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WO2025090985A1 - Compositions comprenant des récepteurs antigéniques chimériques ciblant une vimentine citrullinée et leurs procédés d'utilisation - Google Patents

Compositions comprenant des récepteurs antigéniques chimériques ciblant une vimentine citrullinée et leurs procédés d'utilisation Download PDF

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WO2025090985A1
WO2025090985A1 PCT/US2024/053144 US2024053144W WO2025090985A1 WO 2025090985 A1 WO2025090985 A1 WO 2025090985A1 US 2024053144 W US2024053144 W US 2024053144W WO 2025090985 A1 WO2025090985 A1 WO 2025090985A1
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Jose CONEJO-GARCIA
Scott Antonia
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Duke University
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Duke University
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    • 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
    • A61K40/42—Cancer 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
    • A61K40/428—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • 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
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
    • 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
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/50—Colon
    • 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
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/55—Lung
    • 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
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • 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

  • FIG. 1A-FIG. ID show that neoadjuvant pembrolizumab was associated with increased frequency of TLS and tumor infiltrating plasma cells in resected NSCLC tumors.
  • Co-detection by indexing (CODEX) multiplex immunofluorescent microscopy was performed on patient matched pre- and post-pembrolizumab-treated NSCLC tumor. Numerous lymphoid aggregates and mature tertiary lymphoid structures containing germinal centers are seen in resected post treatment but not pre-treatment biopsy specimens. Representative images are shown at lower magnification (FIG. 1A) and higher magnification higher magnification (FIG. IB). White arrows highlight a few examples of TLS.
  • FIG. 1C-FIG. ID Increased plasma cell infiltrates (CD138+CD38+) were seen in the stroma and leading tumor edge (CK5+) in posttreatment resected NSCLC tumors compared to pre-treatment biopsy. Representative images are shown at lower magnification (FIG. 1C) and higher magnification higher magnification (FIG. ID). White arrows highlight areas of plasma cell infiltration within the stroma and tumor leading edge.
  • FIG. 2A-FIG. 2C show that TIL-Bs demonstrated features of active immunity.
  • FIG. 2B is the SHM frequency shown as a fraction of nucleotide mutations in BCRs for tumor-associated VH, VK, VZ chain genes.
  • FIG. 2C shows the frequency of isotypes used by BCRs in tumor indicative of class-switch recombination.
  • FIG. 3 shows the multistep pipeline for high-throughput isolation and characterization of tumor-reactive antibodies from surgically resected tumors.
  • FIG. 4A-FIG. 4C show tumor reactivity of mAbs derived from clonally -expanded TIL-B cell clones.
  • FIG. 4A shows analysis of patient-derived tumor mAbs binding to indicated targets by ELISA. The heatmap depicts the optical density' (OD) reflective of two independent ELISAs at 0.67 pM. 17b. an anti-HIV-1 gpl20 mAb was used as a negative control.
  • FIG. 4B shows fixed calu-6 cells were stained with representative tumor-derived mAbs and detected with AF488 antihuman IgG (green). Staining for cell membrane (Cell Mask) is in red and nuclei (DAPI) in blue.
  • FIG. 4A shows analysis of patient-derived tumor mAbs binding to indicated targets by ELISA. The heatmap depicts the optical density' (OD) reflective of two independent ELISAs at 0.67 pM. 17b. an anti-HIV-1 gpl20 m
  • FIG. 4C shows binding of the same representative tumor mAbs to fixed Calu-6 cells by FACS.
  • FIG. 5A-FIG. 5B show Ab4 bound to cVIM.
  • FIG. 5A shows proteins eluted from beads coated with Ab4 were resolved on a Western blot imaged with Ab4. Mass spec analysis of the excised 55 kDa band revealed the presence of vimentin.
  • FIG. 5B shows Ab4 binding to unmodified VIM (blue) or cVIM (red) in an ELISA.
  • FIG. 6 shows anti-cVIM antibody concentration correlated with pathologic response.
  • Anti-cVIM Ab concentrations were measured in the plasma of neoadjuvant pembrolizumab treated NSCLC patients who had >90% (Col. A), 50-90% (Col. B), or ⁇ 50% (Col. C) pathologic response in the resection specimens.
  • FIG. 7 shows endocytosis of tumor-reactive mAbs. Calu-6 cells were stained with pHrodo Deep-red-conjugated tumor-reactive mAbs and fluorescent intensity of endocytosed antibody visualized with FACS.
  • FIG. 8A-FIG. 8C shows the expression of cVIM on the surface of human cancer and tumor-promoting myeloid cells.
  • Tumor-derived recombinant cVIM antibodies were produced with the VH/VL sequences of clonally enriched plasma cells on a human IgGl backbone, labelled with APC and used for FACS.
  • FIG. 8A shows representative analysis of 5 freshly dissociated high-grade serous ovarian cancers (gate on live cells).
  • FIG. 8B shows representative analysis of 5 pleural effusions from different patients with NSCLC (gate on live cells).
  • FIG. 8C shows expression of cVIM in tumor-infiltrating T cells is primarily restricted to exhausted CD4+ T cells.
  • FIG. 8D shows a variety of established cancer cell lines or primary tumor cells express cVIM on the cell surface.
  • FIG. 9A shows that most tumor and myelo-monocytic cells in mouse tumors also expressed citrullinated vimentin on the cell surface.
  • FIG. 9B shows that proteonomic analysis confirmed that vimentin was the target of the recombinant Ab.
  • FIG. 10 shows that anti-cVIM CAR T cells effectively killed CV1M+ OVCAR3 cells. Cytotoxicity of anti-cVIM CAR and mock transduced T cells against luciferase-transduced OVCAR3 cells were measured by luciferase signal after co-culture of 24 hours. X axis, target: effector ratios.
  • FIG. 11 the normalized cell index at the indicated target: effector (tumor cell line: CAR T cell) ratios, grown for the indicated times, using T cells from a healthy donor, transduced with the 4-lBB-co-stimulated anti-citrullinated vimentin CAR. Mock transduced T cells from the same donor as shown as controls.
  • a chimeric antigen receptor comprising the sequence set forth in any one of SEQ ID NO: 129-SEQ ID NO: 134 or a fragment thereof.
  • a chimeric antigen receptor comprising the sequence set forth in any one of SEQ ID NO:01-SEQ ID NO: 12 or a fragment thereof.
  • a chimeric antigen receptor comprising fromN-terminus to C- terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co-stimulatory domains.
  • a chimeric antigen receptor comprising from N-terminus to C-terminus a signal peptide; a citrullinated-vimentin binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co-stimulatory domains.
  • a chimeric antigen receptor comprising fromN-terminus to C- terminus a signal peptide; a citrullinated-vimentin binding domain comprising a scFv comprising (i) a VL comprising the sequence set forth in SEQ ID NO: 13 or a fragment thereof and (ii) a VH comprising the sequence set forth in SEQ ID NO: 14 or a fragment thereof; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co-stimulatory domains.
  • CAR chimeric antigen receptor
  • a chimeric antigen receptor comprising from N-terminus to C-terminus an antigen binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co-stimulatory domains.
  • a chimeric antigen receptor comprising from N-terminus to C-terminus a citrullinated- vimentin binding domain; a hinge domain; a transmembrane domain: and an intracellular domain comprising one or more co-stimulatory domains.
  • a chimeric antigen receptor comprising fromN-terminus to C- terminus a citrullinated-vimentin binding domain comprising a scFv comprising (i) a VL comprising the sequence set forth in SEQ ID NO: 13 or a fragment thereof and (ii) a VH comprising the sequence set forth in SEQ ID NO: 14 or a fragment thereof; a hinge domain; a transmembrane domain: and an intracellular domain comprising one or more co-stimulatory domains.
  • CAR chimeric antigen receptor
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising the sequence set forth in any one of SEQ ID NO: 129-SEQ ID NO: 134 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising the sequence set forth in any one of SEQ ID NO:01-SEQ ID NO: 12 or a fragment thereof.
  • a vector comprising a disclosed nucleic acid molecule.
  • a vector comprising a nucleic acid molecule encoding a disclosed CAR.
  • a vector comprising a disclosed nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor.
  • a vector comprising a disclosed nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor targeting citrullinated vimentin (cVIM).
  • a vector comprising a disclosed nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor (CAR) comprising an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising one or more co-stimulatory domains.
  • a vector comprising a disclosed nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor (CAR) comprising an extracellular domain comprising an antigen binding domain targeting citrullinated vimentin, a transmembrane domain, and an intracellular domain comprising one or more co-stimulatory domains.
  • a pharmaceutical formulation comprising a nucleic acid molecule comprising a nucleic acid sequence encoding any disclosed CAR.
  • a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR specific for citrullinated vimentin (cVIM).
  • cVIM citrullinated vimentin
  • a pharmaceutical formulation a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR comprising an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising one or more immunostimulatory domains.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells transduced with a disclosed recombinant vector or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells transformed with a disclosed plasmid or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of treating cancer comprising treating a subj ect in need thereof by administering to the subj ect in need thereof a therapeutically effective amount of one or more genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • cVIM citrullinated vimentin
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more immune cells transduced with a recombinant vector or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more immune cells transformed with a disclosed plasmid or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • cVIM citrullinated vimentin
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)-expressing tumor cells or a pharmaceutical formulation thereof.
  • cVIM citrullinated vimentin
  • a method of slowing disease progression comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells transduced with a disclosed recombinant vector or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of slowing disease progression the method comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells transformed with a disclosed plasmid or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of slowing disease progression comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • cVIM citrullinated vimentin
  • a method of slowing disease progression the method comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more immune cells transduced with a recombinant vector or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • Disclosed herein is a method of slowing disease progression, the method comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more immune cells transformed with a disclosed plasmid or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of slowing disease progression the method comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • cVIM citrullinated vimentin
  • a method of slowing disease progression comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)-expressing tumor cells or a pharmaceutical formulation thereof.
  • cVIM citrullinated vimentin
  • compositions compounds, kits, capsules, containers, and/or methods thereof. It is to be understood that the inventive aspects of which are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.
  • Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about.” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
  • references in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed.
  • X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
  • a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.
  • operably linked refers to a juxtaposition where the components described are in a relationship permitting them to function in their intended manner.
  • a control element “operably linked” to a functional element is associated in such a way that expression and/or activity of the functional element is achieved under conditions compatible with the control element.
  • a promotor is operably linked to nucleic acids.
  • the term “subject” refers to the target of administration, e.g., a human being.
  • the term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
  • the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, areptile, or an amphibian.
  • the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent.
  • the term does not denote a particular age or sex, and thus, adult and child subjects, as well as fetuses, whether male or female, are intended to be covered.
  • a subject can be a human patient.
  • a subject can have cancer, be suspected of having cancer, or be at risk of developing cancer.
  • the term “diagnosed” means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed cells (e.g.. disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK. cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)), disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods.
  • disclosed nucleic acid molecules e.g.. disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK. cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)
  • disclosed pharmaceutical formulations or a combination thereof, or by
  • '‘diagnosed with a disease or disorder’ means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as cancer) that can be treated by one or more the disclosed nucleic acid molecules, disclosed vectors, disclosed cells (e.g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)), disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods.
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • “suspected of having a disease or disorder’ can mean having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as cancer) that can likely be treated by one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed cells (e.g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)), disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods.
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • disclosed pharmaceutical formulations or a combination thereof, or by one or more of the disclosed methods.
  • an examination can be physical, can involve various tests (e.g., blood tests, genoty ping, biopsies, etc.), scans (e.g., CT scans, PET scans, etc.), and assays (e.g., enzymatic assay), or a combination thereof.
  • tests e.g., blood tests, genoty ping, biopsies, etc.
  • scans e.g., CT scans, PET scans, etc.
  • assays e.g., enzymatic assay
  • Vimentin (also known as Epididymis Secretory Sperm Binding Protein) can be the targeted antigen.
  • the vimentin gene (VIM) encodes a type III intermediate filament protein that with microtubules and actin microfilaments comprise the cytoskeleton. Vimentin is responsible for maintaining cell shape and integrity' of the cytoplasm, and stabilizing cytoskeletal interactions. Vimentin is usually attached to the nucleus, endoplasmic reticulum, and mitochondria, either laterally or terminally. Vimentin is known to the art as HGNC (12692), NCBI Gene (7431), Ensembl (ENSG00000026025), OMIM (193060), and UniProtKB/Swiss-Prot (P08670).
  • a “patient” refers to a subject afflicted with a disease or disorder (e.g., cancer).
  • a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder such as cancer.
  • a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder and is seeking treatment or receiving treatment for a disease or disorder (such as cancer).
  • the phrase “identified to be in need of treatment for a disease or disorder,”’ or the like refers to selection of a subject based upon need for treatment of the disease or disorder.
  • a subject can be identified as having a need for treatment of a disease or disorder (e.g., cancer) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the cancer.
  • the identification can be performed by a person different from the person making the diagnosis.
  • the administration can be performed by one who performed the diagnosis.
  • activated and activation can refer to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production and detectable effector functions.
  • the term “activated T cells” can refer to T cells that are proliferating. Signals generated through the TCR alone may be insufficient for full activation of the T cell and one or more secondary or costimulatory signals may also be required.
  • T cell activation comprises a primary stimulation signal through the TCR/CD3 complex and one or more secondary costimulatory signals. Costimulation can be evidenced by proliferation and/or cytokine production by T cells that have received a primary activation signal, such as stimulation through the TCR/CD3 complex.
  • inhibitor means to diminish or decrease an activity, level, response, condition, severity, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, level, response, condition, severity, disease, or other biological parameter.
  • a 10% inhibition or reduction in the activity 7 , level, response, condition, severity 7 , disease, or other biological parameter as compared to the native or control level e.g., a subject not receiving the disclosed nucleic acid molecules, disclosed vectors, disclosed cells (e.g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the
  • the inhibition or reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of reduction in between as compared to native or control levels.
  • the inhibition or reduction can be 10-20%, 20-30%, 30- 40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% as compared to a native or control level (e.g., a subject not receiving the disclosed nucleic acid molecules, disclosed vectors, disclosed cells (e.g..).
  • a native or control level can be a pre-disease or pre-disorder level (such as a precancer state).
  • treat or “treating” or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • the terms cover any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, z.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease.
  • a mammal e.g., a human
  • treating a disease or disorder can reduce the severity of an established a disease or disorder in a subject by 1%-100% as compared to a control (such as, for example, an individual not having cancer).
  • treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a disease or disorder (such as cancer).
  • treating a disease or disorder can reduce one or more symptoms of a disease or disorder in a subject by l%-100% as compared to a control (such as, for example, an individual not having cancer).
  • treating can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms of an established a disease or disorder. It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of a disease or disorder. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of a disease or disorder (such as cancer).
  • the term “prevent” or “preventing” or “prevention” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing a disease or disorder having chromatin deregulation and/or chromatin dysregulation is intended.
  • prevent also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., an individual) not having a given a disease or disorder (such as cancer) or related complication from progressing to that complication. In an aspect, preventing metastasis is intended.
  • administering and “‘administration” refer to any method of providing one or more of the disclosed interfering molecules, the disclosed anti-PDl molecules, the disclosed pharmaceutical formulations, or a combination thereof to a subject.
  • Such methods include, but are not limited to, the following: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intra-tumoral administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intra-aural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intraarterial administration or administration through the hepatic portal vein (HPV).
  • HPV hepatic portal vein
  • Administration of a disclosed composition, a disclosed pharmaceutical composition, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed small molecule, a disclosed endonuclease, a disclosed oligonucleotide, a disclosed RNA therapeutic, or any combination thereof can comprise administration directly into the CNS or the PNS. Administration can be continuous or intermittent. Administration can comprise a combination of one or more routes.
  • the skilled person can determine an efficacious dose, an efficacious schedule, and an efficacious route of administration for the disclosed nucleic acid molecules, disclosed vectors, disclosed cells (e.g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)), disclosed pharmaceutical formulations, or a combination thereof to treat or prevent a disease or disorder (such as cancer).
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • disclosed pharmaceutical formulations or a combination thereof to treat or prevent a disease or disorder (such as cancer).
  • the skilled person can also alter, change, or modify an aspect of an administering step to improve efficacy of the disclosed nucleic acid molecules, disclosed vectors, disclosed cells (e.g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)), disclosed pharmaceutical formulations, or a combination thereof.
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • determining the amount is meant both an absolute quantification of a particular analyte (e.g., biomarker for cancer, for example) or a determination of the relative abundance of a particular analyte (e.g., a cancer biomarker).
  • the phrase includes both direct or indirect measurements of abundance or both.
  • 'modifying the method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method.
  • a method can be altered by changing the amount of the disclosed nucleic acid molecules, disclosed vectors, disclosed cells (e.g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)), disclosed pharmaceutical formulations, or a combination thereof administered to a subject, or by changing the frequency of administration of the disclosed nucleic acid molecules, disclosed vectors, disclosed cells (e g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)), disclosed pharmaceutical formulations, or a combination thereof to a subject, by changing the duration of time that the disclosed nucleic acid molecules, disclosed vectors, disclosed cells (e.g., disclosed
  • nucleic acid molecules e.g., disclosed vectors, disclosed cells (e.g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)), disclosed pharmaceutical formulations, or combinations thereof.
  • disclosed vectors e.g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)
  • disclosed pharmaceutical formulations e.g., disclosed pharmaceutical formulations, or combinations thereof.
  • the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • a pharmaceutical carrier employed can be a solid, liquid, or gas.
  • examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid.
  • examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water.
  • examples of gaseous carriers can include carbon dioxide and nitrogen.
  • oral liquid preparations such as suspensions, elixirs and solutions
  • carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like
  • oral solid preparations such as powders, capsules and tablets.
  • tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed.
  • tablets can be coated by standard aqueous or nonaqueous techniques.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption.
  • Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use.
  • Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.
  • the term ‘excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyd sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.).
  • proteins e.g., serum albumin, etc.
  • amino acids e.g., aspartic acid, glutamic acid, lysine,
  • “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.
  • “contacting” refers to bringing one or more of the disclosed CARs, the disclosed nucleic acid molecules, the disclosed vectors, the disclosed pharmaceutical formulations, the disclosed anti-chemokines, the disclosed anti-cancer agents, the disclosed chemotherapeutics, or a combination thereof together with a target area or intended target area in such a manner that one or more of the disclosed CARs, the disclosed nucleic acid molecules, the disclosed vectors, the disclosed pharmaceutical formulations, the disclosed anti-chemokines, the disclosed anti-cancer agents, the disclosed chemotherapeutics, or a combination thereof can exert an effect on the intended target or targeted area either directly or indirectly.
  • a target area or intended target area can be one or more of a subject’s organs (e.g., lungs, heart, liver, kidney, brain, etc.) hosting cancerous cells expressing cVIM.
  • a target area or intended target area can be any cell or any organ infected by a disease or disorder (such as cancer).
  • a target area or intended target area can be any organ, tissue, or cells that are affected by a disease or disorder (such as cancer).
  • determining can refer to measuring or ascertaining the presence and severity of a disease or disorder, such as, for example, cancer or cancer cells expressing cVIM.
  • Methods and techniques used to determine the presence and/or severity of a disease or disorder are typically known to the medical arts.
  • the art is familiar with the ways to identify and/or diagnose the presence, severity, or both of a disease or disorder (such as, for example, cancer).
  • “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and/or prevention of a disease or disorder (e.g., a cancer) or a suspected disease or disorder.
  • the terms “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired an effect on an undesired condition (e.g., a cancer).
  • a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects.
  • “therapeutically effective amount” means an amount of a disclosed nucleic acid molecule, a disclosed vector, a disclosed cell, or a disclosed pharmaceutical formulation; that (i) treats the particular disease, condition, or disorder (e.g., a cancer), (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder e.g., cancer), or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein (e.g., cancer).
  • the particular disease, condition, or disorder e.g., a cancer
  • iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein (e.g., cancer).
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the disclosed nucleic acid molecules, disclosed vectors, disclosed cells (e.g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)), disclosed pharmaceutical formulations, or a combination thereof employed; the disclosed methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the disclosed nucleic acid molecules, disclosed vectors, disclosed cells (e.g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (
  • disclosed nucleic acid molecules for example, it is well within the skill of the art to start doses of the disclosed nucleic acid molecules, disclosed vectors, disclosed cells (e.g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)), disclosed pharmaceutical formulations, or a combination thereof at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, then the effective daily dose can be divided into multiple doses for purposes of administration.
  • disclosed vectors e.g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)
  • disclosed pharmaceutical formulations or a combination
  • a single dose of the disclosed nucleic acid molecules, disclosed vectors, disclosed cells e.g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)
  • disclosed pharmaceutical formulations, or a combination thereof can contain such amounts or submultiples thereof to make up the daily dose.
  • the dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • a preparation can be administered in a 'prophylaclically effective amount”; that is, an amount effective for prevention of a disease or condition, such as, for example, a disease or disorder due to a missing, deficient, and/or mutant protein or enzyme.
  • polynucleotide or nucleic acid can be used interchangeably, and refer to polymers of nucleotides of any length and includes DNA and RNA.
  • nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction.
  • the direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as "‘upstream sequences” ; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3 ? to the 3 ? end of the RNA transcript are referred to as ‘'downstream sequences”.
  • the term “antibody” includes, without limitation, a glycoprotein immunoglobulin that binds specifically to an antigen.
  • An antibody can comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof.
  • Each H chain can comprise a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region can comprise three constant domains, CHI, CH2 and CH3.
  • Each light chain can comprise a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region can comprise one constant domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL can comprise three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1. CDR1. FR2. CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains can contain a binding domain that interacts with an antigen.
  • the constant regions of the Abs can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g.. effector cells) and the first component (Clq) of the classical complement system.
  • human antibodies can be approximately 150 kD tetrameric agents composed of two identical heavy (H) chain polypeptides (about 50 kD each) and tw o identical light (L) chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a "Y-shaped" structure.
  • the heavy and light chains can be linked or connected to one another by a single disulfide bond and two other disulfide bonds can connect the heavy chain hinge regions to one another, so that the dimers can be connected to one another and the tetramer can be formed.
  • Naturally produced antibodies are also glycosylated, e.g., on the CH2 domain.
  • antibody is used to mean an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing etc., through at least one antigen recognition site within the variable region of the immunoglobulin molecule.
  • the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.
  • antibody fragments such as Fab, Fab', F(ab')2, and Fv fragments
  • scFv single chain Fv mutants
  • multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.
  • An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), based on the identity of their heavy -chain constant domains referred to as alpha, delta, gamma, epsilon, and mu, respectively.
  • the different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations.
  • Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc. In an aspect, any disclosed antibody can be humanized.
  • Antibody humanization is the process of replacing non-human antibody frameworks with human ones. Successful antibody humanization depends on maintaining the affinity after replacing residues.
  • humanized antibodies can be antibody molecules in which at least part of the sequence of both the light chain and the heavy chain arise from human genes. Such antibodies are termed “humanized antibodies”, “human antibodies”, or “fully human antibodies” herein.
  • variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen.
  • CDRs complementarity determining regions
  • FR framework regions
  • the variable region comprises rodent or murine CDRs and human framework regions (FRs).
  • the variable region is a primate (e.g.. non-human primate) variable region.
  • the variable region comprises rodent or murine CDRs and primate framework regions (FRs).
  • VL and VL domain are used interchangeably to refer to the light chain variable region of an antibody or an antigen-binding molecule thereof.
  • VH and VH domain are used interchangeably to refer to the heavy chain variable region of an antibody or an antigen-binding molecule thereof.
  • the terms “constant region” and “constant domain” are interchangeable and have a meaning common in the art.
  • the constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor.
  • the constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.
  • the term “heavy chain” when used in reference to an antibody can refer to any distinct type, e g., alpha (a), delta (5), epsilon (e), gamma (y) and mu (p), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgGi, IgG , IgG? and IgG4.
  • light chain when used in reference to an antibody can refer to any distinct ty pe, e.g., kappa (K) or lambda (A) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In an aspect, the light chain is a human light chain.
  • Endogenous with reference to a gene, protein, and/or nucleic acid refers to the natural presence of that gene, protein, and/or nucleic acid in a cell, such as an immune cell.
  • Exogenous refers to an introduced agent, such as a nucleic acid. gene, or protein, into a cell, for example from an outside source.
  • a nucleic acid introduced into a cell is exogenous even if it encodes a protein which is naturally found in the cell.
  • exogenous introduction of a nucleic acid encoding a protein can be used to increase the expression of the protein over the level that would naturally be found in the cell under similar conditions, e.g., without introduction of the exogenous nucleic acid.
  • T cell receptor or “TCR” refers to antigen-recognition molecules present on the surface of T cells. During normal T cell development, each of the four TCR genes can rearrange leading to highly diverse TCR proteins.
  • effector function can refer to a biological result of interaction of an antibody Fc region with an Fc receptor or ligand.
  • Effector functions comprise, without limitation, antibodydependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement mediated cytotoxicity (CMC).
  • An effector function may be antigen binding dependent, antigen binding independent, or both.
  • ADCC refers to lysis of antibody -bound target cells by immune effector cells. ADCC is generally understood to involve Fc receptor (FcR)- bearing effector cells recognizing and subsequently killing antibody-coated target cells (e.g., cells that express on their surface antigens to which an antibody is bound).
  • Effector cells that mediate ADCC may comprise immune cells, comprising yet not limited to, one or more of natural killer (NK) cells, macrophages, neutrophils, eosinophils.
  • NK natural killer
  • immunotherapy refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response.
  • immunotherapy can include, but are not limited to. NK cells and T cell therapies.
  • T cell therapy can include adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACTTM), and allogeneic T cell transplantation.
  • TIL tumor-infiltrating lymphocyte
  • eACTTM engineered autologous cell therapy
  • allogeneic T cell transplantation eACTTM
  • Adoptive T cell therapies including chimeric antigen receptor (CAR) T cells, have revolutionized cancer therapy.
  • CAR chimeric antigen receptor
  • impressive responses are limited to a subset of patients with hematological cancers and have not been unlocked in patients with solid tumors, which represent 90% of adult cancers.
  • adoptive T cell therapy is limited by a complex combination of factors including fitness of engineered T cells in tumors, T cell exhaustion, poor in vivo persistence and immunosuppressive environmental factors.
  • rational design has failed to overcome the problems associated with such factors.
  • the T cells or NK cells of the immunotherapy can come from any source known in the art.
  • T cells and NK cells can be differentiated in vitro from a hematopoietic stem cell population (for example iPSCs) or can be obtained from a subject.
  • T cells and NK cells can be obtained from, e.g., peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • PBMCs peripheral blood mononuclear cells
  • the T cells can be derived from one or more T cell lines available in the art.
  • T cells can also be obtained from a unit of blood collected from a subject using any number of techniques know n to the skilled artisan.
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
  • antibody fragments include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multi-specific antibodies formed from antibody fragments.
  • monoclonal antibody refers to homogenous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants.
  • monoclonal antibody encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2. Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal antibody” refers to such antibodies made in any number of manners including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.
  • humanized antibody refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences.
  • humanized antibodies are human immunoglobulins in which residues from the complementary 7 determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster, etc.) that have the desired specificity, affinity, and capability.
  • CDR complementary 7 determining region
  • the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity 7 , affinity 7 , and capability 7 .
  • the humanized antibody can be further modified by the substitution of additional residue either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity 7 , and/or capability.
  • the humanized antibody will comprise substantially all of at least one, and ty pically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), ty pically that of a human immunoglobulin.
  • That an antibody “selectively binds” or “specifically binds” to an epitope or receptor means that the antibody reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope or receptor than with alternative substances, including unrelated proteins. “Selectively binds” or “specifically binds” means, for instance, that an antibody binds to a protein w ith a KD of about 0. 1 mM or less, more usually about 1 pM or less. “Selectively binds” or “specifically binds” means at times that an antibody binds to a protein with a KD of about 0.
  • an antibody or binding moiety 7 that specifically binds to a first target may or may not specifically bind to a second target.
  • “‘specific binding” does not necessarily require (although it can include) exclusive binding, e.g., binding to a single target.
  • an antibody may, in an aspect, specifically bind to more than one target (e.g., human citrullinated vimentin (cVIM)).
  • the multiple targets may be bound by the same antigen-binding site on the antibody.
  • an “antigen” refers to a compound, composition, or substance that may stimulate the production of antibodies or a T cell response in a human or animal, including compositions (such as one that includes a tumor-specific protein) that are injected or absorbed into a human or animal.
  • An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens.
  • a “target antigen” is an antigen that is not substantially found on the surface of other normal (desired) cells and to which a binding domain of disclosed TCR or disclosed CAR can bind (e.g. cVIM on cancer cells).
  • a “target” expressing cVIM can bind to a disclosed binding motif, a disclosed CAR, a disclosed TCR, or a disclosed antigen binding agent.
  • Antigen-specific targeting region refers to the region of the CAR or TCR which targets specific antigens.
  • the targeting regions on the CAR or TCR are extracellular (for example, citrullinated vimentin (cVIM)).
  • the antigen-specific targeting regions comprise an antibody or a functional equivalent thereof or a fragment thereof or a derivative thereof and each of the targeting regions target a different antigen.
  • the targeting regions may comprise full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies, each of which are specific to the target antigen.
  • autologous refers to any material derived from the same individual to which it is later to be re-introduced.
  • a subject s own cells can be obtained, made to express one or more disclosed CARs, and then administered to the same subject.
  • antibody production can have both general and specific meanings. In the broad sense, it can refer to the entire process of creating a usable specific antibody, including steps of immunogen preparation, immunization, hybridoma creation, collection, screening, isotyping, purification, and labeling for direct use in a particular method. In the more restricted sense, antibody production refers to the steps leading up to antibody generation but does not include various forms of purifying and labeling the antibody for particular uses. Antibody production involves preparation of antigen samples and their safe injection into laboratory' or farm animals to evoke high expression levels of antigen-specific antibodies in the serum, which can then be recovered from the animal. Polyclonal antibodies are recovered directly from serum (bleeds).
  • Monoclonal antibodies are produced by fusing antibody-secreting spleen cells from immunized mice with immortal myeloma cell to create monoclonal hybridoma cell lines that express the specific antibody in cell culture supernatant.
  • Successful antibody production depends upon careful planning and implementation with respect to several important steps and considerations: (i) synthesize or purify the target antigen (e.g., peptide or hapten); (ii) choose an appropriate immunogenic carrier protein; (iii) conjugate the antigen and carrier protein to create the immunogen; immunize animals using appropriate schedule and adjuvant formula; and screen serum (or hybridoma) for antibody titer and isotype (also called antibody characterization).
  • target antigen e.g., peptide or hapten
  • cancer and “cancerous” refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • proliferative disorder and “proliferative disease” refer to disorders associated with abnormal cell proliferation such as cancer.
  • Tumor and “neoplasm” refer to any mass of tissue that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-cancerous lesions.
  • Metalastasis refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location.
  • a “metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures.
  • cancer stem cell or “tumor stem cell” or “solid tumor stem cell” are used interchangeably herein and refer to a population of cells from a solid tumor that: ( 1 ) have extensive proliferative capacity; (2) are capable of asymmetric cell division to generate one or more kinds of differentiated progeny with reduced proliferative or developmental potential; and (3) are capable of symmetric cell divisions for self-renewal or self-maintenance.
  • cancer stem cells or “tumor stem cells” or “solid tumor stem cells” confer on those cancer stem cells the ability to form palpable tumors upon serial transplantation into an immunocompromised mouse compared to the majority of tumor cells that fail to form tumors. Cancer stem cells undergo self-renewal versus differentiation in a chaotic manner to form tumors with abnormal cell types that can change over time as mutations occur.
  • cancer cell or “tumor cell” and grammatical equivalents refer to the total population of cells derived from a tumor including both non-tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells).
  • tumorigenic refers to the functional features of a solid tumor stem cell including the properties of self-renewal (giving rise to additional tumorigenic cancer stem cells) and proliferation to generate all other tumor cells (giving rise to differentiated and thus non- tumorigenic tumor cells) that allow solid tumor stem cells to form a tumor.
  • the “tumorigenicity” of a tumor refers to the ability’ of a random sample of cells from the tumor to form palpable tumors upon serial transplantation into immunocompromised mice.
  • lipid nanoparticles can deliver nucleic acid (e.g., DNA or RNA), protein (e.g.. RNA-guided DNA binding agent), or nucleic acid together with protein.
  • LNPs can comprise biodegradable, ionizable lipids.
  • LNPs can comprise (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-
  • cationic and ionizable in the context of LNP lipids can be use interchangeably, e.g., wherein ionizable lipids are cationic depending on the pH.
  • sequence identity and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they are optimally aligned. For example, sequence similarity or identity can be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences can have “substantial sequence identity” if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%. 99% or more, preferably 90%, 95%. 98%.
  • immunomodulating refers to the ability of a disclosed nucleic acid molecules, a disclosed vector, a disclosed pharmaceutical formulation, or a disclosed agent to alter (modulate) one or more aspects of the immune system.
  • the immune system functions to protect the organism from infection and from foreign antigens by cellular and humoral mechanisms involving lymphocytes, macrophages, and other antigen-presenting cells that regulate each other by means of multiple cell-cell interactions and by elaborating soluble factors, including lymphokines and antibodies, that have autocrine, paracrine, and endocrine effects on immune cells.
  • immune modulator refers to an agent that is capable of adjusting a given immune response to a desired level (e.g.. as in immunopotentiation, immunosuppression, or induction of immunologic tolerance).
  • immune modulators include but are not limited to, a disclosed immune modulator can comprise aspirin, azathioprine, belimumab, betamethasone dipropionate, betamethasone valerate, bortezomib, bredinin, cyazathioprine, cyclophosphamide, cyclosporine, deoxyspergualin, didemnin B, fluocinolone acetonide, folinic acid, ibuprofen, IL6 inhibitors (such as sarilumab) indomethacin, inebilizumab, intravenousy globulin (IVIG), methotrexate, methylprednisolone, mycophenolate mofeti
  • a disclosed immune modulator can comprise one or more Treg (regulatory T cells) infusions (e.g., antigen specific Treg cells to AAV).
  • a disclosed immune modulator can be bortezomib or SVP-Rapamycin.
  • an immune modulator can be administered by any suitable route of administration including, but not limited to, in utero, intra-CSF, intrathecally, intravenously, subcutaneously, transdermally, intradermally, intramuscularly, orally, transcutaneously, intraperitoneally (IP), or intravaginally.
  • a disclosed immune modulator can be administered using a combination of routes. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV). Administration of an immune modulator can be continuous or intermittent, and administration can comprise a combination of one or more routes.
  • the term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications. usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • the term “in combination” in the context of the administration of other therapies includes the use of more than one therapy (e.g., drug therapy).
  • Administration “in combination with” one or more further therapeutic agents includes simultaneous (e.g., concurrent) and consecutive administration in any order.
  • the use of the term “in combination” does not restrict the order in which therapies are administered to a subject.
  • a first therapy e.g., the disclosed nucleic acid molecules, disclosed vectors, disclosed cells (e.g., disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)), disclosed pharmaceutical formulations, or a combination thereof) can be administered prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours. 12 hours, 24 hours, 48 hours.
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • disclosed pharmaceutical formulations e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours. 12 hours, 24 hours, 48 hours.
  • these and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein.
  • chimeric antigen receptors Disclosed herein are chimeric antigen receptors. Disclosed herein are chimeric antigen receptors targeting citrullinated vimentin (cVIM). Disclosed herein is a chimeric antigen receptor (CAR), comprising an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising one or more co-stimulatory domains. Disclosed herein is a chimeric antigen receptor (CAR), comprising an extracellular domain comprising an antigen binding domain targeting citrullinated vimentin, a transmembrane domain, and an intracellular domain comprising one or more co-stimulatory domains.
  • CAR citrullinated vimentin
  • CAR chimeric antigen receptor
  • CAR chimeric antigen receptor
  • a disclosed CAR can target citrullinated vimentin (cVIM).
  • a disclosed CAR can target citrullinated vimentin (cVIM) expressed on the cell surface of a cancer cell.
  • a disclosed CAR can target citrullinated vimentin (cVIM) expressed on the cell surface of an ovarian cancer cell.
  • a CAR targeting cVIM can be used in conjunction with a CAR targeting any other cancer antigen.
  • a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 129 or a fragment thereof.
  • a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 129 or a fragment thereof.
  • a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 130 or a fragment thereof.
  • a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%. at least 90%. or at least 95% identity 7 to the sequence set forth in SEQ ID NO: 130 or a fragment thereof.
  • a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:01 or a fragment thereof.
  • a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. or at least 95% identity to the sequence set forth in SEQ ID NO: 01 or a fragment thereof.
  • a chimeric antigen receptor encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to sequence set forth in SEQ ID NO: 02 or a fragment thereof.
  • a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:03 or a fragment thereof.
  • a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity 7 to the sequence set forth in SEQ ID NO: 03 or a fragment thereof.
  • a chimeric antigen receptor encoded by the sequence set forth in SEQ ID NO:04 or a fragment thereof Disclosed herein is a chimeric antigen receptor encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity 7 to a sequence set forth in SEQ ID NO: 04 or a fragment thereof.
  • a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 131 or a fragment thereof.
  • a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 131 or a fragment thereof.
  • a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 132 or a fragment thereof.
  • a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. or at least 95% identity ⁇ to the sequence set forth in SEQ ID NO: 132 or a fragment thereof.
  • a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:05 or a fragment thereof.
  • a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. or at least 95% identity to the sequence set forth in SEQ ID NO: 05 or a fragment thereof.
  • a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:07 or a fragment thereof.
  • a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 07 or a fragment thereof.
  • a chimeric antigen receptor encoded by the sequence set forth in SEQ ID NO:08 or a fragment thereof Disclosed herein is a chimeric antigen receptor encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. or at least 95% identity to the sequence set forth in SEQ ID NO: 08 or a fragment thereof.
  • a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 133 or a fragment thereof.
  • a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 133 or a fragment thereof.
  • a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 134 or a fragment thereof.
  • a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 134 or a fragment thereof.
  • a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:09 or a fragment thereof.
  • a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 09 or a fragment thereof.
  • a chimeric antigen receptor encoded by the sequence set forth in SEQ ID NO: 10 or a fragment thereof Disclosed herein is a chimeric antigen receptor encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 10 or a fragment thereof.
  • a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 11 or a fragment thereof.
  • a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 11 or a fragment thereof.
  • a chimeric antigen receptor encoded by the sequence set forth in SEQ ID NO: 12 or a fragment thereof Disclosed herein is a chimeric antigen receptor encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity 7 to the sequence set forth in SEQ ID NO: 12 or a fragment thereof.
  • a chimeric antigen receptor targeting cVIM on the surface of cancer cells comprising a light chain comprising the sequence of SEQ ID NO: 13, a heavy chain comprising the sequence of SEQ ID NO: 15, a CD8 hinge domain comprising the sequence of SEQ ID NO:24, a CD8 transmembrane domain comprising the sequence of SEQ ID NO:29, a 4-1BB signaling domain comprising the sequence of SEQ ID NO:33, and a CD3zeta domain comprising the sequence of SEQ ID NONE
  • a chimeric antigen receptor targeting cVIM on the surface of cancer cells comprising a light chain comprising the sequence of SEQ ID NO: 13, a heavy chain comprising the sequence of SEQ ID NO: 15, a hinge domain comprising the sequence of SEQ ID NO:24, a CD8 transmembrane domain comprising the sequence of SEQ ID NO:29, a CD28 signaling domain comprising the sequence of SEQ ID NO:36, and
  • a chimeric antigen receptor comprising the scFV of Ab#4 as described in the Examples.
  • a disclosed extracellular domain can comprise a citrullinated vimentin binding domain.
  • a disclosed vimentin binding domain can comprise a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)3 fragment, a Fv, a single-chain variable fragment (scFv).
  • Vimentin (also known as Epididymis Secretory Sperm Binding Protein) can be the targeted antigen.
  • the vimentin gene (VIM) encodes a type III intermediate filament protein that with microtubules and actin microfilaments comprise the cytoskeleton. Vimentin is responsible for maintaining cell shape and integrity of the cytoplasm, and stabilizing cytoskeletal interactions. Vimentin is usually attached to the nucleus, endoplasmic reticulum, and mitochondria, either laterally or terminally. Vimentin is known to the art as HGNC (12692), NCBI Gene (7431), Ensembl (ENSG00000026025), OMIM (193060), and UniProtKB/Swiss-Prot (P08670).
  • a disclosed scFv can comprise the sequence set forth in any one of SEQ ID NO: 149-SEQ ID NO: 154.
  • a disclosed scFv can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in forth in any one of SEQ ID NO: 149-SEQ ID NO: 154.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 113, SEQ ID NO: 1 15, or SEQ ID NO: 117.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 114, SEQ ID NO: 116, or SEQ ID NO: 118.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 113 or a fragment thereof.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 113 or a fragment thereof.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 114 or a fragment thereof.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 114 or a fragment thereof.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 115 or a fragment thereof.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 115 or a fragment thereof.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 116 or a fragment thereof.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 116 or a fragment thereof.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 117 or a fragment thereof.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise a sequence having at least 70%, at least 75%. at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 117 or a fragment thereof.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 118 or a fragment thereof.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%. at least 90%. or at least 95% identity to the sequence set forth in SEQ ID NO: 118 or a fragment thereof.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise a disclosed signal / leader peptide, a disclosed VH, a disclosed linker / spacer, a disclosed VL, and a disclosed hinge domain.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise a disclosed signal / leader peptide, a disclosed VL, a disclosed linker / spacer, a disclosed VH, and a disclosed hinge domain.
  • a disclosed vimentin binding domain can comprise a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs).
  • a disclosed vimentin binding domain can comprise a light chain variable region (VL) comprising three complementarity determining regions (CDRs).
  • a disclosed CDR in the VL can comprise the sequence set forth in any one of SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.
  • a disclosed CDR in the VL can comprise a sequence having at least 70%, at least 75%, or at least 80% identity to the sequence set forth in any one of SEQ ID NO:21, SEQ ID NO: 22, and SEQ ID NO: 23.
  • a disclosed CDR in the VH can comprise the sequence set forth in any one of
  • a disclosed CDR in the VH can comprise a sequence having at least 70%, at least 75%, or at least 80% identity to the sequence set forth in any one of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
  • a disclosed CDR comprising 3 amino acids can comprise 1 or more substitutions.
  • a disclosed CDR comprising 8 amino acids can comprise 2 or more substitutions.
  • a disclosed CDR comprising 9 amino acids can comprise 2 or more substitutions.
  • a disclosed CDR comprising 11 amino acids can comprise 3 or more substitutions.
  • a disclosed CDR comprising 17 amino acids can comprise 4 or more substitutions.
  • a disclosed CDR comprising the sequence set forth in SEQ ID NO:22 can further comprise 1 or more substitutions.
  • a disclosed CDR comprising the sequence set forth in SEQ ID NO: 18 can further comprise 2 or more substitutions.
  • a disclosed CDR comprising the sequence set forth in SEQ ID NO: 19 and/or SEQ ID NO:23 can further 2 or more substitutions.
  • a disclosed CDR comprising the sequence set forth in SEQ ID NO:21 can further comprise 3 or more substitutions.
  • a disclosed CDR comprising the sequence set forth in SEQ ID NO: 18 can further comprise 2 or more substitutions.
  • a disclosed CDR comprising the sequence set forth in SEQ ID NO: 19 and/or SEQ ID NO:23 can further 2 or more substitutions.
  • a disclosed CDR comprising the sequence set forth in SEQ ID NO:21 can further comprise 3 or more substitutions.
  • NO:20 can further comprise 4 or more substitutions.
  • a disclosed vimentin binding domain can comprise a CDR that is evolved to differ by at least 20% from the sequence set forth in any one of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
  • a disclosed vimentin binding domain can comprise a CDR that is evolved to differ by at least 20% from the sequence set forth in any one of SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.
  • choosing the amino acid substitutions for a disclosed CDR can be informed by Gonzalez-Munoz et al. (2012) MAbs. 4(6):664-672, which is incorporated herein in its entirety for its teaching of tailoring amino acid diversity for the evolution of antibody affinity.
  • a disclosed VL can be located at the N-terminus of the VH.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise SEQ ID NO: 13 and SEQ ID NO: 15.
  • a disclosed VH can be located at the N-terminus of the VL.
  • a disclosed extracellular domain comprising a vimentin binding domain can comprise SEQ ID NO: 15 and SEQ ID NO: 13.
  • a disclosed VL can comprise the sequence of SEQ ID NO: 13.
  • a disclosed VL can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 13 or a fragment thereof.
  • a disclosed VL can be encoded by the sequence set forth in SEQ ID NO: 14.
  • a disclosed VL can be encoded by a sequence having at least 70%, at least 75%, at least 80%. at least 85%. at least 90%. or at least 95% identity to the sequence set forth in SEQ ID NO: 14 or a fragment thereof.
  • a disclosed VH can comprise the sequence of SEQ ID NO: 15.
  • a disclosed VH can comprise a sequence having at least 70%, at least 75%, at least 80%. at least 85%. at least 90%. or at least 95% identity to the sequence set forth in SEQ ID NO: 15 or a fragment thereof.
  • a disclosed VH can be encoded by the sequence set forth in SEQ ID NO: 16.
  • a disclosed VH can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity 7 to the sequence set forth in SEQ ID NO: 16 or a fragment thereof.
  • a disclosed VH can be encoded by the sequence set forth in SEQ ID NO: 17.
  • a disclosed VH can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 17 or a fragment thereof.
  • a disclosed antigen binding domain can be a scFv and wherein the scFV can comprise a linker and/or a spacer.
  • a disclosed linker and/or linker can join the VH and VL regions of the scFv.
  • a disclosed scFv can be a fusion protein of variable regions of the VH chain and the VL chain that are connected by a short flexible peptide linker and/or spacer.
  • a disclosed scFv can provide one or more advantages conveyed, in part, by its size.
  • a disclosed extracellular domain can further comprise a signal / leader peptide.
  • a disclosed extracellular domain comprising a vimentin binding domain can further comprise a signal / leader peptide.
  • a disclosed signal / leader peptide can comprise any known signal / leader peptide or any known signal / leader peptide known to function in mammalian cells.
  • a disclosed signal / leader peptide can be the signal / leader peptide of Human BM40 (osteonectin SPARC) (SEQ IDNO:48), Human IgKV III (SEQ ID NO:49), Human IgG V (SEQ ID NO:50), Human Chymotry psinogen (SEQ ID NO:51), Human Trypsinogen-2 (SEQ ID NO:52), Human Insulin (SEQ ID NO:53), Human IL-2 (SEQ ID NO: 54), Human Serum Albumin (SEQ ID NO:55).
  • a disclosed signal / leader peptide can comprise the sequence set forth in SEQ ID NO:46 or a fragment thereof. In an aspect, a disclosed signal / leader peptide can be encoded by the sequence set forth in SEQ ID NO:47.
  • a disclosed signal / leader peptide can be cleaved upon the nascent protein crossing the ER membrane.
  • a disclosed signal / leader peptide can be cleaved prior to protein assembly and folding by, for example, a signal peptide peptidase.
  • a disclosed VL and a disclosed VH can directly fused to each other via a peptide bond and/or peptide spacer or linked to each other via a peptide linker and/or peptide spacer.
  • a disclosed linker can comprise a rigid linker.
  • a disclosed linker can comprise a Whitlow linker.
  • a disclosed chimeric antigen receptor can further comprise a spacer domain between the extracellular domain and the transmembrane domain.
  • a disclosed chimeric antigen receptor can further comprise a hinge region betw een the extracellular domain and the transmembrane domain.
  • a disclosed spacer domain can comprise an immunoglobulin hinge region, an extracellular region of a type 1 membrane proteins, a part or all of an immunoglobulin constant region, or any combination thereof.
  • a disclosed vimentin binding domain can be following by one or more hinge domains.
  • a disclosed hinge region can play a role in positioning the antigen binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding and activation.
  • a spacer can connect a disclosed scFv to a disclosed transmembrane domain lend flexibility to the scFv and help improve efficacy.
  • a disclosed appropriate spacer domain engineering can enable recognition of target epitopes that are otherwise sterically inaccessible.
  • a disclosed spacer domain modulation can also be used to regulate synaptic cleft distances and hence signaling phenomena such as kinetic segregation.
  • membrane-distal epitopes usually require shorter spacers whereas membrane-proximal epitopes require longer spacers.
  • the highly dense immune synapse can hinder diffusion of lytic granules, which can enhance pore formation by perforins and granzyme delivery
  • the hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
  • a disclosed hinge region can be referred to as a stalk domain.
  • the size of a hinge region can be altered or varied.
  • a disclosed hinge region can comprise a hinge region of CD4, CD8a, CD28, IgGl, IgG2, IgG3, IgG4, IgA, IgD. or any combination thereof.
  • a disclosed CD8 hinge domain can comprise the sequence set forth in SEQ ID NO:24, SEQ ID NO:25, or a fragment thereof.
  • a disclosed CD8 hinge domain can be encoded by the sequence set forth in SEQ ID NO:26 or a fragment thereof.
  • a disclosed hinge region can be from or can be derived from CD2, CD35, CD3s, CD3y, CD4, CD7, CD8ot, CD813, CD1 la (ITGAL), CDl lb (ITGAM).
  • CDl lc (ITGAX), CDl ld (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3). CD66e (CEACAM5).
  • CD69 (CLEC2).
  • CD79A B-cell antigen receptor complex-associated alpha chain
  • CD79B B-cell antigen receptor complex-associated beta chain
  • CD84 SLAMF5
  • CD96 Tactile
  • CD100 SEMA4D
  • CD103 IGAE
  • CD134 0.X40
  • CD137 4-1BB
  • CD150 SLAMF1
  • CD158A KIR2DL1
  • CD158B1 KIR2DL2
  • CD158B2 KIR2DL3
  • CD158C KIR3DP1
  • CD158D KIRDL4
  • CD158F1 KIR2DL5A
  • CD158F2 KJR2DL5B
  • CD158K KIR3DL2
  • CD160 BY55
  • CD162 SELPLG
  • CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRT AM), CD357 (TNFRSF18), inducible T cell co-stimulator (ICOS), LFA-1 (CDl la/CD18), NKG2C, DAP- 10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2Ry, IL- 7R alpha, LFA1-1, SLAMF9, LAT, GADS (GrpL), S
  • a disclosed transmembrane can further comprise a transmembrane domain of CD2, CD3y. CD3s, CD35, CD3 , CD4, CD8, CD8a, CD9, CD16, CD22. CD25. CD27, CD28, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD79B, CD80, CD86, CD95 (FAS), CD134 (0X40), CD137 (4-1BB), CD152, CD154, CD278(ICOS), TCRa, TCR , NKG2D, 2B4, PD1, or any combination thereof.
  • a disclosed transmembrane domain can be from or can be derived from the alpha, beta or zeta chain of a T-cell receptor, 2B4, CD28.
  • LAT LFA-1, LFA-1, a ligand that binds with CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CDl-la/CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp.
  • PD-1 programmed death-1
  • PSGL1, SELPLG CD162
  • SLAM proteins Signaling Lymphocytic Activation Molecules
  • SLAMF1 SLAMF1; CD150; IPO-3
  • SLAMF4 CD244; 2B4
  • SLAMF6 NTB-A; Lyl08
  • SLAMF7 SLP-76
  • TNF receptor proteins TNFR2, TNFSF14, a Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or a combination thereof, or a fragment/truncation thereof, or a combination of fragments and/or truncations thereof.
  • a disclosed CD8 transmembrane domain can comprise the sequence set forth in SEQ ID NO:27 or a fragment thereof.
  • a disclosed CD8 transmembrane domain can comprise the sequence set forth in SEQ ID NO:28 or a fragment thereof.
  • a disclosed CD8 transmembrane domain can be encoded by the sequence set forth in SEQ ID NO:29 or a fragment thereof.
  • a disclosed CD28 transmembrane domain can comprise the sequence set forth in SEQ ID NO:30 or a fragment thereof.
  • a disclosed NKG2D transmembrane domain can comprise the sequence set forth in SEQ ID NO:31 or a fragment thereof.
  • a disclosed NKG2D transmembrane domain can be encoded by the sequence set forth in SEQ ID NO: 32 or a fragment thereof.
  • a disclosed intracellular domain can be referred to synonymously as an intracellular signaling domain.
  • a disclosed intracellular domain can be responsible for the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed (such, as, for example, activation, cytokine production, proliferation and/or cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, other cellular responses elicited with antigen binding to the extracellular CAR domain, or any combination thereof).
  • the normal effector functions of the immune cell in which the CAR is expressed such, as, for example, activation, cytokine production, proliferation and/or cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, other cellular responses elicited with antigen binding to the extracellular CAR domain, or any combination thereof.
  • T cell activation can be mediated by two distinct classes of signaling domains: intracellular signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., a TCR/CD3 complex) and co-stimulatory signaling domains that act in an antigen-independent manner to provide a secondary or co-stimulatory signal.
  • intracellular signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., a TCR/CD3 complex)
  • co-stimulatory signaling domains that act in an antigen-independent manner to provide a secondary or co-stimulatory signal.
  • a disclosed intracellular domain can comprise one or more immunoreceptor tyrosine-based activation domains (ITAM).
  • ITAM immunoreceptor tyrosine-based activation domains
  • a disclosed ITAM can comprise the signaling domain of DAP10, DAP12, TCR FcRy, FcR0, CD3y, CD3 , CD3s, CD35, CD3 , CD5, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), or any combination thereof.
  • a disclosed intracellular region can comprise a CD3 ⁇ signaling domain.
  • a disclosed CD3 signaling domain can comprise the sequence set forth in SEQ ID NO:41.
  • a disclosed CD3 ⁇ signaling domain can comprise the sequence set forth in SEQ ID NO:42.
  • a disclosed CD3 ⁇ signaling domain can be encoded by the sequence set forth in SEQ ID NO:43.
  • a disclosed CD3 ⁇ can comprise three immunoreceptor ty rosine activation motifs (IT AMs), which can assist in triggering downstream signaling events in response to receptor clustering.
  • ITAM can be on the C-terminal end (distal to the transmembrane domain).
  • increasing the number of disclosed ITAMs in a disclosed intracellular domain can increase the number or fraction of activated cells, can increase T-cell proliferation, can increase IL-2 production, can increase the in vivo anti-tumor response, or any combination thereof.
  • a disclosed intracellular domain can comprise one or more co-stimulatory domains or co-stimulatory signaling domains.
  • the order of one or more co- stimulatory domains in a disclosed intracellular domain can be varied.
  • the order of the one or more co-stimulatory domains can be varied and/or can influence the resulting effector function.
  • CD28-CD3 ⁇ can elicit higher level of IL-2.
  • a disclosed co- stimulatory domain can be on the N-terminal end (proximal to the transmembrane domain).
  • a disclosed intracellular domain can further comprise one or more co-stimulatory signaling domain or co-stimulatory domains.
  • a disclosed co-stimulatory molecule can comprise B7-H2, BAFFR, BLAME (SLAMF8), BTLA, CARD11, CD100 (SEMA4D), CD103, CDl-la. CDl-lb, CDl-lc, CDl-ld, CD134 (0X40). CD137 (4-1BB), CD154, CD16, CD160 (BY55), CD18, CD19.
  • CDS CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fey receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICOS, Iga (CD79a), IL2R0, IL2Ry, IL7Ra, integnn, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1 (CDlla/CD18), LIGHT, LTBR, Ly9 (CD229), MHC class I molecule, NKD2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), 0X40, PAG/Cbp.
  • PD- I PSGL1.
  • SELPLG CD 162
  • signaling lymphocytic activation molecule SLAM (SLAMF1; CD 150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; LylO8), SLAMF7, SLP76, TLR1, TLR10, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE/RANKL, TRIM, VLA1, or VLA-6, ZAP70, or fragments, truncations, or combinations thereof.
  • a disclosed intracellular domain can comprise one or more ITAMs and one or more co-stimulatory domains, wherein the IT AMs can comprise the signaling domain of FcRy, FcRB, CD3y, CD3 ⁇ , CD3s, CD35, CD3 ⁇ , CD5, CD22, CD66d, CD79a. CD79b, CD278 (ICOS), or any combination thereof, and wherein the co- stimulatory domains can comprise the signaling domain of CD27. CD28, 4- IBB. 0X40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7 LIGHT, NKG2C, B7-H3, or any combination thereof.
  • a disclosed intracellular domain can comprise one or more co-stimulatory domains.
  • a disclosed co-stimulatory domain can comprise 4-1BB.
  • a disclosed 4-1BB co-stimulatory domain can comprise the sequence set forth in SEQ ID NO:33 or a fragment thereof.
  • a disclosed 4-1 BB co-stimulatory domain can comprise the sequence set forth in SEQ ID NO: 34 or a fragment thereof.
  • a disclosed 4- IBB co- stimulatory’ domain can be encoded by the sequence set forth in SEQ ID NO: 35 or a fragment thereof.
  • a disclosed co-stimulatory domain can comprise CD28.
  • a disclosed CD28 co-stimulatory domain can comprise the sequence set forth in SEQ ID NO:36 or a fragment thereof.
  • a disclosed CD28 co-stimulatory domain can comprise the sequence set forth in SEQ ID NO:37 or a fragment thereof.
  • a disclosed CD28 co- stimulatory domain can be encoded by the sequence set forth in SEQ ID NO:38 or a fragment thereof.
  • a disclosed co-stimulatory domain can comprise NKG2D.
  • a disclosed NKG2D co-stimulatory domain can comprise the sequence set forth in SEQ ID NO: 39 or a fragment thereof.
  • a disclosed NKG2D co-stimulatory domain can be encoded by the sequence set forth in SEQ ID NO:40 or a fragment thereof.
  • a disclosed co-stimulatory domain can comprise OX-40.
  • a disclosed OX-40 co-stimulatory domain can comprise the sequence set forth in SEQ ID NO: 147 or a fragment thereof.
  • a disclosed OX-40 co-stimulatory domain can be encoded by the sequence set forth in SEQ ID NO: 148 or a fragment thereof.
  • a disclosed co- stimulatory signaling domain can enhance the efficacy and expansion of immune cells expressing CAR receptors.
  • a disclosed CAR can comprise a first-generation CAR, a second-generation CAR, a third-generation CAR, a fourth-generation CAR, or a fifth-generation CAR.
  • a disclosed intracellular domain can comprise a gene cassette for one or more cytokines.
  • a disclosed gene cassette for one or more cytokines can be operably linked to one or more regulatory elements.
  • a disclosed regulatory element can comprise a promoter or a transcription factor.
  • a disclosed transcription factor can be nuclear factor of the activated T cell (NF AT) or a minimal promoter.
  • a disclosed cytokine can comprise, for example, IL-7, IL-12, IL-15, IL-18, IL-23, or any combination thereof.
  • engineering such a construct can comprise use of two transgenic cassettes (e.g., one for the structure of a disclosed CAR and one for the inducible cytokines).
  • a disclosed intracellular domain can comprise a JAK-STAT activation domain.
  • a disclosed JAK- STAT activation domain can be derived, for example, from IL-2R
  • a disclosed JAK-STAT activation can stimulate cell proliferation, can prevent terminal differentiation, can perform better, or any combination thereof.
  • activation of JAK/STAT pathwayactivation can be in an antigen-dependent manner.
  • a disclosed intracellular signaling domain and a disclosed co-stimulatory signaling domain can be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.
  • a disclosed CD28 co-stimulatory domain can demonstrate one or more functional aspects: (i) lower persistence and differentiation towards effector memory phenotype compared to 4- IBB second-generation CARs, (ii) more prone to tonic signaling and causes early exhaustion, (iii) imparts resistance to Tregs in-vitro, in-vivo models show that CD28 costimulation causes increased infiltration of Tregs and were less effective against tumors in presence of Tregs, (iv) resistant to CTLA4 inhibition, (v) faster and higher signaling intensity, (vi) does not alter scFv "‘affinity ceiling” (e.g.. affinity beyond which IFNy, IL2 secretion and cytotoxicity do not increase), or (vii) any combination thereof.
  • affinity ceiling e.g. affinity beyond which IFNy, IL2 secretion and cytotoxicity do not increase
  • a disclosed intracellular domain can further comprise a self-cleaving peptide.
  • a disclosed self-cleaving peptide can comprise a T2A, a P2A. a E2A. or F2A peptide.
  • a GSG linker can be added to the N-terminus of a disclosed 2A peptide.
  • a disclosed P2A peptide can comprise the sequence set forth in SEQ ID NO: 15 or SEQ ID NO:102 or a fragment thereof.
  • a disclosed T2A peptide can comprise the sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104 or a fragment thereof.
  • a disclosed E2A peptide can comprise the sequence set forth in SEQ ID NO: 105 or SEQ ID NO: 106 or a fragment thereof.
  • a disclosed F2A peptide can comprise the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108 or a fragment thereof.
  • a disclosed intracellular domain can comprise a disclosed 4-1BB co- stimulatory domain and a disclosed CD3 ⁇ domain.
  • a disclosed intracellular domain can comprise a disclosed CD28 co-stimulatory domain and a disclosed CD3 ⁇ domain.
  • a disclosed intracellular domain can comprise a disclosed NKG2D co-stimulatory.
  • a disclosed intracellular domain can comprise a disclosed 4-1BB co-stimulatory domain comprising the sequence set forth in SEQ ID NO:33 or SEQ ID NO:34 or a fragment thereof and a disclosed CD3 domain comprising the sequence set forth in SEQ ID NO:41 or SEQ ID NO:42 or a fragment thereof.
  • a disclosed intracellular domain can comprise a disclosed CD28 co- stimulatory domain comprising the sequence set forth in SEQ ID NO:36 or a fragment thereof and a disclosed CD3 domain comprising the sequence set forth in SEQ ID NO:41 or SEQ ID NO:42 or a fragment thereof.
  • a disclosed intracellular domain can comprise a disclosed NKG2D co-stimulatory domain comprising the sequence set forth in SEQ ID NO:29 or a fragment thereof and a disclosed CD3 ⁇ domain comprising the sequence set forth in SEQ ID NO:41 or SEQ ID NO:42 or a fragment thereof.
  • a disclosed intracellular domain can comprise the sequence set forth in SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or a fragment thereof.
  • a disclosed intracellular domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%. at least 90%. at least 95%. or more than 95% identity to the sequence set forth in SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or a fragment thereof.
  • a disclosed CAR can stimulate an effector cell mediated immune modulator response to citrullinated-vimentin-expressing tumor cells.
  • a disclosed CAR can induce a tumor reducing immune response.
  • a disclosed CAR can induce phagocytosis of cancer cells in the subject.
  • a disclosed CAR can cross-prime an antitumor T cell response.
  • a disclosed CAR can induce a tumor eliminating immune response.
  • a disclosed CAR can treat cancer.
  • CAR chimeric antigen receptor
  • a signal peptide comprising fromN-terminus to C- terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co-stimulatory domains.
  • a chimeric antigen receptor comprising fromN-terminus to C- terminus a signal peptide; a citrullinated-vimentin binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co-stimulatory domains.
  • a chimeric antigen receptor comprising from N-terminus to C-terminus a signal peptide; a citrullinated-vimentin binding domain comprising a scFv comprising (i) a VL comprising the sequence set forth in SEQ ID NO: 13 or a fragment thereof and (ii) a VH comprising the sequence set forth in SEQ ID NO: 14 or a fragment thereof; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co- stimulatory domains.
  • CAR chimeric antigen receptor
  • a chimeric antigen receptor comprising from N- terminus to C-terminus an antigen binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co-stimulatory domains.
  • a chimeric antigen receptor comprising from N-terminus to C-terminus a citrullinated- vimentin binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co-stimulatory domains.
  • a chimeric antigen receptor comprising from N-terminus to C-terminus a citrullinated-vimentin binding domain comprising a scFv comprising (i) a VL comprising the sequence set forth in SEQ ID NO: 13 or a fragment thereof and (ii) a VH comprising the sequence set forth in SEQ ID NO: 14 or a fragment thereof; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co-stimulatory domains.
  • CAR chimeric antigen receptor
  • a disclosed CAR can be introduced to T cells and/or NK and/or macrophages or any other immune system.
  • a disclosed CAR can be used to activated one or more types of immune cells (e.g., naive T cells, central memory T cells, effector memory T cells, NK cells or combination thereof) upon antigen binding.
  • sources for the T cells include, but are not limited to, peripheral blood, bone marrow, or other sources of hematopoietic cells.
  • T cells can be isolated by methods well known in the art, including commercially available isolation methods.
  • disclosed herein are methods for making or generating immune effector cells that express one or more disclosed CARs.
  • a disclosed polynucleotide and/or a disclosed vector can be introduced into a disclosed immune effector cell.
  • immune effector cells isolated from an individual in need thereof can be transfected and/or transduced with one or more one disclosed polynucleotides and/or disclosed vectors encoding one or more disclosed CARs.
  • immune effector cells can be isolated from an individual in need thereof and can be genetically modified without further manipulation in vitro.
  • genetically modified immune effector cells can be expanded and/or readministered into the individual.
  • disclosed immune effector cells can be activated and stimulated to proliferate in vitro prior to being genetically modified to express one or more disclosed CARs.
  • disclosed immune effector cells can be genetically modified to express one or more disclosed CARs prior to being activated and stimulated to proliferate.
  • disclosed immune effector cells can be cultured before and/or after being genetically modified (i.e. , transduced or transfected to express one or more disclosed CARs).
  • immune effector cells can be stimulated and/or induced to proliferate by contacting the cells with antibodies or antigen binding fragments that bind CD3 and/or antibodies or antigen binding fragments that bind to CD28; thereby generating a population of immune effector cells.
  • generating disclosed immune effector cells can comprise stimulating the immune effector cell and inducing the cells to proliferate by contacting the cell with antibodies or antigen binding fragments that bind CD3 and antibodies or antigen binding fragments that bind to CD28; thereby generating a population of immune effector cells.
  • the immune effector cells prior to in vitro manipulation or genetic modification of disclosed immune effector cells, can be obtained from a subject.
  • disclosed CAR-modified immune effector cells can comprise T cells.
  • peripheral blood mononuclear cells PBMCs
  • PBMCs peripheral blood mononuclear cells
  • T lymphocytes after isolation of PBMCs, T lymphocytes can be further isolated.
  • both cytotoxic and helper T lymphocytes can be sorted into naive, memory', and effector T cell subpopulations either before or after genetic modification and/or expansion.
  • disclosed immune effector cells can be genetically modified following isolation using known methods, or the immune effector cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified.
  • disclosed immune effector cells can be genetically modified with one or more disclosed CARs (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAR) and then can be activated and expanded in vitro.
  • CARs e.g., transduced with a viral vector comprising a nucleic acid encoding a CAR
  • T cells can be activated and expanded before or after genetic modification to express a CAR.
  • a disclosed vector carrying a disclosed nucleic acid sequence encoding a disclosed CAR can be introduced into a population of human donor T cells, NK cells, or NKT cells.
  • successfully transduced T cells that carry' the expression vector can be sorted using flow cytometry to isolate CD3 positive T cells and then can be further propagated to increase the number of these CAR protein expressing T cells.
  • standard procedures can be used for cry opreservation of T cells expressing the CAR protein for storage and/or preparation for use in a human subject.
  • one or more disclosed vectors can be used in genetically modify ing a donor population of immune effector cells.
  • one or more disclosed vectors can encode the same CAR or can encode different CARs.
  • resulting modified immune effector cells can form a mixed population of modified cells with a proportion of the modified cells expressing more than one different CAR proteins.
  • T cells manufactured by the methods can provide improved adoptive immunotherapy compositions.
  • disclosed T cell compositions manufactured by disclosed methods are imbued with superior properties, including increased survival, expansion in the relative absence of differentiation, persistence in vivo and superior anti-exhaustion properties.
  • a disclosed CAR can be subjected to a validation process or can be subjected to validating the efficacy of one or more of CARs to, for example, bind to a cVIM-expressing cancer cell.
  • one or more domains can be derived from a natural source, from a synthetic source, or a combination thereof.
  • one or more domains can be recombinant domains.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor.
  • a nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor targeting citrullinated vimentin (cVIM).
  • cVIM citrullinated vimentin
  • a nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor (CAR) comprising an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising one or more co-stimulatory domains.
  • CAR chimeric antigen receptor
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor (CAR) comprising an extracellular domain comprising an antigen binding domain targeting citrullinated vimentin, a transmembrane domain, and an intracellular domain comprising one or more costimulatory domains.
  • CAR chimeric antigen receptor
  • a disclosed encoded CAR can target citrullinated vimentin (cVIM). In an aspect, a disclosed encoded CAR can target citrullinated vimentin (cVIM) expressed on the cell surface of a cancer cell. In an aspect, a disclosed encoded CAR can target citrullinated vimentin (cVIM) expressed on the cell surface of an ovarian cancer cell.
  • cVIM citrullinated vimentin
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 129 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 129 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 130 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 130 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:01 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity’ to the sequence set forth in SEQ ID NO: 01 or a fragment thereof.
  • nucleic acid molecule comprising the sequence set forth in SEQ ID NO:02 or a fragment thereof.
  • nucleic acid molecule comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 02 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:03 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 03 or a fragment thereof.
  • nucleic acid molecule comprising the sequence set forth in SEQ ID NO:04 or a fragment thereof.
  • nucleic acid molecule comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity 7 to the sequence set forth in SEQ ID NO: 04 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 131 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising a sequence having at least 70%, at least 75%. at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 131 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 132 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 132 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:05 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 05 or a fragment thereof.
  • nucleic acid molecule comprising the sequence set forth in SEQ ID NO:06 or a fragment thereof.
  • nucleic acid molecule comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 06 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:07 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 07 or a fragment thereof.
  • nucleic acid molecule comprising the sequence set forth in SEQ ID NO:08 or a fragment thereof.
  • nucleic acid molecule comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 08 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 133 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 133 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 134 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising a sequence having at least 70%, at least 75%. at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 134 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:09 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 09 or a fragment thereof.
  • nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 10 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 11 or a fragment thereof.
  • nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 11 or a fragment thereof.
  • an encoded extracellular domain can comprise a citrullinated vimentin binding domain.
  • the vimentin gene encodes a type III intermediate filament protein that with microtubules and actin microfilaments comprise the cytoskeleton. Vimentin is responsible for maintaining cell shape and integrity of the cytoplasm and stabilizing cytoskeletal interactions. Vimentin is usually attached to the nucleus, endoplasmic reticulum, and mitochondria, either laterally or terminally. Vimentin is known to the art as HGNC (12692), NCBI Gene (7431), Ensembl (ENSG00000026025), OMIM (193060), and UniProtKB/Swiss-Prot (P08670).
  • an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 115 or a fragment thereof.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 115 or a fragment thereof.
  • a CDR can be encoded by the sequence set forth in any one SEQ ID NO: 135-SEQ ID NO: 140.
  • SEQ ID NO: 135 can encode a disclosed CDR.
  • SEQ ID NO: 136 can encode a disclosed CDR.
  • SEQ ID NO: 137 can encode a disclosed CDR.
  • SEQ ID NO: 138 can encode a disclosed CDR.
  • SEQ ID NO: 139 can encode a disclosed CDR.
  • SEQ ID NO: 140 can encode a disclosed CDR.
  • SEQ ID NO: 135 can comprise one or more nucleotide substitutions.
  • an encoded VH can comprise the sequence of SEQ ID NO: 15.
  • an encoded VH can comprise a sequence having at least 70%, at least 75%. at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 15 or a fragment thereof.
  • a VH in an aspect of a disclosed nucleic acid molecule, can be encoded by the sequence set forth in SEQ ID NO: 16. In an aspect of a disclosed nucleic acid molecule, a VH can be encoded by a sequence having at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 16 or a fragment thereof.
  • a VH in an aspect of a disclosed nucleic acid molecule, can be encoded by the sequence set forth in SEQ ID NO: 17. In an aspect of a disclosed nucleic acid molecule, a VH can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 17 or a fragment thereof.
  • nucleotide sequence of SEQ ID NO: 14 can encode a disclosed light chain or a fragment thereof.
  • nucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 17 can encode a disclosed heavy chain or a fragment thereof.
  • an encoded antigen binding domain can be a scFv and wherein the scFV can comprise a linker and/or a spacer.
  • a disclosed encoded linker and/or linker can join the Vnand VL regions of the scFv.
  • an encoded scFv can be a fusion protein of variable regions of the VH chain and the VL chain that are connected by a short flexible peptide linker and/or spacer.
  • an encoded scFv can provide one or more advantages conveyed, in part, by its size.
  • an encoded extracellular domain can further comprise a signal / leader peptide.
  • an encoded extracellular domain comprising a vimentin binding domain can further comprise a signal / leader peptide.
  • Signal / leader peptides are known to the art.
  • a disclosed signal / leader peptide can comprise any known signal / leader peptide or any known signal / leader peptide known to function in mammalian cells.
  • a disclosed encoded signal / leader peptide can be the signal / leader peptide of Human BM40 (osteonectin SPARC) (SEQ ID NO:48), Human IgKV III (SEQ ID NO:49), Human IgG V (SEQ ID NO: 50), Human Chymotrypsinogen (SEQ ID NO:51), Human Trypsinogen-2 (SEQ ID NO:52), Human Insulin (SEQ ID NO:53), Human IL-2 (SEQ ID NO: 54), Human Serum Albumin (SEQ ID NO: 55), Human Tissue Plasminogen Activator (SEQ ID NO:56), Secrecon (SEQ ID NO:57), CD33 (SEQ ID NO:58), Vesicular stomatitis virus G protein (VSV-G) (SEQ ID NO:59), Gaussia luc (SEQ ID NO:60), Influenza Haemagglutinin (SEQ ID NO:61).
  • Silkworm Fibroin LC SEQ ID NO:48
  • a disclosed encoded signal / leader peptide can comprise the sequence set forth in SEQ ID NO:46 or a fragment thereof.
  • a disclosed signal / leader peptide can be encoded by the sequence set forth in SEQ ID NO:47.
  • an encoded signal / leader peptide can be cleaved upon the nascent protein crossing the ER membrane. In an aspect of a disclosed nucleic acid molecule, an encoded signal / leader peptide can be cleaved prior to protein assembly and folding by, for example, a signal peptide peptidase.
  • an encoded extracellular domain can be encoded by the sequence set forth in SEQ ID NO: 141 - SEQ ID NO: 146.
  • an encoded extracellular domain can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 141 - SEQ ID NO: 146.
  • a disclosed nucleic acid molecule can further comprise the nucleotide sequence for a signal peptide.
  • a disclosed encoded signal peptide can comprise the sequence set forth in SEQ ID NO:46 or a fragment thereof.
  • a disclosed encoded signal peptide can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:46 or a fragment thereof.
  • a disclosed signal peptide can be encoded by the sequence set forth in SEQ ID NO:47 or a fragment thereof.
  • a disclosed signal peptide can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%. at least 90%. or at least 95% identity to the sequence set forth in SEQ ID NO:47 or a fragment thereof.
  • a disclosed nucleic acid molecule can further comprise the nucleotide sequence for the signal peptide set forth in any one of SEQ ID NO:46-SEQ ID NO:66.
  • vimentin can be encoded by one or more exons of the genomic DNA sequences (i.e., the sequence set forth in SEQ ID NO:70 or part thereof). In an aspect of a disclosed nucleic acid molecule, vimentin can be encoded by the sequence set forth in SEQ ID NO:71. In an aspect of a disclosed nucleic acid molecule, vimentin can comprise the sequence set forth in SEQ ID NO: 72.
  • an encoded hinge region can comprise the sequence set forth in SEQ ID NO:26.
  • an encoded transmembrane region can comprise the sequence set forth in SEQ ID NO:29.
  • an encoded transmembrane region can comprise the sequence set forth in SEQ ID NO:32.
  • an encoded 4-1BB signaling domain can comprise the sequence set forth in SEQ ID NO:33 or SEQ ID NO:34.
  • a disclosed nucleic acid molecule encoding a 4-1 BB signaling domain can comprise the sequence set forth in SEQ ID NO:35.
  • an encoded CD28 signaling domain can comprise the sequence set forth in SEQ ID NO:38 or SEQ ID NO:37.
  • a disclosed nucleic acid molecule encoding a CD28 signaling domain can comprise the sequence set forth in SEQ ID NO: 38.
  • an encoded NKG2D signaling domain can comprise the sequence set forth in SEQ ID NO:40.
  • a disclosed nucleic acid molecule encoding a NK.G2D signaling domain can comprise the sequence set forth in SEQ ID NO:40.
  • an encoded CD3 ⁇ signaling domain can be encoded by the sequence set forth in SEQ ID NO:41.
  • a disclosed nucleic acid molecule encoding a CD3 ⁇ signaling domain can comprise the sequence set forth in SEQ ID NO:42.
  • an encoded spacer can comprise the sequence set forth in SEQ ID NO:44.
  • a disclosed nucleic acid molecule encoding a spacer can comprise the sequence set forth in SEQ ID NO:45.
  • an encoded spacer can comprise the sequence set forth in any one of SEQ ID NO: 73
  • a disclosed encoded linker can comprise a flexible linker.
  • an encoded linker can comprise a rigid linker.
  • an encoded linker can comprise a Whitlow linker.
  • Linkers are known to the skilled person in the art.
  • an encoded chimeric antigen receptor can further compnse a spacer or a hinge region between the extracellular domain and the transmembrane domain.
  • a disclosed spacer domain can comprise an immunoglobulin hinge region, an extracellular region of a type 1 membrane proteins, a part or all of an immunoglobulin constant region, or any combination thereof. Spacers and hinge domain are discussed supra.
  • a disclosed nucleic acid molecule encoding one or more CARs can be introduced to T cells and/or NK and/or macrophages or any other immune system.
  • a disclosed encoded CAR can be used to activated one or more types of immune cells (e.g., naive T cells, central memory T cells, effector memory T cells, NK cells or combination thereof) upon antigen binding.
  • sources for the T cells include, but are not limited to, peripheral blood, bone marrow, or other sources of hematopoietic cells. T cells can be isolated by methods well known in the art, including commercially available isolation methods.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:01 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising a sequence having at least 70%. at least 75%. at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:01 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor and comprising the sequence set forth in SEQ ID NO:02 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:03 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:03 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor and comprising the sequence set forth in SEQ ID NO:04 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 131 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 131 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor having the sequence set forth in SEQ ID NO: 132 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 132 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:05 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. or at least 95% identity to the sequence set forth in SEQ ID NO:05 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor and comprising the sequence set forth in SEQ ID NO:06 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:07 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity' to the sequence set forth in SEQ ID NO:07 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor and comprising the sequence set forth in SEQ ID NO:08 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 133 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%. at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 133 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 134 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor compnsing a sequence having at least 70%. at least 75%. at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 134 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:09 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. or at least 95% identity to the sequence set forth in SEQ ID NO:09 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor and comprising the sequence set forth in SEQ ID NO: 10 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor compnsing the sequence set forth in SEQ ID NO: 11 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 11 or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor and comprising the sequence set forth in SEQ ID NO: 12 or a fragment thereof.
  • vector encoding a chimeric antigen receptor comprising the scFV of Ab#4 as described in the Examples.
  • an encoded extracellular domain can comprise a citrullinated vimentin binding domain.
  • an encoded vimentin binding domain can comprise a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)3 fragment, a Fv, a single-chain variable fragment (scFv), a bis-scFv, a (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), or a single-domain antibody (sdAb).
  • Vimentin (also known as Epididymis Secretory Sperm Binding Protein) can be the targeted antigen.
  • the vimentin gene (VIM) encodes a type III intermediate filament protein that with microtubules and actin microfilaments comprise the cytoskeleton. Vimentin is responsible for maintaining cell shape and integrity of the cytoplasm, and stabilizing cytoskeletal interactions. Vimentin is usually attached to the nucleus, endoplasmic reticulum, and mitochondria, either laterally or terminally. Vimentin is known to the art as HGNC (12692), NCBI Gene (7431), Ensembl (ENSG00000026025), OMIM (193060), and UniProtKB/Swiss-Prot (P08670).
  • a disclosed encoded scFv can comprise the sequence set forth in any one of SEQ ID NO: 149-SEQ ID NO: 154.
  • a disclosed encoded scFv can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in forth in any one of SEQ ID NO:149-SEQ ID NO: 154.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 113, SEQ ID NO: 1 15, or SEQ ID NO: 117.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 114, SEQ ID NO: 116, or SEQ ID NO: 1 18.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 113 or a fragment thereof.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. or at least 95% identity to the sequence set forth in SEQ ID NO: 113 or a fragment thereof.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 114 or a fragment thereof.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 114 or a fragment thereof.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 115 or a fragment thereof.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity 7 to the sequence set forth in SEQ ID NO: 115 or a fragment thereof.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 116 or a fragment thereof.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 116 or a fragment thereof.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 117 or a fragment thereof.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 117 or a fragment thereof.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 118 or a fragment thereof.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 118 or a fragment thereof.
  • vector encoding a chimeric antigen receptor targeting cVIM on the surface of cancer cells comprising a light chain comprising the sequence of SEQ ID NO: 13, a heavy chain comprising the sequence of SEQ ID NO: 15, a CD8 hinge domain comprising the sequence of SEQ ID NO:24, a CD8 transmembrane domain comprising the sequence of SEQ ID NO:29, a 4-1BB signaling domain comprising the sequence of SEQ ID NO:33, and a CD3zeta domain comprising the sequence of SEQ ID NO:41.
  • vector encoding a chimeric antigen receptor targeting cVIM on the surface of cancer cells comprising a light chain comprising the sequence of SEQ ID NO: 13, a heavy chain comprising the sequence of SEQ ID NO: 15, a hinge domain comprising the sequence of SEQ ID NO:24, a CD8 transmembrane domain comprising the sequence of SEQ ID NO:29, a CD28 signaling domain comprising the sequence of SEQ ID NO:36, and a CD3zeta domain comprising the sequence of SEQ ID NO:41.
  • vector encoding a chimeric antigen receptor targeting cVIM on the surface of cancer cells comprising a light chain comprising the sequence of SEQ ID NO: 13, a heavy chain comprising the sequence of SEQ ID NO: 15, a hinge domain comprising the sequence of SEQ ID NO:24, aNKG2D transmembrane domain comprising the sequence of SEQ ID NO:31, aNKG2D signaling domain comprising the sequence of SEQ ID NO: 39.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise a disclosed signal / leader peptide, a disclosed VH, a disclosed linker / spacer, a disclosed VL, and a disclosed hinge domain.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise a disclosed signal I leader peptide, a disclosed VL, a disclosed linker / spacer, a disclosed VH, and a disclosed hinge domain.
  • an encoded vimentin binding domain can comprise a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs).
  • an encoded vimentin binding domain can comprise a light chain variable region (VL) comprising three complementarity determining regions (CDRs).
  • an encoded CDR in the VL can comprise the sequence set forth in any one of SEQ ID NO:21. SEQ ID NO:22. and SEQ ID NO:23.
  • an encoded CDR in the VL can comprise a sequence having at least 70%, at least 75%, or at least 80% identity to the sequence set forth in any one of SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.
  • an encoded CDR in the VH can comprise the sequence set forth in any one of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:20.
  • an encoded CDR in the VH can comprise a sequence having at least 70%, at least 75%, or at least 80% identity to the sequence set forth in any one of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:20.
  • an encoded CDR comprising 3 amino acids can comprise 1 or more substitutions.
  • an encoded CDR comprising 8 amino acids can comprise 2 or more substitutions.
  • an encoded CDR comprising 9 amino acids can comprise 2 or more substitutions.
  • an encoded CDR comprising 11 amino acids can comprise 3 or more substitutions.
  • an encoded CDR comprising 17 amino acids can comprise 4 or more substitutions.
  • an encoded CDR comprising the sequence set forth in SEQ ID NO:22 can further comprise 1 or more substitutions.
  • an encoded CDR comprising the sequence set forth in SEQ ID NO: 18 can further comprise 2 or more substitutions.
  • an encoded CDR comprising the sequence set forth in SEQ ID NO: 19 and/or SEQ ID NO:23 can further 2 or more substitutions.
  • an encoded CDR comprising the sequence set forth in SEQ ID NO:21 can further comprise 3 or more substitutions.
  • an encoded CDR comprising the sequence set forth in SEQ ID NO:20 can further comprise 4 or more substitutions.
  • an encoded vimentin binding domain can comprise a CDR that is evolved to differ by at least 20% from the sequence set forth in any one of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
  • an encoded vimentin binding domain can comprise a CDR that is evolved to differ by at least 20% from the sequence set forth in any one of SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.
  • an encoded CDR can be informed by Gonzalez-Munoz et al. (2012) MAbs. 4(6):664-672, which is incorporated herein in its entirety for its teaching of tailoring amino acid diversity for the evolution of antibody affinity.
  • a CDR can be encoded by the sequence set forth in any one SEQ ID NO: 135-SEQ ID NO: 140.
  • SEQ ID NO: 135 can encode a disclosed CDR.
  • SEQ ID NO: 136 can encode a disclosed CDR.
  • SEQ ID NO: 137 can encode a disclosed CDR.
  • SEQ ID NO: 138 can encode a disclosed CDR.
  • SEQ ID NO: 139 can encode a disclosed CDR.
  • SEQ ID NO: 140 can encode a disclosed CDR.
  • SEQ ID NO: 135 can comprise one or more nucleotide substitutions.
  • SEQ ID NO: 136 can comprise one or more nucleotide substitutions.
  • SEQ ID NO: 137 can comprise one or more nucleotide substitutions.
  • SEQ ID NO: 138 can comprise one or more nucleotide substitutions.
  • SEQ ID NO: 139 can comprise one or more nucleotide substitutions.
  • SEQ ID NO: 140 can comprise one or more nucleotide substitutions.
  • SEQ ID NO: 135 can comprise one or more nucleotide substitutions thereby causing the encoded CDR to differ by at least 20% from the sequence set forth in SEQ ID NO:18.
  • SEQ ID NO: 136 can comprise one or more nucleotide substitutions can comprise one or more nucleotide substitutions thereby causing the encoded CDR to differ by at least 20% from the sequence set forth in SEQ ID NO: 19.
  • SEQ ID NO: 137 can comprise one or more nucleotide substitutions can comprise one or more nucleotide substitutions thereby causing the encoded CDR to differ by at least 20% from the sequence set forth in SEQ ID NO:20.
  • SEQ ID NO: 138 can comprise one or more nucleotide substitutions can comprise one or more nucleotide substitutions thereby causing the encoded CDR to differ by at least 20% from the sequence set forth in SEQ ID NO:21.
  • SEQ ID NO: 139 can comprise one or more nucleotide substitutions can comprise one or more nucleotide substitutions thereby causing the encoded CDR to differ by at least 20% from the sequence set forth in SEQ ID NO:22.
  • SEQ ID NO: 140 can comprise one or more nucleotide substitutions can comprise one or more nucleotide substitutions thereby causing the encoded CDR to differ by at least 20% from the sequence set forth in SEQ ID NO:23.
  • an encoded VL can be located at the N-terminus of the VH.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise SEQ ID NO: 13 and SEQ ID NO: 15.
  • an encoded VH can be located at the N-terminus of the VL.
  • an encoded extracellular domain comprising a vimentin binding domain can comprise SEQ ID NO: 15 and SEQ ID NO: 13.
  • an encoded VL can comprise the sequence of SEQ ID NO: 13.
  • an encoded VL can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 13 or a fragment thereof.
  • VL can be encoded by the sequence set forth in SEQ ID NO: 14.
  • VL can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 14 or a fragment thereof.
  • VH can comprise the sequence of SEQ ID NO: 15.
  • VH can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 15 or a fragment thereof.
  • VH can be encoded by the sequence set forth in SEQ ID NO: 16.
  • VH can be encoded by a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 16 or a fragment thereof.
  • a VH can be encoded by the sequence set forth in SEQ ID NO: 17.
  • a VH can be encoded by a sequence having at least 70%, at least 75%. at least 80%. at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 17 or a fragment thereof.
  • the nucleotide sequence of SEQ ID NO: 14 can encode a disclosed light chain or a fragment thereof.
  • the nucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 17 can encode a disclosed heavy chain or a fragment thereof.
  • an encoded antigen binding domain can be a scFv and wherein the scFV can comprise a linker and/or a spacer.
  • a disclosed encoded linker and/or linker can join the VH and VL regions of the scFv.
  • an encoded scFv can be a fusion protein of variable regions of the VH chain and the VL chain that are connected by a short flexible peptide linker and/or spacer.
  • an encoded scFv can provide one or more advantages conveyed, in part, by its size.
  • an encoded extracellular domain can further comprise a signal / leader peptide.
  • an encoded extracellular domain comprising a vimentin binding domain can further comprise a signal / leader peptide.
  • Signal / leader peptides are known to the art.
  • a disclosed signal / leader peptide can comprise any known signal / leader peptide or any known signal / leader peptide known to function in mammalian cells.
  • a disclosed encoded signal I leader peptide can be the signal / leader peptide of Human BM40 (osteonectin SPARC) (SEQ ID NO:48), Human IgKV III (SEQ ID NO: 49), Human IgG V (SEQ ID NO: 50), Human Chymotrypsinogen (SEQ ID NO:51), Human Trypsinogen-2 (SEQ ID NO:52), Human Insulin (SEQ ID NO:53), Human IL-2 (SEQ ID NO: 54), Human Serum Albumin (SEQ ID NO: 55), Human Tissue Plasminogen Activator (SEQ ID NO:56), Secrecon (SEQ ID NO:57).
  • CD33 SEQ ID NO:58).
  • VSV-G Vesicular stomatitis virus G protein
  • VSV-G Vesicular stomatitis virus G protein
  • Gaussia luc SEQ ID NO:60
  • Influenza Haemagglutinin SEQ ID NO:61
  • Silkworm Fibroin LC SEQ ID NO:62
  • Mouse Ig Kappa SEQ ID NO:63
  • Mouse Ig Heavy SEQ ID NO:64
  • CD8a SEQ ID NO:66
  • a disclosed encoded signal / leader peptide can comprise the sequence set forth in SEQ ID NO:46 or a fragment thereof.
  • a disclosed signal / leader peptide can be encoded by the sequence set forth in SEQ ID NO:47.
  • an encoded signal / leader peptide can be cleaved upon the nascent protein crossing the ER membrane.
  • an encoded signal / leader peptide can be cleaved prior to protein assembly and folding by, for example, a signal peptide peptidase.
  • an encoded extracellular domain can be encoded by the sequence set forth in SEQ ID NO: 141 - SEQ ID NO: 146.
  • an encoded extracellular domain can be encoded by a sequence having at least 70%, at least 75%, at least 80%. at least 85%. at least 90%. or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 141 - SEQ ID NO: 146.
  • a disclosed vector can further comprise the nucleotide sequence for a signal peptide.
  • a disclosed encoded signal peptide can comprise the sequence set forth in SEQ ID NO:46 or a fragment thereof.
  • a disclosed encoded signal peptide can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:46 or a fragment thereof.
  • a disclosed signal peptide can be encoded by the sequence set forth in SEQ ID NO:47 or a fragment thereof.
  • a disclosed signal peptide can be encoded by a sequence having at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:47 or a fragment thereof.
  • a disclosed vector can further comprise the nucleotide sequence for the signal peptide set forth in any one of SEQ ID NO:46-SEQ ID NO:66.
  • vimentin can be encoded by one or more exons of the genomic DNA sequences (i. e. , the sequence set forth in SEQ ID NO:70 or part thereof). In an aspect of a disclosed vector, vimentin can be encoded by the sequence set forth in SEQ ID NO:71. In an aspect of a disclosed vector, vimentin can comprise the sequence set forth in SEQ ID NO:72. [0294] In an aspect of a disclosed vector, an encoded hinge region can comprise the sequence set forth in SEQ ID NO:26. In an aspect of a disclosed vector, an encoded transmembrane region can comprise the sequence set forth in SEQ ID NO:29. In an aspect of a disclosed vector, an encoded transmembrane region can comprise the sequence set forth in SEQ ID NO: 32
  • an encoded 4-1BB signaling domain can comprise the sequence set forth in SEQ ID NO:33 or SEQ ID NO:34.
  • a disclosed nucleic acid molecule encoding a 4- IBB signaling domain can comprise the sequence set forth in SEQ ID NO:35.
  • an encoded CD28 signaling domain can comprise the sequence set forth in SEQ ID NO:38 or SEQ ID NO:37.
  • a disclosed nucleic acid molecule encoding a CD28 signaling domain can comprise the sequence set forth in SEQ ID NO:38.
  • an encoded NK.G2D signaling domain can comprise the sequence set forth in SEQ ID NO:40.
  • a disclosed nucleic acid molecule encoding a NKG2D signaling domain can comprise the sequence set forth in SEQ ID NO:40.
  • an encoded CD3 ⁇ signaling domain can be encoded by the sequence set forth in SEQ ID NO:41.
  • a disclosed nucleic acid molecule encoding a CD3 signaling domain can comprise the sequence set forth in SEQ ID NO:42.
  • an encoded spacer can comprise the sequence set forth in SEQ ID NO:44.
  • a disclosed nucleic acid molecule encoding a spacer can comprise the sequence set forth in SEQ ID NO:45.
  • an encoded spacer can comprise the sequence set forth in any one of SEQ ID NO:73 - SEQ ID NO:112.
  • an encoded chimeric antigen receptor can further comprise a spacer or a hinge region between the extracellular domain and the transmembrane domain.
  • a disclosed spacer domain can comprise an immunoglobulin hinge region, an extracellular region of a type 1 membrane proteins, a part or all of an immunoglobulin constant region, or any combination thereof. Spacers and hinge domain are discussed supra.
  • an encoded CD8 hinge domain can comprise the sequence set forth in SEQ ID NO:24, SEQ ID NO:25, or a fragment thereof.
  • a disclosed CD8 hinge domain can be encoded by the sequence set forth in SEQ ID NO:26 or a fragment thereof.
  • an encoded hinge region can be from or can be derived from CD2, CD35, CD3e, CD3y, CD4, CD7, CD8a, CD813.
  • CDl la (ITGAL), CDl lb (ITGAM).
  • CD18 (1TGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B- cell antigen receptor complex-associated alpha chain), CD79B (B-cell antigen receptor complex- associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3).
  • CD158C KIR3DP1
  • CD158D KIRDL4
  • CD158F1 KIR2DL5A
  • CD158F2 KIR2DL5B
  • CD158K KIR3DL2
  • CD160 BY55
  • CD162 SELPLG
  • CD226 DNAM1
  • CD229 SLAMF3
  • CD244 SLAMF4
  • CD247 CD3-zeta
  • CD258 LIGHT
  • CD268 BAFFR
  • CD270 TNFSF14
  • CD272 BTLA
  • CD276 B7-H3
  • CD279 PD-1
  • CD314 CD319
  • CD319 SLAMF7.
  • CD335 NK-p46
  • CD336 NK-p44
  • CD337 NK-p30
  • CD352 SLAMF6
  • CD353 SLAMF8
  • CD355 CD355
  • CD357 TNFRSF18
  • inducible T cell co-stimulator ICOS
  • LFA-1 CD1 la/CD18
  • NKG2C DAP-10
  • ICAM-1 ICAM-1
  • NKp80 KLRF1
  • IL-2R beta IL-2R beta
  • IL-2Ry IL-7R alpha
  • LFA1-1 SLAMF9
  • SLAMF9 LAT
  • GADS GrpL
  • SLP-76 LCP2
  • PAG1/CBP a CD83 ligand
  • Fey receptor MHC class 1 molecule
  • MHC class 2 molecule
  • TNF receptor protein an immunoglobulin protein
  • a cytokine receptor an integrin
  • activating NK cell receptors activating NK cell receptors.
  • an encoded transmembrane can further comprise a transmembrane domain of CD2, CD3y, CD3s, CD35.
  • an encoded transmembrane domain can be from or can be derived from the alpha, beta or zeta chain of a T-cell receptor.
  • 2B4. CD28, CD3s . CD3 5, CD3y y. CD45.
  • CD4. CD5, CD7, CD8, CD8 alpha, CD8P, CD9, CDl l a, CD 11b, CD1 1c, CDl ld, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB/CD137, activating NK cell receptors, an Immunoglobulin protein.
  • LFA-1 LFA-1, a ligand that binds with CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD1- la/CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1.
  • SELPLG CD162
  • Signaling Lymphocytic Activation Molecules SLAM proteins
  • SLAMF1 Signaling Lymphocytic Activation Molecules
  • CD 150 CD 150; IPO-3
  • SLAMF4 CD244; 2B4
  • SLAMF6 NTB-A; Lyl08
  • SLAMF7 SLP-76
  • TNF receptor proteins TNFR2, TNFSF14, a Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or a combination thereof, or a fragment/truncation thereof, or a combination of fragments and/or truncations thereof.
  • an encoded CD8 transmembrane domain can comprise the sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or a fragment thereof.
  • a disclosed CD8 transmembrane domain can be encoded by the sequence set forth in SEQ ID NO:29 or a fragment thereof.
  • an encoded CD28 transmembrane domain can comprise the sequence set forth in SEQ ID NO:30 or a fragment thereof.
  • an encoded NKG2D transmembrane domain can comprise the sequence set forth in SEQ ID NO: 31 or a fragment thereof.
  • a disclosed NKG2D transmembrane domain can be encoded by the sequence set forth in SEQ ID NO:32 or a fragment thereof.
  • an encoded intracellular domain can be referred to synonymously as an intracellular signaling domain. Intracellular domains are discussed supra.
  • an encoded intracellular domain can comprise one or more immunoreceptor tyrosine-based activation domains (ITAM).
  • ITAM immunoreceptor tyrosine-based activation domains
  • a disclosed ITAM can comprise the signaling domain of DAP10, DAP12, TCR ⁇ , FcRy, FcR[3, CD3y, CD3q, CD3s, CD35, CD3( ⁇ , CD5, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), or any combination thereof.
  • an encoded intracellular region can comprise a CD3 ⁇ signaling domain.
  • a disclosed CD3 ⁇ signaling domain can comprise the sequence set forth in SEQ ID NO:41.
  • a disclosed CD3 signaling domain can comprise the sequence set forth in SEQ ID NO:42.
  • a disclosed CD3q signaling domain can be encoded by the sequence set forth in SEQ ID NO:43.
  • a disclosed CD3 ⁇ can comprise three immunoreceptor ty rosine activation motifs (IT AMs), which can assist in triggering downstream signaling events in response to receptor clustering.
  • ITAM can be on the C-terminal end (distal to the transmembrane domain).
  • an encoded intracellular domain can comprise a gene cassette for one or more cytokines.
  • a disclosed gene cassette for one or more cytokines can be operably linked to one or more regulatory elements.
  • a disclosed regulatory element can comprise a promoter or a transcription factor.
  • a disclosed transcription factor can be nuclear factor of the activated T cell (NF AT) or a minimal promoter.
  • a disclosed cytokine can comprise, for example, IL-7, IL- 12, IL-15, IL- 18. IL-23, or any combination thereof.
  • an encoded intracellular domain can comprise one or more co-stimulatory domains or co-stimulatory signaling domains.
  • the order of one or more co-stimulatory domains in a disclosed intracellular domain can be varied.
  • the order of the one or more co-stimulatory domains can be varied and/or can influence the resulting effector function.
  • CD28-CD3 ⁇ can elicit higher level of IL-2.
  • a disclosed co-stimulatory domain can be on the N -terminal end (proximal to the transmembrane domain).
  • an encoded intracellular domain can further comprise one or more co-stimulatory signaling domain or co-stimulatory domains.
  • a disclosed co-stimulatory molecule can be cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen.
  • an encoded co-stimulatory domain can comprise B7-H2, BAFFR, BLAME (SLAMF8), BTLA, CARD11, CD100 (SEMA4D), CD103, CDl-la, CDl-lb, CDl-lc, CDl-ld, CD134 (0X40).
  • CD137 (4-1BB), CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7- H3), CD278 (ICOS), CD28, CD29, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD30, CD33, CD37, CD4, CD40, CD45, CD49a, CD49D, CD49f, CD5, CD54 (ICAM), CD64, CD69, CD7 LIGHT, CD7, CD80, CD83 ligand, CD84, CD86, CD8a, CD80.
  • IAM CD54
  • NKp46, NKp80 KLRF1.
  • an encoded intracellular domain can comprise one or more ITAMs and one or more co-stimulatory domains, wherein the ITAMs can comprise the signaling domain of FcRy, FcRB, CD3y, CD3q, CD3s. CD35, CD3 ⁇ , CD5, CD22, CD66d, CD79a, CD79b, CD278 (ICOS). or any combination thereof, and wherein the co-stimulatory domains can comprise the signaling domain of CD27, CD28, 4-1 BB, 0X40, CD30, CD40, PD-1, ICOS, LFA- 1, CD2, CD7 LIGHT, NKG2C, B7-H3, or any combination thereof.
  • ITAMs can comprise the signaling domain of FcRy, FcRB, CD3y, CD3q, CD3s.
  • the co-stimulatory domains can comprise the signaling
  • an encoded intracellular domain can comprise one or more co-stimulatory domains.
  • a disclosed co-stimulatory domain can comprise 4- 1BB.
  • an encoded 4- IBB co-stimulatory domain can comprise the sequence set forth in SEQ ID NO:33, SEQ ID NO:34, or a fragment thereof.
  • a disclosed 4- IBB co-stimulatory domain can be encoded by the sequence set forth in SEQ ID NO:35 or a fragment thereof.
  • an encoded 4-1BB co-stimulatory domain can demonstrate one or more functional aspects: (i) greater persistence and differentiation towards central memory phenotype compared to CD28 second-generation CARs, (ii) can reduce tonic signaling at optimal expression levels and decrease exhaustion, (iii) slower and less intense signaling, or (iv) any combination thereof.
  • an encoded co-stimulatory' domain can comprise OX- 40.
  • a disclosed OX-40 co-stimulatory domain can comprise the sequence set forth in SEQ ID NO: 147 or a fragment thereof.
  • a disclosed OX-40 co-stimulatory domain can be encoded by the sequence set forth in SEQ ID NO: 148 or a fragment thereof.
  • an encoded co-stimulatory signaling domain can enhance the efficacy and expansion of immune cells expressing CAR receptors.
  • a disclosed nucleic acid molecule can encode a first-generation CAR, a second-generation CAR, a third-generation CAR. a fourth-generation CAR. or a fifth-generation CAR, or a fragment thereof.
  • an encoded intracellular domain can comprise a JAK-STAT activation domain.
  • a disclosed JAK-STAT activation domain can be derived, for example, from I L -2 R
  • a disclosed JAK-STAT activation can stimulate cell proliferation, can prevent terminal differentiation, can perform better, or any combination.
  • an encoded intracellular signaling domain and a disclosed co-stimulatory signaling domain can be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.
  • an encoded CD28 co-stimulatory domain can demonstrate one or more functional aspects: (i) lower persistence and differentiation towards effector memory phenotype compared to 4- IBB second-generation CARs, (ii) more prone to tonic signaling and causes early exhaustion, (iii) imparts resistance to Tregs in-vitro, in-vivo models indicated that CD28 co-stimulation causes increased infiltration of Tregs and were less effective against tumors in presence of Tregs, (iv) resistant to CTLA4 inhibition, (v) faster and higher signaling intensity, (vi) does not alter scFv “affinity ceiling”’ (e.g., affinity' beyond which IFNy, IL-2 secretion and cytotoxicity do not increase), or (vii) any combination thereof.
  • scFv “affinity ceiling” e.g., affinity' beyond which IFNy, IL-2 secretion and cytotoxicity do not increase
  • an encoded intracellular domain can further comprise a self-cleaving peptide.
  • a disclosed self-cleaving peptide can comprise a T2A, a P2A, a E2A, or F2A peptide.
  • a GSG linker can be added to the N-terminus of a disclosed 2A peptide.
  • a disclosed P2A peptide can comprise the sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 102 or a fragment thereof.
  • a disclosed T2A peptide can comprise the sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104 or a fragment thereof.
  • a disclosed E2A peptide can comprise the sequence set forth in SEQ ID NO: 105 or SEQ ID NO: 106 or a fragment thereof.
  • a disclosed F2A peptide can comprise the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108 or a fragment thereof.
  • an encoded intracellular domain can comprise(i) a disclosed 4-1BB co-stimulatory' domain and a disclosed CD3 ⁇ domain, (ii) a disclosed CD28 co- stimulatory domain and a disclosed CD3 ⁇ domain, or (iii) a disclosed NKG2D co-stimulatory, (iv) a disclosed 4-1BB co-stimulatory domain comprising the sequence set forth in SEQ ID NO:33 or SEQ ID NO:34 or a fragment thereof and a disclosed CD3 domain comprising the sequence set forth in SEQ ID NO:41 or SEQ ID NO:42 or a fragment thereof, (v) a disclosed CD28 co- stimulatory domain comprising the sequence set forth in SEQ ID NO:36 or a fragment thereof and a disclosed CD3 domain comprising the sequence set forth in SEQ ID NO:41 or SEQ ID NO:42 or a fragment thereof, or (vi) a disclosed NK.G2D co-stimulatory domain comprising the sequence set forth in SEQ ID
  • an encoded intracellular domain can comprise the sequence set forth in SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128. or a fragment thereof.
  • an encoded intracellular domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% identity to the sequence set forth in SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or a fragment thereof.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising from N-terminus to C-terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more immunoreceptor tyrosine-based activation domains (ITAMs) and/or one or more costimulatory domains.
  • ITAMs immunoreceptor tyrosine-based activation domains
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising from N-terminus to C-terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co-stimulatory domains.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising from N-terminus to C-terminus a signal peptide; a citrullinated- vimentin binding domain; a hinge domain; a transmembrane domain: and an intracellular domain comprising one or more co-stimulatory domains.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising from N-terminus to C-terminus a signal peptide; a citrullinated- vimentin binding domain comprising a scFv comprising (i) a VL comprising the sequence set forth in SEQ ID NO: 13 or a fragment thereof and (ii) a VH comprising the sequence set forth in SEQ ID NO: 14 or a fragment thereof; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co-stimulatory domains.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising from N-terminus to C-terminus an antigen binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co- stimulatory domains.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising from N-terminus to C-terminus a citrullinated-vimentin binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co-stimulatory domains.
  • a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising from N-terminus to C-terminus a citrullinated-vimentin binding domain comprising a scFv comprising (i) a VL comprising the sequence set forth in SEQ ID NO: 13 or a fragment thereof and (ii) a VH comprising the sequence set forth in SEQ ID NO: 14 or a fragment thereof; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co-stimulatory domains.
  • a disclosed vector can be introduced to T cells and/or NK and/or macrophages or any other immune system.
  • a disclosed encoded CAR can be used to activated one or more types of immune cells (e.g., naive T cells, central memory T cells, effector memory' T cells, NK cells, or combination thereof) upon antigen binding.
  • sources for the T cells include, but are not limited to, peripheral blood, bone marrow, or other sources of hematopoietic cells. T cells can be isolated by methods well known in the art, including commercially available isolation methods.
  • disclosed immune effector cells can be cultured before and/or after being genetically modified (i.e.. transduced or transfected to express one or more disclosed CARs).
  • disclosed immune effector cells can be stimulated and/or induced to proliferate by contacting the cells with antibodies or antigen binding fragments that bind CD3 and/or antibodies or antigen binding fragments that bind to CD28; thereby generating a population of immune effector cells.
  • generating disclosed immune effector cells can comprise stimulating the immune effector cell and inducing the cells to proliferate by contacting the cell with antibodies or antigen binding fragments that bind CD3 and antibodies or antigen binding fragments that bind to CD28; thereby generating a population of immune effector cells.
  • the immune effector cells prior to in vitro manipulation or genetic modification of disclosed immune effector cells, can be obtained from a subject.
  • disclosed CAR-modified immune effector cells can comprise T cells.
  • peripheral blood mononuclear cells PBMCs
  • PBMCs peripheral blood mononuclear cells
  • T lymphocytes can be further isolated.
  • both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and/or expansion.
  • disclosed immune effector cells can be genetically modified following isolation using known methods, or the immune effector cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified.
  • disclosed immune effector cells can be genetically modified with one or more disclosed CARs (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAR) and then can be activated and expanded in vitro.
  • T cells can be activated and expanded before or after genetic modification to express a CAR.
  • a disclosed vector carrying a disclosed nucleic acid sequence encoding a disclosed CAR can be introduced into a population of human donor T cells. NK cells or NKT cells.
  • successfully transduced T cells that carry the expression vector can be sorted using flow cy tometry to isolate CD3 positive T cells and then can be further propagated to increase the number of these CAR protein expressing T cells.
  • standard procedures can be used for cry opreservation of T cells expressing the CAR protein for storage and/or preparation for use in a human subject.
  • one or more disclosed vectors can be used in genetically modifying a donor population of immune effector cells.
  • one or more disclosed vectors can encode the same CAR or can encode different CARs.
  • resulting modified immune effector cells can form a mixed population of modified cells with a proportion of the modified cells expressing more than one different CAR proteins.
  • T cells manufactured by the methods can provide improved adoptive immunotherapy compositions.
  • disclosed T cell compositions manufactured by disclosed methods in contemplated herein are imbued with superior properties, including increased survival, expansion in the relative absence of differentiation, persistence in vivo and superior anti-exhaustion properties.
  • a disclosed vector can further comprise a polyadenylation sequence.
  • a disclosed vector can further comprise a sequence for a promoter.
  • a disclosed 3’ hemi intron can be recognized by nuclear splicing components within a host cell.
  • a disclosed vector can further comprise a nuclear localization signal (NLS).
  • a disclosed vector can further comprise one or more nuclear retention elements (NRE). NRE are known to the skilled person in the art.
  • a disclosed NRE can comprise SIRLOIN or BORG.
  • a disclosed vector can comprise the sequence for one or more regulatory elements.
  • a disclosed regulatory element can comprise promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as poly adenylation signals and poly-U sequences).
  • Regulatory elements can include those that direct constitutive expression of a nucleotide sequence in many t pes of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • a disclosed regulatory element can comprise Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulator Element (WPRE), triplex from MALAT1, the PRE of Hepatitis B virus (HPRE). an iron response element, or any combination thereof.
  • WPRE Woodchuck Hepatitis Virus
  • WPRE Posttranscriptional Regulator Element
  • HPRE Hepatitis B virus
  • an iron response element or any combination thereof.
  • a disclosed regulatory element can comprise a promoter operably linked to a disclosed nucleic acid molecule, wherein the promoter drives the expression of a disclosed variant capsid protein, a disclosed encoded polypeptide, a disclosed encoded therapeutic agent, or both.
  • a disclosed promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the expression desired.
  • a promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the w ild-ty pe host into w hich the transcriptional initiation region is introduced.
  • a disclosed promoter can be a promoter/enhancer.
  • a disclosed promoter for the disclosed nucleic acid molecule can be an endogenous promoter.
  • a disclosed endogenous promoter can be an endogenous promoter/enhancer.
  • a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from anon-coding region upstream of a transcription initiation site of a gene of interest.
  • a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed gene.
  • a disclosed promoter for the one or more disclosed guide RNA sequences can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art.
  • a disclosed vector can be an integrating vector or a non-integrating vector.
  • integration can mean that the nucleotides of nucleic acid sequence can be stably inserted into the cellular genome (e.g., covalently linked to the nucleic acid sequence within the cell’s chromosomal DNA).
  • a disclosed vector can be a viral vector or a non-viral vector.
  • a disclosed non-viral vector can be a polymer-based vector, a peptide-based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid-based vector.
  • a disclosed vector can comprise lipid and/or polymer-based nanoparticles loaded with mRNA encoding a disclosed CAR.
  • a disclosed vector can comprise lipid and/or polymer-based nanoparticles loaded with transposon-based plasmids such as, for example, transposon-based plasmids comprising a sequence encoding a disclosed CAR.
  • a disclosed vector can comprise mRNA encoding a disclosed CAR.
  • a disclosed vector can comprise lipid and/or polymer-based nanoparticles loaded with mRNA encoding a disclosed CAR.
  • a disclosed viral vector can be an adenovirus vector, an AAV vector, a herpes simplex virus vector, a retrovirus vector, a lentivirus vector, and alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease viral vector, a poxvirus vector, or a picomavirus vector.
  • a disclosed viral vector can be an adeno-associated vims (AAV) vector
  • AAV adeno-associated vims
  • a disclosed AAV vector can include naturally isolated serotypes including, but not limited to, AAV1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAVrh39, AAVrh43.
  • an AAV capsid can be a chimera either created by capsid evolution or by rational capsid engineering from a naturally isolated AAV variants to capture desirable serotype features such as enhanced or specific tissue tropism and/or a host immune response escape.
  • Naturally isolated AAV variants include, but not limited to, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y/F, AAV2 T/V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A- String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV- PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81.
  • a disclosed AAV vector can be AAV-Rh74 or a related variant (e.g., capsid variants like RHM4-1).
  • a disclosed AAV vector can be a self-complementary AAV as disclosed herein.
  • a disclosed vector can be a recombinant vector comprising a disclosed nucleic acid sequence. Recombinant vectors (such as recombinant viral vectors) are known to the art.
  • a disclosed promoter can comprise a ubiquitous promoter, a constitutive promoter, or a tissue specific promoter.
  • a disclosed promoter can be operably linked to a disclosed nucleic acid sequence encoding a disclosed CAR.
  • a disclosed promoter can be operably linked to a disclosed nucleic acid sequence encoding a disclosed tumor antigen. Promoters are known to the art.
  • a disclosed promoter can be a promoter/ enhancer. Promoter/ enhancers are know n to the art.
  • a disclosed promoter can be an endogenous promoter.
  • a disclosed endogenous promoter can be an endogenous promoter/enhancer.
  • a disclosed promoter or a disclosed promoter/enhancer can be used for constitutive and efficient expression of a disclosed CAR.
  • a disclosed promoter or a disclosed promoter/enhancer can be used for constitutive and efficient expression of a disclosed tumor antigen.
  • a disclosed vector can be used to engineer cells to express a disclosed CAR.
  • a disclosed vector can further comprise a nucleic acid encoding a second CAR that is specific for a tumor antigen.
  • a disclosed tumor antigen comprises can comprise HPV-16 E6 and HPV-16 E7, alpha folate receptor, 5T4, avp6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19. CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b. CD123, CD137 (4-1BB), CD138.
  • NCAM NKG2D Ligands, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TACI, TAG72, TEMs, VEGFRII, or any combination thereof.
  • a disclosed vector can be a transposon-based vector such as Sleeping Beauty and PiggyBac, both of which are known in the art.
  • a first plasmid can be loaded w ith a disclosed nucleic acid sequence encoding a disclosed CAR, named transposon, surrounded by inverted repeats (IRs) that contain short direct repeats (DRs), while a second plasmid encodes the enzyme (transposase) that can recognize the sequences from the first plasmid and cut the transposon out of the first plasmid.
  • IRs inverted repeats
  • DRs short direct repeats
  • the disclosed CAR sequence can be successfully- delivered into the targeted cell (e.g., a T cell or a NK cell or a macrophage) cytoplasm and inserted randomly into TA dinucleotide base pairs of the recipient DNA sequence.
  • the targeted cell e.g., a T cell or a NK cell or a macrophage
  • stable integration and reliable long-term expression of the disclosed CAR sequence can be achieved.
  • a disclosed vector can stimulate an effector cell-mediated immune modulator response to cVIM-expressing tumor cells.
  • a disclosed vector can induce a tumor reducing immune response.
  • a disclosed vector can induce phagocytosis of cancer cells in the subject.
  • a disclosed vector can cross-prime an anti -tumor T cell response.
  • a disclosed vector can induce a tumor eliminating immune response.
  • a disclosed vector can treat cancer.
  • a disclosed vector comprising a disclosed nucleic acid molecule and/or encoding a disclosed CAR can be subjected to a validation process or can be subjected to validating the efficacy of one or more of nucleic acid molecules to, for example, generate a functional CAR targeting a cVIM-expressing cancer cell by transfecting and/or transducing an immune cell (e.g., T cell, NK cell, macrophage, etc ).
  • an immune cell e.g., T cell, NK cell, macrophage, etc.
  • a disclosed vector can be a recombinant vector.
  • plasmid comprising a disclosed nucleic acid molecule.
  • plasmid comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising the scFV of Ab#4 as described in the Examples.
  • a disclosed plasmid can be used to introduce a disclosed nucleic acid molecule or disclosed nucleic acid sequence to one or more host cells.
  • host cells are discussed infra and can comprise T cells, NK cells, macrophages, or iPSCs.
  • a disclosed plasmid can be used to introduce a disclosed nucleic acid molecule encoding a disclosed CAR or disclosed nucleic acid sequence encoding a disclosed CAR to one or more host cells.
  • a disclosed plasmid can be used to introduce a disclosed nucleic acid molecule or disclosed nucleic acid sequence to one or more T cells or NK cells or macrophages.
  • a disclosed plasmid can be used to introduce a disclosed nucleic acid molecule encoding a disclosed CAR or disclosed nucleic acid sequence encoding a disclosed CAR to one or more T cells or NK cells or macrophages.
  • a disclosed plasmid can be an integrating vector or a non-integrating vector.
  • integration can mean that the nucleotides of nucleic acid sequence can be stably inserted into the cellular genome (e.g., covalently linked to the nucleic acid sequence within the cell’s chromosomal DNA).
  • a disclosed plasmid can be a recombinant vector comprising a disclosed nucleic acid sequence. Recombinant plasmids are known to the art.
  • a disclosed promoter can comprise a ubiquitous promoter, a constitutive promoter, or a tissue specific promoter.
  • a disclosed promoter can be operably linked to a disclosed nucleic acid sequence encoding a disclosed CAR.
  • a disclosed promoter can be operably linked to a disclosed nucleic acid sequence encoding a disclosed tumor antigen. Promoters are known to the art.
  • a disclosed promoter can be a promoter/ enhancer. Promoter/ enhancers are known to the art.
  • a disclosed promoter can be an endogenous promoter.
  • a disclosed endogenous promoter can be an endogenous promoter/enhancer.
  • a disclosed promoter or a disclosed promoter/enhancer can be used for constitutive and efficient expression of a disclosed CAR.
  • a disclosed promoter or a disclosed promoter/enhancer can be used for constitutive and efficient expression of a disclosed tumor antigen.
  • a disclosed plasmid can be used to engineer cells to express a disclosed CAR.
  • a disclosed plasmid can further comprise a nucleic acid encoding a second CAR that is specific for a tumor antigen.
  • a disclosed tumor antigen comprises can comprise HPV-16 E6 and HPV-16 E7, alpha folate receptor, 5T4, avp6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19. CD20.
  • a disclosed vector can be a viral vector or a non-viral vector.
  • a disclosed non-viral vector can be a polymer-based vector, a peptide-based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid-based vector.
  • a disclosed plasmid can comprise a transposon such as, for example, Sleeping Beauty and PiggyBac. both of which are known in the art.
  • a first plasmid can be loaded with a disclosed nucleic acid sequence encoding a disclosed CAR, named transposon, surrounded by inverted repeats (IRs) that contain short direct repeats (DRs).
  • IRs inverted repeats
  • DRs short direct repeats
  • a second plasmid encodes the enzyme (transposase) that can recognize the sequences from the first plasmid and cut the transposon out of the first plasmid.
  • a disclosed plasmid can stimulate an effector cell mediated immune modulator response to PS-expressing tumor cells.
  • a disclosed plasmid can induce a tumor reducing immune response.
  • a disclosed plasmid can induce phagocytosis of cancer cells in the subject.
  • a disclosed plasmid can cross-prime an anti-tumor T cell response.
  • a disclosed plasmid can induce a tumor eliminating immune response.
  • a disclosed plasmid can treat cancer.
  • Disclosed herein are cells transformed or transfected by one or more disclosed nucleic acid molecules. Disclosed herein are cells transformed or transfected by a nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed CAR. Disclosed herein are cells transformed or transfected by a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR specific for citrullinated vimentin (cVIM).
  • cVIM citrullinated vimentin
  • nucleic acid molecule comprising a nucleic acid sequence encoding a CAR comprising an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising one or more immunostimulatory domains.
  • a nucleic acid molecule comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), comprising from N-terminus to C-terminus a signal peptide: an antigen binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more immunoreceptor tyrosine-based activation domains (ITAMs) and/or one or more co-stimulatory domains.
  • CAR chimeric antigen receptor
  • a nucleic acid molecule comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), comprising from N-terminus to C-terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; an intracellular domain, a cleavage signal; a signal peptide; and a tumor specific antigen or fragment thereof.
  • CAR chimeric antigen receptor
  • Disclosed herein are cells transduced by one or more disclosed viral vectors. Disclosed herein are cells transduced by a vector comprising a disclosed nucleic acid molecule. Disclosed herein are cells transduced by a vector comprising a disclosed nucleic acid sequence. Disclosed herein are cells transduced by a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed CAR. Disclosed herein are cells transduced by a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR specific for citrullinated vimentin (cVIM).
  • cVIM citrullinated vimentin
  • a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR comprising an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising one or more immunostimulatory domains.
  • a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR comprising from N-terminus to C-terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more immunoreceptor tyrosine-based activation domains (ITAMs) and/or one or more co-stimulatory domains.
  • ITAMs immunoreceptor tyrosine-based activation domains
  • a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR comprising from N-terminus to C-terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; an intracellular domain, a cleavage signal; a signal peptide; and a tumor specific antigen or fragment thereof.
  • a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a cVIM-specific CAR comprising an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising one or more immunostimulatory domains.
  • a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a cVIM-specific CAR comprising from N-terminus to C-terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more immunoreceptor tyrosine-based activation domains (ITAMs) and/or one or more co-stimulatory domains.
  • ITAMs immunoreceptor tyrosine-based activation domains
  • a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a cVIM-specific CAR comprising from N-terminus to C-terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; an intracellular domain, a cleavage signal; a signal peptide; and a tumor specific antigen or fragment thereof.
  • cells transduced by a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:01. SEQ ID NO:02, or SEQ ID NO:03.
  • cells transduced by a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:05, SEQ ID NO:06, or SEQ ID NO:07.
  • a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), comprising from N-terminus to C-terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more immunoreceptor tyrosine-based activation domains (ITAMs) and/or one or more co-stimulatory domains.
  • CAR chimeric antigen receptor
  • a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), comprising from N-terminus to C-terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; an intracellular domain, a cleavage signal; a signal peptide; and a tumor specific antigen or fragment thereof.
  • CAR chimeric antigen receptor
  • a cell expressing an anti-citrullinated vimentin (cVIM) chimeric antigen receptor Disclosed herein is an engineered immune effector cell comprising a disclosed chimeric antigen receptor.
  • a disclosed engineered immune effector cell can comprise a T cell, a cytotoxic T lymphocyte, a natural killer T lymphocyte cell, a macrophage, and a natural killer cell.
  • an engineered T cell expressing a disclosed chimeric antigen receptor can comprise a T cell, a cytotoxic T lymphocyte, a natural killer T lymphocyte cell, a macrophage, and a natural killer cell.
  • an engineered T cell expressing a disclosed chimeric antigen receptor Disclosed herein is an engineered cytotoxic T lymphocyte expressing a disclosed chimeric antigen receptor.
  • an engineered natural killer T lymphocyte cell expressing a disclosed chimeric antigen receptor.
  • an engineered macrophage expressing a disclosed chimeric antigen receptor.
  • a disclosed cell can be transduced and/or transfected with one or more recombinant nucleic acid molecules and/or one or more recombinant vectors.
  • cells engineered to expression one or more disclosed CARs can comprise T cells or NK cells or macrophages.
  • disclosed cells engineered to expression one or more disclosed CARs can be immune cells.
  • disclosed cells engineered to expression one or more disclosed CARs can comprise T cells, B cells, natural killer (NK) cells, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes, macrophages, platelets, thymocytes, myeloid cells, or any combination thereof.
  • NK natural killer
  • T cells and NK cells can be differentiated in vitro from a hematopoietic stem cell population (for example iPSCs) or can be obtained from a subject.
  • T cells and NK cells can be obtained from, for example, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, tumors, or any combination thereof.
  • PBMCs peripheral blood mononuclear cells
  • disclosed T cells can be derived from one or more T cell lines available in the art.
  • disclosed T cells can also be obtained from a unit of blood collected from a subject in need thereof using any number of techniques known to the skilled person.
  • a disclosed cell can be transformed or transfected by a nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed CAR.
  • a disclosed cell can be transduced by a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed CAR.
  • a disclosed cell can express a disclosed CAR.
  • disclosed host cells expressing a disclosed CAR and/or a disclosed tumor antigen can be administered to a subject.
  • the transformed, transfected, and/or transduced, disclosed host cells expressing a disclosed CAR and/or a disclosed tumor antigen can be autologous to the receiving subject.
  • a disclosed cell can stimulate an effector cell-mediated immune modulator response to cVIM-expressing tumor cells.
  • a disclosed cell can induce a tumorreducing immune response.
  • a disclosed cell can induce phagocytosis of cancer cells in the subject.
  • a disclosed cell can cross-prime an anti -tumor T cell response.
  • a disclosed cell can induce a tumor-eliminating immune response.
  • a disclosed cell can treat cancer.
  • a therapeutically effective amount of the disclosed genetically modified cells expressing a disclosed chimeric antigen receptor targeting citrullinated vimentin (cVIM) can be about 1 x 10 4 to about 1 x 10 9 cells/kg per subject.
  • a therapeutically effective amount of the disclosed genetically modified cells expressing a disclosed chimeric antigen receptor targeting citrullinated vimentin (cVIM) can be about 1 x 10 5 cells/kg per subject, about 1 x 10 6 cells/kg per subject, about I x 10 7 cells/kg per subject, about 1 x 10 8 cells/kg per subject, or about 1 x 10 9 cells/kg per subject.
  • cVIM citrullinated vimentin
  • a disclosed cell can be engineered to express one or more CARs including a CAR targeting cVIM and a CAR targeting any other cancer antigen.
  • Disclosed herein is a pharmaceutical formulation comprising a disclosed nucleic acid molecule; and one or more pharmaceutically acceptable carriers.
  • a pharmaceutical formulation comprising a disclosed vector comprising a pharmaceutically acceptable carriers.
  • a pharmaceutical formulation comprising a disclosed plasmid comprising a pharmaceutically acceptable carriers.
  • a pharmaceutical formulation comprising a disclosed cell comprising a pharmaceutically acceptable carriers.
  • a pharmaceutical formulation comprising a nucleic acid molecule comprising a nucleic acid sequence encoding any disclosed CAR.
  • a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR specific for citrullinated vimentin (cVIM).
  • cVIM citrullinated vimentin
  • a pharmaceutical formulation a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR comprising an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising one or more immunostimulatory domains.
  • a pharmaceutical formulation comprising a vector comprising a disclosed nucleic acid sequence.
  • a pharmaceutical formulation comprising a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding any disclosed CAR.
  • Disclosed herein is a pharmaceutical formulation comprising a plasmid comprising a disclosed nucleic acid molecule.
  • a pharmaceutical formulation comprising a plasmid comprising a disclosed nucleic acid sequence Disclosed herein is a pharmaceutical formulation comprising a plasmid comprising a nucleic acid molecule comprising a nucleic acid sequence encoding any disclosed CAR.
  • a pharmaceutical formulation comprising a plasmid comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR comprising an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising one or more immunostimulatory domains.
  • a disclosed pharmaceutical formulation can comprise (i) one or more active agents, (ii) biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof.
  • a disclosed pharmaceutical formulation can further comprise one or more anti-inflammatory agents.
  • 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 my cophenolate.
  • steroids and glucocorticoids including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinol
  • NSAIDs can comprise ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as rofecoxib and celecoxib, sialylates, or any combination thereof.
  • analgesics can comprise acetaminophen, oxycodone, tramadol, proporxyphene hydrochloride, or any combination thereof.
  • glucocorticoids can comprise cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, or any combination thereof.
  • biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists (e.g., etanercept, adalimumab, and infliximab, chemokine inhibitors and adhesion molecule inhibitors.
  • cytokine inhibitors such as the TNF antagonists (e.g., etanercept, adalimumab, and infliximab, chemokine inhibitors and adhesion molecule inhibitors.
  • TNF antagonists e.g., etanercept, adalimumab, and infliximab
  • chemokine inhibitors e.g., chemokine inhibitors and adhesion molecule inhibitors.
  • biological response modifiers can comprise monoclonal antibodies as well as recombinant forms of molecules.
  • exemplary disease-modifying anti-rheumatic drugs can comprise include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), minocycline, or any combination thereof.
  • a disclosed chemotherapeutic agent in a disclosed pharmaceutical formulation can comprise an anthracycline, a vinca alkaloid, an alkylating agent, an immune cell antibody, an antimetabolite, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor, an immunomodulator, or any combination thereof.
  • GITR TNFR glucocorticoid induced TNFR related protein
  • a disclosed chemotherapeutic agent can comprise 5-fluorouracil (Adrucil, Efudex), 6-mercaptopurine (Purinethol), 6-thioguanine, aclarubicin or aclacinomycin A, alemtuzamab (Lemtrada), anastrozole (Arimidex), bicalutamide (Casodex), bleomycin sulfate (Blenoxane), bortezomib (Velcade), busulfan (Myleran), busulfan injection (Busulfex), capecitabine (Xeloda), carboplatin (Paraplatin), carmustine (BiCNU), chlorambucil (Leukeran), cisplatin (Platinol), cladribine (Leustatin), Cosmegan, cyclophosphamide (Cytoxan or Neosar), cyclophosphamide, cytarabine
  • fludarabine phosphate Fludara
  • flutamide Eulexin
  • folic acid antagonists gemcitabine (difluorodeoxycitidine), gemtuzumab, gliotoxin, hydroxyurea (Hydrea), Idarubicin (Idamycin), ifosfamide (IFEX), ifosfamide, irinotecan (Camptosar), L-asparaginase (ELSPAR), lenalidomide), leucovorin calcium, melphalan (Alkeran), melphalan, methotrexate (Fol ex), mitoxantrone (Novantrone), my lotarg, N4-pentoxycarbonyl-5 deoxy-5-fluorocytidine, nab-paclitaxel (Abraxane), paclitaxel (Taxol), pentostatin, phoenix (Yttrium90/MX-DTPA), polifeprosan 20 with
  • thalidomide or a thalidomide derivative thiotepa, tirapazamine (Tirazone), topotecan hydrochloride for injection (Hy camptin), tositumomab), vinblastine (Velban), vinblastine, vincristine (Oncovin), vindesine, vinorelbine (Navelbine), or any combination thereof.
  • a disclosed pharmaceutical formulation can comprise an anti-chemokine therapy that enhances the resident memory T cell formations in tumor-free tissues.
  • a disclosed anti-chemokine therapy can comprise one or more antibodies against CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL25.
  • a disclosed pharmaceutical formulation can further comprise abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomabm bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab.
  • clivatuzumab conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farietuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab,
  • a disclosed pharmaceutical formulation can stimulate an effector cell mediated immune response to cVIM-expressing tumor cells.
  • a disclosed pharmaceutical formulation can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation in a subject having cancer.
  • metabolic dysregulation can be associated with cancer or cancerous cells.
  • cell death of citrullinated vimentin (cVIM)-expressing cancer cells is effected.
  • a disclosed pharmaceutical formulation can be prepared for systemic or direct administration.
  • a disclosed pharmaceutical formulation can be prepared for oral administration, intravenous administration, intra-tumoral administration, intraperitoneal administration, or any combination thereof.
  • a disclosed pharmaceutical formulation can be prepared for any method of administration disclosed herein.
  • a disclosed pharmaceutical formulation can be prepared for administration via multiple routes either concurrently or sequentially.
  • a disclosed pharmaceutical formulation can be first administered intratumorally and then be administered intravenously.
  • a disclosed pharmaceutical formulation can be first administered intra-tumorally and then be administered orally.
  • a skilled clinical can determine the best route of administration for a subject at a given time.
  • a disclosed pharmaceutical formulation can comprise one or more immune modulators.
  • a disclosed pharmaceutical formulation can comprise one or more proteasome inhibitors.
  • a disclosed pharmaceutical formulation can comprise one or more immunosuppressives or immunosuppressive agents.
  • an immunosuppressive agent can be anti-thymocyte globulin (ATG), cyclosporine (CSP), my cophenolate mofetil (MMF), or a combination thereof.
  • a disclosed pharmaceutical formulation can comprise an anaplerotic agent (such as, for example. C7 compounds like triheptanoin or MCT).
  • a disclosed pharmaceutically acceptable carrier can comprise any disclosed carrier and/or any disclosed excipient.
  • a disclosed pharmaceutical formulation can stimulate an effector cell mediated immune modulator response to citrullinated vimentin (cVIM)-expressing tumor cells.
  • a disclosed pharmaceutical formulation can induce a tumor reducing immune response.
  • a disclosed pharmaceutical formulation can induce phagocytosis of cancer cells in the subject.
  • a disclosed pharmaceutical formulation can cross-prime an antitumor T cell response.
  • a disclosed pharmaceutical formulation can induce a tumor eliminating immune response.
  • a disclosed pharmaceutical formulation can treat cancer.
  • a disclosed pharmaceutical formulation can be subjected to a validation process or can be subjected to validating the efficacy of one or more of nucleic acid molecules and/or vectors to, for example, generate a functional CAR targeting a cVIM-expressing cancer cell by transfecting and/or transducing an immune cell (e g., T cell, NK cell, macrophage, etc.).
  • an immune cell e g., T cell, NK cell, macrophage, etc.
  • a disclosed animal can be treated with one or more disclosed CARs, one or more disclosed cells, one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.
  • animals can be assessed and/or monitored for one or more biological and/or chemical functions prior to treatment, during treatment, after treatment, or any combination thereof.
  • a disclosed treated subject can be a mouse or a rat.
  • a disclosed treated subject can be a transgenic mouse or a transgenic rat.
  • a disclosed treated subject can have one or more types of cancers and/or tumors.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells transduced with a disclosed recombinant vector or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells transformed with a disclosed plasmid or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • cVIM citrullinated vimentin
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more immune cells transduced with a recombinant vector or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more immune cells transformed with a disclosed plasmid or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • cVIM citrullinated vimentin
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)-expressing tumor cells or a pharmaceutical formulation thereof.
  • cVIM citrullinated vimentin
  • a method of slowing disease progression comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells transduced with a disclosed recombinant vector or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • Disclosed herein is a method of slowing disease progression, the method comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells transformed with a disclosed plasmid or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of slowing disease progression the method comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • cVIM citrullinated vimentin
  • Disclosed herein is a method of slowing disease progression, the method comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more immune cells transduced with a recombinant vector or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of slowing disease progression the method comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more immune cells transformed with a disclosed plasmid or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of slowing disease progression comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • a method of slowing disease progression comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)-expressing tumor cells or a pharmaceutical formulation thereof.
  • a disclosed CAR can target citrullinated vimentin (cVIM)-expressing cancer cells.
  • a disclosed CAR can target citrullinated vimentin (cVIM)-expressing ovarian cancer cells.
  • a disclosed CAR can target citrullinated vimentin (cVIM)-expressing colon cancer cells.
  • a disclosed CAR can target citrullinated vimentin (cVIM)-expressing lung cancer cells.
  • disclosed citrullinated vimentin (cVIM)-expressing cancer cells can be in a tumor.
  • disclosed citrullinated vimentin (cVIM)-expressing cancer cells can be in one or more tumors.
  • disclosed cancer cells can be blood borne.
  • a disclosed cell can express a CAR targeting cVIM and at least one CAR targeting another cancer cell antigen.
  • a disclosed cell can express one or more CARs (e.g. targeting cVIM and targeting one or more other cancer cell antigens).
  • a disclosed method of treating cancer and/or slowing disease progression and/or slowing disease progression can comprise stimulating an effector cell mediated immune modulator response to citrullinated vimentin (cVIM)-expressing tumor cells.
  • cVIM citrullinated vimentin
  • a disclosed method of treating cancer and/or slowing disease progression can induce a tumor reducing immune response.
  • a disclosed method of treating cancer and/or slowing disease progression can induce phagocytosis of cancer cells in the subject.
  • a disclosed method of treating cancer and/or slowing disease progression can cross-primer an antitumor T cell response.
  • a disclosed method of treating cancer and/or slowing disease progression can induce a tumor eliminating immune response.
  • a disclosed method of treating cancer and/or slowing disease progression can effect tumor cell death.
  • a disclosed method of treating cancer and/or slowing disease progression can comprise transducing extracted T cells orNK cells or macrophages ex vivo with a disclosed vector or a disclosed nucleic acid molecule such that the T cells, NK cells, or macrophages express a disclosed CAR, and returning the CAR-expressing T cells, NK cells, or macrophages back to the subject.
  • the cells can be autologous to the subject.
  • cancer cells can comprise ovarian cancer, ovarian adenocarcinoma, ovarian teratocarcinoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous cell lung carcinoma, adenocarcinoma, gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, malignant pleomorphic adenoma, sarcoma, synovial sarcoma, carcinosarcoma, bile duct cancer, bladder cancer, transitional cell carcinoma, papillary carcinoma, kidney cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, colon cancer, small bowel cancer, small bowel adenocarcinoma, adenocarcinoma of the ileum, testicular embryonal carcinoma, placental choriocar
  • a subject can have, be diagnosed with, or be suspected of having ovarian cancer, ovarian adenocarcinoma, ovarian teratocarcinoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous cell lung carcinoma, adenocarcinoma, gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, malignant pleomorphic adenoma, sarcoma, synovial sarcoma, carcinosarcoma, bile duct cancer, bladder cancer, transitional cell carcinoma, papillary' carcinoma, kidney cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary' renal cell carcinoma, colon cancer, small bowel cancer, small bowel adenocarcinoma, adenocarcinoma of the ileum, test
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise collecting one or more blood and/or biological samples from a subject at the same time or at different times.
  • a blood sample and/or a biological sample can be collected from a subject at a pre-determined interval.
  • a pre-determined interval can be once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, or at a longer interval.
  • a pre-determined interval can be once a month, once every 2 months, once every' 3 months, once every' 5 months, once every' 5 months, once every' 6 months, or at a longer interval.
  • a blood sample and/or a biological sample can be collected from a subject prior to treatment, during treatment, after treatment, or any combination thereof.
  • a blood and/or a biological sample can be collected from a subject at any time deemed medically and/or clinically appropriate by the skilled clinician.
  • a subject can be a human patient.
  • a subject can be any age (e.g., geriatric, adult, young adult, teenager, tween, adolescent, child, toddler, baby, or infant), can be male or female, can be any nationality, can be of any ethnicity, and/or can be of any race.
  • a subject can be treatment-naive.
  • a subject can have had treatment.
  • a subject can have a terminal cancer.
  • a disclosed subject can have metastatic cancer or onco-metastatic cancer.
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise isolating monocytes from peripheral blood monocular cells in the subject’s blood and/or biological sample.
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise isolating bone marrow derived monocytes from the subject’s blood and/or biological sample.
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise isolating monocytes from the subject’s blood and/or biological sample.
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise isolating naive macrophages (MO) from the subject’s blood and/or biological sample.
  • MO naive macrophages
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise subjecting a disclosed blood sample to centrifugation.
  • a disclosed method can further comprise separating the blood and/or biological sample into its component parts using, for example, centrifugation, apheresis, or any technique to the skilled person.
  • a disclosed separating step can comprise generating a layer of clear fluid, a layer of red fluid, and a thin layer in between the clear fluid layer and the red fluid layer.
  • a disclosed red layer can comprise red blood cells.
  • a disclosed clear layer can comprise plasma.
  • a disclosed thin layer in between the red layer and the clear layer can comprise the buffy coat.
  • a disclosed buffy coat can comprise white blood cells and platelets.
  • a disclosed method can further comprise isolating peripheral blood mononuclear cells (PMBCs) from the buffy coat.
  • PMBCs can comprise lymphocytes, leukocytes, and/or monocytes.
  • macrophages can be derived from monocytes.
  • isolating lymphocytes, leukocytes, and/or monocytes can be done by any method and/or technique known to the skilled person (e.g., leukapheresis).
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise isolating resting or MO macrophages from the buffy 7 coat.
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise differentiating monocytes into resting or MO macrophages.
  • the disclosed macrophages can be resting or MO macrophages.
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise polarizing the resting or MO macrophages into a Ml phenotype or a M2 phenotype or a pro-inflammatory phenotype or an anti-inflammatory phenotype.
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise polarizing the resting or MO macrophages into a classically activated macrophage phenotype.
  • a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of disclosed engineered T cells or NK cells or macrophages can be any amount that, when used alone or in combination with another therapeutic agent, can attack and destroy citrullinated vimentin (cVIM)-expressing tumor cells.
  • cVIM citrullinated vimentin
  • a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of the disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof can be any amount that, when used alone or in combination with another therapeutic agent, can attack and destroy citrullinated vimentin (cVIM)- expressing tumor cells.
  • a therapeutically effective amount of the disclosed genetically modified cells expressing a disclosed chimeric antigen receptor targeting citrullinated vimentin (cVIM) can be about 1 x 10 4 to about 1 x 10 9 cells/kg per subject.
  • a therapeutically effective amount of the disclosed genetically modified cells expressing a disclosed chimeric antigen receptor targeting citrullinated vimentin (cVIM) can be about 1 x 10 5 cells/kg per subject, about 1 x 10 6 cells/kg per subject, about 1 x 10 7 cells/kg per subject, about 1 x 10 8 cells/kg per subject, or about 1 x 10 9 cells/kg per subject.
  • cVIM citrullinated vimentin
  • a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of the disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof can protect a subject against the onset of a disease and/or promotes disease regression evidenced by a decrease in severity' of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • the ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity 7 of the agent in in vitro assays.
  • administering genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or administering engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof can comprise systemic or direct administration.
  • administering can comprise oral administration, intravenous administration, intra-tumoral administration, intraperitoneal administration, or any combination thereof.
  • administering genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or administering engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof can be administered by any method of administration disclosed herein.
  • genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof can be administered via multiple routes either concurrently or sequentially.
  • genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof can be first administered intra-tumorally and then be administered intravenously.
  • genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof can be first administered intra-tumorally and then be administered orally.
  • a skilled clinician can determine the best route of administration for a subject at a given time.
  • a disclosed method can comprise repeating the administering of the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • a disclosed method of treating cancer and/or slowing disease progression can comprise protecting the subject from metastasis. In an aspect, a disclosed method of treating cancer and/or slowing disease progression can comprise reducing the risk of developing metastasis. In an aspect, a disclosed method of treating cancer and/or slowing disease progression can comprise preventing or inhibiting metastasis. [0405] In an aspect, a disclosed method can comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, a disclosed method can comprise continuing to treat the subject.
  • continuing to treat the subject can comprise continuing to administer the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • a disclosed method in the presence of adverse effects, can comprise modifying one or more steps of the method.
  • modifying one or more steps of a disclosed method can comprise modifying the administering step.
  • modifying the administering step can comprise changing the amount of the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof administered to the subject, changing the frequency of administration of the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof, changing the duration of administration of the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise administering to the subject an immune checkpoint inhibitor (e.g.. an anti-PDl molecule).
  • an immune checkpoint inhibitor e.g. an anti-PDl molecule
  • a disclosed anti-PDl molecule can comprise an anti-PDl antibody, an anti-PDLl antibody, or any combination thereof.
  • a disclosed anti-PDl antibody can comprise a monoclonal antibody, a humanized monoclonal antibody, or a fragment thereof.
  • a disclosed anti-PDl antibody can comprise a polyclonal antibody, a humanized polyclonal antibody, or a fragment thereof.
  • a disclosed anti-PDl antibody can comprise any antibody or antibody fragment that specifically recognizes PD1.
  • a disclosed anti- PDL1 antibody can comprise a monoclonal antibody, a humanized monoclonal antibody, or a fragment thereof.
  • a disclosed anti-PDLl antibody can comprise a polyclonal antibody, a humanized polyclonal antibody, or a fragment thereof.
  • a disclosed anti- PDLl antibody can comprise any antibody or antibody fragment that specifically recognizes PDL1.
  • Antibodies and methods of preparing antibodies are known in the art.
  • recombinant antibodies and methods of preparing recombinant antibodies are known in the art.
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise repeating the administering of the anti-PDl molecule.
  • a disclosed anti-PDl molecule can be administered prior to, concurrent with, or after the administration of the interfering molecule.
  • administering a disclosed anti-PDl molecule can comprise systemic or direct administration.
  • administering a disclosed anti-PDl molecule can comprise intravenous administration, intra-tumoral administration, intraperitoneal administration, or any combination thereof.
  • administering a disclosed can be administered by any method of administration disclosed herein.
  • a disclosed anti-PDl molecule can be administered via multiple routes either concurrently or sequentially.
  • a disclosed anti-PDl molecule can be first administered intra-tumorally and then be administered intravenously.
  • administering a disclosed anti-PDl molecule can be first administered intra-tumorally and then be administered orally.
  • a skilled clinician can determine the best route of administration for a subject at a given time.
  • a disclosed anti-PDl molecule can be administered about 3 months, about 2 months, or about 1 month prior to the administering of the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • a disclosed anti-PDl molecule can be administered about 8 weeks, about 7 weeks, about 6 weeks.
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • cVIM engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • a disclosed anti-PDl molecule can be administered about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day prior to the administering of the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • a disclosed anti-PDl molecule can be administered about 24, 23, 22, 21, 20, 19, 18, 17, 16. 15. 14. 13.
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • cVIM engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • a disclosed method of treating cancer and/or slowing disease progression can comprise administering to the subject one or more additional anti-cancer therapies.
  • Anti-cancer therapies are known to the art.
  • a disclosed anti-cancer therapy can comprise endocrine therapy, radiotherapy, hormone therapy, gene therapy, thermal therapy, ultrasound therapy, or any combination thereof.
  • a disclosed anti-cancer therapy can comprise one or more chemotherapeutic agents.
  • a disclosed chemotherapeutic agent can comprise an anthracycline, a vinca alkaloid, an alkylating agent, an immune cell antibody, an antimetabolite, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor, an immunomodulator, or any combination thereof.
  • GITR TNFR glucocorticoid induced TNFR related protein
  • a disclosed chemotherapeutic agent can comprise 5 -fluorouracil (Adrucil, Efudex), 6-mercaptopurine (Purinethol), 6-thioguanine, aclarubicin or aclacinomycin A, alemtuzamab (Lemtrada), anastrozole (Arimidex), axitinib (Inlyta), bevacizumab (Avastin), bicalutamide (Casodex), bleomycin sulfate (Blenoxane).
  • 5 -fluorouracil Adrucil, Efudex
  • 6-mercaptopurine Purine
  • 6-thioguanine 6-thioguanine
  • aclarubicin or aclacinomycin A alemtuzamab (Lemtrada)
  • anastrozole Arimidex
  • axitinib Inlyta
  • bevacizumab Avastin
  • bortezomib (Velcade), busulfan (Myleran), busulfan injection (Busulfex), capecitabine (Xeloda), carboplatin (Paraplatin), carmustine (BiCNU), chlorambucil (Leukeran), cisplatin (Platinol), cladribine (Leustatin), Cosmegan, cyclophosphamide (Cytoxan or Neosar), cyclophosphamide, cytarabine liposome injection (DepoCyt), cytarabine, cytosine arabinoside (Cytosar-U), dacarbazine (DTIC-Dome), dactinomycin (Cosmegen), daunorubicin citrate liposome injection (DaunoXome), daunorubicin hydrochloride (Cerubidine), dexamethasone, docetaxel (Taxotere), dox
  • one or more disclosed additional anticancer therapies can be administered one or more times. In an aspect, one or more disclosed additional anti-cancer therapies can be administered by one or more routes of administration.
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise administering to the subject one or more targeted therapies including, but not limited to, abemaciclib, ado-trastuzumab emtansine, afatinib, alectinib, alemtuzumab, alpelisib, atezolizumab, avelumab, axitinib, bevacizumab, binimetinib, blinatumomab, bosutinib, brentuximab, brigatinib, cabozantinib, carfilzomib, cemiplimab, ceritinib, cetuximab, gilteritinib, cobimetinib, copanlisib, crizotinib, dabrafenib, dacomitinib, daratumumab, dasatinib.
  • targeted therapies including, but not limited
  • one or more of these targeted therapies can be administered one or more times. In an aspect, one or more disclosed targeted therapies can be administered by one or more routes of administration.
  • a disclosed method can further comprise administering to the subject one or more biosimilars.
  • biosimilar can refer to a biologic product that is highly similar to the reference product approved by a regulatory agency (e.g., the Federal Drug Administration (FDA) or the European Medicines Agency (EMA)) based on data from (i) analytical studies demonstrating that the biologic product is highly similar to the reference product notwithstanding minor differences in clinically inactive components; (ii) animal studies (including the assessment of toxicity); and/or (iii) a clinical study or studies (including the assessment of immunogenicity and pharmacokinetics or pharmacodynamics) that are sufficient to demonstrate safety, purity, and potency in one or more appropriate conditions of use for which the reference product is approved and intended to be used and for which approval is sought (e.g., that there are no clinically meaningful differences between the biologic product and the reference product in terms of the safety, purity, and potency of the product).
  • the biosimilar product can be an interchangeable product as determined by a regulatory agency (e.g., the Federal
  • a disclosed biosimilar can comprise an FDA approved biosimilar (e.g., Bkemv (eculizumab-aeeb). Yesafili (aflibercept-jbvf). Opuviz (aflibercept-yszy), Hercessi (trastuzumab-strf). Selarsdi (ustekinumab-aekn).
  • FDA approved biosimilar e.g., Bkemv (eculizumab-aeeb). Yesafili (aflibercept-jbvf).
  • Opuviz aflibercept-yszy
  • Hercessi trastuzumab-strf
  • Selarsdi ustekinumab-aekn).
  • Tyenne tocilizumab-aazg
  • Jubbonti denotes the deoxyribonucleic acid
  • Wyost deoxyribonub
  • Simlandi adalimumab-ryvk
  • Avzivi bevacizumab-tnjn
  • Wezlana ustekinumab-auub
  • Tofidence tocilizumab-bavi
  • Tyruko natalizumab-sztn
  • Yuflyma adalimumab-aaty
  • Idacio adalimumab-aacf
  • Vegzelma bevacizumab-adcd
  • Stimufend pegfilgrastim-fpgk
  • Cimerli ranibizumab-eqm
  • Fylnetra pegfilgrastim-pbbk
  • Alymsys bevacizumab-maly
  • Releuko filgra
  • Fulphila pegfilgrastim-jmdb
  • Retacrit epoetin alfa-epbx
  • Ixifi infliximab- qbtx
  • Ogivri trastuzumab-dkst
  • Mvasi bevacizumab-awwb
  • Cyltezo adalimumab-adbm
  • Renflexis infliximab-abda
  • Amj evita adalimumab-atto
  • Erelzi etanercept-szzs
  • Inflectra infliximab-dyyb
  • Zarxio filgrastim-sndz
  • one or more of biosimilars can be administered one or more times.
  • one or more disclosed biosimilars can be administered by one or more routes of administration.
  • a disclosed method of treating cancer and/or slowing disease progression can comprise administering to the subject an anti-chemokine therapy.
  • a disclosed anti- chemokine therapy can comprise one or more antibodies against CCL1, CCL2, CCL4. CCL17, CCL19, CCL21, CCL22. CCL25, CXCL9. CXCL10, CXCL11. CXCL12, CXCL13, CCR2, CCR5, CCR7, CCR8, CCR9, CXCR3, CXCR4, CXCR5, CX3CL1, CX3CR1, or any combination thereof.
  • a disclosed method of treating cancer and/or slowing disease progression can comprise administering an oligonucleotide therapeutic agent.
  • a disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof.
  • a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi.
  • a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable.
  • a disclosed oligonucleotide therapeutic agent can comprise a CRISPR- based endonuclease.
  • a disclosed endonuclease can be Cas9.
  • CRISPR/Cas9 systems and methods are know n to the art.
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise preventing or inhibiting metastasis of cancer cells.
  • a disclosed method can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of decrease or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof)).
  • a control subject such as, for example, a subject that has not received a disclosed treatment (e.g., genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation
  • preventing or inhibiting metastasis of cancer cells can comprise a 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% or any amount of decrease or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g.. genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof).
  • a control subject such as, for example, a subject that has not received a disclosed treatment (e.g.. genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells
  • a disclosed method of treating cancer and/or slowing disease progression can comprise surgically resecting the tumor and/or cancer cells from the subject.
  • a disclosed method of treating cancer and/or slowing disease progression can comprise continuing to administer to the subject a therapeutically effective amount of the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof and continuing to administer to the subject a therapeutically effective amount of an anti-PDl molecule, a disclosed anti-chemokine therapy, a disclosed chemotherapeutic agent, any disclosed therapeutic agent, or any combination thereof.
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise subjecting the subject to one or more invasive or non -invasive diagnostic assessments. Diagnostic assessments are known to the art.
  • a disclosed non-invasive diagnostic assessment can comprise x-rays, computerized tomography (CT) scans, magnetic resonance imaging (MRI) scans, ultrasounds, positron emission tomography (PET) scans, or any combination thereof.
  • a disclosed invasive diagnostic assessment can comprise a tissue biopsy (including a liquid biopsy and/or a solid tissue biopsy) or exploratory surgery.
  • a disclosed method of treating cancer and/or slowing disease progression can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation in a subject, such as, for example, a subject having cancer or cancerous cells expressing cVIM.
  • a disclosed interfering molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation in a subject having cancer.
  • metabolic dysregulation can be associated with cancer or cancerous cells.
  • restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of a cancer; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a cancer, or (viii) any combination thereof.
  • restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.
  • restoration can be a partial or incomplete restoration.
  • restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.
  • restoring one or more aspects of cellular homeostasis and/or cellular functionality 7 can comprise preventing or inhibiting metastasis of cancer cells in the subject.
  • techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example. representative regulated variables and sensors relating to systemic homeostasis known to the skilled person.
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise administering genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof one or more times, administering an anti-PDl molecule one or more times, administering one or more anti-cancer therapies one or more times, or administering any combination thereof one or more time.
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • preventing or inhibiting metastasis of cancer cells can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of decrease or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof).
  • a control subject such as, for example, a subject that has not received a disclosed treatment (e.g., genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor
  • preventing or inhibiting metastasis of cancer cells can comprise a 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%. 80-90%, or 90-100% or any amount of decrease or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof).
  • a control subject such as, for example, a subject that has not received a disclosed treatment (e.g., genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells
  • a disclosed method of treating cancer and/or slowing disease progression can comprise surgically resecting the tumor and/or cancer cells from the subject.
  • a disclosed method of treating cancer and/or slowing disease progression can comprise continuing to administer to the subject a therapeutically effective amount of a disclosed pharmaceutical formulation.
  • a disclosed method of treating cancer and/or slowing disease progression can further comprise diagnosing the subject as have cancer or cancerous cells.
  • I l l method of treating cancer and/or slowing disease progression can stimulate an effector cell mediated immune modulator response to cVIM-expressing tumor cells.
  • a disclosed method of treating cancer and/or slowing disease progression can induce a tumor reducing immune response.
  • a disclosed method of treating cancer and/or slowing disease progression can induce phagocytosis of cancer cells in the subject.
  • a disclosed method of treating cancer and/or slowing disease progression can cross-prime an anti-tumor T cell response.
  • a disclosed method of treating cancer and/or slowing disease progression can induce a tumor eliminating immune response.
  • a disclosed pharmaceutical formulation can treat cancer.
  • a disclosed method of treating cancer and/or slowing disease progression can comprise administering to the subject a chimeric fusion receptor comprising a cVIM-binding domain.
  • a disclosed method of treating cancer and/or slowing disease progression can further comprising sequencing one or more cancer cells and identifying one or more genomic aberrations in one or more genes.
  • one or more genes can comprise ABCB1, ABCC3, ABLE ABL2, ABRAXAS!, ACTA2, ACVR1, (ALK2), ACVR1B, AGO!.
  • HLA- DRB6, HLA-E, HLA-F HLA-G, HNF1A, HNF1B, HOXA11, HOXB13, HRAS, HSD11B2, HSD3B1, HSD3B2, HSP90AA1, HSPH1, IDH1, IDH2, IDO1, IFIT1, IFIT2, IFIT3, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNL3, IKBKE, IKZF1, IL10RA, IL15, IL2RA, IL6R, IL7R, ING1, INPP4B, IRF1, IRF2, IRF4, 1RS2, 1TPKB, JAK1, JAK2, JAK3, JUN.
  • KDR KEAP1, KEL, KIF1B, KIT.
  • KLF4 KLHL6, KEEN, KMT2A, KMT2B, KMT2C, KMT2D, KRAS, L2HGDH, LAG3, LATS1, LCK, EDER LEF1, LMNA, LMO1, LRP1B, LYN, LZTR1.
  • RICTOR RINT1, RIT1, RNF139, RNF43.
  • ROS1, RPL5, RPS15, RPS6KBL RPTOR RRM1, RSF1, RUNX1, RUNX1T1, RXRA, SCG5, SDHA, SDHAF2, SDHB, SDHC, SDHD, SEC23B, SEMA3C, SETBP1, SETD2, SF3B1, SGK1, SH2B3, SHH SLC26A3, SLC47A2, SLC9A3R1, SLIT2, SLX4, SM, AD2, SMAD3, SMAD4, SMARCA1, SMARCA4, SMARCB1, SMARCE1, SMC1A, SMC3, SMO, SOCS1, SOD2, SOXIO.
  • identifying one or more genomic aberrations can inform a treatment decision and/or treatment regimen and/or can influence a treatment decision and/or treatment regimen.
  • Disclosed herein is a method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of one or more cells transduced with a disclosed recombinant vector or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells the method comprising administering to a subject in need thereof therapeutically effective amount of one or more cells transformed with a disclosed plasmid or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • Disclosed herein is a method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of one or more genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • cVIM citrullinated vimentin
  • a method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells the method comprising administering to a subject in need thereof a therapeutically effective amount of one or more immune cells transduced with a recombinant vector or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • Disclosed herein is a method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of one or more immune cells transformed with a disclosed plasmid or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR.
  • a method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells the method comprising administering to a subject in need thereof a therapeutically effective amount of one or more cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • cVIM citrullinated vimentin
  • cVIM-expressing cancer cells can be in a tumor.
  • disclosed cVIM-expressing cancer cells can be in one or more tumors.
  • disclosed cancer cells can be blood borne.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cell can treat cancer.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can induce a tumor reducing immune response.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can induce phagocytosis of cancer cells in the subject.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can cross-primer an anti-tumor T cell response.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells cancer can induce a tumor eliminating immune response.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can comprise transducing extracted T cells or NK cells or macrophages ex vivo with a disclosed vector or a disclosed nucleic acid molecule such that the T cells, NK cells, or macrophages express a disclosed CAR, and returning the CAR- expressing T cells, NK cells, or macrophages back to the subject.
  • the cells can be autologous to the subject.
  • cancer cells can comprise ovarian cancer, ovarian adenocarcinoma, ovarian teratocarcinoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous cell lung carcinoma, adenocarcinoma, gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, malignant pleomorphic adenoma, sarcoma, synovial sarcoma, carcinosarcoma, bile duct cancer, bladder cancer, transitional cell carcinoma, papillary carcinoma, kidney cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, colon cancer, small bowel cancer, small bowel adenocarcinoma, adenocarcinoma of the ileum, testicular embry onal carcinoma, placental chorio
  • a subject can have, be diagnosed with, or be suspected of having ovarian cancer, ovarian adenocarcinoma, ovarian teratocarcinoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous cell lung carcinoma, adenocarcinoma, gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, malignant pleomorphic adenoma, sarcoma, synovial sarcoma, carcinosarcoma, bile duct cancer, bladder cancer, transitional cell carcinoma, papillary carcinoma, kidney cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, colon cancer, small bowel cancer, small bowel adenocarcinoma, adenocarcinoma of the ileum, testicular
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise collecting one or more blood and/or biological samples from a subject at the same time or at different times.
  • a blood sample and/or a biological sample can be collected from a subject at a predetermined interval.
  • a pre-determined interval can be once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, or at a longer interval.
  • a pre-determined interval can be once a month, once every 2 months, once every 3 months, once every 5 months, once every 5 months, once every 6 months, or at a longer interval.
  • a blood sample and/or a biological sample can be collected from a subject prior to treatment, during treatment, after treatment, or any combination thereof.
  • a blood and/or a biological sample can be collected from a subject at any time deemed medically and/or clinically appropriate by the skilled clinician.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise isolating monocytes from peripheral blood monocular cells in the subject’s blood and/or biological sample.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise isolating bone marrow derived monocytes from the subj ect’s blood and/or biological sample.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise isolating monocytes from the subject’s blood and/or biological sample.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM- expressing tumor cells can further comprise isolating naive macrophages (MO) from the subject’s blood and/or biological sample.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise subjecting a disclosed blood sample to centrifugation.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise separating the blood and/or biological sample into its component parts using, for example, centrifugation, apheresis, or any technique to the skilled person.
  • a disclosed separating step can comprise generating a layer of clear fluid, a layer of red fluid, and a thin layer in between the clear fluid layer and the red fluid layer.
  • a disclosed red layer can comprise red blood cells.
  • a disclosed clear layer can comprise plasma.
  • a disclosed thin layer in between the red layer and the clear layer can comprise the buffy coat.
  • a disclosed buffy coat can comprise white blood cells and platelets.
  • a disclosed method can further comprise isolating peripheral blood mononuclear cells (PMBCs) from the buffy coat.
  • PMBCs can comprise lymphocytes, leukocytes, and/or monocytes.
  • macrophages can be derived from monocytes.
  • isolating lymphocytes, leukocytes, and/or monocytes can be done by any method and/or technique known to the skilled person (e.g., leukapheresis).
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise isolating resting or MO macrophages from the buffy coat.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise differentiating monocytes into resting or MO macrophages.
  • the disclosed macrophages can be resting or MO macrophages.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise polarizing the resting or MO macrophages into a Ml phenotype or a M2 phenotype or a pro-inflammatory phenotype or an anti-inflammatory phenotype.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise polarizing the resting or MO macrophages into a classically activated macrophage phenotype.
  • a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of disclosed engineered T cells or NK cells or macrophages can be any amount that, when used alone or in combination with another therapeutic agent, can attack and destroy cVIM-expressing tumor cells.
  • a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of the disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof can be any amount that, when used alone or in combination with another therapeutic agent, can attack and destroy cVIM-expressing tumor cells.
  • a therapeutically effective amount of the disclosed genetically modified cells expressing a disclosed chimeric antigen receptor targeting citrullinated vimentin (cVIM) can be about 1 x 10 4 to about 1 x 10 9 cells/kg per subject.
  • a therapeutically effective amount of the disclosed genetically modified cells expressing a disclosed chimeric antigen receptor targeting citrullinated vimentin (cVIM) can be about 1 x 10 5 cells/kg per subject, about 1 x 10 6 cells/kg per subject, about 1 x 10 7 cells/kg per subject, about 1 x 10 8 cells/kg per subject, or about 1 x 10 9 cells/kg per subject.
  • cVIM citrullinated vimentin
  • a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of the disclosed genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or the disclosed engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof can protect a subject against the onset of a disease and/or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • the ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
  • administering genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or administering engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof can comprise systemic or direct administration.
  • administering can comprise oral administration, intravenous administration, intra-tumoral administration, intraperitoneal administration, or any combination thereof.
  • administering genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or administering engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof can be administered by any method of administration disclosed herein.
  • genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof can be administered via multiple routes either concurrently or sequentially.
  • genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof can be first administered intra-tumorally and then be administered intravenously.
  • genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof can be first administered intra-tumorally and then be administered orally.
  • a skilled clinician can determine the best route of administration for a subject at a given time.
  • a disclosed method can comprise repeating the administering of the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can comprise protecting the subject from metastasis. In an aspect, a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can comprise reducing the risk of developing metastasis. In an aspect, a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can comprise preventing or inhibiting metastasis. In an aspect, a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can effect tumor cell death.
  • a disclosed method can comprise monitoring the subject for adverse effects.
  • a disclosed method can comprise continuing to treat the subject.
  • continuing to treat the subject can comprise continuing to administer the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • a disclosed method in the presence of adverse effects, can comprise modifying one or more steps of the method.
  • modifying one or more steps of a disclosed method can comprise modifying the administering step.
  • modifying the administering step can comprise changing the amount of the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof administered to the subject, changing the frequency of administration of the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof, changing the duration of administration of the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise administering to the subject an immune checkpoint inhibitor (e.g., an anti-PDl molecule).
  • an immune checkpoint inhibitor e.g., an anti-PDl molecule.
  • a disclosed anti-PDl molecule can comprise an anti-PDl antibody, an anti-PDLl antibody, or any combination thereof.
  • a disclosed anti-PDl antibody can comprise a monoclonal antibody, a humanized monoclonal antibody, or a fragment thereof.
  • a disclosed anti- PDl antibody can comprise a polyclonal antibody, a humanized polyclonal antibody, or a fragment thereof.
  • a disclosed anti-PDl antibody can comprise any antibody or antibody fragment that specifically recognizes PD1.
  • a disclosed anti-PDLl antibody can comprise a monoclonal antibody, a humanized monoclonal antibody, or a fragment thereof.
  • a disclosed anti-PDLl antibody can comprise a polyclonal antibody, a humanized polyclonal antibody, or a fragment thereof.
  • a disclosed anti-PDLl antibody can comprise any antibody or antibody fragment that specifically recognizes PDL1.
  • Antibodies and methods of preparing antibodies are known in the art.
  • recombinant antibodies and methods of preparing recombinant antibodies are known in the art.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise repeating the administering of the anti-PDl molecule.
  • a disclosed anti-PDl molecule can be administered prior to, concurrent with, or after the administration of the interfering molecule.
  • administering a disclosed anti-PDl molecule can comprise systemic or direct administration.
  • administering a disclosed anti-PDl molecule can comprise intravenous administration, intra-tumoral administration, intraperitoneal administration, or any combination thereof.
  • administering a disclosed can be administered by any method of administration disclosed herein.
  • a disclosed anti- PD1 molecule can be administered via multiple routes either concurrently or sequentially.
  • a disclosed anti-PDl molecule can be first administered intra-tumorally and then be administered intravenously.
  • administering a disclosed anti-PDl molecule can be first administered intra-tumorally and then be administered orally.
  • a skilled clinician can determine the best route of administration for a subject at a given time.
  • a disclosed anti-PDl molecule can be administered about 3 months, about 2 months, or about 1 month prior to the administering of the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • a disclosed anti-PDl molecule can be administered about 8 weeks, about 7 weeks, about 6 weeks, about 5 weeks, about 4 weeks, about 3 weeks, about 2 weeks, or about 1 week prior to the administering of the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • a disclosed anti-PDl molecule can be administered about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19. 18. 17. 16, 15, 14, 13, 12, 11, 10, 9, 8. 7, 6, 5, 4.
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • cVIM engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can comprise administering to the subject one or more additional anti-cancer therapies.
  • Anti-cancer therapies are known to the art.
  • a disclosed anti-cancer therapy can comprise endocrine therapy, radiotherapy, hormone therapy, gene therapy, thermal therapy, ultrasound therapy, or any combination thereof.
  • a disclosed anti-cancer therapy can comprise one or more chemotherapeutic agents.
  • a disclosed chemotherapeutic agent can comprise an anthracy cline, a vinca alkaloid, an alkylating agent, an immune cell antibody, an antimetabolite, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor, an immunomodulator, or any combination thereof.
  • a disclosed chemotherapeutic agent can be any disclosed chemotherapeutic, such as those, for example, provided supra.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can comprise administering to the subject an anti-chemokine therapy.
  • a disclosed anti-chemokine therapy can comprise one or more antibodies against CCL1, CCL2.
  • CCR9. CXCR3, CXCR4, CXCR5, CX3CL1, CX3CR1, or any combination thereof.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can comprise administering an oligonucleotide therapeutic agent.
  • a disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA. mRNA, non-coding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof.
  • a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi.
  • a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable.
  • a disclosed oligonucleotide therapeutic agent can comprise a CRISPR-based endonuclease.
  • a disclosed endonuclease can be Cas9.
  • CRISPR/Cas9 systems and methods are known to the art.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise preventing or inhibiting metastasis of cancer cells.
  • a disclosed method can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of decrease or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g.. genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof)).
  • a disclosed treatment e.g. genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof
  • a control subject such as, for example, a subject that has not received a disclosed treatment (e.g., genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof).
  • a disclosed treatment e.g., genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof).
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can comprise surgically resecting the tumor and/or cancer cells from the subject.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can comprise continuing to administer to the subject a therapeutically effective amount of the genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof and continuing to administer to the subject a therapeutically effective amount of an anti-PDl molecule, a disclosed anti-chemokine therapy, a disclosed chemotherapeutic agent, any disclosed therapeutic agent, or any combination thereof.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise subjecting the subject to one or more invasive or non-invasive diagnostic assessments. Diagnostic assessments are known to the art.
  • a disclosed non-invasive diagnostic assessment can comprise x- rays, computerized tomography (CT) scans, magnetic resonance imaging (MRI) scans, ultrasounds, positron emission tomography (PET) scans, or any combination thereof.
  • a disclosed invasive diagnostic assessment can comprise a tissue biopsy or exploratory surgery.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation in a subject, such as, for example, a subject having cancer or cancerous cells.
  • a disclosed interfering molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation in a subject having cancer.
  • metabolic dysregulation can be associated with cancer or cancerous cells.
  • restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of a cancer; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a cancer, or (viii) any combination thereof.
  • restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.
  • restoration can be a partial or incomplete restoration.
  • restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.
  • restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise preventing or inhibiting metastasis of cancer cells in the subject.
  • techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are discussed supra.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise administering genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof one or more times, administering an anti-PDl molecule one or more times, administering one or more anti-cancer therapies one or more times, or administering any combination thereof one or more time.
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • cVIM chimeric antigen receptor targeting citrullinated vimentin
  • preventing or inhibiting metastasis of cancer cells can comprise a 10%, 20%, 30%. 40%, 50%. 60%. 70%. 80%. 90%. 100%, or any amount of decrease or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof).
  • a control subject such as, for example, a subject that has not received a disclosed treatment (e.g., genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor
  • preventing or inhibiting metastasis of cancer cells can comprise a 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%. 80-90%, or 90-100% or any amount of decrease or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof).
  • a control subject such as, for example, a subject that has not received a disclosed treatment (e.g., genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof or engineered T cells or NK cells
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can comprise surgically resecting the tumor and/or cancer cells from the subject.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can comprise continuing to administer to the subject a therapeutically effective amount of a disclosed pharmaceutical formulation.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can further comprise diagnosing the subject as have cancer or cancerous cells.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can improve and/or extend the survivability of the subject, can improve a subject’s quality of life, can increase and/or prolong a subject's life span, or any combination thereof.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can stimulate an effector cell mediated immune modulator response to cVIM-expressing tumor cells.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM- expressing tumor cells can induce a tumor reducing immune response.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can induce phagocytosis of cancer cells in the subject.
  • a disclosed method stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can cross-prime an anti-tumor T cell response.
  • a disclosed method of stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells induce a tumor eliminating immune response.
  • a disclosed pharmaceutical formulation can treat cancer.
  • a disclosed method stimulating an effector cell mediated immune modulator response to cVIM-expressing tumor cells can comprise administering to the subject a chimeric fusion receptor comprising a cVIM-binding domain.
  • kits comprising one or more of a disclosed CAR, a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed host cell, or any combination thereof.
  • a kit comprising one or more disclosed CARs, one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, one or more disclosed cells, or any combination thereof.
  • a CAR can target cVIM expressed on one or more cancer cells.
  • a disclosed kit can comprise one or more additional active agents and/or therapeutic agents.
  • the one or more agents can treat, prevent, inhibit, and/or ameliorate one or more comorbidities in a subject.
  • one or more active agents can treat, inhibit, prevent, and/or ameliorate cellular and/or metabolic complications related to cancer or cancer cells or cancerous cells.
  • a disclosed kit can comprise at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose (such as, for example, treating a subject diagnosed with or suspected of having a disease or disorder such as cancer). Individual member components may be physically packaged together or separately.
  • a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on a computer readable memory device or downloaded from an internet website, or as recorded presentation.
  • a kit for use in a disclosed method can comprise one or more containers holding a disclosed CAR, a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed host cell, or any combination thereof, and a label or package insert with instructions for use.
  • suitable containers include, for example, bottles, vials, syringes, blister pack, etc.
  • the containers can be formed from a variety of materials such as glass or plastic.
  • the container can hold a disclosed CAR, a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed host cell, or any combination thereof, and can have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the label or package insert can indicate a disclosed CAR, a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed host cell, or any combination thereof can be used for treating, preventing, inhibiting, and/or ameliorating a disease or disorder or complications and/or symptoms associated with a disease or disorder such as cancer or metastatic cancer.
  • a kit can comprise additional components necessary for administration such as, for example, other buffers, diluents, filters, needles, and syringes.
  • a disclosed kit can be used to treat cancer.
  • a disclosed kit can be used to stimulate an effector cell mediated immune modulator response to cVIM-expressing tumor cells.
  • a disclosed kit can be used to preventing or inhibiting metastasis of cancer cells.
  • a disclosed kit can be used to risk of developing metastases.
  • a disclosed kit can be used to validate the efficacy and/or toxicity of a disclosed CAR, a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed host cell, or any combination thereof.
  • EMT epithelial-to-mesenchymal transition
  • Vimentin a major constituent of the intermediate filament family of proteins, is ubiquitously expressed in normal mesenchymal cells and is known to maintain cellular integrity and provide resistance against stress. Vimentin is overexpressed in various epithelial cancers, including prostate cancer, gastrointestinal tumors, tumors of the central nervous system, breast cancer, malignant melanoma, and lung cancer. Vimentin was also shown to be a target for other types of post-translational modifications, including citrullination, sumoylation, and O-GlcNAc modification.
  • citrullination is a post-translational modification in which the arginine residues are enzymatically deiminated to citrulline by peptidylargmme deiminase.
  • TIL-Bs are known to spatially interact with T cells in germinal centers (GCs) within TLS, a characteristic that is critical for affinity maturation of antigen-specific B cell receptors and diversification into antibody-secreting plasma cells (Laumont CM, et al. Cancer Cell, 2023. 41(3):466-489; Ma J, et al. Science, 2024. 384(6695): eadj4857; Ruffin AT, et al. Nat Commun, 2021. 12(1):3349; Sato Y, et al. Nat Rev Nephrol, 2023. 19(8):525-537).
  • GCs germinal centers
  • Paired VH and VK/V BCR sequencing was performed using the 10X Genomics single cell immune profiling assay on CD138 and CD19-enriched TIL-Bs.
  • BCR annotation and cl on o type identification was performed using the CellRanger software suite (10X Genomics) and Cloanalyst package using human immunoglobulin gene reference libraries.
  • BCRs with identical or similar paired HCDR3 and LCDR3 were deemed to be clonally related (“clonotypes”). Each of these samples demonstrated a subset of expanded clonotypes as defined by TIL-B clones containing identical or similar paired HCDR3 and LCDR3 (FIG. 2A).
  • VH, VK, and Vx genes isolated from CD138-enriched plasma cells were 5.6%, 3.1%, and 3.6%, (Patient 1), and 4.6%, 2.8%, and 3.4% (Patient 3) (FIG. 2B).
  • Tumor infiltrating plasma cells were primarily class switched; IgGl was the most frequent isotype (60.5% and 43.3%) followed by IgAl (23.8% and 13.5%) for Patient 1 and Patient 3, respectively (FIG. 2C).
  • CD19-enriched TIL-Bs in Patient 2 were less frequently mutated (0.3%, 0.7%, and 0.9% for VH, VK, and Vx genes), (FIG. 2B, FIG. 2C) and less frequently class-switched (12.4% IgGl and 6.5% IgAl) than those enriched for CD138 expression.
  • FIG. 3 shows a graphical summary' of the high-throughput pipeline for isolation of tumor- reactive antibodies.
  • clonotypes from highest frequency (most expanded) to lowest frequency (least or nonexpanded) were ranked and then the most expanded clonotypes not found in control patient matched PBMC BCR sequencing were selected.
  • the consensus BCRs of the most expanded TIL- B clonotypes were used to generate recombinant monoclonal antibodies (mAbs) which were then tested for tumor reactivity.
  • mAbs monoclonal antibodies
  • Ab4 was labeled as the lead tumor-reactive antibody candidate.
  • amurinized version of Ab4 was tested using immunohistochemistry in aNSCLC tissue microarray that was comprised of adenocarcinoma and squamous cell carcinoma core biopsies from different patients.
  • Ab4 bound 63% of tumors tested (60 out of 95) (Table 1), indicating that the target of Ab4 was a shared tumor antigen in a diverse patient population.
  • citrullinated vimentin was determined to be a target of Ab4. Proteins eluted from a column containing beads to which Ab4 was bound were resolved using a Western blot (FIG. 5A). The 55 kDa band was cut out of the gel and subjected to mass spectroscopy, which identified vimentin (MW 54 kDa). Binding of Ab4 to the citrullinated - but not unmodified vimentin - was confirmed using commercially available proteins in ELISA (FIG. 5B). As discussed above, citrullinated vimentin is a tumor-specific cell surface protein.
  • Anti-cVIM antibody concentrations were measured in the plasma of all patients treated on the pembrolizumab neoadjuvant trial using an ELISA (FIG. 6). The patients who had a >90% pathologic response had significantly higher concentrations anti-cVIM antibodies. This indicates that these antibodies have a positive functional consequence.
  • compositions are novel and are nonobvious over the state of the art for several reasons.
  • CAR T cells are re-directed using the scFv of a novel antibody. This scFv was connected to the hinge, transmembrane, co-stimulatory and CD3zeta domains and to assemble a disclosed CAR, which killed tumor cells.
  • the work described herein represents the first time CAR T cells have generated to target citrullinated vimentin of the surface of glioblastomas and other cancer cells.
  • the data provided herein demonstrate cytotoxic activity against tumor cells derived from ovarian, colon, and lung cancers (see, e.g., FIG. 11).
  • the novel CAR T cells disclosed herein target not only tumor but also tumor-promoting myeloid cells, which reduces immunosuppression at tumor beds, thereby unleashing the effector activity of endogenous polyclonal tumor-reactive T cells at tumor beds.

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Abstract

L'invention concerne des récepteurs antigéniques chimériques ciblant cVIM sur la surface de cellules cancéreuses. L'invention concerne également des compositions comprenant des CAR ciblant cVIM et des procédés d'utilisation de ces compositions pour traiter un cancer et/ou ralentir la progression d'une maladie chez un sujet.
PCT/US2024/053144 2023-10-27 2024-10-26 Compositions comprenant des récepteurs antigéniques chimériques ciblant une vimentine citrullinée et leurs procédés d'utilisation Pending WO2025090985A1 (fr)

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