EP4676499A2 - Methods and compositions for cellular immunotherapy- fib4 - Google Patents

Methods and compositions for cellular immunotherapy- fib4

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Publication number
EP4676499A2
EP4676499A2 EP24771517.0A EP24771517A EP4676499A2 EP 4676499 A2 EP4676499 A2 EP 4676499A2 EP 24771517 A EP24771517 A EP 24771517A EP 4676499 A2 EP4676499 A2 EP 4676499A2
Authority
EP
European Patent Office
Prior art keywords
cell
seq
car
cells
binding domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24771517.0A
Other languages
German (de)
French (fr)
Inventor
Katerina Akassoglou
Jae Kyu RYU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
J David Gladstone Institutes
Original Assignee
J David Gladstone Institutes
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Filing date
Publication date
Application filed by J David Gladstone Institutes filed Critical J David Gladstone Institutes
Publication of EP4676499A2 publication Critical patent/EP4676499A2/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/29—Multispecific CARs
    • 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

  • Fibrinogen is an abundant protein synthesized in the liver, present in human blood plasma at concentrations ranging from 1.5-4 g/L in healthy individuals with a normal half-life of 3-5 days. With fibrin, produced by thrombin-mediated cleavage, fibrinogen plays important roles in many physiological processes. Indeed, the formation of a stable blood clot, containing polymerized and cross-linked fibrin, is crucial to prevent blood loss. A balance between clotting, notably the conversion of fibrinogen to fibrin, and fibrinolysis, the proteolytic degradation of fibrin, is essential. Disruption of this equilibrium can cause disease in distinct manners.
  • Fibrinogen regulates blood coagulation by engaging ⁇ IIb ⁇ 3 integrin receptor via its ⁇ 408-411 epitope. Conversion of fibrinogen to fibrin exposes cryptic inflammatory epitopes. Fibrin induces inflammatory processes by engaging the Mac-1 receptor (other names CD11b/CD18, Complement receptor 3) via the fibrin ⁇ 377-395 epitope.
  • Mac-1 receptor other names CD11b/CD18, Complement receptor 3
  • Embodiments of the disclosure include a novel class of chimeric antigen receptor (CAR) T cells to specifically recognize fibrin and guide T cells at sites of lesions in the brain and periphery.
  • Fibrin CAR-T cells are engineered to recognize fibrin in lesions in autoimmune, neurodegenerative, inflammatory, and infectious diseases, cancer, and trauma.
  • a bispecific chimeric antigen receptor comprises a first antigen specific binding domain, a second antigen specific binding domain, a transmembrane domain(s), at least one co-stimulatory domain(s), and a CD3 ⁇ signaling domain, wherein the first antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof. In certain embodiments, the antigen specific binding domain specifically binds to a fibrin domain or fragment.
  • the antigen specific binding domain binds to a fibrin domain or fragment with higher affinity than fibrinogen. In certain embodiments, the antigen specific binding domain binds to an epitope of fibrin or fibrinogen ⁇ C domain. In certain embodiments, the antigen specific binding domain is mouse, human or humanized. In certain embodiments, the antigen specific binding domain blocks binding of fibrin to Mac-1. In certain embodiments, the antigen specific binding domain blocks macrophage or microglia activation. In certain embodiments, the first and second antigen specific binding domains comprise an antibody, antibody fragment, a camelid nanobody or aptamer. In certain embodiments, the antibody fragment is a single chain fragment.
  • the single chain fragment is a single chain variable fragment (scFv).
  • the at least one co-stimulatory domain comprises a cluster of differentiation antigen 28 (CD28), 41BB domain, an ICOS (Inducible T cell Co-stimulator) (CD278), OX40 (CD134), Glucocorticoid-induced Tumor Necrosis Factor Receptor (GITR), CD40 or CD27.
  • the second antigen specific binding domain specifically binds to tumor antigens, exogenous antigens, endogenous antigens or autoantigens.
  • endogenous antigens comprise cluster of differentiation antigens (CD), cell surface molecules or antigens associated with neurodegenerative diseases.
  • the cluster of differentiation antigens comprise CD1a, CD2, CD3, CD5, CD10, CD11c, CD13, CD15, CD19, CD20, CD25, CD30, CD33, CD99, CD103, CD117, Docket No.: 354406.00202 CD123 or CD207.
  • the cluster of differentiation antigen is CD19.
  • the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 9.
  • the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 9.
  • the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 11.
  • the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 11.
  • the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 10.
  • the second antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 10.
  • the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 12.
  • the second antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 12.
  • the CAR further comprises a hinge region.
  • the bispecific chimeric antigen receptor comprises at least a 75% sequence identity to SEQ ID NO: 13.
  • the bispecific chimeric antigen receptor comprises SEQ ID NO: 13.
  • a bispecific chimeric CAR comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 13.
  • the bispecific chimeric CAR comprises an amino acid sequence comprising SEQ ID NO: 13.
  • the CAR further comprises at least one hinge region.
  • a chimeric antigen receptor (CAR) comprises an amino acid sequence comprising at least a 75% sequence identity to SEQ ID NO: 1 or 5.
  • the chimeric antigen receptor (CAR) comprises an having an amino acid sequence SEQ ID NO: 1 or 5.
  • the CAR further comprises at least one hinge region.
  • a chimeric antigen receptor comprises an amino acid sequence comprising at least a 75% sequence identity to SEQ ID NO: 2 or 6.
  • the chimeric antigen receptor comprises an amino acid sequence SEQ ID NO: 2 or 6.
  • the CAR further comprises at least one hinge region.
  • a chimeric antigen receptor comprises an amino acid sequence comprising at least a 75% sequence identity to SEQ ID NO: 3 or 7.
  • the Docket No.: 354406.00202 chimeric antigen receptor (CAR) comprises an amino acid sequence SEQ ID NO: 3 or 7.
  • an expression vector encodes an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, the vector encodes an amino acid sequence comprising SEQ ID NO: 4, 8, or 14. In certain embodiments, the vector comprises adenovirus, adeno-associated virus (AAV), herpes simplex virus, lentivirus, gammaretrovirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus or vesicular stomatitis virus. In certain embodiments, the vector is a lentivirus.
  • AAV adeno-associated virus
  • the vector is a lentivirus.
  • the vector further comprises an inducible promoter, a cell specific promoter, a tissue specific promoter or a constitutive promoter. In certain embodiments, the vector further comprises one or more enhancer or regulatory sequences. In certain embodiments, the vector further comprises an inducible suicide gene. In certain embodiments, the vector further comprises a nucleic acid sequence encoding for one or more cytokines, chemokines, hormones, growth factors or combinations thereof.
  • an isolated cell comprises a chimeric antigen receptor or an expression vector embodied herein.
  • the isolated cell comprises a T cells, B cells, natural killer (NK) cells, macrophages, stem cells, induced pluripotent stem cells (iPSCs) or combinations thereof.
  • the T cell is a CD8 + T cell, a CD4 + T cell, a regulatory T cell (Treg), gamma delta T cells ( ⁇ T cells), or a tumor infiltrating T lymphocyte (TIL).
  • an isolated T cell is transduced with a vector to express a first antigen specific binding domain, a second antigen specific binding domain, a transmembrane domain(s), co-stimulatory domain(s), and a CD3 ⁇ signaling domain, wherein the first antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof and the second antigen domain binds to a cluster of differentiation antigen 19 (CD19).
  • the co-stimulatory domain comprises a CD28, a 41BB polypeptide or the combination thereof.
  • the first antigen specific binding domain wherein the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 9.
  • the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 9. In certain embodiments, the first antigen specific binding domain Docket No.: 354406.00202 comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 11. In certain embodiments, the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 11. In certain embodiments, the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 10. In certain embodiments, the second antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 10. In certain embodiments, the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 12.
  • the second antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 12.
  • the CAR further comprises a hinge region.
  • the bispecific chimeric antigen receptor comprises at least a 75% sequence identity to SEQ ID NO: 13.
  • the bispecific chimeric antigen receptor comprises SEQ ID NO: 13.
  • the CAR further comprises at least one hinge region.
  • an isolated cell comprises a chimeric antigen receptor (CAR) comprises an amino acid sequence comprising at least a 75% sequence identity to SEQ ID NO: 1 or 5.
  • the chimeric antigen receptor (CAR) SEQ ID NO: 1 or 5.
  • an isolated cell comprises a chimeric antigen receptor (CAR) comprises an amino acid sequence comprising at least a 75% sequence identity to SEQ ID NO: 2 or 6. In certain embodiments, the chimeric antigen receptor (CAR) comprises SEQ ID NO: 2 or 6. In certain embodiments, the CAR further comprises at least one hinge region. [0016] In another aspect, an isolated cell comprises a chimeric antigen receptor (CAR) comprises an amino acid sequence comprising at least a 75% sequence identity to SEQ ID NO: 3 or 7. In certain embodiments, the chimeric antigen receptor (CAR) comprises SEQ ID NO: 3 or 7.
  • an isolated cell comprises an expression vector encodes an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 4, 8 or 14.
  • the vector encodes an amino acid sequence comprising SEQ ID NO: 4, 8, or 14.
  • the vector comprises adenovirus, adeno-associated virus (AAV), herpes simplex virus, lentivirus, gammaretrovirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles Docket No.: 354406.00202 virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus or vesicular stomatitis virus.
  • AAV adeno-associated virus
  • the vector is a lentivirus. In certain embodiments, the vector further comprises an inducible promoter, a cell specific promoter, a tissue specific promoter or a constitutive promoter. In certain embodiments, the vector further comprises one or more enhancer or regulatory sequences. In certain embodiments, the vector further comprises an inducible suicide gene. In certain embodiments, the vector further comprises a nucleic acid sequence encoding for one or more cytokines.
  • an isolated cell comprises a bispecific chimeric antigen receptor comprising a first antigen specific binding domain, a second antigen specific binding domain, a transmembrane domain(s), co-stimulatory domain(s), and a CD3 ⁇ signaling domain, wherein the first antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof and the second antigen domain binds to a cluster of differentiation antigen 19 (CD19).
  • the co-stimulatory domain comprises a CD28, a 41BB polypeptide or the combination thereof.
  • the first antigen specific binding domain wherein the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 9.
  • the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 9. In certain embodiments, the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 11. In certain embodiments, the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 11. In certain embodiments, the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 10. In certain embodiments, the second antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 10. In certain embodiments, the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 12.
  • the second antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 12.
  • the CAR further comprises a hinge region.
  • the bispecific chimeric antigen receptor comprises at least a 75% sequence identity to SEQ ID NO: 13.
  • the bispecific chimeric antigen receptor comprises SEQ ID NO: 13.
  • the CAR further comprises at least one hinge region.
  • a method of treating a subject in need of immunotherapy comprises isolating T lymphocytes from a biological sample obtained from the subject; transducing the T lymphocytes with an expression vector encoding a chimeric antigen receptor (CAR) which specifically binds to a fibrin antigenic epitope; expanding the transduced T lymphocytes at least once ex vivo to obtain expanded T lymphocytes specific for the fibrin antigen; and reinfusing the T lymphocytes into the subject, thereby treating the subject.
  • the T lymphocytes are autologous cells.
  • the CAR comprises an antigen binding domain linked to at least one co-stimulatory domain and a CD3 signaling domain, wherein the antigen binding domain comprises a single chain variable fragment (scFv) which specifically binds to fibrin.
  • the co-stimulatory domain comprises a CD28, a 41BB polypeptide or the combination thereof.
  • the CAR comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 1 or 5.
  • the CAR comprises SEQ ID NO: 1, or 5.
  • the CAR comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 2 or 6.
  • the CAR comprises SEQ ID NO: 2 or 6.
  • the CAR comprises an scFv amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 3 or 7. In certain embodiments, the CAR comprises SEQ ID NO: 3 or 7.
  • the T lymphocyte is a CD8 + T lymphocyte, a CD4 + T lymphocyte, a regulatory T lymphocyte (Treg) or a tumor infiltrating T lymphocyte (TIL).
  • the immunotherapy is administered to a subject diagnosed with cancer, a neurodegenerative disorder or an autoimmune disorder.
  • a method of treating a subject in need of immunotherapy comprises isolating T lymphocytes from a biological sample obtained from the subject; transducing the T lymphocytes with an expression vector encoding a bispecific chimeric antigen receptor (CAR) comprising a first antigen specific binding domain, a second antigen specific binding domain, a transmembrane domain(s), a co-stimulatory domain(s), and a CD3 ⁇ signaling domain, wherein the first antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof and the second antigen domain binds to a cluster of differentiation antigen 19 (CD19), expanding the transduced T lymphocytes at least once ex vivo to obtain expanded T lymphocytes expressing the bispecific CAR; and reinfusing the T lymphocytes into the subject, thereby treating the subject.
  • CAR bispecific chimeric antigen receptor
  • the co-stimulatory domain comprises a CD28, a 41BB polypeptide or the Docket No.: 354406.00202 combination thereof.
  • the T lymphocytes are autologous cells.
  • the T lymphocyte is a CD8 + T lymphocyte, a CD4 + T lymphocyte, a regulatory T lymphocyte (Treg) or a tumor infiltrating T lymphocyte (TIL).
  • the immunotherapy is administered to a subject diagnosed with cancer, a neurodegenerative disorder or an autoimmune disorder.
  • an isolated natural killer (NK) cell comprises a chimeric antigen receptor (CAR) wherein the CAR comprises an intracellular activating signaling domain, a transmembrane region and an extracellular antigen binding domain.
  • the intracellular activating signaling domain comprises a CD28 or CD137 molecule.
  • the extracellular antigen binding domain is a single-chain variable fragment which specifically binds to a tumor antigen.
  • an isolated natural killer (NK) cell comprises a chimeric antigen receptor (CAR) wherein the CAR comprises an intracellular activating signaling domain, a transmembrane region, a first extracellular antigen binding domain and a second antigen binding domain.
  • the intracellular activating signaling domain comprises a CD28 or CD137 molecule.
  • the first extracellular antigen binding domain is a single-chain variable fragment which specifically binds to fibrin.
  • the second extracellular antigen binding domain is a single-chain variable fragment which specifically binds to a tumor antigen.
  • a method of treating cancer comprises administering the isolated T lymphocytes embodied herein or the NK cells embodied herein.
  • a chimeric antigen receptor comprises an antigen specific binding domain, a transmembrane domain(s), a co-stimulatory domain(s), and a CD3 ⁇ signaling domain, wherein the antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof.
  • the antigen specific binding domain comprises an antibody, antibody fragment, a camelid nanobody or aptamer.
  • the antibody fragment is a single chain fragment.
  • the single chain fragment is a single chain variable fragment (scFv).
  • the co-stimulatory domain(s) comprise a cluster of differentiation antigen 28 (CD28), 41BB domain, an ICOS (Inducible T cell Co-stimulator) (CD278), OX40 (CD134), Glucocorticoid-induced Tumor Necrosis Factor Docket No.: 354406.00202 Receptor (GITR), CD40 or CD27.
  • the antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 1 or 5.
  • the antigen specific binding domain comprises SEQ ID NO: 1 or 5.
  • the antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 2 or 6.
  • the antigen specific binding domain comprises SEQ ID NO: 2 or 6. In certain embodiments, the antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 3 or 7. In certain embodiments, the antigen specific binding domain comprises SEQ ID NO: 3 or 7. In certain embodiments, the CAR further comprises a hinge region.
  • a chimeric antigen receptor comprises an antigen specific binding domain, a transmembrane domain(s), a co-stimulatory domain(s), and a CD3 ⁇ signaling domain, wherein the antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof.
  • the antigen specific binding domain comprises an antibody, antibody fragment, a camelid nanobody or aptamer.
  • the antibody fragment is a single chain fragment.
  • the single chain fragment is a single chain variable fragment (scFv).
  • the co-stimulatory domain(s) comprise a cluster of differentiation antigen 28 (CD28), 41BB domain, an ICOS (Inducible T cell Co-stimulator) (CD278), OX40 (CD134), Glucocorticoid-induced Tumor Necrosis Factor Receptor (GITR), CD40 or CD27.
  • the antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In certain embodiments, the antigen specific binding domain comprises SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • a pharmaceutical composition comprises the chimeric antigen receptor (CAR) embodied herein, or the expression vector embodied herein, or the isolated cell embodied herein, or the isolated T cell embodied herein, or the isolated NK cell embodied herein.
  • CAR chimeric antigen receptor
  • the chimeric antigen receptor (CAR) embodied herein, or the expression vector embodied herein, or the isolated cell embodied herein, or the isolated T cell embodied herein, or the isolated NK cell embodied herein are administered as a combination therapy with one or more other therapeutic agents.
  • the scFv which specifically binds to fibrin comprises SEQ ID NO: 1 (FIB4scFv, PMC 1694 Retro vector): [0029] DIVMTQAAFSNPITLGTSASMSCRSSKSLLHSSGITYLSWYLQKPGQSPQLLIYQ MSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKGG GGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWIHWVKQRPGQG LEWIGLIDPSDSYTNYNQKFRGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCASSDPTGC WGQGTTLTVSP [0030] In certain embodiments, the scFv-CAR which specifically binds to fibrin further includes SEQ ID NO: 2 (mCD3z CAR PMC 1694 Retro vector)
  • the bispecific chimeric antigen receptor comprises SEQ ID NO: 13 (PMC 1792, bi-specific, Lenti vector CAR): [0053] DIVMTQAAFSNPITLGTSASMSCRSSKSLLHSSGITYLSWYLQKPGQSPQLLIYQ MSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKGG GGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWIHWVKQRPGQG LEWIGLIDPSDSYTNYNQKFRGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCASSDPTGC WGQGTTLTVSPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC
  • a vector encodes a fluorescent peptide, e.g. green fluorescent protein, comprising SEQ ID NO: 16: [0059] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK LPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRA EVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRH NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAG ITLGMDELYK [0060] In certain embodiments, a vector encodes a signal peptide, comprising SEQ ID NO: 17: [0061] MALPVTALLLPLALLLHAARP.
  • a vector encodes a transferrin peptide, comprising SEQ ID NO: 18: [0063] KNPDPWAKNLNEKDY [0064] In certain embodiments, a vector encodes a T2A ribosome skip sequence comprising SEQ ID NO: 19: [0065] EGRGSLLTCGDVEENPGP [0066] In certain embodiments, a vector encodes a FLAG sequence comprising SEQ ID NO: 20: [0067] DYKDDDDK [0068] In certain embodiments, the chimeric antigen receptor (CAR) binds an ⁇ 377-395 epitope of the fibrin or fibrinogen ⁇ C domain.
  • CAR chimeric antigen receptor
  • the CAR can alternatively bind an ⁇ 190-202 epitope of the fibrin or fibrinogen ⁇ C domain.
  • Such antibodies are monoclonal antibodies, and in various aspects humanized antibodies or human antibodies.
  • a fibrin ⁇ 377-395 epitope comprises, CKKTTMKIIPFNRLTIG (SEQ ID NO: 21), or a biologically active derivative thereof.
  • the CARs comprise sequences derived from antibodies comprising a light chain with three complementarity determining regions comprising an amino acid sequence including RSSKSLLHSSGITYLS (SEQ ID NO: 22), QMSNLAS (SEQ ID NO: 23), and AQNLELPLT (SEQ ID NO: 24).
  • the CARs comprise sequences Docket No.: 354406.00202 derived from antibodies comprising a heavy chain with three complementarity determining regions comprising an amino acid sequence including GYTFTSYWIH (SEQ ID NO: 25), LIDPSDSYTNYNQKFRG (SEQ ID NO: 26), and SDPTGC (SEQ ID NO: 27).
  • the CARs comprise sequences derived from antibodies comprising a light chain with three complementarity determining regions comprising an amino acid sequence including RSSKSLLHSSGITYLS (SEQ ID NO: 22), QMSNLAS (SEQ ID NO: 23), and AQNLELPLT (SEQ ID NO: 24) and a heavy chain with three complementarity determining regions comprising an amino acid sequence including GYTFTSYWIH (SEQ ID NO: 25), LIDPSDSYTNYNQKFRG (SEQ ID NO: 26), and SDPTGC (SEQ ID NO: 27).
  • the CARs comprise sequences derived from antibodies comprising the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:28: [0071] Asn Thr Ala Phe Ala Gly Phe Gly Arg Asn Met Arg Ser Leu Phe Ser Leu Gln Leu Leu Ser Thr Gln Asp Leu Ala Met Gly Trp Ser Cys Ile Ile Val Leu Leu Val Ser Thr Ala Thr Gly Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Thr Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Leu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Leu Thr
  • the antigen binding domains, e.g. scFvs, of the CARs are derived from antibodies which bind human fibrin or fibrinogen ⁇ C domain, and inhibits Mac-1 binding to fibrin or fibrinogen ⁇ C domain, and wherein the antibody light chain variable domain comprises the amino acid sequence of SEQ ID NO: 29: [0073] Thr Phe Asp Ser Pro Tyr Gln Val Arg Arg Met Arg Phe Ser Ala Gln Leu Leu Gly Leu Leu Val Leu Trp Ile Pro Gly Ser Thr Ala Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Ile Thr Leu Gly Thr Ser Ala Ser Met Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Ser Gly Ile Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 1.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 2.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 3.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 3.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 3.
  • an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, Docket No.: 354406.00202 sequence identity to SEQ ID NO: 3.
  • an amino acid sequence comprises SEQ ID NO: 3.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 4.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 5.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 5.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 5. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 5. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 5.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 6.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 6.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 6. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, Docket No.: 354406.00202 sequence identity to SEQ ID NO: 6. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 6.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 7.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 7.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 7. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 7. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 7.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 8.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 8.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 8. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 8. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 8.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 9.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 9.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 9.
  • an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, Docket No.: 354406.00202 sequence identity to SEQ ID NO: 9.
  • an amino acid sequence comprises SEQ ID NO: 9.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 10.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 11.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 11.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 11. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 11. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 11.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 12.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 12.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 12. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, Docket No.: 354406.00202 sequence identity to SEQ ID NO: 12. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 12.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 13.
  • a vector comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14.
  • a vector comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14.
  • a vector comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, a vector comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, a vector comprises SEQ ID NO: 4, 8 or 14.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, Docket No.: 354406.00202 sequence identity to SEQ ID NO: 15. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 15.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 15.
  • an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NOS: 16-28 or 29.
  • an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NOS: 16-28 or 29.
  • an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NOS: 16-28 or 29. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NOS: 16-28 or 29. In certain embodiments, an amino acid sequence comprises any one of SEQ ID NOS: 16-28 or 29.
  • the amino acid sequences comprising SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16-28 or 29 comprise one or more modified amino acids, non- natural amino acids, variants or combinations thereof.
  • a method of treating a demyelination disorder in a subject comprises administering a pharmaceutical composition comprising a therapeutically effective amount of an isolated T cell that is modified or transduced with a vector to express a bispecific chimeric antigen receptor (CAR) comprising a first antigen specific binding domain, a second antigen specific Docket No.: 354406.00202 binding domain, a transmembrane domain(s), a co-stimulatory domain(s), and a CD3 ⁇ signaling domain, wherein the first antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof.
  • CAR bispecific chimeric antigen receptor
  • the first and second antigen specific binding domains comprise an antibody, antibody fragment, a camelid nanobody or aptamer.
  • the antibody fragment is a single chain fragment.
  • the single chain fragment is a single chain variable fragment (scFv).
  • the co- stimulatory domain comprises a cluster of differentiation antigen 28 (CD28), 41BB domain, an ICOS (Inducible T cell Co-stimulator) (CD278), OX40 (CD134), Glucocorticoid-induced Tumor Necrosis Factor Receptor (GITR), CD40 or CD27.
  • the second antigen specific binding domain specifically binds to tumor antigens, exogenous antigens, endogenous antigens or autoantigens.
  • endogenous antigens comprise cluster of differentiation antigens (CD) or cell surface molecules.
  • the cluster of differentiation antigens (CD) comprise CD1a, CD2, CD3, CD5, CD10, CD11c, CD13, CD15, CD19, CD20, CD25, CD30, CD33, CD99, CD103, CD117, CD123 or CD207.
  • the antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • the antigen specific binding domain comprises SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NOs: 9 or 11.
  • the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NOs: 9 or 11.
  • the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NOs: 10 or 12.
  • the second antigen specific binding domain comprises SEQ ID NOs: 10 or 12.
  • the bispecific chimeric antigen receptor further comprises a hinge region.
  • the bispecific chimeric antigen receptor comprises at least a 75% sequence identity to SEQ ID NO: 13. In certain embodiments, the bispecific chimeric antigen receptor comprises SEQ ID NO: 13.
  • the isolated T cell expressing the CAR is a regulatory T (CAR-Treg) cell.
  • CAR-Treg regulatory T
  • CC1 + Olig2 + oligodendrocytes are increased in number as compared to baseline controls.
  • the CAR-Treg cells inhibit demyelination as compared to baseline controls.
  • the CAR-Treg cells remyelination as compared to Docket No.: 354406.00202 baseline controls.
  • the Treg cell comprises an autologous cell, an allogeneic cell, a haplotype matched cell, a haplotype mismatched cell, a haplo-identical cell, a xenogeneic cell, cell lines or combinations thereof.
  • the demyelinating disorder comprises: periventricular leukomalacia, multiple sclerosis, acute disseminated encephalomyelitis, chronic inflammatory demyelinating polyneuropathy, adrenoleukodystrophy, adenomyeloneuropathy, Leber's hereditary optic atrophy, HTLV-associated myelopathy, Guillain- Barre syndrome, phenylketonuria, Tay-Sachs disease, Niemann-Pick disease, Gaucher's disease, Hurler's syndrome, Krabbe's disease, Pelizaeus-Merzbacher disease, cerebral palsy, traumatic brain injury or neonatal brain injury or a combination thereof.
  • the method further comprises administering immature oligodendrocytes, pluripotent stem cells or the combination thereof.
  • immature oligodendrocytes pluripotent stem cells or the combination thereof.
  • the term can mean within an order of magnitude, preferably within 5-fold, and also preferably within 2- fold, of a value.
  • the term “about” meaning within an acceptable error range for the particular value should be assumed. All numeric values are herein assumed to be modified by the term “about”, whether or not explicitly indicated.
  • the recitation of numerical ranges by endpoints includes all numbers within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5). [0099] In the description and in the claims, phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features.
  • affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. Affinity also includes the term “avidity,” which refers to the strength of the antigen-antibody bond after formation of reversible complexes. Methods for calculating the affinity of an antibody for an antigen are known in the art, including use of binding experiments to calculate affinity. Antibody activity in functional assays (e.g., flow cytometry assay) is also reflective of antibody affinity. Antibodies and affinities can be phenotypically characterized and compared using functional assays (e.g., flow cytometry assay).
  • the term “agent” is meant to encompass any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent capable of preventing, ameliorating, or treating a disease or other medical condition.
  • the term includes small molecule compounds, antisense oligonucleotides, siRNA reagents, antibodies, antibody fragments bearing epitope recognition sites, such as Fab, Fab ⁇ , F(ab ⁇ )2 fragments, Fv fragments, single chain antibodies, antibody mimetics (such as DARPins, affibody molecules, affilins, affitins, anticalins, avimers, fynomers, Kunitz domain peptides and monobodies), peptoids, aptamers; enzymes, peptides organic or inorganic molecules, natural or synthetic compounds and the like.
  • an agent can be assayed in accordance with the methods of the invention at any stage during clinical trials, during pre-trial testing, or following FDA-approval.
  • ameliorate is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • amino acid refers to naturally occurring and synthetic ⁇ , ⁇ , ⁇ , and ⁇ amino acids, and includes but is not limited to, amino acids found in proteins, i.e.
  • Non-naturally occurring amino acids include, for example, E-alanine (E-Ala), norleucine (Nle), norvaline (Nva), homoarginine (Har), 4-aminobutyric acid (J-Abu), 2- aminoisobutyric acid (Aib), 6-aminohexanoic acid (H-Ahx), ornithine (orn), sarcosine, D-amino isobutyric acid, 3-aminopropionic acid, 2,3-diaminopropionic acid (2,3-diaP), D- or L- phenylglycine, D-(trifluoromethyl)-phenylalanine, and D-p-fluorophenylalanine.
  • antibody includes monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules, as well as antibody fragments (e.g., Fab, F(ab') 2 , and Fv).
  • immunoglobulin Ig
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain.
  • the 4-chain unit is generally about 150,000 daltons.
  • Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each Docket No.: 354406.00202 other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N-terminus, a variable domain (V H ) followed by three constant domains (C H ) for each of the ⁇ and ⁇ chains and four C H domains for ⁇ and ⁇ isotypes.
  • Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (C H1 ). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a V H and V L together forms a single antigen-binding site.
  • the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.
  • the intact antibody may have one or more effector functions.
  • An “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng.8(10): 1057-1062 [1995]); single-chain antibody molecules and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produced two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily.
  • the Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant Docket No.: 354406.00202 domain of one heavy chain (CH1).
  • VH variable region domain of the H chain
  • CH1 first constant Docket No.: 354406.00202 domain of one heavy chain
  • Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.
  • Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen.
  • autoimmune disease refers to a disease or condition in which a subject's immune system has an aberrant immune response against a substance that does not normally elicit an immune response in a healthy subject.
  • autoimmune diseases include Acute Disseminated Encephalomyelitis (ADEM), Acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome (APS), Autoimmune angioedema, Autoimmune aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia, Autoimmune immunodeficiency, Autoimmune inner ear disease (AIED), Autoimmune myo
  • Acute Disseminated Encephalomyelitis Acute necrotizing hemorrhagic le
  • chimeric antigen receptor refers to an antigen- binding domain that is fused to an intracellular signaling domain capable of activating or stimulating an immune cell, and in certain embodiments, the CAR also comprises a transmembrane domain.
  • the CAR's extracellular antigen-binding domain is composed of a single chain variable fragment (scFv) derived from fusing the variable heavy and light regions Docket No.: 354406.00202 of a murine or humanized monoclonal antibody.
  • scFvs may be used that are derived from Fab's (instead of from an antibody, e.g., obtained from Fab libraries).
  • a CAR-T cell is a T cell that expresses a chimeric antigen receptor.
  • chimeric antigen receptor refers to a recombinant fusion protein that has an antigen-specific extracellular domain coupled to an intracellular domain that directs the cell to perform a specialized function upon binding of an antigen to the extracellular domain.
  • the terms “artificial T-cell receptor,” “chimeric T-cell receptor,” and “chimeric immunoreceptor” may each be used interchangeably herein with the term “chimeric antigen receptor.”
  • combination therapy refers to those situations in which two or more different agents are administered in overlapping regimens so that the subject is simultaneously exposed to both agents. When used in combination therapy, two or more different agents may be administered simultaneously or separately. This administration in combination can include simultaneous administration of the two or more agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, two or more agents can be formulated together in the same dosage form and administered simultaneously. Alternatively, two or more agents can be simultaneously administered, wherein the agents are present in separate formulations.
  • a first agent can be administered just Docket No.: 354406.00202 followed by one or more additional agents.
  • two or more agents may be administered a few minutes apart, or a few hours apart, or a few days apart.
  • the transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.
  • the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having.”
  • the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim.
  • diabodies refers to small antibody fragments prepared by constructing sFv fragments with short linkers (about 5-10) residues) between the VH and VL domains such that inter- chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites.
  • Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.
  • Diabodies are described in greater detail in, for example, EP 404,097; WO 93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). [00113] “Diagnostic” or “diagnosed” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of “true positives”).
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) can recognize and bind antigen, although at a lower affinity than the entire binding site.
  • “hinge” or “hinge region” refers to a flexible connector region, e.g. natural or synthetic polypeptides, or any other type of molecule, providing structural flexibility and spacing to flanking polypeptide regions.
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR (hereinafter defined) of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and/or capacity.
  • donor antibody such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and/or capacity.
  • framework (“FR”) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc.
  • the number of these amino acid substitutions in the FR are typically no more than 6 in the H chain, and in the L chain, no more than 3.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a “human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos.6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et al., Proc. Natl. Acad. Sci.
  • HVR hypervariable region
  • VL VL1 L2, L3
  • H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies.
  • the Kabat Complementarity Determining Regions are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Docket No.: 354406.00202 Mol. Biol. 196:901-917 (1987)).
  • the AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.
  • the “contact” HVRs are based on an analysis of the available complex crystal structures.
  • immune cells refers to any cells of the immune system that are involved in mediating an immune response.
  • Non-limiting examples of immune cells include a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell, neutrophil, or combination thereof.
  • an immune cell expresses CD3.
  • the CD3-expressing immune cells are T cells (e.g., CD4 + T cells or CD8 + T cells).
  • an immune cell that can be targeted with a targeting moiety comprises a naive CD4 + T cell.
  • an immune cell comprises a memory CD4 + T cell. In some aspects, an immune cell comprises an effector CD4 + T cell. In some aspects, an immune cell comprises a na ⁇ ve CD8 + T cell. In some aspects, an immune cell comprises a memory CD8 + T cell. In some aspects, an immune cell comprises an effector CD8 + T cell. In some aspects, an immune cell comprises a gamma delta T cell. In some aspects, an immune cell is a dendritic cell.
  • a dendritic cell comprises a plasmacytoid dendritic cell (pDC), a conventional dendritic cell 1 (cDC1), a conventional dendritic cell 2 (cDC2), inflammatory monocyte derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDCs), Kupffer cells, or any combination thereof.
  • pDC plasmacytoid dendritic cell
  • cDC1 conventional dendritic cell 1
  • cDC2 conventional dendritic cell 2
  • inflammatory monocyte derived dendritic cells Langerhans cells
  • dermal dendritic cells lysozyme-expressing dendritic cells
  • Kupffer cells or any combination thereof.
  • Engineered immunoglobulin domains can include components of monoclonal antibodies, antibody fragments such as Fab fragment, a F(ab')2 fragment, a single-chain variable fragment (scFv), a single-chain antibody fragment (scAb), a single domain heavy chain antibody, and a single domain light chain antibody, VHH antibodies (or nanobodies), and other single domain antibodies (sdAbs).
  • Antigen fragments such as Fab fragment, a F(ab')2 fragment, a single-chain variable fragment (scFv), a single-chain antibody fragment (scAb), a single domain heavy chain antibody, and a single domain light chain antibody, VHH antibodies (or nanobodies), and other single domain antibodies (sdAbs).
  • Natural immunoglobulin and immunoglobulin-like domains are found in cell surface receptors, co-receptors, and co-activators and in soluble proteins involved in the recognition, binding and adhesion processes of cells.
  • inflammatory disease refers to a disease or condition characterized by aberrant inflammation (e.g., an increased level of inflammation compared to a control such as a healthy person not suffering from a disease).
  • inflammatory diseases include autoimmune diseases, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, Behcet's disease, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, inflammatory bowel disease, Addison's disease, Vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory
  • a “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other Docket No.: 354406.00202 immunoglobulins.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present disclosure may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd ed.1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat.
  • the hybridoma method e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual
  • phage-display technologies see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol.222: 581-597 (1992); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol.
  • neurodegenerative disorder refers to a disease or condition in which the function of a subject's nervous system becomes impaired.
  • Examples of neurodegenerative diseases that may be treated with a compound, pharmaceutical composition, or method described herein include Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer- Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, chronic fatigue syndrome, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, frontotemporal dementia, Gerstmann-St Hurssler-Scheinker syndrome, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, kuru, Lewy body Docket No.: 354406.00202 dementia, Machado-Joseph disease (S)
  • naked antibody refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel.
  • a “natural amino acid” refers to the twenty genetically encoded alpha- amino acids. See, e.g., Biochemistry by L. Stryer, 3 rd ed.1988, Freeman and Company, New York for structures of the twenty natural amino acids.
  • nucleic acid As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer.
  • Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.
  • “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Docket No.: 354406.00202 [00131] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
  • parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), intravitreal (i.v.i), intracisterna magna (i.e.m), or intrasternal injection, or infusion techniques.
  • patient or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred.
  • methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • a polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA).
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • T thymine
  • polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids.
  • the terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers.
  • a “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed or chemically synthesized as a single moiety.
  • Polypeptide fragment refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, in which the remaining amino acid sequence is usually identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long, or at least 70 amino acids long.
  • Single-chain Fv also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the V H and V L antibody domains connected into a single polypeptide chain.
  • the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding.
  • “Functional fragments” of the antibodies of the disclosure comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the Fc region of an antibody which retains or has modified FcR binding capability.
  • antibody fragments include linear antibody, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.
  • an “unnatural amino acid,” “non-natural”, “modified amino acid” or “chemically modified amino acid” refers to any amino acid, modified amino acid, or amino acid analogue other than the twenty genetically encoded alpha-amino acids.
  • Unnatural amino acids have side chain groups that distinguish them from the natural amino acids, although unnatural amino acids can be naturally occurring compounds other than the twenty proteinogenic alpha- Docket No.: 354406.00202 amino acids. In addition to side chain groups that distinguish them from the natural amino acids, unnatural amino acids may have an extended backbone such as beta-amino acids.
  • Non-limiting examples of non-natural amino acids include selenocysteine, pyrrolysine, homocysteine, an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAc ⁇ -serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L- phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine,
  • variable region refers to the amino-terminal domains of the heavy or light chain of the antibody.
  • the variable domains of the heavy chain and light chain may be referred to as “V H ” and “V L ”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the Docket No.: 354406.00202 antigen binding sites. However, the variability is not evenly distributed across the entire span of the variable domains.
  • variable domains hypervariable regions
  • FR framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • variants refers to an amino acid sequence that is altered by one or more amino acid residues.
  • the variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have “nonconservative” changes (e.g., replacement of glycine with tryptophan).
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • virus includes any type of virus or virus vector.
  • adenovirus adeno-associated virus (AAV), recombinant adeno-associated virus (rAAV), herpes simplex virus, lentivirus, gammaretrovirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus, vesicular stomatitis virus, picornavirus.
  • AAV adeno-associated virus
  • rAAV recombinant adeno-associated virus
  • herpes simplex virus lentivirus
  • gammaretrovirus retrovirus
  • alphavirus alphavirus
  • flavivirus rhabdovirus
  • measles virus Newcastle disease virus
  • poxvirus poxvirus
  • vaccinia virus modified Ankara virus
  • vesicular stomatitis virus picornavirus.
  • the virus is a chimeric virus, a synthetic virus, a recombinant virus, a mosaic virus or a pseudotyped virus.
  • range format various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically Docket No.: 354406.00202 disclosed all the possible subranges as well as individual numerical values within that range.
  • a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6.
  • the recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.
  • genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable.
  • the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates.
  • the genes or gene products disclosed herein which in some embodiments relate to mammalian nucleic acid and amino acid sequences, are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds.
  • the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides and proteins are human.
  • the term “gene” is also intended to include variants.
  • the practice of the present disclosure employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed.
  • FIG.1 is a schematic representation showing the design of FIB4 fibrin-specific human CAR construct.
  • PMC 1947 Amino-acid sequence of the FIB4-CAR (top).
  • FIG. 2 is a schematic representation showing the design of FIB4 fibrin-specific mouse CAR construct.
  • FIG.3 is a series of plots showing a flow cytometry analysis of FIB4-CAR expression assessed by GFP and F(ab ⁇ )2 antibody.
  • FIG.4 is a series of photographs of cell stains and a graph showing results from a FIB4- CAR cell adhesion assay to detect Hoechst stained cells transduced with fibrin-specific FIB4-CAR or control-CAR cells on fibrin-coated plates.
  • FIGS. 5A and 5B show the in vivo target recognition of FIB4-CAR in fibrinogen- injected mouse brain. Microscopy of GFP-expressing FIB4-CAR or GFP-expressing control-CAR transfected HEK293 recruited into the corpus callosum following injection with vehicle control artificial cerebrospinal fluid (ACSF) or fibrinogen.
  • ACSF cerebrospinal fluid
  • FIG. 6 is a schematic showing the design of bi-specific fibrin/CD19-CAR construct.
  • PMC 1792 Amino-acid sequence of the FIB4-CAR (top).
  • FIG.7 shows a series of in vivo imaging of bi-specific FIB4/CD19-CAR human T cells.
  • FIG.8 is a series of immunostains showing fibrin engagement of bi-specific FIB4/CD19- CAR human T cells after traumatic brain injury.
  • Microscopy of coronal brain slices from TBI animals treated with bi-specific FIB4/CD19-or CD19-CAR T cells (GFP + cells, green) and immunostained with fibrinogen antibody (red) reveals substantial recruitment of bi-specific FIB4/CD19-CAR T cells, but not control CD19-CAR T cells, to sites of fibrin deposition.
  • FIG.9 is a plot showing a flow cytometry analysis of FIB4-CAR expression assessed by GFP in human Treg cells.
  • FIG.10 is a series of immunostains and a graph demonstrating detection of FIB4-CAR Treg cells in the LPC-lesioned site.
  • FIG.11 is a series of immunostains and a graph demonstrating the effect of FIB4-CAR Treg cells on remyelinating oligodendrocyte in LPC-injected brain.
  • Right graph Quantification of CC1+OLIG2+ oligodendrocytes in corpus callosum area demyelinated by LPC injection.
  • FIG. 12 is a series of immunostains and a graph demonstrating the remyelination by FIB4-CAR Treg cells in the brain area demyelinated by LPC injection. Microscopy of coronal brain sections from LPC-injected mice treated with vehicle (PBS), Treg cells, and FIB4-CAR Treg cells at 14 d post-LPC injection immunostained for MBP. Right graph: Quantification of demyelinated area in the corpus callosum of LPC injected mice.
  • the present disclosure is directed to chimeric antigen receptors (CARs) targeting fibrin proteins and peptides.
  • Fibrin is deposited only in disease tissues in a wide range of neurological, autoimmune, inflammatory diseases and cancer. Fibrin-CARs serve as a guide to direct T cells in diseased tissues and enables the recruitment of CAR-T cells in solid organs. Previous approaches have failed to guide CART cells, in tissues, such as solid tumors, due to lack of a target antigen.
  • Fibrin CARs overcome these challenges, as they target fibrin deposits in tissues and accumulate specifically at lesion sites with increased selectivity and reduce toxic off-target effects of immunotherapy.
  • a novel class of fibrin CAR-T cells were engineered to express antigen-specific binding domains to recognize fibrin using the variable sequences from a mouse monoclonal fibrin- specific antibody (FIB4) raised against the fibrin epitope ⁇ 377-395 within the fibrinogen ⁇ C domain.
  • Antibodies directed against the ⁇ 377-395 domain blocks the damaging effects of fibrin in the nervous system without affecting its beneficial effects in blood coagulation. These monoclonal antibodies can block formation of MS plaques, neurodegeneration in AD models, and certain Docket No.: 354406.00202 cancers.
  • Exemplary antibodies of the invention include, for example, the FIB4 antibody (targeting the ⁇ 377-395 epitope). See, US Patent 8877195, incorporated herein in its entirety.
  • the CARs can be transduced into T cells, such as cytotoxic T cells or regulatory T cells. These fibrin-specific CARs are used to direct the antigen specificity of regulatory T cells, cytotoxic T cells to target the lesion environment at sites of fibrin deposition. As fibrin is deposited early in disease and persist in chronic stages of inflammation and tissue damage, fibrin-CAR-T cells can be used to guide T cells in disease tissue early and persist during the course of disease. [00165] Fibrin [00166] The activation of innate immunity, disruption of the blood brain barrier (BBB) and deposition of fibrin are intimately linked in neurological diseases.
  • BBB blood brain barrier
  • the blood-coagulation factor fibrinogen extravasates into the central nervous system (CNS) parenchyma after disruption of the BBB and is converted into insoluble fibrin, a key proinflammatory matrix that activates innate immune responses (Petersen, M. A., Ryu, J. K. & Akassoglou, K. Fibrinogen in neurological diseases: mechanisms, imaging and therapeutics. Nat. Rev. Neurosci.19, 283–301 (2016). Davalos, D. & Akassoglou, K. Fibrinogen as a key regulator of inflammation in disease. Semin. Immopathol.34, 43–62 (2012). Both references incorporated herein by reference in their entireties).
  • fibrinogen into fibrin exposes amino acids 377–395 in the fibrinogen ⁇ -chain that bind to the CD11b I- domain of the CD11b-CD18 integrin receptor (also known as Mac-1, complement receptor 3 (CR3), or ⁇ M ⁇ 2 ) and induces the activation of microglia and macrophages (Adams, R. A. et al.
  • the fibrin- derived ⁇ 377-395 peptide inhibits microglia activation and suppresses relapsing paralysis in central nervous system autoimmune disease. J. Exp. Med.204, 571–582 (2007).
  • Sequence ⁇ 377-395(P2), but not ⁇ 190-202(P1), is the binding site for the alpha MI-domain of integrin ⁇ M ⁇ 2 in the ⁇ C-domain of fibrinogen. Biochemistry 42, 9365–9373 (2003). Davalos, D. et al. Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation. Nat. Commun. 3, 1227 (2012). Ryu, J. K. et al. Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation. Nat. Commun.6, 8164 (2015)).
  • Fibrin is deposited in AD and MS lesions at Docket No.: 354406.00202 sites of microglial activation and macrophage infiltration. Fibrin is detected in progressive MS and in active and chronic lesions. In progressive MS, deposition of fibrin in the cortex correlates with neuronal loss and inhibition of fibrinolysis (Yates, R. L. et al. Fibrin(ogen) and neurodegeneration in the progressive multiple sclerosis cortex. Ann. Neurol.82, 259–270 (2017)). Disruption of the BBB and deposition of fibrin also occur early in MS and precede demyelination.
  • Fibrinogen has been proposed as a cerebrospinal fluid and plasma biomarker for AD and mild cognitive impairment, and increased concentrations of fibrinogen are considered a predictor of brain atrophy in AD.
  • Depletion of fibrin either genetically in fibrinogen-deficient mice or by anticoagulants decreases neuroinflammation, demyelination and axonal damage in animal models of MS and reduces the activation of microglia, damage to white matter and cognitive decline in animal models of AD.
  • Fibrin induces rapid and sustained microglial responses and infiltration of macrophages into the CNS (Ryu, J.K., et al. Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration. Nat Immunol 19, 1212–1223 (2016).
  • a TCR is a heterodimeric cell surface protein of the immunoglobulin super-family, which is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. TCRs exist in ⁇ and ⁇ forms, which are structurally similar but have quite distinct anatomical locations and probably functions. The extracellular portion of native heterodimeric ⁇ TCR and ⁇ TCR each contain two polypeptides, each of which has a membrane-proximal constant region, and a membrane-distal variable region. Each of the constant and variable regions include an intra-chain disulfide bond.
  • variable regions contain the highly polymorphic loops analogous to the complementarity determining regions (CDRs), also known as hypervariable regions, of antibodies.
  • CDRs complementarity determining regions
  • the variable regions of both the TCR ⁇ and TCR ⁇ chain each have three CDRs, numbered CDR1, CDR2, and CDR3 in the direction from the amino terminal end to the Docket No.: 354406.00202 carboxy terminal end.
  • CDR3 is the main CDR responsible for recognizing processed antigen.
  • the TCR ⁇ CDR3 has been recognized as more structurally diverse than the other CDRs. [00170]
  • the techniques for determining CDRs are generally known in the art. In some embodiments, the CDRs can be determined by approaches based on cross-species sequence variability.
  • the CDRs can be determined by approaches based on crystallographic studies of antigen-antibody complexes. In addition, combinations of these approaches are sometimes used in the art to determine CDRs.
  • CDRs can be determined using sequence-based prediction tools. Such tools are generally available in the art, e.g., the Loupe V(D)J Browser provided by 10 ⁇ Genomics® (Pleasanton, Calif.). For instance, in one embodiment, the single cell TCR sequencing of epitope reactive T-cell population can be conducted using the 10 ⁇ Genomics® platform. Then the sequence can be processed using the Loupe V(D)J Browser to identify the clonotypes, V(D)J genes, and the CDR motifs, etc.
  • CARs consist of three major domains: 1) extracellular domain (ectodomain), which can be further divided into an antigen recognition domain, a single peptide on the cell surface cleaved from the mature CAR cell (Goulart L.
  • the antigen recognition domain is a single-chain fragment variant (scFV) chiefly comprising heavy and variable light chain regions composed of an antigen-specific immunoglobin separated by a flexible linker and attached to the transmembrane domain by a spacer (hinge) responsible for the transmission of receptor binding signals (Zhang C., et al. (2017). Engineering CAR-T Cells. Biomark. Res. 5, 22. Docket No.: 354406.00202 doi:10.1186/s40364-017-0102-y).
  • scFV single-chain fragment variant
  • a transmembrane domain is essential for receptor stability and surface expression; it is a hydrophobic alpha helix that extends in the cell membrane (Zhang et al., 2017). 3) An intracellular domain (endo-domain), which upon stimulation, clusters and undergoes conformational changes, thus enabling the recruitment and phosphorylation of downstream signaling proteins (Su, X., and Vale, R. (2018). Mechanisms of Chimeric Antigen Receptor (CAR) Signaling during T Cell Activation. Biophysical J. 114, 107a–108a. doi:10.1016/j.bpj.2017.11.625).
  • CAR Chimeric Antigen Receptor
  • the CAR comprises one or more co-stimulatory domains comprising: CD28, ICOS, OX-40 or 41BB.
  • the intracellular signaling region of a CAR or cell of the disclosure may comprise signaling regions from one, two, three, four or all five of these proteins in addition to the other regions specified herein.
  • the co-stimulatory domains of a CAR or cell of the disclosure may comprise co- stimulatory domains from both 41BB and CD28.
  • the 41BB co-stimulatory domain can be downstream of the CD28 co-stimulatory domains.
  • a CD28 and CD3 ⁇ sequence comprises 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15.
  • a CD28 and CD3 ⁇ sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15.
  • a CD28 and CD3 ⁇ sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15.
  • a CD28 and CD3 ⁇ sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15.
  • a CD28 and CD3 ⁇ sequence comprises SEQ ID NO: 15.
  • the CAR may also comprise a spacer or hinge region situated between the antigen binding region and T cell plasma membrane.
  • a spacer or hinge is a sequence derived from IgG subclass IgG1, IgG4, IgD or CD8.
  • the hinge region comprises a CD28 motif.
  • the hinge region can have any length.
  • the hinge region comprises 1 amino acid or 10 amino acids or 20 amino acids or 50 amino acids or 60 amino acids or 70 amino acids or 80 amino acids or 100 amino acids or 120 amino acids or 140 amino acids or Docket No.: 354406.00202 160 amino acids or 180 amino acids or 200 amino acids or 250 amino acids or 300 amino acids or any number therebetween.
  • a CAR may further comprise a linker region. This may be rich in glycine for flexibility.
  • the linker region may be rich in serine and threonine for solubility.
  • the linker region can connect to N-terminus of variable heavy (VH) chain with the C-terminus of the variable light (VL) chain or vice versa.
  • the CARs can be encoded by a vector and/or encompassed in one or more delivery vehicles and formulations as described in detail below.
  • Antigen Binding Domain Numerous antigen-binding domains are known in the art, including those based on the antigen binding site of an antibody, antibody mimetics, nanobodies, and T-cell receptor fragments.
  • the antigen-binding domain may comprise: a single- chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder such as a camelid; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor.
  • the antigen specific binding domain includes, without limitation, an antibody, a T cell receptor fragment, a soluble T cell receptor, nanobody, aptamer, syn/notch recognition domain/effector domain pair, receptors, fragments or combinations thereof.
  • the antigen specific binding domain is a T cell variable region fragments.
  • the antigen specific binding domain is an antibody or fragment thereof.
  • the CAR can include single chains of T cell receptors and antibodies.
  • the antigen binding domain is a single chain variable fragment (scFv).
  • the antigen binding domain is or comprises an antibody or antibody fragment, aptamers, proteins and the like.
  • the antibodies are human antibodies, including any known to bind a targeting molecule.
  • antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab ⁇ )2 fragments, Fab ⁇ fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • Fab fragment antigen binding
  • rIgG Fab ⁇ fragments
  • VH variable heavy chain
  • the term encompasses genetically engineered Docket No.: 354406.00202 and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • antibody should be understood to encompass functional antibody fragments thereof.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • the antigen-binding domain is a humanized antibody of fragments thereof.
  • a humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • the heavy and light chains of an antibody can be full-length or can be an antigen-binding portion (a Fab, F(ab ⁇ ) 2 , Fv or a single chain Fv fragment (scFv)).
  • the antibody heavy chain constant region is chosen from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly chosen from, e.g., IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1).
  • the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa.
  • antibody fragments include but are not limited to Fv, Fab, Fab ⁇ , Fab ⁇ -SH, F(ab ⁇ ) 2 ; diabodies; linear antibodies; variable heavy chain (VH) regions, single-chain antibody molecules such as scFvs and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments.
  • the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody. Docket No.: 354406.00202 [00185]
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells.
  • the antibodies are recombinantly produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody.
  • the antibody fragments are scFvs.
  • the antibody or antibody fragments of the CAR have high binding affinity for fibrin and/or a specific target antigen. In embodiments, the increased binding affinity is greater than effected by a reference antigen.
  • the scFv fragments which specifically bind fibrin are engineered from antibodies obtained from a biological sample. For example, antibodies obtained from multiple sclerosis (MS) patients’ cerebrospinal fluid (CSF) plasma cells were isolated. The antibodies were screened to identify target antigens, such as by immunoprecipitation followed by mass spectrometry. Eighteen antibodies with fibrinogen as a potential target antigen were identified.
  • an scFv fragment which specifically binds to fibrin comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1 or 5.
  • an scFv fragment comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1 or 5. In certain embodiments, an scFv fragment comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1 or 5.
  • an scFv fragment comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1 or 5. In certain embodiments, an scFv fragment comprises SEQ ID NO: 1 or 5. [00189] In certain embodiments, a CAR includes a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID Docket No.: 354406.00202 NO: 2 or 6.
  • a CAR includes at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2 or 6. In certain embodiments, a CAR includes at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2 or 6.
  • a CAR includes at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2 or 6.
  • an scFv which specifically binds to fibrin comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 9 or 11.
  • an scFv fragment which specifically binds to fibrin comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 9 or 11.
  • an scFv fragment which specifically binds to fibrin comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 9 or 11.
  • an scFv fragment which specifically binds to fibrin comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 9 or 11.
  • an scFv fragment which specifically binds to fibrin comprises SEQ ID NO: 9 or 11.
  • an scFv which specifically binds to CD19 comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10 or 12.
  • an scFv fragment which specifically binds to CD19 comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10 or 12.
  • an scFv fragment which specifically binds to CD19 comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10 or 12.
  • an scFv fragment which specifically binds to CD19 comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10 or 12. In certain embodiments, an scFv fragment which specifically binds to CD19 comprises SEQ ID NO: 10 or 12.
  • a CAR which specifically binds to fibrin comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or Docket No.: 354406.00202 99%, sequence identity to SEQ ID NO: 3, 7 or 13.
  • a CAR which specifically binds to fibrin comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 3, 7 or 13.
  • a CAR which specifically binds to fibrin comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 3, 7 or 13. In certain embodiments, a CAR which specifically binds to fibrin comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 3, 7 or 13. In certain embodiments, a CAR which specifically binds to fibrin comprises SEQ ID NO: 3, 7 or 13.
  • a bispecific chimeric antigen receptor which binds to fibrin and CD19 comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13.
  • a bispecific chimeric antigen receptor which binds to fibrin and CD19 comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13.
  • a bispecific chimeric antigen receptor which binds to fibrin and CD19 comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13.
  • a bispecific chimeric antigen receptor which binds to fibrin and CD19 comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13.
  • a bispecific chimeric antigen receptor which binds to fibrin and CD19 comprises SEQ ID NO: 13.
  • a vector comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14.
  • a vector comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14.
  • a vector comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, a vector comprises at least a 90%, 91%, 92%, Docket No.: 354406.00202 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, a vector comprises SEQ ID NO: 4, 8 or 14.
  • a vector or genetic construct is selected from the group comprising adenovirus, adeno-associated virus (AAV), herpes simplex virus, lentivirus, gammaretrovirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus, vesicular stomatitis virus.
  • AAV adeno-associated virus
  • Vectors can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers.
  • a marker gene can confer a selectable phenotype on a host cell.
  • a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin).
  • An expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide.
  • Tag sequences such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAGTM tag (Kodak, New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide.
  • GFP green fluorescent protein
  • GST glutathione S-transferase
  • polyhistidine polyhistidine
  • c-myc hemagglutinin
  • FLAGTM tag FLAGTM tag
  • Additional expression vectors also can include, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.
  • coli plasmids col E1, pCR1, pBR322, pMal-C2, pET, pGEX, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 ⁇ plasmid or derivatives thereof, vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences.
  • phage DNAs e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentous
  • the vector can also include a regulatory region.
  • regulatory region refers to nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5 ⁇ and 3 ⁇ untranslated regions (UTRs), Docket No.: 354406.00202 transcriptional start sites, termination sequences, polyadenylation sequences, nuclear localization signals, and introns.
  • operably linked refers to positioning of a regulatory region and a sequence to be transcribed in a nucleic acid so as to influence transcription or translation of such a sequence.
  • the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter.
  • a promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site or about 2,000 nucleotides upstream of the transcription start site.
  • a promoter typically comprises at least a core (basal) promoter.
  • a promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
  • control element such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
  • the choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the coding sequence.
  • Vectors include, for example, viral vectors (such as adenoviruses Ad, AAV, lentivirus, and vesicular stomatitis virus (VSV) and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell.
  • viral vectors such as adenoviruses Ad, AAV, lentivirus, and vesicular stomatitis virus (VSV) and retroviruses
  • liposomes and other lipid-containing complexes such as liposomes and other lipid-containing complexes
  • macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell.
  • Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells.
  • components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide.
  • Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector.
  • Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities.
  • a “recombinant viral vector” refers to a viral vector comprising one or more heterologous gene products or sequences. Since many viral vectors exhibit size- constraints associated with packaging, the heterologous gene products or sequences are typically introduced by replacing one or more portions of the viral genome.
  • Such viruses may become replication-defective, requiring the deleted function(s) to be provided in trans during viral replication and encapsidation (by using, e.g., a helper virus or a packaging cell line carrying gene products necessary for replication and/or encapsidation).
  • Modified viral vectors in which a polynucleotide to be delivered is carried on the outside of the viral particle have also been described (see, e.g., Curiel, D. T., et al. PNAS 88: 8850-8854, 1991).
  • Additional vectors include viral vectors, fusion proteins and chemical conjugates.
  • Retroviral vectors include Moloney murine leukemia viruses and HIV-based viruses.
  • One HIV based viral vector comprises at least two vectors wherein the gag and pol genes are from an HIV genome and the env gene is from another virus.
  • DNA viral vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector [Geller, A. I. et al., J. Neurochem, 64: 487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad.
  • HSV herpes simplex I virus
  • the genetic construct which includes the nucleotide sequence encoding the desired protein operably linked to the regulatory elements may remain present in the cell as a functioning extrachromosomal molecule or it may integrate into the cell's chromosomal DNA.
  • DNA may be introduced into cells where it remains as separate genetic material in the form of a plasmid.
  • linear DNA which can integrate into the chromosome may be introduced into the cell.
  • reagents which promote DNA integration into chromosomes may be added.
  • DNA sequences which are useful to promote integration may also be included in the DNA molecule.
  • RNA may be administered to the cell.
  • the regulatory elements necessary for gene expression of a DNA molecule include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal.
  • enhancers are often required for gene expression. It is necessary that these elements be operable linked to the sequence that encodes the desired proteins and that the regulatory elements are operably in the individual to whom they are administered.
  • Initiation codons and stop codon are generally considered to be part of a nucleotide sequence that encodes the desired protein.
  • Promoters and polyadenylation signals used must be functional within the cells of the individual.
  • Examples of promoters useful to practice the present disclosure, especially in the production of a genetic vaccine for humans include but are not limited to promoters from Simian Virus 40 (SV40, Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human Actin, human Myosin, human Hemoglobin, human muscle creatine and human metalothionein.
  • Simian Virus 40 SV40, Mouse Mammary Tumor Virus (MMTV) promoter
  • HIV Human Immunodeficiency Virus
  • LTR HIV Long Terminal Repeat
  • ALV Cytomegalovirus
  • a promoter comprises a MNDU3 promoter, a PGK promoter or the combination thereof.
  • polyadenylation signals useful to practice the present disclosure, especially in the production of a genetic vaccine for humans, include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals.
  • additional elements include enhancers.
  • the enhancer may be selected from the group including but not limited to: human Actin, human Myosin, human Hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.
  • a genetic construct may include a suicide gene.
  • An example of a suicide gene is inducible human caspase-9 transgene (iC9), which is dimerized and is activated by the administration of an otherwise bioinert small-molecule drug, AP1903 (Spencer DM, et al.
  • a vector or genetic construct encodes a cytokine.
  • the cytokine and/or suicide gene may be encoded by the same or different vector or included with the vector encoding for a CAR.
  • a vector encodes for a CAR and a suicide gene.
  • the vector encodes a CAR and at least one cytokine.
  • the vector encodes a CAR, a suicide gene and at least one cytokine, or at least one chemokine or at least a hormone, or a growth factor, or combinations thereof.
  • the vector encodes for a CAR and a second vector encodes for a suicide gene.
  • the vector encodes a CAR and a second vector encodes a suicide gene and at least one cytokine.
  • a combination of a plurality of vectors encode CARs, suicide genes and cytokines.
  • cytokine as used herein is meant a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, chemokines, and traditional polypeptide hormones.
  • cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- Docket No.: 354406.00202 beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF- beta; insulin-like growth factor-I and -II; erythrop
  • cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines.
  • a lentiviral gene delivery system may be utilized. Such a system offers stable, long term presence of the gene in dividing and non-dividing cells with broad tropism and the capacity for large DNA inserts. (Dull et al, J Virol, 72:8463-8471 1998).
  • adeno- associated virus AAV may be utilized as a delivery method.
  • AAV is a non-pathogenic, single- stranded DNA virus that has been actively employed in recent years for delivering therapeutic gene in in vitro and in vivo systems (Choi et al, Curr Gene Ther, 5:299-310, 2005).
  • AAV include serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ or DJ/8.
  • An example of non-viral delivery method may utilize nanoparticle technology. This platform has demonstrated utility as a pharmaceutical in vivo. Nanotechnology has improved transcytosis of drugs across tight epithelial and endothelial barriers. It offers targeted delivery of its payload to cells and tissues in a specific manner (Allen and Cullis, Science, 303:1818-1822, 1998).
  • the polynucleotides embodied herein may be used with a microdelivery vehicle such as cationic liposomes and adenoviral vectors.
  • a microdelivery vehicle such as cationic liposomes and adenoviral vectors.
  • Another method is to use single stranded DNA producing vectors which can produce the expressed products intracellularly. See for example, Chen et al.,BioTechniques, 34: 167-171 (2003), which is incorporated herein, by reference, in its entirety.
  • the nucleic acid sequences of the disclosure can be delivered to an appropriate cell of a subject.
  • This can be achieved by, for example, the use of a polymeric, biodegradable microparticle or microcapsule delivery vehicle, sized to optimize phagocytosis by phagocytic cells such as macrophages.
  • a polymeric, biodegradable microparticle or microcapsule delivery vehicle sized to optimize phagocytosis by phagocytic cells such as macrophages.
  • PLGA poly-lacto-co-glycolide
  • the polynucleotide is encapsulated in these microparticles, which are taken up by macrophages and gradually biodegraded within the cell, thereby releasing the polynucleotide. Once released, the DNA is expressed within the cell.
  • a second type of microparticle is intended not to be taken up directly by cells, but rather to serve primarily as a slow-release reservoir of nucleic acid that is taken up by cells only upon release from the micro-particle through biodegradation.
  • These polymeric particles should therefore be large enough to preclude phagocytosis (i.e., larger than 5 ⁇ m and preferably larger than 20 ⁇ m).
  • Another way to achieve uptake of the nucleic acid is using liposomes, prepared by standard methods.
  • the nucleic acids can be incorporated alone into these delivery vehicles or co- incorporated with cell- or tissue-specific antibodies, for example, specific for Treg cells or delivery to tumor cells as a target.
  • a molecular complex composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces.
  • Poly-L-lysine binds to a ligand that can bind to a receptor on target cells.
  • Delivery of “naked DNA” i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site, is another means to achieve in vivo expression.
  • the nucleic acid sequence encoding an isolated nucleic acid sequence comprising a sequence encoding a CAR as described above.
  • compositions of the disclosure can be formulated as a nanoparticle, for example, nanoparticles comprised of a core of high molecular weight linear polyethyleneimine (LPEI) complexed with DNA and surrounded by a shell of polyethylene glycol modified (PEGylated) low molecular weight LPEI.
  • LPEI high molecular weight linear polyethyleneimine
  • PEGylated polyethylene glycol modified low molecular weight LPEI.
  • the nucleic acids and vectors may also be Docket No.: 354406.00202 applied to a surface of a device (e.g., a catheter) or contained within a pump, patch, or other drug delivery device.
  • nucleic acids and vectors disclosed herein can be administered alone, or in a mixture, in the presence of a pharmaceutically acceptable excipient or carrier (e.g., physiological saline).
  • a pharmaceutically acceptable excipient or carrier e.g., physiological saline
  • the excipient or carrier is selected on the basis of the mode and route of administration.
  • Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences (E. W. Martin), a well-known reference text in this field, and in the USP/NF (United States Pharmacopeia and the National Formulary).
  • the compositions can be formulated as a nanoparticle encapsulating the compositions embodied herein.
  • compositions are administered as nucleic acids or polypeptides, they are formulated in such a way as to promote uptake by the mammalian cell.
  • Useful vector systems and formulations are described above.
  • the vector can deliver the compositions to a specific cell type.
  • the disclosure is not so limited however, and other methods of DNA delivery such as chemical transfection, using, for example calcium phosphate, DEAE dextran, liposomes, lipoplexes, surfactants, and perfluoro chemical liquids are also contemplated, as are physical delivery methods, such as electroporation, micro injection, ballistic particles, and “gene gun” systems.
  • Methods for Isolation of Cells Any number of methods known in the art can be used to isolate cells, such as T cells, or any other cell type that may be used in carrying out the treatment of a subject.
  • T cells any number of methods known in the art can be used to isolate cells, such as T cells, or any other cell type that may be used in carrying out the treatment of a subject.
  • various other genetically engineered cells expressing the chimeric antigen receptors e.g., CARs are also provided.
  • the cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells.
  • the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells.
  • Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs).
  • the cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen.
  • the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4 + Docket No.: 354406.00202 cells, CD8 + cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
  • the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods.
  • the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs).
  • the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.
  • T cells and/or of CD4 + and/or of CD8 + T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (T SCMX central memory T (T CM effector memory T (T EM ), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MATT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as T H 1 cells, T H 2 cells, T H 3 cells, T H 17 cells, T H 9 cells, T H 22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
  • T SCMX central memory T T CM effector memory T (T EM )
  • TIL tumor-infiltrating lymphocytes
  • the cells are natural killer (NK) cells.
  • the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
  • the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids.
  • the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived.
  • the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types. Docket No.: 354406.00202 [00225]
  • preparation of the engineered cells includes one or more culture and/or preparation steps.
  • the cells for introduction of the CAR may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • a sample such as a biological sample, e.g., one obtained from or derived from a subject.
  • the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells in some embodiments are primary cells, e.g., primary human cells.
  • the samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector), washing, and/or incubation.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
  • Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
  • the sample from which the cells are derived or isolated is blood or a blood-derived sample or is or is derived from an apheresis or leukapheresis product.
  • Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), brain, central nervous system (CNS), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.
  • Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
  • the cells are derived from cell lines, e.g., T cell lines.
  • the cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non- human primate, or pig.
  • isolation of the cells includes one or more preparation and/or non- affinity based cell separation steps.
  • cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, Docket No.: 354406.00202 enrich for desired components, lyse, or remove cells sensitive to particular reagents.
  • cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
  • cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis.
  • the samples contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
  • the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • the wash solution lacks calcium and/or magnesium and/or many or all divalent cations.
  • a washing step is accomplished a semi-automated “flow- through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions.
  • a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions.
  • the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca ++ /Mg ++ free PBS.
  • components of a blood cell sample are removed, and the cells directly resuspended in culture media.
  • the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
  • the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used.
  • the separation is affinity- or immunoaffinity-based separation.
  • the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by Docket No.: 354406.00202 washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
  • Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use.
  • negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
  • the separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker.
  • positive selection of or enrichment for cells of a particular type, such as those expressing a marker refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker.
  • negative selection, removal, or depletion of cells of a particular type refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
  • multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.
  • a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection.
  • multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
  • T cells can be isolated by positive or negative selection techniques.
  • CD3 + , CD28 + T cells can be positively selected using anti-CD3/anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
  • isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection.
  • positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker“1”) at a relatively higher level (marker high ) on the positively or negatively selected cells, respectively.
  • T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14.
  • a CD4 + or CD8 + selection step is used to separate CD4 + helper and CD8 + cytotoxic T cells.
  • CD4 + and CD8 + populations can be further sorted into sub- populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
  • CD8 + cells are further enriched for or depleted of na ⁇ ve, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation.
  • enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations.
  • T CM - enriched CD8 + T cells and CD4 + T cells further enhances efficacy.
  • the enrichment for central memory T (T CM ) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B.
  • a CD4 expression-based selection step is used to generate the CD4 + cell population or sub-population, such that both the positive and negative fractions from the CD4- based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
  • a sample of PBMCs or other white blood cell sample is subjected to selection of CD4 + cells, where both the negative and positive fractions are retained.
  • CD4 + T helper cells are sorted into na ⁇ ve, central memory, and effector cells by identifying cell populations that have cell surface antigens.
  • CD4 + lymphocytes can be obtained by standard methods.
  • na ⁇ ve CD4 + T lymphocytes are CD45RO + , CD45RA + , CD62L + , CD4 + T cells.
  • central memory CD4 + cells are CD62L + and CD45RO + .
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.
  • the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection.
  • the cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol.58: Metastasis Research Protocols, Vol.2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher ⁇ Humana Press Inc., Totowa, N.J.).
  • the cells are NK cells wherein the NK cells are transduced with the CARs of the disclosure.
  • NK cells have typically been identified in flow cytometry by first excluding other lymphocyte markers such as the CD3 T cell marker.
  • NK cell markers two markers have become well-established as standard NK cell markers, CD56 (neural cell adhesion molecule- 1, NCAM1) and CD16 (low affinity Fc gamma receptor 3A, FCGR3A, Fc ⁇ RIII).
  • CD56 neural cell adhesion molecule- 1, NCAM1
  • CD16 low affinity Fc gamma receptor 3A, FCGR3A, Fc ⁇ RIII.
  • the differential expression of these two surface proteins defines the two main subsets of conventional NK cells, CD56 bright CD16 lo/ ⁇ and CD56 dim CD16 + , often simplified as CD56 bright and CD56 dim , respectively.
  • CD56 bright is less abundant, estimated to comprise only 5–10% of the population.
  • CD56 dim represents greater than 90% of NK cells.
  • CD56 bright has been noted to be abundant in certain tissues, including secondary lymphoid tissues.
  • the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as DYNABEADS or MACS beads).
  • the magnetically responsive material, e.g., particle generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, Docket No.: 354406.00202 present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
  • the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner.
  • a specific binding member such as an antibody or other binding partner.
  • Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference.
  • Colloidal sized particles such as those described in Owen U.S. Pat. No.4,795,698, and Liberti et al., U.S. Pat. No.5,200,084 are other examples.
  • the incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
  • the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained.
  • the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin.
  • the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers.
  • the cells, rather than the beads are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added.
  • streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
  • the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles Docket No.: 354406.00202 are left attached to the cells for administration to a patient.
  • the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.
  • the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto.
  • MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered.
  • the non-target cells are labelled and depleted from the heterogeneous population of cells.
  • the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods.
  • the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination.
  • the system is a system as described in International Patent Application, Publication Nos. WO2009/072003, or US 20110003380 A1.
  • the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion.
  • the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.
  • the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system.
  • Components can include an integrated microcomputer, magnetic Docket No.: 354406.00202 separation unit, peristaltic pump, and various pinch valves.
  • the integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence.
  • the magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column.
  • the peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.
  • the CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution.
  • peripheral blood may be automatically separated into erythrocytes, white blood cells and plasma layers.
  • the CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture.
  • Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother.35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and Wang et al. (2012) Immunother.35(9):689-701.
  • a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream.
  • a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting.
  • FACS preparative scale
  • a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al.
  • MEMS microelectromechanical systems
  • the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies.
  • separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system.
  • FACS fluorescence-activated cell sorting
  • MEMS microelectromechanical systems
  • the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering.
  • the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population.
  • the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets.
  • a freezing solution e.g., following a washing step to remove plasma and platelets.
  • Any of a variety of known freezing solutions and parameters in some aspects may be used.
  • PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively.
  • the cells are then frozen to ⁇ 80° C. at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank.
  • the provided methods include cultivation, incubation, culture, and/or genetic engineering steps.
  • the cell populations are incubated in a culture-initiating composition.
  • the incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.
  • the cells are incubated and/or cultured prior to or in connection with genetic engineering.
  • the incubation steps can include culture, cultivation, stimulation, activation, and/or propagation.
  • the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent.
  • stimulating conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
  • the conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
  • Oligodendrocytes The adult vertebrate central nervous system (CNS) mainly consists of neurons, astrocytes, oligodendrocytes, and microglia cells. Oligodendrocytes, the myelin forming cells of the CNS, are subjected to cell stress or even death in a number of metabolic or inflammatory disorders (Armati P, Mathey E (2010) The biology of oligodendrocytes. Cambridge University Press; Bradl M, Lassmann H (2010) Oligodendrocytes: biology and pathology. Acta Neuropathol 119:37–53; Caprariello AV, et al.
  • MS multiple sclerosis
  • OLGs Oligodendrocytes
  • CNS central nervous system
  • OLGs are sensitive to ischemic insult because they are vulnerable to energy failure. In white matter, the saltatory conduction is highly dependent on ATP supply. Pathological Ca 2+ entry occurs in part because of increased intracellular Na + and membrane depolarization in OLG after stroke (Stys PK. Anoxic and ischemic injury of myelinated axons in CNS white matter: from mechanistic concepts to therapeutics. J Cereb Blood Flow Metab. 1998;18(1):2–25). OLG swelling and death occur within minutes to hours after ischemic insult (L. Jiang et al. Transl Stroke Res.2011 Sep; 2(3): 366–375. doi: 10.1007/s12975-011-0078-0).
  • OLG death causes demyelination of neurofibers that leads to neurologic deficits in stroke patients.
  • oligodendrocytes are closely related to nerve cells and provide a supporting role for neurons.
  • the nervous system of mammals depends on myelin sheaths, which reduce ion leakage and decrease the capacitance of the cell membrane.
  • Myelin also increases impulse speed, as saltatory propagation of action potentials occurs at the nodes of Ranvier in between Schwann cells (of the PNS) and oligodendrocytes (of the CNS).
  • Myelinating oligodendrocytes are a part of the white matter and myelination is an important component of intelligence.
  • Biomarkers of Oligodendrogenesis may be determined by measuring the concentration of certain biomarkers in tissue. These biomarkers include Oligodendrocyte Transcription Factor (OLIG2), 2 ⁇ ,3 ⁇ -Cyclic-Nucleotide 3 ⁇ -Phosphodiesterase (CNPase), and Myelin Basic Protein (MBP). The presence of, or an increase in the concentration of these biomarkers may indicate oligodendrocyte formation.
  • Oligodendrocyte Transcription Factor Oligodendrocyte transcription factor (OLIG2) is a basic helix-loop-helix transcription factor encoded by the Olig2 gene.
  • the protein is of 329 amino acids in length, 32 kDa in size and contains 1 basic helix-loop-helix DNA-binding domain.
  • the expression of OLIG2 is mostly restricted in central nervous system and is well known for determining oligodendrocyte differentiation. Docket No.: 354406.00202 [00271]
  • OLIG2 is mostly expressed in restricted domains of the brain and spinal cord ventricular zone which give rise to oligodendrocytes and specific types of neurons. During embryogenesis, OLIG2 first directs motor neuron fate by establishing a ventral domain of motor neuron progenitors and promoting neuronal differentiation.
  • the present disclosure provides a method for reducing demyelination, inducing remyelination, promoting oligodendroglial progenitor cell (OPC) proliferation, and/or promoting oligodendrocyte differentiation in a subject, the method comprising administering to the subject a therapeutically effective amount of CAR-T cells, CAR- NK cells embodied herein.
  • administration comprises transplanting these cells into injured tissue in the subject, e.g intracranially.
  • the FIB2 specific CAR cells embodied herein are suspended in a pharmaceutically acceptable carrier prior to administration.
  • the pharmaceutically acceptable carrier comprises phosphate- buffered saline.
  • pluripotent stem cells or oligodendrocyte precursors, immature oligodendrocytes or combinations thereof are administered as part of the therapy.
  • the subject has one or more risk factors for a demyelinating disease.
  • reducing demyelination, inducing remyelination, promoting oligodendroglial progenitor cell (OPC) proliferation, and/or promoting oligodendrocyte differentiation in the subject reduces or eliminates one or more signs or symptoms of a demyelinating disease. In other embodiments, the subject does not have signs or symptoms of a demyelinating disease.
  • OPC oligodendroglial progenitor cell
  • the demyelinating disease is selected from the group consisting of periventricular leukomalacia, multiple sclerosis, acute disseminated encephalomyelitis, chronic inflammatory demyelinating polyneuropathy, adrenoleukodystrophy, adenomyeloneuropathy, Leber's hereditary optic atrophy, HTLV-associated myelopathy, Guillain- Barre syndrome, phenylketonuria, Tay-Sachs disease, Niemann-Pick disease, Gaucher's disease, Hurler's syndrome, Krabbe's disease, Pelizaeus-Merzbacher disease, cerebral palsy, Alzheimer’s disease, COVID-19, chemo-brain, neurodegenerative disorders, or combination thereof.
  • a method of treating a subject requiring immunotherapy comprises isolating T lymphocytes and/or NK cells from a biological sample obtained from the subject; transducing the T lymphocytes and/or NK cells with an expression vector encoding a chimeric antigen receptor (CAR) which specifically binds to fibrin and/or CD19; stimulating the transduced T lymphocytes and/or NK cells with fibrin and/or CD19 at least once ex vivo to obtain cells specific for the fibrin and/or NK cells; and reinfusing the cells into the subject, thereby treating the subject.
  • CAR chimeric antigen receptor
  • the cells are autologous cells.
  • CAR-T cells may be generated from any suitable source of T cells known in the art including, but not limited to, T cells collected from a subject.
  • the subject may be a patient with an autoimmune disease such as rheumatoid arthritis, or cancer in need of CAR-T cell or CAR- NK cell therapy or a subject of the same species as the subject with the disease in need of cell therapy.
  • the collected cells may be expanded ex vivo using methods commonly known in the art before transduction with a CAR-To generate a CAR-T cell and/or CAR-NK cells.
  • Methods for CAR design, delivery and expression in T cells, and the manufacturing of clinical-grade CAR-T cell populations are known in the art.
  • the engineered CARs may be introduced into T cells using retroviruses, which efficiently and stably integrate a nucleic acid sequence encoding the chimeric antigen receptor into the target cell genome.
  • the CAR-T cells once they have been expanded ex vivo in response to, for example, an autoimmune disease antigen, can be reinfused into the subject in a therapeutically effective amount.
  • compositions of the present disclosure may be prepared in a manner known in the art and in a manner suitable for parenteral administration to mammals, particularly humans, comprising a therapeutically effective amount of the composition alone, with one or more pharmaceutically acceptable carriers or diluents.
  • compositions of the invention may also include other supplementary physiologically active agents.
  • compositions include those suitable for parenteral administration, including subcutaneous, intramuscular, intravenous and intradermal administration.
  • the compositions may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. Such methods include preparing the carrier for association with the CAR-T cells and/or CAR-NK cells. In general, the compositions are prepared by uniformly and intimately bringing into association any active ingredients with liquid carriers.
  • the composition is suitable for parenteral administration. In another embodiment, the composition is suitable for intravenous administration.
  • compositions suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes, which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the compositions comprise a cell which has been transformed or transfected with one or more vectors or nucleic acids encoding one or more CARs.
  • the methods of the disclosure can be applied ex vivo.
  • a subject's cells can be removed from the body and transduced with the compositions in culture with a desired target antigen, expand target-antigen specific, e.g. T cells and the expanded cells returned to the subject's body.
  • the cells can be the subject's cells or they can be haplotype matched or a cell line.
  • the cells can be irradiated to prevent replication.
  • the cells are human leukocyte Docket No.: 354406.00202 antigen (HLA)-matched, autologous, cell lines, or combinations thereof.
  • the cells can be a stem cell.
  • Embryonic stem cells and artificial pluripotent stem cells (induced pluripotent stem cell, iPS cells) have been established from many animal species, including humans. These types of pluripotent stem cells would be the most useful source of cells for regenerative medicine because these cells are capable of differentiation into almost all of the organs by appropriate induction of their differentiation, with retaining their ability of actively dividing while maintaining their pluripotency.
  • iPS cells in particular, can be established from self-derived somatic cells, and therefore are not likely to cause ethical and social issues, in comparison with ES cells which are produced by destruction of embryos. Further, iPS cells, which are self-derived cell, make it possible to avoid rejection reactions, which are the biggest obstacle to regenerative medicine or transplantation therapy.
  • the CARs can be easily delivered to a subject by methods known in the art, for example, methods which deliver siRNA.
  • the CAR molecules can be used clinically, similar to the approaches taken by current gene therapy.
  • a CAR stable expression stem cell or iPS cells for cell transplantation therapy as well as vaccination can be developed for use in subjects.
  • the CAR-T cells and/or CAR-NK cells, once they have been expanded ex vivo in response to an autoimmune disease antigen, or tumor antigen etc., are reinfused into the subject in a therapeutically effective amount.
  • terapéuticaally effective amount means the amount of CAR-T cells and/or CAR-NK cells when administered to a mammal, in particular a human, in need of such treatment, is sufficient to treat the disease.
  • the precise amount of CAR-T cells and/or CAR-NK cells to be administered can be determined by a physician with consideration of individual differences in age, weight, extent of disease and condition of the subject.
  • administration of T cell therapies is defined by number of cells per kilogram of body weight. However, because T cells will replicate and expand after transfer, the administered cell dose will not resemble the final steady-state number of cells.
  • a pharmaceutical composition comprising the CAR-T cells and/or CAR-NK cells of the present disclosure may be administered at a dosage of 10 4 to 10 9 cells/kg body weight.
  • a pharmaceutical composition comprising the CAR-T cells Docket No.: 354406.00202 and/or CAR-NK cells of the present disclosure may be administered at a dosage of 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges.
  • Compositions comprising the CAR-T cells and/or CAR-NK cells of the present disclosure may also be administered multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are known in the art (see, for example, Rosenberg et al., 1988, New England Journal of Medicine, 319: 1676).
  • the optimal dosage and treatment regimen for a particular subject can be readily determined by one skilled in the art by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • Combination Therapies [00289] The disclosure also contemplates the combination of the composition of the present disclosure with other drugs and/or in addition to other treatment regimens or modalities such as surgery. When the composition of the present disclosure is used in combination with known therapeutic agents the combination may be administered either in sequence (either continuously or broken up by periods of no treatment) or concurrently or as an admixture.
  • chemotherapeutic agents may be administered as part of the combination therapy.
  • a combination therapy may include CAR-T cells and/or CAR- NK cells and one or more anti-inflammatory agents and/or therapeutic agents.
  • the one or more anti-inflammatory agents and/or therapeutic agents may be administered simultaneously or sequentially.
  • the anti-inflammatory agents comprise one or more antibodies which specifically bind to pro-inflammatory cytokines, e.g. pro-inflammatory cytokines such as IL-1, IL-4, IL-5, IL-6, IL-8, IL-10, GM-CSF, TNF ⁇ IFN- ⁇ and MIP-1a.
  • the antibodies are anti-TNF ⁇ , anti-IL-6 or combinations thereof.
  • one or more agents, other than antibodies can be administered which decrease pro-inflammatory cytokines, e.g. non-steroidal anti-inflammatory drugs (NSAIDs). Any combination of antibodies and one or more agents can be administered which decrease pro-inflammatory cytokines.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • Anti- inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti- inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.
  • steroids and glucocorticoids including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone
  • NSAIDS nonsteroidal anti- inflammatory drugs
  • the CAR-T cells and/or CAR-NK cells contemplated herein are administered in conjunction with a cytokine.
  • the CAR-T cells and/or CAR- NK cells are administered in conjunction with a cancer therapy.
  • cancer therapy refers to a therapy useful in treating cancer.
  • anti-cancer therapeutic agents include, but are not limited to, e.g., surgery, chemotherapeutic agents, immunotherapy, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, such as anti-HER-2 antibodies (e.g., HERCEPTIN TM ), anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (TARCEVA TM )), platelet derived growth factor inhibitors (e.g., GLEEVEC TM (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, Erb
  • a “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include Erlotinib (TARCEVA TM , Genentech/OSI Pharm.), Bortezomib (VELCADE TM , Millennium Pharm.), Fulvestrant (FASLODEX TM , Astrazeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARA TM , Novartis), Imatinib mesylate (GLEEVEC TM , Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin TM , Sanofi), 5-FU (5- fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE TM , Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonaf
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN TM doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin
  • chemotherapeutic agent also included in this definition of “chemotherapeutic agent” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX TM (tamoxifen)), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON TM (toremifene); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE TM (megestrol acetate), AROMASIN TM (exemestane), formestanie, fadrozole, RIVISOR TM (vorozole), FEMARA
  • the cancer therapeutic is an immunotherapy selected from the group comprising oncolytic virus, bacteria, oncolytic bacteria or other bacterial compositions, Bacillus Calmette-Guerin (BCG), a microbiome modulator, and/or a toll-like receptor (TLR) Docket No.: 354406.00202 agonist.
  • the TLR agonist is a TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and/or TLR13 agonist.
  • the TLR agonist is derived from virus, bacteria and/or made synthetically.
  • the immunotherapy is a is a stimulator of interferon genes (STING) pathway modulator.
  • STING interferon genes
  • other complementary immune therapies may be added to the regimens described above to further enhance their efficacy including but not limited to GM-CSF to increase the number of myeloid derived innate immune system cells, low dose cyclophosphamide or PI3K inhibitors (e.g., PI3K delta inhibitors) to eliminate T regulatory cells that inhibit innate and adaptive immunity and 5FU (e.g., capecitabine), PI3K inhibitors or histone deacetylase inhibitors to remove inhibitory myeloid derived suppressor cells.
  • GM-CSF to increase the number of myeloid derived innate immune system cells
  • PI3K inhibitors e.g., PI3K delta inhibitors
  • 5FU e.g., capecitabine
  • PI3K inhibitors or histone deacetylase inhibitors to remove inhibitory myeloid derived suppressor cells.
  • PI3K inhibitors include, but are not limited to, LY294002, Perifosine, BKM120, Duvelisib, PX-866, BAY 80-6946, BEZ235, SF1126, GDC-0941, XL147, XL765, Palomid 529, GSK1059615, PWT33597, IC87114, TG100-15, CAL263, PI-103, GNE- 477, CUDC-907, and AEZS-136.
  • the PI3K inhibitor is a PI3K delta inhibitor such as, but not limited to, Idelalisib, RP6530, TGR1202, and RP6503.
  • PI3K inhibitors are disclosed in U.S. Patent Application Nos. US20150291595, US20110190319, and International Patent Application Nos. WO2012146667, WO2014164942, WO2012062748, and WO2015082376.
  • the immunotherapy may also comprise the administration of an interleukin such as IL-2, or an interferon such as INF ⁇ .
  • the CAR-T cells and/or CAR- NK cells are administered with one or more immune checkpoint modulators.
  • Immune checkpoints refer to inhibitory pathways of the immune system that are responsible for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses.
  • checkpoint inhibitor examples include, without limitation an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR- ⁇ .
  • the term “checkpoint inhibitor” means a group of molecules on the cell surface of CD4 + and/or CD8 + T cells that fine-tune immune responses by down-modulating or inhibiting an anti- tumor immune response.
  • Immune checkpoint proteins are well known in the art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, Docket No.: 354406.00202 BTLA, SIRP ⁇ (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, and A2aR (see, for example, WO 2012/177624).
  • Anti-immune checkpoint inhibitor therapy refers to the use of agents that inhibit immune checkpoint inhibitors. Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer.
  • agents useful for inhibiting immune checkpoint inhibitors include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and/or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and/or activity of immune checkpoint inhibitor nucleic acids, or fragments thereof.
  • Exemplary agents for upregulating an immune response include antibodies against one or more immune checkpoint inhibitor proteins block the interaction between the proteins and its natural receptor(s); a non-activating form of one or more immune checkpoint inhibitor proteins (e.g., a dominant negative polypeptide); small molecules or peptides that block the interaction between one or more immune checkpoint inhibitor proteins and its natural receptor(s); fusion proteins (e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fe portion of an antibody or immunoglobulin) that bind to its natural receptor(s); nucleic acid molecules that block immune checkpoint inhibitor nucleic acid transcription or translation; and the like.
  • a non-activating form of one or more immune checkpoint inhibitor proteins e.g., a dominant negative polypeptide
  • small molecules or peptides that block the interaction between one or more immune checkpoint inhibitor proteins and its natural receptor(s)
  • fusion proteins e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fe portion of an antibody or immunoglobulin
  • agents can directly block the interaction between the one or more immune checkpoint inhibitors and its natural receptor(s) (e.g., antibodies) to prevent inhibitory signaling and upregulate an immune response.
  • agents can indirectly block the interaction between one or more immune checkpoint proteins and its natural receptor(s) to prevent inhibitory signaling and upregulate an immune response.
  • a soluble version of an immune checkpoint protein ligand such as a stabilized extracellular domain can binding to its receptor to indirectly reduce the effective concentration of the receptor to bind to an appropriate ligand.
  • anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies either alone or used in combination. [00299] .
  • such therapy involves blockade of programmed cell death 1 (PD- 1).
  • such therapy involves treatment with an agent that interferes with an interaction involving PD-1 (e.g., with PD-L1).
  • such therapy involves administration of an antibody agent that specifically interacts with PD-1 or with PD-L1.
  • such therapy involves administration of one or more of nivolumab (BMS-936558, Docket No.: 354406.00202 MDX-1106, ONO-4538, a fully human Immunoglobulin G4 (IgG4) monoclonal PD-1 antibody), pembrolizumab (MK-3475, a humanized monoclonal IgG4 anti-PD-1 antibody), BMS-936559 (a fully human IgG4 PD-L1 antibody), MPDL3280A (a humanized engineered IgG1 monoclonal PD- L1 antibody) and/or MEDI4736 (a humanized engineered IgG1 monoclonal PD-L1 antibody).
  • BMS-936558 Docket No.: 354406.00202 MDX-1106, ONO-4538
  • IgG4 Immunoglobulin G4
  • MK-3475 a humanized monoclonal IgG4 anti-PD-1 antibody
  • BMS-936559 a fully human Ig
  • the lentiviral vector also encodes a MNDU3 promoter, a PGK promoter, and green fluorescent protein (GFP) for in vivo detection (PMC 1947, FIG.1).
  • the FIB4 ScFvs were also introduced into a second-generation murine CAR cassette incorporating a signaling peptide, the murine CD28 transmembrane domain, and the murine CD3zeta activation domain for the generation of murine FIB4.
  • the retroviral vector also encodes a CMV promoter, green fluorescent protein (GFP) and a T2A sequence upstream of the CAR cassette (PMC 1694, FIG.2).
  • HEK293 cells were transduced with each construct and FIB4-CAR expression was validated by flow cytometry using the expression of GFP and a F(ab ⁇ )2 antibody recognizing FIB4, which showed 69.8% transduction efficiency (FIG.3).
  • F(ab ⁇ )2 antibody recognizing FIB4 which showed 69.8% transduction efficiency (FIG.3).
  • F(ab ⁇ )2 antibody recognizing FIB4-CAR [00311] To validate target binding, the fibrin-specific FIB4-CAR construct was transfected into HEK293 cells, and a cell adhesion assay was performed on fibrin-coated plates. Cells were incubated on fibrin for 30 minutes and nonadherent cells were washed off with PBS.
  • FIB4-CAR Adhered cells were stained with Hoechst dye and detected using the CellInsight CX7 microscope.
  • FIB4-CAR increased adhesion to fibrin by 9.8-fold compared to the control-CAR construct, indicating highly increased fibrin-specific binding (FIG.4).
  • FIG.4 In Vivo Target Recognition of FIB4-CAR in Fibrinogen-Injected Mouse Brain Docket No.: 354406.00202
  • In vivo target selectivity of the FIB4-CAR (FIB4-CAR-T2A-GFP) was evaluated in fibrinogen- injected mice.
  • mice received a single injection of FIB4-CAR or CAR control-transduced HEK293 cells into the cerebral ventricle.
  • GFP-positive FIB4-CAR-Transduced cells were detected in coronal sections from the fibrinogen-injected side but not on the ACSF-injected side (FIG.5A). No preference was observed for fibrinogen or ACSF-injection side in mice that received Control CAR (CAR-T2A-GFP) control cells (FIG.5B).
  • FIB4/CD19-CAR-T Cells [00314] Generation and Validation of Bi-Specific FIB4/CD19-CAR-T Cells [00315] Fibrin accumulation has been detected in the brains of individuals with several neurological diseases involving pathogenic CD19 + B cells, and this microenvironment can hinder the success of B cell-directed CAR-T cell therapies. To overcome this limitation, bispecific FIB4/CD19-CARs were generated by combining the FIB4 scFvs, CD19 scFv, the human 4-1BB co-stimulatory domain, and human CD3 zeta (CD3 ⁇ ) activation signaling domain (PMC1792, FIG. 6).
  • EXAMPLE 2 FIB4 CAR-TREG CELLS Docket No.: 354406.00202
  • a novel class of fibrin CAR human Treg cells were generated by engineering the antigen- specific binding domain to recognize fibrin using the variable sequences from a mouse monoclonal fibrin-specific antibody (FIB4) raised against the fibrin epitope ⁇ 377-395 within the ⁇ C fibrin region.
  • the FIB4 fibrin antibody variable sequence (5B8) is described in the inventors’ prior publication and patent (Ryu et al., Nat Immunol 2018 PMID: 30323343 and US8877195, both of which are incorporated herein in their entirety).
  • fibrin-CAR human T reg cells can be used to guide human T reg cells in disease tissue early and persist during the course of disease.
  • Generation of FIB4-CAR Treg cells Human regulatory T cells were isolated from PBMCs derived from a healthy donor using the Regulatory T cell Separation Kit. After confirming their purity with FACS analysis for CD4, CD25, and CD127, these cells were transduced with the FIB4scFv-Flag-CAR-GFP vector to generate Fibrin CAR human Treg (FIB4-CAR Treg) cells.
  • FIB4-CAR Treg show increased brain infiltration and therapeutic efficacy
  • LPC lysolecithin
  • FIB4-CAR Treg cells increased the number of CC1 + Olig2 + oligodendrocytes in the demyelinated area via Docket No.: 354406.00202 double immunostaining of brain sections with anti-CC1 and anti-Olig2 antibodies. A significantly higher number of CC1 + Olig2 + oligodendrocytes was found in the corpus callosum of LPC-injected mice that received FIB4-CAR Treg cells compared to vehicle or Treg cells (FIG.11). [00326] To test efficacy in increasing myelin, brain sections were immunostained with anti-MBP antibody, and the total demyelinated area was measured at the lesion site.

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Abstract

Fibrin-specific antibodies are used to generate chimeric antigen receptors (CARs) which specifically recognize fibrin, which is deposited only in disease tissues in a wide range of neurological, autoimmune, inflammatory diseases and cancer. Fibrin-CARs serve as a guide to direct T cells in diseased tissues and enables the recruitment of CAR-T cells in solid organs.

Description

Docket No.: 354406.00200 METHODS AND COMPOSITIONS FOR CELLULAR IMMUNOTHERAPY – FIB4 CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/489,506 filed March 10, 2023. The entire contents of this application are incorporated herein by reference in its entirety. STATEMENT OF SPONSORED RESEARCH [0002] Research for this invention was supported by funding from FFBW. FIELD [0003] Immune cells expressing chimeric antigen receptors that specifically target fibrin and their uses in therapy. BACKGROUND [0004] Fibrinogen is an abundant protein synthesized in the liver, present in human blood plasma at concentrations ranging from 1.5-4 g/L in healthy individuals with a normal half-life of 3-5 days. With fibrin, produced by thrombin-mediated cleavage, fibrinogen plays important roles in many physiological processes. Indeed, the formation of a stable blood clot, containing polymerized and cross-linked fibrin, is crucial to prevent blood loss. A balance between clotting, notably the conversion of fibrinogen to fibrin, and fibrinolysis, the proteolytic degradation of fibrin, is essential. Disruption of this equilibrium can cause disease in distinct manners. While some pathological conditions are the consequence of altered levels of fibrinogen, others are related to structural properties of the molecule. Fibrinogen regulates blood coagulation by engaging ĮIIbȕ3 integrin receptor via its Ȗ408-411 epitope. Conversion of fibrinogen to fibrin exposes cryptic inflammatory epitopes. Fibrin induces inflammatory processes by engaging the Mac-1 receptor (other names CD11b/CD18, Complement receptor 3) via the fibrin Ȗ377-395 epitope. Upon vascular injury fibrin forms in tissues and is deposited in brain and periphery in autoimmune, neurodegenerative, traumatic diseases, cancer, and COVID-19. Fibrin has inflammatory properties by activating innate immune and autoimmune responses. Fibrin deposits at aberrant sites including the central nervous system, joint, oral mucosa, muscle or kidney, can also be pathological due to induction of toxic inflammation and inhibition of repair processes. Docket No.: 354406.00200 SUMMARY [0005] Embodiments of the disclosure include a novel class of chimeric antigen receptor (CAR) T cells to specifically recognize fibrin and guide T cells at sites of lesions in the brain and periphery. Fibrin CAR-T cells are engineered to recognize fibrin in lesions in autoimmune, neurodegenerative, inflammatory, and infectious diseases, cancer, and trauma. These cells can be further engineered, inter alia, to a) selectively kill neurotoxic immune cells, b) increase killing of tumor cells in solid tumors; c) deliver neuroprotective molecules to promote repair at sites of brain damage. [0006] Accordingly, in certain aspects a bispecific chimeric antigen receptor (CAR) comprises a first antigen specific binding domain, a second antigen specific binding domain, a transmembrane domain(s), at least one co-stimulatory domain(s), and a CD3ȗ signaling domain, wherein the first antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof. In certain embodiments, the antigen specific binding domain specifically binds to a fibrin domain or fragment. In certain embodiments, the antigen specific binding domain binds to a fibrin domain or fragment with higher affinity than fibrinogen. In certain embodiments, the antigen specific binding domain binds to an epitope of fibrin or fibrinogen ȖC domain. In certain embodiments, the antigen specific binding domain is mouse, human or humanized. In certain embodiments, the antigen specific binding domain blocks binding of fibrin to Mac-1. In certain embodiments, the antigen specific binding domain blocks macrophage or microglia activation. In certain embodiments, the first and second antigen specific binding domains comprise an antibody, antibody fragment, a camelid nanobody or aptamer. In certain embodiments, the antibody fragment is a single chain fragment. In certain embodiments, the single chain fragment is a single chain variable fragment (scFv). In certain embodiments, the at least one co-stimulatory domain comprises a cluster of differentiation antigen 28 (CD28), 41BB domain, an ICOS (Inducible T cell Co-stimulator) (CD278), OX40 (CD134), Glucocorticoid-induced Tumor Necrosis Factor Receptor (GITR), CD40 or CD27. In certain embodiments, the second antigen specific binding domain specifically binds to tumor antigens, exogenous antigens, endogenous antigens or autoantigens. In certain embodiments, endogenous antigens comprise cluster of differentiation antigens (CD), cell surface molecules or antigens associated with neurodegenerative diseases. In certain embodiments, the cluster of differentiation antigens (CD) comprise CD1a, CD2, CD3, CD5, CD10, CD11c, CD13, CD15, CD19, CD20, CD25, CD30, CD33, CD99, CD103, CD117, Docket No.: 354406.00202 CD123 or CD207. In certain embodiments, the cluster of differentiation antigen is CD19. In certain embodiments, the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 9. In certain embodiments, the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 9. In certain embodiments, the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 11. In certain embodiments, the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 11. In certain embodiments, the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 10. In certain embodiments, the second antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 10. In certain embodiments, the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 12. In certain embodiments, the second antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 12. In certain embodiments, the CAR further comprises a hinge region. In certain embodiments, the bispecific chimeric antigen receptor comprises at least a 75% sequence identity to SEQ ID NO: 13. In certain embodiments, the bispecific chimeric antigen receptor comprises SEQ ID NO: 13. [0007] In certain embodiments, a bispecific chimeric CAR comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 13. In certain embodiments, the bispecific chimeric CAR comprises an amino acid sequence comprising SEQ ID NO: 13. In certain embodiments, the CAR further comprises at least one hinge region. [0008] In another aspect, a chimeric antigen receptor (CAR) comprises an amino acid sequence comprising at least a 75% sequence identity to SEQ ID NO: 1 or 5. In certain embodiments, the chimeric antigen receptor (CAR) comprises an having an amino acid sequence SEQ ID NO: 1 or 5. In certain embodiments, the CAR further comprises at least one hinge region. [0009] In another aspect, a chimeric antigen receptor (CAR) comprises an amino acid sequence comprising at least a 75% sequence identity to SEQ ID NO: 2 or 6. In certain embodiments, the chimeric antigen receptor (CAR) comprises an amino acid sequence SEQ ID NO: 2 or 6. In certain embodiments, the CAR further comprises at least one hinge region. [0010] In another aspect, a chimeric antigen receptor (CAR) comprises an amino acid sequence comprising at least a 75% sequence identity to SEQ ID NO: 3 or 7. In certain embodiments, the Docket No.: 354406.00202 chimeric antigen receptor (CAR) comprises an amino acid sequence SEQ ID NO: 3 or 7. In certain embodiments, the CAR further comprises at least one hinge region. [0011] In another aspect, an expression vector encodes an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, the vector encodes an amino acid sequence comprising SEQ ID NO: 4, 8, or 14. In certain embodiments, the vector comprises adenovirus, adeno-associated virus (AAV), herpes simplex virus, lentivirus, gammaretrovirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus or vesicular stomatitis virus. In certain embodiments, the vector is a lentivirus. In certain embodiments, the vector further comprises an inducible promoter, a cell specific promoter, a tissue specific promoter or a constitutive promoter. In certain embodiments, the vector further comprises one or more enhancer or regulatory sequences. In certain embodiments, the vector further comprises an inducible suicide gene. In certain embodiments, the vector further comprises a nucleic acid sequence encoding for one or more cytokines, chemokines, hormones, growth factors or combinations thereof. [0012] In another aspect, an isolated cell comprises a chimeric antigen receptor or an expression vector embodied herein. In certain embodiments, the isolated cell comprises a T cells, B cells, natural killer (NK) cells, macrophages, stem cells, induced pluripotent stem cells (iPSCs) or combinations thereof. In certain embodiments, the T cell is a CD8+ T cell, a CD4+ T cell, a regulatory T cell (Treg), gamma delta T cells (Ȗį T cells), or a tumor infiltrating T lymphocyte (TIL). [0013] In another aspect, an isolated T cell is transduced with a vector to express a first antigen specific binding domain, a second antigen specific binding domain, a transmembrane domain(s), co-stimulatory domain(s), and a CD3ȗ signaling domain, wherein the first antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof and the second antigen domain binds to a cluster of differentiation antigen 19 (CD19). In certain embodiments, the co-stimulatory domain comprises a CD28, a 41BB polypeptide or the combination thereof. In certain embodiments, the first antigen specific binding domain wherein the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 9. In certain embodiments, the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 9. In certain embodiments, the first antigen specific binding domain Docket No.: 354406.00202 comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 11. In certain embodiments, the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 11. In certain embodiments, the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 10. In certain embodiments, the second antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 10. In certain embodiments, the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 12. In certain embodiments, the second antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 12. In certain embodiments, the CAR further comprises a hinge region. In certain embodiments, the bispecific chimeric antigen receptor comprises at least a 75% sequence identity to SEQ ID NO: 13. In certain embodiments, the bispecific chimeric antigen receptor comprises SEQ ID NO: 13. In certain embodiments, the CAR further comprises at least one hinge region. [0014] In another aspect, an isolated cell comprises a chimeric antigen receptor (CAR) comprises an amino acid sequence comprising at least a 75% sequence identity to SEQ ID NO: 1 or 5. In certain embodiments, the chimeric antigen receptor (CAR) SEQ ID NO: 1 or 5. In certain embodiments, the CAR further comprises at least one hinge region. [0015] In another aspect, an isolated cell comprises a chimeric antigen receptor (CAR) comprises an amino acid sequence comprising at least a 75% sequence identity to SEQ ID NO: 2 or 6. In certain embodiments, the chimeric antigen receptor (CAR) comprises SEQ ID NO: 2 or 6. In certain embodiments, the CAR further comprises at least one hinge region. [0016] In another aspect, an isolated cell comprises a chimeric antigen receptor (CAR) comprises an amino acid sequence comprising at least a 75% sequence identity to SEQ ID NO: 3 or 7. In certain embodiments, the chimeric antigen receptor (CAR) comprises SEQ ID NO: 3 or 7. In certain embodiments, the CAR further comprises at least one hinge region. [0017] In another aspect, an isolated cell comprises an expression vector encodes an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, the vector encodes an amino acid sequence comprising SEQ ID NO: 4, 8, or 14. In certain embodiments, the vector comprises adenovirus, adeno-associated virus (AAV), herpes simplex virus, lentivirus, gammaretrovirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles Docket No.: 354406.00202 virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus or vesicular stomatitis virus. In certain embodiments, the vector is a lentivirus. In certain embodiments, the vector further comprises an inducible promoter, a cell specific promoter, a tissue specific promoter or a constitutive promoter. In certain embodiments, the vector further comprises one or more enhancer or regulatory sequences. In certain embodiments, the vector further comprises an inducible suicide gene. In certain embodiments, the vector further comprises a nucleic acid sequence encoding for one or more cytokines. [0018] In another aspect, an isolated cell comprises a bispecific chimeric antigen receptor comprising a first antigen specific binding domain, a second antigen specific binding domain, a transmembrane domain(s), co-stimulatory domain(s), and a CD3ȗ signaling domain, wherein the first antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof and the second antigen domain binds to a cluster of differentiation antigen 19 (CD19). In certain embodiments, the co-stimulatory domain comprises a CD28, a 41BB polypeptide or the combination thereof. In certain embodiments, the first antigen specific binding domain wherein the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 9. In certain embodiments, the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 9. In certain embodiments, the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 11. In certain embodiments, the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 11. In certain embodiments, the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 10. In certain embodiments, the second antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 10. In certain embodiments, the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 12. In certain embodiments, the second antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 12. In certain embodiments, the CAR further comprises a hinge region. In certain embodiments, the bispecific chimeric antigen receptor comprises at least a 75% sequence identity to SEQ ID NO: 13. In certain embodiments, the bispecific chimeric antigen receptor comprises SEQ ID NO: 13. In certain embodiments, the CAR further comprises at least one hinge region. Docket No.: 354406.00202 [0019] In another aspect, a method of treating a subject in need of immunotherapy, comprises isolating T lymphocytes from a biological sample obtained from the subject; transducing the T lymphocytes with an expression vector encoding a chimeric antigen receptor (CAR) which specifically binds to a fibrin antigenic epitope; expanding the transduced T lymphocytes at least once ex vivo to obtain expanded T lymphocytes specific for the fibrin antigen; and reinfusing the T lymphocytes into the subject, thereby treating the subject. In certain embodiments, the T lymphocytes are autologous cells. In certain embodiments, the CAR comprises an antigen binding domain linked to at least one co-stimulatory domain and a CD3 signaling domain, wherein the antigen binding domain comprises a single chain variable fragment (scFv) which specifically binds to fibrin. In certain embodiments, the co-stimulatory domain comprises a CD28, a 41BB polypeptide or the combination thereof. In certain embodiments, the CAR comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 1 or 5. In certain embodiments, the CAR comprises SEQ ID NO: 1, or 5. In certain embodiments, the CAR comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 2 or 6. In certain embodiments, the CAR comprises SEQ ID NO: 2 or 6. In certain embodiments, the CAR comprises an scFv amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 3 or 7. In certain embodiments, the CAR comprises SEQ ID NO: 3 or 7. In certain embodiments, the T lymphocyte is a CD8+ T lymphocyte, a CD4+ T lymphocyte, a regulatory T lymphocyte (Treg) or a tumor infiltrating T lymphocyte (TIL). In certain embodiments, the immunotherapy is administered to a subject diagnosed with cancer, a neurodegenerative disorder or an autoimmune disorder. [0020] In another aspect, a method of treating a subject in need of immunotherapy, comprises isolating T lymphocytes from a biological sample obtained from the subject; transducing the T lymphocytes with an expression vector encoding a bispecific chimeric antigen receptor (CAR) comprising a first antigen specific binding domain, a second antigen specific binding domain, a transmembrane domain(s), a co-stimulatory domain(s), and a CD3ȗ signaling domain, wherein the first antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof and the second antigen domain binds to a cluster of differentiation antigen 19 (CD19), expanding the transduced T lymphocytes at least once ex vivo to obtain expanded T lymphocytes expressing the bispecific CAR; and reinfusing the T lymphocytes into the subject, thereby treating the subject. In certain embodiments, the co-stimulatory domain comprises a CD28, a 41BB polypeptide or the Docket No.: 354406.00202 combination thereof. In certain embodiments, the T lymphocytes are autologous cells. In certain embodiments, the T lymphocyte is a CD8+ T lymphocyte, a CD4+ T lymphocyte, a regulatory T lymphocyte (Treg) or a tumor infiltrating T lymphocyte (TIL). In certain embodiments, the immunotherapy is administered to a subject diagnosed with cancer, a neurodegenerative disorder or an autoimmune disorder. [0021] In another aspect, an isolated natural killer (NK) cell comprises a chimeric antigen receptor (CAR) wherein the CAR comprises an intracellular activating signaling domain, a transmembrane region and an extracellular antigen binding domain. In certain embodiments, the intracellular activating signaling domain comprises a CD28 or CD137 molecule. In certain embodiments, the extracellular antigen binding domain is a single-chain variable fragment which specifically binds to a tumor antigen. [0022] In another aspect, an isolated natural killer (NK) cell comprises a chimeric antigen receptor (CAR) wherein the CAR comprises an intracellular activating signaling domain, a transmembrane region, a first extracellular antigen binding domain and a second antigen binding domain. In certain embodiments, the intracellular activating signaling domain comprises a CD28 or CD137 molecule. In certain embodiments, the first extracellular antigen binding domain is a single-chain variable fragment which specifically binds to fibrin. In certain embodiments, the second extracellular antigen binding domain is a single-chain variable fragment which specifically binds to a tumor antigen. [0023] In another aspect, a method of treating cancer comprises administering the isolated T lymphocytes embodied herein or the NK cells embodied herein. [0024] In another aspect, a chimeric antigen receptor (CAR) comprises an antigen specific binding domain, a transmembrane domain(s), a co-stimulatory domain(s), and a CD3ȗ signaling domain, wherein the antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof. In certain embodiments, the antigen specific binding domain comprises an antibody, antibody fragment, a camelid nanobody or aptamer. In certain embodiments, the antibody fragment is a single chain fragment. In certain embodiments, the single chain fragment is a single chain variable fragment (scFv). In certain embodiments, the co-stimulatory domain(s) comprise a cluster of differentiation antigen 28 (CD28), 41BB domain, an ICOS (Inducible T cell Co-stimulator) (CD278), OX40 (CD134), Glucocorticoid-induced Tumor Necrosis Factor Docket No.: 354406.00202 Receptor (GITR), CD40 or CD27. In certain embodiments, the antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 1 or 5. In certain embodiments, the antigen specific binding domain comprises SEQ ID NO: 1 or 5. In certain embodiments, the antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 2 or 6. In certain embodiments, the antigen specific binding domain comprises SEQ ID NO: 2 or 6. In certain embodiments, the antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 3 or 7. In certain embodiments, the antigen specific binding domain comprises SEQ ID NO: 3 or 7. In certain embodiments, the CAR further comprises a hinge region. [0025] In another aspect, a chimeric antigen receptor (CAR) comprises an antigen specific binding domain, a transmembrane domain(s), a co-stimulatory domain(s), and a CD3ȗ signaling domain, wherein the antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof. In certain embodiments, the antigen specific binding domain comprises an antibody, antibody fragment, a camelid nanobody or aptamer. In certain embodiments, the antibody fragment is a single chain fragment. In certain embodiments, the single chain fragment is a single chain variable fragment (scFv). In certain embodiments, the co-stimulatory domain(s) comprise a cluster of differentiation antigen 28 (CD28), 41BB domain, an ICOS (Inducible T cell Co-stimulator) (CD278), OX40 (CD134), Glucocorticoid-induced Tumor Necrosis Factor Receptor (GITR), CD40 or CD27. In certain embodiments, the antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In certain embodiments, the antigen specific binding domain comprises SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. [0026] In another aspect, a pharmaceutical composition comprises the chimeric antigen receptor (CAR) embodied herein, or the expression vector embodied herein, or the isolated cell embodied herein, or the isolated T cell embodied herein, or the isolated NK cell embodied herein. [0027] In another aspect, the chimeric antigen receptor (CAR) embodied herein, or the expression vector embodied herein, or the isolated cell embodied herein, or the isolated T cell embodied herein, or the isolated NK cell embodied herein, are administered as a combination therapy with one or more other therapeutic agents. Docket No.: 354406.00202 [0028] In certain embodiments, the scFv which specifically binds to fibrin comprises SEQ ID NO: 1 (FIB4scFv, PMC 1694 Retro vector): [0029] DIVMTQAAFSNPITLGTSASMSCRSSKSLLHSSGITYLSWYLQKPGQSPQLLIYQ MSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKGG GGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWIHWVKQRPGQG LEWIGLIDPSDSYTNYNQKFRGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCASSDPTGC WGQGTTLTVSP [0030] In certain embodiments, the scFv-CAR which specifically binds to fibrin further includes SEQ ID NO: 2 (mCD3z CAR PMC 1694 Retro vector): [0031] RAKFSRSAETAANLQDPNQLFNELNLGRREEFDVLEKKRARDPEMGGKQQRR RNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLFQGLSTATKDTFDALHMQT LAPR [0032] In certain embodiments, the CAR which specifically binds to fibrin includes SEQ ID NO: 3 (scFv-CAR-CD28- mCD3z PMC 1694 Retro vector): [0033] DIVMTQAAFSNPITLGTSASMSCRSSKSLLHSSGITYLSWYLQKPGQSPQLLIYQ MSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKGG GGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWIHWVKQRPGQG LEWIGLIDPSDSYTNYNQKFRGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCASSDPTGC WGQGTTLTVSPIEFMYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKLFWALVVVAGVLFC YGLLVTVALCVIWTNSRRNRGGQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRPRA KFSRSAETAANLQDPNQLFNELNLGRREEFDVLEKKRARDPEMGGKQQRRRNPQEGVY NALQKDKMAEAYSEIGTKGERRRGKGHDGLFQGLSTATKDTFDALHMQTLAPREGRGS LLTCGDVEENPGPAAAPVAT [0034] In certain embodiments, a vector encoding a CAR which specifically binds to fibrin comprises SEQ ID NO: 4 (PMC 1694 (SP-FIB4scFv-mCD28-mCD3z-T2A-GFP), Retro vector): [0035] MGVPTQLLGLLLLWITDAICDIVMTQAAFSNPITLGTSASMSCRSSKSLLHSSGI TYLSWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCA QNLELPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKAS GYTFTSYWIHWVKQRPGQGLEWIGLIDPSDSYTNYNQKFRGKATLTVDTSSSTAYMQLS Docket No.: 354406.00202 SLTSEDSAVYYCASSDPTGCWGQGTTLTVSPIEFMYPPPYLDNERSNGTIIHIKEKHLCHT QSSPKLFWALVVVAGVLFCYGLLVTVALCVIWTNSRRNRGGQSDYMNMTPRRPGLTR KPYQPYAPARDFAAYRPRAKFSRSAETAANLQDPNQLFNELNLGRREEFDVLEKKRAR DPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLFQGLSTAT KDTFDALHMQTLAPREGRGSLLTCGDVEENPGPAAAPVAT [0036] In certain embodiments, the scFv which specifically binds to fibrin comprises SEQ ID NO: 5 (FIB4scFv PMC 1947 Lenti vector): [0037] DIVMTQAAFSNPITLGTSASMSCRSSKSLLHSSGITYLSWYLQKPGQSPQLLIYQ MSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKGG GGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWIHWVKQRPGQG LEWIGLIDPSDSYTNYNQKFRGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCASSDPTGC WGQGTTLTVSP [0038] In certain embodiments, the scFv CAR includes SEQ ID NO: 6 (CAR PMC 1947 Lenti vector): [0039] ASAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLA CYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [0040] In certain embodiments, the scFv-CAR which specifically binds to fibrin comprises SEQ ID NO: 7 (FIB4scFv-CAR PMC 1947 Lenti vector): [0041] DIVMTQAAFSNPITLGTSASMSCRSSKSLLHSSGITYLSWYLQKPGQSPQLLIYQ MSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKGG GGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWIHWVKQRPGQG LEWIGLIDPSDSYTNYNQKFRGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCASSDPTGC WGQGTTLTVSPASAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVV GGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Docket No.: 354406.00202 [0042] In certain embodiments, a vector encoding a CAR which specifically binds to fibrin comprises SEQ ID NO: 8 (FIB4scFv-CAR PMC 1947 Lenti vector): [0043] MALPVTALLLPLALLLHAARPDIVMTQAAFSNPITLGTSASMSCRSSKSLLHSS GITYLSWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYC AQNLELPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKA SGYTFTSYWIHWVKQRPGQGLEWIGLIDPSDSYTNYNQKFRGKATLTVDTSSSTAYMQL SSLTSEDSAVYYCASSDPTGCWGQGTTLTVSPDYKDDDDKASAIEVMYPPPYLDNEKSN GTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNL GRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLY QGLSTATKDTYDALHMQALPPR [0044] In certain embodiments, a fibrin specific scFv comprises SEQ ID NO: 9 (PMC 1792, bi- specific, Lenti vector CAR): [0045] DIVMTQAAFSNPITLGTSASMSCRSSKSLLHSSGITYLSWYLQKPGQSPQLLIYQ MSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKGG GGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWIHWVKQRPGQG LEWIGLIDPSDSYTNYNQKFRGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCASSDPTGC WGQGTTLTVSP [0046] In certain embodiments, a CD19 specific scFv comprises SEQ ID NO: 10 (PMC 1792, bi-specific, Lenti vector CAR): [0047] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRL HSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGK PGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWL GVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYA MDYWGQGTSVTVSS [0048] In certain embodiments, a fibrin specific scFv -CAR comprises SEQ ID NO: 11 (PMC 1792, bi-specific, Lenti vector CAR): [0049] DIVMTQAAFSNPITLGTSASMSCRSSKSLLHSSGITYLSWYLQKPGQSPQLLIYQ MSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKGG Docket No.: 354406.00202 GGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWIHWVKQRPGQG LEWIGLIDPSDSYTNYNQKFRGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCASSDPTGC WGQGTTLTVSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR [0050] In certain embodiments, a CD19 specific scFv -CAR comprises SEQ ID NO: 12 (PMC 1792, bi-specific, Lenti vector CAR): [0051] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRL HSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGK PGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWL GVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYA MDYWGQGTSVTVSSASAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV LVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR. [0052] In certain embodiments, the bispecific chimeric antigen receptor comprises SEQ ID NO: 13 (PMC 1792, bi-specific, Lenti vector CAR): [0053] DIVMTQAAFSNPITLGTSASMSCRSSKSLLHSSGITYLSWYLQKPGQSPQLLIYQ MSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGAGTKLELKGG GGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWIHWVKQRPGQG LEWIGLIDPSDSYTNYNQKFRGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCASSDPTGC WGQGTTLTVSPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEG STKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE Docket No.: 354406.00202 TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSSASAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVL ACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [0054] In certain embodiments, a vector encodes for amino acid sequences comprising SEQ ID NO: 14 (PMC 1792, bi-specific, Lenti vector CAR): [0055] MALPVTALLLPLALLLHAARPDIVMTQAAFSNPITLGTSASMSCRSSKSLLHSS GITYLSWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYC AQNLELPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKA SGYTFTSYWIHWVKQRPGQGLEWIGLIDPSDSYTNYNQKFRGKATLTVDTSSSTAYMQL SSLTSEDSAVYYCASSDPTGCWGQGTTLTVSPKNPDPWAKNLNEKDYTTTPAPRPPTPA PTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRG RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMLLLV TSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTV KLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLE ITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQP PRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY YYGGSYAMDYWGQGTSVTVSSDYKDDDDKASAIEVMYPPPYLDNEKSNGTIIHVKGK HLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR [0056] In certain embodiments, a mCD28-mCD3z amino acid sequence comprises SEQ ID NO: 15 (PMC 1694): [0057] MYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKLFWALVVVAGVLFCYGLLVTVA LCVIWTNSRRNRGGQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRPRAKFSRSAETA Docket No.: 354406.00202 ANLQDPNQLFNELNLGRREEFDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKM AEAYSEIGTKGERRRGKGHDGLFQGLSTATKDTFDALHMQTLAPR. [0058] In certain embodiments, a vector encodes a fluorescent peptide, e.g. green fluorescent protein, comprising SEQ ID NO: 16: [0059] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK LPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRA EVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRH NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAG ITLGMDELYK [0060] In certain embodiments, a vector encodes a signal peptide, comprising SEQ ID NO: 17: [0061] MALPVTALLLPLALLLHAARP. [0062] In certain embodiments, a vector encodes a transferrin peptide, comprising SEQ ID NO: 18: [0063] KNPDPWAKNLNEKDY [0064] In certain embodiments, a vector encodes a T2A ribosome skip sequence comprising SEQ ID NO: 19: [0065] EGRGSLLTCGDVEENPGP [0066] In certain embodiments, a vector encodes a FLAG sequence comprising SEQ ID NO: 20: [0067] DYKDDDDK [0068] In certain embodiments, the chimeric antigen receptor (CAR) binds an Ȗ377-395 epitope of the fibrin or fibrinogen ȖC domain. The CAR can alternatively bind an Ȗ190-202 epitope of the fibrin or fibrinogen ȖC domain. Such antibodies are monoclonal antibodies, and in various aspects humanized antibodies or human antibodies. In certain embodiments, a fibrin Ȗ377-395 epitope comprises, CKKTTMKIIPFNRLTIG (SEQ ID NO: 21), or a biologically active derivative thereof. [0069] In certain embodiments, the CARs comprise sequences derived from antibodies comprising a light chain with three complementarity determining regions comprising an amino acid sequence including RSSKSLLHSSGITYLS (SEQ ID NO: 22), QMSNLAS (SEQ ID NO: 23), and AQNLELPLT (SEQ ID NO: 24). In certain embodiments, the CARs comprise sequences Docket No.: 354406.00202 derived from antibodies comprising a heavy chain with three complementarity determining regions comprising an amino acid sequence including GYTFTSYWIH (SEQ ID NO: 25), LIDPSDSYTNYNQKFRG (SEQ ID NO: 26), and SDPTGC (SEQ ID NO: 27). In certain embodiments, the CARs comprise sequences derived from antibodies comprising a light chain with three complementarity determining regions comprising an amino acid sequence including RSSKSLLHSSGITYLS (SEQ ID NO: 22), QMSNLAS (SEQ ID NO: 23), and AQNLELPLT (SEQ ID NO: 24) and a heavy chain with three complementarity determining regions comprising an amino acid sequence including GYTFTSYWIH (SEQ ID NO: 25), LIDPSDSYTNYNQKFRG (SEQ ID NO: 26), and SDPTGC (SEQ ID NO: 27). [0070] In certain embodiments, the CARs comprise sequences derived from antibodies comprising the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:28: [0071] Asn Thr Ala Phe Ala Gly Phe Gly Arg Asn Met Arg Ser Leu Phe Ser Leu Gln Leu Leu Ser Thr Gln Asp Leu Ala Met Gly Trp Ser Cys Ile Ile Val Leu Leu Val Ser Thr Ala Thr Gly Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Thr Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Leu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Ser Ser Asp Pro Thr Gly Cys Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Pro Ala Ser Thr Thr Pro Pro. [0072] In certain embodiments, the antigen binding domains, e.g. scFvs, of the CARs are derived from antibodies which bind human fibrin or fibrinogen ȖC domain, and inhibits Mac-1 binding to fibrin or fibrinogen ȖC domain, and wherein the antibody light chain variable domain comprises the amino acid sequence of SEQ ID NO: 29: [0073] Thr Phe Asp Ser Pro Tyr Gln Val Arg Arg Met Arg Phe Ser Ala Gln Leu Leu Gly Leu Leu Val Leu Trp Ile Pro Gly Ser Thr Ala Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Ile Thr Leu Gly Thr Ser Ala Ser Met Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Ser Gly Ile Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Leu Thr Phe Gly Docket No.: 354406.00202 Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ala Cys Thr Lys Gly Glu Phe. [0074] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 1. [0075] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 2. [0076] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 3. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 3. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 3. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, Docket No.: 354406.00202 sequence identity to SEQ ID NO: 3. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 3. [0077] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 4. [0078] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 5. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 5. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 5. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 5. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 5. [0079] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 6. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 6. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 6. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, Docket No.: 354406.00202 sequence identity to SEQ ID NO: 6. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 6. [0080] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 7. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 7. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 7. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 7. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 7. [0081] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 8. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 8. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 8. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 8. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 8. [0082] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 9. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 9. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 9. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, Docket No.: 354406.00202 sequence identity to SEQ ID NO: 9. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 9. [0083] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 10. [0084] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 11. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 11. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 11. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 11. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 11. [0085] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 12. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 12. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 12. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, Docket No.: 354406.00202 sequence identity to SEQ ID NO: 12. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 12. [0086] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 13. [0087] In certain embodiments, a vector comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, a vector comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, a vector comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, a vector comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, a vector comprises SEQ ID NO: 4, 8 or 14. [0088] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, Docket No.: 354406.00202 sequence identity to SEQ ID NO: 15. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 15. [0089] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15. In certain embodiments, an amino acid sequence comprises SEQ ID NO: 15. [0090] In certain embodiments, an amino acid sequence comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NOS: 16-28 or 29. In certain embodiments, an amino acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NOS: 16-28 or 29. In certain embodiments, an amino acid sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NOS: 16-28 or 29. In certain embodiments, an amino acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NOS: 16-28 or 29. In certain embodiments, an amino acid sequence comprises any one of SEQ ID NOS: 16-28 or 29. [0091] In another aspect, the amino acid sequences comprising SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16-28 or 29 comprise one or more modified amino acids, non- natural amino acids, variants or combinations thereof. [0092] In another aspect, a method of treating a demyelination disorder in a subject, comprises administering a pharmaceutical composition comprising a therapeutically effective amount of an isolated T cell that is modified or transduced with a vector to express a bispecific chimeric antigen receptor (CAR) comprising a first antigen specific binding domain, a second antigen specific Docket No.: 354406.00202 binding domain, a transmembrane domain(s), a co-stimulatory domain(s), and a CD3ȗ signaling domain, wherein the first antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof. In certain embodiments, the first and second antigen specific binding domains comprise an antibody, antibody fragment, a camelid nanobody or aptamer. In certain embodiments, the antibody fragment is a single chain fragment. In certain embodiments, the single chain fragment is a single chain variable fragment (scFv). In certain embodiments, the co- stimulatory domain comprises a cluster of differentiation antigen 28 (CD28), 41BB domain, an ICOS (Inducible T cell Co-stimulator) (CD278), OX40 (CD134), Glucocorticoid-induced Tumor Necrosis Factor Receptor (GITR), CD40 or CD27. In certain embodiments, the second antigen specific binding domain specifically binds to tumor antigens, exogenous antigens, endogenous antigens or autoantigens. In certain embodiments, endogenous antigens comprise cluster of differentiation antigens (CD) or cell surface molecules. In certain embodiments, the cluster of differentiation antigens (CD) comprise CD1a, CD2, CD3, CD5, CD10, CD11c, CD13, CD15, CD19, CD20, CD25, CD30, CD33, CD99, CD103, CD117, CD123 or CD207. In certain embodiments, the antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In certain embodiments, the antigen specific binding domain comprises SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In certain embodiments, the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NOs: 9 or 11. In certain embodiments, the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NOs: 9 or 11. In certain embodiments, the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NOs: 10 or 12. In certain embodiments, the second antigen specific binding domain comprises SEQ ID NOs: 10 or 12. In certain embodiments, the bispecific chimeric antigen receptor further comprises a hinge region. In certain embodiments, the bispecific chimeric antigen receptor comprises at least a 75% sequence identity to SEQ ID NO: 13. In certain embodiments, the bispecific chimeric antigen receptor comprises SEQ ID NO: 13. In certain embodiments, the isolated T cell expressing the CAR (CAR-T cell) is a regulatory T (CAR-Treg) cell. In certain embodiments, CC1+Olig2+ oligodendrocytes are increased in number as compared to baseline controls. In certain embodiments, the CAR-Treg cells inhibit demyelination as compared to baseline controls. In certain embodiments, the CAR-Treg cells remyelination as compared to Docket No.: 354406.00202 baseline controls. In certain embodiments, the Treg cell comprises an autologous cell, an allogeneic cell, a haplotype matched cell, a haplotype mismatched cell, a haplo-identical cell, a xenogeneic cell, cell lines or combinations thereof. In certain embodiments, the demyelinating disorder comprises: periventricular leukomalacia, multiple sclerosis, acute disseminated encephalomyelitis, chronic inflammatory demyelinating polyneuropathy, adrenoleukodystrophy, adenomyeloneuropathy, Leber's hereditary optic atrophy, HTLV-associated myelopathy, Guillain- Barre syndrome, phenylketonuria, Tay-Sachs disease, Niemann-Pick disease, Gaucher's disease, Hurler's syndrome, Krabbe's disease, Pelizaeus-Merzbacher disease, cerebral palsy, traumatic brain injury or neonatal brain injury or a combination thereof. In certain embodiments, the method further comprises administering immature oligodendrocytes, pluripotent stem cells or the combination thereof. [0093] Definitions [0094] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. Definitions of common terms can be found in Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons New York, NY (2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons New York, NY (2001); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012); Jon Lorsch (ed.) Laboratory Methods in Enzymology: DNA, Elsevier, (2013); Frederick M. Ausubel (ed.), Current Protocols in Molecular Biology (CPMB), John Wiley and Sons, (2014); John E. Coligan (ed.), Current Protocols in Protein Science (CPPS), John Wiley and Sons, Inc., (2005); and Ethan M Shevach, Warren Strobe, (eds.) Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, John Wiley and Sons, Inc., (2003); each of which provide one skilled in the art with a general guide to many of the terms used in the present application. Docket No.: 354406.00202 [0095] Standard nomenclature is used for the natural amino acids and their abbreviations. For example, L-alanine is represented with the three-letter abbreviation Ala, or one-letter abbreviation “A”. Where indicated, the “D” stereoisomer of alanine is represented as D-Ala. [0096] Standard nomenclature is used for the bases of DNA, with cytosine, guanosine, adenine, and thymine indicated as “C”, “G”, “A”, and “T”, and codons that encode DNA follow the standard genetic code, for example the amino acid Leu is encoded by TTA, TTG, CTT, CTC, CTA or CTG, and Asp is encoded by GAT or GAC. [0097] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” [0098] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value or range. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and also preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. All numeric values are herein assumed to be modified by the term “about”, whether or not explicitly indicated. The recitation of numerical ranges by endpoints includes all numbers within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5). [0099] In the description and in the claims, phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features. The term “and/or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” Docket No.: 354406.00202 “one or more of A and B;” and “A and/or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C”, “one or more of A, B, and C” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible. [00100] As used herein, the term “affinity” is meant as a measure of binding strength. Without being bound to theory, affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. Affinity also includes the term “avidity,” which refers to the strength of the antigen-antibody bond after formation of reversible complexes. Methods for calculating the affinity of an antibody for an antigen are known in the art, including use of binding experiments to calculate affinity. Antibody activity in functional assays (e.g., flow cytometry assay) is also reflective of antibody affinity. Antibodies and affinities can be phenotypically characterized and compared using functional assays (e.g., flow cytometry assay). [00101] As used herein, the term “agent” is meant to encompass any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent capable of preventing, ameliorating, or treating a disease or other medical condition. The term includes small molecule compounds, antisense oligonucleotides, siRNA reagents, antibodies, antibody fragments bearing epitope recognition sites, such as Fab, Fabƍ, F(abƍ)2 fragments, Fv fragments, single chain antibodies, antibody mimetics (such as DARPins, affibody molecules, affilins, affitins, anticalins, avimers, fynomers, Kunitz domain peptides and monobodies), peptoids, aptamers; enzymes, peptides organic or inorganic molecules, natural or synthetic compounds and the like. An agent can be assayed in accordance with the methods of the invention at any stage during clinical trials, during pre-trial testing, or following FDA-approval. [00102] By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. [00103] The term “amino acid” as used herein refers to naturally occurring and synthetic Į, ȕ, Ȗ, and į amino acids, and includes but is not limited to, amino acids found in proteins, i.e. glycine, Docket No.: 354406.00202 alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine. Alternatively, the amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, ȕ-alanyl, ȕ- valinyl, ȕ-leucinyl, ȕ-isoleucinyl, ȕ-prolinyl, ȕ-phenylalaninyl, ȕ-tryptophanyl, ȕ-methioninyl, ȕ- glycinyl, ȕ-serinyl, ȕ-threoninyl, ȕ-cysteinyl, ȕ-tyrosinyl, ȕ-asparaginyl, ȕ-glutaminyl, ȕ- aspartoyl, ȕ-glutaroyl, ȕ-lysinyl, ȕ-argininyl or ȕ-histidinyl. The amino acids can be non-naturally occurring amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, D-amino acids (i.e. an amino acid of an opposite chirality to the naturally-occurring form), N-D-methyl amino acids, C-D-methyl amino acids, E-methyl amino acids and D- or L-E- amino acids. Other non-naturally occurring amino acids include, for example, E-alanine (E-Ala), norleucine (Nle), norvaline (Nva), homoarginine (Har), 4-aminobutyric acid (J-Abu), 2- aminoisobutyric acid (Aib), 6-aminohexanoic acid (H-Ahx), ornithine (orn), sarcosine, D-amino isobutyric acid, 3-aminopropionic acid, 2,3-diaminopropionic acid (2,3-diaP), D- or L- phenylglycine, D-(trifluoromethyl)-phenylalanine, and D-p-fluorophenylalanine. When the term amino acid is used, it is considered to be a specific and independent disclosure of each of the esters of Į, ȕ, Ȗ, and į glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine in the D and L-configurations. [00104] The term “antibody” includes monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules, as well as antibody fragments (e.g., Fab, F(ab')2, and Fv). The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each Docket No.: 354406.00202 other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the Į and Ȗ chains and four CH domains for μ and İ isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Ten and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated Į, į, İ, Ȗ and μ, respectively. The Ȗ and Į classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2. The terms “full-length antibody,” “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions. [00105] An “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng.8(10): 1057-1062 [1995]); single-chain antibody molecules and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produced two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant Docket No.: 354406.00202 domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the C H1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells. [00106] As used herein, the term “autoimmune disease” refers to a disease or condition in which a subject's immune system has an aberrant immune response against a substance that does not normally elicit an immune response in a healthy subject. Examples of autoimmune diseases that may be treated with a compound, pharmaceutical composition, or method described herein include Acute Disseminated Encephalomyelitis (ADEM), Acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome (APS), Autoimmune angioedema, Autoimmune aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia, Autoimmune immunodeficiency, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic purpura (ATP), Autoimmune thyroid disease, Autoimmune urticaria, Axonal or neuronal neuropathies, Balo disease, Behcet's disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus, Dressler's Docket No.: 354406.00202 syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture's syndrome, Granulomatosis with Polyangiitis (GPA) (formerly called Wegener's Granulomatosis), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (SLE), Lyme disease, chronic, Meniere's disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Reiter's syndrome, Relapsing polychondritis, Restless legs syndrome, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren's syndrome, Sperm & testicular autoimmunity, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, Transverse myelitis, Type 1 diabetes, Ulcerative colitis, Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vesiculobullous dermatosis, Vitiligo, or Wegener's granulomatosis (i.e., Granulomatosis with Polyangiitis (GPA). Docket No.: 354406.00202 [00107] The term “bispecific” as provided herein is used according to its conventional meaning well known in the art and refers to a bispecific recombinant protein capable of simultaneously binding to two different antigens. In contrast to traditional monoclonal antibodies, bispecific antibodies consist of two independently different antibody regions (e.g., two single-chain variable fragments (scFv)), each of which binds a different antigen. [00108] By “cancer” as used herein is meant, a disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art; including colorectal cancer, as well as, for example, leukemias, e.g., acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia, AIDS related cancers such as Kaposi's sarcoma; breast cancers; bone cancers such as Osteosarcoma, Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors, Adamantinomas, and Chordomas; Brain cancers such as Meningiomas, Glioblastomas, Lower- Grade Astrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas, and Metastatic brain cancers; cancers of the head and neck including various lymphomas such as mantle cell lymphoma, non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder and bile duct cancers, cancers of the retina such as retinoblastoma, cancers of the esophagus, gastric cancers, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, lung cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervical cancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrug resistant cancers; and proliferative diseases and conditions, such as neovascularization associated with tumor angiogenesis, macular degeneration (e.g., wet/dry AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration and other proliferative diseases and conditions such as restenosis and polycystic kidney disease, and other cancer or proliferative disease, condition, trait, genotype or phenotype. [00109] The term “chimeric antigen receptor” or “CAR” as used herein refers to an antigen- binding domain that is fused to an intracellular signaling domain capable of activating or stimulating an immune cell, and in certain embodiments, the CAR also comprises a transmembrane domain. In certain embodiments the CAR's extracellular antigen-binding domain is composed of a single chain variable fragment (scFv) derived from fusing the variable heavy and light regions Docket No.: 354406.00202 of a murine or humanized monoclonal antibody. Alternatively, scFvs may be used that are derived from Fab's (instead of from an antibody, e.g., obtained from Fab libraries). In various embodiments, the scFv is fused to the transmembrane domain and then to the intracellular signaling domain. “First-generation” CARs include those that solely provide CD3ȗ signals upon antigen binding, “Second-generation” CARs include those that provide both co-stimulation (e.g., CD28 or CD137) and activation (CD3ȗ). “Third-generation” CARs include those that provide multiple co-stimulation (e.g. CD28 and CD137) and activation (CD3ȗ). “Fourth generation” of CARs have been described as CAR-T cells redirected for cytokine killing (TRUCKS) where the vector containing the CAR construct possesses a cytokine cassette. When the CAR is ligated, the CAR-T cell deposits a pro-inflammatory cytokine into the tumor lesion. “Fifth generation” of CARs are mainly designed based on the second generation. However, these CAR-T cells contain a truncated cytoplasmic receptor (IL-12) and a ȕ-chain domain (IL-2Rȕ truncated intracellular interleukin 2ȕ chain receptor) along with the transcription factor STAT3/5 binding motif (Tokarew et al., 2019. Teaching an Old Dog New Tricks: Next-Generation CAR-T Cells. Br. J. Cancer 120, 26–37. doi:10.1038/s41416-018-0325-1). A CAR-T cell is a T cell that expresses a chimeric antigen receptor. The phrase “chimeric antigen receptor (CAR),” as used herein and generally used in the art, refers to a recombinant fusion protein that has an antigen-specific extracellular domain coupled to an intracellular domain that directs the cell to perform a specialized function upon binding of an antigen to the extracellular domain. The terms “artificial T-cell receptor,” “chimeric T-cell receptor,” and “chimeric immunoreceptor” may each be used interchangeably herein with the term “chimeric antigen receptor.” [00110] The term “combination therapy”, as used herein, refers to those situations in which two or more different agents are administered in overlapping regimens so that the subject is simultaneously exposed to both agents. When used in combination therapy, two or more different agents may be administered simultaneously or separately. This administration in combination can include simultaneous administration of the two or more agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, two or more agents can be formulated together in the same dosage form and administered simultaneously. Alternatively, two or more agents can be simultaneously administered, wherein the agents are present in separate formulations. In another alternative, a first agent can be administered just Docket No.: 354406.00202 followed by one or more additional agents. In the separate administration protocol, two or more agents may be administered a few minutes apart, or a few hours apart, or a few days apart. [00111] As used herein, the transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having.” By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure. [00112] The term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments with short linkers (about 5-10) residues) between the VH and VL domains such that inter- chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in greater detail in, for example, EP 404,097; WO 93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). [00113] “Diagnostic” or “diagnosed” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of “true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay, are termed “true negatives.” The “specificity” of a diagnostic assay is 1 minus the false positive rate, where the “false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. [00114] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate. Docket No.: 354406.00202 [00115] “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) can recognize and bind antigen, although at a lower affinity than the entire binding site. [00116] The term “hinge” or “hinge region” refers to a flexible connector region, e.g. natural or synthetic polypeptides, or any other type of molecule, providing structural flexibility and spacing to flanking polypeptide regions. [00117] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR (hereinafter defined) of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and/or capacity. In some instances, framework (“FR”) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR are typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol.1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Docket No.: 354406.00202 Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409. [00118] A “human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos.6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology. [00119] The term “hypervariable region,” “HVR,” or “HV,” when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996). A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Docket No.: 354406.00202 Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below. [00120] As used herein, the term “immune cells” refers to any cells of the immune system that are involved in mediating an immune response. Non-limiting examples of immune cells include a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell, neutrophil, or combination thereof. In some aspects, an immune cell expresses CD3. In certain aspects, the CD3-expressing immune cells are T cells (e.g., CD4+ T cells or CD8+ T cells). In some aspects, an immune cell that can be targeted with a targeting moiety (e.g., anti-CD3) comprises a naive CD4+ T cell. In some aspects, an immune cell comprises a memory CD4+ T cell. In some aspects, an immune cell comprises an effector CD4+ T cell. In some aspects, an immune cell comprises a naïve CD8+ T cell. In some aspects, an immune cell comprises a memory CD8+ T cell. In some aspects, an immune cell comprises an effector CD8+ T cell. In some aspects, an immune cell comprises a gamma delta T cell. In some aspects, an immune cell is a dendritic cell. In certain aspects, a dendritic cell comprises a plasmacytoid dendritic cell (pDC), a conventional dendritic cell 1 (cDC1), a conventional dendritic cell 2 (cDC2), inflammatory monocyte derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDCs), Kupffer cells, or any combination thereof. [00121] As used herein, the terms “immunoglobulin domain” and “immunoglobulin-like domain” are synonymous with “antibody” and refer to a polypeptide from the immunoglobulin superfamily of proteins with a specific secondary and tertiary structure derived from proteins found in nature. Engineered immunoglobulin domains can include components of monoclonal antibodies, antibody fragments such as Fab fragment, a F(ab')2 fragment, a single-chain variable fragment (scFv), a single-chain antibody fragment (scAb), a single domain heavy chain antibody, and a single domain light chain antibody, VHH antibodies (or nanobodies), and other single domain antibodies (sdAbs). Natural immunoglobulin and immunoglobulin-like domains are found in cell surface receptors, co-receptors, and co-activators and in soluble proteins involved in the recognition, binding and adhesion processes of cells. Docket No.: 354406.00202 [00122] As used herein, the term “inflammatory disease” refers to a disease or condition characterized by aberrant inflammation (e.g., an increased level of inflammation compared to a control such as a healthy person not suffering from a disease). Examples of inflammatory diseases include autoimmune diseases, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, Behcet's disease, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, inflammatory bowel disease, Addison's disease, Vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, ischemia reperfusion injury, stroke, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, scleroderma, and atopic dermatitis. [00123] A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. [00124] The term “linker”, also referred to as a “spacer” or “spacer domain” as used herein, refers to an amino acid or sequence of amino acids that that is optionally located between two amino acid sequences in a fusion protein of the invention. [00125] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other Docket No.: 354406.00202 immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol.222: 581-597 (1992); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and U.S. Pat. No.5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol.14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995). [00126] As used herein, the term “neurodegenerative disorder” refers to a disease or condition in which the function of a subject's nervous system becomes impaired. Examples of neurodegenerative diseases that may be treated with a compound, pharmaceutical composition, or method described herein include Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer- Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, chronic fatigue syndrome, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, frontotemporal dementia, Gerstmann-Sträussler-Scheinker syndrome, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, kuru, Lewy body Docket No.: 354406.00202 dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, myalgic encephalomyelitis, Narcolepsy, Neuroborreliosis, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Prion diseases, Refsum's disease, Sandhoff s disease, Schilder's disease, Subacute combined degeneration of spinal cord secondary to Pernicious Anaemia, Schizophrenia, Spinocerebellar ataxia (multiple types with varying characteristics), Spinal muscular atrophy, Steele-Richardson-Olszewski disease, progressive supranuclear palsy, or Tabes dorsalis. [00127] The term “naked antibody” refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel. [00128] As used herein, a “natural amino acid” refers to the twenty genetically encoded alpha- amino acids. See, e.g., Biochemistry by L. Stryer, 3rd ed.1988, Freeman and Company, New York for structures of the twenty natural amino acids. [00129] As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer. Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences. [00130] “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Docket No.: 354406.00202 [00131] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. [00132] As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise and should be understood to mean “either or both” of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases. [00133] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), intravitreal (i.v.i), intracisterna magna (i.e.m), or intrasternal injection, or infusion techniques. [00134] The terms “patient” or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates. [00135] “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In embodiments, the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. [00136] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as Docket No.: 354406.00202 [00137] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers. A “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed or chemically synthesized as a single moiety. [00138] “Polypeptide fragment” refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, in which the remaining amino acid sequence is usually identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long, or at least 70 amino acids long. [00139] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of the sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994). “Functional fragments” of the antibodies of the disclosure comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the Fc region of an antibody which retains or has modified FcR binding capability. Examples of antibody fragments include linear antibody, single-chain antibody molecules and multispecific antibodies formed from antibody fragments. [00140] As used herein, an “unnatural amino acid,” “non-natural”, “modified amino acid” or “chemically modified amino acid” refers to any amino acid, modified amino acid, or amino acid analogue other than the twenty genetically encoded alpha-amino acids. Unnatural amino acids have side chain groups that distinguish them from the natural amino acids, although unnatural amino acids can be naturally occurring compounds other than the twenty proteinogenic alpha- Docket No.: 354406.00202 amino acids. In addition to side chain groups that distinguish them from the natural amino acids, unnatural amino acids may have an extended backbone such as beta-amino acids. [00141] Non-limiting examples of non-natural amino acids include selenocysteine, pyrrolysine, homocysteine, an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcȕ-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L- phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; an amino acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and/or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto containing amino acid; amino acids comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid, e.g., a sugar substituted serine or the like; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an Į-hydroxy containing acid; an amino thio acid containing amino acid; an Į,Į disubstituted amino acid; a ȕ-amino acid; and a cyclic amino acid other than proline. In an embodiment of the helicases described herein, one or more amino acids of the helicase are substituted with one or more unnatural amino acids and/or one or more natural amino acids. [00142] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the Docket No.: 354406.00202 antigen binding sites. However, the variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. [00143] As used herein, “variant” of polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have “nonconservative” changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR). [00144] As used herein, the term “virus” includes any type of virus or virus vector. For example, adenovirus, adeno-associated virus (AAV), recombinant adeno-associated virus (rAAV), herpes simplex virus, lentivirus, gammaretrovirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus, vesicular stomatitis virus, picornavirus. In various embodiments the virus is a chimeric virus, a synthetic virus, a recombinant virus, a mosaic virus or a pseudotyped virus. [00145] Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically Docket No.: 354406.00202 disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range. [00146] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes or gene products disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences, are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In preferred embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides and proteins are human. The term “gene” is also intended to include variants. [00147] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel et al., 1992), Current Protocols in Molecular Biology (John Wiley & Sons, including periodic updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.1984); Transcription Docket No.: 354406.00202 And Translation (B. D. Hames & S. J. Higgins eds.1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ. of Oregon Press, Eugene, 2000). [00148] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. BRIEF DESCRIPTION OF THE DRAWINGS [00149] FIG.1 is a schematic representation showing the design of FIB4 fibrin-specific human CAR construct. PMC 1947: Amino-acid sequence of the FIB4-CAR (top). Schematic of lentiviral vector map encoding the fibrin- specific targeting CAR FIB4 with variations in co-stimulatory domains (bottom). [00150] FIG. 2 is a schematic representation showing the design of FIB4 fibrin-specific mouse CAR construct. PMC 1694: Amino-acid sequence of the FIB4 mouse CAR. Schematic of retroviral vector map encoding the fibrin- specific targeting FIB4-CAR with variations in co-stimulatory domains (bottom). [00151] FIG.3 is a series of plots showing a flow cytometry analysis of FIB4-CAR expression assessed by GFP and F(ab´)2 antibody. [00152] FIG.4 is a series of photographs of cell stains and a graph showing results from a FIB4- CAR cell adhesion assay to detect Hoechst stained cells transduced with fibrin-specific FIB4-CAR or control-CAR cells on fibrin-coated plates. Quantification of adhered cells transduced with control-CAR or fibrin-specific FIB4- CAR. *P = 0.05 by one-tailed Mann-Whitney test. Docket No.: 354406.00202 [00153] FIGS. 5A and 5B show the in vivo target recognition of FIB4-CAR in fibrinogen- injected mouse brain. Microscopy of GFP-expressing FIB4-CAR or GFP-expressing control-CAR transfected HEK293 recruited into the corpus callosum following injection with vehicle control artificial cerebrospinal fluid (ACSF) or fibrinogen. Higher magnification images of the fibrinogen or ACSF injection area (middle and right panels) shows parenchymal FIB4-CAR-transduced cells. ctx, cortex; cc, corpus callosum. [00154] FIG. 6 is a schematic showing the design of bi-specific fibrin/CD19-CAR construct. PMC 1792: Amino-acid sequence of the FIB4-CAR (top). Schematic of lentiviral vector encoding the fibrin- and CD- 19-bispecific CARs and co-stimulatory domains (bottom). [00155] FIG.7 shows a series of in vivo imaging of bi-specific FIB4/CD19-CAR human T cells. In vivo imaging in the cortex of CCI mice at five days post-lesion identifies sites of active BBB disruption correlating with perivascular accumulation of bi-specific FIB4/CD19-CAR T cells. Imaging started immediately after a bolus i.v. injection of a 70 kDa rhodamine dextran solution (red). Extensive bi-specific CAR T cell accumulation overlaps with areas of perivascular dye leaked (top panels). In CCI mice injected with control CD19-CAR T cells, dye continuously leaked in the perivascular tissue and there was not accumulation of control CD19-CAR T cells (bottom panels). [00156] FIG.8 is a series of immunostains showing fibrin engagement of bi-specific FIB4/CD19- CAR human T cells after traumatic brain injury. Microscopy of coronal brain slices from TBI animals treated with bi-specific FIB4/CD19-or CD19-CAR T cells (GFP+ cells, green) and immunostained with fibrinogen antibody (red) reveals substantial recruitment of bi-specific FIB4/CD19-CAR T cells, but not control CD19-CAR T cells, to sites of fibrin deposition. [00157] FIG.9 is a plot showing a flow cytometry analysis of FIB4-CAR expression assessed by GFP in human Treg cells. [00158] FIG.10 is a series of immunostains and a graph demonstrating detection of FIB4-CAR Treg cells in the LPC-lesioned site. Microscopy of coronal brain sections from LPC-injected mice treated with Treg cells and FIB4-CAR Treg cells at 14 d post-LPC injection showing immunoreactivity for a human nuclear marker (green). Right graph: Quantification of cells with a human nucleus in the LPC lesions of corpus callosum. **P < 0.01, two-tailed Mann-Whitney test. n.s., not significant. All data area mean ± s.e.m. Docket No.: 354406.00202 [00159] FIG.11 is a series of immunostains and a graph demonstrating the effect of FIB4-CAR Treg cells on remyelinating oligodendrocyte in LPC-injected brain. Microscopy of coronal brain sections from LPC-injected mice treated with PBS, Treg cells, and FIB4-CAR Treg cells at 14 d post-LPC injection showing immunoreactivity for CC1 (green) and OLIG2 (red). Right graph: Quantification of CC1+OLIG2+ oligodendrocytes in corpus callosum area demyelinated by LPC injection. ***P < 0.001, **P < 0.0001 one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. All data area mean ± s.e.m. [00160] FIG. 12 is a series of immunostains and a graph demonstrating the remyelination by FIB4-CAR Treg cells in the brain area demyelinated by LPC injection. Microscopy of coronal brain sections from LPC-injected mice treated with vehicle (PBS), Treg cells, and FIB4-CAR Treg cells at 14 d post-LPC injection immunostained for MBP. Right graph: Quantification of demyelinated area in the corpus callosum of LPC injected mice. *P < 0.05, one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. All data area mean ± s.e.m. DETAILED DESCRIPTION [00161] The present disclosure is directed to chimeric antigen receptors (CARs) targeting fibrin proteins and peptides. Fibrin is deposited only in disease tissues in a wide range of neurological, autoimmune, inflammatory diseases and cancer. Fibrin-CARs serve as a guide to direct T cells in diseased tissues and enables the recruitment of CAR-T cells in solid organs. Previous approaches have failed to guide CART cells, in tissues, such as solid tumors, due to lack of a target antigen. Targeting approaches using antigens expressed on cells has resulted in toxic effects, as the CAR- T cells kill the cells expressing the target antigen. Fibrin CARs overcome these challenges, as they target fibrin deposits in tissues and accumulate specifically at lesion sites with increased selectivity and reduce toxic off-target effects of immunotherapy. [00162] As described in detail in the examples section, to overcome these challenges, a novel class of fibrin CAR-T cells were engineered to express antigen-specific binding domains to recognize fibrin using the variable sequences from a mouse monoclonal fibrin- specific antibody (FIB4) raised against the fibrin epitope Ȗ377-395 within the fibrinogen ȖC domain. [00163] Antibodies directed against the Ȗ377-395 domain blocks the damaging effects of fibrin in the nervous system without affecting its beneficial effects in blood coagulation. These monoclonal antibodies can block formation of MS plaques, neurodegeneration in AD models, and certain Docket No.: 354406.00202 cancers. Exemplary antibodies of the invention include, for example, the FIB4 antibody (targeting the Ȗ377-395 epitope). See, US Patent 8877195, incorporated herein in its entirety. [00164] Further described are bispecific fibrin-CD19 CARs having a dual activity of fibrin recognition and B cell toxicity and to selective kill B cells in tumors or autoimmune diseases, such as multiple sclerosis. The CARs can be transduced into T cells, such as cytotoxic T cells or regulatory T cells. These fibrin-specific CARs are used to direct the antigen specificity of regulatory T cells, cytotoxic T cells to target the lesion environment at sites of fibrin deposition. As fibrin is deposited early in disease and persist in chronic stages of inflammation and tissue damage, fibrin-CAR-T cells can be used to guide T cells in disease tissue early and persist during the course of disease. [00165] Fibrin [00166] The activation of innate immunity, disruption of the blood brain barrier (BBB) and deposition of fibrin are intimately linked in neurological diseases. The blood-coagulation factor fibrinogen extravasates into the central nervous system (CNS) parenchyma after disruption of the BBB and is converted into insoluble fibrin, a key proinflammatory matrix that activates innate immune responses (Petersen, M. A., Ryu, J. K. & Akassoglou, K. Fibrinogen in neurological diseases: mechanisms, imaging and therapeutics. Nat. Rev. Neurosci.19, 283–301 (2018). Davalos, D. & Akassoglou, K. Fibrinogen as a key regulator of inflammation in disease. Semin. Immopathol.34, 43–62 (2012). Both references incorporated herein by reference in their entireties). The conversion of fibrinogen into fibrin exposes amino acids 377–395 in the fibrinogen ƣ-chain that bind to the CD11b I- domain of the CD11b-CD18 integrin receptor (also known as Mac-1, complement receptor 3 (CR3), or ơMƢ2) and induces the activation of microglia and macrophages (Adams, R. A. et al. The fibrin- derived Ȗ 377-395 peptide inhibits microglia activation and suppresses relapsing paralysis in central nervous system autoimmune disease. J. Exp. Med.204, 571–582 (2007). Ugarova, T. P. et al. Sequence Ȗ 377-395(P2), but not Ȗ 190-202(P1), is the binding site for the alpha MI-domain of integrin Į Mȕ 2 in the Ȗ C-domain of fibrinogen. Biochemistry 42, 9365–9373 (2003). Davalos, D. et al. Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation. Nat. Commun. 3, 1227 (2012). Ryu, J. K. et al. Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation. Nat. Commun.6, 8164 (2015)). Fibrin is deposited in AD and MS lesions at Docket No.: 354406.00202 sites of microglial activation and macrophage infiltration. Fibrin is detected in progressive MS and in active and chronic lesions. In progressive MS, deposition of fibrin in the cortex correlates with neuronal loss and inhibition of fibrinolysis (Yates, R. L. et al. Fibrin(ogen) and neurodegeneration in the progressive multiple sclerosis cortex. Ann. Neurol.82, 259–270 (2017)). Disruption of the BBB and deposition of fibrin also occur early in MS and precede demyelination. Fibrinogen has been proposed as a cerebrospinal fluid and plasma biomarker for AD and mild cognitive impairment, and increased concentrations of fibrinogen are considered a predictor of brain atrophy in AD. Depletion of fibrin either genetically in fibrinogen-deficient mice or by anticoagulants decreases neuroinflammation, demyelination and axonal damage in animal models of MS and reduces the activation of microglia, damage to white matter and cognitive decline in animal models of AD. Fibrin induces rapid and sustained microglial responses and infiltration of macrophages into the CNS (Ryu, J.K., et al. Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration. Nat Immunol 19, 1212–1223 (2018). doi.org/10.1038/s41590-018-0232-x). [00167] T-Cell Receptors [00168] The chains of the T-cell antigen receptor (TCR) of a T-cell clone are each composed of a unique combination of domains designated variable (V), [diversity (D),] joining (J), and constant (C). In each T-cell clone, the combination of V, D, and J domains of both the alpha and the beta chains or of both the delta and gamma chains participates in antigen recognition in a manner which is uniquely characteristic of that T-cell clone and defines a unique binding site, also known as the idiotype of the T-cell clone. In contrast, the C domain does not participate in antigen binding. [00169] A TCR is a heterodimeric cell surface protein of the immunoglobulin super-family, which is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. TCRs exist in Įȕ and Ȗį forms, which are structurally similar but have quite distinct anatomical locations and probably functions. The extracellular portion of native heterodimeric ĮȕTCR and ȖįTCR each contain two polypeptides, each of which has a membrane-proximal constant region, and a membrane-distal variable region. Each of the constant and variable regions include an intra-chain disulfide bond. The variable regions contain the highly polymorphic loops analogous to the complementarity determining regions (CDRs), also known as hypervariable regions, of antibodies. The variable regions of both the TCRĮ and TCRȕ chain each have three CDRs, numbered CDR1, CDR2, and CDR3 in the direction from the amino terminal end to the Docket No.: 354406.00202 carboxy terminal end. CDR3 is the main CDR responsible for recognizing processed antigen. The TCRȕ CDR3 has been recognized as more structurally diverse than the other CDRs. [00170] The techniques for determining CDRs are generally known in the art. In some embodiments, the CDRs can be determined by approaches based on cross-species sequence variability. In some embodiments, the CDRs can be determined by approaches based on crystallographic studies of antigen-antibody complexes. In addition, combinations of these approaches are sometimes used in the art to determine CDRs. In certain embodiments, CDRs can be determined using sequence-based prediction tools. Such tools are generally available in the art, e.g., the Loupe V(D)J Browser provided by 10× Genomics® (Pleasanton, Calif.). For instance, in one embodiment, the single cell TCR sequencing of epitope reactive T-cell population can be conducted using the 10× Genomics® platform. Then the sequence can be processed using the Loupe V(D)J Browser to identify the clonotypes, V(D)J genes, and the CDR motifs, etc. More detailed information about the Loupe V(D)J Browser is available over the world-wide-web at site: support.10×genomics.com/single-cell-vdj/software/visualization/latest/tutorial-clonotypes, which is herein incorporated by reference in its entirety. [00171] Chimeric Antigen Receptors [00172] The structure and design of CARs contribute to their signaling mechanisms, effector functions, efficacy, and toxicity. The ligand recognition and signaling of CARs are affected by both the single chain variable fragment (scFv) and cytoplasmic domains; however, the transmembrane and spacer domains (nonsignaling) affect the function of CARs (Jayaraman, J., et al. (2020). CAR-T Design: Elements and Their Synergistic Function. EBioMedicine 58, 102931. doi:10.1016/j.ebiom.2020.1029312020). [00173] CARs consist of three major domains: 1) extracellular domain (ectodomain), which can be further divided into an antigen recognition domain, a single peptide on the cell surface cleaved from the mature CAR cell (Goulart L. et al. (2017). Cpd 22, 6560–6576. doi:10.2174/1381612822666160923112816). The antigen recognition domain is a single-chain fragment variant (scFV) chiefly comprising heavy and variable light chain regions composed of an antigen-specific immunoglobin separated by a flexible linker and attached to the transmembrane domain by a spacer (hinge) responsible for the transmission of receptor binding signals (Zhang C., et al. (2017). Engineering CAR-T Cells. Biomark. Res. 5, 22. Docket No.: 354406.00202 doi:10.1186/s40364-017-0102-y). 2) A transmembrane domain is essential for receptor stability and surface expression; it is a hydrophobic alpha helix that extends in the cell membrane (Zhang et al., 2017). 3) An intracellular domain (endo-domain), which upon stimulation, clusters and undergoes conformational changes, thus enabling the recruitment and phosphorylation of downstream signaling proteins (Su, X., and Vale, R. (2018). Mechanisms of Chimeric Antigen Receptor (CAR) Signaling during T Cell Activation. Biophysical J. 114, 107a–108a. doi:10.1016/j.bpj.2017.11.625). [00174] In certain embodiments, the CAR comprises one or more co-stimulatory domains comprising: CD28, ICOS, OX-40 or 41BB. The intracellular signaling region of a CAR or cell of the disclosure may comprise signaling regions from one, two, three, four or all five of these proteins in addition to the other regions specified herein. [00175] The co-stimulatory domains of a CAR or cell of the disclosure may comprise co- stimulatory domains from both 41BB and CD28. The 41BB co-stimulatory domain can be downstream of the CD28 co-stimulatory domains. [00176] In certain embodiments, a CD28 and CD3ȗ sequence comprises 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15. In certain embodiments, a CD28 and CD3ȗ sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15. In certain embodiments, a CD28 and CD3ȗ sequence comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15. In certain embodiments, a CD28 and CD3ȗ sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 15. In certain embodiments a CD28 and CD3ȗ sequence comprises SEQ ID NO: 15. [00177] The CAR may also comprise a spacer or hinge region situated between the antigen binding region and T cell plasma membrane. Commonly a spacer or hinge is a sequence derived from IgG subclass IgG1, IgG4, IgD or CD8. In certain embodiments, the hinge region comprises a CD28 motif. The hinge region can have any length. In some embodiments, the hinge region comprises 1 amino acid or 10 amino acids or 20 amino acids or 50 amino acids or 60 amino acids or 70 amino acids or 80 amino acids or 100 amino acids or 120 amino acids or 140 amino acids or Docket No.: 354406.00202 160 amino acids or 180 amino acids or 200 amino acids or 250 amino acids or 300 amino acids or any number therebetween. [00178] A CAR may further comprise a linker region. This may be rich in glycine for flexibility. The linker region may be rich in serine and threonine for solubility. The linker region can connect to N-terminus of variable heavy (VH) chain with the C-terminus of the variable light (VL) chain or vice versa. [00179] The CARs can be encoded by a vector and/or encompassed in one or more delivery vehicles and formulations as described in detail below. [00180] Antigen Binding Domain: Numerous antigen-binding domains are known in the art, including those based on the antigen binding site of an antibody, antibody mimetics, nanobodies, and T-cell receptor fragments. For example, the antigen-binding domain may comprise: a single- chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder such as a camelid; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor. Accordingly, the antigen specific binding domain includes, without limitation, an antibody, a T cell receptor fragment, a soluble T cell receptor, nanobody, aptamer, syn/notch recognition domain/effector domain pair, receptors, fragments or combinations thereof. In certain embodiments, the antigen specific binding domain is a T cell variable region fragments. In other embodiments, the antigen specific binding domain is an antibody or fragment thereof. The CAR can include single chains of T cell receptors and antibodies. In certain embodiments, the antigen binding domain is a single chain variable fragment (scFv). [00181] In certain embodiments, the antigen binding domain is or comprises an antibody or antibody fragment, aptamers, proteins and the like. In certain embodiments, the antibodies are human antibodies, including any known to bind a targeting molecule. The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(abƍ)2 fragments, Fabƍ fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered Docket No.: 354406.00202 and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. [00182] In some embodiments, the antigen-binding domain is a humanized antibody of fragments thereof. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity. [00183] In some embodiments, the heavy and light chains of an antibody can be full-length or can be an antigen-binding portion (a Fab, F(abƍ)2, Fv or a single chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is chosen from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly chosen from, e.g., IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1). In another embodiment, the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa. [00184] Among the provided antibodies are antibody fragments. Examples of antibody fragments include but are not limited to Fv, Fab, Fabƍ, Fabƍ-SH, F(abƍ)2; diabodies; linear antibodies; variable heavy chain (VH) regions, single-chain antibody molecules such as scFvs and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs. Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody. Docket No.: 354406.00202 [00185] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs. [00186] In certain embodiments, the antibody or antibody fragments of the CAR have high binding affinity for fibrin and/or a specific target antigen. In embodiments, the increased binding affinity is greater than effected by a reference antigen. [00187] In certain embodiments, the scFv fragments which specifically bind fibrin are engineered from antibodies obtained from a biological sample. For example, antibodies obtained from multiple sclerosis (MS) patients’ cerebrospinal fluid (CSF) plasma cells were isolated. The antibodies were screened to identify target antigens, such as by immunoprecipitation followed by mass spectrometry. Eighteen antibodies with fibrinogen as a potential target antigen were identified. These antibodies were further screened for their binding potential. One antibody (antibody G or 45-C1C3) was determined to bind fibrinogen and fibrin, bind the fibrin epitope P2, and inhibit inflammatory properties of fibrinogen, without anticoagulant properties. [00188] Accordingly, in certain embodiments, an scFv fragment which specifically binds to fibrin comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1 or 5. In certain embodiments, an scFv fragment comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1 or 5. In certain embodiments, an scFv fragment comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1 or 5. In certain embodiments, an scFv fragment comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1 or 5. In certain embodiments, an scFv fragment comprises SEQ ID NO: 1 or 5. [00189] In certain embodiments, a CAR includes a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID Docket No.: 354406.00202 NO: 2 or 6. In certain embodiments, a CAR includes at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2 or 6. In certain embodiments, a CAR includes at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2 or 6. In certain embodiments, a CAR includes at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2 or 6. [00190] In certain embodiments, an scFv which specifically binds to fibrin comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 9 or 11. In certain embodiments, an scFv fragment which specifically binds to fibrin comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 9 or 11. In certain embodiments, an scFv fragment which specifically binds to fibrin comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 9 or 11. In certain embodiments, an scFv fragment which specifically binds to fibrin comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 9 or 11. In certain embodiments, an scFv fragment which specifically binds to fibrin comprises SEQ ID NO: 9 or 11. [00191] In certain embodiments, an scFv which specifically binds to CD19 comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10 or 12. In certain embodiments, an scFv fragment which specifically binds to CD19 comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10 or 12. In certain embodiments, an scFv fragment which specifically binds to CD19 comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10 or 12. In certain embodiments, an scFv fragment which specifically binds to CD19 comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 10 or 12. In certain embodiments, an scFv fragment which specifically binds to CD19 comprises SEQ ID NO: 10 or 12. [00192] In certain embodiments, a CAR which specifically binds to fibrin comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or Docket No.: 354406.00202 99%, sequence identity to SEQ ID NO: 3, 7 or 13. In certain embodiments, a CAR which specifically binds to fibrin comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 3, 7 or 13. In certain embodiments, a CAR which specifically binds to fibrin comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 3, 7 or 13. In certain embodiments, a CAR which specifically binds to fibrin comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 3, 7 or 13. In certain embodiments, a CAR which specifically binds to fibrin comprises SEQ ID NO: 3, 7 or 13. [00193] In certain embodiments, a bispecific chimeric antigen receptor which binds to fibrin and CD19 comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13. In certain embodiments, a bispecific chimeric antigen receptor which binds to fibrin and CD19 comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13. In certain embodiments, a bispecific chimeric antigen receptor which binds to fibrin and CD19 comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13. In certain embodiments, a bispecific chimeric antigen receptor which binds to fibrin and CD19 comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 13. In certain embodiments, a bispecific chimeric antigen receptor which binds to fibrin and CD19 comprises SEQ ID NO: 13. [00194] Vectors [00195] In certain embodiments, a vector comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, a vector comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, a vector comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, a vector comprises at least a 90%, 91%, 92%, Docket No.: 354406.00202 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 4, 8 or 14. In certain embodiments, a vector comprises SEQ ID NO: 4, 8 or 14. [00196] In various embodiments, a vector or genetic construct is selected from the group comprising adenovirus, adeno-associated virus (AAV), herpes simplex virus, lentivirus, gammaretrovirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus, vesicular stomatitis virus. [00197] Vectors can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers. A marker gene can confer a selectable phenotype on a host cell. For example, a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin). An expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak, New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus. [00198] Additional expression vectors also can include, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col E1, pCR1, pBR322, pMal-C2, pET, pGEX, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2^ plasmid or derivatives thereof, vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences. [00199] The vector can also include a regulatory region. The term “regulatory region” refers to nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5ƍ and 3ƍ untranslated regions (UTRs), Docket No.: 354406.00202 transcriptional start sites, termination sequences, polyadenylation sequences, nuclear localization signals, and introns. [00200] The term “operably linked” refers to positioning of a regulatory region and a sequence to be transcribed in a nucleic acid so as to influence transcription or translation of such a sequence. For example, to bring a coding sequence under the control of a promoter, the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter. A promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site or about 2,000 nucleotides upstream of the transcription start site. A promoter typically comprises at least a core (basal) promoter. A promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). The choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the coding sequence. [00201] Vectors include, for example, viral vectors (such as adenoviruses Ad, AAV, lentivirus, and vesicular stomatitis virus (VSV) and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell. Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells. Other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide. Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. Other vectors include those described by Chen et al.; Bio Techniques, 34: 167-171 (2003). A large variety of such vectors are known in Docket No.: 354406.00202 the art and are generally available. A “recombinant viral vector” refers to a viral vector comprising one or more heterologous gene products or sequences. Since many viral vectors exhibit size- constraints associated with packaging, the heterologous gene products or sequences are typically introduced by replacing one or more portions of the viral genome. Such viruses may become replication-defective, requiring the deleted function(s) to be provided in trans during viral replication and encapsidation (by using, e.g., a helper virus or a packaging cell line carrying gene products necessary for replication and/or encapsidation). Modified viral vectors in which a polynucleotide to be delivered is carried on the outside of the viral particle have also been described (see, e.g., Curiel, D. T., et al. PNAS 88: 8850-8854, 1991). [00202] Additional vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia viruses and HIV-based viruses. One HIV based viral vector comprises at least two vectors wherein the gag and pol genes are from an HIV genome and the env gene is from another virus. DNA viral vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector [Geller, A. I. et al., J. Neurochem, 64: 487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad. Sci.: U.S.A.:907603 (1993); Geller, A. I., et al., Proc Natl. Acad. Sci USA: 87:1149 (1990)], Adenovirus Vectors [LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet.3: 219 (1993); Yang, et al., J. Virol.69: 2004 (1995)] and Adeno-associated Virus Vectors [Kaplitt, M. G., et al., Nat. Genet.8:148 (1994)]. [00203] When taken up by a cell, the genetic construct which includes the nucleotide sequence encoding the desired protein operably linked to the regulatory elements may remain present in the cell as a functioning extrachromosomal molecule or it may integrate into the cell's chromosomal DNA. DNA may be introduced into cells where it remains as separate genetic material in the form of a plasmid. Alternatively, linear DNA which can integrate into the chromosome may be introduced into the cell. When introducing DNA into the cell, reagents which promote DNA integration into chromosomes may be added. DNA sequences which are useful to promote integration may also be included in the DNA molecule. Alternatively, RNA may be administered to the cell. It is also contemplated to provide the genetic construct as a linear minichromosome including a centromere, telomeres and an origin of replication. Docket No.: 354406.00202 [00204] The regulatory elements necessary for gene expression of a DNA molecule include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for gene expression. It is necessary that these elements be operable linked to the sequence that encodes the desired proteins and that the regulatory elements are operably in the individual to whom they are administered. [00205] Initiation codons and stop codon are generally considered to be part of a nucleotide sequence that encodes the desired protein. However, it is necessary that these elements are functional in the individual to whom the gene construct is administered. The initiation and termination codons must be in frame with the coding sequence. [00206] Promoters and polyadenylation signals used must be functional within the cells of the individual. Examples of promoters useful to practice the present disclosure, especially in the production of a genetic vaccine for humans, include but are not limited to promoters from Simian Virus 40 (SV40, Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human Actin, human Myosin, human Hemoglobin, human muscle creatine and human metalothionein. [00207] In certain embodiments, a promoter comprises a MNDU3 promoter, a PGK promoter or the combination thereof. [00208] Examples of polyadenylation signals useful to practice the present disclosure, especially in the production of a genetic vaccine for humans, include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals. [00209] In addition to the regulatory elements required for DNA expression, other elements may also be included in the DNA molecule. Such additional elements include enhancers. The enhancer may be selected from the group including but not limited to: human Actin, human Myosin, human Hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV. Genetic constructs can be provided with mammalian origin of replication in order to maintain the construct extrachromosomally and produce multiple copies of the construct in the cell. Plasmids pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr virus origin of Docket No.: 354406.00202 replication and nuclear antigen EBNA-1 coding region which produces high copy episomal replication without integration. [00210] In certain embodiments, a genetic construct may include a suicide gene. An example of a suicide gene is inducible human caspase-9 transgene (iC9), which is dimerized and is activated by the administration of an otherwise bioinert small-molecule drug, AP1903 (Spencer DM, et al. Controlling signal transduction with synthetic ligands., Science, 1993, vol. 2625136(pg. 1019- 1024. Fan L, et al. Improved artificial death switches based on caspases and FADD., Hum Gene Ther, 1999, vol.1014, pg.2273-2285. Straathof KC, et al. An inducible caspase 9 safety switch for T-cell therapy., Blood, 2005, vol.10511, pg.4247-4254. Tey SK, et al. Inducible caspase 9 suicide gene to improve the safety of allodepleted T cells after haploidentical stem cell transplantation., Biol Blood Marrow Transplant, 2007, vol. 138, pg. 913-924). Other examples are cytosine deaminase/5-fluorocytosine and the herpes simplex virus/ganciclovir. [00211] In certain embodiments, a vector or genetic construct encodes a cytokine. The cytokine and/or suicide gene may be encoded by the same or different vector or included with the vector encoding for a CAR. Accordingly, in certain embodiments a vector encodes for a CAR and a suicide gene. In certain embodiments, the vector encodes a CAR and at least one cytokine. In certain embodiments, the vector encodes a CAR, a suicide gene and at least one cytokine, or at least one chemokine or at least a hormone, or a growth factor, or combinations thereof. In certain embodiments, the vector encodes for a CAR and a second vector encodes for a suicide gene. In certain embodiments the vector encodes a CAR and a second vector encodes a suicide gene and at least one cytokine. In certain embodiments, a combination of a plurality of vectors encode CARs, suicide genes and cytokines. By “cytokine” as used herein is meant a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, chemokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- Docket No.: 354406.00202 beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF- beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G- CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-11, IL-12; IL-15, IL-17, IL-18, a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines. [00212] Several delivery methods may be utilized in conjunction with the isolated nucleic acid sequences for in vitro (cell cultures) and in vivo (animals and patients) systems. In one embodiment, a lentiviral gene delivery system may be utilized. Such a system offers stable, long term presence of the gene in dividing and non-dividing cells with broad tropism and the capacity for large DNA inserts. (Dull et al, J Virol, 72:8463-8471 1998). In an embodiment, adeno- associated virus (AAV) may be utilized as a delivery method. AAV is a non-pathogenic, single- stranded DNA virus that has been actively employed in recent years for delivering therapeutic gene in in vitro and in vivo systems (Choi et al, Curr Gene Ther, 5:299-310, 2005). AAV include serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ or DJ/8. An example of non-viral delivery method may utilize nanoparticle technology. This platform has demonstrated utility as a pharmaceutical in vivo. Nanotechnology has improved transcytosis of drugs across tight epithelial and endothelial barriers. It offers targeted delivery of its payload to cells and tissues in a specific manner (Allen and Cullis, Science, 303:1818-1822, 1998). [00213] The polynucleotides embodied herein may be used with a microdelivery vehicle such as cationic liposomes and adenoviral vectors. For a review of the procedures for liposome preparation, targeting and delivery of contents, see Mannino and Gould-Fogerite, Bio Techniques, 6:682 (1988). See also, Felgner and Holm, Bethesda Res. Lab. Focus, 11(2):21 (1989) and Maurer, R. A., Bethesda Res. Lab. Focus, 11(2):25 (1989). [00214] Replication-defective recombinant adenoviral vectors can be produced in accordance with known techniques. See, Quantin, et al., Proc. Natl. Acad. Sci. USA, 89:2581-2584 (1992); Docket No.: 354406.00202 Stratford-Perricadet, et al., J. Clin. Invest., 90:626-630 (1992); and Rosenfeld, et al., Cell, 68:143- 155 (1992). [00215] Another method is to use single stranded DNA producing vectors which can produce the expressed products intracellularly. See for example, Chen et al.,BioTechniques, 34: 167-171 (2003), which is incorporated herein, by reference, in its entirety. [00216] The nucleic acid sequences of the disclosure, e.g., vectors, can be delivered to an appropriate cell of a subject. This can be achieved by, for example, the use of a polymeric, biodegradable microparticle or microcapsule delivery vehicle, sized to optimize phagocytosis by phagocytic cells such as macrophages. For example, PLGA (poly-lacto-co-glycolide) microparticles approximately 1-10 ^m in diameter can be used. The polynucleotide is encapsulated in these microparticles, which are taken up by macrophages and gradually biodegraded within the cell, thereby releasing the polynucleotide. Once released, the DNA is expressed within the cell. A second type of microparticle is intended not to be taken up directly by cells, but rather to serve primarily as a slow-release reservoir of nucleic acid that is taken up by cells only upon release from the micro-particle through biodegradation. These polymeric particles should therefore be large enough to preclude phagocytosis (i.e., larger than 5 ^m and preferably larger than 20 ^m). Another way to achieve uptake of the nucleic acid is using liposomes, prepared by standard methods. The nucleic acids can be incorporated alone into these delivery vehicles or co- incorporated with cell- or tissue-specific antibodies, for example, specific for Treg cells or delivery to tumor cells as a target. Alternatively, one can prepare a molecular complex composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine binds to a ligand that can bind to a receptor on target cells. Delivery of “naked DNA” (i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site, is another means to achieve in vivo expression. In the relevant polynucleotides (e.g., expression vectors) the nucleic acid sequence encoding an isolated nucleic acid sequence comprising a sequence encoding a CAR, as described above. [00217] In some embodiments, the compositions of the disclosure can be formulated as a nanoparticle, for example, nanoparticles comprised of a core of high molecular weight linear polyethyleneimine (LPEI) complexed with DNA and surrounded by a shell of polyethylene glycol modified (PEGylated) low molecular weight LPEI. The nucleic acids and vectors may also be Docket No.: 354406.00202 applied to a surface of a device (e.g., a catheter) or contained within a pump, patch, or other drug delivery device. The nucleic acids and vectors disclosed herein can be administered alone, or in a mixture, in the presence of a pharmaceutically acceptable excipient or carrier (e.g., physiological saline). The excipient or carrier is selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences (E. W. Martin), a well-known reference text in this field, and in the USP/NF (United States Pharmacopeia and the National Formulary). [00218] In some embodiments, the compositions can be formulated as a nanoparticle encapsulating the compositions embodied herein. [00219] Regardless of whether compositions are administered as nucleic acids or polypeptides, they are formulated in such a way as to promote uptake by the mammalian cell. Useful vector systems and formulations are described above. In some embodiments the vector can deliver the compositions to a specific cell type. The disclosure is not so limited however, and other methods of DNA delivery such as chemical transfection, using, for example calcium phosphate, DEAE dextran, liposomes, lipoplexes, surfactants, and perfluoro chemical liquids are also contemplated, as are physical delivery methods, such as electroporation, micro injection, ballistic particles, and “gene gun” systems. [00220] Methods for Isolation of Cells [00221] Any number of methods known in the art can be used to isolate cells, such as T cells, or any other cell type that may be used in carrying out the treatment of a subject. Thus, also provided are various other genetically engineered cells expressing the chimeric antigen receptors e.g., CARs. The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ Docket No.: 354406.00202 cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation. [00222] Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCMX central memory T (TCM effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MATT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells. [00223] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils. [00224] In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types. Docket No.: 354406.00202 [00225] In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the CAR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered. [00226] Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom. [00227] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), brain, central nervous system (CNS), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources. [00228] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non- human primate, or pig. [00229] In some embodiments, isolation of the cells includes one or more preparation and/or non- affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, Docket No.: 354406.00202 enrich for desired components, lyse, or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components. [00230] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets. [00231] In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow- through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed, and the cells directly resuspended in culture media. [00232] In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient. [00233] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by Docket No.: 354406.00202 washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. [00234] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population. [00235] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells. [00236] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types. Markers, of various T cells have been reviewed by Tantalo D.G. et al. (Understanding T cell phenotype for the design of effective chimeric antigen receptor T cell therapies Journal for ImmunoTher. of Cancer 2021; 9:e002555. doi:10.1136/jitc-2021-002555). T cells can be isolated by positive or negative selection techniques. For example, CD3+, CD28+ T cells can be positively selected using anti-CD3/anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander). [00237] In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. Docket No.: 354406.00202 In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker“1”) at a relatively higher level (markerhigh) on the positively or negatively selected cells, respectively. [00238] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub- populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations. [00239] In some embodiments, CD8+ cells are further enriched for or depleted of naïve, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother.35(9):689-701. In some embodiments, combining TCM- enriched CD8+ T cells and CD4+ T cells further enhances efficacy. [00240] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. [00241] In some aspects, a CD4 expression-based selection step is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4- based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps. [00242] In one example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of, for example, CD 14 and Docket No.: 354406.00202 CD45RA, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order. [00243] CD4+ T helper cells are sorted into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naïve CD4+ T lymphocytes are CD45RO+, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. [00244] In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol.58: Metastasis Research Protocols, Vol.2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, N.J.). [00245] In certain embodiments, the cells are NK cells wherein the NK cells are transduced with the CARs of the disclosure. NK cells have typically been identified in flow cytometry by first excluding other lymphocyte markers such as the CD3 T cell marker. After this sorting, two markers have become well-established as standard NK cell markers, CD56 (neural cell adhesion molecule- 1, NCAM1) and CD16 (low affinity Fc gamma receptor 3A, FCGR3A, FcȖRIII). The differential expression of these two surface proteins defines the two main subsets of conventional NK cells, CD56brightCD16lo/í and CD56dimCD16+, often simplified as CD56bright and CD56dim, respectively. Among circulating cells in peripheral blood, CD56bright is less abundant, estimated to comprise only 5–10% of the population. CD56dim represents greater than 90% of NK cells. However, CD56bright has been noted to be abundant in certain tissues, including secondary lymphoid tissues. [00246] In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as DYNABEADS or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, Docket No.: 354406.00202 present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select. [00247] In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No.4,795,698, and Liberti et al., U.S. Pat. No.5,200,084 are other examples. [00248] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample. [00249] In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps. [00250] In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies. [00251] In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles Docket No.: 354406.00202 are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable. [00252] In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells. [00253] In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Nos. WO2009/072003, or US 20110003380 A1. [00254] In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps. [00255] In some aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic Docket No.: 354406.00202 separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells. [00256] The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field and are collected within the cell collection bag. [00257] In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood may be automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother.35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and Wang et al. (2012) Immunother.35(9):689-701. Docket No.: 354406.00202 [00258] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton.1(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity. [00259] In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously. [00260] In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to í80° C. at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. [00261] In some embodiments, the provided methods include cultivation, incubation, culture, and/or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and/or engineering the depleted cell populations and culture-initiating compositions. Docket No.: 354406.00202 [00262] Thus, in some embodiments, the cell populations are incubated in a culture-initiating composition. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells. [00263] In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. [00264] The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. [00265] Oligodendrocytes [00266] The adult vertebrate central nervous system (CNS) mainly consists of neurons, astrocytes, oligodendrocytes, and microglia cells. Oligodendrocytes, the myelin forming cells of the CNS, are subjected to cell stress or even death in a number of metabolic or inflammatory disorders (Armati P, Mathey E (2010) The biology of oligodendrocytes. Cambridge University Press; Bradl M, Lassmann H (2010) Oligodendrocytes: biology and pathology. Acta Neuropathol 119:37–53; Caprariello AV, et al. (2012) Apoptosis of oligodendrocytes in the central nervous system results in rapid focal demyelination. Ann Neurol 72:395–405; Jäkel S, Dimou L (2017) Glial cells and their function in the adult brain: a journey through the history of their ablation. Front Cell Neurosci 11:24; Traka M, et al. (2016) Oligodendrocyte death results in immune- mediated CNS demyelination. Nat Neurosci 19:65–74). An example of a demyelinating disorder is multiple sclerosis (MS). MS is associated with the development of large demyelinated plaques, oligodendrocyte destruction, and axonal degeneration. This pathology is paralleled by the activation of astrocytes and microglia as well as the recruitment of peripheral immune cells to the site of tissue injury. Demyelination is also a feature of many neurological diseases including Docket No.: 354406.00202 Alzheimer’s disease, chemo brain and COVID-19 neuropathology (REFs PMID: 29499767; PMID: 37258678; PMID: 36288726). [00267] Oligodendrocytes (OLGs) are one of the major cell types in the white matter in the central nervous system (CNS). OLG-produced myelin enwraps axons facilitating axonal conduction. The differentiation and maturation of OLGs in the adult brain maintain white matter integrity. OLGs are sensitive to ischemic insult because they are vulnerable to energy failure. In white matter, the saltatory conduction is highly dependent on ATP supply. Pathological Ca2+ entry occurs in part because of increased intracellular Na+ and membrane depolarization in OLG after stroke (Stys PK. Anoxic and ischemic injury of myelinated axons in CNS white matter: from mechanistic concepts to therapeutics. J Cereb Blood Flow Metab. 1998;18(1):2–25). OLG swelling and death occur within minutes to hours after ischemic insult (L. Jiang et al. Transl Stroke Res.2011 Sep; 2(3): 366–375. doi: 10.1007/s12975-011-0078-0). OLG death causes demyelination of neurofibers that leads to neurologic deficits in stroke patients. [00268] As part of the nervous system, oligodendrocytes are closely related to nerve cells and provide a supporting role for neurons. In addition, the nervous system of mammals depends on myelin sheaths, which reduce ion leakage and decrease the capacitance of the cell membrane. Myelin also increases impulse speed, as saltatory propagation of action potentials occurs at the nodes of Ranvier in between Schwann cells (of the PNS) and oligodendrocytes (of the CNS). Myelinating oligodendrocytes are a part of the white matter and myelination is an important component of intelligence. [00269] Biomarkers of Oligodendrogenesis: Oligodendrogenesis may be determined by measuring the concentration of certain biomarkers in tissue. These biomarkers include Oligodendrocyte Transcription Factor (OLIG2), 2ƍ,3ƍ-Cyclic-Nucleotide 3ƍ-Phosphodiesterase (CNPase), and Myelin Basic Protein (MBP). The presence of, or an increase in the concentration of these biomarkers may indicate oligodendrocyte formation. [00270] Oligodendrocyte Transcription Factor: Oligodendrocyte transcription factor (OLIG2) is a basic helix-loop-helix transcription factor encoded by the Olig2 gene. The protein is of 329 amino acids in length, 32 kDa in size and contains 1 basic helix-loop-helix DNA-binding domain. The expression of OLIG2 is mostly restricted in central nervous system and is well known for determining oligodendrocyte differentiation. Docket No.: 354406.00202 [00271] OLIG2 is mostly expressed in restricted domains of the brain and spinal cord ventricular zone which give rise to oligodendrocytes and specific types of neurons. During embryogenesis, OLIG2 first directs motor neuron fate by establishing a ventral domain of motor neuron progenitors and promoting neuronal differentiation. OLIG2 then switches to promoting the formation of oligodendrocyte precursors and oligodendrocyte differentiation at later stages of development. [00272] Accordingly, in another aspect, the present disclosure provides a method for reducing demyelination, inducing remyelination, promoting oligodendroglial progenitor cell (OPC) proliferation, and/or promoting oligodendrocyte differentiation in a subject, the method comprising administering to the subject a therapeutically effective amount of CAR-T cells, CAR- NK cells embodied herein. In some embodiments, administration comprises transplanting these cells into injured tissue in the subject, e.g intracranially. In some embodiments, the FIB2 specific CAR cells embodied herein are suspended in a pharmaceutically acceptable carrier prior to administration. In some instances, the pharmaceutically acceptable carrier comprises phosphate- buffered saline. In some embodiments, pluripotent stem cells or oligodendrocyte precursors, immature oligodendrocytes or combinations thereof are administered as part of the therapy. [00273] In other embodiments, the subject has one or more risk factors for a demyelinating disease. In some embodiments, reducing demyelination, inducing remyelination, promoting oligodendroglial progenitor cell (OPC) proliferation, and/or promoting oligodendrocyte differentiation in the subject reduces or eliminates one or more signs or symptoms of a demyelinating disease. In other embodiments, the subject does not have signs or symptoms of a demyelinating disease. In some instances, the demyelinating disease is selected from the group consisting of periventricular leukomalacia, multiple sclerosis, acute disseminated encephalomyelitis, chronic inflammatory demyelinating polyneuropathy, adrenoleukodystrophy, adenomyeloneuropathy, Leber's hereditary optic atrophy, HTLV-associated myelopathy, Guillain- Barre syndrome, phenylketonuria, Tay-Sachs disease, Niemann-Pick disease, Gaucher's disease, Hurler's syndrome, Krabbe's disease, Pelizaeus-Merzbacher disease, cerebral palsy, Alzheimer’s disease, COVID-19, chemo-brain, neurodegenerative disorders, or combination thereof. [00274] Methods of Treatment Docket No.: 354406.00202 [00275] In certain embodiments, a method of treating a subject requiring immunotherapy such as for example, cancer, autoimmunity, and the like comprises isolating T lymphocytes and/or NK cells from a biological sample obtained from the subject; transducing the T lymphocytes and/or NK cells with an expression vector encoding a chimeric antigen receptor (CAR) which specifically binds to fibrin and/or CD19; stimulating the transduced T lymphocytes and/or NK cells with fibrin and/or CD19 at least once ex vivo to obtain cells specific for the fibrin and/or NK cells; and reinfusing the cells into the subject, thereby treating the subject. In certain embodiments, the cells are autologous cells. CAR-T cells may be generated from any suitable source of T cells known in the art including, but not limited to, T cells collected from a subject. The subject may be a patient with an autoimmune disease such as rheumatoid arthritis, or cancer in need of CAR-T cell or CAR- NK cell therapy or a subject of the same species as the subject with the disease in need of cell therapy. The collected cells may be expanded ex vivo using methods commonly known in the art before transduction with a CAR-To generate a CAR-T cell and/or CAR-NK cells. [00276] Methods for CAR design, delivery and expression in T cells, and the manufacturing of clinical-grade CAR-T cell populations are known in the art. See, for example, Lee et al., Clin. Cancer Res.2012, 18(10): 2780-90, hereby incorporated by reference in its entirety. For example, the engineered CARs may be introduced into T cells using retroviruses, which efficiently and stably integrate a nucleic acid sequence encoding the chimeric antigen receptor into the target cell genome. The CAR-T cells, once they have been expanded ex vivo in response to, for example, an autoimmune disease antigen, can be reinfused into the subject in a therapeutically effective amount. The term “therapeutically effective amount” as used herein means the amount of CAR-T or CAR-NK cells when administered to a mammal, in particular a human, in need of such treatment, is sufficient to treat autoimmune diseases, or prevent organ rejection etc. In certain embodiments, administration of any of the compositions embodied herein, can be combined with other cell-based therapies, for example, stem cells, antigen presenting cells, pancreatic islets etc. [00277] The composition of the present disclosure may be prepared in a manner known in the art and in a manner suitable for parenteral administration to mammals, particularly humans, comprising a therapeutically effective amount of the composition alone, with one or more pharmaceutically acceptable carriers or diluents. Docket No.: 354406.00202 [00278] The term “pharmaceutically acceptable carrier” as used herein means any suitable carriers, diluents or excipients. These include all aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers and solutes, which render the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic and absorption agents and the like. It will be understood that compositions of the invention may also include other supplementary physiologically active agents. [00279] The carrier must be pharmaceutically “acceptable” in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. Compositions include those suitable for parenteral administration, including subcutaneous, intramuscular, intravenous and intradermal administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. Such methods include preparing the carrier for association with the CAR-T cells and/or CAR-NK cells. In general, the compositions are prepared by uniformly and intimately bringing into association any active ingredients with liquid carriers. [00280] In an embodiment, the composition is suitable for parenteral administration. In another embodiment, the composition is suitable for intravenous administration. [00281] Compositions suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes, which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. [00282] In other embodiments, the compositions comprise a cell which has been transformed or transfected with one or more vectors or nucleic acids encoding one or more CARs. In some embodiments, the methods of the disclosure can be applied ex vivo. That is, a subject's cells can be removed from the body and transduced with the compositions in culture with a desired target antigen, expand target-antigen specific, e.g. T cells and the expanded cells returned to the subject's body. The cells can be the subject's cells or they can be haplotype matched or a cell line. The cells can be irradiated to prevent replication. In some embodiments, the cells are human leukocyte Docket No.: 354406.00202 antigen (HLA)-matched, autologous, cell lines, or combinations thereof. In other embodiments the cells can be a stem cell. For example, an embryonic stem cell or an artificial pluripotent stem cell (induced pluripotent stem cell (iPS cell)). Embryonic stem cells (ES cells) and artificial pluripotent stem cells (induced pluripotent stem cell, iPS cells) have been established from many animal species, including humans. These types of pluripotent stem cells would be the most useful source of cells for regenerative medicine because these cells are capable of differentiation into almost all of the organs by appropriate induction of their differentiation, with retaining their ability of actively dividing while maintaining their pluripotency. iPS cells, in particular, can be established from self-derived somatic cells, and therefore are not likely to cause ethical and social issues, in comparison with ES cells which are produced by destruction of embryos. Further, iPS cells, which are self-derived cell, make it possible to avoid rejection reactions, which are the biggest obstacle to regenerative medicine or transplantation therapy. [00283] The CARs can be easily delivered to a subject by methods known in the art, for example, methods which deliver siRNA. Thus, the CAR molecules can be used clinically, similar to the approaches taken by current gene therapy. In particular, a CAR stable expression stem cell or iPS cells for cell transplantation therapy as well as vaccination can be developed for use in subjects. [00284] The CAR-T cells and/or CAR-NK cells, once they have been expanded ex vivo in response to an autoimmune disease antigen, or tumor antigen etc., are reinfused into the subject in a therapeutically effective amount. The term “therapeutically effective amount” as used herein means the amount of CAR-T cells and/or CAR-NK cells when administered to a mammal, in particular a human, in need of such treatment, is sufficient to treat the disease. [00285] The precise amount of CAR-T cells and/or CAR-NK cells to be administered can be determined by a physician with consideration of individual differences in age, weight, extent of disease and condition of the subject. Typically, administration of T cell therapies is defined by number of cells per kilogram of body weight. However, because T cells will replicate and expand after transfer, the administered cell dose will not resemble the final steady-state number of cells. [00286] In an embodiment, a pharmaceutical composition comprising the CAR-T cells and/or CAR-NK cells of the present disclosure may be administered at a dosage of 104 to 109 cells/kg body weight. In another embodiment, a pharmaceutical composition comprising the CAR-T cells Docket No.: 354406.00202 and/or CAR-NK cells of the present disclosure may be administered at a dosage of 105 to 106 cells/kg body weight, including all integer values within those ranges. [00287] Compositions comprising the CAR-T cells and/or CAR-NK cells of the present disclosure may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are known in the art (see, for example, Rosenberg et al., 1988, New England Journal of Medicine, 319: 1676). The optimal dosage and treatment regimen for a particular subject can be readily determined by one skilled in the art by monitoring the patient for signs of disease and adjusting the treatment accordingly. [00288] Combination Therapies [00289] The disclosure also contemplates the combination of the composition of the present disclosure with other drugs and/or in addition to other treatment regimens or modalities such as surgery. When the composition of the present disclosure is used in combination with known therapeutic agents the combination may be administered either in sequence (either continuously or broken up by periods of no treatment) or concurrently or as an admixture. For example, in the case of cancer chemotherapeutic agents may be administered as part of the combination therapy. [00290] In the case of autoimmune diseases, a combination therapy may include CAR-T cells and/or CAR- NK cells and one or more anti-inflammatory agents and/or therapeutic agents. The one or more anti-inflammatory agents and/or therapeutic agents may be administered simultaneously or sequentially. The anti-inflammatory agents comprise one or more antibodies which specifically bind to pro-inflammatory cytokines, e.g. pro-inflammatory cytokines such as IL-1, IL-4, IL-5, IL-6, IL-8, IL-10, GM-CSF, TNFĮ IFN-Ȗ and MIP-1a. In certain embodiments, the antibodies are anti-TNFĮ, anti-IL-6 or combinations thereof. In certain embodiments, one or more agents, other than antibodies can be administered which decrease pro-inflammatory cytokines, e.g. non-steroidal anti-inflammatory drugs (NSAIDs). Any combination of antibodies and one or more agents can be administered which decrease pro-inflammatory cytokines. Anti- inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti- inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate. Docket No.: 354406.00202 [00291] .In certain embodiments, the CAR-T cells and/or CAR-NK cells contemplated herein are administered in conjunction with a cytokine. [00292] In certain embodiments, the CAR-T cells and/or CAR- NK cells are administered in conjunction with a cancer therapy. As used herein, the term “cancer therapy” refers to a therapy useful in treating cancer. Examples of anti-cancer therapeutic agents include, but are not limited to, e.g., surgery, chemotherapeutic agents, immunotherapy, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, such as anti-HER-2 antibodies (e.g., HERCEPTINTM), anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (TARCEVATM)), platelet derived growth factor inhibitors (e.g., GLEEVECTM (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGF receptor(s), TRAIL/Apo2, and other bioactive and organic chemical agents, etc. Combinations thereof are also contemplated for use with the methods described herein. [00293] A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include Erlotinib (TARCEVATM, Genentech/OSI Pharm.), Bortezomib (VELCADETM, Millennium Pharm.), Fulvestrant (FASLODEXTM, Astrazeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARATM, Novartis), Imatinib mesylate (GLEEVECTM, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin (EloxatinTM, Sanofi), 5-FU (5- fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNETM, Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs.), and Gefitinib (IRESSATM, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as Thiotepa and CYTOXANTM cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozcicsin, carzcicsin and bizcicsin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; Docket No.: 354406.00202 pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin Ȗ1 and calicheamicin omega 1 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCINTM doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, strcptonigrin, strcptozocin, tubcrcidin, ubenimcx, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKTM polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosinc; arabinoside (“Ara-C”); cyclophosphamidc; thiotcpa; taxoids, e.g., TAXOLTM paclitaxel (Bristol-Myers Squibb Docket No.: 354406.00202 Oncology, Princeton, N.J.), ABRAXANETM Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERETM doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZARTM gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINETM vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. [00294] Also included in this definition of “chemotherapeutic agent” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEXTM (tamoxifen)), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTONTM (toremifene); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASETM (megestrol acetate), AROMASINTM (exemestane), formestanie, fadrozole, RIVISORTM (vorozole), FEMARATM (letrozole), and ARIMIDEXTM (anastrozole); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (viii) ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYMETM (ribozyme)) and a HER2 expression inhibitor; (ix) vaccines such as gene therapy vaccines, for example, ALLOVECTINTM vaccine, LEUVECTINTM vaccine, and VAXIDTM vaccine; PROLEUKINTM rIL-2; LURTOTECANTM topoisomerase 1 inhibitor; ABARELIXTM rmRH; (x) anti-angiogenic agents such as bevacizumab (AVASTINTM, Genentech); and (xi) pharmaceutically acceptable salts, acids or derivatives of any of the above. [00295] In various embodiments, the cancer therapeutic is an immunotherapy selected from the group comprising oncolytic virus, bacteria, oncolytic bacteria or other bacterial compositions, Bacillus Calmette-Guerin (BCG), a microbiome modulator, and/or a toll-like receptor (TLR) Docket No.: 354406.00202 agonist. In various embodiments, the TLR agonist is a TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and/or TLR13 agonist. In various embodiments, the TLR agonist is derived from virus, bacteria and/or made synthetically. In various embodiments, the immunotherapy is a is a stimulator of interferon genes (STING) pathway modulator. [00296] It will be appreciated by those skilled in the art of cancer immunotherapy that other complementary immune therapies may be added to the regimens described above to further enhance their efficacy including but not limited to GM-CSF to increase the number of myeloid derived innate immune system cells, low dose cyclophosphamide or PI3K inhibitors (e.g., PI3K delta inhibitors) to eliminate T regulatory cells that inhibit innate and adaptive immunity and 5FU (e.g., capecitabine), PI3K inhibitors or histone deacetylase inhibitors to remove inhibitory myeloid derived suppressor cells. For example, PI3K inhibitors include, but are not limited to, LY294002, Perifosine, BKM120, Duvelisib, PX-866, BAY 80-6946, BEZ235, SF1126, GDC-0941, XL147, XL765, Palomid 529, GSK1059615, PWT33597, IC87114, TG100-15, CAL263, PI-103, GNE- 477, CUDC-907, and AEZS-136. In some aspects, the PI3K inhibitor is a PI3K delta inhibitor such as, but not limited to, Idelalisib, RP6530, TGR1202, and RP6503. Additional PI3K inhibitors are disclosed in U.S. Patent Application Nos. US20150291595, US20110190319, and International Patent Application Nos. WO2012146667, WO2014164942, WO2012062748, and WO2015082376. The immunotherapy may also comprise the administration of an interleukin such as IL-2, or an interferon such as INFĮ. [00297] In certain embodiments, the CAR-T cells and/or CAR- NK cells are administered with one or more immune checkpoint modulators. Immune checkpoints refer to inhibitory pathways of the immune system that are responsible for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses. Examples of checkpoint inhibitor include, without limitation an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-ȕ. [00298] The term “checkpoint inhibitor” means a group of molecules on the cell surface of CD4+ and/or CD8+ T cells that fine-tune immune responses by down-modulating or inhibiting an anti- tumor immune response. Immune checkpoint proteins are well known in the art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, Docket No.: 354406.00202 BTLA, SIRPĮ (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, and A2aR (see, for example, WO 2012/177624). “Anti-immune checkpoint inhibitor therapy” refers to the use of agents that inhibit immune checkpoint inhibitors. Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer. Exemplary agents useful for inhibiting immune checkpoint inhibitors include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and/or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and/or activity of immune checkpoint inhibitor nucleic acids, or fragments thereof. Exemplary agents for upregulating an immune response include antibodies against one or more immune checkpoint inhibitor proteins block the interaction between the proteins and its natural receptor(s); a non-activating form of one or more immune checkpoint inhibitor proteins (e.g., a dominant negative polypeptide); small molecules or peptides that block the interaction between one or more immune checkpoint inhibitor proteins and its natural receptor(s); fusion proteins (e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fe portion of an antibody or immunoglobulin) that bind to its natural receptor(s); nucleic acid molecules that block immune checkpoint inhibitor nucleic acid transcription or translation; and the like. Such agents can directly block the interaction between the one or more immune checkpoint inhibitors and its natural receptor(s) (e.g., antibodies) to prevent inhibitory signaling and upregulate an immune response. Alternatively, agents can indirectly block the interaction between one or more immune checkpoint proteins and its natural receptor(s) to prevent inhibitory signaling and upregulate an immune response. For example, a soluble version of an immune checkpoint protein ligand such as a stabilized extracellular domain can binding to its receptor to indirectly reduce the effective concentration of the receptor to bind to an appropriate ligand. In one embodiment, anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies, either alone or used in combination. [00299] . In some embodiments, such therapy involves blockade of programmed cell death 1 (PD- 1). In some embodiments, such therapy involves treatment with an agent that interferes with an interaction involving PD-1 (e.g., with PD-L1). In some embodiments, such therapy involves administration of an antibody agent that specifically interacts with PD-1 or with PD-L1. In some embodiments, such therapy involves administration of one or more of nivolumab (BMS-936558, Docket No.: 354406.00202 MDX-1106, ONO-4538, a fully human Immunoglobulin G4 (IgG4) monoclonal PD-1 antibody), pembrolizumab (MK-3475, a humanized monoclonal IgG4 anti-PD-1 antibody), BMS-936559 (a fully human IgG4 PD-L1 antibody), MPDL3280A (a humanized engineered IgG1 monoclonal PD- L1 antibody) and/or MEDI4736 (a humanized engineered IgG1 monoclonal PD-L1 antibody). [00300] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the disclosure. [00301] This disclosure is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the figures and the sequence listing, are hereby incorporated by reference. EXAMPLES [00302] EXAMPLE 1: FIBRIN SPECIFIC CAR-T CELLS [00303] This disclosure describes the generation of CARs specifically recognizing fibrin, an antigen present only in diseased tissues. Fibrin is deposited at sites of damage in the brain and other tissues in a wide range of autoimmune, inflammatory, infectious diseases and cancer diseases, such as multiple sclerosis, Alzheimer’s disease, cancer, rheumatoid arthritis, SARS- COV-2 infection, periodontitis, etc. Since fibrin is not present in normal tissues, but is deposited only in disease, it was hypothesized that it would be an ideal antigen to guide CAR-T cell recruitment at sites of damage. [00304] CAR-T cells are an FDA-approved treatment for certain types of lymphoma and leukemia and have revolutionized the treatment of cancer. However, there are challenges delivering CAR-T cells to solid tumors and tissues, such as the brain. CAR-T cell therapies have not yet been developed for neurological and autoimmune diseases. Furthermore, lack of selectivity and off target effects induces adverse toxicity. [00305] The main challenges with CAR-T cell therapies are recruitment to the diseased tissue and elimination of off-target cytotoxicity. Previous approaches have largely failed to guide CAR-T cells in tissues, such as solid tumors. Approaches using tissue-specific antigens expressed on cells has resulted in toxic effects, as the CAR-T cells kill the cells expressing the target antigen. In Docket No.: 354406.00202 addition to cytotoxic T cells, regulatory T cells (Tregs) also have the undesired potential to destroy tissue cells expressing target antigens. These challenges are overcome as described herein by generation of fibrin CAR-T cells. [00306] Design of Fibrin-Specific Chimeric Antigen Receptor (CAR) Constructs [00307] Chimeric antigen receptors targeting fibrin were established to guide CAR-T cells selectivity in tissues at sites of fibrin deposition, while reducing off-target effects. An ScFv-based human CAR was generated comprising the variable domain of a murine fibrin-specific antibody (FIB4) in a lentiviral or retroviral vector (herein named ‘FIB4-CAR’). The FIB4 ScFv was introduced into a second- generation CAR cassette incorporating a human CD8a signal peptide, the human 4-1BB co- stimulatory domain, and human CD3 zeta activation signaling domain. The lentiviral vector also encodes a MNDU3 promoter, a PGK promoter, and green fluorescent protein (GFP) for in vivo detection (PMC 1947, FIG.1). [00308] The FIB4 ScFvs were also introduced into a second-generation murine CAR cassette incorporating a signaling peptide, the murine CD28 transmembrane domain, and the murine CD3zeta activation domain for the generation of murine FIB4. The retroviral vector also encodes a CMV promoter, green fluorescent protein (GFP) and a T2A sequence upstream of the CAR cassette (PMC 1694, FIG.2). [00309] HEK293 cells were transduced with each construct and FIB4-CAR expression was validated by flow cytometry using the expression of GFP and a F(ab´)2 antibody recognizing FIB4, which showed 69.8% transduction efficiency (FIG.3). [00310] In Vitro Fibrin Target Binding of FIB4-CAR [00311] To validate target binding, the fibrin-specific FIB4-CAR construct was transfected into HEK293 cells, and a cell adhesion assay was performed on fibrin-coated plates. Cells were incubated on fibrin for 30 minutes and nonadherent cells were washed off with PBS. Adhered cells were stained with Hoechst dye and detected using the CellInsight CX7 microscope. FIB4-CAR increased adhesion to fibrin by 9.8-fold compared to the control-CAR construct, indicating highly increased fibrin-specific binding (FIG.4). [00312] In Vivo Target Recognition of FIB4-CAR in Fibrinogen-Injected Mouse Brain Docket No.: 354406.00202 [00313] In vivo target selectivity of the FIB4-CAR (FIB4-CAR-T2A-GFP) was evaluated in fibrinogen- injected mice. Two days after bilateral injection of fibrinogen or ACSF into the corpus callosum, mice received a single injection of FIB4-CAR or CAR control-transduced HEK293 cells into the cerebral ventricle. Four days later, GFP-positive FIB4-CAR-Transduced cells were detected in coronal sections from the fibrinogen-injected side but not on the ACSF-injected side (FIG.5A). No preference was observed for fibrinogen or ACSF-injection side in mice that received Control CAR (CAR-T2A-GFP) control cells (FIG.5B). [00314] Generation and Validation of Bi-Specific FIB4/CD19-CAR-T Cells [00315] Fibrin accumulation has been detected in the brains of individuals with several neurological diseases involving pathogenic CD19+ B cells, and this microenvironment can hinder the success of B cell-directed CAR-T cell therapies. To overcome this limitation, bispecific FIB4/CD19-CARs were generated by combining the FIB4 scFvs, CD19 scFv, the human 4-1BB co-stimulatory domain, and human CD3 zeta (CD3ȗ) activation signaling domain (PMC1792, FIG. 6). [00316] Human T cells were transduced with lentiviral vectors carrying the FIB4/CD19-CAR and validated FIB4 and CD19 CAR expression using flow cytometry. In vivo target engagement was then evaluated in the controlled cortical impact (CCI) model of traumatic brain injury. CCI was induced in Rag2 knockout mice, and three days later, mice received intravenous injection of bi- specific FIB4/CD19-CAR human T cells (5 x 105 cells). Five days later, two-photon in vivo imaging in the brain was performed following retro-orbital injection of 100 ^L of 10-kDa Alexa 647–labeled dextran dissolved in ACSF. Imaging was performed 70 to 100 ^m below the dura mater in the somatosensory cortex. Rapid and progressive dye leakage was observed from locally disrupted blood vessels that spatially correlated with the location of FIB4/CD19-CAR-T cells, while human CD19-CAR-T cells at adjacent perivascular areas with dye leakage exhibited little cell density (FIG.7). [00317] Immunohistochemical staining with a fibrinogen antibody confirmed fibrin-specific engagement of FIB4/CD19-CAR human T cells after CCI. As shown in FIG. 8, coronal mouse brain sections showed marked spatial colocalization of fibrinogen deposits (red) with GFP-positive bi-specific CAR-T cells (green) compared with human CD19-CAR-T cells. [00318] EXAMPLE 2: FIB4 CAR-TREG CELLS Docket No.: 354406.00202 [00319] A novel class of fibrin CAR human Treg cells were generated by engineering the antigen- specific binding domain to recognize fibrin using the variable sequences from a mouse monoclonal fibrin-specific antibody (FIB4) raised against the fibrin epitope Ȗ377-395 within the ȖC fibrin region. The FIB4 fibrin antibody variable sequence (5B8) is described in the inventors’ prior publication and patent (Ryu et al., Nat Immunol 2018 PMID: 30323343 and US8877195, both of which are incorporated herein in their entirety). As fibrin is deposited early in disease and persist in chronic stages of inflammation and tissue damage, fibrin-CAR human T reg cells can be used to guide human T reg cells in disease tissue early and persist during the course of disease. [00320] Generation of FIB4-CAR Treg cells [00321] Human regulatory T cells were isolated from PBMCs derived from a healthy donor using the Regulatory T cell Separation Kit. After confirming their purity with FACS analysis for CD4, CD25, and CD127, these cells were transduced with the FIB4scFv-Flag-CAR-GFP vector to generate Fibrin CAR human Treg (FIB4-CAR Treg) cells. Validation of FIB4-CAR expression in human Treg cells was performed by flow cytometry, demonstrating an 82.7% transduction efficiency (FIG.9). [00322] FIB4-CAR Treg show increased brain infiltration and therapeutic efficacy [00323] To assess the infiltration efficiency and therapeutic efficacy of Fibrin CAR human Tregs, the lysolecithin (LPC) model was employed to induce focal demyelination. LPC was injected stereotactically into the corpus callosum of Rag2-deficient mice lacking T and B cells, and FIB4- CAR Treg cells administered intravenously 4 days post-LPC injection. Mice were sacrificed 14 days post-LPC injection and brain sections prepared for histological analysis. [00324] First, the infiltration of FIB4-CAR Treg cells into the brain was tested using a human nuclear antibody (Abcam, AB84680) in the LPC-injected demyelinated area, indicative of infiltrated human FIB4-CAR Treg cells (FIG.10). These findings show that FIB4-CAR increases the infiltration of Tregs in the brain at lesion sites compared to control Treg cells. [00325] Remyelination following LPC-induced myelin injury correlates with increased numbers of myelinating oligodendrocytes expressing the CC1 and Olig2 in the focal demyelinated area (Saraswat et al., Nat Commun 2021). It was therefore assessed whether injection of FIB4-CAR Treg cells increased the number of CC1+Olig2+ oligodendrocytes in the demyelinated area via Docket No.: 354406.00202 double immunostaining of brain sections with anti-CC1 and anti-Olig2 antibodies. A significantly higher number of CC1+Olig2+ oligodendrocytes was found in the corpus callosum of LPC-injected mice that received FIB4-CAR Treg cells compared to vehicle or Treg cells (FIG.11). [00326] To test efficacy in increasing myelin, brain sections were immunostained with anti-MBP antibody, and the total demyelinated area was measured at the lesion site. Mice injected with control Treg cells showed slightly reduced demyelination compared to mice injected with vehicle only (PBS), although this reduction did not reach statistical significance. However, animals receiving FIB4-CAR Treg cells exhibited markedly decreased demyelination compared to those receiving PBS or Treg cell injection (FIG. 12), providing evidence that FIB4-CAR Treg cells improved remyelination in vivo. OTHER EMBODIMENTS [00327] From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. [00328] All citations to sequences, patents and publications in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

Docket No.: 354406.00202 What is claimed: 1. A bispecific chimeric antigen receptor (CAR) comprising a first antigen specific binding domain, a second antigen specific binding domain, a transmembrane domain(s), a co-stimulatory domain(s), and a CD3ȗ signaling domain, wherein the first antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof. 2. The bispecific chimeric antigen receptor of claim 1, wherein the first and second antigen specific binding domains comprise an antibody, antibody fragment, a camelid nanobody or aptamer. 3. The bispecific chimeric antigen receptor of claim 2, wherein the antibody fragment is a single chain fragment. 4. The bispecific chimeric antigen receptor of claim 3, wherein the single chain fragment is a single chain variable fragment (scFv). 5. The bispecific chimeric antigen receptor of any one of claims 1-4, wherein the co- stimulatory domain comprises a cluster of differentiation antigen 28 (CD28), 41BB domain, an ICOS (Inducible T cell Co-stimulator) (CD278), OX40 (CD134), Glucocorticoid-induced Tumor Necrosis Factor Receptor (GITR), CD40 or CD27. 6. The bispecific chimeric antigen receptor of any one of claims 1-5, wherein the second antigen specific binding domain specifically binds to tumor antigens, exogenous antigens, endogenous antigens or autoantigens. 7. The bispecific chimeric antigen receptor of claim 6, wherein endogenous antigens comprise cluster of differentiation antigens (CD), cell surface molecules or antigens associated with neurodegenerative diseases. 8. The bispecific chimeric antigen receptor of claim 7, wherein the cluster of differentiation antigens (CD) comprise CD1a, CD2, CD3, CD5, CD10, CD11c, CD13, CD15, CD19, CD20, CD25, CD30, CD33, CD99, CD103, CD117, CD123 or CD207. 9. The bispecific chimeric antigen receptor of claim 8, wherein the cluster of differentiation antigen is CD19. Docket No.: 354406.00202 10. The bispecific chimeric antigen receptor of any one of claims 1-9, wherein the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 9. 11. The bispecific chimeric antigen receptor of any one of claims 1-10, wherein the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 9. 12. The bispecific chimeric antigen receptor of any one of claims 1-11, wherein the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 11. 13. The bispecific chimeric antigen receptor of any one of claims 1-12, wherein the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 11. 14. The bispecific chimeric antigen receptor of any one of claims 1-13, wherein the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 10. 15. The bispecific chimeric antigen receptor of any one of claims 1-14, wherein the second antigen specific binding domain comprises SEQ ID NO: 10. 16. The bispecific chimeric antigen receptor of any one of claims 1-15, wherein the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 12. 17. The bispecific chimeric antigen receptor of any one of claims 1-16, wherein the second antigen specific binding domain comprises SEQ ID NO: 12. 18. The bispecific chimeric antigen receptor of any one of claims 1-17, further comprising a hinge region. 19. The bispecific chimeric antigen receptor of any one of claims 1-18, wherein the bispecific chimeric antigen receptor comprises at least a 75% sequence identity to SEQ ID NO: 13. 20. The bispecific chimeric antigen receptor of any one of claims 1-19, wherein the bispecific chimeric antigen receptor comprises SEQ ID NO: 13. 21. An expression vector comprising SEQ ID NOS: 4, 8 or 14. Docket No.: 354406.00202 22. The expression vector of claim 21, wherein the vector comprises adenovirus, adeno- associated virus (AAV), herpes simplex virus, lentivirus, gammaretrovirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus or vesicular stomatitis virus. 23. The expression vector of claim 22, wherein the vector is a lentivirus. 24. The expression vector of any one of claims 21-23, further comprising an inducible promoter, cell specific promoter, a tissue specific promoter or a constitutive promoter. 25. The expression vector of any one of claims 21-24, further comprising one or more enhancer or regulatory sequences. 26. The expression vector of any one of claims 21-25, further comprising an inducible suicide gene. 27. The expression vector of any one of claims 21-26, further comprising a nucleic acid sequence encoding for one or more cytokines. 28. An isolated cell comprising the chimeric antigen receptor of any one of claims 1-20 or the expression vector of any one of claims 21-27. 29. The isolated cell of claim 28, wherein the isolated cell comprises a T cells, B cells, natural killer (NK) cells, stem cells, induced pluripotent stem cells (iPSCs) or combinations thereof. 30. The isolated cell of claim 29, wherein the T cell is a CD8+ T cell, a CD4+ T cell, a regulatory T cell (Treg) or a tumor infiltrating T lymphocyte (TIL). 31. An isolated T cell that is modified or transduced with a vector to express a first antigen specific binding domain, a second antigen specific binding domain, a transmembrane domain(s), co-stimulatory domain(s), and a CD3ȗ signaling domain, wherein the first antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof and the second antigen domain binds to a cluster of differentiation antigen 19 (CD19). 32. The isolated T cell of claim 31, wherein the co-stimulatory domain comprises a CD28 or a 41BB polypeptide. 33. The isolated T cell of claim 31, wherein the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 9. Docket No.: 354406.00202 34. The isolated T cell of claim 31, wherein the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 9. 35. The isolated T cell of claim 31, wherein the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 11. 36. The isolated T cell of claim 31, wherein the first antigen specific binding domain comprises SEQ ID NO: 11. 37. The isolated T cell of claim 31, wherein the second antigen specific binding domain comprises an scFv amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 10. 38. The isolated T cell of claim 31, wherein the second antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 10. 39. The isolated T cell of claim 31, wherein the second antigen specific binding domain comprises an scFv amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 12. 40. The isolated T cell of claim 31, wherein the second antigen specific binding domain has an amino acid sequence comprising SEQ ID NO: 12. 41. The isolated T cell of claim 31, wherein the bispecific chimeric antigen receptor comprises SEQ ID NO: 13. 42. A method of treating a subject in need of immunotherapy, comprising: isolating T lymphocytes from a biological sample obtained from the subject; transducing the T lymphocytes with an expression vector encoding a chimeric antigen receptor (CAR) which specifically binds to a fibrin antigenic epitope; expanding the transduced T lymphocytes at least once ex vivo to obtain expanded T lymphocytes specific for the fibrin antigen; and reinfusing the T lymphocytes into the subject, thereby treating the subject. 43. The method of claim 42, wherein the T lymphocytes are autologous cells. 44. The method of claim 42 or 43, wherein the CAR comprises an antigen binding domain linked to at least one co-stimulatory domain and a CD3 signaling domain, wherein the antigen Docket No.: 354406.00202 binding domain comprises a single chain variable fragment (scFv) which specifically binds to fibrin. 45. The method of claim 44, wherein the co-stimulatory domain comprises a CD28 or a 41BB polypeptide. 46. The method of claim 42, wherein the CAR comprises an scFv amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 1 or 5. 47. The method of claim 42, wherein the CAR comprises SEQ ID NO: 1 or 5. 48. The method of claim 42, wherein the CAR comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 2 or 6. 49. The method of claim 42, wherein the CAR comprises SEQ ID NO: 2 or 6. 50. The method of claim 42, wherein the CAR comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 3 or 7. 51. The method of claim 42, wherein the CAR comprises SEQ ID NO: 3 or 7. 52. The method of claim 42, wherein the CAR comprises a bispecific chimeric antigen receptor having at least a 75% sequence identity to SEQ ID NO: 13. 53. The method of claim 52, wherein the bispecific chimeric antigen receptor comprises SEQ ID NO: 13. 54. The method of claim 42, wherein the T lymphocyte is a CD8+ T lymphocyte, a CD4+ T lymphocyte, a regulatory T lymphocyte (Treg) or a tumor infiltrating T lymphocyte (TIL). 55. The method of claim 42, wherein the immunotherapy is administered to a subject diagnosed with cancer, a neurodegenerative disorder or an autoimmune disorder. 56. A method of treating a subject in need of immunotherapy, comprising: isolating T lymphocytes from a biological sample obtained from the subject; transducing the T lymphocytes with an expression vector encoding a bispecific chimeric antigen receptor (CAR) comprising a first antigen specific binding domain, a second antigen specific binding domain, a transmembrane domain(s), a co-stimulatory domain(s), and a CD3ȗ signaling domain, wherein the first antigen specific binding domain specifically binds to fibrin Docket No.: 354406.00202 polypeptides or peptides thereof and the second antigen domain binds to a cluster of differentiation antigen 19 (CD19), expanding the transduced T lymphocytes at least once ex vivo to obtain expanded T lymphocytes expressing the bispecific CAR; and reinfusing the T lymphocytes into the subject, thereby treating the subject. 57. The method of claim 56, wherein the co-stimulatory domain comprises a CD28 or a 41BB polypeptide. 58. The method of claim 56, wherein the T lymphocytes are autologous cells. 59. The method of claim 56, wherein the T lymphocyte is a CD8+ T lymphocyte, a CD4+ T lymphocyte, a regulatory T lymphocyte (Treg) or a tumor infiltrating T lymphocyte (TIL). 60. The method of claim 56, wherein the immunotherapy is administered to a subject diagnosed with cancer, a neurodegenerative disorder, demyelinating disorder or an autoimmune disorder. 61. An isolated natural killer (NK) cell comprising a chimeric antigen receptor (CAR) wherein the CAR comprises an intracellular activating signaling domain, a transmembrane region and an extracellular antigen binding domain. 62. The isolated NK cell of claim 61, wherein the intracellular activating signaling domain comprises a CD28 or CD137 molecule. 63. The isolated NK cell of claim 61, wherein the extracellular antigen binding domain is a single-chain variable fragment which specifically binds to a tumor antigen. 64. An isolated natural killer (NK) cell comprising a chimeric antigen receptor (CAR) wherein the CAR comprises an intracellular activating signaling domain, a transmembrane region, a first extracellular antigen binding domain and a second antigen binding domain. 65. The isolated NK cell of claim 64, wherein the intracellular activating signaling domain comprises a CD28 or CD137 molecule. 66. The isolated NK cell of claim 64, wherein the first extracellular antigen binding domain is a single-chain variable fragment which specifically binds to fibrin. Docket No.: 354406.00202 67. The isolated NK cell of claim 64, wherein the second extracellular antigen binding domain is a single-chain variable fragment which specifically binds to a tumor antigen. 68. A method of treating cancer comprising administering the isolated T lymphocytes of claims 31-41 or the NK cells of claims 61 -67. 69. A chimeric antigen receptor (CAR) comprising an antigen specific binding domain, a transmembrane domain(s), a co-stimulatory domain(s), and a CD3ȗ signaling domain, wherein the antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof. 70. The chimeric antigen receptor of claim 70, wherein the antigen specific binding domain comprises an antibody, antibody fragment, a camelid nanobody or aptamer. 71. The chimeric antigen receptor of claim 70, wherein the antibody fragment is a single chain fragment. 72. The chimeric antigen receptor of claim 71, wherein the single chain fragment is a single chain variable fragment (scFv). 73. The chimeric antigen receptor of any one of claims 69-72, wherein the co-stimulatory domain(s) comprise a cluster of differentiation antigen 28 (CD28), 41BB domain, an ICOS (Inducible T cell Co-stimulator) (CD278), OX40 (CD134), Glucocorticoid-induced Tumor Necrosis Factor Receptor (GITR), CD40 or CD27. 74. The chimeric antigen receptor of any one of claims 69-72, wherein the antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. 75. The chimeric antigen receptor of any one of claims 69-74, wherein the antigen specific binding domain comprises SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. 76. The chimeric antigen receptor of any one of claims 69-73, wherein the antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 3 or 7. 77. The chimeric antigen receptor of any one of claims 69-73, wherein the antigen specific binding domain comprises SEQ ID NO: 3 or 7. Docket No.: 354406.00202 78. The chimeric antigen receptor of any one of claims 64-72, further comprising a hinge region. 79. A chimeric antigen receptor (CAR) comprising an amino acid sequence derived from an isolated antibody, wherein the antibody binds human fibrin or fibrinogen ȖC domain, and inhibits Mac-1 binding to fibrin or fibrinogen ȖC domain, and wherein the antibody light chain variable domain comprises the amino acid sequence of SEQ ID NO:29. 80. The CAR of claim 79, wherein the amino acid sequence derived from the antibody which binds human fibrin or fibrinogen ȖC domain comprises an amino acid sequence of SEQ ID NO:28. 81. The CAR of claim 79, wherein an antigen binding domain of the CAR binds a Ȗ377-395 epitope (SEQ ID NO: 21) of the fibrin or fibrinogen ȖC domain. 82. The CAR of claim 81, wherein the isolated antibody, from which the CAR is derived, exhibits greater than 20% inhibition of microglial adhesion to the fibrin or fibrinogen ȖC domain. 83. The CAR of claim 81, wherein the isolated antibody, from which the CAR is derived, exhibits greater than 50% inhibition of Mac-1 binding to the fibrin or fibrinogen ȖC domain. 84. A pharmaceutical composition comprising the chimeric antigen receptor (CAR) of any one of claims 1-20, 69-78 or 79-83, or the expression vector of any one of claims 21-27, or the isolated cell of any one of claims 28-30, or the isolated T cell of any one of claims 31-41, or the isolated NK cell of any one of claims 61-67. 85. A method of treating a demyelination disorder in a subject, comprising administering a pharmaceutical composition comprising a therapeutically effective amount of an isolated T cell that is modified or transduced with a vector to express a bispecific chimeric antigen receptor (CAR) comprising a first antigen specific binding domain, a second antigen specific binding domain, a transmembrane domain(s), a co-stimulatory domain(s), and a CD3ȗ signaling domain, wherein the first antigen specific binding domain specifically binds to fibrin polypeptides or peptides thereof. 86. The method of claim 85, wherein the first and second antigen specific binding domains comprise an antibody, antibody fragment, a camelid nanobody or aptamer. 87. The method of claim 85, wherein the antibody fragment is a single chain fragment. Docket No.: 354406.00202 88. The method of claim 87, wherein the single chain fragment is a single chain variable fragment (scFv). 89. The method of any one of claims 85-88, wherein the co-stimulatory domain comprises a cluster of differentiation antigen 28 (CD28), 41BB domain, an ICOS (Inducible T cell Co- stimulator) (CD278), OX40 (CD134), Glucocorticoid-induced Tumor Necrosis Factor Receptor (GITR), CD40 or CD27. 90. The method of any one of claims 85-89, wherein the second antigen specific binding domain specifically binds to tumor antigens, exogenous antigens, endogenous antigens or autoantigens. 91. The method of any one of claims 85-89, wherein endogenous antigens comprise cluster of differentiation antigens (CD) or cell surface molecules. 92. The method of claim 91, wherein the cluster of differentiation antigens (CD) comprise CD1a, CD2, CD3, CD5, CD10, CD11c, CD13, CD15, CD19, CD20, CD25, CD30, CD33, CD99, CD103, CD117, CD123 or CD207. 93. The method of any one of claims 85-92, wherein the antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. 94. The method of any one of claims 85-92, wherein the antigen specific binding domain comprises SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. 95. The method of any one of claims 85-94, wherein the first antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NOs: 9 or 11. 96. The method of any one of claims 85-95, wherein the first antigen specific binding domain has an amino acid sequence comprising SEQ ID NOs: 9 or 11. 97. The method of any one of claims 85-96, wherein the second antigen specific binding domain comprises an amino acid sequence having at least a 75% sequence identity to SEQ ID NOs: 10 or 12. Docket No.: 354406.00202 98. The method of any one of claims 85-96, wherein the second antigen specific binding domain comprises SEQ ID NOs: 10 or 12. 99. The method of any one of claims 85-98, wherein the bispecific chimeric antigen receptor further comprises a hinge region. 100. The method of any one of claims 85-96, wherein the bispecific chimeric antigen receptor comprises at least a 75% sequence identity to SEQ ID NO: 13. 101. The method of any one of claims 85-96, wherein the bispecific chimeric antigen receptor comprises SEQ ID NO: 13. 102. The method of claim 85, wherein the isolated T cell expressing the CAR (CAR-T cell) is a regulatory T (CAR-Treg) cell. 103. The method of claim 102, wherein CC1+Olig2+ oligodendrocytes are increased in number as compared to baseline controls. 104. The method of any one of claims 85-103, wherein the CAR-Treg cells inhibit demyelination as compared to baseline controls. 105. The method of any one of claims 85-104, wherein the CAR-Treg cells remyelination as compared to baseline controls. 106. The method of any one of claims 85-105, wherein the Treg cell comprises an autologous cell, an allogeneic cell, a haplotype matched cell, a haplotype mismatched cell, a haplo-identical cell, a xenogeneic cell, cell lines or combinations thereof. 107. The method of claim 85, wherein the demyelinating disorder comprises: periventricular leukomalacia, multiple sclerosis, acute disseminated encephalomyelitis, chronic inflammatory demyelinating polyneuropathy, adrenoleukodystrophy, adenomyeloneuropathy, Leber's hereditary optic atrophy, HTLV-associated myelopathy, Guillain-Barre syndrome, phenylketonuria, Tay- Sachs disease, Niemann-Pick disease, Gaucher's disease, Hurler's syndrome, Krabbe's disease, Pelizaeus-Merzbacher disease, cerebral palsy, traumatic brain injury or neonatal brain injury, Alzheimer’s disease, chemo brain, COVID-19 neuropathology or a combination thereof. 108. The method of any one of claims 85-107, further comprising administering immature oligodendrocytes, pluripotent stem cells or the combination thereof.
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