Compositions comprising chimeric antigen receptors targeting citrullinated vimentin and methods of use thereof

Abstract

Disclosed herein are chimeric antigen receptors targeting cVIM on the surface of cancer cells. Also disclosed are compositions comprising CARs targeting cVIM and methods of using these compositions to treat cancer and / or slowing disease progression in a subject.

Description

COMPOSITIONS COMPRISING CHIMERIC ANTIGEN RECEPTORS TARGETING CITRULLINATED VIMENTIN AND METHODS OF USE THEREOFI. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 546,043 filed 27 October 2023 and to U.S. Provisional Application No. 63 / 703,742 filed 4 October 2024, each of which is incorporated herein in its entirety.II. REFERENCE TO THE SEQUENCE LISTING

[0002] The Sequence Listing submitted 26 October 2024 as an XML file named “24-3015- WO_Sequence-Listing’‘, created on 26 October 2024 and having a size of 171,912 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).III. BACKGROUND

[0003] For decades, the foundations of cancer treatment have been surgery7, chemotherapy, and radiation therapy. These continue to be critical mainstays of treatment, but new categories of treatment have recently helped transform the treatment picture for people with cancer. For example, it is highly desirable to exploit cancer-specific markers for therapeutic development.

[0004] Thus, there remains a need for treatment that is focused on a cancer cell marker. The compositions and methods disclosed herein meet this need.IV. BRIEF DESCRIPTION OF THE FIGURES

[0005] 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. for reference. (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.

[0006] FIG. 2A-FIG. 2C show that TIL-Bs demonstrated features of active immunity. FIG. 2A shows the distribution of TIL-B clonal expansion for recovered clonotypes as identified in CellRanger. Each colored slice represents a single clonal family. White slices represent all unique(n = 1) clonotypes. Total number of clonotypes is highlighted in the center of the pie. 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.

[0007] FIG. 3 shows the multistep pipeline for high-throughput isolation and characterization of tumor-reactive antibodies from surgically resected tumors.

[0008] 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. 4C shows binding of the same representative tumor mAbs to fixed Calu-6 cells by FACS.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] FIG. 9A shows that most tumor and myelo-monocytic cells in mouse tumors also expressed citrullinated vimentin on the cell surface. C57BL / 6 mice (n = 9) were challenged IP with ID8-Defb29 / Vegf-a ovarian cancer cells. Peritoneal wash (ascites) and spleens were analyzed by flow cytometry 3 weeks later. FIG. 9B shows that proteonomic analysis confirmed that vimentin was the target of the recombinant Ab.

[0014] 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.

[0015] 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.V. BRIEF SUMMARY

[0016] Disclosed herein is 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. Disclosed herein is 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.

[0017] Disclosed herein is a chimeric antigen receptor (CAR), 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. Disclosed herein is a chimeric antigen receptor (CAR), 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.

[0018] Disclosed herein is a chimeric antigen receptor (CAR), 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. Disclosed herein is a chimeric antigen receptor (CAR), 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. Disclosed herein is a chimeric antigen receptor (CAR), 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.

[0019] Disclosed herein is a chimeric antigen receptor (CAR), 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.

[0020] Disclosed herein is a 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.

[0021] Disclosed herein is a 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.

[0022] Disclosed herein is a vector comprising a disclosed nucleic acid molecule. Disclosed herein is a vector comprising a nucleic acid molecule encoding a disclosed CAR. Disclosed herein is a vector comprising a disclosed nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor. Disclosed herein is a vector comprising a disclosed nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor targeting citrullinated vimentin (cVIM). Disclosed herein is 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. Disclosed herein is 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.

[0023] Disclosed herein is a pharmaceutical formulation comprising a nucleic acid molecule comprising a nucleic acid sequence encoding any disclosed CAR. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR specific for citrullinated vimentin (cVIM). Disclosed herein is a pharmaceutical formulation a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR comprising an extracellular domaincomprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising one or more immunostimulatory domains.

[0024] Disclosed herein is a method of treating cancer, 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 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 treating cancer, 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. Disclosed herein is a method of treating cancer, the method 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. Disclosed herein is a method of treating cancer, 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 treating cancer, 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. Disclosed herein is a method of treating cancer, 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. Disclosed herein is a method of treating cancer, 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)-expressing tumor cells or a pharmaceutical formulation thereof.

[0025] 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 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 ofone 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 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. 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. 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. 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 expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof. 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 expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM)-expressing tumor cells or a pharmaceutical formulation thereof.VI. DETAILED DESCRIPTION

[0026] The present disclosure describes formulations, compounded compositions, 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.

[0027] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.A. Definitions

[0028] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they 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.

[0029] This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure.

[0030] As used in the specification and the appended claims, the singular forms “a”, "an", and "the” include plural referents unless the context clearly dictates otherwise.

[0031] The phrase “consisting essentially of’ limits the scope of a claim to the recited components in a composition or the recited steps in a method as well as those that do not materially affect the basic and novel characteristic or characteristics of the claimed composition or claimed method. The phrase “consisting of’ excludes any component, step, or element that is not recited in the claim. The phrase “comprising” is synonymous with “including”, “containing”, or “characterized by”, and is inclusive or open-ended. “Comprising” does not exclude additional, unrecited components or steps.

[0032] In an aspect, when referring to any numerical value, the term “about” means a value falling within a range that is ± 10% of the stated value.

[0033] 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 betweentwo particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0034] 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. Thus, in a compound containing 2 parts by weight component X and 5 parts by weight component Y, 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.

[0035] In an aspect, the terms ‘‘optional’' or “optionally"’ means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. In an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.

[0036] In an aspect, “operably linked” refers to a juxtaposition where the components described are in a relationship permitting them to function in their intended manner. For example, 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. In an aspect, a promotor is operably linked to nucleic acids.

[0037] In an aspect, 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.). Thus, the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, areptile, or an amphibian. Alternatively, 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. In an aspect, a subject can be a human patient. In an aspect, a subject can have cancer, be suspected of having cancer, or be at risk of developing cancer.

[0038] In an aspect, 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)), disclosedpharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. For example, '‘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. For example, “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. In an aspect, 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.

[0039] 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).

[0040] A “patient” refers to a subject afflicted with a disease or disorder (e.g., cancer). In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder such as cancer. In an aspect, 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).

[0041] In an aspect, 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. For example, 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 totreatment for the cancer. In an aspect, the identification can be performed by a person different from the person making the diagnosis. In an aspect, the administration can be performed by one who performed the diagnosis.

[0042] In an aspect, “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. Thus, 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.

[0043] In an aspect, “inhibit,” “inhibiting”, and “inhibition” mean 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. This can also include, for example, a 10% inhibition or reduction in the activity7, level, response, condition, severity7, 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 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). Thus, in an aspect, 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. In an aspect, 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.. 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). In an aspect, the inhibition or reduction can be 0-25%, 25-50%. 50-75%. or 75-100% as compared to native or control levels. In an aspect, a native or control level can be a pre-disease or pre-disorder level (such as a precancer state).

[0044] The words "‘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. In an aspect, 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. For example, in an aspect, 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). In an aspect, 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). For example, 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). In an aspect, 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).

[0045] In an aspect, 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. The words “prevent”, “preventing”, and “prevention” 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.

[0046] In an aspect, the terms ’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 are well known to those skilled in the art and 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). 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.

[0047] In an aspect, 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). In an aspect, 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.

[0048] By “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.

[0049] In an aspect, 'modifying the method” can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. In an aspect, 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 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 is administered to a subject, or by substituting for one or more of the disclosed components and / or reagents with a similar or equivalent component and / or reagent. The same applies to all 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 combinations thereof.

[0050] In an aspect, 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. Examples of 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. In an aspect, a pharmaceutical carrier employed can be a solid, liquid, or gas. In an aspect, examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. In an aspect, examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water. In an aspect, examples of gaseous carriers can include carbon dioxide and nitrogen. In preparing a disclosed composition for oral dosageform, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, 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.

[0051] In an aspect, 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.). See, also, for reference, Remington’s Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, Pa., which is hereby incorporated by reference in its entirety.

[0052] In an aspect, “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.

[0053] In an aspect, “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. In an aspect, a target area or intended target area can be any cell or any organ infected by a disease or disorder (such as cancer). In an aspect, 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).

[0054] In an aspect, “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. For example, 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).

[0055] In an aspect, “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. In an aspect, 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). For example, 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. In an aspect, “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 specific therapeutically effective dose level for any particularpatient 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 (cVIM)), disclosed pharmaceutical formulations, or a combination thereof employed; the duration of the treatment; drugs used in combination or coincidental with 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, and other like factors well known in the medical arts. 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. Consequently, 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. In further various aspects,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.

[0056] In an aspect, polynucleotide or nucleic acid can be used interchangeably, and refer to polymers of nucleotides of any length and includes DNA and RNA. In an aspect, 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. In an aspect, a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. Unless specified otherwise, 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”.

[0057] The term “antibody” (Ab) 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). 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. Generally, 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 iscommonly 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. The term "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. In an aspect, 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. 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. In an aspect, 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.

[0058] The term “variable region” or “variable domain” is used interchangeably. The 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. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). In an aspect, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In an aspect, the variable region is a primate (e.g..non-human primate) variable region. In an aspect, the variable region comprises rodent or murine CDRs and primate framework regions (FRs).

[0059] The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody or an antigen-binding molecule thereof. The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody or an antigen-binding molecule thereof.

[0060] 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.

[0061] 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.

[0062] The term “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.

[0063] “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. Such 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.

[0064] In an aspect. “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.

[0065] In an aspect, “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 antigenbinding 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.

[0066] The term “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. Examples of 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. However, one of skill in the art would recognize that the conditioning methods disclosed herein would enhance the effectiveness of any transplanted T cell therapy.

[0067] Adoptive T cell therapies, including chimeric antigen receptor (CAR) T cells, have revolutionized cancer therapy. However, 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. In both treatment-resistant hematological and solid 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. Despite significant recent advances, rational design has failed to overcome the problems associated with such factors.

[0068] The T cells or NK cells of the immunotherapy can come from any source known in the art. For example, 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. In addition, 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.

[0069] In an aspect, the term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of 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.

[0070] In an aspect, “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. The term “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.

[0071] In an aspect, the term “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. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary7determining 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. In some instances, 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 specificity7, affinity7, and capability7. 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, affinity7, and / or capability. In general, 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.

[0072] 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. 1 mM or less, at times about 1 pM or less, at times about 0. 1 pM or less, at times about 0.01 pM or less, and at times about 1 nM or less. It is understood that, in an aspect, an antibody or binding moiety7that specifically binds to a first targetmay or may not specifically bind to a second target. As such, "‘specific binding” does not necessarily require (although it can include) exclusive binding, e.g., binding to a single target. Thus, an antibody may, in an aspect, specifically bind to more than one target (e.g., human citrullinated vimentin (cVIM)). In an aspect, the multiple targets may be bound by the same antigen-binding site on the antibody.

[0073] 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).

[0074] In an aspect, a “target” expressing cVIM can bind to a disclosed binding motif, a disclosed CAR, a disclosed TCR, or a disclosed antigen binding agent.

[0075] “Antigen-specific targeting region” (ASTR) 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)). In an aspect, 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.

[0076] The term “autologous” refers to any material derived from the same individual to which it is later to be re-introduced. For example, a subject’ s own cells can be obtained, made to express one or more disclosed CARs, and then administered to the same subject.

[0077] In an aspect, the term “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 fromimmunized 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).

[0078] In an aspect, the terms ‘‘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. More particular examples of such 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.

[0079] The terms “proliferative disorder” and “proliferative disease” refer to disorders associated with abnormal cell proliferation such as cancer.

[0080] “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. “Metastasis” 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.

[0081] The terms “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. These properties of “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 undergoself-renewal versus differentiation in a chaotic manner to form tumors with abnormal cell types that can change over time as mutations occur.

[0082] The terms “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).

[0083] In an aspect “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.

[0084] In an aspect, 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.

[0085] In an aspect, “lipid nanoparticles” or “LNPs” 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. For example, 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-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9, 12-di enoate) or another ionizable lipid. In an aspect, the term cationic and ionizable in the context of LNP lipids can be use interchangeably, e.g., wherein ionizable lipids are cationic depending on the pH.

[0086] “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%. 99% or more. Such sequences are also referred to as “variants” herein, e.g., other variants of a missing, deficient, and / or mutant protein or enzyme. It should be understood that sequence with substantial sequence identity do not necessarily have the same length and may differ in length. For example, sequences that have the same nucleotide sequence but of which one has additional nucleotides on the 3’- and / or 5’-side are 100% identical.

[0087] In an aspect, “immune-modulating” 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.

[0088] In an aspect, “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). Examples of 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 mofetil, naproxen, prednisolone, prednisone, prednisolone indomethacin, rapamycin, rituximab, sirolimus, sulindac, synthetic vaccine particles containing rapamycin (SVP-Rapamycin or ImmTOR), thalidomide, tocilizumab. tolmetin, triamcinolone acetonide, anti-CD3 antibodies, anti-CD4 antibodies. anti-CD19 antibodies, anti- CD20 antibodies, anti-CD22 antibodies, anti-CD40 antibodies, anti-FcRN antibodies, anti-IL6 antibodies, anti-IGFIR antibodies, an IL2 mutein, a BTK inhibitor, or a combination thereof. In an aspect, a disclosed immune modulator can comprise one or more Treg (regulatory T cells) infusions (e.g., antigen specific Treg cells to AAV). In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, 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. In an aspect, 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.

[0089] In an aspect, 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.

[0090] In an aspect, the term “in combination” in the context of the administration of other therapies (e.g., other agents) 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. By way of non-limiting example, 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. 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks), concurrently, or after (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, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer) the administration of a second therapy to a subject having or diagnosed with cancer.

[0091] Disclosed are the components to be used to prepare the disclosed chimeric antigen receptors, disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations as well as the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations used within the methods disclosed herein. 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. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A. B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E. B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B- F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific aspects or combination of aspects of the methods of the invention.B. Compositions for Use in the Disclosed Methods1. Chimeric Antigen Receptors

[0092] 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.

[0093] In an aspect, a disclosed CAR can target citrullinated vimentin (cVIM). In an aspect, a disclosed CAR can target citrullinated vimentin (cVIM) expressed on the cell surface of a cancer cell. In an aspect, a disclosed CAR can target citrullinated vimentin (cVIM) expressed on the cell surface of an ovarian cancer cell. In an aspect, a CAR targeting cVIM can be used in conjunction with a CAR targeting any other cancer antigen.

[0094] Disclosed herein is a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 129 or a fragment thereof. Disclosed herein is 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.

[0095] Disclosed herein is a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 130 or a fragment thereof. Disclosed herein is 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% identity7to the sequence set forth in SEQ ID NO: 130 or a fragment thereof.

[0096] Disclosed herein is a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:01 or a fragment thereof. Disclosed herein is 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.

[0097] Disclosed herein is a chimeric antigen receptor encoded by the sequence set forth in SEQ ID NO:02 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 sequence set forth in SEQ ID NO: 02 or a fragment thereof.

[0098] Disclosed herein is a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:03 or a fragment thereof. Disclosed herein is 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% identity7to the sequence set forth in SEQ ID NO: 03 or a fragment thereof.

[0099] Disclosed herein is 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% identity7to a sequence set forth in SEQ ID NO: 04 or a fragment thereof.

[0100] Disclosed herein is a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 131 or a fragment thereof. Disclosed herein is 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.

[0101] Disclosed herein is a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 132 or a fragment thereof. Disclosed herein is 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.

[0102] Disclosed herein is a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:05 or a fragment thereof. Disclosed herein is 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.

[0103] Disclosed herein is a chimeric antigen receptor encoded by' the sequence set forth in SEQ ID NO:06 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 sequence set forth in SEQ ID NO:06 or a fragment thereof.

[0104] Disclosed herein is a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:07 or a fragment thereof. Disclosed herein is 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.

[0105] Disclosed herein is 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 asequence 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.

[0106] Disclosed herein is a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 133 or a fragment thereof. Disclosed herein is 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.

[0107] Disclosed herein is a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 134 or a fragment thereof. Disclosed herein is 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.

[0108] Disclosed herein is a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO:09 or a fragment thereof. Disclosed herein is 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.

[0109] Disclosed herein is 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.

[0110] Disclosed herein is a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 11 or a fragment thereof. Disclosed herein is 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.

[0111] Disclosed herein is 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% identity7to the sequence set forth in SEQ ID NO: 12 or a fragment thereof.

[0112] Disclosed herein is 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 Disclosed herein is 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 thesequence 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. Disclosed herein is 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 NKG2D transmembrane domain comprising the sequence of SEQ ID NO: 31, a NKG2D signaling domain comprising the sequence of SEQ ID NO:39.

[0113] Disclosed herein is a chimeric antigen receptor comprising the scFV of Ab#4 as described in the Examples.

[0114] In an aspect, a disclosed extracellular domain can comprise a citrullinated vimentin binding domain. In an aspect, 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). 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).

[0115] In an aspect, a disclosed scFv can comprise the sequence set forth in any one of SEQ ID NO: 149-SEQ ID NO: 154. In an aspect, 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.

[0116] In an aspect, 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. In an aspect, 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. In an aspect, a disclosed extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 113 or a fragment thereof. In an aspect, 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 SEQID NO: 113 or a fragment thereof. In an aspect, a disclosed extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 114 or a fragment thereof. In an aspect, 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. In an aspect, a disclosed extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 115 or a fragment thereof. In an aspect, 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. In an aspect, a disclosed extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 116 or a fragment thereof. In an aspect, 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. In an aspect, a disclosed extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 117 or a fragment thereof. In an aspect, 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. In an aspect, a disclosed extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 118 or a fragment thereof. In an aspect, 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.

[0117] In an aspect, 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. In an aspect, 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.

[0118] In an aspect, a disclosed vimentin binding domain can comprise a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs). In an aspect, a disclosed vimentin binding domain can comprise a light chain variable region (VL) comprising three complementarity determining regions (CDRs).

[0119] In an aspect, 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. In an aspect, 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.

[0120] In an aspect, a disclosed CDR in the VH can comprise the sequence set forth in any one ofSEQ ID NO: 18. SEQ ID NO: 19, and SEQ ID NO:20. In an aspect, 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.

[0121] In an aspect, a disclosed CDR comprising 3 amino acids can comprise 1 or more substitutions. In an aspect, a disclosed CDR comprising 8 amino acids can comprise 2 or more substitutions. In an aspect, a disclosed CDR comprising 9 amino acids can comprise 2 or more substitutions. In an aspect, a disclosed CDR comprising 11 amino acids can comprise 3 or more substitutions. In an aspect, a disclosed CDR comprising 17 amino acids can comprise 4 or more substitutions. In an aspect, a disclosed CDR comprising the sequence set forth in SEQ ID NO:22 can further comprise 1 or more substitutions.

[0122] In an aspect, a disclosed CDR comprising the sequence set forth in SEQ ID NO: 18 can further comprise 2 or more substitutions. In an aspect, 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. In an aspect, a disclosed CDR comprising the sequence set forth in SEQ ID NO:21 can further comprise 3 or more substitutions. In an aspect, a disclosed CDR comprising the sequence set forth in SEQ IDNO:20 can further comprise 4 or more substitutions.

[0123] In an aspect, 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. In an aspect, 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. In an aspect, 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.

[0124] In an aspect, a disclosed VL can be located at the N-terminus of the VH. In an aspect, a disclosed extracellular domain comprising a vimentin binding domain can comprise SEQ ID NO: 13 and SEQ ID NO: 15. In an aspect, a disclosed VH can be located at the N-terminus of the VL. In an aspect, a disclosed extracellular domain comprising a vimentin binding domain can comprise SEQ ID NO: 15 and SEQ ID NO: 13.

[0125] In an aspect, a disclosed VL can comprise the sequence of SEQ ID NO: 13. In an aspect, 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. In an aspect, a disclosed VL can be encoded by the sequence set forth in SEQ ID NO: 14. In an aspect, 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. In an aspect, a disclosed VH can comprise the sequence of SEQ ID NO: 15. In an aspect, 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. In an aspect, a disclosed VH can be encoded by the sequence set forth in SEQ ID NO: 16. In an aspect, 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% identity7to the sequence set forth in SEQ ID NO: 16 or a fragment thereof. In an aspect, a disclosed VH can be encoded by the sequence set forth in SEQ ID NO: 17. In an aspect, 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.

[0126] In an aspect, a disclosed antigen binding domain can be a scFv and wherein the scFV can comprise a linker and / or a spacer. In an aspect, a disclosed linker and / or linker can join the VH and VL regions of the scFv. In an aspect, 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. In an aspect, a disclosed scFv can provide one or more advantages conveyed, in part, by its size.

[0127] In an aspect, a disclosed extracellular domain can further comprise a signal / leader peptide. In an aspect, a disclosed extracellular domain comprising a vimentin binding domain can further comprise a signal / leader peptide.

[0128] Signal / leader peptides are known to the art. In an aspect, a disclosed signal / leader peptide can comprise any known signal / leader peptide or any known signal / leader peptide known to function in mammalian cells. For example, in an aspect, 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). 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 N0:61), Silkworm Fibroin LC (SEQ ID NO:62), Mouse Ig Kappa (SEQ ID NO:63), Mouse Ig Heavy (SEQ ID NO:64), or CD8a (SEQ ID NO:66). In an aspect, 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.

[0129] In an aspect, a disclosed signal / leader peptide can be cleaved upon the nascent protein crossing the ER membrane. In an aspect, a disclosed signal / leader peptide can be cleaved prior to protein assembly and folding by, for example, a signal peptide peptidase. In an aspect, 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.

[0130] In an aspect, a disclosed peptide bond and / or peptide spacer can comprise the sequence set forth in SEQ ID NO:44 or SEQ ID NO:45. In an aspect, a disclosed peptide bond and / or peptide spacer can comprise the sequence set forth in any one of SEQ ID NO:73 - SEQ ID NO: 112. In an aspect, a disclosed VL and a disclosed VH can be connected by a linker. In an aspect, a disclosed linker can comprise a flexible linker. In an aspect, a flexible linker can comprise (GGGGS)n, wherein n = 1 - 8 (SEQ ID NO:73 - SEQ ID NO:80). In an aspect, a flexible linker can comprise (G)n, wherein n = 1 - 8 (SEQ ID NO:81 - SEQ ID NO:88). In an aspect, a disclosed linker can comprise a rigid linker. In an aspect, a flexible linker can comprise (EAAAK)n, wherein n = 1 - 8 (SEQ ID NO:89 - SEQ ID NO:96). In an aspect, a disclosed linker can comprise a Whitlow linker. In an aspect, a Whitlow linker can comprise (GSTSGSGKSSEGKG)n, wherein n = 1 - 8 (SEQ ID NO:97 - SEQ ID NO: 104). In an aspect, a Whitlow linker can comprise (GSTSGSGKPGSGEGSTKG)n, wherein n = 1 - 8 (SEQ ID NO: 105 - SEQ ID NO: 112). Linkers are known to the skilled person in the art.

[0131] In an aspect, a disclosed chimeric antigen receptor can further comprise a spacer domain between the extracellular domain and the transmembrane domain. In an aspect, a disclosed chimeric antigen receptor can further comprise a hinge region betw een the extracellular domain and the transmembrane domain. In an aspect, 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. In an aspect, a disclosed vimentin binding domain can be following by one or more hinge domains. In an aspect, 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.

[0132] In an aspect, a spacer can connect a disclosed scFv to a disclosed transmembrane domain lend flexibility to the scFv and help improve efficacy. In an aspect, a disclosed appropriate spacerdomain engineering can enable recognition of target epitopes that are otherwise sterically inaccessible. In an aspect a disclosed spacer domain modulation can also be used to regulate synaptic cleft distances and hence signaling phenomena such as kinetic segregation. In an aspect, to maintain optimal synapse distance, membrane-distal epitopes usually require shorter spacers whereas membrane-proximal epitopes require longer spacers. In an aspect, apart from limiting exclusion of inhibitory phosphatases, increasing epitope-paratope distance can also result in impaired delivery of granzymes and perforins to the target cell, thus reducing lytic efficiency. In an aspect, a disclosed physiological T-cell setting, the highly dense immune synapse can hinder diffusion of lytic granules, which can enhance pore formation by perforins and granzyme delivery

[0133] The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In an aspect, a disclosed hinge region can be referred to as a stalk domain. In an aspect, the size of a hinge region can be altered or varied. In an aspect, a disclosed hinge region can comprise a hinge region of CD4, CD8a, CD28, IgGl, IgG2, IgG3, IgG4, IgA, IgD. or any combination thereof. In an aspect, a disclosed CD8 hinge domain can comprise the sequence set forth in SEQ ID NO:24, SEQ ID NO:25, or a fragment thereof. In an aspect, a disclosed CD8 hinge domain can be encoded by the sequence set forth in SEQ ID NO:26 or a fragment thereof.

[0134] In an aspect, 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 (ITGAE), CD134 (0X40), 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), SLP-76 (LCP2), PAG1 / CBP, a CD83 ligand,Fey receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors. Toll ligand receptor, or any combination thereof, or any fragment / truncation thereof, or any combination of fragments / truncations thereof.

[0135] In an aspect, 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. In an aspect, 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. CD3e, CD35, CD3yy, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD80, CD9, CDl la, CDl lb, CDl lc, CDl ld, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4- 1BB / CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18. CD19, CD19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DAP-12, DNAM1 (CD226), Fey receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Iga (CD79a), IL-2R0, IL-2Ry, IL-7Ra, inducible T cell costimulator (ICOS), integrins, ITGA4. ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX. ITGB2, ITGB7. ITGB1, KIRDS2. 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. programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (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.

[0136] In an aspect, a disclosed CD8 transmembrane domain can comprise the sequence set forth in SEQ ID NO:27 or a fragment thereof. In an aspect, a disclosed CD8 transmembrane domain can comprise the sequence set forth in SEQ ID NO:28 or a fragment thereof. In an aspect, a disclosed CD8 transmembrane domain can be encoded by the sequence set forth in SEQ ID NO:29 or a fragment thereof. In an aspect, a disclosed CD28 transmembrane domain can comprise the sequence set forth in SEQ ID NO:30 or a fragment thereof. In an aspect, a disclosed NKG2D transmembrane domain can comprise the sequence set forth in SEQ ID NO:31 or a fragmentthereof. In an aspect, a disclosed NKG2D transmembrane domain can be encoded by the sequence set forth in SEQ ID NO: 32 or a fragment thereof.

[0137] In an aspect, a disclosed intracellular domain can be referred to synonymously as an intracellular signaling domain. In an aspect, 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). In an aspect. 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.

[0138] In an aspect, a disclosed intracellular domain can comprise one or more immunoreceptor tyrosine-based activation domains (ITAM). In an aspect, 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.

[0139] In an aspect, a disclosed intracellular region can comprise a CD3^ signaling domain. In an aspect, a disclosed CD3 signaling domain can comprise the sequence set forth in SEQ ID NO:41. In an aspect, a disclosed CD3^ signaling domain can comprise the sequence set forth in SEQ ID NO:42. In an aspect, a disclosed CD3^ signaling domain can be encoded by the sequence set forth in SEQ ID NO:43. In an aspect, 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. In an aspect, a disclosed ITAM can be on the C-terminal end (distal to the transmembrane domain). In an aspect, 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.

[0140] In an aspect, a disclosed intracellular domain can comprise one or more co-stimulatory domains or co-stimulatory signaling domains. In an aspect, the order of one or more co- stimulatory domains in a disclosed intracellular domain can be varied. In an aspect, the order of the one or more co-stimulatory domains can be varied and / or can influence the resulting effector function. For example, CD28-CD3^ can elicit higher level of IL-2. In an aspect, a disclosed co- stimulatory domain can be on the N-terminal end (proximal to the transmembrane domain). In an aspect, a disclosed intracellular domain can further comprise one or more co-stimulatory signalingdomain or co-stimulatory domains. In an aspect, 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. In an aspect, a disclosed 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. 1!

[0141] In an aspect, 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. 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, CD9, CD96 (Tactile). 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.

[0142] In an aspect of a disclosed chimeric antigen receptor, 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.

[0143] In an aspect, a disclosed intracellular domain can comprise one or more co-stimulatory domains. In an aspect, a disclosed co-stimulatory domain can comprise 4-1BB. In an aspect, a disclosed 4-1BB co-stimulatory domain can comprise the sequence set forth in SEQ ID NO:33 or a fragment thereof. In an aspect, a disclosed 4-1 BB co-stimulatory domain can comprise the sequence set forth in SEQ ID NO: 34 or a fragment thereof. In an aspect, a disclosed 4- IBB co-stimulatory’ domain can be encoded by the sequence set forth in SEQ ID NO: 35 or a fragment thereof.

[0144] In an aspect, a disclosed co-stimulatory domain can comprise CD28. In an aspect, a disclosed CD28 co-stimulatory domain can comprise the sequence set forth in SEQ ID NO:36 or a fragment thereof. In an aspect, a disclosed CD28 co-stimulatory domain can comprise the sequence set forth in SEQ ID NO:37 or a fragment thereof. In an aspect, a disclosed CD28 co- stimulatory domain can be encoded by the sequence set forth in SEQ ID NO:38 or a fragment thereof. In an aspect, a disclosed co-stimulatory domain can comprise NKG2D. In an aspect, a disclosed NKG2D co-stimulatory domain can comprise the sequence set forth in SEQ ID NO: 39 or a fragment thereof. In an aspect, a disclosed NKG2D co-stimulatory domain can be encoded by the sequence set forth in SEQ ID NO:40 or a fragment thereof.

[0145] In an aspect, a disclosed co-stimulatory domain can comprise OX-40. In an aspect, a disclosed OX-40 co-stimulatory domain can comprise the sequence set forth in SEQ ID NO: 147 or a fragment thereof. In an aspect, a disclosed OX-40 co-stimulatory domain can be encoded by the sequence set forth in SEQ ID NO: 148 or a fragment thereof. In an aspect, a disclosed co- stimulatory signaling domain can enhance the efficacy and expansion of immune cells expressing CAR receptors.

[0146] In an aspect, 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. In an aspect, a disclosed intracellular domain can comprise a gene cassette for one or more cytokines. In an aspect, a disclosed gene cassette for one or more cytokines can be operably linked to one or more regulatory elements. In an aspect, for example, a disclosed regulatory element can comprise a promoter or a transcription factor. In an aspect, a disclosed transcription factor can be nuclear factor of the activated T cell (NF AT) or a minimal promoter. In an aspect, a disclosed cytokine can comprise, for example, IL-7, IL-12, IL-15, IL-18, IL-23, or any combination thereof. In an aspect, 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). In an aspect, a disclosed intracellular domain can comprise a JAK-STAT activation domain. In an aspect, a disclosed JAK- STAT activation domain can be derived, for example, from IL-2R|3. In an aspect, a disclosed JAK-STAT activation can stimulate cell proliferation, can prevent terminal differentiation, can perform better, or any combination thereof. In an aspect, activation of JAK / STAT pathwayactivation can be in an antigen-dependent manner.

[0147] In an aspect 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.

[0148] In an aspect, 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.

[0149] In an aspect of a chimeric antigen receptor, a disclosed intracellular domain can further comprise a self-cleaving peptide. In an aspect, a disclosed self-cleaving peptide can comprise a T2A, a P2A. a E2A. or F2A peptide. In an aspect, a GSG linker can be added to the N-terminus of a disclosed 2A peptide. In an aspect, a disclosed P2A peptide can comprise the sequence set forth in SEQ ID NO: 15 or SEQ ID NO:102 or a fragment thereof. In an aspect, a disclosed T2A peptide can comprise the sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104 or a fragment thereof. In an aspect, a disclosed E2A peptide can comprise the sequence set forth in SEQ ID NO: 105 or SEQ ID NO: 106 or a fragment thereof. In an aspect, a disclosed F2A peptide can comprise the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108 or a fragment thereof.

[0150] In an aspect, a disclosed intracellular domain can comprise a disclosed 4-1BB co- stimulatory domain and a disclosed CD3^ domain. In an aspect, a disclosed intracellular domain can comprise a disclosed CD28 co-stimulatory domain and a disclosed CD3^ domain. In an aspect, a disclosed intracellular domain can comprise a disclosed NKG2D co-stimulatory. In an aspect, 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.

[0151] In an aspect, 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. In an aspect, a disclosed intracellular domain can comprise a disclosed NKG2D co-stimulatory domain comprising the sequence set forth in SEQ ID NO:29 or a fragmentthereof and a disclosed CD3^ domain comprising the sequence set forth in SEQ ID NO:41 or SEQ ID NO:42 or a fragment thereof.

[0152] In an aspect, 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. In an aspect, 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.

[0153] In an aspect, a disclosed CAR can stimulate an effector cell mediated immune modulator response to citrullinated-vimentin-expressing tumor cells. In an aspect, a disclosed CAR can induce a tumor reducing immune response. In an aspect, a disclosed CAR can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed CAR can cross-prime an antitumor T cell response. In an aspect, a disclosed CAR can induce a tumor eliminating immune response. In an aspect, a disclosed CAR can treat cancer.

[0154] Disclosed herein is a chimeric antigen receptor (CAR), 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.

[0155] Disclosed herein is a chimeric antigen receptor (CAR), 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. Disclosed herein is a chimeric antigen receptor (CAR), 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. Disclosed herein is a chimeric antigen receptor (CAR), 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. Disclosed herein is a chimeric antigen receptor (CAR), 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. Disclosed herein is a chimeric antigen receptor (CAR), 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 fragmentthereof; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more co-stimulatory domains.

[0156] In an aspect, a disclosed CAR can be introduced to T cells and / or NK and / or macrophages or any other immune system. In an aspect, 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. In an aspect, 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. In an aspect, disclosed herein are methods for making or generating immune effector cells that express one or more disclosed CARs. For example, in an aspect, a disclosed polynucleotide and / or a disclosed vector can be introduced into a disclosed immune effector cell.

[0157] In an aspect, 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. In an aspect, immune effector cells can be isolated from an individual in need thereof and can be genetically modified without further manipulation in vitro. In an aspect, genetically modified immune effector cells can be expanded and / or readministered into the individual.

[0158] In an aspect, 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. In an aspect, disclosed immune effector cells can be genetically modified to express one or more disclosed CARs prior to being activated and stimulated to proliferate. In an aspect, 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). In an aspect, 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.

[0159] In an aspect, 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. In an aspect, prior to in vitro manipulation or genetic modification of disclosed immune effector cells, the immune effector cells can be obtained from a subject. In an aspect, disclosed CAR-modified immune effector cells can comprise T cells.

[0160] In an aspect, peripheral blood mononuclear cells (PBMCs) can be directly genetically modified to express one or more disclosed CARs using methods known to the skilled person in the art. For example, in an aspect, after isolation of PBMCs, T lymphocytes can be further isolated. In an aspect, 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. In an aspect, 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. In an aspect, 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. In an aspect, T cells can be activated and expanded before or after genetic modification to express a CAR. In an aspect, 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.

[0161] In an aspect, 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. In an aspect, 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. In an aspect, one or more disclosed vectors can be used in genetically modify ing a donor population of immune effector cells. In an aspect, one or more disclosed vectors can encode the same CAR or can encode different CARs. In an aspect, 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.

[0162] Methods of manufacturing T cells for human therapy are known in the art. In an aspect, disclosed T cells manufactured by the methods can provide improved adoptive immunotherapy compositions. In an aspect, 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. In an aspect, 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.

[0163] In an aspect of a disclosed CAR, one or more domains (e.g., extracellular, transmembrane, and / or intracellular) can be derived from a natural source, from a synthetic source, or a combination thereof. In an aspect of a disclosed CAR, one or more domains can be recombinant domains.2. Nucleic Acid Molecules

[0164] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor targeting citrullinated vimentin (cVIM). Disclosed herein is 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. Disclosed herein is 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 targeting citrullinated vimentin, a transmembrane domain, and an intracellular domain comprising one or more costimulatory domains.

[0165] In an aspect, 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.

[0166] Disclosed herein is a 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. Disclosed herein is a 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.

[0167] Disclosed herein is a 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. Disclosed herein is a 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.

[0168] Disclosed herein is a 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. Disclosed herein is a 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.

[0169] Disclosed herein is a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:02 or a fragment thereof. Disclosed herein is a 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.

[0170] Disclosed herein is a 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. Disclosed herein is a 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.

[0171] Disclosed herein is a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:04 or a fragment thereof. Disclosed herein is a 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% identity7to the sequence set forth in SEQ ID NO: 04 or a fragment thereof.

[0172] Disclosed herein is a 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. Disclosed herein is a 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.

[0173] Disclosed herein is a 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. Disclosed herein is a 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.

[0174] Disclosed herein is a 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. Disclosed herein is a 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.

[0175] Disclosed herein is a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:06 or a fragment thereof. Disclosed herein is a nucleic acid molecule comprising a sequencehaving 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.

[0176] Disclosed herein is a 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. Disclosed herein is a 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.

[0177] Disclosed herein is a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:08 or a fragment thereof. Disclosed herein is a 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.

[0178] Disclosed herein is a 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. Disclosed herein is a 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.

[0179] Disclosed herein is a 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. Disclosed herein is a 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.

[0180] Disclosed herein is a 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. Disclosed herein is a 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.

[0181] Disclosed herein is a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 10 or a fragment thereof.

[0182] Disclosed herein is a 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 afragment thereof. Disclosed herein is a 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.

[0183] Disclosed herein is a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 12 or a fragment thereof. Disclosed herein is a 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: 12 or a fragment thereof.

[0184] Disclosed herein is a nuclic acid molecule encoding a chimeric antigen receptor comprising the scFV of Ab#4 as described in the Examples.

[0185] In an aspect of a disclosed nucleic acid molecule, an encoded extracellular domain can comprise a citrullinated vimentin binding domain.

[0186] In an aspect of a disclosed nucleic acid molecule, 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).

[0187] In an aspect, a disclosed encoded scFv can comprise the sequence set forth in any one of SEQ ID NO: 149-SEQ ID NO: 154. In an aspect, 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.

[0188] In an aspect of a disclosed nucleic acid molecule, an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 113, SEQ ID NO: 115, or SEQ ID NO: 117. In an aspect of a disclosed nucleic acid molecule, 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: 118.

[0189] In an aspect of a disclosed nucleic acid molecule, an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 113 ora fragment thereof. In an aspect of a disclosed nucleic acid molecule, 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.

[0190] In an aspect of a disclosed nucleic acid molecule, an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 114 or a fragment thereof. In an aspect of a disclosed nucleic acid molecule, 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.

[0191] In an aspect of a disclosed nucleic acid molecule, an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 115 or a fragment thereof. In an aspect of a disclosed nucleic acid molecule, 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.

[0192] In an aspect of a disclosed nucleic acid molecule, an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 116 or a fragment thereof. In an aspect of a disclosed nucleic acid molecule, 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.

[0193] In an aspect of a disclosed nucleic acid molecule, an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 117 or a fragment thereof. In an aspect of a disclosed nucleic acid molecule, 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.

[0194] In an aspect of a disclosed nucleic acid molecule, an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 118 or a fragment thereof. In an aspect of a disclosed nucleic acid molecule, 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.

[0195] In an aspect of a disclosed nucleic acid molecule, 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. In an aspect of a disclosed nucleic acid molecule, an encoded 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.

[0196] In an aspect of a disclosed nucleic acid molecule, an encoded vimentin binding domain can comprise a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs). In an aspect of a disclosed nucleic acid molecule, an encoded vimentin binding domain can comprise a light chain variable region (VL) comprising three complementarity determining regions (CDRs). In an aspect of a disclosed nucleic acid molecule, 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. In an aspect of a disclosed nucleic acid molecule, 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:2L SEQ ID NO:22, and SEQ ID NO:23. In an aspect of a disclosed nucleic acid molecule, 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. In an aspect of a disclosed nucleic acid molecule, 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.

[0197] In an aspect of a disclosed nucleic acid molecule, an encoded CDR comprising 3 amino acids can comprise 1 or more substitutions. In an aspect of a disclosed nucleic acid molecule, an encoded CDR comprising 8 amino acids can comprise 2 or more substitutions. In an aspect of a disclosed nucleic acid molecule, an encoded CDR comprising 9 amino acids can comprise 2 or more substitutions. In an aspect of a disclosed nucleic acid molecule, an encoded CDR comprising 11 amino acids can comprise 3 or more substitutions. In an aspect of a disclosed nucleic acid molecule, an encoded CDR comprising 17 amino acids can comprise 4 or more substitutions. In an aspect of a disclosed nucleic acid molecule, an encoded CDR comprising the sequence set forth in SEQ ID NO:22 can further comprise 1 or more substitutions. In an aspect of a disclosed nucleic acid molecule, an encoded CDR comprising the sequence set forth in SEQ ID NO: 18 can further comprise 2 or more substitutions. In an aspect of a disclosed nucleic acid molecule, an CDR comprising the sequence set forth in SEQ ID NO: 19 and / or SEQ ID NO:23 can further 2 or more substitutions. In an aspect of a disclosed nucleic acid molecule, an encoded CDR comprising the sequence set forth in SEQ ID NO:21 can further comprise 3 or more substitutions. In an aspectof a disclosed nucleic acid molecule, an encoded CDR comprising the sequence set forth in SEQ ID NO:20 can further comprise 4 or more substitutions.

[0198] In an aspect of a disclosed nucleic acid molecule, 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. In an aspect of a disclosed nucleic acid molecule, 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. In an aspect, choosing the amino acid substitutions for a disclosed 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.

[0199] In an aspect of a disclosed nucleic acid molecule, a CDR can be encoded by the sequence set forth in any one SEQ ID NO: 135-SEQ ID NO: 140. In an aspect, SEQ ID NO: 135 can encode a disclosed CDR. In an aspect, SEQ ID NO: 136 can encode a disclosed CDR. In an aspect, SEQ ID NO: 137 can encode a disclosed CDR. In an aspect, SEQ ID NO: 138 can encode a disclosed CDR. In an aspect, SEQ ID NO: 139 can encode a disclosed CDR. In an aspect, SEQ ID NO: 140 can encode a disclosed CDR. In an aspect, SEQ ID NO: 135 can comprise one or more nucleotide substitutions. In an aspect, SEQ ID NO: 136 can comprise one or more nucleotide substitutions. In an aspect. SEQ ID NO: 137 can comprise one or more nucleotide substitutions. In an aspect, SEQ ID NO: 138 can comprise one or more nucleotide substitutions. In an aspect, SEQ ID NO: 139 can comprise one or more nucleotide substitutions. In an aspect, SEQ ID NO: 140 can comprise one or more nucleotide substitutions. In an aspect, 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. In an aspect, 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. In an aspect. 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. In an aspect, 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. In an aspect. 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. In an aspect, SEQ ID NO: 140 can comprise one or morenucleotide 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.

[0200] In an aspect of a disclosed nucleic acid molecule, an encoded VL can be located at the N- terminus of the VH. In an aspect of a disclosed nucleic acid molecule, an encoded extracellular domain comprising a vimentin binding domain can comprise SEQ ID NO: 13 and SEQ ID NO: 15. In an aspect of a disclosed nucleic acid molecule, an encoded VH can be located at the N-termmus of the VL. In an aspect of a disclosed nucleic acid molecule, an encoded extracellular domain comprising a vimentin binding domain can comprise SEQ ID NO:15 and SEQ ID NO: 13. In an aspect of a disclosed nucleic acid molecule, an encoded VL can comprise the sequence of SEQ ID NO: 13. In an aspect of a disclosed nucleic acid molecule, 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.

[0201] In an aspect, a disclosed VL can be encoded by the sequence set forth in SEQ ID NO: 14. In an aspect, 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.

[0202] In an aspect of a disclosed nucleic acid molecule, an encoded VH can comprise the sequence of SEQ ID NO: 15. In an aspect of a disclosed nucleic acid molecule, 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.

[0203] In an aspect of a disclosed nucleic acid molecule, a VH 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.

[0204] In an aspect of a disclosed nucleic acid molecule, a VH 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.

[0205] In an aspect, the nucleotide sequence of SEQ ID NO: 14 can encode a disclosed light chain or a fragment thereof.

[0206] In an aspect, the nucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 17 can encode a disclosed heavy chain or a fragment thereof.

[0207] In an aspect of a disclosed nucleic acid molecule, an encoded antigen binding domain can be a scFv and wherein the scFV can comprise a linker and / or a spacer. In an aspect, a disclosedencoded linker and / or linker can join the Vnand VL regions of the scFv. In an aspect of a disclosed nucleic acid molecule, 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. In an aspect of a disclosed nucleic acid molecule, an encoded scFv can provide one or more advantages conveyed, in part, by its size.

[0208] In an aspect of a disclosed nucleic acid molecule, an encoded extracellular domain can further comprise a signal / leader peptide.

[0209] In an aspect of a disclosed nucleic acid molecule, an encoded extracellular domain comprising a vimentin binding domain can further comprise a signal / leader peptide. Signal / leader peptides are known to the art. In an aspect, a disclosed signal / leader peptide can comprise any known signal / leader peptide or any known signal / leader peptide known to function in mammalian cells. For example, in an aspect, 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:62), Mouse Ig Kappa (SEQ ID NO:63), Mouse Ig Heavy (SEQ ID NO:64), or CD8a (SEQ ID NO:66).

[0210] In an aspect, a disclosed encoded 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.

[0211] In an aspect of a disclosed nucleic acid molecule, 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.

[0212] In an aspect of a disclosed nucleic acid molecule, an encoded extracellular domain can be encoded by the sequence set forth in SEQ ID NO: 141 - SEQ ID NO: 146. In an aspect of a disclosed nucleic acid molecule, 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.

[0213] In an aspect, a disclosed nucleic acid molecule can further comprise the nucleotide sequence for a signal peptide. In an aspect, a disclosed encoded signal peptide can comprise thesequence set forth in SEQ ID NO:46 or a fragment thereof. In an aspect, 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. In an aspect, a disclosed signal peptide can be encoded by the sequence set forth in SEQ ID NO:47 or a fragment thereof. In an aspect, 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.

[0214] In an aspect, 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.

[0215] In an aspect of a disclosed nucleic acid molecule, 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.

[0216] In an aspect of a disclosed nucleic acid molecule, an encoded hinge region can comprise the sequence set forth in SEQ ID NO:26. In an aspect of a disclosed nucleic acid molecule, an encoded transmembrane region can comprise the sequence set forth in SEQ ID NO:29. In an aspect of a disclosed nucleic acid molecule, an encoded transmembrane region can comprise the sequence set forth in SEQ ID NO:32.

[0217] In an aspect of a disclosed nucleic acid molecule, an encoded 4-1BB signaling domain can comprise the sequence set forth in SEQ ID NO:33 or SEQ ID NO:34. In an aspect, a disclosed nucleic acid molecule encoding a 4-1 BB signaling domain can comprise the sequence set forth in SEQ ID NO:35. In an aspect of a disclosed nucleic acid molecule, an encoded CD28 signaling domain can comprise the sequence set forth in SEQ ID NO:38 or SEQ ID NO:37. In an aspect, a disclosed nucleic acid molecule encoding a CD28 signaling domain can comprise the sequence set forth in SEQ ID NO: 38. In an aspect of a disclosed nucleic acid molecule, an encoded NKG2D signaling domain can comprise the sequence set forth in SEQ ID NO:40. In an aspect, a disclosed nucleic acid molecule encoding a NK.G2D signaling domain can comprise the sequence set forth in SEQ ID NO:40. In an aspect of a disclosed nucleic acid molecule, an encoded CD3^ signaling domain can be encoded by the sequence set forth in SEQ ID NO:41. In an aspect, a disclosed nucleic acid molecule encoding a CD3^ signaling domain can comprise the sequence set forth in SEQ ID NO:42.

[0218] In an aspect of a disclosed nucleic acid molecule, an encoded spacer can comprise the sequence set forth in SEQ ID NO:44. In an aspect, a disclosed nucleic acid molecule encoding aspacer can comprise the sequence set forth in SEQ ID NO:45. In an aspect of a disclosed nucleic acid molecule, an encoded spacer can comprise the sequence set forth in any one of SEQ ID NO: 73- SEQ ID NO: 112.

[0219] In an aspect, a disclosed encoded linker can comprise a flexible linker. In an aspect, an encoded flexible linker can comprise (GGGGS)n, wherein n = 1 - 8 (SEQ ID NO: 73 - SEQ ID NO: 80). In an aspect, an encoded flexible linker can comprise (G)n. wherein n = 1 - 8 (SEQ ID NO: 81 - SEQ ID NO: 88). In an aspect, an encoded linker can comprise a rigid linker. In an aspect, an encloded flexible linker can comprise (EAAAK)n, wherein n = 1 - 8 (SEQ ID NO: 89- SEQ ID NO:96). In an aspect, an encoded linker can comprise a Whitlow linker. In an aspect, a Whitlow linker can comprise (GSTSGSGKSSEGKG)n, wherein n = 1 - 8 (SEQ ID NO:97 - SEQ ID NO: 104). In an aspect, a Whitlow linker can comprise (GSTSGSGKPGSGEGSTKG)n, wherein n = 1 - 8 (SEQ ID NO: 105 - SEQ ID NO: 112). Linkers are known to the skilled person in the art.

[0220] In an aspect of a disclosed nucleic acid molecule, an encoded chimeric antigen receptor can further compnse a spacer or a hinge region between the extracellular domain and the transmembrane domain. In an aspect, 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.

[0221] In an aspect of a disclosed nucleic acid molecule, an encoded CD8 hinge domain can comprise the sequence set forth in SEQ ID NO:24, SEQ ID NO:25, or a fragment thereof. In an aspect, a disclosed CD8 hinge domain can be encoded by the sequence set forth in SEQ ID NO:26 or a fragment thereof. In an aspect of a disclosed nucleic acid molecule, 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), CDl lc (ITGAX), CDl ld (ITGAD), CD 18 (ITGB2), CD 19 (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), CD 162 (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 (CRTAM), CD357 (TNFRSF18), inducible T cell co-stimulator (ICOS), LFA-1 (CD1 la / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2Ry, IL-7R alpha, LFA1-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP. a CD83 ligand. Fey receptor. MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, Toll ligand receptor, or any combination thereof, or any fragment / truncation thereof, or any combination of fragments / truncations thereof.

[0222] In an aspect of a disclosed nucleic acid molecule, an encoded 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[3, NK.G2D, 2B4, PD1, or any combination thereof. In an aspect of a disclosed nucleic acid molecule, 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, CD3c , CD3 5, CD3y y, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD8 , CD9, CDl la, CDllb, CDl lc, CDl ld, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137. TNFSFR25, CD154, 4-1BB / CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 100 (SEMA4D), CD 103, CD 160 (BY55), CD 18, CD 19, CD 19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DAP-12, DNAM1 (CD226), Fey y receptor, GADS, GITR. HVEM (LIGHTR), IA4, ICAM-1. ICAM-1. Iga (CD79a), IL-2RP , IL-2Ry y, IL- 7Ra , inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, 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. programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (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.

[0223] In an aspect of a disclosed nucleic acid molecule, an encoded CD8 transmembrane domain can comprise the sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or a fragment thereof. In an aspect, a disclosed CD8 transmembrane domain can be encoded by the sequence set forth in SEQ ID NO:29 or a fragment thereof. In an aspect of a disclosed nucleic acid molecule, an encoded CD28 transmembrane domain can comprise the sequence set forth in SEQ ID NO:30 or a fragment thereof. In an aspect of a disclosed nucleic acid molecule, an encoded NK.G2D transmembrane domain can comprise the sequence set forth in SEQ ID NO:31 or a fragment thereof. In an aspect, a disclosed NKG2D transmembrane domain can be encoded by the sequence set forth in SEQ ID NO:32 or a fragment thereof.

[0224] In an aspect of a disclosed nucleic acid molecule, an encoded intracellular domain can be referred to synonymously as an intracellular signaling domain. Intracellular domains are discussed supra. In an aspect of a disclosed nucleic acid molecule, an encoded intracellular domain can comprise one or more immunoreceptor tyrosine-based activation domains (IT AM). In an aspect, a disclosed ITAM can comprise the signaling domain of DAP10. DAP12, TCR^, FcRy, FcR0, CD3y, CD3 , CD3e, CD35, CD3 CD5, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), or any combination thereof. In an aspect of a disclosed nucleic acid molecule, an encoded intracellular region can comprise a CD3^ signaling domain. In an aspect, a disclosed CD3^ signaling domain can comprise the sequence set forth in SEQ ID NO:41. In an aspect, a disclosed CD3 signaling domain can comprise the sequence set forth in SEQ ID NO:42. In an aspect, a disclosed CD3^ signaling domain can be encoded by the sequence set forth in SEQ ID NO:43.

[0225] In an aspect, a disclosed CD3^ can comprise three immunoreceptor tyrosine activation motifs (ITAMs), which can assist in triggering downstream signaling events in response to receptor clustering. In an aspect, a disclosed ITAM can be on the C-terminal end (distal to the transmembrane domain).

[0226] In an aspect of a disclosed nucleic acid molecule, an encoded intracellular domain can comprise one or more co-stimulatory domains or co-stimulatory signaling domains. In an aspect, the order of one or more co-stimulatory domains in a disclosed intracellular domain can be varied. In an aspect, the order of the one or more co-stimulatory domains can be varied and / or can influence the resulting effector function. For example, CD28-CD3^ can elicit higher level of IL- 2. In an aspect, a disclosed co-stimulatory domain can be on the N-terminal end (proximal to the transmembrane domain).

[0227] In an aspect of a disclosed nucleic acid molecule, an encoded intracellular domain can further comprise one or more co-stimulatory signaling domain or co-stimulatory domains. In an aspect, a disclosed co-stimulatory molecule can be cell surface molecules other than antigenreceptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen.

[0228] In an aspect of a disclosed nucleic acid molecule, an encoded co-stimulalory 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 (1C0S), 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, CD9, CD96 (Tactile), CDS, CEACAM1, CRT AM, DAP- 10. DNAM1 (CD226), Fey receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM- 1, ICOS, Iga (CD79a), IL2R , IL2Ry, IL7Ra, integrm, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1 (CD1 la / CD18), LIGHT, LTBR, Ly9 (CD229), MHC class I molecule, NKD2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), 0X40, PAG / Cbp, PD-1, PSGLL SELPLG (CD162), signaling lymphocytic activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), 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.

[0229] In an aspect of a disclosed nucleic acid molecule, an encoded 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^, CD3E, CD3S, CD3^, CD5, CD22, CD66d, CD79a. CD79b, CD278 (ICOS), or any combination thereof, and wherein the costimulatory 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.

[0230] In an aspect of a disclosed nucleic acid molecule, an encoded intracellular domain can comprise one or more co-stimulatory domains. _In an aspect, a disclosed co-stimulatory domain can comprise 4-1BB. In an aspect of a disclosed nucleic acid molecule, an encoded 4-1BB co- stimulatory domain can comprise the sequence set forth in SEQ ID NO:33, SEQ ID NO:34, or a fragment thereof. In an aspect of a disclosed nucleic acid molecule, a disclosed 4- IBB co- stimulatory domain can be encoded by the sequence set forth in SEQ ID NO:35 or a fragment thereof.

[0231] In an aspect of a disclosed nucleic acid molecule, an encoded 4- IBB 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 reducetonic signaling at optimal expression levels and decrease exhaustion, (iii) slower and less intense signaling, or (iv) any combination thereof, lln an aspect of a disclosed nucleic acid molecule, an encoded co-stimulatory domain can comprise CD28. In an aspect, a disclosed CD28 costimulatory domain can comprise the sequence set forth in SEQ ID NO:36, SEQ ID NO:37, or a fragment thereof. In an aspect, a disclosed CD28 co-stimulatory domain can be encoded by the sequence set forth in SEQ ID NO: 38 or a fragment thereof. In an aspect of a disclosed nucleic acid molecule, an encoded CD28 co-stimulatory domain can comprise the sequence set forth co- stimulatory domain can comprise NKG2D. In an aspect, a disclosed NKG2D co-stimulatory domain can comprise the sequence set forth in SEQ ID NO:39 or a fragment thereof. In an aspect, a disclosed NKG2D co-stimulatory domain can be encoded by the sequence set forth in SEQ ID NQ:40 or a fragment thereof.

[0232] In an aspect of a disclosed nucleic acid molecule, an encoded co-stimulatory domain can comprise OX-40. In an aspect, a disclosed OX-40 co-stimulatory domain can comprise the sequence set forth in SEQ ID NO: 147 or a fragment thereof. In an aspect, a disclosed OX-40 co- stimulatory domain can be encoded by the sequence set forth in SEQ ID NO: 148 or a fragment thereof.

[0233] In an aspect of a disclosed nucleic acid molecule, an encoded co-stimulatory signaling domain can enhance the efficacy and expansion of immune cells expressing CAR receptors.

[0234] In an aspect, 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.

[0235] In an aspect of a disclosed nucleic acid molecule, an encoded intracellular domain can comprise a gene cassette for one or more cytokines. In an aspect, a disclosed gene cassette for one or more cytokines can be operably linked to one or more regulatory elements. In an aspect, for example, a disclosed regulatory element can comprise a promoter or a transcription factor. In an aspect, a disclosed transcription factor can be nuclear factor of the activated T cell (NF AT) or a minimal promoter. In an aspect, a disclosed cytokine can comprise, for example, IL-7, IL- 12, IL-15, IL-18, IL-23, or any combination thereof. In an aspect, 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). In an aspect of a disclosed nucleic acid molecule, an encoded intracellular domain can comprise a JAK-STAT activation domain. In an aspect, a disclosed JAK- STAT activation domain can be derived, for example, from IL-2RJ3. In an aspect, a disclosed JAK-STAT activation can stimulate cell proliferation, can prevent terminal differentiation, can perform better, or any combination thereof. In an aspect of a disclosed nucleic acid molecule, anencoded 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.

[0236] In an aspect of a disclosed nucleic acid molecule, 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.

[0237] In an aspect of a disclosed nucleic acid molecule, an encoded intracellular domain can further comprise a self-cleaving peptide. In an aspect, a disclosed self-cleaving peptide can comprise a T2A, a P2A, a E2A, or F2A peptide. In an aspect, a GSG linker can be added to the N-terminus of a disclosed 2A peptide. In an aspect, a disclosed P2A peptide can comprise the sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 102 or a fragment thereof. In an aspect, a disclosed T2A peptide can comprise the sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104 or a fragment thereof. In an aspect, a disclosed E2A peptide can comprise the sequence set forth in SEQ ID NO: 105 or SEQ ID NO: 106 or a fragment thereof. In an aspect, a disclosed F2A peptide can comprise the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108 or a fragment thereof.

[0238] In an aspect of a disclosed nucleic acid molecule, an encoded intracellular domain can comprise a disclosed 4- IBB co-stimulatory domain and a disclosed CD3^ domain. In an aspect of a disclosed nucleic acid molecule, an encoded intracellular domain can comprise a disclosed CD28 co-stimulatory domain and a disclosed CD3 domain. In an aspect of a disclosed nucleic acid molecule, an encoded intracellular domain can comprise a disclosed NKG2D co-stimulatory. In an aspect of a disclosed nucleic acid molecule, an encoded 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.

[0239] In an aspect of a disclosed nucleic acid molecule, an encoded 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 CD3C domain comprising the sequence set forth in SEQ ID NO:41 or SEQ ID NO:42 or a fragment thereof. In an aspect of a disclosed nucleic acid molecule, an encoded intracellular domain can comprise a disclosed NKG2D co-stimulatorydomain comprising the sequence set forth in SEQ ID NO:29 or a fragment thereof and a disclosed CD3ij domain comprising the sequence set forth in SEQ ID NO:41 or SEQ ID NO:42 or a fragment thereof.

[0240] In an aspect of a disclosed nucleic acid molecule, 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. In an aspect of a disclosed nucleic acid molecule, 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.

[0241] In an aspect of a disclosed nucleic acid molecule, an encoded CAR can stimulate an effector cell mediated immune modulator response to citrullinated-vimentin-expressing tumor cells. In an aspect of a disclosed nucleic acid molecule, an encoded CAR can induce a tumor reducing immune response. In an aspect of a disclosed nucleic acid molecule, an encoded CAR can induce phagocytosis of cancer cells in the subject. In an aspect of a disclosed nucleic acid molecule, an encoded CAR can cross-prime an anti-tumor T cell response. In an aspect of a disclosed nucleic acid molecule, an encoded CAR can induce a tumor eliminating immune response. In an aspect, a disclosed CAR can treat cancer.

[0242] Disclosed herein is a nucleic acid molecule 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.

[0243] Disclosed herein is a nucleic acid molecule 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 costimulatory domains. Disclosed herein is a nucleic acid molecule encoding a chimeric antigen receptor (CAR), 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. Disclosed herein is a nucleic acid molecule encoding a chimeric antigen receptor (CAR), 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-stimulatorydomains. Disclosed herein is a nucleic acid molecule encoding a chimeric antigen receptor (CAR), 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. Disclosed herein is a nucleic acid molecule encoding a chimeric antigen receptor (CAR), 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 costimulatory domains. Disclosed herein is a nucleic acid molecule encoding a chimeric antigen receptor (CAR), 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.

[0244] In an aspect, 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. In an aspect, 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. In an aspect, 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.

[0245] In an aspect, disclosed herein is a method for making or generating immune effector cells that express one or more disclosed CARs. For example, in an aspect, a disclosed polynucleotide and / or a disclosed vector can be introduced into a disclosed immune effector cell. In an aspect, 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. In an aspect, immune effector cells can be isolated from an individual in need thereof and can be genetically modified without further manipulation in vitro. In an aspect, genetically modified immune effector cells can be expanded and / or re-administered into the individual.

[0246] In an aspect, 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. In an aspect, disclosed immune effector cells can be genetically modified to express one or more disclosed CARs prior to being activated and stimulated to proliferate in vitro. In an aspect, 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).

[0247] In an aspect, 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. In an aspect, 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.

[0248] In an aspect, prior to in vitro manipulation or genetic modification of disclosed immune effector cells, the immune effector cells can be obtained from a subject. In an aspect, disclosed CAR-modified immune effector cells can comprise T cells. In an aspect, peripheral blood mononuclear cells (PBMCs) can be directly genetically modified to express one or more disclosed CARs using methods known to the skilled person in the art. For example, in an aspect, after isolation of PBMCs, T lymphocytes can be further isolated. In an aspect, 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.

[0249] In an aspect, 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. In an aspect, 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. In an aspect, T cells can be activated and expanded before or after genetic modification to express a CAR.

[0250] In an aspect, a disclosed nucleic acid molecule encoding a disclosed CAR can be introduced into a population of human donor T cells, NK cells or NKT cells. In an aspect, 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. In an aspect, 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.

[0251] In an aspect, a disclosed nucleic acid molecule can be used in genetically modifying a donor population of immune effector cells. In an aspect, a disclosed nucleic acid molecule can encode the same CAR or can encode different CARs. In an aspect, 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.

[0252] Methods of manufacturing T cells for human therapy are know n in the art. In an aspect, disclosed T cells manufactured by the methods can provide improved adoptive immunotherapy compositions. In an aspect, 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.

[0253] In an aspect, a disclosed nucleic acid molecule can be incorporated into a non-viral or a viral vector. In an aspect, a disclosed nucleic acid molecule can be packaged into a viral vector. In an aspect, a disclosed viral vector can comprise an AAV vector. In an aspect, a disclosed nucleic acid molecule can be packaged into a non-viral carrier. In an aspect, a disclosed nucleic acid molecule can be incorporated into a plasmid. In an aspect, a disclosed nucleic acid molecule can be incorporated into lipid nanoparticles.

[0254] In an aspect, a disclosed nucleic acid molecule can be isolated. In an aspect, isolated can mean that the nucleic acid can be substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. For example, in an aspect, a disclosed nucleic acid molecule can be one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. In an aspect, a disclosed nucleic acid molecule can be a recombinant nucleic acid molecule. In an aspect, a disclosed nucleic acid molecule encoding a disclosed CAR can be a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced.

[0255] In an aspect, a disclosed nucleic acid molecule can further comprise a polyadenylation sequence. In an aspect, a disclosed nucleic acid molecule can further comprise a sequence for a promoter. In an aspect, a disclosed 3’ hemi intron can be recognized by nuclear splicing components within a host cell. In an aspect, a disclosed exogenous RNA can induce a splice event. In an aspect, a disclosed nucleic acid molecule can further comprise a spacer region. In an aspect, a disclosed spacer region can separate the 5’ splice region from the one or more RNA structures. In an aspect, a disclosed spacer region can comprise any known spacer. In an aspect, a disclosed spacer region can comprise a consensus splicing motif (e.g., such as LU or U2). In an aspect, a disclosed spacer region can comprise a limited number of consensus splicing motifs (e.g., such as U1 or U2). In an aspect, a disclosed nucleic acid molecule can further comprise a nuclear localization signal (NLS). In an aspect, a disclosed nucleic acid molecule can further comprise one or more nuclear retention elements (NRE). NRE are known to the skilled person in the art. In an aspect, a disclosed NRE can comprise SIRLOIN or BORG.

[0256] In an aspect, a disclosed nucleic acid molecule can comprise the sequence for one or more regulatory elements. In an aspect, 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 polyadenylation signals and poly-U sequences). Regulatory elements can include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e g., tissue-specific regulatory sequences). In an aspect, a disclosed regulatory element can comprise Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulator Element (WPRE), triplex from MALAT1, the PRE of Hepatitis B virus (HP RE), an iron response element, or any combination thereof. For example, 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.

[0257] In an aspect of a disclosed nucleic acid molecule, a disclosed promoter can be tissuespecific 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 wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter / enhancer. In an aspect, a disclosed promoter for the disclosed nucleic acid molecule can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter / enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter / enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter / enhancer can be used for constitutive and efficient expression of a disclosed gene. In an aspect, 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 promo ters / enhancers are ell known to the art.

[0258] In an aspect, a disclosed nucleic acid molecule encoding 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. In an aspect, a disclosed nucleic acid molecule 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.

[0259] In an aspect, a disclosed nucleic acid can be a recombinant nucleic acid.3. Vectors

[0260] Disclosed herein is a vector comprising a disclosed nucleic acid molecule. Disclosed herein is a vector comprising a nucleic acid molecule encoding a disclosed CAR. Disclosed herein is a vector comprising a disclosed nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor. Disclosed herein is a vector comprising a disclosed nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric antigen receptor targeting citrullinated vimentin (cVIM). Disclosed herein is 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. Disclosed herein is 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.

[0261] Disclosed herein is a nucleic acid molecule encoding 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. Disclosed herein is a nucleic acid molecule 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. Disclosed herein is a nucleic acid molecule 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 NKG2D transmembrane domain comprising the sequence of SEQ ID NO: 31, a NKG2D signaling domain comprising the sequence of SEQ ID NO:39.

[0262] In an aspect of a disclosed vector, an encoded CAR can target citrullinated vimentin (cVIM). In an aspect of a disclosed vector, an encoded CAR can target citrullinated vimentin(cVIM) expressed on the cell surface of a cancer cell. In an aspect of a disclosed vector, a disclosed encoded CAR can target citrullinated vimentin (cVIM) expressed on the cell surface of an ovarian cancer cell.

[0263] Disclosed herein is a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 129 or a fragment thereof. Disclosed herein is 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: 129 or a fragment thereof.

[0264] Disclosed herein is a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising the sequence set forth in SEQ ID NO: 130 or a fragment thereof. Disclosed herein is 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: 130 or a fragment thereof.

[0265] Disclosed herein is 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. Disclosed herein is 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. Disclosed herein is 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.

[0266] Disclosed herein is 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. Disclosed herein is 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. Disclosed herein is 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.

[0267] Disclosed herein is 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. Disclosed herein is 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 fragmentthereof. Disclosed herein is 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. Disclosed herein is 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.

[0268] Disclosed herein is 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. Disclosed herein is 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. Disclosed herein is 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.

[0269] Disclosed herein is 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. Disclosed herein is 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. Disclosed herein is 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.

[0270] Disclosed herein is 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. Disclosed herein is 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. Disclosed herein is 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. Disclosed herein is 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.

[0271] Disclosed herein is 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. Disclosed herein is 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%, atleast 90%. or at least 95% identity to the sequence set forth in SEQ ID NO:09 or a fragment thereof.

[0272] Disclosed herein is 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. Disclosed herein is 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. Disclosed herein is 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.

[0273] Disclosed herein is 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.

[0274] Disclosed herein is vector encoding a chimeric antigen receptor comprising the scFV of Ab#4 as described in the Examples.

[0275] In an aspect of a disclosed vector, an encoded extracellular domain can comprise a citrullinated vimentin binding domain. In an aspect of a disclosed vector, 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).

[0276] In an aspect of a disclosed vector, a disclosed encoded scFv can comprise the sequence set forth in any one of SEQ ID NO: 149-SEQ ID NO: 154. In an aspect of a disclosed vector, 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.

[0277] In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, an encoded extracellular domain comprising avimentin binding domain can comprise the sequence set forth in SEQ ID NO: 114, SEQ ID NO: 116, or SEQ ID NO: 1 18. In an aspect of a disclosed vector, an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 113 or a fragment thereof. In an aspect of a disclosed vector, 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.

[0278] In an aspect of a disclosed vector, an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 114 or a fragment thereof. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 115 or a fragment thereof. In an aspect of a disclosed vector, 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% identity7to the sequence set forth in SEQ ID NO: 115 or a fragment thereof. In an aspect of a disclosed vector, an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 116 or a fragment thereof. In an aspect of a disclosed vector, 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.

[0279] In an aspect of a disclosed vector, an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 117 or a fragment thereof.

[0280] In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, an encoded extracellular domain comprising a vimentin binding domain can comprise the sequence set forth in SEQ ID NO: 118 or a fragment thereof. In an aspect of a disclosed vector, 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.

[0281] Disclosed herein is 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. Disclosed herein is 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. Disclosed herein is 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.

[0282] In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, an encoded vimentin binding domain can comprise a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs). In an aspect of a disclosed vector, an encoded vimentin binding domain can comprise a light chain variable region (VL) comprising three complementarity determining regions (CDRs).

[0283] In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, 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.

[0284] In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, an encoded CDR in the VH can comprise a sequence having at least 70%, atleast 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.

[0285] In an aspect of a disclosed vector, an encoded CDR comprising 3 amino acids can comprise 1 or more substitutions. In an aspect of a disclosed vector, an encoded CDR comprising 8 amino acids can comprise 2 or more substitutions. In an aspect of a disclosed vector, an encoded CDR comprising 9 amino acids can comprise 2 or more substitutions. In an aspect of a disclosed vector, an encoded CDR comprising 11 amino acids can comprise 3 or more substitutions. In an aspect of a disclosed vector, an encoded CDR comprising 17 amino acids can comprise 4 or more substitutions.

[0286] In an aspect of a disclosed vector, an encoded CDR comprising the sequence set forth in SEQ ID NO:22 can further comprise 1 or more substitutions. In an aspect of a disclosed vector, an encoded CDR comprising the sequence set forth in SEQ ID NO: 18 can further comprise 2 or more substitutions. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, an encoded CDR comprising the sequence set forth in SEQ ID NO:21 can further comprise 3 or more substitutions. In an aspect of a disclosed vector, an encoded CDR comprising the sequence set forth in SEQ ID NO:20 can further comprise 4 or more substitutions. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, 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.

[0287] In an aspect of a disclosed vector, a CDR can be encoded by the sequence set forth in any one SEQ ID NO: 135-SEQ ID NO: 140. In an aspect, SEQ ID NO: 135 can encode a disclosed CDR. In an aspect, SEQ ID NO: 136 can encode a disclosed CDR. In an aspect, SEQ ID NO: 137 can encode a disclosed CDR. In an aspect, SEQ ID NO: 138 can encode a disclosed CDR. In an aspect, SEQ ID NO: 139 can encode a disclosed CDR. In an aspect, SEQ ID NO: 140 can encode a disclosed CDR. In an aspect, SEQ ID NO: 135 can comprise one or more nucleotide substitutions. In an aspect, SEQ ID NO: 136 can comprise one or more nucleotide substitutions. In an aspect, SEQ ID NO: 137 can comprise one or more nucleotide substitutions. In an aspect, SEQ ID NO: 138 can comprise one or more nucleotide substitutions. In an aspect, SEQ ID NO: 139can comprise one or more nucleotide substitutions. In an aspect, SEQ ID NO: 140 can comprise one or more nucleotide substitutions. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, 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.

[0288] In an aspect of a disclosed vector, an encoded VL can be located at the N-terminus of the VH. In an aspect of a disclosed vector, an encoded extracellular domain comprising a vimentin binding domain can comprise SEQ ID NO: 13 and SEQ ID NO: 15. In an aspect of a disclosed vector, an encoded VH can be located at the N-terminus of the VL. In an aspect of a disclosed vector, an encoded extracellular domain comprising a vimentin binding domain can comprise SEQ ID NO: 15 and SEQ ID NO: 13. In an aspect of a disclosed vector, an encoded VL can comprise the sequence of SEQ ID NO: 13. In an aspect of a disclosed nucleic acid molecule, 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. In an aspect of a disclosed vector, VL can be encoded by the sequence set forth in SEQ ID NO: 14. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, VH can comprise the sequence of SEQ ID NO: 15. In an aspect of a disclosed vector, 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. In an aspect of a disclosedvector. VH can be encoded by the sequence set forth in SEQ ID NO: 16. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, a VH can be encoded by the sequence set forth in SEQ ID NO: 17. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, the nucleotide sequence of SEQ ID NO: 14 can encode a disclosed light chain or a fragment thereof. In an aspect of a disclosed vector, the nucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 17 can encode a disclosed heavy chain or a fragment thereof.

[0289] In an aspect of a disclosed vector, an encoded antigen binding domain can be a scFv and wherein the scFV can comprise a linker and / or a spacer. In an aspect, a disclosed encoded linker and / or linker can join the VH and VL regions of the scFv. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, an encoded scFv can provide one or more advantages conveyed, in part, by its size.

[0290] In an aspect of a disclosed vector, an encoded extracellular domain can further comprise a signal / leader peptide. In an aspect of a disclosed vector, an encoded extracellular domain comprising a vimentin binding domain can further comprise a signal / leader peptide. Signal / leader peptides are known to the art. In an aspect, a disclosed signal / leader peptide can comprise any known signal / leader peptide or any known signal / leader peptide known to function in mammalian cells. For example, in an aspect, 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). 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:62), Mouse Ig Kappa (SEQ ID NO:63), Mouse Ig Heavy (SEQ ID NO:64), or CD8a (SEQ ID NO:66).

[0291] In an aspect, a disclosed encoded 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. In an aspect of a disclosed vector, an encoded signal / leader peptide can be cleaved upon the nascent protein crossing the ERmembrane. In an aspect of a disclosed vector, an encoded signal / leader peptide can be cleaved prior to protein assembly and folding by, for example, a signal peptide peptidase.

[0292] In an aspect of a disclosed vector, an encoded extracellular domain can be encoded by the sequence set forth in SEQ ID NO: 141 - SEQ ID NO: 146. In an aspect of a disclosed vector, 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. In an aspect, a disclosed vector can further comprise the nucleotide sequence for a signal peptide. In an aspect, a disclosed encoded signal peptide can comprise the sequence set forth in SEQ ID NO:46 or a fragment thereof. In an aspect, 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. In an aspect, a disclosed signal peptide can be encoded by the sequence set forth in SEQ ID NO:47 or a fragment thereof. In an aspect, 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. In an aspect, 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.

[0293] In an aspect of a disclosed vector, 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

[0295] In an aspect of a disclosed vector, an encoded 4-1BB signaling domain can comprise the sequence set forth in SEQ ID NO:33 or SEQ ID NO:34. In an aspect of a disclosed vector, a disclosed nucleic acid molecule encoding a 4- IBB signaling domain can comprise the sequence set forth in SEQ ID NO:35. In an aspect of a disclosed vector, an encoded CD28 signaling domain can comprise the sequence set forth in SEQ ID NO:38 or SEQ ID NO:37. In an aspect of a disclosed vector, a disclosed nucleic acid molecule encoding a CD28 signaling domain can comprise the sequence set forth in SEQ ID NO:38.

[0296] In an aspect of a disclosed vector, an encoded NK.G2D signaling domain can comprise the sequence set forth in SEQ ID NO:40. In an aspect, a disclosed nucleic acid molecule encoding aNKG2D signaling domain can comprise the sequence set forth in SEQ ID NO:40. In an aspect of a disclosed vector, an encoded CD3^ signaling domain can be encoded by the sequence set forth in SEQ ID NO:41. In an aspect, a disclosed nucleic acid molecule encoding a CD3 signaling domain can comprise the sequence set forth in SEQ ID NO:42. In an aspect of a disclosed vector, an encoded spacer can comprise the sequence set forth in SEQ ID NO:44. In an aspect, a disclosed nucleic acid molecule encoding a spacer can comprise the sequence set forth in SEQ ID NO:45. In an aspect of a disclosed nucleic acid molecule, an encoded spacer can comprise the sequence set forth in any one of SEQ ID NO:73 - SEQ ID NO:112.

[0297] In an aspect, a disclosed encoded linker can comprise a flexible linker. In an aspect, an encoded flexible linker can comprise (GGGGS)n, wherein n = 1 - 8 (SEQ ID NO:73 - SEQ ID NO: 80). In an aspect, an encoded flexible linker can comprise (G)n, wherein n = 1 - 8 (SEQ ID NO: 81 - SEQ ID NO: 88). In an aspect, an encoded linker can comprise a rigid linker. In an aspect, an encoded flexible linker can comprise (EAAAK)n, wherein n = 1 - 8 (SEQ ID NO: 89 - SEQ ID NO:96). In an aspect, an encoded linker can comprise a Whitlow linker. In an aspect, a Whitlow linker can comprise (GSTSGSGKSSEGKG)n, wherein n = 1 - 8 (SEQ ID NO:97 - SEQ ID NO: 104). In an aspect, a Whitlow linker can comprise (GSTSGSGKPGSGEGSTKG)n, wherein n = 1 - 8 (SEQ ID NO: 105 - SEQ ID NO: 112). Linkers are known to the art.

[0298] In an aspect of a disclosed vector, an encoded chimeric antigen receptor can further comprise a spacer or a hinge region between the extracellular domain and the transmembrane domain. In an aspect, 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.

[0299] In an aspect of a disclosed vector, an encoded CD8 hinge domain can comprise the sequence set forth in SEQ ID NO:24, SEQ ID NO:25, or a fragment thereof. In an aspect, a disclosed CD8 hinge domain can be encoded by the sequence set forth in SEQ ID NO:26 or a fragment thereof. In an aspect of a disclosed vector, 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). CDl lc (1TGAX), CDl ld (1TGAD). 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 (NKG2D). CD319 (SLAMF7). CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM). CD357 (TNFRSF18), inducible T cell co-stimulator (ICOS), LFA-1 (CD1 la / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2Ry, IL-7R alpha, LFA1-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, a CD83 ligand, Fey receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors. Toll ligand receptor, or any combination thereof, or any fragment / truncation thereof, or any combination of fragments / truncations thereof.

[0300] In an aspect of a disclosed vector, an encoded 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, TCRP, NKG2D, 2B4, PD1, or any combination thereof. In an aspect of a disclosed vector, 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. B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 100 (SEMA4D), CD 103, CD 160 (BY55), CD 18, CD 19, CD 19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DAP-12, DNAM1 (CD226), Fey y receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Iga (CD79a), IL-2RP , IL-2Ry y, IL-7Ra , inducible T cell costimulator (ICOS), integrins. ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, 1TGAX. ITGB2. 1TGB7, 1TGB1, KIRDS2. LAT. 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), SLAM (SLAMF1; 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 acombination thereof, or a fragment / truncation thereof, or a combination of fragments and / or truncations thereof.

[0301] In an aspect of a disclosed vector, an encoded CD8 transmembrane domain can comprise the sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or a fragment thereof. In an aspect, a disclosed CD8 transmembrane domain can be encoded by the sequence set forth in SEQ ID NO:29 or a fragment thereof. In an aspect of a disclosed vector, an encoded CD28 transmembrane domain can comprise the sequence set forth in SEQ ID NO:30 or a fragment thereof. In an aspect of a disclosed vector, an encoded NKG2D transmembrane domain can comprise the sequence set forth in SEQ ID NO: 31 or a fragment thereof. In an aspect, a disclosed NKG2D transmembrane domain can be encoded by the sequence set forth in SEQ ID NO:32 or a fragment thereof.

[0302] In an aspect of a disclosed vector, an encoded intracellular domain can be referred to synonymously as an intracellular signaling domain. Intracellular domains are discussed supra. In an aspect of a disclosed vector, an encoded intracellular domain can comprise one or more immunoreceptor tyrosine-based activation domains (ITAM). In an aspect, 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. In an aspect of a disclosed vector, an encoded intracellular region can comprise a CD3^ signaling domain. In an aspect, a disclosed CD3^ signaling domain can comprise the sequence set forth in SEQ ID NO:41. In an aspect, a disclosed CD3 signaling domain can comprise the sequence set forth in SEQ ID NO:42. In an aspect, a disclosed CD3q signaling domain can be encoded by the sequence set forth in SEQ ID NO:43. In an aspect, 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. In an aspect, a disclosed ITAM can be on the C-terminal end (distal to the transmembrane domain).

[0303] In an aspect of a disclosed vector, an encoded intracellular domain can comprise a gene cassette for one or more cytokines. In an aspect, a disclosed gene cassette for one or more cytokines can be operably linked to one or more regulatory elements. In an aspect, for example, a disclosed regulatory element can comprise a promoter or a transcription factor. In an aspect, a disclosed transcription factor can be nuclear factor of the activated T cell (NF AT) or a minimal promoter. In an aspect, a disclosed cytokine can comprise, for example, IL-7, IL- 12, IL-15, IL- 18. IL-23, or any combination thereof. In an aspect, 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).

[0304] In an aspect of a disclosed vector, an encoded intracellular domain can comprise one or more co-stimulatory domains or co-stimulatory signaling domains. In an aspect, the order of one or more co-stimulatory domains in a disclosed intracellular domain can be varied. In an aspect, the order of the one or more co-stimulatory domains can be varied and / or can influence the resulting effector function. For example, CD28-CD3^ can elicit higher level of IL-2. In an aspect, a disclosed co-stimulatory domain can be on the N -terminal end (proximal to the transmembrane domain).

[0305] In an aspect of a disclosed vector, an encoded intracellular domain can further comprise one or more co-stimulatory signaling domain or co-stimulatory domains. In an aspect, 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. In an aspect of a disclosed nucleic acid molecule, 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. CD9, CD96 (Tactile), CDS, CEACAML CRT AM, DAP-10, DNAM1 (CD226), Fey receptor, GADS. GITR. HVEM (LIGHTR), IA4, ICAM-1, ICOS, Iga (CD79a), IL2R0, IL2Ry, IL7Ra, integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1 (CDl la / CD18), LIGHT, LTBR, Ly9 (CD229), MHC class I molecule, NKD2C, NKG2D, NKp30, NKp44. NKp46, NKp80 (KLRF1). 0X40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocytic activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), 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.

[0306] In an aspect of a disclosed vector, 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.

[0307] In an aspect of a disclosed vector, an encoded intracellular domain can comprise one or more co-stimulatory domains. In an aspect, a disclosed co-stimulatory domain can comprise 4- 1BB. In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, a disclosed 4- IBB co-stimulatory domain can be encoded by the sequence set forth in SEQ ID NO:35 or a fragment thereof.

[0308] In an aspect of a disclosed vector, 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. 1

[0309] In an aspect of a disclosed vector, an encoded co-stimulatory domain can comprise CD28. In an aspect, a disclosed CD28 co-stimulatory domain can comprise the sequence set forth in SEQ ID NO:36. SEQ ID NO:37, or a fragment thereof. In an aspect, a disclosed CD28 co-stimulatory domain can be encoded by the sequence set forth in SEQ ID NO:38 or a fragment thereof. In an aspect of a disclosed vector, an encoded CD28 co-stimulatory domain can comprise the sequence set forth co-stimulatory7domain can comprise NKG2D. In an aspect, a disclosed NKG2D co- stimulatory domain can comprise the sequence set forth in SEQ ID NO:39 or a fragment thereof. In an aspect, a disclosed NK.G2D co-stimulatory domain can be encoded by the sequence set forth in SEQ ID NO:40 or a fragment thereof.

[0310] In an aspect of a disclosed vector, an encoded co-stimulatory' domain can comprise OX- 40. In an aspect, a disclosed OX-40 co-stimulatory domain can comprise the sequence set forth in SEQ ID NO: 147 or a fragment thereof. In an aspect, a disclosed OX-40 co-stimulatory domain can be encoded by the sequence set forth in SEQ ID NO: 148 or a fragment thereof.

[0311] In an aspect of a disclosed vector, an encoded co-stimulatory signaling domain can enhance the efficacy and expansion of immune cells expressing CAR receptors.

[0312] In an aspect, 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. In an aspect of a disclosed vector, an encoded intracellular domain can comprise a JAK-STAT activation domain. In an aspect, a disclosed JAK-STAT activation domain can be derived, for example, from I L -2 R|3 In an aspect, a disclosed JAK-STAT activation can stimulate cell proliferation, can prevent terminal differentiation, can perform better, or any combination. In an aspect of a disclosed vector, an encoded intracellular signaling domain and adisclosed co-stimulatory signaling domain can be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.

[0313] In an aspect of a disclosed nucleic acid molecule, 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.

[0314] In an aspect of a disclosed vector, an encoded intracellular domain can further comprise a self-cleaving peptide. In an aspect, a disclosed self-cleaving peptide can comprise a T2A, a P2A, a E2A, or F2A peptide. In an aspect, a GSG linker can be added to the N-terminus of a disclosed 2A peptide. In an aspect, a disclosed P2A peptide can comprise the sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 102 or a fragment thereof. In an aspect, a disclosed T2A peptide can comprise the sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104 or a fragment thereof. In an aspect, a disclosed E2A peptide can comprise the sequence set forth in SEQ ID NO: 105 or SEQ ID NO: 106 or a fragment thereof. In an aspect, a disclosed F2A peptide can comprise the sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108 or a fragment thereof.

[0315] In an aspect of a disclosed vector, 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 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.

[0316] In an aspect of a disclosed vector, 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. In an aspect of a disclosed vector, 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 morethan 95% identity to the sequence set forth in SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or a fragment thereof.

[0317] In an aspect of a disclosed vector, an encoded CAR (i) can stimulate an effector cell mediated immune modulator response to citrullinated-vimentin-expressing tumor cells; (ii) can induce a tumor reducing immune response; (iii) can induce phagocytosis of cancer cells in the subject; (iv) can cross-prime an anti -tumor T cell response; (v) can induce a tumor eliminating immune response; (vi) can treat cancer; or (vii) or any combination thereof.

[0318] Disclosed herein is 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.

[0319] Disclosed herein is 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.

[0320] Disclosed herein is 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.

[0321] Disclosed herein is 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.

[0322] Disclosed herein is 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.

[0323] Disclosed herein is 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.

[0324] Disclosed herein is 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.

[0325] In an aspect, a disclosed vector can be introduced to T cells and / or NK and / or macrophages or any other immune system. In an aspect, 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. In an aspect, 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.

[0326] In an aspect, disclosed herein are methods for making or generating immune effector cells that express one or more disclosed CARs using one or more disclosed vectors. For example, in an aspect, a disclosed polynucleotide and / or a disclosed vector can be introduced into a disclosed immune effector cell. In an aspect, 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. In an aspect, immune effector cells can be isolated from an individual in need thereof and can be genetically modified without further manipulation in vitro. In an aspect, genetically modified immune effector cells can be expanded and / or re-administered into the individual. In an aspect, 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.

[0327] In an aspect, disclosed immune effector cells can be genetically modified to express one or more disclosed CARs prior to being activated and stimulated to proliferate in vitro.

[0328] In an aspect, 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). In an aspect, 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. In an aspect, generating disclosed immune effector cells can comprise stimulating the immune effector cell and inducing the cells to proliferate by contacting the cell with antibodiesor antigen binding fragments that bind CD3 and antibodies or antigen binding fragments that bind to CD28; thereby generating a population of immune effector cells.

[0329] In an aspect, prior to in vitro manipulation or genetic modification of disclosed immune effector cells, the immune effector cells can be obtained from a subject. In an aspect, disclosed CAR-modified immune effector cells can comprise T cells. In an aspect, peripheral blood mononuclear cells (PBMCs) can be directly genetically modified to express one or more disclosed CARs using methods known to the skilled person in the art. For example, in an aspect, after isolation of PBMCs, T lymphocytes can be further isolated. In an aspect, 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. In an aspect, 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. In an aspect, 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.

[0330] In an aspect, T cells can be activated and expanded before or after genetic modification to express a CAR.

[0331] In an aspect, 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. In an aspect, 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. In an aspect, 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.

[0332] In an aspect, one or more disclosed vectors can be used in genetically modifying a donor population of immune effector cells. In an aspect, one or more disclosed vectors can encode the same CAR or can encode different CARs. In an aspect, 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.

[0333] Methods of manufacturing T cells for human therapy are know n in the art. In an aspect, disclosed T cells manufactured by the methods can provide improved adoptive immunotherapy compositions. In an aspect, disclosed T cell compositions manufactured by disclosed methods in contemplated herein are imbued with superior properties, including increased survival, expansionin the relative absence of differentiation, persistence in vivo and superior anti-exhaustion properties.

[0334] In an aspect, a disclosed vector can further comprise a polyadenylation sequence. In an aspect, a disclosed vector can further comprise a sequence for a promoter. In an aspect, a disclosed 3’ hemi intron can be recognized by nuclear splicing components within a host cell. In an aspect, a disclosed vector can further comprise a nuclear localization signal (NLS). In an aspect, a disclosed vector can further comprise one or more nuclear retention elements (NRE). NRE are known to the skilled person in the art. In an aspect, a disclosed NRE can comprise SIRLOIN or BORG.

[0335] In an aspect, a disclosed vector can comprise the sequence for one or more regulatory elements. In an aspect, 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). In an aspect, 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. For example, 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.

[0336] In an aspect of a disclosed vector, 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. In an aspect, a disclosed promoter can be a promoter / enhancer. In an aspect, a disclosed promoter for the disclosed nucleic acid molecule can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter / enhancer. In an aspect, 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. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter / enhancer can be used for constitutive and efficient expression of a disclosed gene. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences canbe a CMV promoter or a CMV promoter / enhancer. CMV promoters and CMV promoters / enhancers are well known to the art.

[0337] In an aspect, a disclosed vector can be an integrating vector or a non-integrating vector. In an aspect, 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). In an aspect, a disclosed vector can be a viral vector or a non-viral vector. In an aspect, 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.

[0338] In an aspect, a disclosed vector can comprise lipid and / or polymer-based nanoparticles loaded with mRNA encoding a disclosed CAR. In an aspect, 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. In an aspect, a disclosed vector can comprise mRNA encoding a disclosed CAR. In an aspect, a disclosed vector can comprise lipid and / or polymer-based nanoparticles loaded with mRNA encoding a disclosed CAR.

[0339] In an aspect, 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. In an aspect, a disclosed viral vector can be an adeno-associated vims (AAV) vector In an aspect, 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. AAVcy.7 as well as bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, non-primate AAV, and any other virus classified by the International Committee on Taxonomy of Viruses (ICTV) as an AAV. In an aspect, 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. In an aspect, a disclosed AAV vector can be AAV-Rh74 or a related variant (e.g., capsid variants like RHM4-1). In an aspect, a disclosed AAV vector can be a self-complementary AAV as disclosed herein.

[0340] In an aspect, 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.

[0341] In an aspect, a disclosed promoter can comprise a ubiquitous promoter, a constitutive promoter, or a tissue specific promoter. In an aspect, a disclosed promoter can be operably linked to a disclosed nucleic acid sequence encoding a disclosed CAR. In an aspect, a disclosed promoter can be operably linked to a disclosed nucleic acid sequence encoding a disclosed tumor antigen. Promoters are known to the art. In an aspect, a disclosed promoter can be a promoter / enhancer. Promoter / enhancers are know n to the art. In an aspect, a disclosed promoter can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter / enhancer. In an aspect, a disclosed promoter or a disclosed promoter / enhancer can be used for constitutive and efficient expression of a disclosed CAR. In an aspect, a disclosed promoter or a disclosed promoter / enhancer can be used for constitutive and efficient expression of a disclosed tumor antigen. In an aspect, a disclosed vector can be used to engineer cells to express a disclosed CAR.

[0342] In an aspect, a disclosed vector can further comprise a nucleic acid encoding a second CAR that is specific for a tumor antigen. In an aspect, 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. CD171, CEA, CSPG4, CLL-1, CS1, EGFR, EGFR family including ErbB2 (HERII), EGFRvIll, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, Flt3, GD2, GD3, Glypican-3 (GPC3), HLAA1+MAGEI, HLA- A2+MAGE1, HLAA3+MAGE1, HLA-A1+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NYES0- 1, IL-l lRa, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Mud, Mucl6. NCAM, NKG2D Ligands, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TACI, TAG72, TEMs, VEGFRII, or any combination thereof.

[0343] In an aspect, a disclosed vector can be a transposon-based vector such as Sleeping Beauty and PiggyBac, both of which are known in the art. In an aspect, 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. Then 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. In an aspect, using thisvector, stable integration and reliable long-term expression of the disclosed CAR sequence can be achieved.

[0344] In an aspect, a disclosed vector can stimulate an effector cell-mediated immune modulator response to cVIM-expressing tumor cells. In an aspect, a disclosed vector can induce a tumor reducing immune response. In an aspect, a disclosed vector can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed vector can cross-prime an anti -tumor T cell response. In an aspect, a disclosed vector can induce a tumor eliminating immune response. In an aspect, a disclosed vector can treat cancer.

[0345] In an aspect, 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 ).

[0346] In an aspect, a disclosed vector can be a recombinant vector.4. Plasmids

[0347] Disclosed herein is a plasmid comprising a disclosed nucleic acid molecule.

[0348] Disclosed herein is a plasmid comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising the scFV of Ab#4 as described in the Examples.

[0349] In an aspect, a disclosed plasmid can be used to introduce a disclosed nucleic acid molecule or disclosed nucleic acid sequence to one or more host cells. In an aspect, host cells are discussed infra and can comprise T cells, NK cells, macrophages, or iPSCs. In an aspect, 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. In an aspect, 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. In an aspect, 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. In an aspect, a disclosed plasmid can be an integrating vector or a non-integrating vector. In an aspect, 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). In an aspect, a disclosed plasmid can be a recombinant vector comprising a disclosed nucleic acid sequence. Recombinant plasmids are known to the art.

[0350] In an aspect, a disclosed promoter can comprise a ubiquitous promoter, a constitutive promoter, or a tissue specific promoter. In an aspect, a disclosed promoter can be operably linkedto a disclosed nucleic acid sequence encoding a disclosed CAR. In an aspect, a disclosed promoter can be operably linked to a disclosed nucleic acid sequence encoding a disclosed tumor antigen. Promoters are known to the art. In an aspect, a disclosed promoter can be a promoter / enhancer. Promoter / enhancers are known to the art. In an aspect, a disclosed promoter can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter / enhancer. In an aspect, a disclosed promoter or a disclosed promoter / enhancer can be used for constitutive and efficient expression of a disclosed CAR. In an aspect, a disclosed promoter or a disclosed promoter / enhancer can be used for constitutive and efficient expression of a disclosed tumor antigen. In an aspect, a disclosed plasmid can be used to engineer cells to express a disclosed CAR.

[0351] In an aspect, a disclosed plasmid can further comprise a nucleic acid encoding a second CAR that is specific for a tumor antigen. In an aspect, 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, CD171, CEA, CSPG4, CLL-1, CS1, EGFR, EGFR family including ErbB2 (HERB), EGFRvIll, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, Flt3, GD2, GD3, Glypican-3 (GPC3), HLAA1+MAGEI, HLA- A2+MAGE1, HLAA3+MAGE1, HLA-A1+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NYES0- 1, IL-l lRa, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Mud, Mucl6. NCAM, NKG2D Ligands, NYE-S0-1, PRAME, PSCA, PSMA, RORL SSX, Survivin, TACI, TAG72, TEMs, VEGFRII, or any combination thereof.

[0352] In an aspect, a disclosed vector can be a viral vector or a non-viral vector. In an aspect, 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.

[0353] In an aspect, a disclosed plasmid can comprise a transposon such as, for example, Sleeping Beauty and PiggyBac. both of which are known in the art. In an aspect, 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). 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. Then 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. In an aspect, using this vector, stable integration and reliable long-term expression of the disclosed CAR sequence can be achieved.

[0354] In an aspect, a disclosed plasmid can stimulate an effector cell mediated immune modulator response to PS-expressing tumor cells. In an aspect, a disclosed plasmid can induce a tumor reducing immune response. In an aspect, a disclosed plasmid can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed plasmid can cross-prime an anti-tumor T cell response. In an aspect, a disclosed plasmid can induce a tumor eliminating immune response. In an aspect, a disclosed plasmid can treat cancer.5. Cells

[0355] 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).

[0356] Disclosed herein are cells transformed or transfected by 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.

[0357] Disclosed herein are cells transformed or transfected by 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.

[0358] Disclosed herein are cells transformed or transfected by 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.

[0359] 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).

[0360] Disclosed herein are cells transduced by 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. Disclosed herein are cells transduced by 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.

[0361] Disclosed herein are cells transduced by 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. Disclosed herein are cells transduced by 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. Disclosed herein are cells transduced by 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. Disclosed herein are cells transduced by 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.

[0362] Disclosed herein are cells transduced by a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR comprising the sequence set forth in SEQ ID NO:01, SEQ ID NO:02, or SEQ ID NO:03. Disclosed herein are cells transduced by a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR comprising the sequence set forth in SEQ ID NO:05, SEQ ID NO:06, or SEQ ID NO:07. Disclosed herein are 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 SEQID NO:01. SEQ ID NO:02, or SEQ ID NO:03. Disclosed herein are 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.

[0363] Disclosed herein are cells transduced by 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. Disclosed herein are cells transduced by 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.

[0364] Disclosed herein is 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. In an aspect, 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. Disclosed herein is an engineered T cell expressing a disclosed chimeric antigen receptor. Disclosed herein is an engineered cytotoxic T lymphocyte expressing a disclosed chimeric antigen receptor. Disclosed herein is an engineered natural killer T lymphocyte cell expressing a disclosed chimeric antigen receptor. Disclosed herein is an engineered macrophage expressing a disclosed chimeric antigen receptor. Disclosed herein is an engineered natural killer cell expressing a disclosed chimeric antigen receptor.

[0365] In an aspect, a disclosed cell can be transduced and / or transfected with one or more recombinant nucleic acid molecules and / or one or more recombinant vectors. In an aspect, cells engineered to expression one or more disclosed CARs can comprise T cells or NK cells or macrophages. In an aspect, disclosed cells engineered to expression one or more disclosed CARs can be immune cells. In an aspect, 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. In an aspect, disclosed T cells and NK cells can be differentiated in vitro from a hematopoietic stem cell population (for example iPSCs) or can be obtained from asubject. In an aspect, 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. In an aspect, disclosed T cells can be derived from one or more T cell lines available in the art. In an aspect, 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.

[0366] In an aspect, a disclosed cell can be transformed or transfected by a nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed CAR. In an aspect, a disclosed cell can be transduced by a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed CAR. In an aspect, following transformation, transfection, and / or transduction, a disclosed cell can express a disclosed CAR. In an aspect, following transformation, transfection, and / or transduction, disclosed host cells expressing a disclosed CAR and / or a disclosed tumor antigen can be administered to a subject. In an aspect, 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.

[0367] In an aspect, a disclosed cell can stimulate an effector cell-mediated immune modulator response to cVIM-expressing tumor cells. In an aspect, a disclosed cell can induce a tumorreducing immune response. In an aspect, a disclosed cell can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed cell can cross-prime an anti -tumor T cell response. In an aspect, a disclosed cell can induce a tumor-eliminating immune response. In an aspect, a disclosed cell can treat cancer.

[0368] In an aspect, a therapeutically effective amount of the disclosed genetically modified cells expressing a disclosed chimeric antigen receptor targeting citrullinated vimentin (cVIM) (e.g., T cells, NK cells, and / or macrophages) can be about 1 x 104to about 1 x 109cells / kg per subject. In an aspect, a therapeutically effective amount of the disclosed genetically modified cells expressing a disclosed chimeric antigen receptor targeting citrullinated vimentin (cVIM) (e.g., T cells, NK cells, and / or macrophages) can be about 1 x 105cells / kg per subject, about 1 x 106cells / kg per subject, about I x 107cells / kg per subject, about 1 x 108cells / kg per subject, or about 1 x 109cells / kg per subject.

[0369] In an aspect, 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.6. Pharmaceutical Formulations

[0370] Disclosed herein is a pharmaceutical formulation comprising a disclosed nucleic acid molecule; and one or more pharmaceutically acceptable carriers. Disclosed herein is apharmaceutical formulation comprising a disclosed vector; and one or more pharmaceutically acceptable carriers. Disclosed herein is a pharmaceutical formulation comprising a disclosed plasmid; and one or more pharmaceutically acceptable carriers. Disclosed herein is a pharmaceutical formulation comprising a disclosed cell; and one or more pharmaceutically acceptable carriers.

[0371] Disclosed herein is a pharmaceutical formulation comprising a nucleic acid molecule comprising a nucleic acid sequence encoding any disclosed CAR. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR specific for citrullinated vimentin (cVIM). Disclosed herein is 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.

[0372] Disclosed herein is a pharmaceutical formulation comprising a vector comprising a disclosed nucleic acid sequence. Disclosed herein is a pharmaceutical formulation comprising a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding any disclosed CAR. Disclosed herein a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR specific for citrullinated vimentin (cVIM).

[0373] Disclosed herein is a pharmaceutical formulation comprising a plasmid comprising a disclosed nucleic acid molecule. Disclosed herein is 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. Disclosed herein is a pharmaceutical formulation comprising a plasmid comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR specific for citrullinated vimentin (cVIM). Disclosed herein is 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.

[0374] In an aspect, 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.

[0375] In an aspect, 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.

[0376] In an aspect, NSAIDs can comprise ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as rofecoxib and celecoxib, sialylates, or any combination thereof. In an aspect, analgesics can comprise acetaminophen, oxycodone, tramadol, proporxyphene hydrochloride, or any combination thereof. In an aspect, glucocorticoids can comprise cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, or any combination thereof. Exemplary 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. In an aspect, biological response modifiers can comprise monoclonal antibodies as well as recombinant forms of molecules. In an aspect, exemplary disease-modifying anti-rheumatic drugs (DMARDs) can comprise include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), minocycline, or any combination thereof.

[0377] In an aspect, 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. In an aspect, 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 liposome injection (DepoCyt), cytarabine, cytosine arabinoside (Cytosar-U), dacarbazine (DTIC- Dome), dactinomycin (Cosmegen), daunorubicin citrate liposome injection (DaunoXome), daunorubicin hydrochloride (Cerubidine), dexamethasone, docetaxel (Taxotere), doxorubicin hydrochloride (Adriamycin, Rubex), etoposide (Vepesid). 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 carmustine implant (Gliadel), purine analogs and adenosine deaminase inhibitors (fludarabine), pyrimidine analogs, rituximab, tamoxifen citrate (Nolvadex), temozolomide), teniposide (Vumon), tezacitibine. 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.

[0378] In an aspect, a disclosed pharmaceutical formulation can comprise an anti-chemokine therapy that enhances the resident memory T cell formations in tumor-free tissues. In an aspect, 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.

[0379] In an aspect, 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, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, namatumab, naptumomab, necitumumab, nimotuzumab, nofetumomab, ocaratuzumab. ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab, pamtumumab. parsatuzumab, patritumab. pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab, rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab, siltuximab, simtuzumab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, vorsetuzumab. votumumab, zalutumumab, CC49, 3F8, or any combination thereof.

[0380] In an aspect, a disclosed pharmaceutical formulation can stimulate an effector cell mediated immune response to cVIM-expressing tumor cells. In an aspect, a disclosedpharmaceutical formulation can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation in a subject having cancer. In an aspect, metabolic dysregulation can be associated with cancer or cancerous cells. In an aspect, following administration of a disclosed pharmaceutical formulation, cell death of citrullinated vimentin (cVIM)-expressing cancer cells is effected.

[0381] In an aspect, a disclosed pharmaceutical formulation can be prepared for systemic or direct administration. In an aspect, a disclosed pharmaceutical formulation can be prepared for oral administration, intravenous administration, intra-tumoral administration, intraperitoneal administration, or any combination thereof. In an aspect, a disclosed pharmaceutical formulation can be prepared for any method of administration disclosed herein. In an aspect, a disclosed pharmaceutical formulation can be prepared for administration via multiple routes either concurrently or sequentially. For example, in an aspect, a disclosed pharmaceutical formulation can be first administered intratumorally and then be administered intravenously. In an aspect, 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.

[0382] In an aspect, a disclosed pharmaceutical formulation can comprise one or more immune modulators. In an aspect, a disclosed pharmaceutical formulation can comprise one or more proteasome inhibitors. In an aspect, a disclosed pharmaceutical formulation can comprise one or more immunosuppressives or immunosuppressive agents. In an aspect, an immunosuppressive agent can be anti-thymocyte globulin (ATG), cyclosporine (CSP), my cophenolate mofetil (MMF), or a combination thereof. In an aspect, a disclosed pharmaceutical formulation can comprise an anaplerotic agent (such as, for example. C7 compounds like triheptanoin or MCT).

[0383] In an aspect, a disclosed pharmaceutically acceptable carrier can comprise any disclosed carrier and / or any disclosed excipient.

[0384] In an aspect, a disclosed pharmaceutical formulation can stimulate an effector cell mediated immune modulator response to citrullinated vimentin (cVIM)-expressing tumor cells. In an aspect, a disclosed pharmaceutical formulation can induce a tumor reducing immune response. In an aspect, a disclosed pharmaceutical formulation can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed pharmaceutical formulation can cross-prime an antitumor T cell response. In an aspect a disclosed pharmaceutical formulation can induce a tumor eliminating immune response. In an aspect, a disclosed pharmaceutical formulation can treat cancer.

[0385] In an aspect, 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.).7. Animals

[0386] Disclosed herein are animals used to validate the efficacy and / or safety of 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. In an aspect, 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. In an aspect, 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. In an aspect, a disclosed treated subject can be a mouse or a rat. In an aspect, a disclosed treated subject can be a transgenic mouse or a transgenic rat. In an aspect, a disclosed treated subject can have one or more types of cancers and / or tumors.C. Methods of Treating Cancer and / or Slowing Disease Progression

[0387] Disclosed herein is a method of treating cancer, 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 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 treating cancer, 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. Disclosed herein is a method of treating cancer, 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. Disclosed herein is a method of treating cancer, 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 treating cancer, 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 adisclosed plasmid or a pharmaceutical formulation thereof, wherein the one or more cells express a CAR. Disclosed herein is a method of treating cancer, 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. Disclosed herein is a method of treating cancer, 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)-expressing tumor cells or a pharmaceutical formulation thereof. 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 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. 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 genetically modified cells expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof. 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. 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. 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 expressing a chimeric antigen receptor targeting citrullinated vimentin (cVIM) or a pharmaceutical formulation thereof. 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 expressing achimeric antigen receptor targeting citrullinated vimentin (cVIM)-expressing tumor cells or a pharmaceutical formulation thereof.

[0388] In an aspect, a disclosed CAR can target citrullinated vimentin (cVIM)-expressing cancer cells. In an aspect, a disclosed CAR can target citrullinated vimentin (cVIM)-expressing ovarian cancer cells. In an aspect, a disclosed CAR can target citrullinated vimentin (cVIM)-expressing colon cancer cells. In an aspect, a disclosed CAR can target citrullinated vimentin (cVIM)- expressing lung cancer cells. In an aspect, disclosed citrullinated vimentin (cVIM)-expressing cancer cells can be in a tumor. In an aspect, disclosed citrullinated vimentin (cVIM)-expressing cancer cells can be in one or more tumors. In an aspect, disclosed cancer cells can be blood borne.

[0389] In an aspect, a disclosed cell can express a CAR targeting cVIM and at least one CAR targeting another cancer cell antigen. In an aspect, a disclosed cell can express one or more CARs (e.g. targeting cVIM and targeting one or more other cancer cell antigens).

[0390] In an aspect, 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. In an aspect, a disclosed method of treating cancer and / or slowing disease progression can induce a tumor reducing immune response. In an aspect, a disclosed method of treating cancer and / or slowing disease progression can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed method of treating cancer and / or slowing disease progression can cross-primer an antitumor T cell response. In an aspect, a disclosed method of treating cancer and / or slowing disease progression can induce a tumor eliminating immune response. In an aspect, a disclosed method of treating cancer and / or slowing disease progression can effect tumor cell death.

[0391] In an aspect, 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. In an aspect, the cells can be autologous to the subject.

[0392] In an aspect, 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 cellcarcinoma, papillary renal cell carcinoma, colon cancer, small bowel cancer, small bowel adenocarcinoma, adenocarcinoma of the ileum, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, testicular seminoma, testicular teratoma, embryonic testicular cancer, uterine cancer, teratocarcinoma, embryonal carcinoma, or any combination thereof.

[0393] Thus, in an aspect, 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 embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, testicular seminoma, testicular teratoma, embryonic testicular cancer, uterine cancer, teratocarcinoma, embryonal carcinoma, or any combination thereof. In an aspect, a subject can have ovarian cancer, colon cancer, lung cancer, or any combination.

[0394] In an aspect, 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. For example, in an aspect, a blood sample and / or a biological sample can be collected from a subject at a pre-determined interval. In an aspect, 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. In an aspect, 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. In an aspect, 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. In an aspect, 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.

[0395] In an aspect, a subject can be a human patient. In an aspect 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. In an aspect, a subject can be treatment-naive. Or, in an aspect, a subject can have had treatment. In anaspect, a subject can have a terminal cancer. In an aspect, a disclosed subject can have metastatic cancer or onco-metastatic cancer.

[0396] In an aspect, 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. In an aspect, 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. In an aspect, 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. In an aspect, 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. In an aspect, a disclosed method of treating cancer and / or slowing disease progression can further comprise subjecting a disclosed blood sample to centrifugation. In an aspect, 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. In an aspect, 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. In an aspect, a disclosed red layer can comprise red blood cells. In an aspect, a disclosed clear layer can comprise plasma. In an aspect, a disclosed thin layer in between the red layer and the clear layer can comprise the buffy coat. In an aspect, a disclosed buffy coat can comprise white blood cells and platelets. In an aspect, a disclosed method can further comprise isolating peripheral blood mononuclear cells (PMBCs) from the buffy coat. In an aspect, PMBCs can comprise lymphocytes, leukocytes, and / or monocytes. In an aspect, macrophages can be derived from monocytes. In an aspect, isolating lymphocytes, leukocytes, and / or monocytes can be done by any method and / or technique known to the skilled person (e.g., leukapheresis).

[0397] In an aspect, a disclosed method of treating cancer and / or slowing disease progression can further comprise isolating resting or MO macrophages from the buffy7coat. In an aspect, a disclosed method of treating cancer and / or slowing disease progression can further comprise differentiating monocytes into resting or MO macrophages. In an aspect of a disclosed method of treating cancer and / or slowing disease progression, the disclosed macrophages can be resting or MO macrophages. In an aspect, 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. In an aspect, 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.

[0398] In an aspect, a therapeutically effective amount or effective dose or effective amount or therapeutically effectiv...

Claims

VIII. CLAIMSWhat is claimed is:

1. A chimeric antigen receptor (CAR), comprising: an extracellular domain comprising a citrullinated vimentin (cVIM) antigen binding domain, a transmembrane domain; and an intracellular domain comprising one or more immunostimulatory domains.

2. The CAR of Claim 1, wherein the citrullinated vimentin (cVIM) antigen binding domain comprises a scFv.

3. The CAR of Claim 2, wherein the scFv comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and the VL are separated by a spacer.

4. The CAR of Claim 2, wherein the scFV comprises the sequence set forth in any one SEQ IDNO: 149-SEQ ID NO: 154.

5. The CAR of Claim 3, wherein the VH comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 13.

6. The CAR of Claim 3, wherein the VL comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 15.

7. The CAR of Claim 3, wherein the VH comprises three complementarity determining regions(CDRs).

8. The CAR of Claim 7, wherein the first heavy chain CDR (VH-CDR1) comprises the sequence set forth in SEQ ID NO: 18, wherein the second heavy chain CDR (VH-CDR2) comprises the sequence set forth in SEQ ID NO: 19, and wherein the third heavy chain CDR (VH- CDR3) comprise the sequence set forth in SEQ ID NO:20.

9. The CAR of Claim 3, wherein the VL comprises three complementarity determining regions(CDRs).

10. The CAR of Claim 9, wherein the first light chain CDR (VL-CDR1) comprises the sequence set forth in SEQ ID NO:21, wherein the second light chain CDR (VL-CDR2) comprises the sequence set forth in SEQ ID NO:22, and wherein the third light chain CDR (VL- CDR3) comprise the sequence set forth in SEQ ID NO:23.

11. The CAR of Claim 1, wherein the CAR further comprises a hinge domain between the extracellular domain and the transmembrane domain.

12. The CAR of Claim 1, wherein the CAR comprises a sequence having at least 80% identity’ to the sequence set forth in any one of SEQ ID NO: 129-SEQ ID NO: 134.

13. The CAR of Claim 1, wherein the transmembrane domain comprises the transmembranedomain of CD8, and wherein the CD8 transmembrane domain comprises the sequence set forth in SEQ ID NO:27.

14. The CAR of Claim 1, wherein the transmembrane domain comprises the transmembrane domain of NKG2D, and wherein the NKG2D transmembrane domain comprises the sequence set forth in SEQ ID NO:31.

15. The CAR of Claim 1, wherein the intracellular domain comprises one or more co-stimulatory domains.

16. The CAR of Claim 1 , wherein the one or more co-stimulatory domains comprise the signaling domain of CD8, 4-1BB, or NKG2D.

17. The CAR of Claim 1, wherein the intracellular domain further comprises a CD3^ domain.

18. The CAR of Claim 18, wherein the CD3^ domain comprises the sequence set forth in SEQ IDNO:41 or SEQ ID NO:42.

19. The CAR of Claim 1, wherein the intracellular domain comprises a CD8 signaling domain and a CD3 domain.

20. The CAR of Claim 1, wherein the intracellular domain comprises a 4- IBB signaling domain and a CD3 domain.

21. A nucleic acid molecule encoding the CAR of any preceding claim.

22. The nucleic acid molecule of Claim 21, comprising a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:02, SEQ ID NO:04, SEQ ID NO:06, SEQ IDNO:08, SEQ ID NO: 10, or SEQ ID NO: 12.

23. A vector comprising the nucleic acid molecule of Claims 21 or 22.

24. An immune cell expressing the CAR of any one of Claims 1-20.

25. An immune cell that has been (i) transfected by the nucleic acid molecule of Claim 21 orClaim 22, or (ii) transduced by the vector of Claim 23.

26. A population of immune cells that have been (i) transfected by the nucleic acid molecule ofClaim 21 or Claim 22, or (ii) transduced by the vector of Claim 23.

27. The immune cell of Claim 25 or the population of immune cells of Claim 26, wherein the immune cell comprises a T cell, an NK cell, a macrophage, or any combination thereof, or wherein the population of immune cells comprise T cells, NK cells, macrophages, or any combination thereof.

28. A pharmaceutical formulation comprising the immune cell of Claim 25 or the population of immune cells of Claim 26. and one or more pharmaceutically acceptable earners.

29. A method of treating cancer and / or slowing disease progression, the method comprising: administering to a subject in need thereof a therapeutically effective amount of thepharmaceutical formulation of Claim 28, wherein, following administration, an effector cell mediated immune modulator response to cVIM-expressing tumor cells is stimulated.

30. The method of Claim 29, wherein administering comprises oral administration, intravenous administration, intra-tumoral administration, intraperitoneal administration, or any combination thereof.

31. The method of Claim 29, further comprising administering to the subject one or more additional anti-cancer therapies.

32. The method of Claim 31, wherein the one or more anti-cancer therapies comprises endocrine therapy, radiotherapy, hormone therapy, gene therapy, thermal therapy, ultrasound therapy, or any combination thereof.

33. The method of Claim 31, further comprising administering to the subject one or more chemotherapeutic agents.

34. The method of any one of Claims 29-33, wherein the subject has been diagnosed with 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, papillary7renal cell carcinoma, colon cancer, small bowel cancer, small bowel adenocarcinoma, adenocarcinoma of the ileum, testicular embry onal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, testicular seminoma, testicular teratoma, embryonic testicular cancer, uterine cancer, teratocarcinoma, embryonal carcinoma, or any combination thereof.

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