . ⁇ - ⁇ refers to an MHC class I cell surface receptor associated with this name and any other molecules that have analogous biological function that share at least 80% amino acid sequence identity, preferably 90% sequence identity, or alternatively at least 95% sequence identity with any HLA-B variant, including but not limited to any one of its several variants, including but not limited to IILA-B serotypes Bl to B83.
  • the gene locus of HLA-B is located at chromosome 6:31.35-36 (mRNA: NM_005514 included herein as SEQ ID NO: 21 ).
  • HLA-B is a heterodimer composed of an a chain (encoded by the I ILA-B gene) and a p chain.
  • the ⁇ chain ( H ffe microglobulin") is encoded by the B2M gene located at chromosome 15:44.71 -44.72 in humans and chromosome 2: 122.15 in mice (mRNA: NM_004048, SEQ ID NO: 20).
  • HLA-B sequences arc known in the art and a non-limited example of an HLA-B protein sequence is provided herein as SEQ 10 NO: 23: MLVMAPRTVLLLLSAALALTETWAGSIISMRYFYTSVSRPGRGEPRFISVGYVDDTQFV RFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRGYYNQSEAGS I ITLQSMYGCDVGPDGRLLRG1 IDQYAYDGKDYI ALNEDLRSWTAADTAAQITQRKWEA AREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHP1SDHEATLRCWALG FYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVWPSGEEQRYTCl IVQI IEG LPKPLTLRWEPSSQSTVPIVGIVAGLAVLAVVVIGAVVAAVMCRRKSSGGKGGSYSQAA CSDS
  • HLA-B serotypes and/or alleles are known in the art to be associated with disease.
  • B27 is associated with ankylosing spondylitis, inflammatory joint diseases, psoriasis, inflammatory bowel disorders, reactive arthritis.
  • ⁇ . ⁇ - ⁇ is also associated with HLA graft compatibility (e.g. HLA-A*02:01 to ⁇ . ⁇ - A*02:426).
  • HLA-C refers to an MHC class I cell surface receptor associated with this name and any other molecules that have analogous biological function that share at least 80% amino acid sequence identity, preferably 90% sequence identity, or alternatively at least 95% sequence identity with any HLA-C variant, including but not limited to any one of its several variants, including but not limited to HLA-C serotypes Cw I to Cwl I and CI 2 to CI 8.
  • the gene locus of HLA-C is located at chromosome 6:31.21 (mRNA: NM 0021 17, included herein as SEQ ⁇ NO: 24. and NM_001243042, included herein as SEQ ID NO: 25).
  • HLA-C is a hctcrodimcr composed of an a chain (encoded by the HLA-C gene) and a ⁇ chain.
  • the p chain (“
  • HLA-C sequences are known in the art and a non-limited example is HLA-Cw-l precursor included herein as SEQ ID NO: 22: MRVMAPRALL LLLSUGLALT ETWACSHSMR YFDTAVSRPG RGEPRFISVG YVDDTQFVRF DSDAASPRGE PRAPWVEQF.G PEYWDRETQK YKRQAQADRV SLRNLRGYYN QSEDGSHTLQ RMSGCDLGPD GRLLRGYDQS AYDGKDY1AL NEDLRSWTAA DTAAQITQRK LEAARAAEQL RAYLEGTCVE WLRRYLENGK ETLQRAEPPK THVTHHPLSD HEATLRCWAL GFYPAEITLT WQRDGEDQTQ DTELVETRPA GDGTFQKWAA VVVPSGQEQR YTCHMQHEGL QEPLTLSWEP SSQPTIPIMG IVAGLAVLVV LAVLGAVVTA M
  • HLA-C serotypes and/or alleles are known in the art to be associated with disease including but not limited to Cw 1 (multinodular goiters) and C* 16 (chronic B-cell lymphocytic leukemia). I ILA- C is associated with HLA graft compatibility.
  • HI.A-DP refers to an MHC class II cell surface receptor associated with this name and any other molecules that have analogous biological function that share at least 80% amino acid sequence identity, preferably 90% sequence identity, or alternatively at least 95% sequence identity with any HLA-DP variant, including but not limited to any one of its several variants, including but not limited to HI .A-DP serotypes A I and R I and HI .A-DP alleles A 1*01 to A 1*04 and Bl*01 to Bl *l l . In humans, the gene locus of HLA-DP is located at chromosome 6p21.31 (mRNA: NM_0021 17 (SEQ ID NO 24).
  • HLA-DP is a hclcrodimer composed of an u chain (encoded by the HLA-DPA1 gene) and a P chain (encoded by the HLA-DPBI gene).
  • HLA-DPA1 is included herein as SEQ ID NO: 26: RPEDRMFH1RAVILRAI5LAFI.I.SLRGAGAIKADHVSTYAAFVQTHRPTGEFMFEFDEDF.
  • HI.A-DPBI is included herein as SEQ ID NO: 27:
  • HLA-DR refers to an MHC class II cell surface receptor associated with this name and any other molecules that have analogous biological function that share at least 80% amino acid sequence identity, preferably 90% sequence identity, or alternatively at least 95% sequence identity with any HLA-DR variant, including but not limited to any one of its several variants, including but not limited to IILA-DR serotypes DRI to DR 75 comprising a combination of HLA-DRA and HLA-DRB haplotypes.
  • HLA-DR sequences are known in the art and non-limited examples of such are disclosed in Rose, L.M. et al. ( 1996) Cancer Immunol. Immunother.43:26-30: HLA-DRB 1*1001 [DR101 which is included herein as SEQ ID NO: 28:
  • HLA-DRB3*0201 [DR52] which is included herein as SEQ ID NO: 29:
  • HLA-G also known as “MHC-G” refers to a specific molecule associated with this name and any other molecules that have analogous biological function that share at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity with IILA-G, including but not limited to any one of its several isoforms.
  • HLA-G is a nonclassical MIIC class I paralogue consisting of a helerodimer of a heavy chain and a jfc microglobulin. The genetic locus for HLA-G is found at chromosome 6:29.83 in humans and at chromosome 17:37.27 in mice.
  • NM_002I27.5 included herein as SEQ ID NO: 31; XM_006715080.1, included herein as SEQ ID NO: 32; XM_006725041.1 , included herein as SEQ ID NO: 33; XM_006725700.1 , included herein as SEQ ID NO: 34: and XM_006725909.1 , included herein as SRQ ID NO: 35.
  • SEQ ID NO: 36 An example of the protein translation of NM_002127.5 is included herein as SEQ ID NO:
  • nucleic acids or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, e.g., at least 60% identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (eg., nucleotide sequence encoding an antibody described herein or amino acid sequence of an antibody described herein).
  • Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. ⁇ degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
  • the alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel el a I., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1.
  • default parameters are used for alignment.
  • a preferred alignment program is BLAST, using default parameters.
  • the terms also include sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is at least 50- 100 amino acids or nucleotides in length.
  • An "unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences disclosed herein.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozymc.
  • Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C: hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%: and wash solutions from about 4x SSC to about 8x SSC.
  • Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about S0°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about Sx SSC to about 2x SSC.
  • high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about Ix SSC to about O.lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about Ix SSC, O.lx SSC. or deionized water.
  • hybridization incubation limes are from 5 minutes to 24 hours, with 1 , 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
  • SSC is 0.15 M NaCI and 15 mM citrate buffer. It is understood that equivalents ofSSC using other buffer systems can be employed.
  • the term "1COS coslimulalory signaling region” refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, preferably 00% sequence identity, more preferably at least 05% sequence identity with the ( COS coslimulalory signaling region sequence as shown herein.
  • Non-limiting example sequences of the ICOS costimulatory signaling region are provided in U.S.
  • immune response refers to the development of a cell-mediated response (e.g. mediated by antigen-specific T cells or their secretion products).
  • a cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules, to treat or prevent a viral infection, expand antigen-specific Breg cells.
  • the response may also involve activation of other components.
  • the term “immune response” may be used to encompass the formation of a regulatory network of immune cells.
  • regulatory network formation may refer to an immune response elicited such that an immune cell, preferably a T eel I. more preferably a T regulatory cell, triggers further differentiation of other immune cells, such as but not limited to, D cells or antigen-presenting cells - non limiting examples of which include dendritic cells, monocytes, and macrophages.
  • regulatory network formation involves B cells being differentiated into regulatory B cells; in certain embodiments, regulatory network formation involves the formation of tolerogenic antigen- presenting cells.
  • Immuno cells include all cells that arc produced by hematopoietic stem cells (lISC) including, but not limited to, HSCs, white blood cells (leukocytes), lymphocytes (including T cells, 13 cells, and natural killer (NK) cells) and mycloid-dcrived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells).
  • LISC hematopoietic stem cells
  • Leukocytes include but are not limited to lymphocytes, granulocytes, monocytes, and macrophages.
  • inflammatory response and "inflammation” as used herein indicate the complex biological response of immune cells, humoral factors, and vascular tissues of an individual or subject to exogenous or endogenous stimuli, such as pathogens, damaged cells, or irritants, and/or inflammatory signals such as pro-inflammatory cytokines.
  • the inflammatory response includes secretion of cytokines and, more particularly, of pro-inflammatory cytokines, i.e. cytokines which are produced predominantly by activated immune cells and are involved in the amplification of inflammatory reactions.
  • Exemplary pro-inflammatory cytokines and chemokines include but arc not limited to IL- ⁇ , TNF-o, IFN- ⁇ , IL-8, IL-6, IL-12, IL-15, IL-16, lL-17 (including family members IL17A. 1LI7B. IL-17C, IL-I7D. 1L-I7E, IL-I7F). IL-18. GM- CSF, IL-21, IL-23, IL-27 and TGF- ⁇ .
  • Exemplary anti-inflammatory cytokines include but arc not limited to TGF- ⁇ , IL-IRot. 1L-4. IL-6, IL-10. lL-1 1. IL-13. IL-35. INF-a.
  • a cytokine may have either pro-inflammatory and anti-inflammatory properties depending on the particular biological context (Cavaillon. J.M (2001 ) Cell Mol Biol 47(4): 69S-702).
  • Exemplary inflammations include acute inflammation and chronic inflammation.
  • Acute inflammation indicates a short-term process characterized by the classic signs of inflammation (swelling, redness, pain, heat, and loss of function) due to the infiltration of the tissues by plasma and leukocytes.
  • An acute inflammation typically occurs as long as the injurious stimulus is present and ceases once the stimulus has been removed, broken down, or walled off by scarring (fibrosis).
  • Chronic inflammation indicates a condition characterized by concurrent active inflammation, tissue destruction, and attempts at repair. Chronic inflammation is not characterized by the classic signs of acute inflammation listed above.
  • chronically inflamed tissue is characterized by the infiltration of mononuclear immune cells (monocytes, macrophages, lymphocytes, and plasma cells), tissue destruction, and attempts at healing, which include angiogenesis and fibrosis.
  • An inflammation can be inhibited in the sense of the present disclosure by affecting and in particular inhibiting any one of the events that form the complex biological response associated with an inflammation in an individual.
  • exemplary diseases or conditions associated with or related to inflammation and/or inflammatory responses include but are not limited to multiple sclerosis, muscle injuries, graft versus host disease, Parkinson's disease, Alzheimer's, inflammatory bowel disease, Huntington's disease, amyotrophic lateral sclerosis, Behcet's disease, sarcopenia, aging, spinal cord injury, wound repair, and dysphagia. Additional diseases or conditions associated with or related to inflammation and/or inflammatory responses include autoimmune disease or disorders.
  • autoimmune disease or disorder includes diseases or disorders arising from and directed against an individual's own tissues or organs or manifestation thereof or a condition resulting there from. In one embodiment, it refers to a condition that results from, or is aggravated by, the production by T cells that are reactive with normal body tissues and antigens.
  • autoimmune diseases or disorders include, but are not limited to arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis.
  • autoimmune urticaria myositis, polymyositis/dermatomyositis. juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS.
  • myositis polymyositis/dermatomyositis. juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS.
  • MS multiple sclerosis
  • PPMS primary progressive MS
  • RRMS relapsing remitting MS
  • progressive systemic sclerosis atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica spectrum disorder (NMO, also known as Devic's Disease or Devic's Syndrome), inflammatory bowel disease (IDD)
  • NMO neuromyelitis optica spectrum disorder
  • IBD inflammatory bowel disease
  • Crohn's disease autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease
  • bowel inflammation pyoderma gangrenosum, erythema nodosum
  • respiratory distress syndrome including adult or acute respiratory distress syndrome (ARDS).
  • ARDS adult or acute respiratory distress syndrome
  • meningitis inflammation of all or part of the uvea, ulceris, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition.
  • IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and/or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN.
  • GN
  • balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, eythema multiform, granuloma annulare, lichen nitidus.
  • lichen sclerosus et atrophicus lichen simplex chronicus, lichen spinulosis, lichen planus, lamellar ichthyosis, cpidcrmolytic hyperkeratosis, prcmalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reaction, eczema including allergic or atopic eczema, aslealotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema, asthma such as asthma branchiate, bronchial asthma, and auto-immune asthma, conditions involving infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as fetal A- D-O blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lup
  • NLE neonatal lupus syndrome
  • lupus erythematosus disseminatus Type I diabetes.
  • Type II diabetes latent autoimmune diabetes in adults (or Type l.S diabetes)
  • Diamond Black fan anemia hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer's disease.
  • Parkinson's disease multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, anti-phospholipid syndrome, allergic neuritis, Behcet's disease/syndrome, Castleman's syndrome, Goodpasture's syndrome.
  • Reynaud's syndrome Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermal injury, preeclampsia, an immune complex disorder such as immune complex nephritis, antibody-mediated nephritis, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM-mcdiated neuropathy, autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute 1TP, acquired thrombocytopenic purpura, sclcritis such as
  • autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism. Graves disease.
  • polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as mymoma-associated myasthenia gravis, cerebellar degeneration, neuromyolonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, gianT cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis,
  • IgA nephropathy idiopathic IgA nephropathy
  • linear IgA dermatosis acute febrile neutrophilic dermatosis
  • subcorneal pustular dermatosis subcorneal pustular dermatosis
  • transient acantholytic dermatosis cirrhosis such as primary biliary cirrhosis and pneumonocirrhosis
  • autoimmune enteropathy syndrome autoimmune enteropathy syndrome.
  • Celiac orCoeliac disease Celiac orCoeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome/nonsyphilitic interstitial keratitis. Bell's palsy.
  • ALS amyotrophic lateral sclerosis
  • AIED autoimmune inner ear disease
  • polychondritis such as refractory or relapsed or relapsing polychondritis
  • pulmonary alveolar proteinosis Cogan's syndrome/nonsyphilitic interstitial keratitis. Bell'
  • Sweet's disease/syndrome rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance. MGUS), peripheral neuropathy, paraneoplastic .syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS).
  • FSGS focal or segmental or focal segmental glomerulosclerosis
  • endocrine ophthalmopathy uvcorctinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrcnalitis. gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Drcssler's syndrome, alopecia greata. alopecia totalis. CREST syndrome (calcinosis.
  • Chagas' disease rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post- cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis. aspergillosis. Sampler's syndrome.
  • Caplan's syndrome dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Feltys syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis, Ilenoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SOL), acquired immune deficiency syndrome (AIDS), echovinis infection, sepsis, endotoxemia, pancreatitis, thyroxicosis, parvovirus infection,
  • Epstein-Barr virus infection Epstein-Barr virus infection, mumps. F.van's syndrome, autoimmune gonadal failure, Sydenham's chorea, poststreptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis.
  • gianT cell polymyalgia chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway/pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, asperniogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylaciica.
  • enteritis allergica erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, fcbris rheumatics.
  • pancreatitis polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis. acquired spcnic atrophy, non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen- antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis.
  • autoimmune polyglandular syndrome type I adult- onset idiopathic hypoparathyroidism ( ⁇ )
  • cardiomyopathy such as dilated cardiomyopathy, cpidcrmolisis bullosa acquisita (EBA)
  • hemochromatosis myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis
  • an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Lofllert syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils,
  • Wiskott-Aldrich syndrome ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, repertusion injury, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute serious inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, emphysema,
  • introduce refers to the process whereby a foreign (i.e. extrinsic or extracellular) agent is introduced into a host cell thereby producing a cell comprising the foreign agent.
  • Methods of introducing nucleic acids include but are not limited to transduction, retroviral gene transfer, iransfcction. clcciroporation. transformation, viral infection, and other recombinant DNA techniques known in the art.
  • transduction is done via a vector (e.g. a viral vector).
  • iransfcction is done via a chemical carrier, DNA/liposomc complex, or micelle (e.g.
  • viral infection is done via infecting the cells with a viral particle comprising the polynucleotide of interest (e.g. AAV).
  • introduction further comprises CRISPR mediated gene editing or Transcription activator-like effector nuclease (TALEN) mediated gene editing.
  • CRISPR mediated gene editing or Transcription activator-like effector nuclease (TALEN) mediated gene editing.
  • soluble factors, cytokines, proteins, peptides, enzymes, growth factors, signaling molecules, small molecule inhibitors include but are not limited to culturing the cells in the presence of the foreign agent, contacting the cells with the agent, contacting the cells with a composition comprising the agent and an excipient, and contacting the cells with vesicles or viral particles comprising the agent.
  • isolated refers to molecules or biologicals or cellular materials being substantially free from other materials.
  • isolated refers to nucleic acid, such as DNA or RNA. or protein or polypeptide (e.g.. an antibody or derivative thereof), or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source.
  • isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an "isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • isolated is also used herein to refer to cells or tissues that are isolated from other cells or tissues and substantially separated from other cells of a tissue. "Isolated cell” is meant to encompass both cultured and engineered cells or tissues.
  • linker sequence relates to any amino acid sequence comprising from 1 to 10, or alternatively, 8 amino acids, or alternatively 6 amino acids, or alternatively 5 amino acids that may be repealed from I to 10. or alternatively to about 8, or alternatively to about 6, or alternatively about S, or 4 or alternatively 3, or alternatively 2 times.
  • the linker may comprise up to IS amino acid residues consisting of a pentapeptide repeated three times.
  • the linker sequence is a (Glycine4Serine)3 flexible polypeptide linker comprising three copies of gly-gly-gly-gly-ser, or equivalents thereof.
  • linker sequences are known in the art, e.g., GGGGSGGGGSGGGG (and equivalents thereof): the tripeptide EFM; or Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met, and equivalents of each thereof.
  • a "flexible linker” intends a linker characterized by minimal rigidity.
  • a flexible linker facilitates improved, preferred, or optimal secondary conformation, tertiary conformation, and/or quaternary conformation of the linked protein domains or full length polypeptide.
  • a flexible linker reduces or minimizes negative steric effects.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • MHC class I MHC- I
  • MHC-II MHC class II
  • a particular antigen, peptide, and/or epitope is identified and presented in an antigen-MHC complex in the context of an appropriate MHC class I or II protein.
  • the genetic makeup of a subject may be assessed to determine which MHC allele is suitable for a particular patient, disease, or condition with a particular set of antigens.
  • the MHC genes arc known as the histocompatibility 2 (H-2) genes.
  • Murine classical MHC class I subtypes include II-2D. II-2K, and H-2L.
  • Murine non- classical MHC class I subtypes include H-2Q, H-2M, and H-2T.
  • Murine classical MHC class II subtypes include ⁇ -2 ⁇ ( ⁇ - ⁇ ), and H-2F.
  • Non-classical murine MHC class II subtypes include H-2M and H-20.
  • Canine MHC molecules arc known as Dog Leukocyte Antigens (DLA).
  • Feline MHC molecules are known as Feline Leukocyte Antigens (FI.A).
  • an orthologous or homologous MHC molecule is selected to transition a therapy or treatment involving a specific antigen-MHC complex from one species to a different species.
  • Non-classical MHC molecules are non-polymorphic, conserved among species, and possess narrow, deep, hydrophobic ligand binding pockets. These binding pockets are capable of presenting glycolipids and phospholipids to Natural Killer T (NKT) cells or certain subsets of CD8+ T-cells.
  • NKT Natural Killer T
  • MHCs for use according to the present disclosure may be produced, isolated, or purified through techniques known in the art. Common protocols for obtaining MHCs involve steps such as, but not limited to, electrophoresis or other techniques of charge or size based separation, biotinylation or other tagging methods and purification, or transfection and induction of vector constructs expressing MHC proteins. Purified animal antibodies arc also available through commercially available sources, including retailers such as eBioscience. Biolegend. or Tonbo Biosciences.
  • the MHC may be classical MHC I, non-classical MHC I, classical MHC II. non-classical MHC II. dimers (Fc fusions). MHC tetramers.
  • MHC multimcrs arc generated according to methods well documented in the art. see, e.g., Bakker et al. "MHC Multimer Technology: Current Status and Future Prospects," Current Opinion in Immunology, Vol. 17, No. 4 pp. 428-433 (2005) and references cited therein.
  • MHC restriction refers to an antigen, antigen fragment, peptide, or epitope that is only specifically recognized and bound by an antigen binding domain when the antigen is bound to a particular MHC molecule.
  • an MI IC-reslrictcd antigen is not specifically recognized and bound by an antigen binding domain outside of the context of a particular MHC molecule.
  • the particular MHC molecule is a specific allele or subtype of H LA- A, HLA-B, HLA-C, HLA-b, HLA-F, HLA-G, HLA-DM, HLA- DO.
  • the antigen binding domain is the antigen binding domain of an antibody, an antibody fragment, a CAR, an engineered TCR, or a B- cell receptor ("BCR").
  • the term "monoclonal antibody” refers to an antibody produced by a cell into which the light and heavy chain genes of a single antibody have been transfected or, more traditionally, by a single clone of B-lymphocytcs.
  • Monoclonal antibodies generally have affinity for a single epitope (i.e. they are monovalent) but may be engineered to he specific for two or more epitopes (e.g. bispecific). Methods of producing monoclonal antibodies arc known to those of skill in the art.
  • Monoclonal antibodies include recombinant antibodies, chimeric antibodies, humanized antibodies, and human antibodies.
  • NK cell also known as natural killer cell, refers to a type of lymphocyte that originates in the bone marrow and play a critical role in the innate immune system. NK cells provide rapid immune responses against viral-infected cells, tumor cells or other stressed cell, even in the absence of antibodies and major histocompatibility complex on the cell surfaces. NK. cells may cither be isolated or obtained from a commercially available source. Non-limiting examples of commercial NK cell lines include lines NK-92 (ATCC® CRL-2407TM). NK-92MI (ATCCtt CRL-2408TM). Further examples include but arc not limited to NK lines HANK1, KHYG-I. NKI.. NK-YS. NO 1-90.
  • Non-limiting exemplary sources for such commercially available cell lines include the American Type Culture Collection, or ATCC, (http://www.atcc.org/) and the German Collection of Microorganisms and Cell Cultures (https://www.dsnu.de/).
  • the term "overexpress" with respect to a cell, a tissue, or an organ expresses a protein to an amount that is greater than the amount that is produced in a control cell, a control issue, or an organ.
  • a protein that is overexpressed may be endogenous to the host cell or exogenous to the host cell.
  • OX40 costimulatory signaling region refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, or alternatively 90% sequence identity, or alternatively at least 95% sequence identity with the ⁇ 40 costimulatory signaling region sequence as shown herein.
  • Non-limiting example sequences of the OX40 costimulatory signaling region are disclosed in U.S.
  • a "pathogenic T cell” is a T cell that is harmful to a subject containing the T cell. Whereas, a non-pathogenic T cell is not substantially harmful to a subject, and an anti-pathogenic T cells reduces, ameliorates, inhibits, or negates the harm of a pathogenic T cell.
  • protein refers to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another aspect, the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • a protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • polynucleotide and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonueleolides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any aspect of this technology that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (IJ) for thymine when the polynucleotide is RNA.
  • a polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinrormatics applications such as functional genomics and homology searching.
  • nucleic acid sequence and “polynucleotide” are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • ⁇ polypeptide may contain a contiguous nucleic acid sequence of the following lengths: 10, 20, 30, 40, SO.60, 70. 80, 90. 100. 110. 120, 130, 140, ISO, 160. 170. 180. 190.200. 210. 220. 230, 240, 250, 260, 270, 280. 290, 300, 310, 320, 330, 340, 350, 360, 370. 380. 390. 400, 410, 420, 430. 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530. 540, 550. 560. 570. 580. 590, 600. 610. 620. 630.
  • nucleosides or base pairs.
  • a particular polypeptide from may be encoded by nucleic acids containing natural variations that having slightly di!Tcrcnl nucleic acid sequences but. nonetheless, encode the same or substantially similar protein, polypeptide, or peptide.
  • promoter refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters may be constitutive, inducible, repressive, or tissue-specific, for example.
  • a "promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rale of transcription arc controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RN A polymerase and other transcription factors.
  • Non-limiting exemplary promoters include CMV, U6, EFIa, SV40, PGKI (human or mouse). PS, Ubc. human beta actin, CAG, TRE, IJAS. Ac5. Polyhedrin. CaMKIIa.
  • TEF1 GDS. ADIl l, CaMV35S. IJbi, III. U6. and Alpha- 1 -antitrypsin.
  • Synthetically-derived promoters may be used for ubiquitous or tissue specific expression.
  • virus-derived promoters some of which are noted above, may be useful in the methods disclosed herein, e.g., CMV, HIV, adenovirus, and AAV promoters.
  • purification marker refers to at least one marker useful for purification or identification. ⁇ non-exhaustive list of this marker includes His, lacZ, GST, maltose-binding protein, NusA, BCCP.
  • c-myc CaM, FLAG, GFP, YFP. cherry, thioredoxin. poly(NANP), V5, Snap, HA, chitin-binding protein, Softag I, Soflag 3, Strep, or S-protein.
  • Suitable direct or indirect fluorescence marker comprise FLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy 5, Cy 5.5, Cy 7, DNP, AMCA, Biotin, Digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluors, F1TC. TRITC or any other fluorescent dye or hapten.
  • a purified nucleic acid, peptide, protein, biological complexes or other active compound is one that is isolated in whole or in part from proteins or other contaminants.
  • substantially purified peptides, proteins, biological complexes, or other active compounds for use within the disclosure comprise more than 80% of all macromolccular species present in a preparation prior to admixture or formulation of the peptide, protein, biological complex or other active compound with a pharmaceutical carrier, excipient. buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient in a complete pharmaceutical formulation for therapeutic administration.
  • the peptide, protein, biological complex or other active compound is purified to represent greater than 90%, often greater than 95% of all macromolecular species present in a purified preparation prior to admixture with other formulation ingredients.
  • the purified preparation may be essentially homogeneous, wherein other macromolecular species are not detectable by conventional techniques.
  • the term “recognizes and specifically binds” or “antibody binding” or “specific binding” means the contact between the antigen binding domain of an antibody, antibody fragment.
  • an antigen binding domain binds to both a complex of both an antigen and an MHC molecule.
  • antigen binding domains bind with affinities of less than about 10 ⁇ ' M, 10 7 M.
  • specific binding refers to the binding of an antigen to an MHC molecule, or the binding of an antigen binding domain of an engineered T-cell receptor to an antigen or antigen-MI IC complex.
  • recombinant protein refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%. or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) arc the same in comparing the two sequences.
  • the alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al.. eds. 1987) Supplement 30. section 7.7.18. Table 7.7.1.
  • default parameters arc used for alignment.
  • a preferred alignment program is BLAST, using default parameters.
  • signal peptide or “signal polypeptide” intends an amino acid sequence usually present al the N-lerminal end of newly synthesized secretory or membrane polypeptides or proteins. It acts to direct the polypeptide across or into a cell membrane and is then subsequently removed Examples of such are well known in the art. Non-limiting examples are those described in U.S. Patent Nos. 8,853,381 and 5,958,736.
  • the terms "subject.” “host,” “individual,” and “patient” are as used interchangeably herein to refer to human and veterinary subjects, for example, humans, animals, non-human primates, dogs, cats, sheep, mice, horses, and cows.
  • the subject is a human.
  • the subject is suffering from a disease or condition to be treated by one of the methods disclosed herein.
  • suicide gene refers to a gene encoding a factor that is capable of inducing death in a cell that expresses it.
  • a suicide gene provides a strategy to regulate cell persistence by providing a mechanism for specific depletion of cells expressing the suicide gene. Once activated. the suicide gene kills the cell through, for example, apoptosis or cell-mediated cytotoxicity.
  • Non limiting examples of suicide genes include (1) iCasp9 which is activated by administration of API 903 to cause apoptosis, (2) CD20 which is activated by administration of CD-20 specific antibody rituximab causing depletion through antibody-dependent cellular cytotoxicity, and (3) herpesvirus thymidine kinase which is activated by ganciclovir.
  • T-cell refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and arc distinguished from other lymphocytes, such as R cells, by the presence of a T-cell receptor (TCR) on the cell surface. T- cclls may either be isolated or obtained from a commercially available source. * T cell” includes all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), natural killer T-cclls, naive T cells (CCR7+, CD45RA+), central memory T-cclls (CCR7+, CD45RA-).
  • T-regulatory cells Treg
  • gamma-dclla T cells T-regulatory cells
  • Natural killer T cells TCR co-express NK cell markers and a semi- invariant T cell receptor (TCR). They are implicated in the regulation of immune responses associated with a broad range of diseases.
  • Non-limiting examples of commercially available T- cell lines include lines BCL2 (AAA) Jurkat (ATCCCD CRL-2902TM), BCI.2 (S70A) Jurkat (ATCC® CRL-2900TM), BCL2 (S87A) Jurkat (ATCC® CRL-2901TM), BCL2 Jurkat (ATCC® CRI.-2899TM).Neo Jurkat (ATCCflDCRL-2898TM),TAI.I.-l04 cytotoxic human Tcell line(ATCC # CRL-11386).
  • ⁇ ' -ccll lines e.g., such as Deglis, F.BT-8, HPB-Ml.p-W, HIJT 78, HUT 102, Karpas 384, Ki 225, My-I.a, Sc-Ax, SKW-3, SMZ-I and 134: and immature T- cell lines, e.g., ALL-SIL. Bel 3. CCRF-CKM, CML-Tl, DND- 41, Dl 1.528, F.U-9, HD-Mar, ⁇ - ⁇ . ⁇ ., H-SB2. HT-1, JK-TI, Jurkat, Karpas 45, KF.-37, KOPT- Kl. K-TI.
  • mature ⁇ ' -ccll lines e.g., such as Deglis, F.BT-8, HPB-Ml.p-W, HIJT 78, HUT 102, Karpas 384, Ki 225, My-I.a, Sc-Ax, S
  • J.CaMl .6 (ATCC CRL-2063), RS4;1 1 (ATCC CRL-1873).
  • CCRF-CEM ATCC CRM-CCL-1 19
  • cutaneous T-cell lymphoma lines e.g., HuT78 (ATCC CRM-TIB-161 ).
  • GI 11 (ATCC CRL-8294), Hull 02 (ATCC ⁇ -162).
  • Null leukemia cell lines including but not limited to REIl, NALL-l, KM-3, 1.92-221, are a another commercially available source of immune cells, as arc cell lines derived from other leukemias and lymphomas. such as K562 crythroleukemia, THP-1 monocytic leukemia.
  • HEL erythroleukemia, IIL60 leukemia, HMC-1 leukemia, KG- 1 leukemia, U266 myeloma include the American Type Culture Collection, or ATCC, (http://www.atcc.orgO and the German Collection of Microorganisms and Cell Cultures (https://www.dsmz.de/).
  • T-cell receptor refers to a cell surface molecule found on T-cells that functions to recognize and bind antigens presented by antigen presenting molecules.
  • a TCR is a hetemdimer of an alpha chain (TRA) and a beta chain (TRR).
  • TRU alternative gamma
  • TRD delta
  • T-cells expressing this version of a TCR are known as ⁇ T-cells.
  • TCRs are part of the immunoglobulin supcrfamily. Accordingly, like an antibody, the TCR comprises three hypcrvariablc CDR regions per chain.
  • the TCR hclerodimcr is generally present in an octomcric complex that further comprises three dimcric signaling modules CD3y/e, CD3o7e. and CD247 ⁇ / ⁇ or ⁇ / ⁇ .
  • a nonlimiting exemplary amino acid sequence of the human TCR-alpha chain is included herein as SEQ ID NO: 39: MF.TLLGVSI .VII.WI.QLARVNSQQGEF.DPQAI 51QEGF.NATMNCSYKTS1NNI,QWYRQN SGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCAPVLSGGGAD GI.TFGKGTHI.IIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITD KTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFET DTNI .NFQNI .SVIGFRH .1.1.KVAGFNI .1.MTI .RI .WSS.
  • a Nonlimiting exemplary amino acid sequence of the human ICR-beta chain is included herein as SEQ ID NO: 40:
  • modified TCR refers to a I CR that has been genetically engineered, and/or a transgenic TCR. and/or a recombinant TCR.
  • modified TCRs include single-chain ⁇ TCRs (scTv), full-length TCRs produced through use of a T cell display system, and TCRs wherein the CDR regions have been engineered to recognize a specific antigen, peptide, fragment, and/or MHC molecule.
  • scTv single-chain ⁇ TCRs
  • Methods of developing and engineering modified TCRs are known in the art. For example, see Stone, J.D. et al. Methods in Enzymology 503: 189-222 (2012), PCI ' Application WO2014018863 Al.
  • TRI cells are a subset of CD4+ T cells that have regulatory properties and are able to suppress antigen-specific immune responses in vi/ro and in vivo. These TRI cells are defined by their unique profile of cytokine production and make high levels of IL-IO and TGF-beta, but no IL-4 or IL-2. The IL-10 and TGF-beta produced by these cells mediate the inhibition of primary naive T cells in vitro. There is also evidence that TR cells exist in vivo, and the presence of high IL-10-producing CD4(+) T cells in patients with severe combined immunodeficiency who have received allogeneic stem-cell transplants have been documented.
  • TR 1 cells are involved in the regulation of peripheral tolerance and they could potentially be used as a cellular therapy to modulate immune responses in vivo. See, for example, Levings, M. et al. J. Allergy Clin. Immunol. 106(1 Pt2):SI09-l2 (2000).
  • TRI cells arc defined by their ability to produce high levels of IL-10 and TUF-bcia. Trl cells specific for a variety of antigens arise in vivo, but may also differentiate from naive CD4+ T cells in the presence of IL-10 in vitro. TRI cells have a low proliferative capacity, which can be overcome by 11.-15. TRI cells suppress naive and memory T helper type I or 2 responses via production of IL-10 and TUF-bcta. Further characterization of TRI cells at the molecular level will define their mechanisms of action and clarify their relationship with other subsets of Tr cells.
  • TRI cells to identify novel targets for the development of new therapeutic agents, and as a cellular therapy to modulate peripheral tolerance, can be foreseen. See, for example. Roncarolo. M. ct al. Immunol. Rev. 182:68-79 (2001 ).
  • treating or “treatment” of a disease or condition in a subject refers to ( 1 ) preventing the symptoms or disease or condition from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or condition or arresting its development; or (3) ameliorating or causing regression of the disease or condition or the symptoms of the disease or condition.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.
  • the disease or condition is associated with the immune response, the clinical endpoints will vary by the specific tissue targeted or affected by the immune response.
  • the disease is neurodegeneration, e.g.
  • Parkinson's disease exemplary clinical endpoints for successful treatment include but are not limited to ( I ) improvement or stabilization of the following: unified Parkinson's disease rating scale (UPDKS) rating, motor function, ambulation, and/or speech; (2) decreased time to dementia and/or nursing home placement: (3) decreases or stabilization of autonomic failure. falls, and/or cognitive symptoms. Additional clinical endpoints are described in more detail herein.
  • UPDKS unified Parkinson's disease rating scale
  • unit dose refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen.
  • the quantity to be administered depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations arc easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.
  • vector refers to a nucleic acid construct deigned for transfer between different hosts, including but not limited to a plasmid, a virus, a cosmid, a phage, a BAC, a YAC, etc.
  • a "viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • plasmid vectors may be prepared from commercially available vectors.
  • viral vectors may be produced from baculoviruses, retroviruses, adenoviruses, AAVs, etc.
  • the viral vector is a Ienti viral vector.
  • examples of viral vectors include retroviral vectors, adenovirus vectors, adeno- associated virus vectors, alphavirus vectors and the like.
  • Infectious tobacco mosaic virus (TMV)- based vectors can be used to manufacturer proteins and have been reported to express Griffithsin in tobacco leaves (OTCeefe et al. (2009) Proc. Nat. Acad. Sci. USA ⁇ 06 ⁇ 5):6099-6 ⁇ 04).
  • Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger & Dubensky (1999) Curr. Opin.
  • a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a gene of interest such as a polynucleotide encoding a CAR. Further details as to modern methods of vectors for use in gene transfer may be found in. for example, Kotterman et al. (2015) Viral Vectors for Gene Therapy: Translational and Clinical Outlook Annual Review of Biomedical Engineering 17.
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Pnimega Biotech (Madison, Wis.).
  • an engineered T-cell receptor comprising, consisting essentially of, or yet further consisting ⁇ : (a) an extracellular antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the extracellular antigen binding domain binds an antigen that binds an MIIC molecule, wherein the antigen binds to the MHC molecule with high affinity, and/or wherein the antigen induces a specific cytokine response, optionally an II.- 17, IFNy, and/or II.-5 response.
  • the antigen is bound to the MHC molecule.
  • the engineered T-cell receptor is a modified TCR.
  • the engineered T-cell receptor is a CAR.
  • the polynucleotide is the complement of a polynucleotide encoding an engineered T-cell receptor. Also provided herein are the expression product(s) of the polynucleotide encoding an engineered T-cell receptor.
  • the present disclosure provides an engineered T-cell receptor that comprises, consists, or alternatively consists essentially of an antigen binding domain that specifically recognizes and binds, binds, or is specific to an antigen bound to an MHC molecule.
  • the MHC binds to the antigen with high affinity, optionally an affinity less than 1000 nM.
  • the antigen binds to the MHC with high affinity, optionally an affinity less than 1000 nM.
  • the affinity of the interaction between the antigen and the MHC molecule is characterized by a dissociation constant (Kn) which is less than about 0.1 nM, less than than about I nM, less than about 5 nM. less than about 10 nM. less than about IS nM, less than about 20 nM, less than about SO nM, less than about 100 nM, less than about ISO nM, less than about 200 nM. less than about 250 nM, less than about 300 nM, less than about 350 nM. less than about 400 nM, less than about 450 nM, less than about 500 nM, less than about 550 nM, less than about 600 nM.
  • Kn dissociation constant
  • nM less than about 650 nM, less than about 700 nM, less than about 750 nM, less than about 800 nM, less than about 850 nM, less than about 900 nM, less than about 950 nM, less than about 1000 nM, less than about 1100 nM, less than about 1200 nM, less than about 1300 nM, less than about 1400 nM, less than about 1500 nM, less than about 2000 nM. less than about 2500 nM.
  • the affinity of the interaction between the antigen and the MHC molecule ranges from: about I nM to about 10 nM. about I nM to about 15 nM, about 1 nM to about 50 nM, about I nM to about 100 nM, about I nM to about 200 nM, about I nM to about 300 nM, about 1 nM to about 400 nM. about I nM to about 500 nM.
  • I nM to about 600 nM about I nM to about 700 nM, about I nM to about 800 nM, about I nM to about 900 nM, about I nm to about 1000 nM, about I nM to about 1 100 nM, about I nM to about 1200 nM, about I nM to about 1300 nM, about I nM to about 1400 nM, about 1 nM to about 1500 nM.
  • nM to about 2 uM about 1 nM to about 3 uM, about 1 nM to about 4 ⁇ , about 1 nM to about 5 uM , about 10 nM to about 15 nM, about 10 nM to about 50 nM, about 10 nM to about 100 nM. about 10 nM to about 200 nM. about 10 nM to about 300 nM, about 10 nM to about 400 nM, about 10 nM to about 500 nM, about 10 nM to about 600 nM, about 10 nM to about 700 nM, about 10 nM to about 800 nM. about 10 nM to about 900 nM.
  • about 50 nM to about 1300 nM, about 50 nM to about 1400 nM about 50 nM to about 1500 nM, about 50 nM to about 2 ⁇ .
  • the MHC's affinity for the antigen is less than about 1000 nM.
  • the antigen comprises, consists, or alternatively consists essentially of all or part of an epitope derived from an antigen of the group of: a microbial antigen, a viral antigen, a bacterial antigen, a fungal antigen, a protozoan antigen, an antigen involved in autoimmune disease, a neurodegenerative disease, an autoantigen. an allergy antigen, a graft rejection antigen, a tumor antigen, or a cancer or tumor antigen.
  • the antigen comprises all or part of a toxin.
  • the antigen comprises all or part of an epitope derived from an antigen involved in a neurodegenerative disease or disorder such as a-synucleinopathy, Parkinson's disease. Lewy Body dementia, or Alzheimer's disease.
  • the antigen comprises all or part of an epitope derived from a-synuclein (NM_000345, included herein as SEQ ID NO: 41; NM_001 146054, included herein as SEQ ID NO: 42: NM_001 146055, included herein as SF.Q ID NO: 43: and NM_007308 included herein as SF.Q ID NO: 44), Tau protein (Nl'JJOl 1 16538, included herein as SliQ ID NO: 45: NP_001 1 16539, included herein as SF.Q ID NO: 46: NP_001 190180.
  • a-synuclein NM_000345, included herein as SEQ ID NO: 41; NM_001 146054, included herein as SEQ ID NO: 42: NM_001 146055, included herein as SF.Q ID NO: 43: and NM_007308 included herein as SF.Q ID NO: 44
  • Tau protein Nl'JJOl
  • SF.Q ID NO: 47 NP_00l 190181 , included herein as SEQ ID NO: 48; and NP_005901, included herein as SEQ ID NO: 49), or TAR DNA- binding protein 43 (TDP-43. NP_031401. included herein as SEQ ID NO: 50: and NP_031401 I . included herein as SEQ ID NO: 51 ), or equivalents of each thereof.
  • amino acid sequence of a-synuclein is included herein as SEQ ID NO: 52:
  • the antigen comprises, consists, or alternatively consists essentially of all or part of any one of the peptides listed in the following Table 1. or equivalents thereof.
  • the antigen comprises, consists, or alternatively consists essentially of all or part of the amino acid sequence of the amino acid sequence KTKEGVLY VGSK.TK.E (SEQ ID NO: 55), MPVDPDNF.AYF.MPSE (SEQ ID NO: 56). DNEAYEMPSFEGYQD (SEQ ID NO: 57), EMPSEEGYQDYEPEA (SEQ ID NO: 58). VLYVGSKTK (SEQ ID NO: 59). or an equivalent of each thereof.
  • the length of the antigen disclosed herein may vary based on the particular MHC allele and/or the specific antigen recognition domain (eg. TCR, seFv, etc.).
  • the length of the antigen peptides according to some embodiments described herein may vary from, for example, at least 5, al least 6, at least 7, at least 8, at least 9, at least 10, at least I I, at least 12, at least 13, at least 14, at least 15. at least 16. at least 17. at least 18. at least 19. at least 20. between 20 - 25. between 20-30, between 30-40 amino acids, or up to 50 amino acids in length.
  • the antigen includes a core of at least at least 5 amino acids, at least 6, at least 7. at least 8, al least 9 and more.
  • the length of the autoantigenic peptide does not exceed about 100 amino acids, does not exceed about 50 amino acids, does not exceed about 30 amino acids, or docs not exceed 20 amino acids. According to some embodiments of the invention, the length of the autoantigenic peptide includes at least 5 and no more than 35 amino acids.
  • antigens or equivalents thereof that exhibit sequence identity to a reference a-synuclein, Tau, or TDP-43.
  • the antigens or equivalents thereof comprise, consist or consist essentially of a sequence at least 60% or more (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. etc.) identical to a reference peptide of Table I. or sub-sequence, portion, homologuc, variant thereof.
  • the antigens disclosed herein have one or more modifications, such as an amino acid addition to, deletion of, or substitution of any amino acid residue in any peptide set forth in Table 1 or to the reference sequence of ⁇ -symic!ein, Tau, or TDP-43.
  • a modified sequence is at least 80% or more, e.g., 80-85%, 85-90%, 90-95%, 95-100% identical to a Table I peptide or to the reference sequence of a-synuclein, Tau. or TDP- 43, or sub-sequence, portion, homologuc or derivative thereof or has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12. 13, 14. 15. 16. 17. 18. 19.20. 20-25. 25-30.30-50. 50-100. or more, additions to. deletions of. or substitutions.
  • antigen binding domains that recognize and/or bind modified and variant forms of antigens including but not limited to ⁇ -synuclein, Tau, TDP-43 peptides, or equivalents thereof.
  • modified and variant forms of antigens include but not limited to ⁇ -synuclein, Tau, TDP-43 peptides, or equivalents thereof.
  • modifications intend an antigen or equivalent thereof that deviates from a reference sequence.
  • modifications may have greater or less activity or function than a reference peptide such as ability to elicit, stimulate, induce, promote, increase or enhance a T-cell response or immune or inflammatory response.
  • the antigens or equivalents disclosed herein include sequences having substantially the same, greater or less relative activity or function as a T-cell epitope than a reference epitope set forth in in Table 1 , for example, an ability to elicit, stimulate, induce, promote, increase or enhance an immune response in vitro or in vivo.
  • Non-limiting examples of modifications include one or more amino acid substitutions (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9. 10, 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50-100, or more residues), additions and insertions (eg., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50-100, or more residues) and deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9. 10, 1, 12, 13, 14, 15, 16, 17. 18, 19, 20, 20-25, 25-30.30-50, 50-100) ofa reference antigen.
  • amino acid substitutions e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9. 10, 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50-100, or more residues
  • additions and insertions eg., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30.30-50,
  • antigens and peptides contemplated herein include but are not limited to acetylation, amidalion, azido group conjugated to the primary epsilon amino group on an inserted lysine or as 5-azidopentanoic acid on the N-terminus, biotinylation.
  • carrier proteins and MAP peptides e.g.
  • KLII or BSA conjugated peptides KLII or BSA conjugated peptides
  • MAP peptides modifications to increase cell penetration
  • conjugation to 5-azidopentanoic acid azidogroup conjugated to lysine or propargylglycine
  • F1TC 5,6 FAM, Rhodamine B
  • fluorescence/quencher pairs for FRET analysis e.g. Abz/Unp and EDANS/Uabcyl
  • formylation methylation.
  • phosphorylation of tyrosine, serine or threonine, conjugated to resin, DTT can be added if peptides contain several cysteines or other amino acids that are easily oxidized, sulfation of tyrosine, Tyr(S03H2), and unnatural amino acids: D-amino acids, Aib, Abu, Ahx, Om, pGlu, Nle, DAB, Cit, Hyp, Tyr(3- N02), or Met sulfoxide or sulfone.
  • antigens or equivalents thereof may further comprise independently at least 2, or alternatively at least 3, or alternatively at least 4, or alternatively at least 5. or at least 6, or alternatively at least 7. or alternatively at least 8. or alternatively at least 9 or alternatively at least 10 amino acids at the amino and/or carboxyl terminus of the polypeptide.
  • the antigens listed in Table I or equivalents thereof further comprise independently at least 2, or alternatively at least 3, or alternatively at least 4, or alternatively at least 5, or at least 6, or alternatively at least 7, or alternatively at least 8, or alternatively at least 9 or alternatively at least 10 amino acids at the amino and/or carboxyl terminus of the polypeptide.
  • antigens or equivalents thereof can be a part of or contained within a larger molecule, such as another peptide sequence, such as a fusion, heterologous domain, or chimera.
  • an addition is a chimeric fusion sequence or heterologous domain (i.e. an amino acid sequence having one or more molecules not normally present in a reference endogenous sequence covalently attached to the sequence).
  • the antigen is an Ml IC-restricted antigen.
  • the antigen binding domain specifically binds to both the antigen and the MHC molecule.
  • the antigen binding domain specifically binds a region spanning the antigen and MIIC bound to the antigen (i.e. the antigen-MI IC complex).
  • the M1IC molecule comprises, consists, or alternatively consists essentially of all or part of an MHC class I molecule (e.g. 1ILA-A, IILA-B, IILA-C, I1LA-E, I1LA-F, IILA-G, or CDI molecule).
  • the MHC molecule comprises, consists of, or alternatively consists essentially of an MIIC class II molecule (e.g. IILA-DM, IILA-DO, IILA-DP, IILA-DQ, and IILA-DR).
  • MIIC class II molecule e.g. IILA-DM, IILA-DO, IILA-DP, IILA-DQ, and IILA-DR.
  • the MHC molecule is a classical MHC molecule.
  • the MHC molecule is a non-classical MIIC molecule.
  • the extracellular antigen binding domain specifically binds and recognizes an antigen bound to a specific MHC allele or mutation. Additional information regarding HI .A alleles and their association with Parkinson's disease is available in Wissemann et al. (2013) Am J Hum Genet.93:984-993. PMC38241 16.
  • the antigen is bound to an MHC molecule comprising, consisting, or consisting essentially of all or part ⁇ HLA-A*I 1 :01, HLA-DRB5*01 :01, HLA- DRBI* I5:0I . HLA-DQB 1*03:04. HLA-DRB 1 *07:01. HI. A-DRB 1*09:01, or HI.A- DQB 1*03:01.
  • a nonlimiting exemplary amino acid sequence of HLA-A*1 1 :01 is provided herein as SEQ ID NO: 206:
  • a nonlimiting exemplary amino acid sequence of HI .A-DRB5*01 :01 is provided herein as SEQ ID NO: 207:
  • HLA-DQB 1*03:04 A nonlimiting exemplary sequence of HLA-DQB 1*03:04 is provided herein as SEQ ID NO: 209:
  • a nonlimiting exemplary sequence of II LA- DRD 1*09:01 is provided herein as SEQ ID NO: 21 1 :
  • the antigen bound to an MHC comprises, consists, or consists essentially of a peptide comprising the sequence KTKEGVLYVGSKTKE (SEQ ID NO: 55) or an equivalent thereof bound to DRB1* 15:01 or DRB5*01 :01.
  • a peptide comprising the sequence MPVDPDNEAYEMPSE (SEQ ID NO: 56) or an equivalent thereof " bound to an MHC molecule
  • a peptide comprising the sequence DNEAYEMPSEEGYQD (SEQ ID NO: 57) or an equivalent thereof bound to DRB5*01 :01
  • a peptide comprising the sequence EMPSEEGYQDYEPEA (SEQ ID NO: 58) or an equivalent thereof bound to an MHC molecule
  • a peptide comprising the sequence VLYVGSKTK (SEQ ID NO: 59) or an equivalent thereofbound to A*l 1 :01.
  • the antigen binding domain comprises, consists, or consists essentially of Fab, variable regions of a TCR, BCR, or Ig, or a fragment of an scFv (e.g. a VH or VL chain).
  • An scFv region can comprise the variable regions of the heavy (Vn) and light chains (VL) of immunoglobulins, connected with a short linker peptide.
  • the linker peptide may be from 1 to 50 amino acids, for instance. 1.2, 3, 4, 5. 6. 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17. 18, 19, 20, 21, 22, 23. 24, 25, 26. 27, 28. 29, 30, 31. 32.
  • the linker is glycine rich, although it may also contain serine or threonine.
  • the heavy chain variable region of the Ig comprises, or consists essentially thereof, or consists of those disclosed herein or an equivalent of each thereof and/or comprises one or more CDR regions comprising those disclosed herein or an equivalent of each thereof.
  • the light chain variable region of the Ig comprises, or consists essentially thereof, or consists of those disclosed herein or an equivalent of each thereof and/or comprises one or more CDR regions comprising those disclosed herein or an equivalent of each thereof.
  • Transmembrane Domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in this disclosure may be derived from the following:
  • the transmembrane domain is a CD3. CD8, or a CD28 transmembrane domain.
  • the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the engineered t-cell receptor.
  • a glycine-serine doublet provides a particularly suitable linker.
  • intracellular signaling domain (or cytoplasmic domain) of the engineered T-cell receptor is responsible for activation of at least one of the traditional cITcctor functions of an immune cell in which an engineered T-ccII receptor has been introduced.
  • the intracellular signaling domain comprises, consists, or consists essentially of the intracellular signaling domain of a co-stimulatory molecule.
  • the intracellular signaling domain refers to a portion of a protein which transmits the effector function signal and directs the immune cell to perform its specific function. An entire signaling domain or a portion thereof may be used so long as the portion is sufficient to transmit the effector function signal.
  • Cytoplasmic sequences of the T-cell receptor (TCR) and co-rcccptors, as well as derivatives or variants thereof, can function as intracellular signaling domains for use in a CAR or modified TCR.
  • Intracellular signaling domains of particular use in this disclosure may be derived from FcR (e.g. NM_000566 (SEQ ID NO: 242)).
  • CD3 (NM_000732 (SEQ ID NO: 218), NM_000733 (SF.Q ID NO: 219), NM_000073 (SEQ ID NO: 220)), phosphatide cylidylyltransfcrasc 1 (CDS, NM_001263 (SEQ ID NO: 232)), CD22 (NM_024916 (SF.Q ID NO: 236)), CD79a (NM_021601 , (SEQ ID NO: 252). NM_00I783 (SEQ ID NO: 253)), CD79B (NM_000626 (SEQ ID NO: 254)).
  • CD66d (NM_001277163 (SF.Q ID NO: 255).
  • the intracellular signaling domain of the engineered l-ccll receptor can comprise the signaling domain of CD28, 4- IBB, CD3 zeta, CD27 (NM_001242 (SEQ ID NO: 263)). ICOS. or OX40.
  • the intracellular signaling domain is derived from a protein of the same species as the subject In other embodiments, the intracellular signaling domain is derived from a protein of a different cell (e.g. macrophage, B cell) or a different species than the subject.
  • a second co-stimulatory signal may also be required.
  • the intracellular region of at least one co-stimulatory signaling molecule including but not limited to CD27 (NM 001242 (SEQ ID NO: 263)), CD28, 4- IBB, OX40, CD30 (NM_00I243 (SEQ ID NO: 257)), CD40 (NM_001250 (SEQ ID NO: 258)). programmed cell death protein 1 (PD- 1. NM_005018 (SEQ ID NO: 259)), ICOS.
  • lymphocyte function-associated antigen- 1 (LFA-l, NM_001 1 14380 (SEQ ID NO: 260)), CD2 (NM_001767 (SEQ ID NO: 261)), CD7 (NM_006137 (SEQ ID NO: 262)), CD27 (NM_001242 (SEQ ID NO: 263)), CD276 (NM_001024736 (SEQ ID NO: 264)), or a ligand that specifically binds with CD83, may also be included in the cytoplasmic domain of the engineered T-cell receptor.
  • the engineered T-cell receptor of the present disclosure can comprise one or more co- stimulatory domain.
  • a CAR may comprise one. two, or more co-stimulatory domains.
  • the costimulatory domain can be derived from the costimulatory domain of CD28, 4-1 BB, CD3 zeta, CD27, ICOS, or OX40. In some embodiments, the costimulatory domain is derived from a protein of the same species as the subject. In other embodiments, the costimulatory domain is derived from a protein of a different species than the subject.
  • the polynucleotide can further comprise a detectable marker or purification marker and/or a polynucleotide encoding a detectable marker or a purification marker, each conjugated to the polynucleotide.
  • the engineered T-cell receptor may optionally further comprise a spacer domain of up to 300 amino acids, preferably 5 to 100 amino acids, more preferably 25 to 50 amino acids.
  • the spacer may be 1. 2. 3. 4. 5. 6. 7, 8. 9. 10. 1 1. 12. 13. 14. 15. 16, 17. 18. 19, 20, 21, 22. 23, 24, 25. 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41.42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.
  • a spacer domain may comprise, for example, a portion of a human Fc domain, a CI 13 domain, or the hinge region of any immunoglobulin, such as IgA. IgD. IgE, IgG, or IgM, or variants thereof. Additional spacers include, but are not limited to, CD4, CD8, and CD28 hinge regions.
  • present disclosure provides vectors comprising, consisting, or alternatively consisting essential of a polynucleotide according to any of the embodiments described herein.
  • the polynucleotide is operatively linked to a promoter.
  • the vector is from the group of: a plasmid, a retroviral vector, a lentiviral vector. an adenoviral vector, or an adeno-associated viral vector.
  • the vector is an expression vector.
  • the vector is useful for integration in genomic DNA, replication, viral particle production, and/or infection or transduction with high efficiency.
  • the disclosed vectors comprise an element that enhances or induces a regulatory T-cell or a memory regulatory T-cell phenotype. In other embodiments, the disclosed vectors comprise an element that reduces or inhibit effector T-cell phenotype.
  • the isolated nucleic acid sequence is comprised in a vector.
  • the vector is a plasmid.
  • the vector is a viral vector.
  • the vector is a lcnliviral vector.
  • the term "vector” intends a recombinant vector that retains the ability to infect and transduce non-dividing and/or slowly-dividing cells and integrate into the target cell's genome.
  • the vector is derived from or based on a wild-type virus.
  • the vector is derived from or based on a wild-type lcnlivirus. Examples of such, include without limitation, human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV). simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV).
  • retrovirus can be used as a basis for a vector backbone such murine leukemia virus (MLV).
  • MLV murine leukemia virus
  • a viral vector according to the disclosure need not be confined to the components of a particular virus.
  • the viral vector may comprise components derived from two or more different viruses, and may also comprise synthetic components. Vector components can be manipulated to obtain desired characteristics, such as target cell specificity.
  • the recombinant vectors of this disclosure may be derived from primates and non- primates.
  • primate Icntiviruscs include the human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV).
  • the non-primate lcnliviral group includes the prototype "slow vims" visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
  • Recombinant lentiviral vectors are known in the art, c.g.. sec US Patent Nos. 6.924,123; 7,056.699; 7,07,993; 7,419,829 and 7,442,551.
  • Retroviral vectors for use in this disclosure include, hut are not limited to pl.enti series versions 4, 6, and 6.2 (Invilrogen); "ViraPower” system (Lenligen Corp.), plIIV-7-GFP, "Lenli- X”. pLVX, (Clontcch). pLKO.l-puro (Sigma-Aldrich), pLemiR (Open Biosystems), and pLV (Charitt. Medical School, Institute of Virology (CBF), Berlin, Germany).
  • assays include, for example, Southern and Northern blotting. RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (F.I.ISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.
  • assays include, for example, Southern and Northern blotting. RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (F.I.ISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.
  • the disclosed vectors can further comprise a regulatory element such as an enhancer element.
  • enhancers include, for example, WPRE, the human cytomegalovirus (HCMV) immediate early (IB) enhancer, the enhancer of the Moloney Murine Sarcoma Virus (MMSV), the U3 region of Rous Sarcoma Virus (RSV), the U3 region of Spleen Focus Forming Virus (SFFV), and the HCMV IE enhancer.
  • the disclosed vectors can further comprise a polynucleotide encoding all or part of forkhcad box P3 ("FoxP3," NM_001 1 14377. (SEQ ID NO: 265); and NM_014009. (SEQ ID NO: 266)) or an equivalent thereof.
  • FoxP3 may he operatively linked to a regulatory control element such as a promoter or an internal ribosomc entry site (IRES).
  • IRS internal ribosomc entry site
  • FoxP3 is fused to a detectable marker such as GFP.
  • the ubiquitin binding sites in FoxP3 arc mutated to reduce degradation and/or stabilize the protein.
  • the STUBl gene (NM_005861, (SF.Q ID NO: 267); and NM_001293I97 (SF.Q ID NO: 268) or its equivalent) is included in the vector to reduce degradation and/or stabilize the protein.
  • SF.Q ID NO: 269 A nonlimiting example of the nucleotide sequence of FoxP3 is included herein as SF.Q ID NO: 269:
  • AGTCCACTTC ACCAAGCCTG CCCTTGGACA AGGACCCGAT GCCCAACCCC
  • ATCCCAGTGC ACCCAGGAAG GACAGCACCC TTTCGGCTGT GCCCCAGAGC
  • CAACATGGAC TACTTCAAGT TCCACAACAT GCGACCCCCT TTCACCTACG
  • the disclosed vectors can further comprise a polynucleotide encoding all or part of II.- 10 (NM_000572 (SliQ ID NO: 270)) or an equivalent thereof.
  • IL-10 may be operatively linked to a regulatory control element such as a promoter or an internal ribosome entry site (IRES).
  • IL-10 is (used to a detectable marker such as GFP.
  • the IL- 10 is activation inducible.
  • expression of 11 -10 may be under the control of a promoter activated by an inflammatory response (e.g. mxl promoter activated by interferon).
  • SEQ ID NO: 271 A nonlimiting example of the nucleotide sequence of IL-10 is included herein as SEQ ID NO: 271:
  • the disclosed vectors can further comprise a suicide gene to induce cell death in cells comprising and/or expressing the vector.
  • the suicide gene is operatively linked to a promoter.
  • the promoter is inducible (e.g. tetracycline-inducible).
  • the suicide gene is triggered following adoptive cell treatment (Buddee et al., Pl.oS One, (2013)).
  • the suicide gene may function to downrcgulatc expression of the engineered T-cell receptor following binding to the target antigen (WO 2016/01 1210).
  • Non- limiting examples of suicide genes include caspasc-9 (NM_00I229, (SEQ ID NO: 272): NM_001278054 (SEQ ID NO: 273): or NM_032996 (SEQ ID NO: 274), or its equivalent) and thymidine kinase ⁇ NM_003258 (SEQ ID NO: 275) or its equivalent).
  • Engineered T-cell receptors described herein may comprise, consist of, or alternatively consist essentially of: (a) an extracellular antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the extracellular antigen binding domain binds an antigen that binds an MIIC molecule, wherein the antigen binds to the MHC molecule with high affinity, and/or wherein the antigen induces a specific cytokine response, optionally an IL- 17, IFNy, and/or IL-5 response.
  • the engineered T-cell receptor is a modified T-cell receptor. In other aspects, the engineered T-cell receptor is a chimeric antigen receptor (CAR). In certain aspects, the extracellular antigen binding domain binds both the antigen and the MHC molecule.
  • the polynucleotide or vector encodes the engineered T cell receptor. In one aspect, the cells express the polynucleotide or vector according to any of the embodiments disclosed herein. In some aspects, the cells comprise two or more polynucleotides encoding distinct engineered T-cell receptors. In some aspects, the cells comprise two or more vectors encoding distinct engineered T-cell receptors.
  • ITic cells comprising two or more vectors or polynucleotides may express engineered T-cell receptors that bind a plurality of antigens (e.g. the cells comprise a plurality of engineered T-cell receptors have distinct antigen specificities).
  • the cell is an isolated cell.
  • the cell is isolated or purified from a subject's peripheral blood mononuclear cells and in other aspects it is a cultured cell from a cell population that optionally is commercially available.
  • the cell is of any appropriate species for the subject being treating, e.g., mammalian, canine, feline, murine or human.
  • the cell is a leukocyte.
  • the leukocyte may be murine, canine, feline, simian, or human.
  • the cell is a T-cell.
  • the T-cell may be a regulatory T-cell. regulatory memory T-cell, a central memory T-cell.
  • the cell Is an NK cell that is isolated or a cultured cell from a cell population that optionally is commercially available.
  • the cell is of any appropriate species for the subject being treating, e.g., mammalian, canine, feline, murine or human.
  • the cell comprises and/or expresses an engineered T-cell receptor on the cell surface.
  • Engineered T-cell receptors described herein may comprise, consist of. or alternatively consist essentially of: (a) an extracellular antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the extracellular antigen binding domain binds an antigen that binds an MHC molecule, wherein the antigen binds lo the MHC molecule with high affinity, and/or wherein the antigen induces a specific cytokine response, optionally an 1L-17. lFNy, and/or 1L-5 response.
  • the engineered T-cell receptor is a modified T-cell receptor.
  • the engineered T-cell receptor is a chimeric antigen receptor (CAR).
  • the extracellular antigen binding domain binds both the antigen and the MHC molecule.
  • the polynucleotide or vector encodes the engineered T cell receptor.
  • the cell further comprises the antigen-MHC complex bound to the extracellular antigen binding domain.
  • the population is substantially homogenous.
  • substantially homogenous intends a plurality of cells of greater than 50%. 60%, 70%. 80%. or 95% purity or homogeneity.
  • the population is a heterogenous mixture of two or more cells comprising and/or expressing distinct engineered T-cell receptors with distinct antigen specificities.
  • non-human animal comprising, consisting, or alternatively consisting essentially of the polynucleotide or vector of any one of the embodiments described herein.
  • compositions comprising, consisting of, or alternatively consisting essentially of a carrier and one or more of the products disclosed herein: a polynucleotide, vector, engineered T cell receptor, cell, modified cell, population comprising said cells or modified cells, or composition according to any of the embodiments described herein.
  • Engineered T-cell receptors described herein may comprise, consist of, or alternatively consist essentially of: (a) an extracellular antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the extracellular antigen binding domain binds an antigen that binds an MHC molecule, wherein the antigen binds to the MHC molecule with high affinity, and/or wherein the antigen induces a specific cytokine response, optionally an IL-17, ⁇ , and/or IL-S response.
  • the engineered T-cell receptor is a modified T-cell receptor.
  • the engineered T-cell receptor is a chimeric antigen receptor (CAR).
  • the extracellular antigen binding domain binds both the antigen and the MHC molecule.
  • the polynucleotide or vector encodes the engineered T cell receptor.
  • the cell or modified cell expresses the polynucleotide or vector and/or comprises an engineered T cell receptor on a surface of the cell or modified cell.
  • the composition comprises one or more of the polynucleotide, vector, engineered T cell receptor, cell, modified cell, population comprising said cells or modified cells and, optionally, a carrier, such as but not limited to a pharmaceutically acceptable carrier
  • the composition comprises a pharmaceutically or physiologically acceptable carrier, diluent, or cxcipiunl.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like: carbohydrates such as glucose, mannose. sucrose or dcxtrans, mannitol; proteins: polypeptides or amino acids such as glycine: antioxidants: chelating agents such as F.DTA or glutathione: adjuvants (e.g.. aluminum hydroxide): and preservatives.
  • compositions of the present disclosure may be formulated for oral, intravenous, intranasal, intramuscular, intrathecal, topical, enteral, and/or parenteral administration.
  • the compositions of the present disclosure arc formulated for intravenous administration.
  • the compositions are administered systemically.
  • the addition of one or more antimicrobial agents such as chlorobulanol, ascorbic acid, parabens. thermerosal. or the like can be used to prevent the growth of microorganisms. It may also be preferable to include agents that alter the tonicity such as sugars or salts.
  • compositions comprising cells or modified cells
  • solutions can be prepared in suitable diluents such as saline, phosphate-buffered saline, hydrogcl, nutrient carrier, albumin, recombinant albumin, Dulhecco's Modified Ragle Medium, glucose, water, ethanol, glycerol, liquid polyethylene glycol(s), various oils, and/or mixtures thereof, and others known to those skilled in the art.
  • suitable diluents such as saline, phosphate-buffered saline, hydrogcl, nutrient carrier, albumin, recombinant albumin, Dulhecco's Modified Ragle Medium, glucose, water, ethanol, glycerol, liquid polyethylene glycol(s), various oils, and/or mixtures thereof, and others known to those skilled in the art.
  • Administration of the cells or compositions can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy and the subject being treated. In some aspects, a matrix and or catheter may be used. Preferably, the administration is in such an amount as will be therapeutically effective and immune modifying. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art.
  • administrations of a composition about, at least about, or at most about 3, 4, 5, 6, 7. 8, 9, 10 or more administrations.
  • the administrations will normally range from 1, 2, 3, 4, 5, 6, or 7 days to annual intervals, more usually from one to two week intervals.
  • Periodic boosters at intervals of every other day, twice a week, weekly, biweekly, monthly, or 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3,4 or 5 years, usually two years, will be desirable to maintain the condition of the immune system.
  • the administration(s) may be followed by assays for autoreactive immune responses, inflammatory cytokine production, cytotoxic cells, and T cell activity.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobulanol, phenol, sorbic acid, Ihimcrosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the cells, populations, vectors, and polynucleotides of the disclosure can be administered in combination with other traditional therapies, These include, but arc not limited to, the administration of immunosuppressive or modulating therapies or treatments and therapies to ameliorate the symptoms of and/or treat neurodegenerative disorders such as Parkinson's disease.
  • immunosuppressive agents or therapies include anti-inflammatory drugs such as sulfasalazine, corticosteroids such as prednisone, and immune system suppressors such as azathioprine and mercaptopurine.
  • Additional classes of immune modulating therapies or treatments include but arc not limited to a calcincurin inhibitor, a chemokine receptor inhibitor, a glucocorticoid, an mTOR inhibitor, an anti-metabolic compound, a phosphodicstcrasc-3 inhibitor, an antibody, or a leukocyte function antigcn-3/Fc fusion protein.
  • The cells and populations of cell are administered to the host using methods known in the art and described, for example, in PC17US2011/064191. This administration of the cells or compositions of the invention can be performed to generate an animal model of the relevant disease, disorder, or condition for experimental assays and screens.
  • kits comprising, consisting of, or alternatively consisting essentially of a composition as described herein and instructions for use. Additional reagents and/or instructions can further be provided as necessary.
  • a method of producing a modified cell comprising, consisting, or alternatively consisting essentially of: (i) introducing a polynucleotide or vector according to any of the embodiments described herein into a cell or a population of cells, and optionally culturing the cell or population of cells under conditions that favor expression of the polynucleotide or the vector; (ii) and further optionally selecting a cell or enriching a cell or a subpopulaiion of cells that have been successfully modified with the polynucleotide or vector of step (i).
  • the cells are selected from or isolated from a group consisting of leukocytes.
  • the T-cclls arc regulatory T-cclls, regulatory memory T-cells, naTve T-cells, central memory T-cells. effector memory T-cells, CD4+ T-cclls. or a CD8+ T-cclls.
  • the cell is isolated from a subject.
  • the subject may he a murine, canine, feline, simian, or a human.
  • step (i) comprises CRISPR mediated gene editing to introduce the polynucleotide into the genome of the target cell.
  • Methods of using CRISPR to perform gene editing are known in the art.
  • the polynucleotide or vector is introduced to the target cell via a viral particle comprising, consisting of, or alternatively consisting essentially of a polynucleotide or vector according to the embodiments described herein.
  • cells expressing the disclosed engineered T-cell receptors may be further modified to express one or more of: FoxP3, II.- 10, a detectable or selectable marker, a suicide gene, and/or an equivalent of each thereof as described herein,.
  • one or more of FoxP3, IL- 10, the detectable or selectable marker, and/or a suicide gene may be encoded on one or more vectors introduced to the cell or subpopulation of modified cells.
  • each gene may be operatively linked to an expression control element such as a promoter.
  • one or more of FoxP3, lL-10. the detectable or selectable marker, and/or a suicide gene are encoded on the same vector.
  • one or one or more of FoxP3, IL- 10, the detectable or selectable marker, and/or a suicide gene are encoded on separate vectors.
  • one or more of FoxP3, 1 L- 10, the delectable or selectable marker, and/or a suicide gene is encoded on the same vector that encodes an engineered T-cell receptor according to any of the embodiments described herein.
  • l ; oxP3 expression is induced by contacting the cell or subpopulation of modified cells with an effective amount of transforming growth factor beta 1-4 ("TUFB," eg. ⁇ ' ⁇ : N1MXW651; available from Pcprolcch rhlGl ⁇ cat# 100-21. I00-21C) and/or II .-10 (available from Peprotech rhll.-IO cat# 200-10), thereby inducing FoxP3 expression in the cell or subpopulation of modi I ted cells.
  • the culture conditions comprise about I to about 10 ng/ml.. or alternatively about 5 to about 20 ng/ml..
  • the culture conditions comprise about 10 ng/mL, or alternatively about 15 ng/mL, or alternatively about 20 ng/ml ., or alternatively about 25 ng/ml ., or alternatively about 30 ng/mL. or alternatively about 40 ng/mL. or alternatively about 50 ng/ml.
  • TGFp * and/or IL-10 is about 5 to about 100 ng/mL.
  • the cell or subpopulation of modified cells is contacted with an effective amount of anti-interferon ⁇ antibody (e.g. ThermoFisher cat# 16-731 l-8IXSkurkovich, S. et al. J. of Immune Based Therapies, Vaccines, and Antimicrobials, 4:1-8 (2015)) anti IL-5 antibody (e.g. mepolizumab (GlaxoSmithKline)) (Mukhergee, M. et al. World Allergy Organ J. 7( 1 ): 32 (2014)), anti-TNF antibody or inhibitor (e.g.
  • anti-interferon ⁇ antibody e.g. ThermoFisher cat# 16-731 l-8IXSkurkovich, S. et al. J. of Immune Based Therapies, Vaccines, and Antimicrobials, 4:1-8 (2015)
  • anti IL-5 antibody e.g. mepolizumab (GlaxoSmithKline)
  • anti-TNF antibody or inhibitor
  • the antibodies are activation-induced.
  • cells Prior to expansion and genetic modification of the cells disclosed herein, cells may be obtained From a subject - for instance, in embodiments involving autologous therapy - or a commercially available culture, that are available from the American Type Culture Collection (ATCC), for example.
  • ATCC American Type Culture Collection
  • Cells can be obtained from a number of sources in a subject, including peripheral blood mononuclear cells (PRMC), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • PRMC 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.
  • Isolation methods for use in relation to this disclosure include, but arc not limited to Life Technologies Dynabeads® system; STEMcell ' Technologies EasySepTM, RoboScpTM, RosetleSepTM, ScpMaleTM: Millenyi Biotec MACSTM cell separation kits, fluorescence activated cell sorting (FACS), and other commercially available cell separation and isolation kits.
  • Particular subpopulations of immune cells may be isolated through the use of beads or other binding agents available in such kits specific to unique cell surface markers. For example, MACSTM CD4+ and CD8-* MicroBcads or complement depletion may be used to isolate CD4» and CD8+ T-cells.
  • cells may be obtained through commercially available cell lines, including but not limited to BCL2 (AAA) Jurkat (ATCC® CRL-2902TM).
  • appropriate cells may be derived from stem cells or lymphoid progenitors including iPS cells, F.S cells, hematopoietic stem cells, common lymphoid progenitors (CI.Ps), DN1, DN2. DN3, DN4, or DP cells.
  • iPS cells including iPS cells, F.S cells, hematopoietic stem cells, common lymphoid progenitors (CI.Ps), DN1, DN2. DN3, DN4, or DP cells.
  • the cells are autologous to the subject being treated. In another aspect, the cells are allogeneic to the subject being treated. Packaging vector and cell tines
  • Polypeptides encoding engineered T-cell receptors can be packaged into a ientiviral or retroviral packaging system by using a packaging vector and cell lines.
  • the packaging plasmid includes, but is not limited to retroviral vector, Ientiviral vector, adenoviral vector, and adeno- assoeiated viral vector.
  • the packaging vector contains elements and sequences that facilitate the delivery of genetic materials into cells.
  • the retroviral constructs are packaging plasmids comprising at least one retroviral helper UNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required to package a replication incompetent retroviral vector, and for producing virion proteins capable of packaging the replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus.
  • the retroviral UNA sequence lacks the region encoding the native enhancer and/or promoter of the viral 5' I.TR of the virus, and lacks both the psi function sequence responsible for packaging helper genome and the 3' LTR, but encodes a foreign polyadenylation site, for example the SV40 polyadenylation site, and a foreign enhancer and/or promoter which directs efficient transcription in a cell type where virus production is desired.
  • the retrovirus is a leukemia vims such as a Moloney Murine Leukemia Virus (MMLV), the Human Immunodeficiency Virus (HIV), or the Gibbon Ape Leukemia virus (GALV).
  • the foreign enhancer and promoter may be the human cytomegalovirus (HCMV) immediate early (IF.) enhancer and promoter, the enhancer and promoter (U3 region) of the Moloney Murine Sarcoma Virus (MMSV), the 1)3 region of Rous Sarcoma Virus (RSV), the U3 region of Spleen Focus Forming Virus (SFFV), or the HCMV IE enhancer joined to the native Moloney Murine Leukemia Virus (MMI.V) promoter.
  • HCMV human cytomegalovirus
  • IF. Enhancer and promoter
  • U3 region of the Moloney Murine Sarcoma Virus
  • RSV Rous Sarcoma Virus
  • SFFV Rous Sarcoma Virus
  • SFFV Spleen Focus Forming Virus
  • HCMV IE enhancer joined to the native Moloney Murine Leukemia Virus
  • the retroviral packaging plasmid may consist of two retroviral helper UNA sequences encoded by plasmid based expression vectors, for example where a first helper sequence contains a cDNA encoding the gag and pol proteins of ecotropic MMLV or GALV and a second helper sequence contains a cUNA encoding the env protein.
  • the Env gene which determines the host range, may be derived from the genes encoding xenotropic, amphotropic, ecotropic.
  • polytropic polytropic (mink focus forming) or 10A1 murine leukemia virus env proteins, or the Gibbon Ape Leukemia Virus (GALV env protein, the Human Immunodeficiency Virus env (gpl60) protein, the Vesicular Stomatitus Virus (VSV) G protein, the Human T cell leukemia (IITLV) type I and II env gene products, chimeric envelope gene derived from combinations of one or more of the aforementioned env genes or chimeric envelope genes encoding the cytoplasmic and transmembrane of the aforementioned env gene products and a monoclonal antibody directed against a specific surface molecule on a desired target cell.
  • GLV env protein Gibbon Ape Leukemia Virus
  • gpl60 Human Immunodeficiency Virus env
  • VSV Vesicular Stomatitus Virus
  • IITLV Human T cell leukemia
  • the cells can be activated and expanded using generally known methods such as those described in U.S. Patent Nos. 6,352,694: 6,534.055; 6.905.680: 6.692,964: 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7, 175,843; 5.883.223: 6.905,874: 6.797.514; 6,867.041.
  • Stimulation with the antigen and/or MHC ex vivo can activate and expand the selected engineered T-cell receptor expressing cell subpopulalion.
  • Isolation methods for use in relation to this disclosure include, but are not limited to Lite Technologies Dynabeads® system activation and expansion kits; RD Biosciences PhosflowTM activation kits, Miltcnyi Biotcc MACS 1 * 1 activation/expansion kits, and other commercially available cell kits specific to activation moieties of the relevant cell.
  • Particular subpopulations of immune cells may be activated or expanded through the use of beads or other agents available in such kits. For example, u-CD3/a-CD28 Dynaheadsffi) may be used to activate and expand a population of isolated T-cclls.
  • the polynucleotides, cells, vectors, populations, and modified cells of the present disclosure may be used to induce an anti-inflammatory response in a cell, tissue, or subject, mediate an immune response in a cell, tissue, or subject or mediate an inflammatory response in a cell tissue, or subject in vitro, ex vivo, or in vivo.
  • the polynucleotides, cells, vectors, populations, and modified cells of the present invention may be administered either alone or in combination with diluents, known anti-cancer therapeutics, and/or with other components such as cytokines or other cell populations that are immunostimulatory to a subject in need thereof.
  • the cell, tissue, or subject may be canine, equine, murine, rat, simian, feline, or human.
  • Method aspects of the present disclosure relate to methods for inducing an antiinflammatory response, mediating an immune response, or mediating an inflammatory response in a subject in need thereof, the method comprising, consisting of, or alternatively consisting essentially of administering to a subject in need thereof an effective amount of the polynucleotides, vectors, cells, modified cells, compositions, or populations disclosed herein.
  • the response is characterized by suppression of pathogenic T-cells.
  • the response is characterized by increased or decreased expression of one or more pro-inflammatory cytokines in the cell, tissue, or subject, and optionally wherein the one or more pro-inflammatory cytokines comprise IL-lp, TNF-a, IFN- ⁇ , 1L-8.
  • the response is characterized by increased or decreased expression of one or more anti-inflammatory cytokines in the cell, tissue, or subject, and optionally wherein the one or more anti-inflammatory cytokines comprise TGF- ⁇ , IL-lRa, IL-4. IL-6, lL-IO. IL-I I. IL-13, 1L-3S, and/or INF-a.
  • the cell, modified cell, or population is autologous to the subject. The subject may he canine, equine, murine, rat, simian, feline, or human.
  • Additional method aspects of the present disclosure relate to methods for enhancing the activity of a regulatory T-ccll or a regulatory memory T-ccll, the methods comprising, consisting of. or alternatively consisting essentially of administering to a subject in need thereof, an effective amount of the polynucleotides, vectors, cells, modified cells, compositions, or populations disclosed herein.
  • the enhanced activity of the regulatory T-cell is characterized by increased expression of IL-10.
  • the subject may be canine, equine, murine, rat, simian, feline, or human.
  • a method of treating a disease or condition involving an inflammatory response or related to inflammation in a subject in need thereof comprising, consisting of, or alternatively consisting essentially of administering to the subject an effective amount of the polynucleotides, vectors, cells, modified cells, compositions, or populations disclosed herein. Success of the treatment can be determined by detecting improvement or stabilization of one or more clinical endpoints.
  • Exemplary clinical endpoints include but are not limited to reduction in expression of pro-inflammatory cytokines in the inflamed tissue (or systemically), increase in the expression of anti-inflammatory cytokines in the inflamed tissue (or systemically), reduction in infiltration of lymphocytes to the inflamed tissue, decreased numbers of circulating or localized pathogenic and/or cytotoxic cells, decreased numbers of auto-reactive pathogenic cells, expansion of regulatory cells, reduced pain, reduced swelling, reduced inflammation, reduced or stabilized neurological damage, and/or increased or stabilized function of the inflamed tissue.
  • the subject may be canine, equine, murine, ral, simian, feline, or human.
  • Also provided herein is a method of treating a neurodegenerative disease or disorder in a subject in need thereof, comprising, consisting of. or alternatively consisting essentially of administering to the subject an effective amount of the polynucleotides, vectors, cells, modified cells, compositions, or populations disclosed herein.
  • Engineered T-cell receptors described herein may comprise, consist of, or alternatively consist essentially of: (a) an extracellular antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the extracellular antigen binding domain binds an antigen that binds an M11C molecule, wherein the antigen binds to the MHC molecule with high affinity, and/or wherein the antigen induces a specific cytokine response, optionally an IL-I7, lFNy, and/or IL-S response.
  • the engineered T-cell receptor is a modified T-cell receptor.
  • the engineered T-cell receptor is a chimeric antigen receptor (CAR).
  • the extracellular antigen binding domain binds both the antigen and the MHC molecule.
  • the polynucleotide or vector encodes the engineered T cell receptor.
  • the cell or modified cell expresses the polynucleotide or vector and/or comprises an engineered T eel I receptor on a surface of the cell or modified cell.
  • the composition comprises one or more of the polynucleotide, vector, engineered T cell receptor, cell, modified cell, population comprising said cells or modified cells and, optionally, a carrier, such as but not limited to a pharmaceutically acceptable carrier.
  • the neurodegenerative disease or disorder is a- synuclcinopathy, Parkinson's disease, Lcwy Body dementia, or Alzheimer's disease.
  • the cell, modified cell, or population is autologous to the subject being treated. Success of the treatment can be determined by detecting improvement or stabilization of one or more clinical endnoints. Fxemplary clinical endpoints include hut are not limited to reduction in expression of pro-inflammatory cytokines in the inflamed tissue (or systemically).
  • the subject may be canine, equine, murine, rat, simian, feline, or human.
  • the peptide-reactive. cytotoxic T-cells identified in the examples below may contribute to the pathogenesis of neurodegenerative disease by targeting neurons and killing them.
  • the polynucleotides, vectors, cells, modified cells, compositions, or populations disclosed herein may function to treat neurodegenerative disease, halt progression of neurodegenerative disease, or prophy tactically prevent the onset of neurodegenerative disease by targeting these pathogencic T-cells and suppressive their inflammatory response.
  • the methods described herein further comprise administration of an effective amount of one or more immunosuppressive therapeutic compounds to the subject in need thereof.
  • the immunosuppressive therapeutic compounds include but are not limited to a calcineurin inhibitor, chemokine receptor inhibitor, a glucocorticoid, an m TOR inhibitor, an anti- metabolic compound, a phosphodicsterasc-5 inhibitor, an antibody, or a leukocyte function antigen-3/Fc fusion protein.
  • the subject may be canine, equine, murine, rat. simian, feline, or human.
  • the endogenous T-cclls and/or other lymphocytes of the patient are depicted prior to administration of an effective amount of the polynucleotides, vectors, cells, modified cells, compositions, or populations disclosed herein.
  • compositions of the present invention may be administered in a manner appropriate to the disease to be treated or prevented.
  • the quantity and frequency of administration will he determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
  • the polynucleotides, vectors, engineered T-cell receptors, cells, modified cells, compositions, or populations disclosed herein may be delivered or administered intravenously, intrathecal ly, intraperitoneally, intranasally, by inhalation, intramuscularly, suhcutaneously, or by other suitable means of administration such as inhalation therapy or intranasally.
  • CARs Chimeric antigen receptors
  • modified 1 -cell receptors offer two distinct approaches to alter immune cell specificity. The primary structural difference between these two receptors is based on their origin. CARs arc typically derived from the small chain variable fragments (scFv) of antibody molecules. In contrast, modified TCRs are typically derived from sequenced, disease-relevant T cell receptors. Generally, CARs recognize surface bound antigens expressed by targeted cells without regard to MHC presentation, while modified TCRs recognize peptides presented by MIIC molecules, similar to endogenous TCRs that recognize cognate antigen-MHC complexes. Because modified TCRs bind to antigen-MHC complexes, they can be used to target both extracellular and intracellular proteins. Doth approaches to develop adoptive cell therapies are described in this example.
  • T-cell epitopes appropriate for modulating the immune response with adoptive cell therapy and MHC haplotypes that can present the eplidopes are identified (e.g. through an epitope screen and next generation sequencing).
  • One method e.g. an epitope screen
  • samples comprising peripheral blood mononuclear cells (PBMCs) arc obtained from the venous blood of human subjects suffering from the disease or condition of interest (e.g. Parkinson's disease, autoimmune disorder, or inflammatory condition).
  • the MHC haplotypes orihe samples are identified by next- generation sequencing.
  • Peptides comprising epitopes derived from autoantigen targets or other disease-relevant antigen targets are synthesized.
  • the peptides are predicted to bind specific subtypes and/or alleles of MHC molecules, e.g. those identified to be associated with disease.
  • the PBMCs arc stimulated with the synthesized peptides in culture for about two weeks or an appropriate period to measure response.
  • inflammatory cytokines e.g. IFNy or 1L-5 arc measured in the samples to delect a specific peplidc-MIIC immune response.
  • Complexes of peptides with MHC molecules are chosen if the affinity of the binding of the peptide to the MHC is less than about 1000 nM or, in some embodiments, if the peptide elicits a cytokine response despite restriction to a particular MHC allele (e.g. binds promiscuously to a panel or several Ml IC alleles).
  • TCR polypeptide e.g. uSyn CD4+ T-cclls, 1ILA-A*1 1:01 CD3+ T-cells
  • T-cells that produce inflammatory cytokines upon stimulation with peptide and/or peptide-MHC complex are selected and sequenced to determine the amino acid sequence of the variable regions of their T-cell receptor (TCR).
  • Peptide-MHC multimers e.g. tetramers, (kxtramers) may be developed to enable cell-tracking and purification.
  • Exemplary uSyn peptides recognized by T-cells and the corresponding MHC allele are provided in Table 7 below:
  • scFv single-chain variable fragments
  • scFv is a fusion protein of the variable regions of immunoglobulin heavy and light chains, connected by linker peptide.
  • scFv can be created from subcloned heavy and light chains derived from a monoclonal antibody or hybridoma, or by phage display.
  • Peptide-MHC complexes identified in the epitope screen are selected for monoclonal antibody development or scFv development. For example, in Parkinson's disease, the following combinations are targeted for development:
  • the selection criteria for optimal scFv or antibodies includes clones that produce soluble recombinant IgG 1 Tor in vitro blocking assays. If a suitable rodent model for a disease or condition exists, labeled recombinant IgG produced is tested to determine cellular localization and efficacy in high-multi-dose PoC experiments. For example, for Parkinson's disease such models include but are not limited to Park2. LRRK2, and Synuclcin mutant mouse strains (available from Jackson Laboratory). Tetramers are developed for cell selection and purification. Once suitable scFv is identified, the amino acid sequence is determined.
  • the vector can further comprise a FoxP3 transcription factor to promote and maintain Trcg function.
  • the FoxP3 can be mutated to deactivate S ' fUBI or ubiquitin binding domains. In some aspects, additional gain of function mutations can he included.
  • the vector can further comprise UFP or another detectable marker.
  • the FoxP3 can be fused to GFP or another delectable marker to allow screening for FoxP3 positive cells.
  • the vector can further comprise a selection marker, killing marker, and/or a suicide gene (e.g. caspase 0) to attenuate the life of a cell transduced with the vector.
  • a selection marker, killing marker, and/or a suicide gene e.g. caspase 0
  • Such marker or gene can be inducible (e.g. rapamycin or doxycycline inducible).
  • the vector can comprise activation-inducible II. -10.
  • the vector is then introduced (e.g. via transduction, CRISPR, transfection) into enriched regulatory T-cells of a subject. Methods of enriching regulatory T-cdls are known in the art and described herein. Alternatively, the vector is introduced into regulatory memory T-cells, NK cells, CD4+ T-cells, CD8+ T-cells, central memory T-cells, naive T-cells. effector T-cells, or cytotoxic T-cells.
  • mice model is used to determine whether cells comprising the engineered T-cell receptor can modulate the immune response in an appropriate disease model.
  • Parkinson's disease such models may include Park2, LRRK2, and Synuclein mutant mouse strains (available from Jackson Laboratory). Additional models for Parkinson's use neurotoxins specific for DA SN neurons, including MPTP and 60HDA, to produce neuron loss.
  • viral vectors that overexpress o-syn may be used to mimic a familial form of Parkinson's disease in humans caused by gene multiplication of u-syn (AAV2-SYN mouse model, Theodore el al., J. Neuropathol Kxp. Neurol. 67: 1 149-58 (2008)).
  • the mice may be on a mixed C57/BL6 (!Ab) or C3II (lAk) background which may affect antigen presentation.
  • the pcptidc-MHC affinity is measured for the model. If the affinity is within an acceptable range (e.g. up to 1,000 nM) or the peptide induces a robust T-cell cytokine response (e.g. ⁇ , IL-S, or IL-17 response), an engineered T-ccll receptor is developed as described above. The CD4+ T-cell responses are characterized and compared to those measured in the human subjects.
  • mice comprising die engineered T-cell receptor are crossed to mice with MHC alleles of interest.
  • uSyn transgenic mice are crossed to DRB5*01 :01 , DRR 1* 15:01. DQBI «03:04.
  • the CD4+ T-cell responses are characterized and compared to those measured in the human cells.
  • T-regulatory cells comprising the engineered T-cells are administered to the mice via any appropriate method.
  • Treatment parameters are measured including decreased expression of proinflammatory cytokines, increased expression of anti-inflammatory cytokines, suppression of cytotoxic cells, expansion in vivo of regulatory T-cells, reduced inflammation in tissue, and/or amelioration of disease-specific symptoms.
  • For example, in Parkinson's disease, a reduction in the amount of neurodegeneration can be determined by assaying for neuronal loss (e.g. by cell counting of brain tissue sections). Behavioral tests can be performed to determine whether neuronal function has improved, remained static, or decreased at a reduced rate relative to untreated or control treated mice. Such behavioral tests include beam traversal, the cylinder test, and the adhesive test to assess the motor abilities of the mice.
  • Additional tests to measure treatment outcome include dopamine release and update assays (decreased dopamine release is associated with neurodegeneration). as well as assays for modulation of inflammatory cytokine expression in neuronal tissue, and assays to detect infiltration of B and T Lymphocytes.
  • adoptive cell therapy is performed in humans or other mammals.
  • the mode of administering the modified cells will vary by condition and target tissue. Administration may occur by any suitable method described herein.
  • the success of the therapy is measured by a reduction in clinical symptoms of the disease or condition, decrease in inflammation, decrease in expression of pro-inflammatory cytokines, increased expression of anti-inflammatory cytokines, reduction or suppression of cytotoxic T-cells, expansion of regulatory T-cells, reduction in the infiltration of B and T-cells into the target tissue, or arresting or suppressing the development of clinical symptoms.

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Abstract

L'invention concerne de nouveaux récepteurs de lymphocytes T modifiés comprenant des TCR et CAR modifiés ciblant les antigènes-MIIC pertinents pour une maladie. L'invention concerne également de nouvelles méthodes de traitement de maladies ou d'états associés à une inflammation et/ou à des troubles neurodégénératifs.
PCT/US2018/038478 2017-06-20 2018-06-20 Récepteurs de lymphocytes t modifiés et méthodes d'utilisation correspondantes WO2018236986A1 (fr)

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WO2020198299A1 (fr) * 2019-03-26 2020-10-01 The Board Of Trustees Of The Leland Stanford Junior University Compositions et procédés de caractérisation et de traitement de la maladie d'alzheimer
US11142570B2 (en) 2017-02-17 2021-10-12 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof

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WO2015157117A2 (fr) * 2014-04-09 2015-10-15 The Trustees Of Columbia University In The City Of New York Utilisation de leucocytes et de nouveaux biomarqueurs dans le diagnostic, la confirmation et le traitement d'un trouble neurologique
US20160175358A1 (en) * 2014-11-17 2016-06-23 Adicet Bio, Inc. Engineered gamma delta t-cells

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Publication number Priority date Publication date Assignee Title
WO2015157117A2 (fr) * 2014-04-09 2015-10-15 The Trustees Of Columbia University In The City Of New York Utilisation de leucocytes et de nouveaux biomarqueurs dans le diagnostic, la confirmation et le traitement d'un trouble neurologique
US20160175358A1 (en) * 2014-11-17 2016-06-23 Adicet Bio, Inc. Engineered gamma delta t-cells

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Publication number Priority date Publication date Assignee Title
US11142570B2 (en) 2017-02-17 2021-10-12 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof
US11827695B2 (en) 2017-02-17 2023-11-28 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof
WO2020198299A1 (fr) * 2019-03-26 2020-10-01 The Board Of Trustees Of The Leland Stanford Junior University Compositions et procédés de caractérisation et de traitement de la maladie d'alzheimer

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