Methods for treating systemic sclerosis using Anti-CD19 car-t cell therapies

Abstract

Provided herein are methods and compositions for treating a subject having systemic sclerosis using T cells engineered with a chimeric antigen receptor that binds CD19.

Description

Attorney Docket No. KVN-011WO METHODS FOR TREATING SYSTEMIC SCLEROSIS USING ANTI-CD19 CAR-T CELL THERAPIES RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No.63 / 587,991, filed on October 4, 2023; U.S. Provisional Patent Application No.63 / 589,588, filed on October 11, 2023; U.S. Provisional Patent Application No. 63 / 597,233, filed on November 8, 2023; and U.S. Provisional Patent Application No. 63 / 632,949, filed on April 11, 2024, the entire contents of each of which are incorporated by reference herein for all purposes. SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on October 2, 2024, is titled KVN-011WO_SL.xml and is 53,575 bytes in size. TECHNICAL FIELD

[0003] The present disclosure generally relates to T cells engineered to express chimeric antigen receptors (CARs) (e.g., fully human anti-CD19 CARs) and their use in the treatment and / or prevention of systemic sclerosis. BACKGROUND

[0004] Systemic sclerosis (SSc), also called scleroderma, is an immune-mediated rheumatic disease that is characterized by fibrosis of the skin and internal organs and vasculopathy. It is classified based on the extent of skin involvement and the associated pattern of clinical manifestations. SSc is associated with high morbidity and mortality (10- year mortality rate of 23% to 45%). Despite recent evidence of some improvements in patient survival, the mortality remains greater than for any other rheumatic disease. SSc is rare and, like many systemic autoimmune diseases, more common in women. The overall incidence rate globally is estimated to be 8 to 56 per million persons / year and the prevalence between 38 to 341 cases per million persons.Attorney Docket No. KVN-011WO

[0005] There are different SSc subtypes associated with different patterns of involvement and prognosis, including limited and diffuse subtypes. Limited cutaneous systemic sclerosis (lcSSc) affects areas below, but not above, the elbows and knees with or without involvement of the face. Diffuse cutaneous SSc (dcSSc) also affects the skin above the elbows and knees, e.g., upper arms, thighs, and / or trunk. Compared with patients with lcSSc, dcSSc patients are more likely to experience rapid progression of skin thickening and to have higher risk of pulmonary, cardiac, renal, and gastrointestinal complications including lung fibrosis, pulmonary arterial hypertension (PAH), renal failure, and esophageal / intestinal dysmotility. Accordingly, patients with limited cutaneous systemic sclerosis have a better prognosis than those with the diffuse form. Regardless, death in all cases is most often caused by lung, heart, and kidney involvement. An increase in the risk of cancer has also been observed.

[0006] Patients with diffuse skin and / or severe inflammatory organ involvement are usually treated aggressively with systemic immunosuppressive therapy because of the increased risk of complications and organ failure. This includes patients with (i) diffuse skin involvement that is severe or progressive; (ii) interstitial lung disease (ILD); (iii) myocarditis, and (iv) severe inflammatory myopathy and / or arthritis. The primary aim of current immunosuppressive therapy is merely to reduce progression or severity of SSc complications. Treatment is started as early as possible in the disease course to slow disease progression before further damage occurs. Immunosuppressive drugs have been tested and used for clinical management with only modest effect and with significant toxicity. Hematopoietic stem cell transplantation has been reported to provide benefit but carries severe treatment- related morbidity and mortality. Nintedanib was recently approved to slow the rate of decline in pulmonary function in patients with SSc associated interstitual lung disease (SSc-ILD), but the effect was modest and no benefit was observed in skin based on the secondary endpoint of modified Rodnan skin score (mRSS). Tocilizumab was also recently approved for the prevention of progression of SSc-ILD, but the primary endpoint of improvement in skin tightening was not met. The etiology and pathogenesis of SSc are poorly understood. As a result, treatment of these conditions is difficult, incomplete, and not curative, and no treatment is currently approved for the condition. There remains a need for effective therapies to treat SSc.Attorney Docket No. KVN-011WO SUMMARY

[0007] The present disclosure encompasses the recognition that B cells play a role in systemic sclerosis (SSc). Indeed, B cell infiltration has been observed in tissue samples from patients with SSc associated interstitial lung disease (SSC-ILD). Further, B cells have been found in the involved skin of patient with early diffuse SSc and correlated with skin symptom progression. Moreover, prospective observational case-control studies of rituximab, a chimeric monoclonal antibody against CD20, in SSc have supported the potential of B cell depletion to stabilize or improve lung function in patients with SSc-ILD and to improve skin fibrosis. More recently, a blinded controlled trial in patients with SSc demonstrated that rituximab treatment led to statistically significant improvement in the skin and in lung function compared with placebo at 6 months, strongly supporting the role of B cells in SSc disease pathogenesis and the therapeutic potential of effective B cell depletion.

[0008] The present disclosure appreciates that while B cell depletion with anti-CD20 antibodies such as rituximab has demonstrated enough efficacy to support the B cell driven- nature of SSc, there are notable drawbacks to using such therapeutic approaches. In particular, the efficacy observed when using anti-CD20 antibodies to treat SSc is variable, and in many cases there is a failure to maintain disease control over time. The present disclosure recognizes that this variable efficacy and failed sustained response is at least in part due to the inability of anti-CD20 therapy to adequately deplete B cells residing in lymphoid tissues outside of the peripheral circulation. The present disclosure encompasses the recognition that a deeper, more complete depletion of B cells, including the disease- driving pathogenic B cells, may provide the prospect of longer-term disease control. The present disclosure provides, among other things, compositions and methods including CD19- targeted CAR T-cell therapy to induce deep B cell depletion in the circulation for the treatment of refractory SSc.

[0009] Provided herein are methods and compositions for treating a subject with systemic sclerosis. Notably, provided herein are methods and compositions for treating a subject with systemic sclerosis for which conventional therapeutic regimens (e.g., corticosteroids, immunomodulators (e.g., immunosuppressants), etc.) have been shown to be ineffective or intolerable (e.g., due to their toxicity profiles or limitations in their pharmacological action, e.g., inability to treat or prevent one or more clinical manifestations of SSc). In some embodiments, conventional therapeutic regimens (i.e., standard of care (SOC) therapy, such as corticosteroids, immunomodulators, etc.) are withdrawn so that patients are free of anyAttorney Docket No. KVN-011WO supportive drugs for a duration of time (e.g., at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 8 months, 1 year, 2 years, 3 years, or more) after receiving provided methods and compositions. In some embodiments, conventional therapeutic regimens (i.e., SOC therapy, such as corticosteroids, immunomodulators, etc.) are withdrawn ahead of collecting host cells (e.g., lymphocytes, such as T cells) to be engineered by any method described herein (e.g., at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 day, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, or more before collecting host cells). Further provided herein are methods and compositions for treating a subject with systemic sclerosis, where said methods and compositions are used in place of known conventional therapeutic regimens. In some embodiments, the systemic sclerosis is limited cutaneous systemic sclerosis, or diffuse cutaneous systemic sclerosis. In some embodiments, the systemic sclerosis is limited cutaneous systemic sclerosis. In some embodiments, the systemic sclerosis is diffuse cutaneous systemic sclerosis. In some embodiments, the systemic sclerosis is refractory systemic sclerosis. In some embodiments, the present disclosure provides for methods and compositions that result in elimination of B cells at tissue sites (e.g., in a subject with systemic sclerosis) normally not accessible to, or targeted by, conventional systemic sclerosis treatments.

[0010] The methods of the present disclosure use T cells engineered to express anti-CD19 CAR constructs, which can reduce or deplete B cells responsible for one or more clinical symptoms of systemic sclerosis. In many embodiments, the anti-CD19 CAR constructs have a lower toxicity profile as compared to conventional treatment (e.g., the anti-CD19 CAR constructs results in fewer adverse side effects as compared to conventional treatment). In some embodiments, an anti-CD19 CAR is substantially non-toxic to a subject receiving treatment with the CAR therapy. In some embodiments, such low toxicity or non-toxic CAR therapies provided by the present disclosure allow for higher doses and / or multiple doses (e.g., chronic administration) which result in depletion of B cells at sites not treatable with conventional autoimmune treatments due to their toxicity profile. Indeed, CAR therapies provided herein remarkably exhibit low levels of toxicity commonly associated with CAR therapy (e.g., anti-CD19 CAR therapy), including cytokine-release syndrome (CRS) and neurologic toxicities. In some embodiments, the present disclosure provides methods of and compositions for treating systemic sclerosis (e.g., limited cutaneous systemic sclerosis, diffuse cutaneous systemic sclerosis, etc.) with an anti-CD19 CAR. In many embodiments of any of the provided methods and compositions described herein, an anti-CD19 CAR comprises a fully human chimeric antigen receptor comprising an extracellular antigen-Attorney Docket No. KVN-011WO binding domain, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises a cytoplasmic signaling domain and one or more co- stimulatory domains. See, e.g., U.S. Patent No.10,287,350, which is incorporated by reference herein in its entirety.

[0011] The present disclosure provides, among other things, a method of treating systemic sclerosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of T cells that comprise a vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises, from N-terminus to C-terminus: (a) an antigen-binding fragment of an anti-CD19 antibody; (b) a transmembrane domain; and (c) an intracellular T cell signaling domain from human CD3ζ.

[0012] In some embodiments, an anti-CD19 antibody is a human antibody.

[0013] In some embodiments, an antigen-binding fragment of the anti-CD19 antibody comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0014] In some embodiments, an antigen-binding fragment of the anti-CD19 antibody comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 25, 26, and 3, respectively, and the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0015] In some embodiments, a heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 7, and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 8. In some embodiments, a heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 8.

[0016] In some embodiments, an antigen-binding fragment of the anti-CD19 antibody comprises the amino acid sequence of SEQ ID NO: 17.

[0017] In some embodiments, a transmembrane domain is from human CD8.

[0018] In some embodiments, a transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 11. In some embodiments, a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 11.Attorney Docket No. KVN-011WO

[0019] In some embodiments, an intracellular T cell signaling domain from human CD3ζ comprises an amino acid sequence at least 90% identical to SEQ ID NO: 23. In some embodiments, an intracellular T cell signaling domain from human CD3ζ comprises the amino acid sequence of SEQ ID NO: 23.

[0020] In some embodiments, a CAR further comprises an intracellular T cell signaling domain from human CD28. In some embodiments, an intracellular T cell signaling domain from human CD28 comprises the amino acid sequence of SEQ ID NO: 21.

[0021] In some embodiments, a CAR does not comprise an intracellular T cell signaling domain from 4-1BB.

[0022] In some embodiments, a CAR comprises an amino acid sequence of SEQ ID NO: 10 or 13.

[0023] In some embodiments, a vector is a lentivirus vector. In some embodiments, a vector further comprises a murine stem cell virus (MSCV) U3 promoter operably linked to the nucleic acid.

[0024] In some embodiments, at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the provided T cells express a CAR (e.g., any CAR provided herein). In some embodiments, provided T cells comprise at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%) of CD8+ cytotoxic T cells. In some embodiments, provided T cells comprise at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%) of CD4+ helper T cells.

[0025] In some embodiments, the systemic sclerosis is limited cutaneous systemic sclerosis or diffuse cutaneous systemic sclerosis. In some embodiments, the systemic sclerosis is limited cutaneous systemic sclerosis. In some embodiments, the systemic sclerosis is diffuse cutaneous systemic sclerosis. In some embodiments, the systemic sclerosis is refractory to a conventional therapeutic regimen such as a corticosteroid, immunomodulatory, or other SOC therapy (e.g., any of those described herein). In some embodiments, the systemic sclerosis is refractory to an immunomodulatory therapy such as an anti-CD20 antibody. In some embodiments, the systemic sclerosis is refractory systemic sclerosis.

[0026] In some embodiments, the treatment (e.g., any T cell treatment provided herein) is administered as a first-line therapy.

[0027] In some embodiments, a therapeutically effective dose is in a range of about 2x107to 5x107, about 2x107to 4x107, about 2x107to 3x107, about 3x107to 5x107, about 4x107toAttorney Docket No. KVN-011WO 5x107, about 5×107to 1×108, about 5×107to 9×107, about 5×107to 8×107, about 5×107to 7×107, about 5×107to 6×107, about 6×107to 1×108, about 7×107to 1×108, about 8×107to 1×108, about 9×107to 1×108, about 6×107to 9×107, or about 7×107to 8×107of the T cells that comprise a vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments, a therapeutically effective dose is in a range of about 5×107to 1×108of such T cells. In some embodiments, a therapeutically effective dose is about 5×107of such T cells. In some embodiments, a therapeutically effective dose is about 1×108of such T cells.

[0028] In some embodiments, a therapeutically effective dose is in a range of about 2x107to 5x107, about 2x107to 4x107, about 2x107to 3x107, about 3x107to 5x107, about 4x107to 5x107, about 5×107to 1×108, about 5×107to 9×107, about 5×107to 8×107, about 5×107to 7×107, about 5×107to 6×107, about 6×107to 1×108, about 7×107to 1×108, about 8×107to 1×108, about 9×107to 1×108, about 6×107to 9×107, or about 7×107to 8×107of the T cells that express the CAR. In some embodiments, a therapeutically effective dose is in a range of about 5×107to 1×108of such T cells. In some embodiments, a therapeutically effective dose is about 5×107of such T cells. In some embodiments, a therapeutically effective dose is about 1×108of such T cells.

[0029] In some embodiments, provided T cells are administered by intravenous infusion.

[0030] In some embodiments, the subject receives a single dose of the T cells.

[0031] In some embodiments, the subject has received a lymphodepletion treatment or a minimized lymphodepletion treatment resulting in about 50% reduction of lymphocytes in the subject relative to the subject before receiving lymphodepletion treatment or another suitable control. In some embodiments, the subject has received a reduced or minimized lymphodepletion treatment. In some embodiments, the subject has not received a lymphodepletion treatment.

[0032] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0033] Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well asAttorney Docket No. KVN-011WO specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in accordance with the present disclosure, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0035] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG.1 is a schematic of a lentivirus vector encoding an anti-CD19 CAR transgene.

[0037] FIG.2 is a series of graphs showing cytolysis of SSc patient-derived and healthy donor-derived KYV-101 against primary B cells and cancer cell lines. Percent cytotoxicity of autologous B cells (panel A), CD19+ NALM6 cells (panel 2B), or CD19- CEM / C1 cells (panel C) measured by flow cytometry when co-cultured with SSc patient-derived and healthy donor (HD)-derived KYV-101 or donor-matched control T cells (UNTD) is shown.

[0038] FIG.3 is a series of graphs showing IFNγ release following co-culture of SSc patient-derived and healthy donor-derived KYV-101 or untransfected control T cells with primary B cells or cancer cell lines. IFNγ production from SSc patient-derived and healthy donor (HD)-derived KYV-101 or untransfected control T cells (UNTD) after overnight co- culture with autologous B cells (panel A), CD19+ NALM6 cells (panel B), or CD19- CEM / C1 cells (panel C) is shown.

[0039] FIG.4 is a series of graphs showing granzyme B release following co-culture of SSc patient-derived and healthy donor-derived KYV-101 or untransfected control T cells with primary B cells or cancer cell lines. Granzyme B production from SSc patient-derived and healthy donor (HD)-derived KYV-101 or untransfected control T cells (UNTD) afterAttorney Docket No. KVN-011WO overnight co-culture with autologous B cells (panel A), CD19+ NALM6 cells (panel B), or CD19- CEM / C1 cells (panel C) is shown.

[0040] FIG.5 is a series of graphs showing percent KYV-101 and donor-matched untransduced control T cells proliferating following 96 hours of co-culture with primary B cells or cancer cell lines. Percentage of SSc patient-derived or healthy donor (HD)-derived KYV-101 or paired untransfected control T cells (UNTD) that were proliferating, indicated by CTV Dim, following co-culture with CD19+ primary B cells (panel A), CD19+ NALM6 cells (panel B), or CD19- CEM / C1 (panel C) at two different E:T ratios is shown. DETAILED DESCRIPTION

[0041] The present disclosure provides methods and compositions that can be used to treat a subject identified as having systemic sclerosis. In some embodiments, the systemic sclerosis is limited cutaneous systemic sclerosis or diffuse cutaneous systemic sclerosis. In some embodiments, the systemic sclerosis is limited cutaneous systemic sclerosis. In some embodiments, the diffuse cutaneous systemic sclerosis. In some embodiments, the systemic sclerosis is refractory systemic sclerosis. In some embodiments, the systemic sclerosis is positive for one or more autoantibodies associated with the disease (e.g., anti-Scl-70, anti- centromere, anti-RNAPIII, anti-anti-Th / To, anti-U1 RNP, anti-U3 RNP, anti-PM / Scl-100, etc.). In some embodiments, the systemic sclerosis is anti-Scl-70 positive (i.e., the subject tests positive for anti-Scl-70 antibodies). In some embodiments, the systemic sclerosis is anti- centromere positive (i.e., the subject tests positive for anti-centromere antibodies). In some embodiments, the systemic sclerosis is anti-RNAPIII positive (i.e., the subject tests positive for anti-RNAPIII antibodies). In some embodiments, the systemic sclerosis is anti-Th / To positive (i.e., the subject tests positive for anti-Th / To antibodies). In some embodiments, the systemic sclerosis is anti-U1 RNP positive (i.e., the subject tests positive for anti-U1 RNP antibodies). In some embodiments, the systemic sclerosis is anti-U3 RNP positive (i.e., the subject tests positive for anti-U3 RNP antibodies). In some embodiments, the systemic sclerosis is anti-PM / Sc-100 positive (i.e., the subject tests positive for anti-PM / Sc-100 antibodies). In some embodiments, the systemic sclerosis is anti-endothelial cell positive (i.e., the subject tests positive for anti-endothelial cell antibodies). In some embodiments, the systemic sclerosis is anti-matrix metalloproteinase positive (i.e., the subject tests positive for anti-matrix metalloproteinase antibodies). In some embodiments, the systemic sclerosis is anti-fibrillin I positive (i.e., the subject tests positive for anti-fibrillin I antibodies). In someAttorney Docket No. KVN-011WO embodiments, the systemic sclerosis is anti-B23 positive (i.e., the subject tests positive for anti-B23 antibodies).

[0042] In some embodiments, a subject to be treated by a provided method or composition is identified as having systemic sclerosis (e.g., limited cutaneous systemic sclerosis or diffuse cutaneous systemic sclerosis) according to the American College of Rheumatology / European League Against Rheumatism (ACR / EULAR) classification criteria (e.g., the 2013 ACR / EULAR classification criteria, with a total score of ≥9).

[0043] The present disclosure provides, among other things, methods and compositions for reducing the number of B cells in a tissue in a subject having systemic sclerosis. The present disclosure also provides engineered T cells (e.g., T cells engineered to express any CAR described herein) and method of making engineered T cells for use in treating systemic sclerosis.

[0044] The present disclosure appreciates that B cells express a wide array of cell surface molecules during their differentiation and proliferation, e.g., CD19. CD19 is widely expressed on B cells during all phases of B cell development from pro-B cells to plasmablasts. The present disclosure further appreciates, that because of the ubiquity of CD19 on B cells, CD19 can function as a therapeutic target for certain provided methods and compositions (e.g., methods and compositions for treating systemic sclerosis). Accordingly, in some embodiments, provided herein are methods and compositions for reducing the number of B cells in a subject (e.g., in a tissue of a subject) via targeting of CD19. In some embodiments, the present disclosure provides methods and compositions for treating a subject having systemic sclerosis via targeting of CD19. In some embodiments, the present disclosure provides engineered T cells that target CD19. In some embodiments, the present disclosure provides engineered nucleic acids that express one or more polypeptides that target CD19. In many embodiments of the present disclosure a CAR that binds to CD19 is used to target cells that express CD19 (e.g., B cells). Definitions

[0045] “About” a number, as used herein, refers to range including the number and ranging from 10% below that number to 10% above that number. “About” a range refers to 10% below the lower limit of the range, spanning to 10% above the upper limit of the range.

[0046] As used herein, the term “antibody” refers to any immunoglobulin, whether naturally occurring or wholly or partially synthetically produced. All derivatives thereof which maintain specific binding ability are also included in the term. In some embodiments,Attorney Docket No. KVN-011WO the term “antibody” refers to any protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain. Antibody proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In certain embodiments, an antibody may be a member of the IgG immunoglobulin class.

[0047] As used herein, “derived from” or “derivative” indicates a structural similarity and a functional similarity between a subject molecule and a reference molecule (e.g., between polynucleotides, polypeptides, etc.). With respect to structural similarity, the subject molecule does not necessarily comprise the same sequence (e.g., nucleic acid sequence, amino acid sequence, etc.) as the reference molecule, but has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the sequence (e.g., nucleic acid sequence, amino acid sequence, etc.) of the reference molecule or a fragment thereof, the fragment comprising at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the sequence of the reference molecule. With respect to functional similarity, the subject molecule has at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of an activity of the reference molecule or the fragment thereof as determined in a suitable assay. For example, a subject polypeptide may be considered to be derived from a reference polypeptide when the subject polypeptide has structural similarity, as defined above, to the reference polypeptide and retains certain function(s), such as certain intermolecular or intramolecular interactions (e.g., binding to a protein, e.g., a particular receptor, or a signaling activity), though such interactions could be stronger, equivalent, or weaker than that of the reference polypeptide. As another non-limiting example, a subject polynucleotide may be considered to be derived from a reference polynucleotide when the subject polynucleotide has structural similarity to the reference polynucleotide, as defined above, and encodes a protein or protein fragment that is a derivative of the protein encoded by the reference polynucleotide, or has the same or similar function (e.g., as a regulatory element, e.g., promoter or enhancer) as the reference polynucleotide. Functional similarity takes into account the context of the disclosure. For example, when applied to a subject intracellular T cell signaling domain derived from a reference protein (e.g., CD3ζ, CD28), the subject intracellular T cell signaling domain has structural and functional similarities to an intracellular T cell signaling domain of the reference protein as known in the art. Similarly, when applied to a subject transmembrane domains derived from a reference protein, theAttorney Docket No. KVN-011WO subject transmembrane domain has structural and functional similarities to a transmembrane domain of the reference protein as known in the art. In one non-limiting example, an intracellular T cell signaling domain derived from a CD3ζ molecule retains sufficient CD3ζ structure such that it has the ability to transduce a signal (e.g., ZAP-70 activation) under appropriate conditions.

[0048] As used herein, the term “functional fragment” of a reference biomolecule, e.g., a polynucleotide or polypeptide, refers to a shorter and / or smaller derivative of the reference biomolecule that has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of a fragment of the reference biomolecule.

[0049] As used herein the term “operably linked” refers to polynucleotide sequences placed into a functional relationship with one another. For instance, a promoter or enhancer is operably linked to a coding sequence if it regulates, or contributes to modulation of, the transcription of a coding sequence. Operably linked DNA sequences encoding regulatory sequences are typically contiguous to a coding sequence. However, enhancers can function when separated from a promoter by up to several kilobases or more. Additionally, multi- cistronic constructs can include multiple coding sequences which use only one promoter by including a 2A self-cleaving peptide, an IRES element, etc. Accordingly, some polynucleotide elements may be operably linked but not contiguous.

[0050] As used herein, the term “patient” or “subject” are used interchangeably to refer to any organism to which a compositions disclosed herein may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, a patient or subject is a human. In many embodiments, a patient is a human subject suffering from systemic sclerosis. Chimeric Antigen Receptors

[0051] In some embodiments, a chimeric antigen receptor (CAR) of the present disclosure comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, an extracellular domain is or comprises an antigen-binding domain (e.g., a CD19 binding domain, such as an anti-CD19 scFv). In some embodiments, a transmembrane domain is or comprises a transmembrane domain or functional fragment thereof derived from any suitable cell membrane-associated polypeptide, e.g., obtained from a membrane-binding polypeptide or transmembrane polypeptide. In some embodiments, aAttorney Docket No. KVN-011WO transmembrane domain is or comprises a transmembrane domain or functional fragment thereof derived from a T cell receptor alpha chain, a T cell receptor beta chain, a CD3 zeta chain, a CD28 polypeptide, or a CD8 polypeptide (e.g., a CD8α polypeptide). In some embodiments, an intracellular domain is or comprises an intracellular signaling domain (e.g., any of intracellular signaling domains described herein, e.g., derived from a CD28 or CD3 polypeptide). In some embodiments, an intracellular signaling domain comprises one or more signaling sequences or motifs. In some embodiments, one or more signaling sequences, or signaling motifs, are essential for the functional signaling capacity of a polypeptide (e.g., an intracellular signaling domain). In some embodiments, a signaling sequence is a sequence derived from a CD3 polypeptide (e.g., a CD3 zeta polypeptide). In some embodiments, a signaling sequence is derived from a CD28 polypeptide. In some embodiments, a signaling sequence is or comprises a co-stimulatory domain (e.g., any co-stimulatory domain described herein, e.g., derived from a CD28 polypeptide). In some embodiments, a CAR of the present disclosure is a human CAR. Extracellular Domain

[0052] In some embodiments, an extracellular domain used in accordance with the present disclosure comprises an antigen-binding domain (e.g., any antigen-binding domain described herein). In some embodiments, an antigen-binding domain is or comprises an antibody sequence (e.g., an immunoglobulin) or antigen-binding fragment thereof (e.g., any antibody or antigen-binding fragment thereof described herein). Anticalins or other alternative scaffolds are also contemplated.

[0053] In some embodiments, the antigen-binding domain comprises one or more Fab, Fab’, F(ab’)2, Fv, domain antibody (dAb), single-chain antibody (scFv), chimeric antibody, diabody, triabody, tetrabody, scAb, or single domain antibody (e.g., VHH or VNAR) polypeptide sequences. In some embodiments, the antigen-binding domain comprises at least a portion of an immunoglobulin that is sufficient to confer specific antigen-binding to a polypeptide (e.g., an antibody fragment comprising an antigen-binding portion). In some embodiments, the antigen-binding domain comprises an scFv. In some embodiments, the scFv comprises a VH and VL domain of an antibody. In some embodiments, the scFv comprises a spacer sequence between the VH and the VL. In some embodiments, the scFv comprises a spacer sequence as set forth in SEQ ID NO: 9 between the VH and the VL. In some embodiments, the antigen-binding domain is humanized, or fully human (e.g., derived from a suitable human polypeptide). Exemplary methods of generating fully human antibodies are described in Lu et al., (2020) J. Biomed. Sci. (2020) 27(1):1.Attorney Docket No. KVN-011WO

[0054] In some embodiments, the antigen-binding domain binds to a target antigen (e.g., a polypeptide). In some embodiments, the antigen-binding domain binds specifically to a target antigen (e.g., a polypeptide). In some embodiments, the antigen-binding domain binds to a CD19 polypeptide (e.g., a CD19 polypeptide present at the surface of a cell, e.g., a B cell). In some embodiments, the antigen-binding domain binds specifically to a CD19 polypeptide. In some embodiments, the antigen-binding domain comprises an antibody, or antigen-binding fragment thereof, that binds to a CD19 polypeptide. In some embodiments, the antigen-binding domain comprises a scFv sequence that binds to a CD19 polypeptide (e.g., an anti-CD19 scFv).

[0055] It is generally understood that CD19 expression is largely restricted to B lymphocytes. CD19 has two N-terminal extracellular Ig-like domains separated by a non-Ig- like domain, a hydrophobic transmembrane domain, and a large C-terminal cytoplasmic domain. The CD19 protein forms a complex with several membrane proteins including complement receptor type 2 (CD21) and tetraspanin (CD81) and this complex reduces the threshold for antigen-initiated B cell activation. Activation of this B-cell antigen receptor complex activates the phosphatidylinositol 3-kinase signaling pathway and the subsequent release of intracellular stores of calcium ions. An example of a human CD19 polypeptide sequence includes, without limitation, NCBI reference sequence: NP_001171569.1, and fragments and derivatives thereof.

[0056] In some embodiments, an antigen-binding domain comprises a variable region of an anti-CD19 antibody. In some embodiments, an antigen-binding domain comprises a variable region of an anti-CD19 monoclonal antibody. In some embodiments, an antigen- binding domain comprises a variable region of a mouse or human anti-CD19 monoclonal antibody. An anti-CD19 monoclonal antibody can be obtained or derived from a subject (e.g., a mouse, a rat, a rabbit, a human, etc.) using any suitable method. In some embodiments, an antigen-binding domain comprises a light chain variable region and a heavy chain variable region of a mouse, human, or humanized anti-CD19 monoclonal antibody. In some embodiments, an antigen-binding domain comprises a light chain variable region of a mouse, human, or humanized anti-CD19 monoclonal antibody. In some embodiments, an antigen- binding domain comprises a heavy chain variable region of a mouse, human, or humanized anti-CD19 monoclonal antibody. The 47G4 antibody (described in U.S. Patent Application Publication No.2010 / 0104509, which is incorporated herein by reference in its entirety) is one example of a human anti-CD19 monoclonal antibody that can be used in accordance with the present disclosure.Attorney Docket No. KVN-011WO

[0057] In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0058] In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 3, respectively, and the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 3, respectively, and the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0059] In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. In some embodiments, the antigen- binding domain that binds CD19 comprises a heavy chain variable domain, the heavy chainAttorney Docket No. KVN-011WO variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0060] In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 3, respectively. In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 3, respectively.

[0061] In some embodiments, the antigen-binding domain that binds CD19 comprises a light chain variable domain, the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. In some embodiments, the antigen- binding domain that binds CD19 comprises a light chain variable domain, the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0062] In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 7, and the light chain variable domain comprising an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 8. In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 7, and the light chain variable domain comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 7, and the light chain variable domain comprising an amino acid sequence with at least 90% sequence identity to SEQ IDAttorney Docket No. KVN-011WO NO: 8. In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 7, and the light chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 8.

[0063] In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain, the heavy chain variable domain comprising an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 7. In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain, the heavy chain variable domain comprising an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain, the heavy chain variable domain comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain, the heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 7.

[0064] In some embodiments, the antigen-binding domain that binds CD19 comprises a light chain variable domain, the light chain variable domain comprising an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 8. In some embodiments, the antigen-binding domain that binds CD19 comprises a light chain variable domain, the light chain variable domain comprising an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, the antigen-binding domain that binds CD19 comprises a light chain variable domain, the light chain variable domain comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, the antigen-binding domain that binds CD19 comprises a light chain variable domain, the light chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 8.

[0065] In some embodiments, the antigen-binding domain that binds CD19 comprises a spacer sequence between two domains or components. In some embodiments, an antigen- biding domain comprises a spacer sequence between a heavy chain variable domain and a light chain variable domain. In some embodiments, a spacer comprises a sequence as set forth in SEQ ID NO: 9.Attorney Docket No. KVN-011WO

[0066] In some embodiments, the antigen-binding domain that binds CD19 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 17. In some embodiments, the antigen-binding domain that binds CD19 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17. In some embodiments, the antigen-binding domain that binds CD19 comprises an amino acid sequence as set forth in SEQ ID NO: 17.

[0067] In some embodiments, the antigen-binding domain that binds CD19 is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 18. In some embodiments, the antigen-binding domain that binds CD19 is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 18. In some embodiments, an antigen-binding domain is encoded a nucleic acid sequence as set forth in SEQ ID NO: 18.

[0068] Other antigen-binding domains that bind CD19 can also be included in the CAR disclosed herein. Exemplary antigen-binding domains are described in International Application Publication No. WO2017062952 and U.S. Application Publication No. US20220220200.

[0069] In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NOs: 35, 36, and 37, respectively, and the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NOs: 38, 39, and 40, respectively. In some embodiments, the heavy chain variable domain comprises an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 29, and the light chain variable domain comprises an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 30.

[0070] In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NOs: 41, 42, and 43, respectively, and the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NOs: 44, 45, and 46,Attorney Docket No. KVN-011WO respectively. In some embodiments, the heavy chain variable domain comprises an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 31, and the light chain variable domain comprises an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 32.

[0071] In some embodiments, the antigen-binding domain that binds CD19 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NOs: 47, 48, and 49, respectively, and the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences as set forth in SEQ ID NOs: 50, 51, and 52, respectively. In some embodiments, the heavy chain variable domain comprises an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 33, and the light chain variable domain comprises an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 34.

[0072] In some embodiments, the extracellular domain of the CAR further comprises a hinge region. In some embodiments, a hinge region is positioned between (e.g., links together), an extracellular domain and a transmembrane domain. In some embodiments, the hinge region is a short sequence of amino acids that can facilitate structural flexibility between polypeptide domains, e.g., between an extracellular domain and a transmembrane domain (see, e.g. Woof et al., Nat. Rev. Immunol.4(2):89-99 (2004)). In some embodiments, a hinge region may include all, or a portion of, an extracellular region of any suitable transmembrane protein (e.g., CD8α).

[0073] In some embodiments, the hinge region is derived from a CD8α protein or a CD28 protein. In some embodiments, a hinge region is derived from a CD8α protein. In some embodiments, the hinge region is derived from a CD28 protein. In some embodiments, a hinge region is or comprises a hinge region or functional fragment thereof from a CD28 protein. In some embodiments, the hinge region is or comprises a hinge region or functional fragment thereof from a CD8α protein. In some embodiments, the hinge region is derived from a human CD8α protein or a human CD28 protein. In some embodiments, the hinge region is derived from a human CD8α protein. In some embodiments, the hinge region is derived from a human CD28 protein. In some embodiments, the hinge region is or comprisesAttorney Docket No. KVN-011WO a hinge region or functional fragment thereof from a human CD28 protein. In some embodiments, the hinge region is or comprises a hinge region or functional fragment thereof from a human CD8α protein.

[0074] In some embodiments, a hinge region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 28. In some embodiments, a hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 28.

[0075] In some embodiments, a hinge region is derived from the same polypeptide as a transmembrane domain. In some embodiments, a hinge region and a transmembrane domain are derived from a CD8 polypeptide. In some embodiments, a hinge region and a transmembrane domain are derived from a CD8α polypeptide. In some embodiments, a hinge region and transmembrane domain comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 19. In some embodiments, a hinge region and transmembrane domain comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19. In some embodiments, a hinge region and transmembrane domain comprise an amino acid sequence as set forth in SEQ ID NO: 19.

[0076] In some embodiments, a hinge region and transmembrane domain are encoded by nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 20. In some embodiments, a hinge region and transmembrane domain are encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 20. In some embodiments, a hinge region and transmembrane domain are encoded by a nucleic acid sequence as set forth in SEQ ID NO: 20. Transmembrane Domain

[0077] In some embodiments, the transmembrane domain of the CAR is derived from a natural source (e.g., a natural or wild-type polypeptide). In some embodiments, the transmembrane domain, as used in accordance with the present disclosure, is derived from any suitable transmembrane protein or polypeptide known in the art. In some embodiments, a transmembrane domain is derived from a CD3 epsilon polypeptide, a CD4 polypeptide, a CD5 polypeptide, a CD8 polypeptide, a CD9 polypeptide, a CD16 polypeptide, a CD22 polypeptide, a CD28 polypeptide, a CD33 polypeptide, a CD37 polypeptide, a CD45Attorney Docket No. KVN-011WO polypeptide, a CD64 polypeptide, a CD80 polypeptide, a CD86 polypeptide, a CD134 polypeptide, a CD137 polypeptide, a CD154 polypeptide, a T cell receptor alpha chain polypeptide, a T cell receptor beta chain polypeptide, a T cell receptor zeta chain polypeptide, or any combination thereof. In some embodiments, a transmembrane is or comprises a transmembrane domain or functional fragment thereof from a CD3 epsilon polypeptide, a CD4 polypeptide, a CD5 polypeptide, a CD8 polypeptide, a CD9 polypeptide, a CD16 polypeptide, a CD22 polypeptide, a CD28 polypeptide, a CD33 polypeptide, a CD37 polypeptide, a CD45 polypeptide, a CD64 polypeptide, a CD80 polypeptide, a CD86 polypeptide, a CD134 polypeptide, a CD137 polypeptide, a CD154 polypeptide, a T cell receptor alpha chain polypeptide, a T cell receptor beta chain polypeptide, a T cell receptor zeta chain polypeptide, or any derivatives thereof and / or any combination thereof. In some embodiments, a transmembrane is synthetically derived, or engineered. In some embodiments, a synthetically derived or engineered transmembrane domain comprises predominantly hydrophobic residues (e.g., leucine, valine, etc.). In some embodiments, an engineered transmembrane domain is or comprises any engineered transmembrane domain known in the field.

[0078] The present disclosure appreciates that CD8 is a transmembrane glycoprotein that functions as a co-receptor for the T-cell receptor (TCR), and is expressed primarily on the surface of T-cells (e.g., cytotoxic T-cells). The most common form of CD8 exists as a dimer composed of a CD8α and CD8β chain. In some embodiments, a transmembrane domain is derived from a CD8α protein. In some embodiments, a transmembrane protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 11. In some embodiments, a transmembrane protein comprises an amino acid sequence as set forth in SEQ ID NO: 11.

[0079] The present disclosure further appreciates that CD28 is expressed on T-cells and provides co-stimulatory signals required for T-cell activation. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2). In some embodiments, a CAR of the present disclosure comprises a CD28 transmembrane domain. In some embodiments, the transmembrane protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 12. In some embodiments, a transmembrane protein comprises an amino acid sequence as set forth in SEQ ID NO: 12.Attorney Docket No. KVN-011WO Intracellular Signaling Domain

[0080] In some embodiments, an intracellular signaling domain of the CAR disclosed herein is derived from a polypeptide found in humans (e.g., an intracellular signaling domain or fragment thereof found in any suitable human polypeptide). In some embodiments, the intracellular signaling domain provided herein is derived from a 4-1BB polypeptide, a B7-H3 polypeptide, a CD2 polypeptide, a CD3 gamma polypeptide, a CD3 delta polypeptide, a CD3 zeta polypeptide, a CD7 polypeptide, a CD27 polypeptide, a CD28 polypeptide, a CD30 polypeptide, a CD40 polypeptide, an FcεRI polypeptide (e.g., an FcεRI gamma chain polypeptide), an FcγRI polypeptide, LIGHT polypeptide, NKG2C polypeptide, OX40 polypeptide, PD-1 polypeptide, or any derivatives thereof or any combination thereof. In some embodiments, the intracellular signaling domain is derived from a CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain is derived from a CD28 polypeptide. In some embodiments, the intracellular signaling domain is derived from a CD28 polypeptide and a CD3 zeta polypeptide.

[0081] In some embodiments, the intracellular signaling domain comprises at least one intracellular signaling domain or functional fragment thereof from a 4-1BB polypeptide, a B7-H3 polypeptide, a CD2 polypeptide, a CD3 gamma polypeptide, a CD3 delta polypeptide, a CD3 zeta polypeptide, a CD7 polypeptide, a CD27 polypeptide, a CD28 polypeptide, a CD30 polypeptide, a CD40 polypeptide, an FcεRI polypeptide (e.g., an FcεRI gamma chain polypeptide), an FcγRI polypeptide, LIGHT polypeptide, NKG2C polypeptide, OX40 polypeptide, PD-1 polypeptide, or any derivatives thereof or any combination thereof. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain or functional fragment thereof from a CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain or functional fragment thereof from a CD28 polypeptide. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain or functional fragment thereof from a CD28 polypeptide and an intracellular signaling domain or functional fragment thereof from a CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain comprises, from N-terminus to C-terminus, an intracellular signaling domain or functional fragment thereof from a CD28 polypeptide and an intracellular signaling domain or functional fragment thereof from a CD3 zeta polypeptide.

[0082] In some embodiments, the intracellular signaling domain of the present disclosure comprises at least one signaling sequence from a 4-1BB polypeptide, a B7-H3 polypeptide, a CD2 polypeptide, a CD3 gamma polypeptide, a CD3 delta polypeptide, a CD3 zetaAttorney Docket No. KVN-011WO polypeptide, a CD7 polypeptide, a CD27 polypeptide, a CD28 polypeptide, a CD30 polypeptide, a CD40 polypeptide, an FcεRI polypeptide (e.g., an FcεRI gamma chain polypeptide), an FcγRI polypeptide, LIGHT polypeptide, NKG2C polypeptide, OX40 polypeptide, PD-1 polypeptide, or any combination thereof. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence from a CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence from a CD28 polypeptide. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence from a CD28 polypeptide and at least one signaling sequence from a CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain comprises, from N-terminus to C-terminus, at least one signaling sequence from a CD28 polypeptide and at least one signaling sequence from a CD3 zeta polypeptide.

[0083] In some embodiments, an intracellular signaling domain is or comprises at least one signaling sequence or signaling motif. In some embodiments, a signaling sequence (or signaling motif) comprises one or more (e.g., two, three, four, five, or more) co-stimulatory domains (e.g., any co-stimulatory domain described herein). In some embodiments, a signaling sequence comprises one co-stimulatory domain. In some embodiments, a signaling sequence comprises two co-stimulatory domains. In some embodiments, a signaling sequence comprises three co-stimulatory domains. In some embodiments, a signaling sequence comprises two or more of the same co-stimulatory domains. In some embodiments, a signaling sequence comprises two or more different co-stimulatory domains.

[0084] In some embodiments, a signaling sequence as used in accordance with the present disclosure is or comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, a signal sequence is or comprises a consensus sequence of YXXL / I, where Y is a tyrosine residue, L / I is a leucine or isoleucine residue, and X is any amino acid residue. In some embodiments, a signaling sequence is or comprises a consensus sequences of YXXL / IX(6-8)YXXL (SEQ ID NO: 54), where Y is a tyrosine residue, L / I is a leucine or isoleucine residue, and X is any amino acid residue. In some embodiments, a signaling sequence comprises a YNMN (SEQ ID NO: 53) motif. In some embodiments, a signaling sequence comprises at least one ITAM sequence from a CD3 polypeptide (e.g., a CD3 zeta polypeptide). In some embodiments, a signaling sequence comprises at least one ITAM sequence from a CD28 polypeptide.

[0085] It is understood that the most common intracellular signaling domain used in CAR therapies is an intracellular signaling domain of CD3 zeta (CD3ζ). CD3 zeta associates withAttorney Docket No. KVN-011WO T cell receptors to produce a signal and contains ITAMs. In some embodiments, an intracellular signaling domain is or comprises a CD3 zeta intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises an intracellular signaling domain or a functional fragment thereof from a CD3 zeta polypeptide.

[0086] In some embodiments, an intracellular signaling domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 23. In some embodiments, an intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NO: 23.

[0087] In some embodiments, an intracellular signaling domain is encoded by nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 24. In some embodiments, an intracellular signaling domain is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 24.

[0088] In some embodiments, an intracellular signaling domain comprises a CD28 intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises an intracellular signaling domain or a functional fragment thereof from a CD28 polypeptide. In some embodiments, a CD28 polypeptide intracellular signaling domain or functional fragment thereof comprises a co-stimulatory domain.

[0089] In some embodiments, an intracellular signaling domain disclosed herein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 21. In some embodiments, the intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NO: 21.

[0090] In some embodiments, an intracellular signaling domain is encoded by nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 22. In some embodiments, an intracellular signaling domain is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 22. Chimeric Antigen Receptors (CARs)

[0091] In some embodiments, a CAR of the present disclosure comprises an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a CAR of the present disclosure comprises a signal peptide sequence (also referred to as a targeting signal, localization signal, localization sequence, leaderAttorney Docket No. KVN-011WO sequence, or leader peptide), an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a CAR of the present disclosure comprises, from N-terminus to C-terminus, an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a CAR of the present disclosure comprises, from N-terminus to C-terminus, a signal peptide sequence, an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the signal peptide sequence is cleaved from the CAR during or after its insertion into a membrane (e.g., ER membrane) during synthesis of the CAR protein. In some embodiments, domains or components (e.g., extracellular domains, hinge regions, transmembrane domains, intracellular signaling domains, etc.) of a CAR are directly linked, or are contiguous. In some embodiments, domains or components of a CAR are not-directly linked, or are non-contiguous.

[0092] In some embodiments, a CAR as described herein comprises an intracellular signaling domain, wherein the intracellular signaling domain comprises: (a) a CD3 zeta intracellular signaling domain or functional fragment thereof; and (b) at least one of a 4-1BB, an OX40, or a CD28 intracellular signaling domain or functional fragment thereof. In some embodiments, a 4-1BB intracellular signaling domain or functional fragment thereof, an OX40 intracellular signaling domain, and / or a CD28 intracellular signaling domain or functional fragment thereof is or comprises a co-stimulatory domain.

[0093] In some embodiments, a CAR of the present disclosure comprises: (a) a CD28 transmembrane domain; and (b) an intracellular signaling domain comprising: (i) a CD3ζ intracellular signaling domain or functional fragment thereof; and (ii) a CD28 intracellular signaling domain or functional fragment thereof. In some embodiments, a CD28 intracellular signaling domain or functional fragment thereof is or comprises a CD28 co-stimulatory domain.

[0094] In some embodiments, a CAR of the present disclosure comprises: (a) a CD8α transmembrane domain; (b) an intracellular signaling domain comprising: (i) a CD3ζ intracellular signaling domain or functional fragment thereof; and (ii) a CD28, an FcεRI gamma chain, and / or a 4-1BB intracellular signaling domain or functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises: (a) a CD8α transmembrane domain; (b) an intracellular signaling domain comprising: (i) a CD3ζ intracellular signaling domain or functional fragment thereof; and (ii) a CD28, an FcεRI gamma chain, and a 4-1BB intracellular signaling domain or functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises: (a) a CD8α transmembraneAttorney Docket No. KVN-011WO domain; (b) an intracellular signaling domain comprising: (i) a CD3ζ intracellular signaling domain or functional fragment thereof; and (ii) an FcεRI gamma chain intracellular signaling domain or functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises: (a) a CD8α transmembrane domain; (b) an intracellular signaling domain comprising: (i) a CD3ζ intracellular signaling domain or functional fragment thereof; and (ii) a 4-1BB intracellular signaling domain or functional fragment thereof. In some embodiments, a CD28 intracellular signaling domain or functional fragment thereof is or comprises a CD28 co-stimulatory domain. In some embodiments, a FcεRI intracellular signaling domain or functional fragment thereof is or comprises a FcεRI co-stimulatory domain. In some embodiments, a 4-1BB intracellular signaling domain or functional fragment thereof is or comprises a 4-1BB co-stimulatory domain.

[0095] In some embodiments, a CAR of the present disclosure comprises (a) a CD8α transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3ζ intracellular signaling domain or functional fragment thereof, and (ii) a CD27 and / or a CD28 intracellular signaling domain or functional fragment thereof. In some embodiments, a CD27 intracellular signaling domain or functional fragment thereof is or comprises a CD27 co- stimulatory domain. In some embodiments, a CD28 intracellular signaling domain or functional fragment thereof is or comprises a CD28 co-stimulatory domain.

[0096] In some embodiments, a CAR of the present disclosure comprises (a) a CD28 transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3ζ intracellular signaling domain or functional fragment thereof; and (ii) a CD27, a 4-1BB, and / or an FcεRI gamma chain intracellular signaling domain or functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a CD28 transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3ζ intracellular signaling domain or functional fragment thereof; and (ii) a CD27, a 4-1BB, and an FcεRI gamma chain intracellular signaling domain or functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a CD28 transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3ζ intracellular signaling domain or functional fragment thereof; and (ii) a CD27 intracellular signaling domain or functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a CD28 transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3ζ intracellular signaling domain or functional fragment thereof; and (ii) a 4-1BB intracellular signaling domain or functional fragment thereof. In some embodiments, a CAR of the present disclosure comprises (a) a CD28 transmembrane domain, and (b) anAttorney Docket No. KVN-011WO intracellular signaling domain comprising: (i) a CD3ζ intracellular signaling domain or functional fragment thereof; and (ii) an FcεRI gamma chain intracellular signaling domain or functional fragment thereof. In some embodiments, a CD27 intracellular signaling domain or functional fragment thereof is or comprises a CD27 co-stimulatory domain. In some embodiments, a FcεRI intracellular signaling domain or functional fragment thereof is or comprises a FcεRI co-stimulatory domain. In some embodiments, a 4-1BB intracellular signaling domain or functional fragment thereof is or comprises a 4-1BB co-stimulatory domain.

[0097] The present disclosure also includes functional variants of any of CAR, or CAR domain / component, described herein. CAR functional variants encompass, for example, variants of a CAR described herein (a parent CAR) that retains the ability to recognize a particular target cell to a similar extent, the same extent, or to a higher extent, as the parent CAR. In reference to a nucleic acid sequence encoding a parent CAR, a nucleic acid sequence encoding a functional variant of the CAR can be for example, about 10% identical, about 25% identical, about 30% identical, about 50% identical, about 65% identical, about 80% identical, about 90% identical, about 95% identical, or about 99% identical to the nucleic acid sequence encoding the parent CAR. In some embodiments, a parent CAR comprises an amino acid sequence as set forth in SEQ ID NO: 10 or 13. Alternatively or additionally, in some embodiments, a CAR functional variant comprises the amino acid sequence of a parent CAR with at least one non-conservative amino acid substitution. In some embodiments, a non-conservative amino acid substitution does not compromise or inhibit a biological activity of a CAR functional variant. In some embodiments, a non- conservative amino acid substitution may enhance a biological activity of a CAR functional variant, such that biological activity of the functional variant is increased relative to its parent CAR.

[0098] The present disclosure further provides for CARs comprising an extracellular domain directed to any target molecule of interest (e.g., comprising any of known antigen- binding domain, e.g., antibody, scFv, etc.), and further comprising any transmembrane domain described herein (including any hinge domain described herein), any intracellular signaling domain described herein (including any signal sequences or motifs, any co- stimulatory domains, etc., described herein), present in any combination.

[0099] In some embodiments, a CAR comprises: (a) a hinge region, (b) a transmembrane domain derived from a human CD8α polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3ζ intracellular signaling domain or fragment thereof; and (ii) aAttorney Docket No. KVN-011WO human CD28 intracellular signaling domain or fragment thereof, wherein the CD28 intracellular signaling domain or fragment thereof is or comprises a co-stimulatory domain. In some embodiments, a CAR comprises: (a) a hinge region derived from a human CD8α polypeptide, (b) a transmembrane domain derived from a human CD8α polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3ζ intracellular signaling domain; and (ii) a human CD28 intracellular signaling domain. In some embodiments, a CAR comprises a sequence as set forth in SEQ ID NO: 27.

[0100] In some embodiments, a CAR comprises: (a) a hinge region, (b) a transmembrane domain derived from a human CD8α polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3ζ intracellular signaling domain or fragment thereof; and (ii) a CD27 and / or a CD28 intracellular signaling domain or fragment thereof, wherein the CD27 and / or CD28 intracellular signaling domain or fragment thereof is or comprises a co- stimulatory domain.

[0101] In some embodiments, a CAR comprises: (a) a hinge region, (b) a transmembrane domain derived from a human CD8α polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3ζ intracellular signaling domain or fragment thereof; and (ii) a human CD28, a human CD27, and / or an FcεRI gamma chain intracellular signaling domain or fragment thereof, wherein the human CD28, the human CD27, and / or the FcεRI gamma chain intracellular signaling domain or fragment thereof are or comprise a co-stimulatory domain.

[0102] In some embodiments, a CAR can comprises: (a) a hinge region, (b) a transmembrane domain derived from a human CD8α polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3ζ intracellular signaling domain; and (ii) a human CD28 and / or an FcεRI gamma chain intracellular signaling domain, wherein the CD28 and / or the FcεRI gamma chain intracellular signaling domain or fragment thereof are or comprise a co-stimulatory domain.

[0103] In some embodiments, a CAR as described herein, further comprises a signal peptide sequence. In some embodiments, a signal peptide is positioned at the amino terminus of an extracellular domain (e.g., at the N-terminus of an antigen-binding domain). A signal peptide as used in accordance with the present disclosure may comprise any suitable signal peptide sequence. In some embodiments, a signal peptide sequence is a human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor signal peptide sequence or a CD8α signal peptide sequence. In some embodiments, a CAR provided herein comprises aAttorney Docket No. KVN-011WO human scFv comprising a CD8α signal peptide sequence. In some embodiments, a signal peptide sequence comprises an amino acid sequence as set forth in SEQ ID NO: 15.

[0104] In some embodiments, a provided CAR comprises: (a) a CD8α hinge region comprising SEQ ID NO: 28, (b) a CD8α transmembrane domain comprising SEQ ID NO: 11, (c) a CD28 intracellular signaling domain comprising SEQ ID NO: 21, and (d) a CD3ζ intracellular signaling domain comprising SEQ ID NO: 23. In some embodiments, a provided CAR comprises, from N-terminus to C-terminus: (a) a CD8α hinge region comprising SEQ ID NO: 28, (b) a CD8α transmembrane domain comprising SEQ ID NO: 11, (c) a CD28 intracellular signaling domain comprising SEQ ID NO: 21, and (d) a CD3ζ intracellular signaling domain comprising SEQ ID NO: 23.

[0105] In some embodiments, a provided CAR comprises: (a) an antigen-binding domain comprising SEQ ID NO: 17, (b) a CD8α hinge region comprising SEQ ID NO: 28, (c) a CD8α transmembrane domain comprising SEQ ID NO: 11, (d) a CD28 intracellular signaling domain comprising SEQ ID NO: 21, and (e) a CD3ζ intracellular signaling domain comprising SEQ ID NO: 23. In some embodiments, a provided CAR comprises, from N- terminus to C-terminus: (a) an antigen-binding domain comprising SEQ ID NO: 17, (b) a CD8α hinge region comprising SEQ ID NO: 28, (c) a CD8α transmembrane domain comprising SEQ ID NO: 11, (d) a CD28 intracellular signaling domain comprising SEQ ID NO: 21, and (e) a CD3ζ intracellular signaling domain comprising SEQ ID NO: 23.

[0106] In some embodiments, a provided CAR comprises: (a) a CD8α signal peptide sequence comprising SEQ ID NO: 15, (b) an antigen-binding domain comprising SEQ ID NO: 17, (c) a CD8α hinge region as set forth in SEQ ID NO: 28, (d) a CD8α transmembrane domain as set forth in SEQ ID NO: 11, (e) a CD28 intracellular signaling domain as set forth in SEQ ID NO: 21, and (f) a CD3ζ intracellular signaling domain as set forth in SEQ ID NO: 23. In some embodiments, a provided CAR comprises, from N-terminus to C-terminus: (a) a CD8α signal peptide sequence comprising SEQ ID NO: 15, (b) an antigen-binding domain comprising SEQ ID NO: 17, (c) a CD8α hinge region as set forth in SEQ ID NO: 28, (d) a CD8α transmembrane domain as set forth in SEQ ID NO: 11, (e) a CD28 intracellular signaling domain as set forth in SEQ ID NO: 21, and (f) a CD3ζ intracellular signaling domain as set forth in SEQ ID NO: 23.

[0107] In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an aminoAttorney Docket No. KVN-011WO acid sequence having at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence as set forth in SEQ ID NO: 10.

[0108] In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence as set forth in SEQ ID NO: 13.

[0109] In some embodiments, a CAR of the present disclosure is encoded by nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at leastAttorney Docket No. KVN-011WO 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 14.

[0110] It has been observed that T cells engineered to express a CD19-CAR that incorporates a 4-1BB costimulatory domain produced substantially higher background levels of IFNγ, in the absence CD19-expressing target cells, than T cells engineered to express a CD19-CAR that incorporates a costimulatory domain derived from CD28, CD27, or FcεRI gamma chain. Without wishing to be bound by theory, it is hypothesized that the higher background level of cytokine production is undesirable for treating autoimmune diseases. Therefore, in certain embodiments, a CAR disclosed herein does not comprise an intracellular T cell signaling domain derived from 4-1BB. Nucleic Acid Constructs

[0111] The present disclosure further provides for an engineered nucleic acid, or nucleic acid construct, comprising a nucleic acid sequence that encodes any polypeptide described herein, e.g., any CAR described herein. In some embodiments, an engineered nucleic acid comprises a promoter operably linked to a nucleic acid sequence that encodes a CAR (e.g., any CAR described herein). Any appropriate promoter may be operably linked to any of the engineered nucleic acid sequences described herein. Non-limiting examples of promoters that may be used in accordance with the present disclosure include EF1a, SFFV, PGK, CMV, CAG, UbC, murine stem cell virus (MSCV), MND, EF1a hybrid promoters, CAG hybrid promoters, or derivatives or functional fragments thereof. In some embodiments, a promoterAttorney Docket No. KVN-011WO is an EF1a promoter. In some embodiments, promoter is a SFFV promoter. In some embodiments, a promoter is a PGK promoter. In some embodiments, a promoter is a CMV promoter. In some embodiments, a promoter is a CAG promoter. In some embodiments, a promoter is a UbC promoter. In some embodiments, a promoter is a MSCV promoter. In some embodiments, a promoter is a MND promoter.

[0112] In some cases, an engineered nucleic acid comprises sufficient cis-acting elements (e.g., a promoter and / or an enhancer) that supplement expression of a provided engineered nucleic acid sequence where the remaining elements needed for expression can be supplied by a host cell (e.g., a mammalian cell, e.g., a T cell) or in an in vitro expression system.

[0113] The present disclosure also provides for vectors, or plasmids, comprising any engineered nucleic acid as described herein. The present disclosure provides for transposons, cosmids, viral vectors (e.g., any adenoviral vectors (e.g., pSV or pCMV vectors), adeno- associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors), and any Gateway® vectors comprising any engineered nucleic acid described herein. In some embodiments, a viral vector is selected from the group consisting of: a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral (AAV) vector. In some embodiments, a viral vector is a lentiviral vector. In some embodiments, a viral vector is a retroviral vector. In some embodiments, a viral vector is an adenoviral vector. In some embodiments, a viral vector is a AAV vector.

[0114] Exemplary lentiviral vectors that may be used in accordance with the present disclosure include vectors derived from human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), and caprine arthritis encephalitis virus (CAEV).

[0115] Retroviral vectors typically are constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by a gene of interest or expression cassette of interest (e.g., an engineered nucleic acid as described here). Most often, the structural genes (i.e., gag, pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal. Accordingly, in some embodiments, a minimum retroviral vector comprises from 5’ to 3’: a 5’ long terminal repeat (LTR), a packaging signal, an optional exogenous promoter and / or enhancer, anAttorney Docket No. KVN-011WO exogenous gene of interest (or engineered nucleic acid), and a 3' LTR. In some embodiments, if no exogenous promoter is provided, gene expression may be driven by the 5' LTR, which is a weak promoter and requires the presence of Tat to activate expression. In many embodiments, structural genes can be provided in separate vectors for manufacture of the lentivirus, rendering the produced virions replication-defective. Specifically, with respect to lentivirus, the packaging system may comprise a single packaging vector encoding the Gag, Pol, Rev, and Tat genes, and a third, separate vector encoding the envelope protein Env (usually VSV‐G due to its wide infectivity). To improve the safety of the packaging system, the packaging vector can be split, expressing Rev from one vector, Gag and Pol from another vector. Tat can also be eliminated from the packaging system by using a retroviral vector comprising a chimeric 5’ LTR, wherein the U3 region of the 5’ LTR is replaced with a heterologous regulatory element.

[0116] Nucleic acids (e.g., genes) to be packaged into a retrovirus (e.g., a lentivirus) can be incorporated into the proviral backbone in several general ways. The most straightforward constructions are ones in which the structural genes of the retrovirus are replaced by a single gene which then is transcribed under the control of the viral regulatory sequences within the LTR. Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.

[0117] Accordingly, nucleic acids (e.g., genes) to be packaged into a retrovirus are flanked by 5′ and 3′ LTRs, which serve to promote transcription and polyadenylation of the virion RNAs, respectively. The term “long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which, in their natural sequence context, are direct repeats and contain U3, R and U5 regions. LTRs generally provide functions fundamental to the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication. The LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals, and sequences needed for replication and integration of the viral genome. The U3 region contains the enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. In certain embodiments, the R region comprises a trans-activation response (TAR) genetic element, which interacts with the trans-activatorAttorney Docket No. KVN-011WO (tat) genetic element to enhance viral replication. This element is not required in embodiments wherein the U3 region of the 5′ LTR is replaced by a heterologous promoter.

[0118] In some embodiments, a retroviral vector comprises a modified 5′ LTR and / or 3′ LTR. Modifications of the 3′ LTR are often made to improve the safety of lentiviral or retroviral systems by rendering viruses replication-defective. In some embodiments, a retroviral vector is a self-inactivating (SIN) vector. As used herein, a SIN retroviral vector refers to a replication-defective retroviral vector in which the 3′ LTR U3 region has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the 3′ LTR U3 region is used as a template for the 5′ LTR U3 region during viral replication and, thus, the viral transcript cannot be made without the U3 enhancer-promoter. In some embodiments, a 3′ LTR is modified such that the U5 region is replaced, for example, with an ideal polyadenylation sequence. It should be noted that modifications to the LTRs such as modifications to the 3′ LTR, the 5′ LTR, or both 3′ and 5′ LTRs, are also included in some embodiments of the present disclosure.

[0119] In some embodiments, the U3 region of the 5′ LTR is replaced with a heterologous promoter to drive transcription of the viral genome during production of viral particles. Examples of heterologous promoters which can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. Typical promoters are able to drive high levels of transcription in a Tat-independent manner. This replacement reduces the possibility of recombination to generate replication-competent virus, because there is no complete U3 sequence in the virus production system.

[0120] Adjacent to a 5′ LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient packaging of viral RNA into particles (the Psi site). As used herein, the term “packaging signal” or “packaging sequence” refers to sequences located within the retroviral genome which are required for encapsidation of retroviral RNA strands during viral particle formation (see e.g., Clever et al., 1995 J. Virology, 69(4):2101-09). The packaging signal may be a minimal packaging signal (also referred to as the psi [Ψ] sequence) needed for encapsidation of the viral genome.

[0121] In some embodiments, a retroviral vector (e.g., lentiviral vector) further comprises a FLAP. As used herein, the term “FLAP” refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Patent No.Attorney Docket No. KVN-011WO 6,682,907 and in Zennou et al. (2000) Cell 101:173. During reverse transcription, central initiation of the plus-strand DNA at the cPPT and central termination at the CTS lead to the formation of a three-stranded DNA structure: a central DNA flap. While not wishing to be bound by any theory, the DNA flap may act as a cis-active determinant of lentiviral genome nuclear import and / or may increase the titer of the virus. In some embodiments, retroviral vector backbones comprise one or more FLAP elements upstream or downstream of the heterologous genes of interest in the vectors. For example, in some embodiments, a transfer plasmid includes a FLAP element. In some embodiments, a vector of the present disclosure comprises a FLAP element isolated from HIV-1.

[0122] In some embodiments, a retroviral vector (e.g., lentiviral vector) further comprises an export element. In some embodiments, retroviral vectors comprise one or more export elements. The term “export element” refers to a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) RRE (see e.g., Cullen et al., (1991) J. Virol.65: 1053; and Cullen et al., (1991) Cell 58: 423) and the hepatitis B virus post-transcriptional regulatory element (HPRE). Generally, the RNA export element is placed within the 3′ UTR of a gene, and can be inserted as one or multiple copies.

[0123] In some embodiments, a retroviral vector (e.g., lentiviral vector) further comprises a posttranscriptional regulatory element. A variety of posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid, e.g., woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; see Zufferey et al., (1999) J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); an optimized posttranscriptional regulatory element (oPRE; see Schambach et al., (2006) Gene Therapy 13, 641–45); and the like (Liu et al., (1995), Genes Dev., 9:1766). The posttranscriptional regulatory element is generally positioned at the 3′ end the heterologous nucleic acid sequence. This configuration results in synthesis of an mRNA transcript whose 5′ portion comprises the heterologous nucleic acid coding sequences and whose 3′ portion comprises the posttranscriptional regulatory element sequence. In some embodiments, vectors of the present disclosure lack or do not comprise a posttranscriptional regulatory element such as a WPRE or HPRE, because in some instances these elements increase the risk of cellular transformation and / or do not substantially or significantly increase the amount of mRNA transcript or increase mRNA stability. Therefore, in certainAttorney Docket No. KVN-011WO embodiments, vectors of the present disclosure lack or do not comprise a WPRE or HPRE as an added safety measure.

[0124] Elements directing the efficient termination and polyadenylation of the heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. Accordingly, in some embodiments, a retroviral vector (e.g., lentiviral vector) further comprises a polyadenylation signal. The term “polyadenylation signal” or “polyadenylation sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase H. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a polyadenylation signal are unstable and are rapidly degraded. Illustrative examples of polyadenylation signals that can be used in a vector of the present disclosure, include an ideal polyadenylation sequence (e.g., AATAAA, ATTAAA AGTAAA), a bovine growth hormone polyadenylation sequence (BGHpA), a rabbit β-globin polyadenylation sequence (rβgpA), or another suitable heterologous or endogenous polyadenylation sequence known in the art.

[0125] In some embodiments, a retroviral vector further comprises an insulator element. Insulator elements may contribute to protecting retrovirus-expressed sequences, e.g., therapeutic genes, from integration site effects, which may be mediated by cis-acting elements present in genomic DNA and lead to deregulated expression of transferred sequences (i.e., position effect; see, e.g., Burgess-Beusse et al., (2002) Proc. Natl. Acad. Sci., USA, 99:16433; and Zhan et al., 2001, Hum. Genet., 109:471). In some embodiments, a retroviral vector comprises an insulator element in one or both LTRs or elsewhere in the region of the vector that integrates into the cellular genome. Suitable insulators for use in the present disclosure include, but are not limited to, the chicken β-globin insulator (see Chung et al., (1993). Cell 74:505; Chung et al., (1997) Proc. Natl. Acad. Sci., USA 94:575; and Bell et al., 1999. Cell 98:387). Examples of insulator elements include, but are not limited to, an insulator from a β-globin locus, such as chicken HS4.

[0126] Non-limiting examples of lentiviral vectors include pLVX-EF1alpha-AcGFP1-C1 (Clontech Catalog #631984), pLVX-EF1alpha-IRES-mCherry (Clontech Catalog #631987), pLVX-Puro (Clontech Catalog #632159), pLVX-IRES-Puro (Clontech Catalog #632186), pLenti6 / V5-DESTTM(Thermo Fisher), pLenti6.2 / V5-DESTTM(Thermo Fisher), pLKO.1 (Plasmid #10878 at Addgene), pLKO.3G (Plasmid #14748 at Addgene), pSico (Plasmid #11578 at Addgene), pLJM1-EGFP (Plasmid #19319 at Addgene), FUGW (Plasmid #14883 at Addgene), pLVTHM (Plasmid #12247 at Addgene), pLVUT-tTR-KRAB (Plasmid #11651Attorney Docket No. KVN-011WO at Addgene), pLL3.7 (Plasmid #11795 at Addgene), pLB (Plasmid #11619 at Addgene), pWPXL (Plasmid #12257 at Addgene), pWPI (Plasmid #12254 at Addgene), EF.CMV.RFP (Plasmid #17619 at Addgene), pLenti CMV Puro DEST (Plasmid #17452 at Addgene), pLenti-puro (Plasmid #39481 at Addgene), pULTRA (Plasmid #24129 at Addgene), pLX301 (Plasmid #25895 at Addgene), pHIV-EGFP (Plasmid #21373 at Addgene), pLV-mCherry (Plasmid #36084 at Addgene), pLionII (Plasmid #1730 at Addgene), pInducer10-mir-RUP- PheS (Plasmid #44011 at Addgene). These vectors can be modified to be suitable for therapeutic use. For example, a selection marker (e.g., puro, EGFP, or mCherry) can be deleted or replaced with a second exogenous gene of interest. Further examples of lentiviral vectors are disclosed in U.S. Patent Nos.7,629,153, 7,198,950, 8,329,462, 6,863,884, 6,682,907, 7,745,179, 7,250,299, 5,994,136, 6,287,814, 6,013,516, 6,797,512, 6,544,771, 5,834,256, 6,958,226, 6,207,455, 6,531,123, and 6,352,694, and PCT Publication No. WO2017 / 091786.

[0127] In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence encoding a CAR, wherein the nucleic acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered nucleic acid of the present disclosure comprises a nucleic acid sequence as set forth in SEQ ID NO: 14.Attorney Docket No. KVN-011WO

[0128] In certain embodiments, a lentivirus vector disclosed herein comprises a truncated 5’ LTR (e.g., with deletion of its U3 region), an HIV-1Ψ packaging sequence, a MSCV promoter operably linked to a nucleic acid encoding a CAR (e.g., any of the CARs as disclosed herein), and a truncated 3’ LTR (e.g., with deletion of its U3 region). In certain embodiments, the lentivirus vector further comprises a RRE, a cPPT / CTS, and / or an oPRE. In certain embodiments, the lentivirus vector comprises a truncated 5’ LTR (e.g., with deletion of its U3 region), an HIV-1Ψ packaging sequence, a RRE, a cPPT / CTS, a MSCV promoter operably linked to a nucleic acid encoding a CAR (e.g., any of the CARs as disclosed herein), an oPRE, and a truncated 3’ LTR (e.g., with deletion of its U3 region). In certain embodiments, the lentivirus vector is pseudotyped with VSV-G envelope protein. Methods of Making Engineered T Cells

[0129] Also provided herein are methods of making an engineered T cell, the methods comprising introducing into a host T cell an engineered nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., any CAR described herein). Accordingly in some embodiments, an engineered T cell refers to a genetically modified T cell that has been modified to express a CAR, e.g., any provided anti-CD19 CAR.

[0130] In some embodiments, a host T cell used to make an engineered T cell can be any T cell such as a cultured T cell, e.g., a primary T cell, or a T cell derived from a cultured T cell line, e.g., a Jurkat, SupT1, etc., or a T cell obtained from a mammal. In some embodiments, a T cell used to make an engineered T cell can be selected from naive T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or sub-populations thereof. In some embodiments, a host T cell used to make an engineered T cell can be a CD3+ cell. In some embodiments, a host T cell can be CD4+, CD8+, or CD4+ and CD8+. In some embodiments, a host T cell can be any type of T cell, e.g., CD4+ / CD8+ double positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), memory T cells, naive T cells, regulatory T cells, γδT cells, etc. In some embodiments, a host T cell used to make an engineered T cell can be any T cell at any stage of development. Additional types of helper T cells include Th3 (Treg) cells, Th17 cells, Th9 cells, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). In some embodiments, obtained host T cells are substantially free of non-T cells.Attorney Docket No. KVN-011WO

[0131] The T cells can be obtained from various biological samples of a subject (e.g., a human subject). Non-limiting examples of biological sample include cells, tissue (e.g., tissue obtained by biopsy), blood, serum, plasma, or any sample derived therefrom. In certain embodiments, the sample is a whole blood sample or an apheresis (e.g., leukapheresis) sample obtained from the subject. In certain embodiments, the method comprises obtaining the sample from the subject. In certain embodiments, the method comprises having obtained the sample from the subject.

[0132] In certain embodiments, the T cells are isolated from the sample. Isolation of T cells may include an initial purification of T cells from a mixture of plasma, lymphocytes, platelets, red blood cells, monocytes, and granulocytes. Methods for isolation of T cells from a biological sample, such as a whole blood sample or a leukapheresis sample, are well- known. Exemplary methods may include leukapheresis, elutriation, density gradient centrifugation, enrichment by selection, and the like. For example, the method may include obtaining or having obtained a biological sample, such as a fresh, refrigerated, frozen, or cryopreserved leukapheresis product or alternative source of hematopoietic tissue, such as a whole blood sample, bone marrow sample, or a tumor or organ biopsy or removal (e.g., thymectomy) from an entity, such as a laboratory, hospital, or healthcare provider, and performing the aforementioned isolation steps to produce an enriched population of T cells (e.g., starting population of T cells) suitable for expression of a heterologous protein.

[0133] Furthermore, the purity of the T cell population can be increased by using one or more selection steps, such as negative selection or positive selection. Negative selection typically involves removal of undesired cell types from a mixed population of cells in a sample using one or more agents that selectively bind to the undesired cell type, whereas positive selection typically involves isolation of the desired cell population using one or more agents that selectively bind to the desired cell type. Enrichment of a T cell population by negative selection can be accomplished, for example, with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immuno-adherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail can include antibodies to CD14, CD20, CDb, CD16, HLA-DR, and CD8. On the other hand, a positive selection step can be used to specifically select for the desired cell type. Positive selection of T cells can, in certain embodiments, include incubation of aAttorney Docket No. KVN-011WO mixed population of cells that contains the T cells with a CD3-binding agent (e.g., anti-CD3 antibody-conjugated beads) for a time sufficient for positive selection of the desired T cells.

[0134] In some embodiments, engineered T cells are made using a mixture of cells (e.g., a mixture of host cells). For example, a mixture of cells may be obtained (e.g., from a subject), and an engineered nucleic acid may be inserted into the mixture of cells such that a mixture of engineered cells is made. In some embodiments, a mixture of cells comprises a mixture of T cells (e.g., any T cells described herein). In some embodiments, a mixture of cells comprises CD4+ and / or CD8+ T cells. In some embodiments, a mixture of cells comprises CD4+ and CD8+ T cells. In some embodiments, a mixture of cells is obtained by enriching for CD4+ and CD8+ T cells, yielding an enriched mixture of CD4+ and CD8+ cells. In certain embodiments, the mixture of cells comprises 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 20-30%, 20-40%, 20-50%, 20- 60%, 30-40%, 30-50%, or 30-60% of CD8+T cells (e.g., CD8+cytotoxic T cells) out of all T cells in the population. In certain embodiments, the mixture of cells further comprises 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1-70%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 30-40%, 30-50%, 30-60%, or 30-70% of CD4+T cells (e.g., CD4+helper T cells) out of all T cells in the population. In certain embodiments, the mixture of cells comprise CD8+T cells (e.g., CD8+cytotoxic T cells) and CD4+T cells (e.g., CD4+helper T cells) at a ratio of 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, 1:5 to 2:1, 1:4 to 2:1, 1:3 to 1:1, or 1:2 to 1:1. In some embodiments, the mixture of cells comprise CD8+ T cells and CD4+ T cells at a ratio of about 1:2. In some embodiments, a host cell or mixture of host cells are expanded before introduction of an engineered nucleic acid or vector or plasmid comprising an engineered nucleic acid. In some embodiments, a host cell or mixture of host cells are allogeneic. In some embodiments, a host cell or mixture of host cells are autologous.

[0135] In some embodiments, introducing an engineered nucleic acid (or a vector or plasmid comprising an engineered nucleic acid) to a host cell comprises contacting the host cell with a viral vector (e.g., any viral vector described herein). In some embodiments, a viral vector is selected from the group consisting of: a lentiviral vector, a retroviral vector, an adenoviral vector, transposons, cosmids, and an AAV vector. In some embodiments, a viral vector is a lentiviral vector. In some embodiments, a step of introducing an engineered nucleic acid (or a vector or plasmid comprising an engineered nucleic acid) to a host cell comprises use of viral transduction. Any known method of introducing nucleic acidsAttorney Docket No. KVN-011WO (including nucleic acid vectors and plasmids) into a host cell may be used in accordance with the present disclosure.

[0136] Methods of introducing nucleic acid constructs into a cell (e.g., a eukaryotic cell) are known in the art. Non-limiting examples of methods that can be used to introduce an engineered nucleic acid or nucleic acid construct (e.g., a vector or plasmid comprising an engineered nucleic acid) into a cell include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalefection, hydrodynamic delivery, magnetofection, viral transduction (e.g., adenoviral and lentiviral transduction), and nanoparticle transfection. As used herein, “transformed” and “transduced” are used interchangeably.

[0137] In some embodiments, an engineered nucleic acid is introduced to a cell using a lentiviral vector. In some embodiments, the lentiviral vector is used at a multiplicity of infection (MOI) of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or greater. In some embodiments, the lentiviral vector is used at an MOI of about 1. In some embodiments, the lentiviral vector is used at an MOI of about 2. In some embodiments, the lentiviral vector is used at an MOI of about 3. In some embodiments, the lentiviral vector is used at an MOI of about 4. In some embodiments, the lentiviral vector is used at an MOI of about 5. In some embodiments, the lentiviral vector is used at an MOI of about 6. In some embodiments, the lentiviral vector is used at an MOI of about 7. In some embodiments, the lentiviral vector is used at an MOI of about 8. In some embodiments, the lentiviral vector is used at an MOI of about 9. In some embodiments, the lentiviral vector is used at an MOI of about 10.

[0138] In some embodiments, a provided method further includes a step of contacting a host T cell with an effective amount of one or more CD3-stimulation agents in the absence of a CD28 stimulating agent under conditions that allow for the stimulation of the host T cell. In other embodiments, a provided method further includes a step of contacting a host T cell with an effective amount of one or more agents that activate both CD3 and CD28 (e.g., a solid surface, such as a polymeric nanomatrix, coated with an anti-CD3 antibody and an anti- CD28 antibody) under conditions that allow for the stimulation of the host T cell.

[0139] In some embodiments, the present disclosure provides a method of making an engineered T cell, the method comprising steps of: (a) obtaining a host T cell from a subject; and (b) introducing an engineered nucleic acid (e.g., any engineered nucleic acid described herein) to the host T cell. In some embodiments, a method of making an engineered T cellAttorney Docket No. KVN-011WO further comprises a step of contacting a host T cell with an effective amount of one or more CD3-stimulation agents in the absence of a CD28 stimulating agent under conditions that allow for the stimulation of the host T cell. In other embodiments, a method of making an engineered T cell further comprises a step of contacting a host T cell with an effective amount of one or more agents that activate both CD3 and CD28 (e.g., a magnetic bead coated with an anti-CD3 antibody and an anti-CD28 antibody) under conditions that allow for the stimulation of the host T cell. In certain embodiments, the stimulation step is taken prior to step (b).

[0140] In some embodiments, the present disclosure provides a method of making an engineered T cell, the method comprising steps of: (a) obtaining a host T cell from a subject; and (b) introducing an engineered nucleic acid (e.g., any engineered nucleic acid described herein, e.g., a vector comprising an engineered nucleic acid, using any method of introducing provided herein) to the host T cell.

[0141] In some embodiments, the present disclosure provides a method of making an engineered T cell, the method comprising steps of: (a) obtaining a host T cell mixture from a subject; and (b) introducing an engineered nucleic acid (e.g., any engineered nucleic acid described herein, e.g., a vector comprising an engineered nucleic acid, using any method of introducing provided herein) to the host T cell mixture. In some embodiments, a method of making an engineered T cell further comprises a step of enriching a host T cell mixture for CD4+ and CD8+ T cells prior to introducing an engineered nucleic acid to the host T cell mixture.

[0142] In some embodiments, a method of making an engineered T cell further comprises a step of expanding a host T cell or T cell mixture before introduction of an engineered nucleic acid or vector or plasmid comprising an engineered nucleic acid. In certain embodiments, the T cells are expanded after introduction of the engineered nucleic acid, while the nucleic acid is still in the cell culture medium. In certain embodiments, the T cells are expanded for at least 3, 4, 5, 6, 7, or 8 days in the presence of one or more cytokines, including but not limited to IL-2, IL-7, and / or IL-15. In certain embodiments, the T cells are expanded for at least 3, 4, 5, 6, 7, or 8 days in the presence of IL-7 and IL-15.

[0143] Also provided herein are engineered T cells produced using any of the methods described herein. The present disclosure provides for engineered T cells comprising an engineered nucleic acid (e.g., any of the engineered nucleic acid described herein). In many embodiments of the present disclosure an engineered T cell comprises an engineered nucleic acid encoding a CAR (e.g., any CAR described herein). In some embodiments, the presentAttorney Docket No. KVN-011WO disclosure provides an engineered T cell comprising a nucleic acid sequence encoding an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, the present disclosure provides an engineered T cell comprising an engineered nucleic acid sequence as set forth in SEQ ID NO: 14.

[0144] The population of engineered T cells produced may include T cells of various phenotypes, such as naive T (TN) cells characterized as CD45RO-, CCR7+, and CD95-, central memory T (TCM) cells characterized as CD45RO+ and CCR7+, effector memory T (TEM) cells characterized as CD45RO+ and CCR7-, stem memory T (TSCM) cells characterized as CD45RO-, CCR7+, and CD95+, and effector memory cells re-expressing CD45RA T (TEMRA) cells characterized as CD45RO- and CCR7-. Alternative characterizations of these T cell subsets include but are not limited to naive T (TN) cells characterized as CD45RA+, CCR7+, and CD95-, central memory T (TCM) cells characterized as CD45RA- and CCR7+, effector memory T (TEM) cells characterized as CD45RA- and CCR7-, stem memory T (TSCM) cells characterized as CD45RA+, CCR7+, and CD95+, and effector memory cells re-expressing CD45RA T (TEMRA) cells characterized as CD45RA+ and CCR7-. In some embodiments, the population comprises at least 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% CD4+ T cells, out of all T cells in the population. In some embodiments, the population comprises at least 20%, 25%, 30%, 40%, 50%, 55%, or 60% CD8+ T cells, out of all T cells in the population. In some embodiments, the population comprises at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7% CD4+ TN, out of all T cells in the population. In some embodiments, the population comprises at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, or 35% CD4+ TSCM, out of all T cells in the population. In some embodiments, the population comprises at least 10%, 15%, 20%, 25%, or 30% CD4+ TCM, out of all T cells in the population. In some embodiments, the population comprises at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% CD4+ TEM, out of all T cells in the population. In some embodiments, the population comprises at least 5%, 10%, 15%, 20%, 25%, or 30% CD4+ TEMRA, out of all T cells in the population. In some embodiments, the population comprises at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 5%, 10%, 15%, 20%, or 25% CD8+ TN, out of all T cells in the population. In some embodiments, the population comprises at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% CD8+ TSCM, out of all T cells in the population. In some embodiments, the population comprises at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%,Attorney Docket No. KVN-011WO 10%, 15%, 20%, 25%, or 30% CD8+ TCM, out of all T cells in the population. In some embodiments, the population comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD8+ TEM, out of all T cells in the population. In some embodiments, the population comprises at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% CD8+ TTEMRA, out of all T cells in the population. It will be understood by a skilled artisan that the percentage of certain T cells present in a population of engineered T cells may vary from patient to patient. Compositions

[0145] Also provided herein are compositions (e.g., pharmaceutical compositions) comprising any engineered T cell described herein or any engineered nucleic acid described herein. In some embodiments, a provided pharmaceutical compositions can be formulated for intravenous administration. In some embodiments, a pharmaceutical compositions can include a pharmaceutically acceptable carrier (e.g., phosphate buffered saline).

[0146] In some embodiments, a provided composition (e.g., pharmaceutical composition) is formulated in a chemically defined freezing medium. In some embodiments, the chemically defined freezing medium comprises about 1% human serum albumin and about 5% dimethyl sulfoxide. In some embodiments, the chemically defined freezing medium comprises about 50% Plasma-Lyte A™ containing about 2% human serum albumin (for final human serum albumin concentration of about 1%) and about 50% CryoStor10 containing dimethyl sulfoxide to a final concentration of about 5%. Methods of Treatment

[0147] Provided herein are methods and compositions for reducing the number of B cells in a tissue in a subject having systemic sclerosis. In some embodiments, the present disclosure provides a method of reducing the number of B cells in a tissue (e.g., any human tissue, such as the skin) in a subject having systemic sclerosis, the method comprising administering a therapeutically effective amount of any engineered T cell described herein to the subject. Also provided herein are methods of treating a subject having systemic sclerosis, the method comprising administering a therapeutically effective amount of an engineered T cell (e.g., any engineered T cell described herein, e.g., an anti-CD19 CAR T cell) to the subject.

[0148] In some embodiments, the systemic sclerosis is limited cutaneous systemic sclerosis or diffuse cutaneous systemic sclerosis. In some embodiments, the systemicAttorney Docket No. KVN-011WO sclerosis is limited cutaneous systemic sclerosis. In some embodiments, the diffuse cutaneous systemic sclerosis. In some embodiments, the systemic sclerosis is refractory systemic sclerosis. In some embodiments, the systemic sclerosis is positive for one or more autoantibodies associated with the disease (e.g., anti-Scl-70, anti-centromere, anti-RNAPIII, etc.). In some embodiments, the systemic sclerosis is anti-Scl-70 positive (i.e., the subject tests positive for anti-Scl-70 antibodies). In some embodiments, the systemic sclerosis is anti- centromere positive (i.e., the subject tests positive for anti-centromere antibodies). In some embodiments, the systemic sclerosis is anti-RNAPIII positive (i.e., the subject tests positive for anti-RNAPIII antibodies). In some embodiments, the systemic sclerosis is anti-Th / To positive (i.e., the subject tests positive for anti-Th / To antibodies). In some embodiments, the systemic sclerosis is anti-U1 RNP positive (i.e., the subject tests positive for anti-U1 RNP antibodies). In some embodiments, the systemic sclerosis is anti-U3 RNP positive (i.e., the subject tests positive for anti-U3 RNP antibodies). In some embodiments, the systemic sclerosis is anti-PM / Sc-100 positive (i.e., the subject tests positive for anti-PM / Sc-100 antibodies). In some embodiments, the systemic sclerosis is anti-endothelial cell positive (i.e., the subject tests positive for anti-endothelial cell antibodies). In some embodiments, the systemic sclerosis is anti-matrix metalloproteinase positive (i.e., the subject tests positive for anti-matrix metalloproteinase antibodies). In some embodiments, the systemic sclerosis is anti-fibrillin I positive (i.e., the subject tests positive for anti-fibrillin I antibodies). In some embodiments, the systemic sclerosis is anti-B23 positive (i.e., the subject tests positive for anti-B23 antibodies).

[0149] In some embodiments, a subject receiving a provided treatment has previously received at least one standard of care or approved treatment (e.g., any standard of care or approved treatment described herein) for systemic sclerosis that was ineffective and / or caused one or more adverse side effects. For example, in some embodiments, a subject receiving a presently provided treatment has previously been treated with an immunosuppressive drug (e.g., azathioprine, mycophenolate mofetil, cyclophosphamide, cyclosporin, tacrolimus, tocilizumab, nintedanib, etc.). In some embodiments, a subject receiving a presently provided treatment has previously been treated with a JAK inhibitor (e.g., baricitinib, tofacitinib, upadacitinib, etc.). In some embodiments, a subject receiving a presently provided treatment has previously been treated for Raynaud syndrome (e.g., with a nifedipine, bosentan, sildenafil, tadalafil, vardenafil, etc.). In some embodiments, a subject receiving a presently provided treatment has previously been treated with corticosteroids (e.g., glucocorticoids such as betamethasone, dexamethasone, cortisone, hydrocortisone,Attorney Docket No. KVN-011WO prednisone, prednisolone, methylprednisolone). In some embodiments, a subject receiving a presently provided treatment has been previously treated with an anti-CD20 antibody therapy (e.g., an obinutuzumab therapy (e.g., Gazyva), an ocrelizumab therapy (e.g., Ocrevus), an ofatumumab therapy (e.g., Kesimpta), a rituximab therapy (e.g., Rituxan, Ruxience, Truxima, Riabni), etc.). In some embodiments, a subject receiving a presently provided treatment has previously been treated with hydroxychloroquine. In some embodiments, a subject receiving a presently provided treatment has previously been treated with leflunomide. In some embodiments, a subject receiving a presently provided treatment has previously been treated with abatacept. In some embodiments, a subject receiving a presently provided treatment has previously been treated with nintedanib (e.g., Ofev). In some embodiments, a subject receiving a presently provided treatment has previously been treated with tocilizumab. In some embodiments, a subject receiving a presently provided treatment has previously been treated with hematopoietic stem cell transplantation. In some embodiments, a subject receiving a presently provided treatment has previously been treated with intravenous immunoglobulin (IVIG) infusion. In some embodiments, a subject with systemic sclerosis (e.g., any form of SSc described herein) is treated with a composition or method of the present disclosure as a first line therapy.

[0150] In some embodiments, a subject receiving a presently provided treatment has previously received a standard of care or approved treatment for systemic sclerosis (e.g., an immunosuppressive drug, or any common treatment described herein, e.g., those discussed above), and the standard of care or approved treatment dose is reduced (e.g., by tapering) or the standard of care or approved treatment is terminated (e.g., by washing out) before T cells are obtained from the subject for making engineered T cells (e.g., via any method described herein). In some embodiments, a standard of care or approved treatment dose is reduced or the standard of care treatment is terminated at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks before T cells are obtained from the subject for making engineered T cells (e.g., by apheresis). In some embodiments, a standard of care or approved treatment dose is reduced or terminated at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks prior to a subject receiving a provided treatment. In some embodiments, a standard of care or approved treatment dose is reduced at least 6 weeks prior to a subject receiving a provided treatment. InAttorney Docket No. KVN-011WO some embodiments, a subject continues to receive a standard of care or approved treatment (e.g., at a standard dose, at a reduced dose, and / or keeping only a limited number of individual therapeutic components from the original SOC treatment) while also receiving the presently provided treatment. In some embodiments, a subject does not continue to receive a standard of care or approved treatment while receiving the presently provided treatment.

[0151] In some embodiments, a subject receiving a presently provided treatment has previously received a standard of care or approved therapy, and the standard of care or approved therapy is washed out over a period of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 10 weeks, about 12 weeks, or greater before T cells are obtained from the subject for making engineered T cells (e.g., via any method described herein, such as apheresis, or any common technique). In some embodiments, after T cells are obtained from the subject for making engineered T cells, the standard of care or approved therapy is resumed if required (e.g., as determined by a clinician to maintain disease stability). In some embodiments, a subject receiving a presently provided treatment has previously received an anti-CD20 monoclonal antibody therapy (e.g., rituximab), and the anti-CD20 monoclonal antibody therapy is washed out over a period of about 3 months or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, a subject receiving a presently provided treatment has previously received cyclophosphamide, and the cyclophosphamide is washed out over a period of about 14 days or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, a subject receiving a presently provided treatment has previously received cyclosporine, and the cyclosporine is washed out over a period of about 7 days or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, after T cells are obtained from the subject for making engineered T cells, treatment with cyclosporine is resumed at the prior stable dose. In some embodiments, a subject receiving a presently provided treatment has previously received tacrolimus, and the tacrolimus is washed out over a period of about 7 days or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, after T cells are obtained from the subject for making engineered T cells, treatment with tacrolimus is resumed at the prior stable dose. In some embodiments, a subject receiving a presently provided treatment has previously received mycophenolate mofetil, and the mycophenolate mofetil is washed out over a period of about 7 days or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, after TAttorney Docket No. KVN-011WO cells are obtained from the subject for making engineered T cells, treatment with mycophenolate mofetil is resumed at the prior stable dose. In some embodiments, a subject receiving a presently provided treatment has previously received hydroxychloroquine, and the hydroxychloroquine is washed out over a period of about 7 days or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, after T cells are obtained from the subject for making engineered T cells, treatment with hydroxychloroquine is resumed at the prior stable dose. In some embodiments, a subject receiving a presently provided treatment has previously received azathioprine, and the azathioprine is washed out over a period of about 7 days or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, after T cells are obtained from the subject for making engineered T cells, treatment with azathioprine is resumed at the prior stable dose. In some embodiments, a subject receiving a presently provided treatment has previously received methotrexate, and the methotrexate is washed out over a period of about 14 days or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, after T cells are obtained from the subject for making engineered T cells, treatment with methotrexate is resumed at the prior stable dose. In some embodiments, a subject receiving a presently provided treatment has previously received leflunomide, and the leflunomide is washed out over a period of about 14 days or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, leflunomide is washed out with cholestyramine. In some embodiments, a subject receiving a presently provided treatment has previously received tofacitinib, and the tofacitinib is washed out over a period of about 7 days or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, after T cells are obtained from the subject for making engineered T cells, treatment with tofacitinib is resumed at the prior stable dose. In some embodiments, a subject receiving a presently provided treatment has previously received upadacitinib, and the upadacitinib is washed out over a period of about 7 days or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, after T cells are obtained from the subject for making engineered T cells, treatment with upadacitinib is resumed at the prior stable dose. In some embodiments, a subject receiving a presently provided treatment has previously received baricitinib, and the baricitinib is washed out over a period of about 7 days or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, after T cells are obtained from the subject for making engineered T cells, treatment with baricitinib is resumed at the prior stable dose. In some embodiments, a subjectAttorney Docket No. KVN-011WO receiving a presently provided treatment has previously received abatacept, and the abatacept is washed out over a period of about 14 days or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, a subject receiving a presently provided treatment has previously received nintedanib, and the nintedanib is washed out over a period of about 3 days or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, after T cells are obtained from the subject for making engineered T cells, treatment with nintedanib is resumed at the prior stable dose. In some embodiments, a subject receiving a presently provided treatment has previously received tocilizumab, and the tocilizumab is washed out over a period of about 14 days or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, a subject receiving a presently provided treatment has previously received intravenous immunoglobulin (IVIG) therapy, and after T cells are obtained from the subject for making engineered T cells, IVIG therapy is resumed at the prior stable dose. In some embodiments, a subject receiving a presently provided treatment has previously received an investigational drug, and the investigational drug is washed out over a period of about 5 half- lives of the investigational drug or greater before T cells are obtained from the subject for making engineered T cells. In some embodiments, a corticosteroid (e.g., a glucocorticoid) treatment dose is reduced to lower than or equal to 10 mg per day, e.g., of prednisone (or equivalent), within 3 days before T cells are obtained from the subject for making engineered T cells (e.g., by apheresis).

[0152] In some embodiments, a subject to receive a provided treatment will undergo a treatment scheme that comprises steps of: (1) reducing and / or washing out one or more previous systemic sclerosis medications (e.g., as described herein); (2) obtaining T cells from the subject for making engineered T cells (e.g., by apheresis); (3) optionally resuming one or more of the previous medications from step (1) if required (e.g., as determined by a clinician to maintain disease stability) for a duration of time; (4) lymphodepletion (e.g., via any method described herein); and (5) administration of the engineered T cells. In some embodiments, the time between step (1) and step (2) is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 3 weeks, about 4 weeks, about 8 weeks, about 10 weeks, about 12 weeks, or more. It will be understood by a skilled artisan that the time to effectively reduce and / or wash out one or more previous medications (e.g., the time between step (1) and step (2)) may vary, e.g., may be longer or shorter, or fall between values provided herein, depending on many factorsAttorney Docket No. KVN-011WO including the type or class of previous medication and its half-life, among other things. In some embodiments, the time between step (2) and step (4) is about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, or more, or within 60 days. It will be understood by a skilled artisan that the time between obtaining T cells from a subject for making engineered T cells (e.g., by apheresis) and lymphodepletion (e.g., the time between step (2) and step (4)) may vary, e.g., may be longer or shorter, or fall between values provided herein, depending on many factors including the health of the subject and the quality of the cells recovered for engineering, among other things. In some embodiments, the time between step (4) and step (5) is about 5 days, about 6 days, about 7 days, or more. In some embodiments, the time between step (4) and step (5) is set to allow for at least 3 consecutive days of lymphodepletion treatment prior to administration of the engineered T cells. It will be understood by a skilled artisan that the time between lymphodepletion and administration of the engineered T cells (e.g., the time between step (4) and step (5)) may vary, e.g., may be longer or shorter, or fall between values provided herein, depending on many factors including the method of lymphodepletion and the health of the subject, among other things. In some embodiments, a treatment scheme does not comprise a lymphodepletion step (e.g., a step (4)). In some embodiments, a treatment scheme comprises a minimized lymphodepletion step (e.g., a step (4)), wherein, for example, minimized lymphodepletion may be carried out using any method described herein.

[0153] The present disclosure appreciates that therapeutic regimens for systemic sclerosis generally require chronic administration or administration of multiple treatment cycles to effectively treat the disease or disorder. The most common treatments include corticosteroids and / or immunosuppressive drugs, which can be very toxic to a subject. In some cases, these drugs can also suppress a subject’s immune system, resulting in serious infections (e.g., blood infections, fungal infections, skin infections, respiratory infections, etc.), malignancy, and / or other adverse side effects, e.g., in bone marrow, liver, kidneys. Accordingly, standard of care treatments such as immunosuppressive drugs present challenges to chronic use. In contrast, use of engineered T cells provided herein offers the premise of a single infusion possibly controlling disease for a prolonged period of time. Use of an engineered T cells as provided herein also permits repeated treatments (e.g., chronic administration, multiple treatment cycles, etc.) due to low toxicity profile and subsequent reduced side effects and low immunogenicity, e.g., as a result of the fully human nature the scFv, among other things. InAttorney Docket No. KVN-011WO many embodiments, use of the provided engineered T cells results in lower toxicity with subsequent reduced adverse effects and low immunogenicity over time and can be repeated in the future as needed.

[0154] In some embodiments of any of the methods described herein, a step of administering comprises administering two or more doses of an engineered T cell (e.g., any engineered T cell described herein). In some embodiments, a step of administering comprises administering five or more doses of an engineered T cell. In some embodiments, a step of administering comprises administering ten or more doses of an engineered T cell. In some embodiments, a dose of an engineered T cell is fractionated such that the total dose is administered over the course at least two, three, four, five, six, seven, or more days.

[0155] In some embodiments, a subject receiving a provided treatment (e.g., any engineered nucleic acid, engineered T cell, or CAR provided herein) has previously been treated with a lymphodepletion agent (e.g., cyclophosphamide and / or fludarabine), to increase the proliferation and persistence of CAR-T cells. In some embodiments, the subject has received a lymphodepletion treatment. In some embodiments, lymphodepletion treatment comprises intravenous administration of cyclophosphamide (e.g., at a dose of 300 mg / m2) and of fludarabine (e.g., at a dose of 30 mg / m2) prior to administration of the T cells, e.g., once every day for 3 days, starting 5 to 7 days prior to administration of the T cells. In some embodiments, the subject does not receive a lymphodepletion treatment prior to administration of the T cells. In some embodiments, the subject has received a minimized lymphodepletion treatment. In some embodiments, a minimized lymphodepletion treatment comprises intravenous administration of cyclophosphamide (e.g., at a dose of 150 mg / m2) and of fludarabine (e.g., at a dose of 15 mg / m2) prior to administration of the T cells, e.g., once every day for 3 days, starting 5 to 7 days prior to administration of the T cells. In some embodiments, a minimized lymphodepletion treatment reduces lymphocytes in a subject by about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% relative to the subject before receiving lymphodepletion treatment or another suitable control. In some embodiments, the subject has received a minimized lymphodepletion treatment resulting in about 50% reduction of lymphocytes in the subject relative to the subject before receiving lymphodepletion treatment or another suitable control.

[0156] The engineered T cells used in the methods of the present disclosure can be generated by a process disclosed herein. In some embodiments, an engineered T cell is generated by introducing into a T cell an engineered nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., any CAR described herein, e.g, an anti-CD19 CAR). InAttorney Docket No. KVN-011WO some embodiments, an engineered nucleic acid further comprises a promoter operably linked to a nucleic sequence encoding a CAR. In some embodiments, an engineered T cell is generated by further contacting the T cell with an effective amount of one or more agents that activate CD3 and CD28 under conditions that allow for stimulation of the T cell. In some embodiments, a T cell is obtained from a subject (e.g., an autologous or allogeneic subject), prior to a step of generating an engineered T cell and a step of administering the engineered T cell. An engineered T cell can be generated or made using any method of making an engineered T cell described herein, e.g., in the “Methods of Making Engineered T Cells” section above.

[0157] In some embodiments, the present disclosure provides for a method of reducing the number of B cells in a tissue in a subject having systemic sclerosis, the method comprising a step of administering to the subject an engineered T cell (e.g., any engineered T cell described herein). In some embodiments, the present disclosure provides for a method of reducing the number of B cells in a tissue in a subject having systemic sclerosis, the method comprising a step of administering to the subject an engineered T cell, wherein the engineered T cell is an anti-CD19 CAR T cell. In some embodiments, an anti-CD19 CAR T cell comprises a nucleic acid encoding an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, an anti-CD19 CAR T cell comprises a nucleic acid as set forth in SEQ ID NO: 14.

[0158] In some embodiments, the present disclosure provides for a method of treating systemic sclerosis, the method comprising a step of administering to a subject in need thereof an engineered T cell (e.g., any engineered T cell described herein). In some embodiments, the present disclosure provides for a method of treating systemic sclerosis, the method comprising a step of administering to a subject in need thereof an engineered T cell, wherein the engineered T cell is an anti-CD19 CAR T cell. In some embodiments, an anti-CD19 CAR T cell comprises a nucleic acid encoding an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, an anti-CD19 CAR T cell comprises a nucleic acid as set forth in SEQ ID NO: 14.

[0159] In some embodiments, the present disclosure provides for a method of treating limited cutaneous systemic sclerosis, the method comprising a step of administering to a subject in need thereof an engineered T cell (e.g., any engineered T cell described herein). In some embodiments, the present disclosure provides for a method of treating limited cutaneous systemic sclerosis, the method comprising a step of administering to a subject in need thereof an engineered T cell, wherein the engineered T cell is an anti-CD19 CAR T cell.Attorney Docket No. KVN-011WO In some embodiments, an anti-CD19 CAR T cell comprises a nucleic acid encoding an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, an anti-CD19 CAR T cell comprises a nucleic acid as set forth in SEQ ID NO: 14.

[0160] In some embodiments, the present disclosure provides for a method of treating diffuse cutaneous systemic sclerosis, the method comprising a step of administering to a subject in need thereof an engineered T cell (e.g., any engineered T cell described herein). In some embodiments, the present disclosure provides for a method of treating diffuse cutaneous systemic sclerosis, the method comprising a step of administering to a subject in need thereof an engineered T cell, wherein the engineered T cell is an anti-CD19 CAR T cell. In some embodiments, an anti-CD19 CAR T cell comprises a nucleic acid encoding an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, an anti-CD19 CAR T cell comprises a nucleic acid as set forth in SEQ ID NO: 14.

[0161] In some embodiments, the present disclosure provides for a method of treating refractory systemic sclerosis, the method comprising a step of administering to a subject in need thereof an engineered T cell (e.g., any engineered T cell described herein). In some embodiments, the present disclosure provides for a method of treating refractory systemic sclerosis, the method comprising a step of administering to a subject in need thereof an engineered T cell, wherein the engineered T cell is an anti-CD19 CAR T cell. The refractory systemic sclerosis can be refractory to any of the previous treatments disclosed herein, such as a corticosteroid (e.g., betamethasone, dexamethasone, cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone), an anti-CD20 antibody (e.g., obinutuzumab (e.g., Gazyva), ocrelizumab (e.g., Ocrevus), ofatumumab (e.g., Kesimpta), rituximab (e.g., Rituxan, Ruxience, Truxima, Riabni), etc.), an immunosuppressive drug (e.g., azathioprine, mycophenolate mofetil, cyclophosphamide, cyclosporin, tacrolimus, tocilizumab, nintedanib, etc.), a JAK inhibitor (e.g., baricitinib, tofacitinib, upadacitinib, etc.), a treatment for Raynaud syndrome (e.g., with a nifedipine, bosentan, sildenafil, tadalafil, vardenafil, etc.), hydroxychloroquine, leflunomide, abatacept, nintedanib, tocilizumab, hematopoietic stem cell transplantation, IVIG, or any combination thereof. In some embodiments, an anti-CD19 CAR T cell comprises a nucleic acid encoding an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, an anti-CD19 CAR T cell comprises a nucleic acid as set forth in SEQ ID NO: 14.

[0162] In some embodiments, provided engineered T cells are administered using parenteral administration (e.g., intravenous administration). Methods and compositions of the present disclosure may be administered using any suitable method.Attorney Docket No. KVN-011WO

[0163] In some embodiments, administering of methods and compositions provided herein to a subject with systemic sclerosis results in a reduction in the number, severity, or frequency of one or more symptoms of systemic sclerosis in the subject (e.g., as compared to the number, severity, or frequency of the one or more symptoms of systemic sclerosis in the subject prior to receiving treatment with provided methods or compositions). In some embodiments, a subject having systemic sclerosis having been administered an engineered T cell as described here can experience a reduction in inflammation and / or auto-antibody production. In some embodiments, a subject having systemic sclerosis having been administered an engineered T cell as described here can experience a remission in clinical manifestations of systemic sclerosis (e.g., calcinosis, Raynaud’s phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia, skin hardening and scarring, and impairment to other organs such as lungs, kidneys, etc.).

[0164] A pharmaceutical composition useful in the method disclosed herein can contain an engineered T cell and a pharmaceutically acceptable carrier or buffer. In some embodiments, the pharmaceutical composition can be formulated in an injectable form (e.g., as solution and / or suspension). In some embodiments, a pharmaceutical composition comprising an engineered T cell as provided herein can further include phosphate buffered saline. Pharmaceutically acceptable carriers, fillers, and vehicles that can be used in a pharmaceutical composition described herein can include, without limitation, ion exchangers, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, and sodium chloride.

[0165] An effective dosage (e.g., for a provided T cell composition) to administer to a patient intravenously can vary depending on the severity of systemic sclerosis, the age and general health condition of a subject, excipient usage, the possibility of co-usage with other therapeutic treatments, and the judgment of the treating physician. An effective amount of an engineered T cell can be any amount that reduces inflammation and auto-antibody production within a subject having systemic sclerosis (e.g., via deletion or reduction of autoreactive B cells) without producing significant toxicity to the subject. In some embodiments, an effective dosage may vary depending on the types of engineered T cells present in a provided engineered T cell composition (e.g., the effective dosage may be higher or lower depending on if the engineered T cell composition comprises more of one engineered T cell type, e.g., any T cell type described herein, such as a TEMRA cell, a Tscm cell, etc.). In manyAttorney Docket No. KVN-011WO embodiments, an effective dosage may also be dependent on the level of CAR expression in the provided engineered T cells and / or the percentage of engineered T cells within a provided composition. In some cases, engineered T cells can be a purified population of engineered T cells generated as described herein. In some cases, the purity of a population of engineered T cells can be assessed using any appropriate method, including, without limitation, flow cytometry. In some embodiments, purity of a population of engineered T cells can be assessed by quantifying the amount of T cells expressing the CAR relative to all the T cells in the population. In some cases, a population of engineered T cells to be administered to a subject can include a range of purities from about 5% to about 80%, about 10% to about 80%, about 15% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, about 5% to about 70%, about 5% to about 60%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 70% to about 100%, from about 70% to about 90%, from about 70% to about 80%, from about 80% to about 90%, from about 90% to about 100%, from about 80% to about 100%, from about 80% to about 90%, or from about 90% to 100%. In some cases, a dosage of a provided therapy (e.g., number of engineered T cells to be administered) can adjusted based on the level of purity of the therapy.

[0166] In some embodiments, provided compositions (e.g., pharmaceutical compositions) of engineered T cells are administered at a dose of about 0.1×108, about 0.2×108, about 0.3×108, about 0.4×108, about 0.5×108, about 0.6×108, about 0.7×108, about 0.8×108, about 0.9×108, about 1.0×108, about 1.1×108, about 1.2×108, about 1.3×108, about 1.4×108, or about 1.5×108engineered cells. In some embodiments, provided compositions (e.g., pharmaceutical compositions) of engineered T cells are administered at a dose of about 0.5×108engineered cells. In some embodiments, provided compositions (e.g., pharmaceutical compositions) of engineered T cells are administered at a dose of about 1.0×108engineered cells.

[0167] The frequency of administration of an engineered T cell can be any frequency that adequately controls disease per clinical assessment and / or reduces inflammation or auto- antibody production (e.g., any SSc associated auto-antibody, including those described herein) within a subject having systemic sclerosis (e.g., via deletion or reduction of autoreactive B cells) without producing toxicity to the subject. In some embodiments, the actual frequency of administration can vary depending on various factors including, without limitation, the effective amount, duration of treatment, use of multiple treatment agents, and severity of the condition may require an increase or decrease in frequency of administration.Attorney Docket No. KVN-011WO

[0168] An effective duration for administering a composition containing an anti-CD19 CAR T cell or a nucleic acid encoding the same can be any duration that reduces inflammation or auto-antibody production within the subject having systemic sclerosis (e.g., via deletion or reduction of autoreactive B cells) without producing toxicity to the subject. In some embodiments, the effective duration can vary from several days to several months. In some embodiments, the effective treatment duration for administering a composition containing an engineered T cell to treat systemic sclerosis can range in duration from about one month to about five years (e.g., from about two months to about five years, from about three months to about five years, from about six months to about five years, from about eight months to about five years, from about one year to about five years, from about one month to about four years, from about one month to about three years, from about one month to about two years, from about six months to about four years, from about six months to about three years, or from about six months to about two years). In some embodiments, the effective treatment duration is at least one year, two years, three years, or more. In some embodiments, a subject receives an infusion of a provided treatment and is cured (e.g., via initiation of an immune reset).

[0169] In some embodiments, a course of treatment and / or the severity of one or more symptoms related to systemic sclerosis can be monitored. Any appropriate method can be used to determine whether systemic sclerosis is being treated. For example, signs of a subject with a refractory form of systemic sclerosis being treated may include the subject entering a period of remission characterized by a reduction or improvement in one or more clinical symptoms of systemic sclerosis that are present at the time of treatment, and / or reduction in the progression or new incidence of one or more clinical symptoms of systemic sclerosis.

[0170] In some embodiments, the modified Rodnan skin score (mRSS) is used to monitor the course of treatment of a subject receiving a provided composition (e.g., lack of disease progression may be indicated by stable mRSS value or mRSS value not exceeding a certain threshold value). The mRSS may be calculated by summation of measurements of skin thickness in 17 different body sites including the face, upper arms, forearms, dorsum of hands, fingers, chest, abdomen, thighs, legs and feet. Each area is graded as follows: 0 = normal skin, 1 = mild skin thickening, 2 = moderate skin thickening with difficulty in making skin folds and no wrinkles, 3 = severe skin thickness with inability to make skin folds between two examining fingers. The total score ranges from 0 to 51, higher score indicates worse disease severity.Attorney Docket No. KVN-011WO

[0171] In some embodiments, a scleroderma health assessment questionnaire (SHAQ) component score (e.g., HAQ-DI, PtGA, HAQ-Pain, vascular, digital ulcers, lung involvement, and gastrointestinal involvement) is used to monitor the course of treatment of a subject receiving a provided composition. The HAQ-DI domain may be assessed by the eight sections of dressing, arising, eating, walking, hygiene, reach, grip, and common activities. There are 2 or 3 questions for each section. Scoring within each section is from 0 (without any difficulty) to 3 (unable to do). For each section, the score given to that section is the worst score within the section, i.e. if one question is scored 1 and another 2, then the score for the section is 2. In addition, if an aide or device is used or if help is required from another individual, then the minimum score for that section is 2. If the section score is already 2 or more then no modification is made. Each section can have one of four responses: 0 (without ANY difficulty), 1(with SOME difficulty), 2 (with MUCH difficulty), or 3 (UNABLE to do).

[0172] Patient global assessment (PtGA) is reported on a visual analogue scale (VAS) 0- 100 mm. PtGA may be assessed as part of the SHAQ assessment. Higher score indicates worse disease severity. Each of the disability sections has a companion aids / devices variable that is used to record what type(s) of assistance, if any, the participant uses for his / her usual activities. These variables are coded as follows: 0 = No assistance is needed.1 = A special device is used by the patient in his / her usual activities.2 = The patient usually needs help from another person.3 = The patient usually needs BOTH a special device AND help from another person. The score of each section is adjusted based on the patient’s use of an aid or device or assistance for that category. If either devices and / or help from another person are checked for a category, the score is set to “2”, unless the score is already “3” (i.e., scores of “0” or “1” are increased to “2”). For example, if the highest score for the dressing category is “1”, and the patient says they use a device for dressing, the computed category score would be “2”.

[0173] Health assessment questionnaire index (HAQ-DI) is calculated as the average of the 8 section scores (sum of the computed section scores divided by the number of section answered). This gives a score in the 0 to 3 range. If less than 6 sections are non-missing, then no HAQ-DI score is calculated. Higher score indicates worse disease severity. HAQ-DI may be assessed as part of the SHAQ assessment.

[0174] In some embodiments, physician global assessment (PhGA) is used to monitor the course of treatment of a subject receiving a provided composition. PhGA is reported on a visual analogue scale (VAS) 0-10 cm. Higher score indicates worse disease severity.Attorney Docket No. KVN-011WO

[0175] In some embodiments, diffusing capacity of lung for carbon monoxide (DLCO) is used to monitor the course of treatment of a subject receiving a provided composition.

[0176] In some embodiments, forced vital capacity percent predicted (FVC% or %FVC) is used to monitor the course of treatment of a subject receiving a provided composition. For example, FVC% may be used as a part of ACR-CRISS and rCRISS components.

[0177] In some embodiments, the American College of Rheumatology Composite Response Index in Systemic Sclerosis (ACR-CRISS) is used to monitor the course of treatment of a subject receiving a provided composition. In some embodiments, the revised ACR-CRISS (rCRISS) is used to monitor the course of treatment of a subject receiving a provided composition. In some embodiments, ACR-CRISS response measurement is based on the probability of improvement from baseline in each patient ranging from 0 to 1 (no to high probability), and includes evaluation of five core items: mRSS, percent predicted forced vital capacity (%FVC), a health assessment questionnaire disability index (HAQ-DI), patient global assessment, and / or clinical global assessment. Revised ACR CRISS (rCRISS) responses, a.k.a. rCRISS 30 / 5 responses, are measured based on the proportion of patients who improve in ≥ 3 / 5 of the core items by certain percentages, for example, 30% (except ≥ 5% in %FVC). In some embodiments, improvement in ≥ 3 / 5 of the core items by at least 30% (except at least 5% in %FVC) is considered a positive response.

[0178] For continued efficacy, a subject can receive 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, or more doses of any engineered T cell described herein. In some embodiments, a subject receives at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 doses of an engineered T cell (e.g., a T cell expressing a CAR, e.g., a CAR comprising an amino acid sequence as set forth in SEQ ID NO: 13).

[0179] In some embodiments, administration of a provided method and composition results in a reduction (e.g., at least a 1% reduction, at least a 5% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, or about a 1% reduction to about a 99% reduction, about a 1% reduction to about aAttorney Docket No. KVN-011WO 90% reduction, about a 1% reduction to about a 80% reduction, about a 1% reduction to about a 70% reduction, about a 1% reduction to about a 60% reduction, about a 1% reduction to about a 50% reduction, about a 1% reduction to about a 45% reduction, about a 1% reduction to about a 40% reduction, about a 1% reduction to about a 35% reduction, about a 1% reduction to about a 30% reduction, about a 1% reduction to about a 25% reduction, about a 1% reduction to about a 20% reduction, about a 1% reduction to about a 15% reduction, about a 1% reduction to about a 10% reduction, about a 1% reduction to about a 5% reduction, about a 5% reduction to about a 99% reduction, about a 5% reduction to about a 90% reduction, about a 5% reduction to about a 80% reduction, about a 5% reduction to about a 70% reduction, about a 5% reduction to about a 60% reduction, about a 5% reduction to about a 50% reduction, about a 5% reduction to about a 45% reduction, about a 5% reduction to about a 40% reduction, about a 5% reduction to about a 35% reduction, about a 5% reduction to about a 30% reduction, about a 5% reduction to about a 25% reduction, about a 5% reduction to about a 20% reduction, about a 5% reduction to about a 15% reduction, about a 5% reduction to about a 10% reduction, about a 10% reduction to about a 99% reduction, about a 10% reduction to about a 90% reduction, about a 10% reduction to about a 80% reduction, about a 10% reduction to about a 70% reduction, about a 10% reduction to about a 60% reduction, about a 10% reduction to about a 50% reduction, about a 10% reduction to about a 45% reduction, about a 10% reduction to about a 40% reduction, about a 10% reduction to about a 35% reduction, about a 10% reduction to about a 30% reduction, about a 10% reduction to about a 25% reduction, about a 10% reduction to about a 20% reduction, about a 10% reduction to about a 15% reduction, about a 15% reduction to about a 99% reduction, about a 15% reduction to about a 90% reduction, about a 15% reduction to about a 80% reduction, about a 15% reduction to about a 70% reduction, about a 15% reduction to about a 60% reduction, about a 15% reduction to about a 50% reduction, about a 15% reduction to about a 45% reduction, about a 15% reduction to about a 40% reduction, about a 15% reduction to about a 35% reduction, about a 15% reduction to about a 30% reduction, about a 15% reduction to about a 25% reduction, about a 15% reduction to about a 20% reduction, about a 20% reduction to about a 99% reduction, about a 20% reduction to about a 90% reduction, about a 20% reduction to about a 80% reduction, about a 20% reduction to about a 70% reduction, about a 20% reduction to about a 60% reduction, about a 20% reduction to about a 50% reduction, about a 20% reduction to about a 45% reduction, about a 20% reduction to about a 40% reduction, about a 20% reduction to about a 35% reduction, about a 20% reduction to about a 30% reduction, about a 20% reduction toAttorney Docket No. KVN-011WO about a 25% reduction, about a 25% reduction to about a 99% reduction, about a 25% reduction to about a 90% reduction, about a 25% reduction to about a 80% reduction, about a 25% reduction to about a 70% reduction, about a 25% reduction to about a 60% reduction, about a 25% reduction to about a 50% reduction, about a 25% reduction to about a 45% reduction, about a 25% reduction to about a 40% reduction, about a 25% reduction to about a 35% reduction, about a 25% reduction to about a 30% reduction, about a 30% reduction to about a 99% reduction, about a 30% reduction to about a 90% reduction, about a 30% reduction to about a 80% reduction, about a 30% reduction to about a 70% reduction, about a 30% reduction to about a 60% reduction, about a 30% reduction to about a 50% reduction, about a 30% reduction to about a 45% reduction, about a 30% reduction to about a 40% reduction, about a 30% reduction to about a 35% reduction, about a 35% reduction to about a 99% reduction, about a 35% reduction to about a 90% reduction, about a 35% reduction to about a 80% reduction, about a 35% reduction to about a 70% reduction, about a 35% reduction to about a 60% reduction, about a 35% reduction to about a 50% reduction, about a 35% reduction to about a 45% reduction, about a 35% reduction to about a 40% reduction, about a 40% reduction to about a 99% reduction, about a 40% reduction to about a 90% reduction, about a 40% reduction to about a 80% reduction, about a 40% reduction to about a 70% reduction, about a 40% reduction to about a 60% reduction, about a 40% reduction to about a 50% reduction, about a 40% reduction to about a 45% reduction, about a 45% reduction to about a 99% reduction, about a 45% reduction to about a 90% reduction, about a 45% reduction to about a 80% reduction, about a 45% reduction to about a 70% reduction, about a 45% reduction to about a 60% reduction, about a 45% reduction to about a 50% reduction, about a 50% reduction to about a 99% reduction, about a 50% reduction to about a 90% reduction, about a 50% reduction to about a 80% reduction, about a 50% reduction to about a 70% reduction, about a 50% reduction to about a 60% reduction, about a 60% reduction to about a 99% reduction, about a 60% reduction to about a 90% reduction, about a 60% reduction to about a 80% reduction, about a 60% reduction to about a 70% reduction, about a 70% reduction to about a 99% reduction, about a 70% reduction to about a 90% reduction, about a 70% reduction to about a 80% reduction, about a 80% reduction to about a 99% reduction, about a 80% reduction to about a 90% reduction, or about a 90% reduction to about a 99% reduction) in the number of B cells in a tissue of the subject (e.g., in peripheral blood) having systemic sclerosis, e.g., as compared to the levels in the subject prior to treatment or the levels in a similar subject not treated or receiving a different treatment. In some embodiments, after deep measured depletion of B cells in a tissue of a subject (e.g., inAttorney Docket No. KVN-011WO peripheral blood), B cell counts may increase and substantially recover to normal levels, e.g., as compared to levels in the subject prior to treatment or the levels in a similar subject not treated or receiving a different treatment or any other suitable control. In some embodiments, B cell counts will substantially recover to normal levels after about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiment, recovered B cells will show a sustained naive phenotype profile, indicating the potential for an immune reset and a long-term functional cure of the subject. Without wishing to be bound by theory, it is contemplated that the B cell depletion caused by the CAR T cells may lead to an immune reset, e.g., as evidenced by the durable absence of symptoms of the disease even in the presence of reconstituted B cell numbers.

[0180] In some embodiments, administration of methods and compositions described herein result in a reduction (e.g., at least a 1% reduction, at least a 5% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, or about a 1% reduction to about a 99% reduction (or any of the subranges of this range described herein) in the level of auto-antibodies in the subject having systemic sclerosis, e.g., as compared to the levels in the subject prior to treatment or the levels in a similar subject not treated or receiving a different treatment. In some embodiments, the level of auto-antibodies (e.g., as determined by any suitable method) in the subject having systemic sclerosis remains stable, e.g., lower than the levels in the subject prior to treatment or the levels in a similar subject not treated or receiving a different treatment. Kits

[0181] Also provided herein are kits that include any of the compositions described herein. For example, a kit can include one or more of any of the nucleic acid constructs described herein. In other examples, a kit can include any of engineered T cell described herein or one or more doses of a composition including any engineered T cell described herein. In some embodiments, a kit can include instructions for performing any of the methods described herein.

[0182] Technologies of the present disclosure will be further described in the following examples, which do not limit the scope of the disclosure described in the claims.Attorney Docket No. KVN-011WO EXAMPLES

[0183] The following examples are put forth to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their invention. Example 1. Preparation of Engineered T Cells Comprising a Lentivirus Vector Encoding Hu19-CD828Z

[0184] A Hu19-CD828Z may be prepared as described in U.S. Patent No.10,287,350. Briefly, fully human anti-CD19 CARs were generated by utilizing sequences of the fully human 47G4 monoclonal antibody (described in U.S. Patent Application Publication No. 2010 / 0104509). The 47G4 antibody was generated by vaccinating mice of the KM strain, which carry a human kappa light chain transgene and a human heavy chain transchromosome. The sequences of the 47G4 antibody light chain and heavy chain variable regions were obtained from U.S. Patent Application Publication No.2010 / 0104509. A 47G4 scFv was designed comprising the following elements from 5' to 3': a CD8 signal sequence, the 47G4 antibody light chain variable region, a linker sequence (encoding a peptide comprising the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 9)) (see Cooper et al., Blood, 101(4): 1637-1644 (2003)), and the 47G4 antibody heavy chain variable region. A DNA sequence encoding a CAR was then designed comprising the following components from 5' to 3': the 47G4 scFv described above, part of the extracellular region and all of the transmembrane region of the human CD8 molecule, and the cytoplasmic (or intracellular) portions of the human CD28 molecule and the human CD3 zeta. molecule. This CAR was designated 47G4-CD828Z (SEQ ID NO: 13), and the sequence was synthesized by Invitrogen (Carlsbad, Calif.).

[0185] Expression of a Hu19-CD828Z polypeptide in a cell can be performed using any suitable method. In these experiments, T cells (CD4+ T cells, CD8+ T cells, or an cell mixture enriched for CD4+ and CD8+ T cells) are transduced with a lentivirus where the lentiviral vector includes a nucleic acid sequence encoding Hu19-CD828Z polypeptide. The lentiviral vector includes an MSCV promoter among other regulatory factors (see, e.g., FIG. 1). Lentivirus is produced in HEK293 cells according to standard protocols. A KL-h198a28z lentiviral vector system may be used to transduce T cells. KL-h198a28z is a self-inactivating (SIN) vesicular stomatitis virus (VSV)-G pseudotyped 3rd generation lentiviral vector expressing human anti-CD19 chimeric antigen receptor (CAR). The lentiviral vector KL-Attorney Docket No. KVN-011WO h198a28z is manufactured using a HEK 293T cell line transiently transfected with a state-of- the-art four-plasmid system. The envelope protein encoding plasmid (pLTG1292) expresses a heterologous spike protein, the VSV-G protein, under control of the cytomegalovirus (CMV) promoter. The VSV-G envelope protein provides broad cell tropism for transduction of a wide variety of mammalian cell types. KL-h198a28z encodes the CAR construct Hu19- CD828Z, and can be used to manufacture CAR T cells for treating patients with B cell- associated diseases.

[0186] Engineered T cells can be produced from blood cells using various known methods, e.g., as described in Ghassemi et al., (2018) Cancer Immunol. Res.6(9) and Mackensen et al., (2022) Nat. Med.28:2124–32. In one example, white blood cells are collected from a patient by apheresis. The cells are enriched for CD4+ and CD8+ T cells and are then activated with CD3 and CD28 agonistic agents, e.g., TransActTM, a polymeric nanomatrix coated. Following activation, the cells are transduced with lentiviral vector KL- h198a28z vector encoding Hu19-CD828Z and expanded in culture. The cells are then harvested and assessed for viability, Hu19-CD828Z expression, T cell phenotype, and potency (e.g., cytotoxicity and cytokine release). Example 2. In Vitro Cytotoxicity of SSc Patient Derived KYV-101 and Accompanying Cytokine Release

[0187] The CAR-mediated and CD19-dependent cytolytic activity and activation of KYV-101, an autologous anti-CD19 CAR T-cell therapy generated from SSc patient PBMCs, was assessed.

[0188] KYV-101 is an autologous CAR T cell therapy consisting of autologous patient- derived CD4+ and CD8+ enriched and expanded T cells genetically modified to express a anti-CD19 CAR, which redirects T cells to target CD19 expressed on normal and autoreactive B cells including SSc patients. The CD19 CAR construct (Hu19-CD828Z) used to produce KYV-101 is further described in Example 1.

[0189] Primary B cells within autologous PBMCs and the NALM6 B cell cancer line represent CD19+ target cells whereas the CEM / C1 T cell cancer line is used to represent CD19- target cells. Three lots of KYV-101 generated from SSc patient PBMCs (2005, 4194, 6940), two lots of KYV-101 generated from healthy donor (HD) PBMCs (817, 1066), or untransduced T cells (UNTD) from the same SSc patients and healthy donors (2005 UNTD, 4194 UNTD, 6940 UNTD, 817 UNTD, 1066 UNTD) were co-cultured for 24 hours withAttorney Docket No. KVN-011WO target cells: either autologous primary B cells within PBMCs (5x105cells / well) that express CD19, the human ALL cell line NALM6 (5x104cells / well) that expresses high levels of CD19, or the human ALL cell line CEM / C1 (5x104cells / well) that does not express CD19 at Effector:Target (E:T) ratios of 1:1, 0.1:1, and 0.02:1 for autologous B cell / PBMC or 3:1, 1:1, 0.5:1 and 0.1:1 for NALM6 and CEM / C1 co-cultures. Effector and target cells alone were included as negative controls. The effector cell number for the E:T ratios was based on the total number of PBMCs in each well for co-cultures with PBMCs. Due to limited availability of SSc patient-derived PBMCs, samples were run in duplicate when possible whereas healthy donor PBMC samples were run in triplicate. The effector cell number for the E:T ratios was based on the total number of effector T cells in each well and all samples were run in triplicate for co-cultures with NALM6 or CEM / C1 cells. After co-incubation, the supernatant was retained for cytokine analysis, and the live target cells were quantified. Concentrations of IFNγ, IL-1β, IL-2, IL4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNFα, Granzyme A and Granzyme B were measured using an electrochemiluminescence-based immunoassay.

[0190] Following overnight co-culture, SSc- and HD-derived KYV-101 induced strong, dose-dependent cytolysis of autologous primary B cells within PBMCs at E:T ratios > 0:1 (FIG.2, panel A). Percent cytolysis plateaued or decreased at the highest E:T ratio with the exception of HD 817-derived KYV-101 which showed a continuous increase in percent cytolysis. Nonetheless, the level of cytotoxicity by KYV-101 was significantly greater than observed for untransduced T cells, demonstrating that the difference in cytotoxicity is mediated by the CAR.

[0191] Similar to primary B cell cytotoxicity, SSc- and HD-derived KYV-101 also induced strong, dose dependent cytotoxicity of CD19+ NALM6 cells at E:T ratios > 0:1 (FIG.2, panel B). The level of cytotoxicity by KYV-101 was significantly greater than that observed for untransduced T cells, demonstrating that the difference in cytotoxicity is due to the presence of CAR. In contrast, little or no difference in cytotoxicity was observed between KYV-101 and untransduced T cells when co-cultured with CD19- target cells (CEM / C1) (FIG.2, panel C). Together, these studies demonstrate CAR-mediated and CD19-dependent cytotoxicity for SSc-derived KYV-101, and that the activity of SSc- and HD-derived KYV- 101 is comparable.

[0192] In addition to direct cytolysis, IFNγ and Granzyme B were the predominant and relevant cytokines detected and are directly related to T cell activation and / or T cell-mediated cytotoxicity. CD19-dependent IFNγ and Granzyme B release was observed for SSc- and HD- derived KYV-101 following co-culture with autologous PBMCs, between the E:T ratios ofAttorney Docket No. KVN-011WO 0.02:1 and 1:1. The IFNγ levels secreted were donor dependent and continue to increase, plateau or decrease at E:T ratio of 1:1 (FIG.3, panel A). Levels of IFNγ were similar and very low in co-cultures with UNTD control T cells at all E:T ratios, confirming the CAR- mediated cytokine secretion by activation of KYV-101. SSc- and HD-derived KYV-101 also exhibited strong, dose-dependent, CAR-mediated secretion of Granzyme B when co-cultured with CD19+ primary B cell containing autologous PBMCs compared to respective UNTD control T cells (FIG.4, panel A). Furthermore, a similar CAR mediated secretion profile was observed for IL-2, IL-13, IL-6, IL-8, TNFα, and IL-1β while IL-4, IL-10, and IL-12p70 were released at very low levels in all samples or comparable to PBMCs alone.

[0193] Moreover, an effector cell dose-dependent increase in release of IFNγ was observed between the E:T ratios of 0.1:1 and 3:1 (FIG.3, panel B). At E:T ratio of 3:1, IFNγ secretion by SSc- and HD-derived KYV-101 began to plateau for all samples except for HD 1066 which slightly decreased. Levels of IFNγ were similar and very low in co-cultures with UNTD control T cells at all E:T ratios, confirming the CAR-mediated cytokine secretion by activation of KYV-101. In contrast, the concentration of IFNg when SSc- and HD-derived KYV-101 were co-cultured with CD19-CEM / C1 cells was several orders of magnitude lower than concentrations observed when KYV-101 was co-cultured with NALM6 cells (FIG.3, panel C). In addition to IFNγ, SSc- and HD derived KYV-101 exhibited strong, dose- dependent, target-dependent and CAR-mediated secretion of Granzyme B when co-cultured with CD19+ NALM6 compared to both UNTD control T cells (FIG.4, panel B) or when co- cultured with CD19- CEM / C1 cells (FIG.4, panel C). A similar CAR mediated, target- specific secretion profile was observed for IL-2, IL-8, IL-13, and TNFα. IL-6, IL-4, IL-10, IL-1β, and IL-12p70 were released at very low levels in all samples. These results further support the conclusion that both KYV-101 cytokine secretion and T cell activation and cytotoxicity are CAR-mediated and CD19-dependent.

[0194] SSc-derived KYV-101 showed CAR-mediated cytotoxicity in a CD19-dependent manner as demonstrated by robust and dose-dependent cytotoxicity against both autologous primary B cells and a CD19+ human B cell cancer cell line (NALM6) and by minimal cytotoxicity against the CEM / C1 CD19- cell line. All three batches of SSc-derived KYV-101 also showed CAR-mediated, CD19-dependent and dose-dependent production of Granzyme B and the cytokines IFNγ, IL-13, IL-2, IL-8, and TNF-α, which are directly related to T cell activation and / or T cell-mediated cytotoxicity, upon KYV-101 co-culture with CD19+ autologous B cells and NALM6 cells, but not with the CD19- CEM / C1 cells. Collectively,Attorney Docket No. KVN-011WO these data support the conclusion that the activity of SSc- and HD-derived KYV-101 is comparable. Example 3. In Vitro SSc-Derived KYV-101 CD19-Dependent Proliferation

[0195] The CAR-mediated and CD19-dependent proliferation of KYV-101 (an autologous anti-CD19 CAR T-cell therapy, see Examples 1 and 2) generated from SSc patient PBMCs was assessed by co-culture with CD19+ and CD19- target cells.

[0196] Human primary B cells and the NALM6 B cell cancer line are used to represent CD19+ target cells whereas the CEM / C1 T cell cancer line is used to represent CD19- target cells. Three batches of KYV-101 generated from SSc patient PBMCs (2005, 4194, 6940), two lots of KYV-101 generated from HD PBMCs (817, 1066), or untransduced T cells from the same SSc patients and healthy donors (2005 UNTD, 4194 UNTD, 6940 UNTD, 817 UNTD, 1066 UNTD) were labeled with CellTrace Violet Dye prior to co-culturing for 4 days at E:T ratios of 0.3:1 and 1:1 with CD19+ healthy donor primary human B cells, CD19+ human NALM6 B cell cancer cells or CD19- human CEM / C1 T cell cancer cells. The target cells were plated at a cell density of 2.5x104cells / well for all testing paradigms. For KYV- 101, the effector cell number for the E:T ratios was adjusted for the percent of CAR+ cells with donor-matched UNTD cells to normalize the number of CAR T cells added across donors. Due to this normalization, the total number of untransduced T cell controls in the assay was consistent across donors at each E:T ratio. After 96 hours of co-culture, the proliferation of the effector cells was assessed by flow cytometry using CellTrace Violet “dim” (CTV Dim) gating, and the subpopulations of cells were determined by flow cytometry using detection reagents for CAR, CD45 (hematopoietic cells), CD3 (T cells), and CD20 (B cells).

[0197] Donor-matched autologous primary B cells were not available in sufficient quantities so allogeneic primary B cells were used as target cells. This was considered representative since the CD19 expression levels (measured by MFI) on SSc- and HD-derived primary B cells within PBMCs are comparable (27,707 ± 6,282 for SSc and 21,919 ± 4,354 for HD).

[0198] T cell proliferation, determined by the dilution of the CellTrace Violet dye (CTV Dim), was observed for SSc- and HD-derived KYV-101 after a 96-hour co-culture with primary allogenic B cells (CTV Dim 69.8 – 91.8% for SSc- and 79.4 – 92.1% for HD-derived KYV-101; FIG.5, panel A). In general, KYV-101 proliferated more at the lower E:T of 0.3:1 (CTV Dim 78.3 – 92.1%) than at the higher E:T of 1:1 (CTV Dim 69.8 – 89.2%). InAttorney Docket No. KVN-011WO contrast to KYV-101, UNTD control T cells showed demonstrably lower levels of proliferation (CTV Dim 18.0 – 45.7% for SSc- and 9.2 – 21.0% for HD-derived). The lower yet measurable levels of UNTD control T cell proliferation were anticipated due to the allogenic reactivity of these cells.

[0199] T cell proliferation was also observed for SSc- and HD-derived KYV-101 after a 96-hour co-culture with the CD19+ NALM6 cell line. SSc- and HD-derived KYV-101 CD3+ T cells proliferated to similar levels (CTV Dim 78.2– 94.4% for SSc- and 79.5 – 93.1% for HD-derived KYV-101). In contrast, much lower levels of CD3+ T cell proliferation were observed in UNTD control T cells co-cultured with NALM6 cells at both E:T ratios tested (CTV Dim 15.2 – 50.4% for SSc- and 10.4 – 28.1% for HD-derived UNTD cells) (FIG.5, panel B).

[0200] When co-cultured with CD19- CEM / C1 cells, KYV-101 demonstrated lower levels of proliferation (CTV Dim 5.9 – 54.8%) (FIG.5, panel C) compared to co-culture with CD19+ NALM6 (CTV Dim 78.2 – 94.4%) (FIG.5, panel B). While SSc 2005-derived KYV-101 proliferated more (CTV Dim 40.7 – 54.8%) than its paired UNTD control T cells (CTV Dim 2.4 - 4.8%), it also proliferated to a similar level when cultured alone (CTV Dim 63.7%) and with CD19- CEM / C1 (CTV Dim 54.8%). Taken together, this data supports the observation of CAR-mediated and CD19-dependent proliferation of KYV-101 derived from both SSc patients and HDs.

[0201] KYV-101 generated from both SSc patients and HDs showed CAR-mediated and CD19-dependent cellular proliferation when co-cultured with primary B cells and CD19+ NALM6 B cell cancer cells but lower levels of proliferation with CD19- CEM / C1 T cell cancer cells. The lower levels of proliferation of the untransduced control T cells that occasionally occurred in response to co-culture with primary B cells and NALM6 cells can be the result of allogeneic reactivity of KYV-101 to these target cells. Example 4. Use of KYV-101 to Treat Systemic Sclerosis in Human Patients

[0202] This example describes a phase 1 / 2 clinical study to assess the safety, tolerability, and clinical activity of KYV-101 (a fully human anti-CD19 CAR T-cell therapy) in adult subjects with diffuse cutaneous systemic sclerosis.

[0203] Systemic sclerosis (SSc) is an immune-mediated rheumatic disease that is characterized by fibrosis of the skin and internal organs and vasculopathy. There are different SSc subtypes characterized by the extent and location of skin involvement as well as other associated manifestations of the disease, including limited cutaneous systemic sclerosisAttorney Docket No. KVN-011WO (lcSSc) and diffuse cutaneous systemic sclerosis (dcSSc). dcSSc is marked by puffy hands and skin thickening that extends proximally to the upper arms, thighs, and / or trunk. Compared with patients with lcSSc, dcSSc patients are more likely to experience rapid progression of skin thickening and to have higher risk of pulmonary, cardiac, renal, and gastrointestinal complications including lung fibrosis, pulmonary arterial hypertension (PAH), renal failure, and esophageal / intestinal dysmotility. B cells play a role in SSc, and the disease is characterized by the presence of autoantibodies such as anti-Scl-70 and anti-RNAP III antibodies.

[0204] KYV-101 consists of autologous CD4+ and CD8+ enriched and expanded T cells genetically engineered to express a chimeric antigen receptor that targets CD19, an antigen expressed on the surface of both normal and autoreactive B cells in patients with autoimmune diseases. CD19-targeted CAR T cells harness the ability of cytotoxic T cells to directly and specifically lyse target cells, leading to effective depletion of B cells in the circulation and in lymphoid and potentially non-lymphoid tissues. The CAR expressed by the KYV-101 cells is Hu19-CD828Z, see, e.g., Example 1. Patient Selection

[0205] Inclusion criteria • ≥18 years of age and classified as diffuse cutaneous SSc • 6 years or less since first non-Raynaud’s sign or symptom • Active disease defined as: mRSS ≥16 with, in the prior 6 months, one or more of the following: o An increase in mRSS of ≥3 points o Involvement of 1 new body area with ≥2 mRSS units o 2 new body areas with ≥1 mRSS unit OR, Progressive ILD meeting all of the following criteria: o Inadequate response or intolerance to at least two medications administered for at least 12 weeks within 24 months prior to screening. These medications include glucocorticoids, tocilizumab, mycophenolate mofetil (MMF) or mycophenolic acid, cyclophosphamide, azathioprine, calcineurin inhibitor, tofacitinib or other JAK-inhibitors, abatacept, methotrexate, nintedanib, rituximab, IVIG, or other immunomodulatory agents AND one of the following: o Evidence of progression on high-resolution computed tomography (HRCT) ORAttorney Docket No. KVN-011WO o Forced vital capacity percent predicted (FVC%) <80% or diffusing capacity of lung for carbon monoxide (DLCO) <80% or evidence of FVC decline of 10% (absolute decline) / FVC decline of 5% to 9% and DLCO 15%

[0206] Key Exclusion Criteria: Patients with ILD with any of the following: • Requiring O2 therapy and / or FVC ≤45% of predicted or DLCO ≤40% of predicted at screening • Evidence of pulmonary arterial hypertension (PAH) as defined as estimated right ventricular systolic pressure (RVSP) of ≥ 45 mmHg or right atrial or ventricular enlargement or dilatation, unless subsequent right heart catheterization (RHC) shows no PAH • PAH on right heart catheterization requiring PAH specific treatment Dosage and Administration

[0207] KYV-101, is an autologous anti-CD19 CAR T-cell immunotherapy. The KYV- 101 structure is composed of a human scFv CD19-targeting domain, a CD8 alpha hinge and transmembrane domains (CD8α hinge + TM), a CD28 cytoplasmic costimulatory domain, and a CD3-zeta cytoplasmic domain. The constitutive expression of the CAR is controlled by a murine stem cell virus (MSCV) promoter. KYV-101 is a cell suspension formulated in a chemically defined freezing medium (50% Plasma-Lyte ATMcontaining 2% human serum albumin [for a final HSA concentration of 1%] + 50% CryoStor10 containing dimethyl sulfoxide [DMSO] to a final concentration of 5%). The finished product is filled in a freezing bag and stored at ≤ -150ºC in the vapor phase of liquid nitrogen. Lymphodepleting chemotherapy consisting of cyclophosphamide (CYC) 300 mg / m2 and fludarabine (FLU) 30 mg / m2 intravenously (IV) daily for 3 days will be administered 5 to 7 days prior to administration of KYV-101. KYV-101 will be administered IV as a single infusion.

[0208] For Phase 1, approximately 6 subjects will be enrolled to evaluate the safety and tolerability of KYV-101 in dcSSc. The first 3 subjects will be enrolled sequentially at a dose of 1x108CAR T cells. Key objectives and endpoints for Phase 1 are detailed in Table 1. Table 1: Phase 1 Objectives and Endpoints Primary Objectives Primary EndpointsAttorney Docket No. KVN-011WO To characterize the pharmacokinetics (PK) CAR-positive T-cell counts, CAR transgene and pharmacodynamics (PD) of KYV-101 level, B-cell counts over time, systemic i bl d t ki t ti - A t

[0209] The Phase 2 study is initiated after the recommended Phase 2 dose (RP2D) has been identified in Phase 1. The RP2D will demonstrate acceptable safety with evidence of sufficient T-cell expansion, B-cell depletion, and other PD activity from at least 28 days post CAR T-cell infusion for all dosed subjects. The Phase 2 portion is designed to evaluate the safety, tolerability, and clinical efficacy of KYV-101 at the RP2D in subjects with dcSSc. Approximately 15 subjects will be enrolled in Phase 2 for primary endpoint analysis. Key objectives and endpoints for Phase 2 are detailed in Table 2. Table 2: Phase 2 Objectives and Endpoints Primary Objectives Primary EndpointsAttorney Docket No. KVN-011WO gastrointestinal involvement), mRSS, PhGA, FVC%, and DLCO over time. T l t i l t i k h i ti L 7 ti RNAP III e

[0210] This example describes treatment of two adult patients having systemic sclerosis, using KYV-101.

[0211] The first patient had diffuse systemic sclerosis with interstitial lung disease. The patient received a single infusion of 1×108anti-CD19 CAR T lymphocytes (KYV-101). After recovering from an initial cytokine release syndrome, the patient showed improvements from systemic sclerosis, including performance of basic activities without requiring oxygen any longer and reduction in fibrosis. The improvement continued even after reconstitution of CD19+B cells.

[0212] The second patient had diffuse systemic sclerosis. The patient received a single infusion of 1×108anti-CD19 CAR T lymphocytes (KYV-101). After recovering from an initial cytokine release syndrome, the patient showed improvements from systemic sclerosis and increased physical activity.

[0213] These results demonstrate responses of systemic sclerosis patients to the KYV- 101 treatment. OTHER EMBODIMENTS

[0214] It is to be understood that while the provided technologies have been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.Attorney Docket No. KVN-011WO SEQUENCE APPENDIX SEQ ID NO: 1 Hu19-CD828Z Heavy Chain CDR1 (Kabat) SYAIS SEQ ID NO: 2 Hu19-CD828Z Heavy Chain CDR2 (Kabat) GIIPIFGTTNYAQQFQG SEQ ID NO: 3 Hu19-CD828Z Heavy Chain CDR3 (Kabat, Chothia) EAVAADWLDP SEQ ID NO: 4 Hu19-CD828Z Light Chain CDR1 (Kabat, Chothia) RASQSVSSSYLA SEQ ID NO: 5 Hu19-CD828Z Light Chain CDR2 (Kabat, Chothia) GASSRAT SEQ ID NO: 6 Hu19-CD828Z Light Chain CDR3 (Kabat, Chothia) QQYGSSRFT SEQ ID NO: 7 Hu19-CD828Z Heavy Chain Variable Region QVQLVQSGAEVKKPGSSVKVSCKDSGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGT TNYAQQFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREAVAADWLDPWGQG TLVTVSS SEQ ID NO: 8 Hu19-CD828Z Light Chain Variable Region EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSRFTFGPGTKVDIK SEQ ID NO: 9 Hu19-CD828Z Peptide Linker GSTSGSGKPGSGEGSTKG SEQ ID NO: 10 Full-length Hu19-CD828Z Chimeric Antigen Receptor without signal peptide EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSRFTFGPGTKVDIKGSTSGSGKPG SGEGSTKGQVQLVQSGAEVKKPGSSVKVSCKDSGGTFSSYAISWVRQAPGQGLEW MGGIIPIFGTTNYAQQFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREAVAAD WLDPWGQGTLVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTP RRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYAttorney Docket No. KVN-011WO DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 11 CD8a transmembrane domain – amino acid sequence IYIWAPLAGTCGVLLLSLVIT SEQ ID NO: 12 CD28 transmembrane domain FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 13 Full-length Hu19-CD828Z Chimeric Antigen Receptor – amino acid sequence MALPVTALLLPLALLLHAARPEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSR FTFGPGTKVDIKGSTSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGSSVKVSCKDSGG TFSSYAISWVRQAPGQGLEWMGGIIPIFGTTNYAQQFQGRVTITADESTSTAYMELSS LRSEDTAVYYCAREAVAADWLDPWGQGTLVTVSSFVPVFLPAKPTTTPAPRPPTPAP TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNH RNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 14 Full-length Hu19-CD828Z Chimeric Antigen Receptor – nucleic acid sequence ATGGCCCTGCCTGTGACAGCTCTGCTGCTGCCCCTGGCCCTGCTGCTGCATGCCG CCAGACCTGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGTCTCTCAGCCCAGG AGAGAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGCGTGTCCAGCAGCTACCT GGCCTGGTATCAGCAGAAGCCCGGACAGGCCCCCAGACTGCTGATCTACGGCGC CAGCTCTAGAGCCACCGGCATCCCCGACAGATTCAGCGGCAGCGGCAGTGGCAC CGACTTCACCCTGACCATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTAC TGCCAGCAGTACGGCAGCAGCCGGTTCACCTTCGGCCCTGGCACCAAGGTGGAC ATCAAGGGCAGCACCTCCGGCAGCGGCAAGCCTGGCTCTGGCGAGGGCTCTACC AAGGGCCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCCGGCTCT AGCGTGAAGGTGTCCTGCAAGGACAGCGGCGGCACCTTCAGCAGCTACGCCATC AGCTGGGTGCGCCAGGCCCCAGGACAGGGGCTGGAATGGATGGGCGGCATCATC CCCATCTTCGGCACCACCAACTACGCCCAGCAGTTCCAGGGCAGAGTGACCATC ACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTGAGCAGCCTGCGGAGC GAGGACACAGCCGTGTATTACTGTGCCCGCGAGGCCGTGGCCGCCGACTGGCTG GATCCTTGGGGACAGGGCACCCTGGTGACAGTGTCCAGCTTCGTGCCCGTGTTCC TGCCCGCCAAGCCTACCACCACCCCTGCCCCTAGACCTCCCACCCCAGCCCCAAC AATCGCCAGCCAGCCTCTGTCCCTGCGGCCCGAAGCCTGTAGACCTGCTGCCGGC GGAGCCGTGCACACCAGAGGCCTGGACTTCGCCTGCGATATCTACATCTGGGCCC CTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTG CAACCACCGGAACAGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATGA ACATGACCCCAAGACGGCCTGGCCCCACCCGGAAGCACTACCAGCCTTACGCCC CTCCCAGAGACTTCGCCGCCTACCGGTCCAGAGTGAAGTTCAGCAGATCCGCCG ACGCCCCTGCCTACCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAACCTGG GCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCCGAG ATGGGCGGAAAGCCCAGACGGAAGAACCCCCAGGAAGGCCTGTATAACGAACT GCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCAttorney Docket No. KVN-011WO GGAGGCGCGGCAAGGGCCACGATGGCCTGTACCAGGGCCTGAGCACCGCCACCA AGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCAGATGA SEQ ID NO: 15 CD8a signal peptide - amino acid sequence MALPVTALLLPLALLLHAARP SEQ ID NO: 16 CD8a signal peptide - nucleic acid sequence ATGGCCCTGCCTGTGACAGCTCTGCTGCTGCCCCTGGCCCTGCTGCTGCATGCCG CCAGACCT SEQ ID NO: 17 Antigen binding domain with scFv 47G4 - amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSRFTFGPGTKVDIKGSTSGSGKPG SGEGSTKGQVQLVQSGAEVKKPGSSVKVSCKDSGGTFSSYAISWVRQAPGQGLEW MGGIIPIFGTTNYAQQFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREAVAAD WLDPWGQGTLVTVSS SEQ ID NO: 18 Antigen binding domain with scFv 47G4 - nucleic acid sequence GAGATCGTGCTGACCCAGTCTCCCGGCACCCTGTCTCTCAGCCCAGGAGAGAGA GCCACCCTGAGCTGCAGAGCCAGCCAGAGCGTGTCCAGCAGCTACCTGGCCTGG TATCAGCAGAAGCCCGGACAGGCCCCCAGACTGCTGATCTACGGCGCCAGCTCT AGAGCCACCGGCATCCCCGACAGATTCAGCGGCAGCGGCAGTGGCACCGACTTC ACCCTGACCATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAG CAGTACGGCAGCAGCCGGTTCACCTTCGGCCCTGGCACCAAGGTGGACATCAAG GGCAGCACCTCCGGCAGCGGCAAGCCTGGCTCTGGCGAGGGCTCTACCAAGGGC CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCCGGCTCTAGCGTG AAGGTGTCCTGCAAGGACAGCGGCGGCACCTTCAGCAGCTACGCCATCAGCTGG GTGCGCCAGGCCCCAGGACAGGGGCTGGAATGGATGGGCGGCATCATCCCCATC TTCGGCACCACCAACTACGCCCAGCAGTTCCAGGGCAGAGTGACCATCACCGCC GACGAGAGCACCAGCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGA CACAGCCGTGTATTACTGTGCCCGCGAGGCCGTGGCCGCCGACTGGCTGGATCCT TGGGGACAGGGCACCCTGGTGACAGTGTCCAGC SEQ ID NO: 19 CD8a hinge and transmembrane domain - amino acid sequence FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRN SEQ ID NO: 20 CD8a hinge and transmembrane domain - nucleic acid sequence TTCGTGCCCGTGTTCCTGCCCGCCAAGCCTACCACCACCCCTGCCCCTAGACCTC CCACCCCAGCCCCAACAATCGCCAGCCAGCCTCTGTCCCTGCGGCCCGAAGCCTG TAGACCTGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGACTTCGCCTGCGA TATCTACATCTGGGCCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTG GTGATCACCCTGTACTGCAACCACCGGAACAttorney Docket No. KVN-011WO SEQ ID NO: 21 CD28 intracellular domain fragment - amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 22 CD28 intracellular domain fragment - nucleic acid sequence AGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCCAAGA CGGCCTGGCCCCACCCGGAAGCACTACCAGCCTTACGCCCCTCCCAGAGACTTCG CCGCCTACCGGTCC SEQ ID NO: 23 CD3 zeta intracellular domain fragment - amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR SEQ ID NO: 24 CD3 zeta intracellular domain fragment - amino acid sequence AGAGTGAAGTTCAGCAGATCCGCCGACGCCCCTGCCTACCAGCAGGGACAGAAC CAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGAC AAGCGGAGAGGCCGGGACCCCGAGATGGGCGGAAAGCCCAGACGGAAGAACCC CCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACA GCGAGATCGGCATGAAGGGCGAGCGGAGGCGCGGCAAGGGCCACGATGGCCTG TACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAG GCCCTGCCCCCCAGA SEQ ID NO: 25 Hu19-CD828Z Heavy Chain CDR1 (Chothia) GGTFSSY SEQ ID NO: 26 Hu19-CD828Z Heavy Chain CDR2 (Chothia) IPIFGT SEQ ID NO: 27 Hinge-Transmembrane-Intracellular portion of Hu19-CD828Z Chimeric Antigen Receptor FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR SEQ ID NO: 28 CD8a hinge domain – amino acid sequence FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO: 29 anti-CD19 Antibody 1 VHAttorney Docket No. KVN-011WO EVQLVQSGAEVKKPGESLKISCKGSGYSFTNSWIGWVRQMPGKGLEWMGLIYPDDS DTRYSPSFQGQVTISADSAINTAYLQWSSLKASDTAMYYCARQSTYIYGGYYDTWG QGTLVTVSS SEQ ID NO: 30 anti-CD19 Antibody 1 VL QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYENTNRPS GVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWRVFGGGTKLTVLG SEQ ID NO: 31 anti-CD19 Antibody 2 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDS DTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSYSWSSWYWDFWG QGTLVTVSS SEQ ID NO: 32 anti-CD19 Antibody 2 VL QAVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVSWYQQLPGKAPKLLIYYDDLLPSG VSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKVTVLG SEQ ID NO: 33 anti-CD19 FMC63 VH EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSS SEQ ID NO: 34 anti-CD19 FMC63 VL DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGV PSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT SEQ ID NO: 35 anti-CD19 Antibody 1 Heavy Chain CDR1 GYSFTNSW SEQ ID NO: 36 anti-CD19 Antibody 1 Heavy Chain CDR2 IYPDDSDT SEQ ID NO: 37 anti-CD19 Antibody 1 Heavy Chain CDR3 ARQSTYIYGGYYDT SEQ ID NO: 38 anti-CD19 Antibody 1 Light Chain CDR1 SSNIGAGYD SEQ ID NO: 39 anti-CD19 Antibody 1 Light Chain CDR2Attorney Docket No. KVN-011WO ENT SEQ ID NO: 40 anti-CD19 Antibody 1 Light Chain CDR3 QSYDSSLSGWRV SEQ ID NO: 41 anti-CD19 Antibody 2 Heavy Chain CDR1 GYSFTSYW SEQ ID NO: 42 anti-CD19 Antibody 2 Heavy Chain CDR2 IYPGDSDT SEQ ID NO: 43 anti-CD19 Antibody 2 Heavy Chain CDR3 ARLSYSWSSWYWDF SEQ ID NO: 44 anti-CD19 Antibody 2 Light Chain CDR1 SSNIGNNA SEQ ID NO: 45 anti-CD19 Antibody 2 Light Chain CDR2 YDD SEQ ID NO: 46 anti-CD19 Antibody 2 Light Chain CDR3 AAWDDSLNGWV SEQ ID NO: 47 anti-CD19 FMC63 Heavy Chain CDR1 GVSLPDY SEQ ID NO: 48 anti-CD19 FMC63 Heavy Chain CDR2 WGSET SEQ ID NO: 49 anti-CD19 FMC63 Heavy Chain CDR3 HYYYGGSYAMDY SEQ ID NO: 50 anti-CD19 FMC63 Light Chain CDR1 RASQDISKYLNAttorney Docket No. KVN-011WO SEQ ID NO: 51 anti-CD19 FMC63 Light Chain CDR2 HTSRLHS SEQ ID NO: 52 anti-CD19 FMC63 Light Chain CDR3 QQGNTLPYT SEQ ID NO: 53 signaling sequence YNMN SEQ ID NO: 54 signaling sequence YXXL / IX(6-8)YXXL

Claims

Attorney Docket No. KVN-011WO WHAT IS CLAIMED IS:

1. A method of treating systemic sclerosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of T cells that comprises a vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises, from N-terminus to C-terminus: (a) an antigen-binding fragment of an anti-CD19 antibody; (b) a transmembrane domain; and (c) an intracellular T cell signaling domain from human CD3ζ.

2. The method of claim 1, wherein the anti-CD19 antibody is a human antibody.

3. The method of claim 1 or 2, wherein the antigen-binding fragment of the anti-CD19 antibody comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 25, 26, and 3, respectively, and the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

4. The method of claim 1 or 2, wherein the antigen-binding fragment of the anti-CD19 antibody comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable domain comprising CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

5. The method of claim 3 or 4, wherein the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 7, and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:

8.

6. The method of claim 5, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

8.

7. The method of claim 6, wherein the antigen-binding fragment of the anti-CD19 antibody comprises the amino acid sequence of SEQ ID NO:

17.

8. The method of any one of claims 1-7, wherein the transmembrane domain is from human CD8.Attorney Docket No. KVN-011WO 9. The method of any one of claims 1-8, wherein the transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:

11.

10. The method of claim 9, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:

11.

11. The method of any one of claims 1-10, wherein the intracellular T cell signaling domain from human CD3ζ comprises an amino acid sequence at least 90% identical to SEQ ID NO:

23.

12. The method of claim 11, wherein the intracellular T cell signaling domain from human CD3ζ comprises the amino acid sequence of SEQ ID NO:

23.

13. The method of any one of claims 1-12, wherein the CAR further comprises an intracellular T cell signaling domain from human CD28.

14. The method of claim 13, wherein the intracellular T cell signaling domain from human CD28 comprises the amino acid sequence of SEQ ID NO:

21.

15. The method of any one of claims 1-12, wherein the CAR does not comprise an intracellular T cell signaling domain from 4-1BB.

16. The method of any one of claims 1-15, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 10 or 13.

17. The method of any one of claims 1-16, wherein the vector is a lentivirus vector.

18. The method of any one of claims 1-17, wherein the vector further comprises a murine stem cell virus (MSCV) U3 promoter operably linked to the nucleic acid.

19. The method of any one of claims 1-18, wherein at least 10% of the T cells express the CAR.

20. The method of any one of claims 1-19, wherein the T cells comprise at least 10% of CD8+cytotoxic T cells.

21. The method of any one of claims 1-20, wherein the T cells comprise at least 10% of CD4+helper T cells.Attorney Docket No. KVN-011WO 22. The method of any one of claims 1-21, wherein the systemic sclerosis is limited cutaneous systemic sclerosis or diffuse cutaneous systemic sclerosis.

23. The method of claim 22, wherein the systemic sclerosis is limited cutaneous systemic sclerosis.

24. The method of claim 22, wherein the systemic sclerosis is diffuse cutaneous systemic sclerosis.

25. The method of any one of claims 1-24, wherein the systemic sclerosis is refractory to an anti-CD20 antibody.

26. The method of any one of claims 1-25, wherein the therapeutically effective dose is in a range of about 5×107to 1×108of the T cells.

27. The method of any one of claims 1-26, wherein the therapeutically effective dose is about 1×108of the T cells.

28. The method of any one of claims 1-27, wherein the T cells are administered by intravenous infusion.

29. The method of any one of claims 1-28, wherein the subject receives a single dose of the T cells.

30. The method of any one of claims 1-29, wherein the subject has received a lymphodepletion treatment.

31. The method of any one of claims 1-30, wherein the subject has received a minimal lymphodepletion treatment resulting in about 50% reduction of lymphocytes in the subject as compared to the amount of lymphocytes in the subject prior to the minimal lymphodepletion treatment.

Images