Methods of treating autoimmune diseases with gamma delta t cells

Abstract

Aspects of the disclosure include methods of treating an autoimmune disease, including reducing the need for chronic immunosuppression, and for effectuating immune reset and / or producing a naive B cell repertoire in a subject in need thereof, the methods comprising administering a therapeutically effective amount of anti-CD20 yδ T cells to the subject, wherein the anti-CD20 yδ T cells express a chimeric antigen receptor (CAR) comprising a binding domain that specifically binds to CD20.

Description

METHODS OF TREATING AUTOIMMUNE DISEASES WITH d T CELTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional Patent Application No.63 / 738,730, filed December 24, 2024, the contents of which are expressly incorporated by reference herein.FIELD OF DISCLOSURE

[0002] The present disclosure relates generally to methods of treating autoimmune diseases using y5 T cells, and anti-CD20 CAR-expressing 51 y5 T cells in particular.BACKGROUND OF THE DISCLOSURE

[0003] Autoreactive B cells play a key role in the pathogenesis of autoimmune disorders, leading to serious and often life-threatening damage to vital organs. Autologous chimeric antigen receptor (CAR)-modified oc|3 T cells that have been genetically engineered to recognize CD 19 and other B-cell surface antigens have emerged as a powerful tool for the treatment of relapsed or refractory B-cell cancers. Based on their success in oncology, CAR-T approaches are being investigated to help patients with serious autoimmune disorders. An autologous CD19-directed CAR-T product has shown some activity in patients with three types of autoimmune disease (severe systemic lupus erythematosus, inflammatory myositis, or systemic sclerosis).

[0004] The CD 19 antigen is a type I transmembrane glycoprotein that belongs to the immunoglobulin Ig superfamily. It is expressed on early pro-B cells, late pro-B cells, memory B cells, plasma blasts and some plasma cells, the latter of which are the main cellular source of protective, highly target-specific antibodies and autoantigen-specific antibodies. Anti-CD19 CAR-T therapies have been associated with various hematologic adverse reactions including decreases in lymphocyte numbers, white blood cell numbers, hemoglobin concentration, neutrophil numbers, and platelet numbers. T-cell malignancies can also develop in patients who received treatment with anti-CD19 or anti-BCMA autologous CAR T cell therapies.

[0005] Therefore, there is a need for improved CAR-T therapies for the treatment of autoimmune diseases.SUMMARY OF DISCLOSURE

[0006] During B cell development, CD 19 is expressed on earlier- and later-stage B cells than CD20 (see e.g., Forsthuber T et al., Ther Adv Neurol Disord. 2018 Mar 21:11:1756286418761697). It has been reported that CD20 antibody can only completely deplete CD19+ cells in peripheral blood but not lymph nodes, while anti-CD19 CAR-T therapy was able to cause complete depletion of CD19+B cells in both peripheral blood and lymph nodes (see e.g.. Tur C et al., CD19-CAR T-cell therapy induces deep tissue depletion of B cells, Ann Rheum Dis. 2024 Sep 11:ard-2024-226142). Importantly, however, and despite the narrower expression pattern of CD20 and apparent lower activity of the anti-CD20 antibody, the inventors surprisingly discovered that anti-CD20 CAR-expressing yo T cells exhibit efficacy and safety profiles that are at least comparable with, if not better than, anti-CD19 CAR-T therapies. Moreover, given that yo T cells preferentially traffic to organs and tissues, the anti-CD20 CAR-expressing y5 T cells provided herein can synergistically target and deplete B cells in the periphery, secondary lymphoid organs, kidneys, and other organs, which is highly desirable in autoimmune diseases.

[0007] The present disclosure provides methods of treating autoimmune diseases with anti-CD20 CAR-expressing y5 T cells, and for reducing the need for and / or reliance on chronic immunosuppression in the management of these types of diseases. In embodiments, the anti-CD20 CAR-expressing yo T cells are allogeneic cell products that do not include gene editing or manipulations to resolve allogeneic histo-incompatibility with the patient’s own immune system. With this incomplete histocompatibility, the anti-CD20 y5 T cells may be subject to elimination and are not expected to persist longer term. The nature of an allogeneic cell therapy which is expected to be eventually rejected by the host support a lower tumorigenicity risk for the anti-CD20 y5 T cells than autologous CAR T therapies (e.g.. those using ot0 T cells) and alternative gene-edited allogeneic therapies.

[0008] Remarkably, and as demonstrated herein for the first time, administration of anti-CD20 CAR-expressing y5 T cells in accordance with the present invention can 1) dramatically reduce clinical indicators and / or symptoms of autoimmune diseases in vivo, 2) result in partial or complete remission of disease activity. 3) reduce the need for chronic immunosuppression in patients, and / or 4) eliminate pathogenic B cells and repopulate a patient’s B cell repertoire with naive cells so as to effectuate immune reset, notwithstanding the aforementioned concerns in the art with targeting CD20. As such, the subject therapies will find advantageous use across a wide range of autoimmune diseases, and in B cell- and antibody-mediated autoimmune diseases in particular.

[0009] In one aspect, the present disclosure provides methods of treating an autoimmune disease, comprising administering a therapeutically effective amount of anti-CD20 y5 T cells to a subject in need thereof, wherein the anti-CD20 y5 T cells express a chimeric antigen receptor (CAR) comprising a binding domain that specifically binds to CD20. In another aspect, the present disclosure provides methods for reducing and / or eliminating a need for chronic immunosuppression in a human subject with an autoimmune disease, comprising administering a therapeutically effective amount of anti-CD20 y6 T cells to the subject, wherein the anti-CD20 y3 T cells express a CAR comprising a binding domain that specifically binds to CD20.

[0010] In a further aspect, methods are provided for effectuating immune reset in a subject with an autoimmune disease, comprising administering a therapeutically effective amount of anti-CD20 y5 T cells to the subject, wherein the anti-CD20 y8 T cells express a CAR comprising a binding domain that specifically binds to CD20. In an additional aspect, methods are provided for producing a naive B cell repertoire in a subject in need thereof, comprising administering a therapeutically effective amount of anti-CD20 y <5 T cells to the subj ect, wherein the anti-CD20 y§ T cells express a CAR comprising a binding domain that specifically binds to CD20. In embodiments, the subject is suffering from an autoimmune disease.

[0011] In embodiments, the subj ect therapies are surprisingly shown herein to achieve deep and broad B cell depletion in vivo despite the previously alleged shortcomings in CD20 expression. In exemplary embodiments, patient B cells are undetectable for at least 3, at least 5, at least 7, at least 10, at least 14, at least 21, or at least 28 days after administration of the anti-CD20 y3 T cells. In embodiments, the subject therapies are effective to eliminate clonally dominant and potentially pathogenic B cell clones. In embodiments, the subj ect therapies result in reconstitution of the patient's immune system driven by naive, non-class switched B cells.

[0012] In embodiments, the autoimmune disease is selected from the group comprising or consisting of lupus, lupus nephritis, systemic sclerosis, antineutrophil cytoplasmic autoantibody-associated vasculitis, and autoimmune muscle disease. In embodiments, the lupus is systemic lupus erythematosus. In embodiments, the systemic lupus erythematosus is systemic lupus erythematosus with extrarenal involvement. In embodiments, the autoimmune muscle disease is idiopathic inflammatory myopathies, dermatomyositis, or stiff person syndrome.

[0013] In embodiments, the therapeutically effective amount of the anti-CD20 CAR y8 T cells is from about 1 x 108to about 1 x 109y5 T cells. In embodiments, the therapeutically effective amount of the anti-CD20 CAR y§ T cells is about 1 x 108y5 T cells. In embodiments,the therapeutically effective amount of the anti-CD20 CAR y8 T cells is about 3 x 108y8 T cells. In embodiments, the therapeutically effective amount of the anti-CD20 CAR y8 T cells is about 1 x 109y8 T cells.

[0014] In embodiments, the anti-CD20 CAR y8 T cells are 81 y8 T cells, 82 y8 T cells, 83 y8 T cells, 84 y8 T cells and / or 85 y8 T cells. In embodiments, the y8 T cells are 81 y8 T cells.

[0015] In embodiments, the methods further comprise administering to the subject a lymphodepletion (LD) regimen prior to administering to the subject a first dose of the therapeutically effective amount of anti-CD20 CAR y8 T cells. In embodiments, the LD regimen comprises administration of fludarabine at about 30 mg / m2 / day and cyclophosphamide at about 500 mg / m2 / day for three days. In embodiments, the LD regimen comprises administration of fludarabine at about 20 mg / m2 / day and cyclophosphamide at about 300 mg / m2 / day for three days. In embodiments, the LD regiment further comprises administration of mesna at about 15 mg / kg with the cyclophosphamide. In embodiments, the LD regimen comprises administration of bendamustine at about 90 mg / m2 / day for 2 days.

[0016] In embodiments, the methods further comprise administering a non-therapeutic dose of the anti-CD20 CAR y6 T cells at least 1. 2, 3, 4. 5, 6, 7, 8. 9, 10, 11, 12. 13. or 14 days prior to administration of the LD regimen, preferably between 3 to 5, 4 to 6 or 7 to 9 days prior to administration. In embodiments, the LD regimen comprises administration of cyclophosphamide at about 50 mg / m2 / day for two days. In embodiments, the LD regimen comprises administration of fludarabine at about 20 mg / m2 / day and cyclophosphamide at about 300 mg / m2 / day for three days. In embodiments, the LD regimen comprises administration of fludarabine at about 30 mg / m2 / day and cyclophosphamide at about 500 mg / m2 / day for three days. In embodiments, the LD regimen comprises administration of fludarabine at about 30 mg / m2 / day and cyclophosphamide at about 1000 mg / m2 / day for three days.

[0017] In embodiments, the methods further comprise administering one or more additional doses of anti-CD20 CAR y8 T cells at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least 28 days, or at least one month after the first dose. In embodiments, the one or more additional doses of anti-CD20 CAR y8 T cells are administered without an additional LD regimen. In embodiments, the one or more additional doses of anti-CD20 CAR y8 T cells are administered following an additional LD regimen.

[0018] In embodiments, the one or more additional doses of anti-CD20 CAR y8 T cells comprise an increased amount of anti-CD20 CARyS T cells, a decreased amount of anti-CD20 CARy8 T cells, or the same amount of anti-CD20 CAR y8 T cells. In embodiments, the one or more additional doses of anti-CD20 CAR y6 T cells comprise anti-CD20 CAR y8 T cellsderived from the same donor. In embodiments, the one or more additional doses of anti-CD20 CAR y6 T cells comprise anti-CD20 CAR y5 T cells derived from a different donor.

[0019] In embodiments, the methods further comprise monitoring the subject for one or more pharmacodynamics / pharmacokinetics biomarkers following administration of the therapeutically effective amount of anti-CD20 CAR y5 T cells, wherein the biomarkers are selected from the group comprising or consisting of CAR transgene expression level, quantitative measurement of CAR+ y5 T cells, and serum level of one or more cytokines and / or serum proteins.

[0020] In embodiments, one or more cytokines and / or serum proteins are selected from the group comprising or consisting of IL-6, TNF-a, IL-8, IL-1, IL-2, GM-CSF, IL-15, IL-17a, IFNy, IL-12, granzyme A, granzyme B, perforin, sFasL, MIP-la. orMCP-1. In embodiments, the method further comprises administering a secondary treatment regimen based at least in part on monitoring one or more of the biomarkers.

[0021] In embodiments, the secondary treatment regimen comprises one or more additional doses of anti-CD20 CAR y5 T cells. In embodiments, the one or more additional doses of anti-CD20 CAR y5 T cells are administered without an additional LD regimen. In embodiments, the one or more additional doses of anti-CD20 CAR y5 T cells are administered following an additional LD regimen. In embodiments, the one or more additional doses comprise an increased amount of anti-CD20 CAR y5 T cells a decreased amount of anti-CD20 CAR y3 T cells, or the same amount of anti-CD20 CAR y6 T cells. In embodiments, the one or more additional doses comprise anti-CD20 CAR y5 T cells derived from the same donor. In embodiments, the one or more additional doses comprise anti-CD20 CAR y5 T cells derived from a different donor.

[0022] In embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 41. In embodiments, the anti-CD20 y5 T cells further comprise an isolated nucleic acid encoding the CAR. In embodiments, isolated nucleic acid encoding the CAR having the sequence of SEQ ID NO: 46.INCORPORATION BY REFERENCE

[0023] 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, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows that ADI-001 exhibited potent killing of patient-derived CD 19+ B cells in multiple autoimmune diseases.

[0025] FIG. 2 shows ADI-001 clinical tissue analyses.

[0026] FIG. 3 shows complete depletion of CD 19+ B cells at day 10 within secondary lymphoid tissue.

[0027] FIG. 4 shows clinical responses to ADI-001 observed in extra-nodal tissue.

[0028] FIG. 5 shows a general design of the clinical study described in Example 2.

[0029] FIG. 6 shows the significant decline in systemic lupus erythematosus disease activity index (SLEDAI) in three patients (2 LN and one SLE) enrolled in the ongoing clinical study.

[0030] FIG. 7 illustrates the rapid and sustained reductions in Physician Global Assessment (PGA) scores across the same patients.

[0031] FIG. 8 illustrates the broad and deep B cell depletion resulting from ADI-001 administration in these patients.

[0032] FIGS. 9A-E provide further detail on the elimination of particular B cell subsets and the reconstitution of naive B cells in these patients. Corresponding phenotypes for individual subsets are Naive (CD19+CD20+IgD+CD27-), Transitional (CD19+CD20+IgD+CD27-CD24+CD38+), Pre-class switched memory (CD19+CD20+IgD+CD27+), Memoty B bells (CD19+CD20+IgD-CD27+), and Plasmablasts (CD19+CD20+ / -IgD-CD27highCD38highCD138-).

[0033] FIG. 10 illustrates that dominant and potentially pathogenic B cell clones were eliminated in the LN patients, and did not persist or return post-treatment. Relative Clonal Frequency = B cell receptor (BCR) read count normalized to total population of BCR's detected.

[0034] FIG. 11 illustrates the tracking of dominant BCR clonotypes pre- and posttreatment. Post Treatment = 3 Months (LN-4) or 6 Months (LN-1); Node sizes are proportional to clone read count at each timepoint. Dominant Clones = Most abundant detectable clones achieving >1 read counts (cutoff of 40 defined by lowest limits for a sample in the cohort), Minor Clones = Detected clones with read counts below those quantified as dominant.DETAILED DESCRIPTION

[0035] The achievement of immune reset is a rapidly emerging objective in the treatment of autoimmune diseases, with the goal of restoring a stable, self-tolerant immune system and increasing the likelihood and / or length of a sustained remission. See, e.g., Junt et al., Defining immune reset: achieving sustained remission in autoimmune diseases. Nat Rev Immunol. (2025) 25:528-41. doi: 10.1038 / s41577-025-01141-w. A drastic disruption of the immune system is generally required to achieve the requisite elimination of pathogenic cells and their subsequent repopulation with naive cells, in the hope of restoring homeostatic immune function.

[0036] Conventional regimens employed for this purpose include myeloablative or non-myeloablative conditioning of patients followed by autologous human stem cell transplantation (aHSCT), which has been tested in autoimmune diseases such as multiple sclerosis. Unfortunately, however, this approach does not produce remission in all patients and remains associated with an unacceptably high non-relapse mortality rate of -1 / 30. Snowden et al., Evolution, trends, outcomes, and economics of hematopoietic stem cell transplantation in severe autoimmune diseases. Blood Adv. (2017) 1:2742-55. doi: 10.1182 / bloodadvances.2017010041. T cell depleters such as alemtuzumab have also been investigated but can result in the emergence of de novo and unrelated autoimmune conditions, likely due to the impact on regulatory' T cell populations. Willis and Robertson, Alemtuzumab for the treatment of multiple sclerosis. Ther Clin Risk Manag. (2015) 11:525-34. doi: 10.2147 / TCRM. S80112. B cell depletion presents as an attractive alternative, but the cunent autologous CD19-directed CAR-T therapies suffer from the toxicity, tumorigenicity, and other concerns discussed previously above.

[0037] As such there remains an unmet need in the art for safer and more effective means for achieving immune reset in patients with autoimmune diseases. The present invention addresses these and other unmet needs.Definitions

[0038] For purposes of interpreting this specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0039] “About'’ as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0040] As used herein, “w / v” refers to the weight of the component in a given volume of solution.

[0041] “Ranges”: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2. 2.7, 3, 4. 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0042] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal amenable to the methods described herein. In embodiments, the patient, subject or individual is a human.

[0043] As used herein, the term “agent” refers to any protein, nucleic acid molecule (including chemically modified nucleic acids), compound, antibody, small molecule, organic compound, inorganic compound, other molecule of interest, or cell (e.g., cell engineered to express a chimeric antigen receptor). Agent can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent. A therapeutic or pharmaceutical agent is one that alone or together with an additional agent induces the desired response (such as inducing a therapeutic or prophylactic effect when administered to a subject, including treating a subject suffering from an autoimmune disease, or other disease / condition.

[0044] The term "therapeutically effective amount", or simply “effective amount” refers to the amount of an agent or composition (e.g., composition comprising an agent) that will elicit a biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes that amount of an agent, or a composition comprising an agent, that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease (e.g.. an autoimmune disease)being treated. The therapeutically effective amount will vary depending on the composition, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0045] To "treat" a disease as the term is used herein, means to decrease or reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. In one example, a therapy (e.g., administration of a therapeutic agent of the present disclosure) treats a disease or condition by decreasing one or more signs or symptoms associated with the disease or condition, for example as compared to the response in the absence of the therapy. For example, administration of a therapeutic agent may provide an effect that decreases one or more signs or symptoms associated with an autoimmune disease.

[0046] The term "immune reset” as used herein refers to the depletion of a subset of autoreactive immune cells and, ideally, the restoration of a stable, self-tolerant immune system. Junt et al., supra. In practice, this can be demonstrated by the elimination of potentially pathogenic cell population(s) in vivo such as, e.g., clonally dominant B cell clones, and the subsequent reconstitution of a patient’s immune system with naive cells such as, e.g., non-class switched B cells. In therapeutic effect, achieving immune reset in a patient can increase the probability of a sustained remission, and / or increase the length of such a remission.

[0047] As used herein, the term “administration” means to provide or give a subject one or more agents, such as an agent that treats one or more signs or symptoms associated with a condi tion / disorder or disease such as an autoimmune disease by any effective route. Exemplary routes of administration include injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes. Administration “in combination with ” one or more further therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0048] The term “pharmaceutically acceptable,” as used herein, refers to a material, including a salt, carrier or diluent, which does not abrogate the biological activity' or properties of a therapeutic agent e.g., the y5 cells, and is relatively non-toxic, e.g., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of one or more agents, such as one or more modulatoryagents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations can include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically neutral carriers, pharmaceutical agents to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate. For example, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and ay5 T cell engineered to express a CAR directed to CD20, as described herein.

[0049] As used herein, the term '‘pharmacodynamic (PD) biomarker’’ and / or “pharmacokinetic (PK) biomarker” refers to one or more measurable indicators associated with administration of a therapeutic agent to a subject. Broadly speaking, a PK marker relates to how the body affects a therapeutic agent, whereas a PD marker relates to how the therapeutic agent affects a subject.

[0050] '‘Activation”, as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

[0051] The term '‘antigen" or " Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both. The skilled artisan will understand that any macromolecule, including proteins or peptides, can serve as an antigen.

[0052] The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with poly epitopic specificity, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity’)- formed from at least two intact antibodies, diabodies, single domain antibodies (sdAbs), as long as they exhibit thedesired biological or immunological activity, Fv, Fab and F(ab), as well as single chain antibodies and humanized antibodies (Harlow et ah, 1999. In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY: Harlow et ah, 1989, In; Antibodies: A Laboratory Manual, Cold Spring Harbor, N. Y.; Houston et ah, 1988, Proc. Nat Acad. Sci. USA 85:5879-5883: Bird et ah, 1988, Science 242:423-426). In embodiments, the antibody is a synthetic antibody. By the term “synthetic antibody” as used herein, is meant an antibody which is generated using recombinant DNA technology, such as. for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0053] The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0054] An “antibody heaw chain,” as used herein, refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations.

[0055] An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, K and X light chains refer to the two major antibody light chain isotypes.

[0056] The term “epitope” includes any protein determinant, lipid or carbohydrate determinant capable of specific binding to an immunoglobulin or receptor, for example a T-cell receptor. Epitopic determinants usually consist of active surface groupings of molecules such as amino acids, lipids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0057] The term “specifically binds” as used herein refers to a receptor (which can include but is not limited to an antibody or antibody fragment) which recognizes a specific molecule / ligand, but does not substantially recognize or bind other molecules in a sample. For example, a receptor that specifically binds to a molecule from one species may also bind to that molecule from one or more other species. But, such cross-species reactivity does not itself alter the classification as specific. In another example, a receptor that specifically binds to a molecule may also bind to different allelic forms of the molecule. However, such crossreactivity does not itself alter the classification as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of a protein (or a peptide) with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, a receptor recognizes and binds to a specific a structure rather than to proteins generally. If receptor is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the receptor, will reduce the amount of labeled A bound to the receptor.

[0058] In embodiments, specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10-8M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0059] As used herein, the term "autologous" is meant to refer to any material derived from an individual which is later to be re-introduced into the same individual.

[0060] As used herein, the term "allogeneic" refers to material derived from an animal which is later introduced into a different animal of the same species.

[0061] The term “yb T cells” or “gamma delta T cells" as used herein refers to a subset of T cells that express a distinct T-cell receptor (TCR), namely yb TCR, on their surface, composed of one y-chain and one b-chain. The term “yb T cells” specifically includes all subsets of yb T-cells, including Vbl, Vb2, and Vb3 yb T cells, as well as naive, effector memory, central memory, and terminally differentiated yb T cells. As a further example, the term “yb T cells” includes Vb4, Vb5, Vb7, and Vb8 yb T cells, as well as Vy2, Vy3, Vy5, Vy8, Vy9, VylO. and Vyl 1 yb T cells. In embodiments, the yb T cells are Vbl (also known as bl) yb T cells. In embodiments, the yb T cells are Vb2 (also known as b2) yb T cells. In embodiments, the yb T cells are Vb3 (also known as b3) yb T cells.

[0062] Antigen recognition by the yb TCR fundamentally differs from the a[3 TCR, in that the yb TCR recognizes native, unprocessed antigens, many of which are induced or upregulated following cellular injury, infection, or transformation, whereas the af> TCR recognizes processed peptide antigens that are presented by major histocompatibility complex (MHC) molecules, yb T cells can also detect stress-induced surface molecules through expression of non-TCR molecules, such as the germline-encoded NK receptors (NKRs). The co-expression of TCR and non-TCR molecules endows yb T cells with theability to monitor and maintain tissue integrity through recognition and destruction of stressed, infected, or malignant cells. Since yb T cells recognize antigen in an MHC-unrestricted manner, they do not initiate TCR-mediated GvHD and therefore do not require gene editing to disrupt TCR expression or TCR signaling. Because they predominantly reside in peripheral tissues, y5 T cells are better adapted than circulating lymphocytes at functioning in hypoxia, a condition often found in disease microenvironment.

[0063] “Encoding’7refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA. can be referred to as encoding the protein or other product of that gene or cDNA.

[0064] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0065] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0066] “Expression cassette” refers to a nucleic acid comprising expression control sequences operatively linked to a nucleic acid encoding a transcript or polypeptide to be expressed. An expression cassette comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression cassettes can be a component of a vector such as a cosmid, a plasmid (e.g, naked or contained in a liposome), or a virus (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus). An expression cassette can be in a host cell, such as ayb T cell.

[0067] The term “chimeric antigen receptors (CARs)” refers to artificial T-cell receptors, T-bodies, single-chain immunoreceptors, chimeric T-cell receptors, or chimericimmunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. In specific embodiments, CARs direct specificity of the cell to a disease associated antigen, (e.g., an antigen on an autoreactive immune cell). In embodiments, CARs comprise an intracellular activation domain (allowing the T cell to activate upon engagement of targeting moiety with target cell, such as a autoreactive immune cell), a transmembrane domain, and an extracellular domain that may vary in length and comprises a disease- or disorder-associated, e.g., an antigen binding region. In embodiments, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta a transmembrane domain and endodomain. The specificity of other CAR designs may be derived from ligands of receptors (e.g., peptides) or from pattern-recognition receptors, such as Dectins. In certain cases, the spacing of the antigen-recognition domain can be modified to reduce activation-induced cell death. In certain cases, CARs comprise domains for additional co-stimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD137. DAP 10 / 12, and / or 0X40. ICOS, TLRs. etc. In some cases, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0068] The term '‘autoimmune disease’’ refers to a disorder that results from an autoimmune response. An autoimmune disease is the result of an inappropriately excessive response to an antigen associated with an autoimmune disease (e.g., a self-antigen).

[0069] The term “autoreactive immune cell” refers to a cell produced by an organism and acting against its own cells or tissues. In embodiments, an autoreactive immune cell may be a B cell. An autoreactive B cell may be at any development stage. For example, an autoreactive B cell may be a pro-B cell, a pre-B cell, an immature B cell, a naive B cell, a mature B cell, a memory B cell, a plasmablast, or a plasma cell. In embodiments, an autoreactive immune cell is aT cell.Chimeric Antigen Receptors

[0070] In embodiments, the y5 T cells herein express a CAR comprising a binding domain that specifically binds to an antigen expressed on a surface of an autoreactive immune cell (e.g., autoreactive B cell). In preferred embodiments, the binding domain is a CD20 binding domain, such as a CD20 binding domain described in U. S. Patent Appl. No. 2009 / 0035322 andW02020 / 072536, the contents of each of which are incorporated by reference in the entirety and for all purposes and in particular for the binding domains, antibodies, antibody fragments, complementarity determining regions, polypeptides containing said complementarity determining regions, nucleic acids encoding for said complementarity determining regions, and epitope specificities and assays for determining epitope specificity described therein.

[0071] In embodiments, the yd T cells comprise nucleic acids encoding the CARs. or constructs and vectors containing such nucleic acids. In some cases, the nucleic acid is a, e.g., heterologous, component of an expression cassette. In embodiments, the nucleic acid is a, e.g., heterologous, component of a retroviral vector. In embodiments, the nucleic acid is a, e.g., heterologous, component of a yd T cell. In embodiments, the nucleic acid is a, e.g., heterologous, component of ay+T cell and / or a d+T cell. In embodiments, the region encoding the binding domain is 5’ of a linker region (e.g., a region encoding a CD 8 a hinge domain).

[0072] Exemplary CD20 binding domains include binding domains that selectively bind to an epitope within CD20 bound by, or that competes for binding with, 3B9, 3H7, 2B7, 9C11, or 10F2; or 3B9, 3H7, 2B7, or 9C11; or 3H7. Additionally or alternatively, the CD20 binding domain can compnse the complementary determining regions of an anti-CD20 antibody selected from the group consisting of 3B9, 3H7, 2B7, 9C11, and 10F2; selected from the group consisting of 3B9, 3H7, 2B7, and 9C11; or comprise the complementary' determining regions of an anti-CD20 antibody selected from the group consisting of 3H7. The present disclosure also contemplates CD20 binding domains that compete for binding with a sequence provided herein. Antibodies 3B9, 9C11, 3H7, 2B7, and 10F2, fragments thereof, and their complementary determining regions, are described in U. S. 2009 / 0035322, where they are referred to as 3B9-10, 9C11-14, 3H7-6, 2B7-7, and 10F2-13 respectively.

[0073] One can determine whether a CD20 binding domain binds to the same epitope as, or competes for binding with, a reference antibody or binding domain by using known methods. For example, to determine if a test antibody binds to the same epitope as a reference binding domain, the reference binding domain can be allowed to bind to CD20 under saturating conditions. Next, the ability of a test binding domain to bind to CD20 molecule can be assessed. If the test binding domain is able to bind to CD20 following saturation binding with the reference binding domain, it can be concluded that the test binding domain binds to a different epitope than the reference binding domain. On the other hand, if the test binding domain is not able to bind to CD20 following saturation binding with the reference binding domain, then the test binding domain may bind to the same epitope as the epitope bound by the reference binding domain.

[0074] To determine if a binding domain competes for binding with a reference binding domain, the above-described binding methodology is performed in two orientations: In a first orientation, the reference binding domain is allowed to bind to CD20 under saturating conditions followed by assessment of binding of the test binding domain to the CD20 molecule. In a second orientation, the test binding domain is allowed to bind to a CD20 molecule under saturating conditions followed by assessment of binding of the reference binding domain to the CD20 molecule. If, in both orientations, only the first (saturating) binding domain is capable of binding to the CD20 molecule, then it is concluded that the test binding domain and the reference binding domain compete for binding to CD20. As will be appreciated by a person of ordinary skill in the art, a binding domain that competes for binding with a reference binding domain may not necessarily bind to the identical epitope as the reference binding domain, but may sterically block binding of the reference binding domain by binding an overlapping or adjacent epitope.

[0075] Two binding domains bind to the same or an overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one binding domain inhibits binding of the other by at least 50%, for example, 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two binding domains have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one binding domain also reduce or eliminate binding of the other. Two binding domains have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one binding domain also reduce or eliminate binding of the other.

[0076] Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test binding domain is in fact due to binding to the same epitope as the reference binding domain or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative binding assay available in the art.

[0077] In embodiments, a CD20 binding domain binds a CD20 epitope that is different from a CD20 epitope bound by rituximab. In embodiments, a CD20 binding domain as herein disclosed binds a CD20 epitope that is different from a CD20 epitope bound by ocrelizumab. In embodiments, a CD20 binding domain as herein disclosed binds a same or overlapping CD20 epitope bound by rituximab or ocrelizumab. In embodiments, a CD20 binding domain as herein disclosed binds a CD20 epitope that is different from a CD20 epitope bound byofatumumab, or obinutuzumab, or veltuzumab. In embodiments, a CD20 binding domain as herein disclosed bind a same or overlapping CD20 epitope bound by ofatumumab. or obinutuzumab, or veltuzumab. In embodiments, a CD20 binding domain as herein disclosed binds a CD20 epitope that is different from one or more anti-CD20 mAbs described by Luo et al. (2021) Scientific Reports, 11(3255). In embodiments, a CD20 binding domain as herein disclosed binds a same or overlapping CD20 epitope bound by one or more anti-CD20 mAbs described by Luo et al. (2021) Scientific Reports, 11(3255). In embodiments, a CD20 binding domain as herein disclosed binds a CD20 epitope that is different from one or more anti-CD20 mAbs described by Casan et al. (2018) Hum Vaccin Immunother, 14(12): 2820-2841. In embodiments, a CD20 binding domain as herein disclosed binds a same or overlapping CD20 epitope bound by one or more anti-CD20 mAbs described by Casan et al. (2018) Hum Vaccin Immunother,, 14(12): 2820-2841.

[0078] In embodiments, a CD20 binding domain expressing in a y5 T cell herein comprises the CD20 binding domain of rituximab, ocrelizumab, ofatumumab, obinutuzumab, or veltuzumab. In embodiments, a CD20 binding domain expressing in a y5 T cell herein comprises the CD20 binding domain in antibodies described in Luo et al. (2021) Scientific Reports, 11(3255); Casan et al. (2018) Hum Vaccin Immunother, 14(12): 2820-2841, each of which is incorporated by reference herein in its entirety.

[0079] The present disclosure provides antibodies and CARs with “substantial identity'’ or “substantial similarity” to the sequences provided herein in the CDR or framework regions. The term “substantial identity” or “substantially identical,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with another nucleic acid (or the complementary strand of the other nucleic acid), there is nucleotide sequence identity in %, for example, at least 80%. at least 81%. at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP. as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may. in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0080] As applied to polypeptides, the term "substantial similarity" or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity. In some aspects, residue positions, which are not identical, differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g.. Pearson (1994) Methods Mol. Biol. 24: 307-331, which is herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysinearginine, alanine-valine. glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0081] Sequence identity and / or similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity' assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance. GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identityof the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Sequences also can be compared using the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty' of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. Another preferred algorithm when comparing a sequence disclosed herein to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g.. Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res.25:3389-3402, each of which is herein incorporated by reference.

[0082] Provided herein are anti-CD20 CARs comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more substitutions (e.g, conservative substitutions). For example, the present disclosure includes anti-CD20 CARs having HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR (e.g, HCDRL HCDR2. HCDR3. LCDR1, LCDR2, or LCDR3) ammo acid sequences disclosed herein. For example, an anti-CD20 CAR can comprise 20, 19, 18, 17, 16, 15, 14 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions (e.g., conservative amino acid substitutions) relative to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein.

[0083] In embodiments, the anti-CD20 binding domain has a heavy chain complementary determining region 3 (HCDR3) and a light chain CDR3 (LCDR3), wherein the HCDR3 and LCDR3 are selected from the group consisting of SEQ ID NO: 1 (AKDPSYGSGSYHSYYGMDV) and 2 (QQRFNWPLT); 3 (VKDFHYGSGSNYGMDV) and 4 (QQSNDWPLT); and 5 (TKDGSYGHFYSGLDV) and 6 (QQRYYWPLT).

[0084] In embodiments, the anti-CD20 binding domain has a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence, wherein the HCVR and LCVR sequences are selected from the group consisting of SEQ ID NO: 7 (EEQLVESGGDLVQPGRSLRLSCAASGFTFHDYTMH WVRQAPGKGLEWVSGISWNSGSLGYADSVKGRFTISRDNAKKSLYLQMNSLRAED TALYYCAKDPSYGSGSYHSYYGMDVWGQGTTVTVSS) and 8 (EIVLTQSPATLSLSPGE RATLSCWASQSISRYLVWYQQKCGQAPRLLIYEASKRATGIPVRFSGSGSGTDFTLTI SSLESEDFAVYYCQQRFNWPLTFGGGTKVEIK); 9(EVQLAESGGDLVQSGRSLRLSCAAS GITFHDYAMHWVRQPPGKGLEWVSGISWNSDYIGYADSVKGRFTISRDNAKKSLYL QMNSLRPDDTALYYCVKDFHYGSGSNYGMDVWGQGTTVTVSP) and 10 (EIVMTQSPATL SMSPGERATLSCRASQSVSRNLAWYQQKVGQAPRLLISGASTRATGIPARFSGSGSG TEFTLTINSLQSEDFAVYYCQQSNDWPLTFGQGTRLEIK); and 11 (EVQLVESGGGLVQPGR SLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSDTIGYADSVKGRFTISR DNAKNSLYLQMNSLRAEDTALYYCTKDGSYGHFYSGLDVWGQGTTVTVSS) and 12 (EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYVASNRATGI PARFSGSGSGTDFTLTISSLEPDDFAVYYCQQRYYWPLTFGGGTKVEIK).

[0085] In embodiments, the anti-CD20 binding domain hasa heavy chain complementary determining region 3 (HCDR3) domain and a light chain CDR3 (LCDR3) domain, wherein the HCDR3 domain comprises an amino acid sequence of the formula XI — X2 — X3 — X4 — X5 — X6— X7— X8— X9— X10— XI 1— X12— X13— X14— X15— X16— X17— X18— X19, wherein XI =A, V or T; X2=K; X3=D; X4=P, F or G; X5=S or H; X6=Y; X7=G; X8=S or H; X9=G or F; X10=S or Y; XI 1=Y, N or S; X12=Y, G or H; X13=G, L or S; X14=Y, M or D; X15=Y, D or V; X16 =G, V or absent; X17=M or absent; X18=D or absent; X19=V or absent; and the LCDR3 domain comprises an amino acid sequence of the formula XI — X2 — X3— X4— X5— X6— X7— X8— X9, wherein X1=Q; X2=Q; X3=R or S; X4=N. Y or F; X5=N, D, or Y; X6=W; X7=P; X8=L; X9=T.

[0086] In embodiments, the anti-CD20 binding domain has a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence, wherein the HCVR and LCVR sequences are SEQ ID NO: 13 (EVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNS GYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSYGKFYYGLDV WGQGTTVTVSS) and 14 (EIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQKPGQAPR LLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEl).

[0087] In embodiments, the anti-CD20 binding domain binds the same epitope as, competes with, or is an anti-CD20 binding domain having heavy chain complementarity determining regions (HCDR) and a light chain complementarity determining regions (LCDR),wherein the HCDR and LCDR sequences are the HCVR sequences of SEQ ID NO: 13 and the LCVR sequences of SEQ ID NO: 14 respectively.

[0088] In embodiments, the anti-CD20 binding domain binds the same epitope as, competes with, or is an anti-CD20 binding domain having an HCDR1 that is or comprises SEQ ID NO: 15 (GFTFYDYA), an HCDR2 that is or comprises SEQ ID NO: 16 (ISWNSGYI), and / or an HCDR3 that is or comprises SEQ ID NO: 17 (AKDNSYGKFYYGLDV). In embodiments, the anti-CD20 binding domain binds the same epitope as. competes with, or is an anti-CD20 binding domain having an LCDR1 that is or comprises SEQ ID NO: 18 (QSVSSN), an LCDR2 that is or comprises SEQ ID NO: 19 (GAS), and / or an LCDR3 that is or comprises SEQ ID NO: 20 (QQYNNWPIT). In embodiments, the anti-CD20 binding domain binds the same epitope as. competes with, or is an anti-CD20 binding domain having an HCDR1 that is or comprises SEQ ID NO: 15, an HCDR2 that is or comprises SEQ ID NO: 16, an HCDR3 that is or comprises SEQ ID NO: 17, an LCDR1 that is or comprises SEQ ID NO: 18, an LCDR2 that is or comprises SEQ ID NO: 19, and an LCDR3 that is or comprises SEQ ID NO: 20. In embodiments, the anti-CD20 binding domain has an HCDR1 comprising SEQ ID NO: 15, an HCDR2 comprising SEQ ID NO: 16, an HCDR3 comprising SEQ ID NO: 17, an LCDR1 comprising SEQ ID NO: 18, an LCDR2 comprising SEQ ID NO: 19, and an LCDR3 comprising SEQ ID NO: 20.

[0089] In embodiments, exemplary' binding domains comprise, in order from the amino to carboxy terminus, a heavy chain region followed by a light chain region (VH-VL). Where a certain order of VH and VL region in the binding domain is explicitly or implicitly described, the present disclosure is also understood to describe the alternate embodiment in which the order of VH and VL regions are reversed, e.g., in an scFV or a CAR comprising an scFv binding domain. Thus, description of a VH-VL order also describes the alternate VL-VH order, e.g, in an scFV or a CAR comprising an scFv binding domain. Moreover, description of a VL-VH order also describes the alternate VH-VL order, e.g., in an scFv or a CAR comprising an scFv binding domain.

[0090] In embodiments, the CAR comprises an extracellular linker portion that encodes a peptide linker that links the binding domain to a transmembrane domain. Exemplary’ linker portions include, without limitation, a linker portion that encodes the CD8a hinge domain, e.g, SEQ ID NO: 21 (PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID NO:22 (TTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY). Typically, the region encoding the peptide linker (e.g, CD8a hinge domain) is 3' of the region encoding the binding domain and 5’ of a region encoding a transmembrane domain.

[0091] In embodiments, the CAR comprises a transmembrane domain. The transmembrane domain can link an extracellular antigen binding domain, e.g, and hinge, to one or more intracellular signaling components. For example, the transmembrane domain can link an antigen binding domain, e.g., and hinge, to a CD3^ signaling domain and optionally with one or two costimulation endodomains. Exemplary’ transmembrane domains include without limitation a CD8a transmembrane domain, e.g., SEQ ID NO: 23 (IWAPLAGTCGVLLLSLVITLYC). Typically, the region encoding the transmembrane domain (e.g., CD8a transmembrane domain) is 3’ of the region encoding the peptide linker (e.g., CD8a hinge domain) and 5’ of a region encoding one or more cytoplasmic domains.

[0092] In embodiments, the CAR comprises a cytoplasmic region containing one or more cytoplasmic domains. The nucleic acid region encoding the cytoplasmic region is typically 3’ of the region encoding the transmembrane domain. The cytoplasmic domains are typically signaling domains that provide an activating signal for y5 T cell proliferation, cytotoxic activity, and / or pro-inflammatory cytokine expression (e.g., TNF-a or IFNy). An exemplary cytoplasmic domain is a CD3^ signaling domain. In embodiments, the CD3^ signaling domain is or comprises SEQ ID NO: 24 (RVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR). In embodiments, the CD3 signaling domain is or comprises SEQ ID NO: 25 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR). In embodiments, the cytoplasmic region contains multiple (e.g., 2, 3, 4, 5, or 6) signaling domains, such as multiple (e.g., 2. 3, 4, 5, or 6) CD3^ signaling domains, e.g., each independently selected from SEQ ID NO: 24 and 25. In embodiments, the cytoplasmic region contains multiple (e.g., 2, 3, 4, 5, or 6) non- CD3C, signaling domains and a CD3^ signaling domain. In embodiments, the cytoplasmic region contains a non- CD3^ signaling domain and multiple (e.g., 2, 3, 4, 5, or 6) CD3^ signaling domains.

[0093] The cytoplasmic region can contain one or more costimulatory domains. A nucleic acid region encoding one or more costimulatory domains can be 5’ or 3’ of a region encoding a signaling domain. In embodiments, the region encoding one or more costimulatory endodomains is 5’ of the region encoding a signaling domain. In embodiments, a region encoding one or more costimulatory endodomains is 5 ’ of a signaling domain and an additional region encoding one or more costimulatory endodomains is 3' of the signaling domain. Exemplary costimulation endodomains include, without limitation, CD28; CD137 (4-1BB);CD278 (ICOS); CD27: CD134 (0X40); DaplO; Dapl2; DNAm-1; 2B4; a SLAM domain; and TLR2 costimulation endodomains, and combinations thereof.

[0094] In embodiments, the CAR comprises at least one 4- 1 BB costimulation endodomain, and optionally a second costimulation endodomain selected from a 4-1BB, 2B4, ICOS, CD28, and CD27 costimulation endodomain. In embodiments, the CAR comprises at least two 4-1BB costimulation endodomains, or two 4-1BB costimulation endodomains in combination with one, two. three, or four, or more, costimulation endodomains selected from a 4- IBB, ICOS, CD28, and CD27. In embodiments, the 4-1 BB costimulation endodomain comprises SEQ ID NO: 26 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL).

[0095] In embodiments, the CAR comprises CD27 costimulation endodomain, and optionally a second costimulation endodomain selected from a 4-1BB. ICOS, CD28, and CD27 costimulation endodomain. In embodiments, the CAR comprises a CD27 costimulation endodomain, and a 4-1BB costimulation endodomain. In embodiments, the CAR comprises two CD27 costimulation endodomains. In embodiments, the CD27 costimulation endodomain comprises SEQ ID NO: 27 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP).

[0096] In embodiments, the CAR comprises a secretion signal, e.g., SEQ ID NO: 28 (MALPVTALLLPLALLLHAARP) operably linked to facilitate secretion of a C-terminal polypeptide, such as a cytokine that supports the activation, cytotoxicity, and / or persistence of a T cell (e.g., CAR-T cell). In embodiments, the CAR comprises a secretion signal, e.g., SEQ ID NO: 28 operably linked to facilitate secretion of a common gamma chain cytokine such as IL- 15 or an active fragment thereof, e.g., SEQ ID NO: 29 (NWVNVISDLKKIED LIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNS LSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS). Exemplary common gamma chain cytokines include IL-2 and IL-15. In embodiments, the common gamma chain cytokine is selected from IL-2, IL-7, and IL-15. In embodiments, the common gamma chain cytokine is IL-15. IL-15 sequences, including codon optimized nucleic acid sequences encoding sIL15, are disclosed herein and in WO 2007 / 037780.

[0097] In embodiments, a nucleic acid encoding the CAR encodes one or more multi-cistronic linker regions, e.g.. between a signaling domain and / or costimulation endodomain and a secretion signal operably linked to facilitate secretion of a cytokine. A multi-cistronic linker region is a region of polypeptide sequence or RNA sequence that facilitates the production of multiple discrete polypeptides from a single transcription product. In embodiments, the multi-cistronic linker region encodes a cleavage sequence. Suitable cleavagesequences include self-cleavage sequences such as a P2A, F2A, E2A, or T2A cleavage sequence and / or sequences that are cleaved by an endogenous protease, such as furin.

[0098] In embodiments, the cleavage sequence is a P2A cleavage sequence. In embodiments, the cleavage sequence is a furin cleavage sequence. In embodiments, the cleavage sequences are a P2A and a furin cleavage sequence. In embodiments, the cleavage sequence is the P2A cleavage sequence of SEQ ID NO: 30 (SGSGATNFSLLKQAGDVEENPGP). In embodiments, the cleavage sequence is a furin cleavage sequence of SEQ ID NO: 31 (RAKR). In embodiments, the cleavage sequence is a P2A+furin cleavage sequence of SEQ ID NO: 32 (RAKRSGSGATNFSLLKQAG DVEENPGP).

[0099] In embodiments, the cleavage sequence is or comprises a P2A cleavage sequence of SEQ ID NO: 33 (ATNFSLLKQAGDVEENPGP). In embodiments, the cleavage sequence is or comprises an F2A cleavage sequence of SEQ ID NO: 34 (VKQTLNNFDLLKLAGDVESNPGP). In embodiments, the cleavage sequence is or comprises an E2A cleavage sequence of SEQ ID NO: 35 (QCTNYALLKLAGDVESNPGP). In embodiments, the cleavage sequence is or comprises an T2A cleavage sequence of SEQ ID NO: 36 (EGRSLLTCGDVEENPGP). In certain aspects, multiple self-cleavage sequences can be encoded carboxy terminal to a signaling and / or costimulatory domain and amino-terminal to an encoded secreted cytokine (e.g., common gamma chain cytokine such as IL-15), preferably wherein the multiple self-cleavage sequences are independently selected from the group consisting of a P2A cleavage sequence, a T2A cleavage sequence, an E2A cleavage sequence, and an F2A cleavage sequence. In certain aspects, one or more self-cleavage sequences and one or more sequences cleaved by an endogenous protease are encoded in a construct described herein. In certain embodiments, a endogenous protease recognition site is encoded amino terminal to a self-cleavage sequence.

[0100] In embodiments, the multi-cistronic linker region encodes an internal ribosome entry site. An exemplary' internal ribosome entry' site is encoded by SEQ ID NO: 37 (CTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATA TGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGG CCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATG CAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGAC AAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACA GGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCAC AACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTAT GGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTA AAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACA CGATGATA).

[0101] Another exemplary internal ribosome entry' site is encoded by SEQ ID NO: 38 (AGCAGGTTTCCCCAACTGACACAAAACGTGCAACTTGAAACTCCGCCTGGTCTT TCCAGGTCTAGAGGGGTAACACTTTGTACTGCGTTTGGCTCCACGCTCGATCCAC TGGCGAGTGTTAGTAACAGCACTGTTGCTTCGTAGCGGAGCATGACGGCCGTGG GAACTCCTCCTTGGTAACAAGGACCCACGGGGCCAAAAGCCACGCCCACACGGG CCCGTCATGTGTGCAACCCCAGCACGGCGACTTTACTGCGAAACCCACTTTAAAG TGACATTGAAACTGGTACCCACACACTGGTGACAGGCTAAGGATGCCCTTCAGG TACCCCGAGGTAACACGCGACACTCGGGATCTGAGAAGGGGACTGGGGCTTCTA TAAAAGCGCTCGGTTTAAAAAGCTTCTATGCCTGAATAGGTGACCGGAGGTCGG CACCTTTCCTTTGCAATTACTGACCAC).

[0102] Further suitable internal ribosome entry sites include, but are not limited to, those described in Nucleic Acids Res. 2010 Jan;38(Database issue): D131-6. doi: 10.1093 / nar / gkp981. Epub 2009 Nov 16, those described at iresite.org, those described in WO 2018 / 215787, the sequence described in GenBank accession No. KP019382.1, and the IRES element disclosed in GenBank accession No. LT727339.1.

[0103] Additional multi-cistronic linker regions, including cleavage, self-cleavage, and IRES elements, are disclosed in US 2018 / 0360992 and U. S. 8,865,467.

[0104] In embodiments, the isolated nucleic acid encodes SEQ ID NO: 39 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWY LQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWP ITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYD YAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSL RAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLS LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCQRRKYRSNK GESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), a 3H7 - CD8 - CD27z polypeptide comprising the following domains in order: a 3H7 binding domain, a CD8a hinge and transmembrane domain, a CD27 costimulation endodomain, and a CD3^ signaling domain.

[0105] In embodiments, the isolated nucleic acid encodes SEQ ID NO: 40 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWY QQKPGQAPRLLIYGTSTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNW PLTFGGGTKVEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCVASGFTFN DYAMHWVRQAPGKGLEWVSVISWNSDSIGYADSVKGRFTISRDNAKNSLYLQMHS LRAEDTALYYCAKDNHYGSGSYYYYQYGMDVWGQGTTVTVSSTTTPAPRPPTPAP TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), a 3B9-CD8-BBz polypeptide comprising the following domains in order: a 3B9 binding domain, a CD8a hinge and transmembrane domain, a 4-1BB costimulation endodomain, and a CD3 signaling domain.

[0106] In embodiments, the isolated nucleic acid encodes SEQ ID NO: 41 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWY LQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWP ITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYD YAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSL RAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLS LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), a 3H7-CD8-BBz polypeptide comprising the following domains in order: a 3H7 binding domain, a CD8a hinge and transmembrane domain, a 4- IBB costimulation endodomain, and a CD3 signaling domain.

[0107] In embodiments, the isolated nucleic acid encodes SEQ ID NO: 42 (MSVPTQVLGLLLLWLTDARCEIVLTQSPATLSLSPGERAALSCRASQSVSNYLAWY QQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNW PLTFGGGTKVEIRGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFR DYTMHWVRQGPGKGLEWVSGISWNSDYIGYADSVKGRFTISRDNAKNSLYLQMNS LRVEDTALYYCAKLSGTYRDYFYGVDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), a 2B7-CD8-BBz polypeptide comprising the following domains in order: a 2B7 binding domain, a CD8a hinge and transmembrane domain, a 4- IBB costimulation endodomain, and a CD3 signaling domain.

[0108] In embodiments, the isolated nucleic acid encodes SEQ ID NO: 43 (MSVPTQVLGLLLLWLTDARCEIVVTQSPATLSLSPGERATLSCRTSQTTTSYLAWYR QKPGQAPRLLIYDASNRAAGIPARFSGSGSGTDFTLTINSLEPEDFAVYYCQLRTNWI TFGQGTRLEIKGGGGSGGGGSGGGGQVQLVESGGDSVKPGGSLRLSCAASGFTFSDS YMTWIRQAPGKGLEWVSFISSSGSTIYYADSVKGRFTISRDNVKKSLYLQMNRLRAE DTAVYYCAREEPGNYVYYGMDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRP EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFK QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNL GRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR), a 9Cll-CD8-BBz polypeptide comprising the following domains in order: a 9C11 binding domain, a CD8a hinge and transmembrane domain, a 4-1BB costimulation endodomain, and a CD3c signaling domain.

[0109] In embodiments, the isolated nucleic acid encodes SEQ ID NO: 44 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWY LQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWP ITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYD YAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSL RAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLS LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), a 3H7-CD3z polypeptide comprising the following domains in order: a 3H7 binding domain, a CD8a hinge and transmembrane domain, and a CD3 signaling domain.

[0110] In embodiments, the isolated nucleic acid encoding a 3H7-CD8-27z polypeptide comprises the sequence of SEQ ID NO: 45 (ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCC AGATGCGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGG AAAGAACCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCT GGTACCTTCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAC CAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTT CATTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCGATCACCTTCGGCCAAGGGACACGGCTGGAGATTAAA GGTGGAGGTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGAAGTGCAACT GGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGT GCAGCCTCTGGATTCACCTTTTATGATTATGCCATGCACTGGGTCCGGCAAGCTC CAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTTACATAG GCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA ACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTA CTGTGCAAAAGATAACAGCTATGGAAAGTTCTACTACGGTTTGGACGTCTGGGG CCAAGGGACCACGGTCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCACC AACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTG CCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGA TATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTG GTTATCACCCTTTACTGCCAACGACGCAAGTACCGCTCCAATAAAGGAGAGTCAC CAGTAGAACCCGCCGAACCTTGTCACTATTCATGTCCACGCGAAGAGGAGGGTT CAACGATCCCTATTCAGGAAGATTACAGAAAGCCGGAACCTGCTTGTAGCCCCA GAGTGAAGTTCAGCCGCAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAACC AGCTGTATAACGAGCTGAACCTGGGCAGGCGGGAGGAATACGACGTGCTGGACA AGCGCAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCCAGAGGCGGAAGAAC CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTAC AGCGAGATCGGCATGAAGGGCGAGCGGCGACGCGGCAAGGGCCACGACGGCCT GTACCAGGGCCTGTCCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCA GGCCCTGCCTCCCCGTTAG).

[0111] In embodiments, the isolated nucleic acid encoding a 3H7-CD8-BBz polypeptide comprises the sequence of SEQ ID NO: 46 (ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCC AGATGCGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGG AAAGAACCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCT GGTACCTTCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAC CAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTT CATTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAG CAGTATAATAACTGGCCGATCACCTTCGGCCAAGGGACACGGCTGGAGATTAAA GGTGGAGGTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGAAGTGCAACT GGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGT GCAGCCTCTGGATTCACCTTTTATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTTACATAG GCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA ACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTA CTGTGCAAAAGATAACAGCTATGGAAAGTTCTACTACGGTTTGGACGTCTGGGG CCAAGGGACCACGGTCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCACC AACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTG CCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGA TATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTG GTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC AACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCC GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGG AGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGAC CCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTA CAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGA AAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTA CAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA).

[0112] In embodiments, the isolated nucleic acid encoding a 3B9-CD8-BBz polypeptide comprises the sequence of SEQ ID NO: 47 (ATGAGCGTTCCAACCCAAGTTCTGGGACTGCTTCTGCTCTGGTTGACTGACGCTA GGTGCGAAATAGTAATGACCCAATCCCCAGCCACTCTCTCCGTTAGCCCAGGTGA AAGAGCCACTCTTAGTTGCAGGGCTAGTCAATCCGTATCTAGCAACCTGGCCTGG TACCAGCAAAAGCCCGGACAAGCGCCGCGGTTGTTGATCTATGGGACGAGCACA CGAGCTACGGGTATTCCGGCCAGGTTCTCAGGGTCTGGCTCCGGAACCGAATTTA CATTGACGATCAGTAGTCTGCAATCAGAGGATTTCGCCGTTTACTATTGCCAACA GTACAATAATTGGCCGCTCACATTCGGGGGAGGAACCAAGGTCGAGATTAAGGG AGGTGGGGGTAGTGGGGGCGGGGGGTCAGGAGGTGGAGGAGAGGTACAGTTGG TAGAAAGCGGCGGGGGGTTGGTTCAACCTGGACGGAGTCTGAGATTGTCTTGCG TGGCTTCCGGCTTTACTTTCAATGATTACGCCATGCACTGGGTACGCCAGGCGCC TGGAAAGGGTCTGGAGTGGGTTTCCGTGATATCCTGGAATAGTGATAGTATAGGC TATGCCGATAGTGTAAAAGGAAGGTTTACAATCTCTAGGGATAACGCTAAGAAC AGCCTGTACCTTCAAATGCATAGTCTCCGGGCTGAGGACACAGCCTTGTACTATT GTGCTAAGGACAATCATTATGGAAGCGGGTCATATTATTACTATCAATATGGGATGGATGTGTGGGGTCAGGGAACGACCGTTACGGTATCCTCAACCACCACCCCTGC ACCAAGGCCCCCGACTCCCGCGCCCACCATCGCGTCACAGCCTCTTAGCCTGCGA CCGGAAGCATGCAGACCAGCTGCCGGGGGGGCCGTGCATACGAGAGGTTTGGAC TTCGCCTGCGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTC TCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTA TATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGG CTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAA GTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTA TAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACG TGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAG ATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCA GGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCT GCCCCCTCGCTAA).

[0113] In embodiments, the isolated nucleic acid encoding a 2B7-CD8-BBz polypeptide comprises the sequence of SEQ ID NO: 48 (ATGTCCGTACCTACCCAGGTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCC AGATGCGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGG AAAGAGCCGCCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAACTACTTAGCCTG GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAAC AGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTC ACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGC AGCGTAGCAACTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAGAG GTGGAGGTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGAAGTGCAGCTG GTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGCGACTCTCCTGTG CAGCCTCTGGATTCACCTTTCGAGATTATACCATGCACTGGGTCCGGCAAGGTCC AGGGAAGGGCCTGGAATGGGTCTCAGGTATTAGTTGGAATAGTGATTACATAGG CTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA CTCCCTGTATCTGCAAATGAACAGTCTGAGAGTTGAGGACACGGCCTTGTATTAC TGTGCAAAGCTCAGTGGGACCTACAGGGACTACTTCTACGGAGTGGACGTCTGG GGCCAAGGGACCACGGTCACCGTCTCCTCAACCACCACCCCTGCACCAAGGCCC CCGACTCCCGCGCCCACCATCGCGTCACAGCCTCTTAGCCTGCGACCGGAAGCAT GCAGACCAGCTGCCGGGGGGGCCGTGCATACGAGAGGTTTGGACTTCGCCTGCG ATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAA CAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGC CGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCT CAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGA CCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGT ACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATG AAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGT ACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCT AA).

[0114] In embodiments, the isolated nucleic acid encoding a 9C1 l-CD8-BBz polypeptide comprises the sequence of SEQ ID NO: 49 (ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCC AGATGCGAAATTGTGGTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGG AAAGAGCCACCCTCTCCTGCAGGACCAGTCAGACTACTACCAGCTACTTAGCCTG GTACCGACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAAC AGGGCCGCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTC ACTCTCACCATCAACAGCCTGGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGC TGCGTACCAACTGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAAGGTG GAGGTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTCAGGTGCAGCTGGTG GAGTCTGGGGGAGACTCGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCA GCCTCTGGATTCACCTTCAGTGACTCCTACATGACTTGGATCCGCCAGGCTCCAG GGAAGGGGCTGGAGTGGGTTTCATTCATTAGTAGTAGTGGAAGTACCATATATTA TGCAGACTCTGTGAAGGGCCGATTCACCATTTCCAGGGACAACGTCAAGAAGTC ATTGTATCTGCAGATGAACAGACTGAGAGCCGAGGACACGGCCGTGTATTACTG TGCGAGAGAAGAACCAGGAAACTACGTCTATTACGGTATGGACGTCTGGGGCCA AGGGACCACGGTCACCGTCTCCTCAACCACCACCCCTGCACCAAGGCCCCCGACT CCCGCGCCCACCATCGCGTCACAGCCTCTTAGCCTGCGACCGGAAGCATGCAGA CCAGCTGCCGGGGGGGCCGTGCATACGAGAGGTTTGGACTTCGCCTGCGATATCT ACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTAT CACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACC ATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATT TCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCG CAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTG AGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAAT GAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGG CGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGC CACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA).

[0115] In embodiments, the isolated nucleic acid comprises a codon optimized sequence encoding a CD8a hinge region. Exemplary codon optimized CD8a hinge region nucleic acid sequences include, without limitation, SEQ ID NO: 50 (ACCACCACCCCTGCACCAAGGCCCCCGACTCCCGCGCCCACCATCGCGTCA CAGCCTCTTAGCCTGCGACCGGAAGCATGCAGACCAGCTGCCGGGGGGGCCGTG CATACGAGAGGTTTGGACTTCGCCTGCGAT). In embodiments, the CD8a hinge region is encoded by the following sequence SEQ ID NO: 51 (ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCA GCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCA CACGAGGGGGCTGGACTTCGCCTGTGAT).

[0116] In embodiments, the isolated nucleic acid encodes a 3B9 binding domain and comprises the following sequence encoding a CD8a hinge domain SEQ ID NO: 50. In embodiments, the isolated nucleic acid encodes a 2B7 binding domain and comprises the following sequence encoding a CD8a hinge domain SEQ ID NO: 50. In embodiments, the isolated nucleic acid encodes a 9C11 binding domain and comprises the following sequence encoding a CD8a hinge domain SEQ ID NO: 50. In embodiments, the isolated nucleic acid encodes a 3H7 binding domain and comprises the following sequence encoding a CD8a hinge domain SEQ ID NO: 51.

[0117] In embodiments, the isolated nucleic acid encodes SEQ ID NO: 52 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWY LQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWP ITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYD YAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSL RAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLS LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPG PMALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKN IKEFLQSFVHIVQMFINTS*), an anti-CD20-CAR polypeptide comprising the following domains in order: a 3H7 binding domain, a CD8a hinge and transmembrane domain, a 4- IBB costimulation endodomain, a CD3^ signaling domain, a P2A cleavage domain (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 53), a secretion signal, and a sIL-15 domain.

[0118] In embodiments, the isolated nucleic acid encodes SEQ ID NO: 54 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWY LQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWP ITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYD YAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSL RAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLS LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPG PMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLK KIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIIL ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*), an anti-CD20-CAR polypeptide comprising the following domains in order: a 3H7 binding domain, a CD8a hinge and transmembrane domain, a 4-1BB costimulation endodomain, a CD3 signaling domain, a P2A cleavage domain of SEQ ID NO: 53, a secretion signal of SEQ ID NO: 55 (MRISKPHLRSISIQCYLCLLLNSHFLTEAG IHVFILGCFSAGLPKTEA), and a sIL-15 domain.

[0119] In embodiments, the isolated nucleic acid encoding an anti-CD20 CAR + sIL15 polypeptide comprises the sequence of SEQ ID NO: 56 (ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCC AGATGCGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGG AAAGAACCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCT GGTACCTTCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAC CAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTT CATTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAG CAGTATAATAACTGGCCGATCACCTTCGGCCAAGGGACACGGCTGGAGATTAAA GGTGGAGGTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGT GCAGCCTCTGGATTCACCTTTTATGATTATGCCATGCACTGGGTCCGGCAAGCTC CAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTTACATAG GCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA ACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTA CTGTGCAAAAGATAACAGCTATGGAAAGTTCTACTACGGTTTGGACGTCTGGGG CCAAGGGACCACGGTCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCACC AACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTG CCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGA TATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTG GTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC AACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCC GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGG AGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGAC CCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTA CAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGA AAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTA CAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCG GTAGCGGGGCTACGAACTTCTCCCTTCTTAAACAAGCGGGAGACGTGGAAGAAA ATCCCGGACCTATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCT GCTCCACGCCGCCAGGCCGAACTGGGTGAATGTAATAAGTGATTTGAAAAAAAT TGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGAT GTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAG TTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGAT CATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGC AAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTT GTACATATTGTCCAAATGTTCATCAACACTTCTTGA).

[0120] In embodiments, the isolated nucleic acid encoding an anti-CD20 CAR + sIL15 polypeptide comprises the sequence of SEQ ID NO: 57 (ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCC AGATGCGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGG AAAGAACCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCT GGTACCTTCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTT CATTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAG CAGTATAATAACTGGCCGATCACCTTCGGCCAAGGGACACGGCTGGAGATTAAA GGTGGAGGTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGAAGTGCAACT GGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGT GCAGCCTCTGGATTCACCTTTTATGATTATGCCATGCACTGGGTCCGGCAAGCTC CAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTTACATAG GCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA ACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTA CTGTGCAAAAGATAACAGCTATGGAAAGTTCTACTACGGTTTGGACGTCTGGGG CCAAGGGACCACGGTCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCACC AACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTG CCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGA TATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTG GTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC AACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCC GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGG AGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGAC CCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTA CAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGA AAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTA CAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCG GTAGCGGGGCTACGAACTTCTCCCTTCTTAAACAAGCGGGAGACGTGGAAGAAA ATCCCGGACCTATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTG CTACTTGTGTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCT TCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAACTGGGTGAA TGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGAT GCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGA AGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATT CATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATG GGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAAT ATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTC TTGA).

[0121] In embodiments, the isolated nucleic acid encodes SEQ ID NO: 58 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWY LQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWP ITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYD YAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSL RAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLS LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*), an anti-CD20-CAR polypeptide comprising the following domains in order: a 3H7 binding domain, a CD8a hinge and transmembrane domain, a 4-1 BB costimulation endodomain, and a CD3 signaling domain; and, via an internal ribosome entry site (e.g., encoded by SEQ ID NO: 37) 3’of the region encoding SEQ ID NO: 58, the isolated nucleic acid further encodes SEQ ID NO: 59 (MALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHP SCKVTAMKCFLLELQVISLESGDASIHDTVENL1ILANNSLSSNGNVTESGCKECEELE EKNIKEFLQSFVHIVQMFINTS*), a secretion signal of SEQ ID NO: 28 and a sIL15 domain.

[0122] In embodiments, the isolated nucleic acid encoding an anti-CD20 CAR + sIL15 polypeptide comprises the sequence of SEQ ID NO: 60 (ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCC AGATGCGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGG AAAGAACCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCT GGTACCTTCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAC CAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTT CATTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAG CAGTATAATAACTGGCCGATCACCTTCGGCCAAGGGACACGGCTGGAGATTAAA GGTGGAGGTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGAAGTGCAACT GGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGT GCAGCCTCTGGATTCACCTTTTATGATTATGCCATGCACTGGGTCCGGCAAGCTC CAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTTACATAG GCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA ACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTA CTGTGCAAAAGATAACAGCTATGGAAAGTTCTACTACGGTTTGGACGTCTGGGG CCAAGGGACCACGGTCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTG CCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGA TATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTG GTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC AACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCC GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGG AGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGAC CCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTA CAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGA AAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTA CAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTA GAGTACTGCGGCCGCTACGTAAATTCCGCCCCTCTCCCTCCCCCCCCCCTAACGT TACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTT TCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCT TCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCT GTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAAC GTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTC TGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAG TGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCG TATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTG ATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACG TCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAT TAATTAAGCCACCGCCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCC TTGCTGCTCCACGCCGCCAGGCCGAACTGGGTGAATGTAATAAGTGATTTGAAA AAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAA GTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTT ACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAAT CTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTG GATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGA GTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTGA).

[0123] In embodiments, the isolated nucleic acid is a linear nucleic acid. In embodiments, the isolated nucleic acid is a circular nucleic acid. In embodiments, the isolated nucleic acid is a vector, such as a plasmid vector, an adenoviral vector, an adeno-associated viral vector, aviral vector, a retroviral vector, or a lentiviral vector. In embodiments, the isolated nucleic acid, or an, e.g, contiguous, portion thereof containing the binding domain transmembrane domain and one or more signaling and / or costimulation endodomains is integrated into the genome of a host cell, such as a host y5 T cell. In an exemplary embodiment, the isolated nucleic acid is retroviral vector.yd T Cells

[0124] In embodiments, the y6 T cells having in vitro or in vivo cytotoxic activity against an autoreactive immune cell (e.g., an autoreactive B cell) that exhibits cell surface expression of CD20. In some cases, the cytotoxic activity is innate activity. In some cases, the cytotoxicity is at least in part, significantly (> about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds CD20 expressed on the surface of the autoreactive immune cell. In some cases, the y8 T cells exhibit autoreactive immune cell killing activity greater than an innate level of in vitro and / or in vivo autoreactive immune cell killing activity in a control y5 T cell. In some cases, the control y5 T cell does not comprise a CAR construct. In some cases, the control y5 T cell comprises a CAR construct lacking a binding domain described herein, a hinge region described herein, a transmembrane domain described herein, a signaling domain described herein, and / or a costimulation endodomain described herein.

[0125] In some cases, the cytotoxicity is at least in part, significantly (> about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds CD20 or an epitope within CD20. In some cases, the y5 T cells functionally express a CD20 specific CAR encoded by an isolated nucleic acid.

[0126] In embodiments, the y5 T cells exhibit HLA-restricted (e.g., HLA class I restricted) cytotoxicity. In other embodiments, most (>50%), substantially all (>90%), or all of the cytotoxic activity is not HLA-restricted (e.g.. HLA class I restricted). HLA-restricted cytotoxic activity’ can be assessed by comparing in vitro cytotoxicity against an HLA (e.g, HLA class I) (null) cell line versus in vitro cytotoxicity against an HLA+ (e.g., HLA class I+) cell line. In embodiments, the HLA-restricted cytotoxic activity is at least in part, significantly (>25%), or entirely, provided by the use of a T cell Receptor-like binding domain. T cell receptor like binding domains are binding domains that specifically recognize the antigen when presented on the surface of a cell in complex with an MHC molecule. T cell Receptor-like binding domains are further described, e.g., in WO 2016 / 199141.

[0127] In embodiments, the y8 T cells exhibit robust and / or persistent killing activity against autoreactive immune cells. In some cases, the autoreactive immune cell killing activitycan persist for at least about 6 days to 120 days, or for at least about 6 days to 180 days, from first contact with an autoreactive immune cell. In some cases, the autoreactive immune cell killing activity of a yb T cell, or a progeny thereof, can persist for at least about 6 days to 120 days, or for at least about 6 days to 180 days, from first contact with an autoreactive immune cell, or from administration of the yb T cell. This persistent autoreactive immune cell killing activity can be exhibited in vitro, in vivo, or both in vitro and in vivo.

[0128] Aspects of the invention can additionally or alternatively include yb T cells that proliferate in response to contact with cells that exhibit cell surface expression, or overexpression, of CD20 on an autoreactive immune cell. In some cases, the proliferation is at least in part, significantly (> about 20% or > about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds CD20 expressed on the surface of the autoreactive immune cell. In some cases, the yb T cells exhibit a greater level of in vitro and / or in vivo proliferation as compared to a control yb T cell. In some cases, the control yb T cell does not comprise a CAR construct. In some cases, the control yb T cell comprises a CAR construct lacking a binding domain described herein, a hinge region described herein, a transmembrane domain described herein, a signaling domain described herein, and / or a costimulation endodomain described herein.

[0129] In some cases, the proliferation is at least in part, significantly (> about 20 or > about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds CD20 or an epitope within CD20. In some cases. y5 T cells exhibiting proliferation in response to contact with an autoreactive immune cell (e.g., an autoreactive B cell) that exhibits cell surface expression of CD20 specific CAR.

[0130] In embodiments, the yo T cells exhibit robust and / or persistent proliferation in a host organism that comprises the autoreactive immune cell (e.g.. an autoreactive B cell) that exhibits cell surface expression, or overexpression, of CD20. In some cases, the proliferation can persist for at least about 6 days to 120 days, or for at least about 6 days to 180 days, from first contact with an autoreactive immune cell or from a date of administration of the yo T cell to the host organism. In some cases, the proliferation of a yb T cell described herein, or a progeny thereof, in the host organism that comprises the autoreactive immune cell that exhibits cell surface expression, or overexpression, of CD20, can persist for at least about 6 days to 120 days, or for at least about 6 days to 180 days, from first contact with an autoreactive immune cell or from the date of first administration of the yb T cell to the host organism. In some cases, the proliferation in the host organism is at least in part, significantly (> about 20% or > about 25%), or entirely, due to the presence of a CAR construct having a binding domain thatspecifically binds CD20 or an epitope within CD20. In some cases. y5 T cells exhibiting proliferation in the host organism comprising an autoreactive immune cell that exhibits cell surface expression of CD20 expresses a CD20 specific CAR.

[0131] In embodiments, the y8 T cell, or a pharmaceutical composition containing the y5 T cell, exhibits essentially no, or no graft versus host response when introduced into an allogeneic host. In embodiments, the y5 T cell, or a pharmaceutical composition containing the y5 T cell, exhibits a clinically acceptable level of graft versus host response when introduced into an allogeneic host. In embodiments, a clinically acceptable level is an amount of graft versus host response that does not require cessation of a y5 T cell treatment to achieve a therapeutically effective treatment. In embodiments, a clinically acceptable level of graft versus host response (GvHD) is an acute response that is less severe than Grade C according to an applicable IBMTR grading scale. The severity of acute graft versus host response is determined by an assessment of the degree of involvement of the skin, liver, and gastrointestinal tract. The stages of individual organ involvement are combined to produce an overall grade, which has prognostic significance. Grade 1(A) GvHD is characterized as mild disease, grade 11(B) GvHD as moderate, grade 111(C) as severe, and grade IV(D) lifethreatening. The IBMTR grading system defines the severity of acute GvHD as follows (Rowlings et al., Br J Haematol 1997; 97:855):•Grade A - Stage 1 skin involvement alone (maculopapular rash over <25 percent of the body) with no liver or gastrointestinal involvement•Grade B - Stage 2 skin involvement; Stage 1 to 2 gut or liver involvement •Grade C - Stage 3 involvement of any organ system (generalized erythroderma; bilirubin 6.1 to 15.0 mg / dL; diarrhea 1500 to 2000 mL / day)•Grade D - Stage 4 involvement of any organ system (generalized erythroderma with bullous formation; bilirubin >15 mg / dL; diarrhea >2000 mL / day OR pain OR ileus). See also, Schoemans et al., Bone Marrow Transplantation volume 53, pages1401–1415 (2018), e.g.. at Tables 1 and 2, which discloses criteria for assessing and grading acute GvHD.

[0132] In embodiments, the y6 T cell, or a pharmaceutical composition containing the y5 T cell, exhibits reduced or substantially reduced graft versus host response when introduced into an allogeneic host as compared to a graft versus host response exhibited by control «P T cells, or a control pharmaceutical composition comprising the control aP T cells, administered to an allogeneic host. In some cases, the control otp T cell is an allogeneic non-engineeredcontrol αβ T cell. In some cases, the control αβ T cell does not comprise a CAR or does not comprise the same CAR as a reference γδ T cell.

[0133] In embodiments, the γδ T cells are δ1, δ2, δ3, or δ4 γδ T cells, or combinations thereof. In embodiments, the γδ T cells are δ1 γδ T cells. In embodiments, the γδ T cells are δ2 γδ T cells. In embodiments, the γδ T cells are δ3 γδ T cells. In embodiments, the γδ T cells are δ4 γδ T cells. In some cases, the y5 T cells are mostly (>50%), substantially (>90%). essentially all. or entirely 52‘ y5 T cells. In some cases, the y5 T cells are mostly (>50%). substantially (>90%), essentially all, or entirely δ1 γδ T cells. In some cases, the y5 T cells are mostly (>50%), substantially (>90%), essentially all, or entirely δ2 γδ T cells. In some cases, the y5 T cells are mostly (>50%), substantially (>90%), essentially all, or entirely δ3 γδ T cells. In some cases, the γδ T cells are mostly (>50%), substantially (>90%), essentially all, or entirely δ4 γδ T cells.

[0134] In embodiments, the y5 T cells are obtained from an allogeneic or an autologous donor. The γδ T cells can be, partially or entirely purified, or not purified, and expanded ex vivo. Methods and compositions for ex vivo expansion include, without limitation, those described in WO 2017 / 197347. The expansion may be performed before or after, or before and after, a CAR construct is introduced into the γδ T cell(s).

[0135] γδ T cells described herein can be stored, e.g., cryopreserved, for use in adoptive cell transfer.Modifications of γδ T cells

[0136] In embodiments, γδ T cells described herein can be modified to comprise one or more gene edits. Discussed herein, gene editing is a type of genetic engineering in which nucleotide(s) / nucleic acid(s) is / are inserted, deleted, and / or substituted in a DNA sequence, such as the genome of a γδ T cell. Targeted gene editing enables insertion, deletion, and / or substitution at pre-selected sites in the genome of a targeted cell. When a sequence of an endogenous gene is edited, for example by deletion, insertion, or substitution of nucleotide(s) / nucleic acid(s), the endogenous gene comprising the affected sequence may be knocked-out or knocked-down due to the sequence alteration. Therefore, targeted editing may be used to disrupt endogenous gene expression. Discussed herein, a "disrupted gene’7refers to a gene comprising an insertion, deletion or substitution relative to an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. As used herein, “disrupting a gene” refers to a method of inserting, deleting, or substituting at least one nucleotide / nucleic acid in an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. Methods of disrupting a gene are known tothose of skill in the art, and described, e.g., in United States Patent No. 11254912, incorporated herein by reference in its entirely.

[0137] In embodiments, a nuclease-dependent approach can be used to conduct targeted gene editing of a y5 T cell. Such a nuclease-dependent approach can achieve targeted editing through the specific introduction of double strand breaks (DSBs) by specific endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms, for example, non-homologous end joining (NHEJ), which occurs in response to DSBs. DNA repair by NHEJ often leads to random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast to NHEJ mediated repair, repair can also occur by a homology directed repair (HDR). When a donor template containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome by HDR, which results in targeted integration of the exogenous genetic material. Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and RNA-guided CRISPR-Cas9 nuclease (CRISPR / Cas9; Clustered Regular Interspaced Short Palindromic Repeats Associated 9). Discussed herein, a CRISPR system, or CRISPR nuclease system can include a non-coding RNA molecule (e.g., guide RNA) that binds DNA and Cas proteins (e.g., Cas9) with nuclease functionality (Sander et al., Nature Biotechnology (2014): 32:347-355; Hsu et al., Cell (2014): 157(6):1262-1278).

[0138] In embodiments, a γδ T cell herein may comprise one or more disrupted genes. For example, one or more genes whose expression is disrupted can comprise adenosine A2a receptor (ADORA), CD276. V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte-activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD244). cytokine inducible SH2-containing protein (CISH), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS SITE (E. G. AAVS1, AAVS2, ETC.)), or chemokine (C C motif) receptor 5 (gene / pseudogene) (CCR5), CD160 molecule (CD 160), T-cell immunoreceptor with Ig and 1TIM domains (TIGIT), CD96 molecule (CD96), cytotoxic and regulatory T-cell molecule (CRT AM), leukocyte associated immunoglobulin like receptor 1 (LAIR1), sialic acid binding Ig like lectin 7 (SIGLEC7), sialic acid binding Ig like lectin 9 (SIGLEC9), tumor necrosis factor receptorsuperfamily member 10b (TNFRSF10B), tumor necrosis factor receptor superfamily member 0a(TNFRSF10A), caspase 8 (CASP8). caspase 10 (CASP10). caspase 3 (CASP3), caspase 6 (CASP6). caspase 7 (CASP7). Fas associated via death domain (FADD), Fas cell surface death receptor (FAS), transforming growth factor beta receptor II (TGFBRII), transforming growth factor beta receptor I (TGFBRI), SMAD family member 2 (SMAD2), SMAD family member 3 (SMAD3), SMAD family member 4 (SMAD4), SKI proto-oncogene (SKI), SKI-like protooncogene (SKIL), TGFB induced factor homeobox 1 (TGIF I), interleukin 10 receptor subunit alpha (IL10RA), interleukin 10 receptor subunit beta (IL10RB), heme oxygenase 2 (HMOX2), interleukin 6 receptor (IL6R), interleukin 6 signal transducer (IL6ST), c-src tyrosine kinase (CSK), phosphoprotein membrane anchor with glycosphingolipid microdomains 1(PAG1), signaling threshold regulating transmembrane adaptor 1 (SIT1), forkhead box P3 (FOXP3), PR domain 1 (PRDMI). basic leucine zipper transcription factor, ATF-like (BATF). guanylate cyclase 1, soluble, alpha 2 (GUCY1A2), guanylate cyclase 1, soluble, alpha 3 (GUCY1A3), guanylate cyclase 1, soluble, beta 2 (GUCY1B2), guanylate cyclase 1, soluble, beta 3 (GUCY1B3), cytokine inducible SH2-containing protein (CISH), prolyl hydroxylase domain (PHD1. PHD2. PHD3) family of proteins, Cbl proto-oncogene B (CBL-B), Zinc Finger Protein 91 (ZFP91), Roquin. CD58. ICAM-1, Regnase-1, RASA2, MED 12, Fas, Arid1a, or any combination thereof.

[0139] In a preferred embodiment, a gene whose expression is disrupted is CISH, a negative regulator of TCR signaling. Disruption of the CISH gene may provide a functional advantage over control cells that have an intact CISH gene in improving the sensitivity to certain cytokines (e.g., IL-2 / IL-15), increasing T cell proliferation, and / or limiting T cell exhaustion. In some examples, the CISH gene may be disrupted by methods described in Daher M. et al, Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells. Blood. 2021 Feb 4;137(5):624-636, which is incorporated by reference herein in its entirety. In one example, the CISH gene is disrupted by gene editing using a CRISPR-Cas system comprising one or more guide RNAs comprising the sequences of any one of SEQ ID NOs: 78-81. In another example, the CISH gene is disrupted by gene editing using a CRISPR- Cas system comprising one or more guide RNAs comprising the sequences of any one of SEQ ID NOs: 85-86.

[0140] In embodiments, a gene whose expression is disrupted is CBL-B, a negative regulator of T cell activation. Disruption of the CBL-B gene may provide a functional advantage over control cells that have an intact CBL-B gene in enhancing T cell activation. In some examples, the CBL-B gene may be disrupted by methods described in Augustin R. et al..Targeting Cbl-b in cancer immunotherapy, J Immunother Cancer. 2023 Feb;ll(2):e006007; Hooper K. et al., Knockout of CBLB Greatly Enhances Anti-Tumor Activity of CAR T Cells, Blood (2018) 132 (Supplement 1): 338; and Guo X. et al., CBLB ablation with CRISPR / Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy, J Immunother Cancer. 2021 Mar;9(3):e001975, each of which is incorporated by reference herein in its entirety. In one example, the CBL-B gene is disrupted by gene editing using a CRISPR-Cas system comprising one or more guide RNAs comprising the sequence of any one of SEQ ID NOs: 82-84.

[0141] In embodiments, a gene whose expression is disrupted is Roquin (e g., Roquin-1). Disruption of the Roquin gene may provide a functional advantage over control cells that have an intact Roquin gene in increasing T cell proliferation and enhancing antitumor activity. In some examples, the Roquin gene may be disrupted by methods described in Mai D et al., Combined disruption of T cell inflammatory regulators Regnase-1 and Roquin-1 enhances antitumor activity of engineered human T cells, Proc Natl Acad Sci U S A. 2023 Mar 21; 120(12): e2218632120, which is incorporated by reference herein in its entirety.

[0142] In embodiments, a gene whose expression is disrupted is ZFP91. Disruption of the ZFP91 gene may provide a functional advantage over control cells that have an intact ZFP91 gene in improving T cell glycolytic fitness and effector function. In some examples, the ZFP91 gene may be disrupted by method described in Wang F. et al.. J Clin Invest. 2021 Oct 1;131(19):e144318, which is incorporated by reference herein in its entirety. In embodiments, a gene whose expression is disrupted is CD58. In embodiments, a gene whose expression is disrupted is ICAM-1. In embodiments, both CD58 and ICAM-1 are disrupted. Disruption of the CD58 and / or ICAM-1 genes may provide a functional advantage over control cells that have an intact CD58 and / or ICAM-1 gene in disrupting T cell adhesion and co-stimulatory interactions to reduce Host vs Graft allocytotoxicity. In one example, the ICAM-1 gene may be disrupted as described in Teo HY et al. IL12 / 18 / 21 Preactivation Enhances the Antitumor Efficacy of Expanded yoT Cells and Overcomes Resistance to Anti-PD-Ll Treatment, Cancer Immunol Res. 2023 Jul 5;ll(7):978-999, which is incorporated by reference in its entirety.

[0143] In embodiments, a gene whose expression is disrupted is Regnase-1 gene. Regnase- 1 (Regulatory RNase 1), also known as ZC3H12A or MCPIP-1, is a ribonuclease that promotes decay of target rnRNA through recognition of 3’ UTR stem loop motifs. Disruption of the Regnase-1 gene may provide a functional advantage over control cells that have an intact Regnase-1 gene in enhancing T ceil expansion and persistence. In one example, the Regnase- 1 gene may be disrupted as described m Jun Wei et al.. Targeting Regnase-1 programs long-lived effector T cells for cancer therapy. Nature. 2019 Dec; 576(7787): 471 -476. which is incorporated by reference in its entirety.

[0144] In embodiments, a gene whose expression is disrupted is RAS p21 protein activator 2 (RASA2) gene. RASA2 is a RAS GTPase-activating protein (RasGAP) that is a signaling checkpoint in T cells, which can be downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. Disruption of RASA2 may enhance MAPK signaling and CAR T cell cytolytic activity in response to target antigen. RASA2-deficient T cells may exhibit increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent disease-associated cell killing. In some examples, the RASA2 gene may be disrupted by the method described in Julia Carnevale et al., RASA2 ablation in T cells boosts antigen sensitivity and long-term function. Nature. 2022 Sep;609(7925): 174-182, which is incorporated by reference herein in its entirety.

[0145] In embodiments, a gene whose expression is disrupted is MED12 gene. Mediator complex subunit 12 (MED12) is a Mediator subunit, and can regulate metabolic activity and fitness, e g., increased glycolysis, oxidative phosphorylation, and spare respiratory capacity. Disruption of the MED 12 gene may provide a functional advantage over control cells that have an intact MED12 gene in enhancing T cell activation and effector function. In one example, the MED12 gene may be disrupted as described in Katherine A Freitas et al., Enhanced T cell effector activity by targeting the Mediator kinase module, Science. 2022 Nov 11;378(6620):eabn5647. which is incorporated by reference in its entirety.

[0146] In embodiments, a gene whose expression is disrupted is Fas gene. Disruption of the Fas gene may provide a functional advantage over control cells that have an intact Fas gene in reducing T cell allocytotoxicity. In one example, the ICAM-1 gene may be disrupted as described in Jiangtao Ren et al., A versatile system for rapid multiplex genome-edited CAR T cell generation, Oncotarget. 2017 Mar 7;8(10):17002–17011, which is incorporated by reference herein in its entirety.

[0147] In embodiments, a gene whose expression is disrupted is Aridla gene. Aridla regulates the acquisition of the epigenetic state of terminal exhaustion. Disruption of the Aridla gene may provide a functional advantage over control cells that have an intact Aridla gene in improving tumor control and enhancing persistence ofT cells. In one example, the Aridla gene may be disrupted as described in Julia A Belk et al., Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence, Cancer Cell.2022 Jul 11;40(7):768-786.e7.

[0148] In embodiments, the δγ T cells can be modified to include a nucleic acid construct that encodes a protein which imparts a desired functionality to the T cells of the present disclosure. For example, such a nucleic acid may encode for a chimeric DAP 10 adaptor polypeptide, described in International Application No. PCT / US2022 / 048097 and International Application No. PCT / US2023 / 024073, the contents of each of which is hereby expressly incorporated herein by reference in their entirety. In embodiments, a chimeric DAP 10 adaptor polypeptide is capable of associating with a chimeric antigen receptor of the present disclosure, for example a CAR of the present disclosure may comprise a DAP10-interacting domain. In additional or alternative examples, a chimeric DAP10 adaptor polypeptide may not associate with a CAR of the present disclosure, but instead may interact with an endogenous or exogenous polypeptide that includes a DAP 10-interacting domain.Methods of treating autoimmune diseases

[0149] In some aspects, the present disclosure provides methods of treating autoimmune diseases. Also provided are methods for reducing and / or eliminating a need for chronic immunosuppression in a human subject with an autoimmune disease, as well as methods for effectuating immune reset and / or for producing a naive B cell repertoire in a subject in need thereof. In general, the methods comprise administering a therapeutically effective amount of the y5 T cells herein to a subject with an autoimmune disease.

[0150] In embodiments, the autoimmune disease is lupus (e.g., systemic lupus erythematosus (SLE) such as systemic lupus erythematosus with extrarenal involvement), lupus nephritis (LN), systemic sclerosis (SSc), antineutrophil cytoplasmic autoantibody-associated vasculitis (AAV), or autoimmune muscle disease (e.g., idiopathic inflammatory myopathies (IIM), dermatomyositis (DM), or stiff person syndrome (SPS)).

[0151] In some examples, the autoimmune disease is lupus. Lupus is to an autoimmune disease in which the body’s immune system mistakenly attacks healthy tissue in many parts of the body. Lupus may be systemic lupus erythematosus, cutaneous lupus erythematosus, neonatal lupus, or drug-induced lupus.

[0152] In some examples, the autoimmune disease is systemic lupus erythematosus (SLE). Systemic lupus erythematosus is the most common type of lupus. SLE is an autoimmune disease characterized by dysfunction of adaptive and innate immune systems, heterogeneous manifestations in different organs and tissues from patient to patient and unpredictability of disease flares. Activated B cells that contribute to inflammation and generate autoantibodies against endogenous nuclear antigens may contribute to the pathogenesis of SLE.

[0153] In some examples, the autoimmune disease is lupus nephritis. Lupus nephritis involves inflammation of the kidneys due to SLE. Lupus nephritis may be a cause of end-stage kidney disease.

[0154] In some examples, the autoimmune disease is systemic lupus erythematosus with extrarenal involvement. Systemic lupus erythematosus with extrarenal involvement is SLE that involves inflammation and damage in organs or tissues other than the kidney.

[0155] In some examples, the autoimmune disease is systemic sclerosis (SSc). SSc is a chronic, systemic autoimmune disease with heterogeneous manifestations and variable disease course. The common and hallmark features in patients with SSc include vasculopathy and fibrosis, particularly involving the skin, lungs, j oints, and tendons. SSc may also affect kidneys, heart, and gastrointestinal tract.

[0156] In some examples, the autoimmune disease is antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). AAV is a grouping of multisystem diseases characterized by a pauci-immune small-vessel vasculitis and the presence of circulating autoantibodies. AAV includes granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic GPA (EGPA).

[0157] In some examples, the autoimmune disease is autoimmune muscle disease. An autoimmune muscle disease refers to an autoimmune disease in which the body’s immune system attacks muscles (e g., skeletal muscles), e.g., resulting inflammation, swelling, pain, and / or weakness of the muscles. Autoimmune muscle diseases include idiopathic inflammatory myopathies and stiff person syndrome. In some examples, the autoimmune disease is idiopathic inflammatory' myopathies (IIM). IIM are a heterogeneous group of disease conditions in which chronic muscle inflammation, or myositis, causes the hallmark symptoms of symmetric, proximal muscle weakness, low muscle endurance and myalgias. IIM include the following subgroups: dermatomyositis (DM), amyopathic dermatomyositis (ADM), polymyositis (PM), antisynthetase syndrome (ASyS), immune-mediated necrotizing myopathy (IMNM), and inclusion body myositis. IIM may also have extramuscular manifestations, including skin rash, interstitial lung disease, arthritis, and cardiac involvement.

[0158] In some examples, the autoimmune disease is stiff person syndrome (SPS). SPS is an autoimmune disorder characterized by muscle rigidity, episodic painful spasms and an exaggerated startle response to visual, auditory' and tactile stimuli. Signs of SPS include lumbar hyperlordosis and increased deep tendon reflexes.

[0159] Further examples of autoimmune diseases suitable for treatment with the present invention include rheumatoid arthritis, rheumatic fever, multiple sclerosis, experimentalautoimmune encephalomyelitis, psoriasis, uveitis, diabetes mellitus, eczema, scleroderma, polymyositis / scleroderma, polymyositis / dermatomyositis, ulcerative proctitis, ulcerative colitis, severe combined immunodeficiency (SCID), DiGeorge syndrome, ataxiatelangiectasia, seasonal allergies, perennial allergies, food allergies, anaphylaxis, mastocytosis, allergic rhinitis, atopic dermatitis, Parkinson's, Alzheimer’s, hypersplenism, leukocyte adhesion deficiency, X-linked lymphoproliferative disease, X-linked agammaglobulinemia, selective immunoglobulin A deficiency, hyper IgM syndrome, HIV, autoimmune lymphoproliferative syndrome, Wiskott-Aldrich syndrome, chronic granulomatous disease, common variable immunodeficiency (CVID), hyperimmunoglobulin E syndrome, Hashimoto’s thyroiditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenia purpura, Sydenham’ a chorea, myasthenia gravis, polyglandular syndromes, bullous pemphigoid, Henoch-Schonlein purpura, poststreptococcal nephritis, erythema nodosum, erythema multiforme, IgA nephropathy, Takayasu’s arteritis, Addison’s disease, sarcoidosis, ulcerative colitis, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangiitis obliterans, Sjogren’s syndrome, primary biliary cirrhosis. Hashimoto’s thyroiditis, thyrotoxicosis, chronic active hepatitis, polychondritis, pemphigus vulgaris, Wegener’s granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis, polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, and fibrosing alveolitis.

[0160] In embodiments, the therapeutically effective amount of y8 T cells that are administered is from about 1×107to about 1×1010γδ T cells, e.g., from about 5×107to about 5×109, from about 7×107to about 3×109, from about 9×107to about 2×109, from about 1×108to about 1×109, from about 1×108to about 3×108, from about 2×108to about 4×108, from about 3×108to about 5×108, from about 4×108to about 6×108, from about 5×108to about 7×108, from about 6×108to about 8×108, from about 7×108to about 9×108, or from about 8×108to about 1×109In some examples, the therapeutically effective amount of y5 T cells is from about 1 x 108to about 1 x 109y5 T cells. In embodiments, the therapeutically effective amount of y5 T cells is about 1×107, about 2×107, about 3×107, about 4×107, about 5×107, about 6×107, about 7×107, about 8×107, about 9×107, about 1×108, about 2×108, about 3×108, about 4×108, about 5×108, about 6×108, about 7×108, about 8×108, about 9×108, about 1×109, about 2×109, about 3×109, about 4×109, about 5×109, about 6×109, about 7×109, about 8×109, about 9×109, or about 1×1010. In some examples, the therapeutically effective amount of γδ T cells is about 1×108γδ T cells. In some examples, the therapeutically effective amount of γδ T cells is about 3×108γδ T cells. In some examples, the therapeutically effective amount of γδ T cells is about 1×109γδ T cells.

[0161] In embodiments, the γδ T cells are administered to a subject in any order or simultaneously. If simultaneously, the γδ T cells may be provided in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions, s.c, injections or pills. The y5 T cells may be packed together or separately, in a single package or in a plurality of packages γδ T cells. In embodiments, the γδ T cells are given in multiple doses. If not simultaneous, the timing between the multiple doses may vary to as much as about a week, a month, two months, three months, four months, five months, six months, or about a year. In some cases, the γδ T cells proliferate within a subject's body, in vivo, after administration to a subject. The γδ T cells may be frozen to provide cells for multiple treatments with the same cell preparation. The γδ T cells, and pharmaceutical compositions comprising the same, may be packaged as a kit. A kit may include instructions (e.g., written instructions) on the use of the γδ T cells, and compositions comprising the same.

[0162] In embodiments, when multiple doses are given, an additional dose (e.g.. the second dose) of the γδ T cells is given 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 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days, at least 1 month, at least 2, month, or at least 3 months after the administration of the previous dose (e.g., the first dose). In some examples, an additional dose (e.g., second dose) is given 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 21 days, 28 days, 1 month, 2, month, or 3 months after the administration of the previous dose (e.g., first dose).

[0163] In embodiments, when multiple doses are given, the one or more additional doses (e.g., the second dose) of the γδ T cells comprise an increased amount of γδ T cells compared to the previous dose (e.g., the first dose) of the γδ T cells. In embodiments, when multiple doses are given, the one or more additional doses (e.g., the second dose) of the γδ T cells comprise a decreased amount of γδ T cells compared to the previous dose (e.g., the first dose) of the γδ T cells. In embodiments, when multiple doses are given, the one or more additional doses (e.g., the second dose) of the γδ T cells comprise the same amount of γδ T cells compared to the previous dose (e.g., the first dose) of the γδ T cells.

[0164] In embodiments, when multiple doses are given, the one or more additional doses (e.g., the second dose) of the γδ T cells comprise γδ T cells derived from the same donor as the previous dose (e.g., the first dose) of the γδ T cells. In embodiments, when multiple doses aregiven, the one or more additional doses (e.g., the second dose) of the γδ T cells comprise γδ T cells derived from a different donor than the previous dose (e.g., the first dose) of the γδ T cells.

[0165] In embodiments the γδ T cells are administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In embodiments the γδ T cells are administered for at least one week. In embodiments the γδ T cells are administered for at least two weeks.

[0166] The γδ T cells may be administered before, during, or after the occurrence of a disease or condition, and the timing of administering a pharmaceutical composition containing the y8 T-cell population can vary. For example, the γδ T cells may be used as a prophylactic and may be administered continuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition. The initial administration may be via any route practical, such as by any route described herein using any formulation described herein. In some examples, the administration of the γδ T cells is an intravenous administration. One or multiple doses of the γδ T cells can be administered as soon as is practicable after the onset of an autoimmune disease and for a length of time necessary for the treatment of the immune disease, such as, for example, from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months. In embodiments, the γδ T cells are be administered years after onset of the autoimmune disease.

[0167] The methods may further comprise administering to the subject one or more lymphodepletion (LD) regimens. In embodiments, the methods comprise administering to the subject an LD regimen prior to the administration of the first dose of the γδ T cells. In embodiments, the methods may further comprise a priming step comprising administering a non-therapeutic dose of the γδ T cells at least 3 to 5, 4 to 6 or 7 to 9 days prior to administration of the LD regimen. Alternatively, in embodiments, a different immune cell or T cell population from the same allogeneic donor may be used for the priming step, e.g., αβ T cells removed from the expanded γδ T cell population. In embodiments, when one or more additional doses of γδ T cells is administered after the first dose, the additional dose(s) is administered following an additional LD regimen. Alternatively or additionally, in embodiments, when one or more additional doses of γδ T cells is administered after the first dose, the additional dose(s) is administered without an additional LD regimen. Preferably, and in contrast with such protocols in the context of oncology, the present invention aims to administer the minimum sufficientlevel of LD to Al patients, as opposed to the maximum tolerated depletion regimens typically administered to oncology patients.

[0168] In embodiments, the LD regimen comprises fludarabine and cyclophosphamide to the subject. In some examples, the fludarabine is administered at from 5 to 50 mg / m2 / day, such as 10 to 40 mg / m2 / day, e.g., about 10 mg / m2 / day, about 12.5 mg / m2 / day, about 15 mg / m2 / day, about 20 mg / m2 / day, about 30 mg / m2 / day, or about 40 mg / m2 / day. In some examples, the fludarabine is administered at about 30 mg / m2 / day. In some examples, the fludarabine is administered at about 20 mg / m2 / day. In some examples, the cyclophosphamide is administered from 200 mg / m2 / day to 600 mg / m2 / day such as from 250 mg / m2 / day to 500 mg / m2 / day, e.g., at about 50 mg / m2 / day, about 100 mg / m2 / day, about 150 mg / m2 / day, about 200 mg / m2 / day, about 250 mg / m2 / day, 300 mg / m2 / day, 400 mg / m2 / day, 500 mg / m2 / day, or 600 mg / m2 / day. In some examples, the cyclophosphamide is administered at about 500 mg / m2 / day. In some examples, the cyclophosphamide is administered at about 500 mg / m2 / day for three days. In some examples, the cyclophosphamide is administered at about 300 mg / m2 / day. In some examples, the cyclophosphamide is administered at about 300 mg / m2 / day for three days. In some examples, the fludarabine is given to the subject for one, two, three, four, or five days. In one example, the fludarabine is given to the subject for three days. In some examples, the cyclophosphamide is given to the subject for one, two, three, four, or five days. In one example, the cyclophosphamide is given to the subject for three days. In some examples, the fludarabine and cyclophosphamide are given to the subject for one, two. three, four, or five days. In one example, the fludarabine and cyclophosphamide are given to the subject for three days. In some examples, the LD regimen comprises administration of cyclophosphamide at about 50 mg / m2 / day for two days. In some examples, the LD regimen comprises administration of fludarabine at from 10 to 40 mg / m2 / day and cyclophosphamide at from 250 to 500 mg / m2 / day for three days. In some examples, the LD regimen comprises administration of fludarabine at about 30 mg / m2 / day and cyclophosphamide at about 500 mg / m2 / day for three days. In some examples, the LD regimen comprises administration of fludarabine at about 20 mg / m2 / day and cyclophosphamide at about 300 mg / m2 / day for three days. In some examples, the LD regimen comprises administration of fludarabine at about 30 mg / m2 / day and cyclophosphamide at about 1000 mg / m2 / day for three days. In embodiments, the dose of fludarabine is reduced, e.g., by up to 50%, if moderate to severe kidney impairment is present.

[0169] In embodiments, the LD regimen comprises or further comprises administration of bendamustine. In some examples, the bendamustine is given at about 70 mg / m2 / day, about 80 mg / m2 / day, about 90 mg / m2 / day, about 100 mg / m2 / day, about 110 mg / m2 / day, or about 120mg / m2 / day. In some examples, the bendamustine is given at about 90 mg / m2 / day. In some examples, the bendamustine is given for one, two. three, four, or five days. In some examples, LD regimen comprises administration of bendamustine at about 90 mg / m2 / day for 2 days.

[0170] In embodiments, the LD regimen further comprises administration of an protective agent, e.g., to protect the subject from risk of a side effect of an agent used in the LD regimen. In embodiments, the LD regimen further comprises administration of mesna. Mesna may protect the subject against the risk of bleeding of the bladder (e.g.. hemorrhagic cystitis) as a side effect (e.g., a side effect of cyclophosphamide). The mesna may be administered with cyclophosphamide. In some examples, the mesna is administered at about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, or about 30 mg / kg. In some examples, the mesna is administered at about 15 mg / kg. In some examples, the LD regiment further comprises administration of mesna at about 15 mg / kg with the cyclophosphamide.

[0171] Additional examples of lymphodepletion methods are disclosed in, for example, Amini, et al., “Preparing for CAR T cell therapy: patient selection, bridging therapies and lymphodepletion,” Nat. Rev. Clin. Oncol. 19(5):342-355 (May 2022) and Bechman, N. and Maher. J., “Lymphodepletion strategies to potentiate adoptive T-cell immunotherapy - what are we doing; where are we going,” Expert Opin. Biol. Ther. 21(5): 627-637 (May 2021), each of which is incorporated herein by reference in its entirety.Biomarkers, Methods of Detection, and Use Thereof

[0172] In embodiments, the methods further comprise monitoring the subject for one or more pharmacodynamics and / or pharmacokinetics biomarkers following administration of the y5 T cells. Examples of the biomarkers include CAR transgene expression level, quantitative measurement of CAR+ y<5 T cells, and serum level of one or more cytokines and / or serum proteins.

[0173] The biomarkers are biological indicators of disease or therapeutic effects that can be measured in vivo by biomedical / molecular imaging, as well as other in vitro or laboratory methodologies. As disclosed herein, one or more biomarkers can advantageously be relied upon to inform cell activation, treatment efficacy and / or follow-on treatment regimens. With respect to administration of the y5 T cells to a subject in need thereof as herein described, one or more biomarkers can be relied upon as indicator(s) of effectiveness, potential for effectiveness, or lack thereof in terms of. In embodiments, one or more biomarkers can be relied upon to determine, for example, whether to administer one or more additional dosing regimens, and if so, whether to adjust a dosage level (e.g., increase, decrease, or maintain the same dosage of the y5 T cells), to include one or more additional or alternative therapies, toadjust a previously planned dosing schedule, to administer the y8 T cells derived from a same or a different donor, or whether to halt / postpone treatment or discontinue treatment altogether, and the like. In embodiments, the biomarker(s) includes the CAR transgene expression level. In embodiments, the biomarker(s) includes a quantitative measurement of CAR+ y8 T cells.

[0174] In embodiments, activation and / or expansion of an administered the y8 T cells can be monitored by way of flow cytometry detection of CAR+ y5 T cells and / or via quantitative polymerase chain reaction (qPCR) detection of the anti-CD20 CAR transgene. Preferably, such methodology(s) are conducted at a number of time points following administration of y8 T cells, for example daily, every other day, every 3 days, every 4 days, etc., up to e.g., 14 days, 28 days, 2 months. 3 months, or more, as it is well known that the presence and the status of CAR-T cells in peripheral blood can vary’ over time (see, e.g.. Shah et al. (2020) Nat Med., 26:1569-75). In the art, PCR results have been reported to correlate with CAR surface expression as monitored by flow cytometry, however flow cytometry has advantages in that it allows for identification and characterization of CAR-T cell subpopulations and of a patient’s immune cells in a fast way and at a single cell level. In addition, flow cytometry detects the CAR at the proteomic level and thus can provide information regarding CAR cell functionality. The experimental manner in how to rely on one or more of flow cytometry, qPCR and / or other detection methodologies to monitor activation and / or expansion of the y6 T cells is readily determined by the skilled artisan, as shown and described, e.g., as described e.g., by Hu and Huang (2020) Front. Immunol., 11(1770).

[0175] In embodiments, the y8 T cells administered to a subject can induce release of one or more cytokines. In embodiments, the one or more cytokines are secreted from the y8 T cells. In additional or alternative embodiments, the one or more cytokines are secreted from cells other than the y8 T cells including, e.g., T cells, NK. cells, dendritic cells, and macrophages.

[0176] In embodiments, one or more cytokines and / or serum proteins are biomarkers of cell activation and / or therapeutic efficacy of the γδ T cell therapy as herein disclosed. Examples of the cytokines and / or serum proteins include IL-6, TNF-a, IL-8, IL-1, IL-2, GM-CSF, IL-15, IL-17a, IFNy, IL-12, granzyme A, granzyme B, perforin, sFasL, MIP-la, and monocyte chemotactic protein- 1 (MCP-1). In some examples, the cytokines and / or serum proteins include one or more of IL-5, IL-6, IL-1β, IL-1RA, IL-8, IL-18, MCP-1, MIP1α, MIP1β, CRP, or ferritin. Further examples of cytokines include INFγ, GM-CSF, IL-7, IL-15, IL-1β, IL-6, IL-10, MIP1β, CRP, CXCL9, CXCL10, CXCL11, CCL5, IL-5, IL-IRA, IL-18, soluble MICA. IL-10, IL-4, IL-13. IL-17, CCL2, CXCL12, CCL17, and CCL22.

[0177] In embodiments, the method comprises or further comprises monitoring CD19+B cell counts in the peripheral blood, e.g., as a pharmacodynamic (PD) biomarker.

[0178] In embodiments, induction of cytokines for use as biomarkers of therapeutic efficacy occurs within a timeframe between one day or less and 28 days following administration of the y5 T cells. In embodiments, said timeframe is between one day or less and 21 days, or 18 days, or 14 days, or 10 days following administration of the y5 T cells. In some additional or alternative embodiments, induction of cytokines for use as biomarkers occurs within a timeframe between one day or less following LD and 28 days following administration of the y5 T cells. In embodiments, such a timeframe is between one day or less following LD and 21 days, or 18 days, or 14 days, or 10 days following administration of the y6 T cells.

[0179] Measurement of serum levels of single cytokines are commonly performed using enzyme-linked immunosorbent assay (ELISA) and / or chemiluminesent assays, and multiplex bead-based assays can be used to determine serum levels of a plurality of cytokines in a single test (Knight et al. (2020) Archives of Pathology & Laboratory Medicine, 144(10)). In embodiments, serum levels of one or more cytokines are measured before administration of the y8 T cells, e.g., before lymphodepletion and / or following / during lymphodepletion but prior to the y8 T cells. In additional or alternative embodiments, serum levels of one or more cytokines are measured following administration of the y5 T cells. In embodiments, serum levels of one or more cytokines for use as biomarkers are measured before administration of the y5 T cells (e.g., between 1 and 7 days prior to administration), and / or are measured one or more times following administration of the y5 T cells up to about 28 days. In embodiments, a plurality of measurements of serum levels of one or more cytokines encompassing a timeframe before and / or following administration of the y6 T cells provide a time course of induction of the one or more cytokines. Such a time course can be used to establish peak serum levels of said one or more cytokines and / or the time course can be used to establish approximate total levels of cytokine induction during the time course. It is within the scope of this disclosure that peak levels of one or more cytokines are used as a biomarker metric. Additionally or alternatively, it is within the scope of this disclosure that total levels of release of one or more cytokines are used as a biomarker metric.

[0180] In embodiments, a presence of a biomarker (e.g., a cytokine) is confirmed in response to said biomarker being measured above some predetermined threshold, for example following administration of the y8 T cells.

[0181] In embodiments, confirmation of the presence of one or more cytokine biomarkers as herein described is used to inform follow-on treatments. For example, in response to cytokine biomarker confirmation follow ing administration of a first dose of the y5 T cells, a second dose may be optional, or a dosage of the corresponding second dose may be adjusted accordingly (e.g., maintained the same as the first dose or decreased). In additional or alternative embodiments, a lack of cytokine biomarker confirmation following administration of a first dose of a the y6 T cells may indicate a need for a second dose (e.g., with or without another lymph odepleti on step), that a cell dosage amount be increased for said second dose, and / or that the second dose comprise the y5 T cells derived from a different donor as compared to the first dose. Similar logic additionally or alternatively applies to an indication of presence or absence of biomarkers indicative of in vivo activation and / or expansion of administered the y8 T cells as described above.

[0182] In embodiments, the methods further comprise administering a secondary treatment regimen based at least in part on monitoring one or more of the biomarkers. In embodiments, the secondary treatment regimen comprises one or more additional doses of yo T cells described herein. In embodiments, the secondary treatment may follow a LD regimen described herein.EXAMPLES

[0183] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.Example 1. Effects of anti-CD20 CAR-expressing y8 T cells on autoimmune diseases

[0184] This example shows that the effects of exemplary anti-CD20 CAR-expressing y5 T cells (“ADI-001"’) on autoimmune disease models. The anti-CD20 CAR-expressing y5 T cells are δ1 γδ T cells expressing anti-CD20 CAR comprising the following domains in order: a 3H7 binding domain, a CD8α hinge and transmembrane domain, a 4-1BB costimulation endodomain, and a CD3ζ signaling domain (SEQ ID NO: 41).

[0185] The y5 T cells exhibited potent killing of patient-derived CD 19+ B cells in multiple autoimmune diseases, including SLE. systemic sclerosis, rheumatoid arthritis, multiple sclerosis, and Sjogren’s syndrome. B cells from 5 SLE patients and 3 patents each for SSc,RA, multiple sclerosis, and Sjogren’s syndrome were co-cultured with ADI-001 manufactured from two independent donors at varying effector-to-target (E: T) ratios for 24 hours and then analyzed by flow cytometry to quantify live B cells relative to negative controls. The results are shown in FIG. 1.

[0186] The y5 T cells demonstrated significant levels of CAR T cell activation and tissue exposure in lymph node biopsies, with a mean exposure of 236,701 CAR T cells per million across all dose levels, representing a range of 27-64% of total cellular material detected by ddPCR in evaluable biopsies at the 1E9 dose, and exceeding levels previously reported (Badbaran A et al., Accurate In-Vivo Quantification of CD19 CAR-T Cells after Treatment with Axicabtagene Ciloleucel (Axi-Cel) and Tisagenlecleucel (Tisa-Cel) Using Digital PCR, Cancers (Basel). 2020 Jul 20; 12(7):1970; Axi-cel= Axicabtagene ciloleucel) for patients who received autologous alpha-beta CAR T therapies. The data is shown in Table 1 below.Table 1. ADI-001 Tissue Trafficking Exceeds Data Reported for Axi-celLymph Node Exposure ADI-001Average CAR T per Million Cells1E8-1E9 Dose Levels 236,7011E9 Dose Level 461,867(276,588 – 647,163)Lymph Node Exposure Axi-celAxi-cel Patient #011 62,948Axi-cel Patient #014 19,647

[0187] RNAscope LS Fluorescent Multiplex assay was used to detect cells positive for ADI-001 CAR transgene and granzyme B mRNAs in formalin-fixed, paraffin-embedded (FFPE) secondary lymphoid tissue. CD 19 was also evaluated both pre- and post-dose with ADI-001. Representative images showing lymph node biopsies from diffuse large B-cell lymphoma and mantle cell lymphoma patients. Activated CAR is depicted by co-expression of CAR transgene and granzy me B in post-treatment samples. Quantitative ADI-001 CAR analyses were conducted in 6 available pairs of patient biopsies using droplet digital PCR (ddPCR) and are represented as the total ADI-001 CAR cells detected per million.

[0188] CAR T cells detected in tissues also demonstrated a robust activation profile, based on in situ detection of granzy me B. The results are shown in FIG. 2. The results show that robust tissue tropism for ADI-001 observed in lymph node biopsies across dose levels. ADI-001 cells represent 27%-64% of total cellular material detected by ddPCR in lymph nodes at 1E9 dose level.

[0189] Recently published studies have demonstrated depletion of CD19+ plasmablasts, memory B cells and naive B cells in peripheral blood using anti-CD20 targeted antibodies, however, these CD20-targeted antibody modalities failed to deplete B cells within secondary lymphoid tissues. See e.g., Tur C et al., CD19-CAR T-cell therapy induces deep tissue depletion of B cells, Ann Rheum Dis. 2024 Sep ll:ard-2024-226142, see e.g.. FIGS. 1A and IE

[0190] Concurrent with ADI-001 tissue trafficking and activation, complete depletion of CD19+ B cells within analyzed secondary lymphoid tissue was also observed. Clinical responses were observed in extra-nodal tissues. The results are shown in FIGS.3 and 4. These results support ADI-001’s potential for achieving complete B-cell depletion in peripheral blood and within tissues.

[0191] In sum, the yd T cells demonstrated robust tissue trafficking resulting in high levels of the yd T cells, significant chimeric antigen receptor (CAR) T cell activation, and complete CD 19+ B cell depletion in secondary lymphoid tissue. The results suggest that the yd T cells provide superior exposure of ADI-001 in secondary lymphoid tissue compared to published third-party data reported for alpha-beta CAR T therapies.Example 2. Clinical study of anti-CD20 CAR-expressing y6 T cells in treating autoimmune diseases

[0192] This Example shows the clinical study of anti-CD20 CAR-expressing y5 T cells in treating autoimmune diseases. The study is an open-label, dose-escalation study with a dose expansion cohort at the selected dose. A general design of the study is shown in FIG.5. Clinical indications encompassed by the study include lupus nephritis (LN), systemic lupus erythematosus (SLE), systemic sclerosis (SSc), idiopathic inflammatory myopathies (IIM), dermatomyositis (DM), stiff person syndrome (SPS), and antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV).

[0193] The anti-CD20 CAR-expressing γδ T cells described in Example 1 are prepared from PBMCs that are obtained via a standard leukapheresis procedure from qualified donors. After activation, transduction, expansion, and αβ T cell depletion, the enriched γδ T cells are washed, formulated in a suspension and cryopreserved in vials. The anti-CD20 CAR-expressing yd T cells are supplied in frozen vial(s) containing 5 mL nominal extractable volume of a cryopreserved cell suspension. Each vial of the cells contains > 1.00E7 viable cells / mL, and the product is stored in liquid nitrogen vapor phase (< -120°C). The product will be thawedin a dry -thawing device or water bath just prior to intravenous (IV) infusion. The thawed cell suspension is translucent to opaque and colorless to slightly yellow in appearance. The anti-CD20 CAR-expressing y5 T cells are administered intravenously. The number of CD20 CAR+ V51 T cells per milliliter of cell suspension, number of vials to thaw, and the volume of drug product to administer is determined for each subject depending on the dose cohort.

[0194] The study includes two parts. Part 1 is an open-label, 3+3 dose-finding study (designed according to Storer BE (1989). Design and analysis of phase I clinical trials, Biometrics, 45(3):925-37, which is incorporated herein by its entirety). The dose-finding algorithm starts at 3E8 CAR+ V51 cells flat dose and either escalate to 1E9 CAR+ V61 cells or de-escalate to 1E8, depending on safety findings. Up to approximately 80 subjects across 4 cohorts are enrolled to evaluate the safety of escalating doses of anti-CD20 CAR-expressing y8 T cells, and to establish the MTD (maximum tolerated dose) / MAD (maximum administered dose), as follows: Cohort 1: up to approximately 20 subjects with either LN or SLE with extrarenal involvement; Cohort 2: up to approximately 20 subjects with SSc; Cohort 3: up to approximately 20 subjects with AAV; Cohort 4: up to approximately 20 subjects with autoimmune muscle diseases. Part 2 includes a dose expansion cohort in up to approximately 100 subjects with autoimmune disease using the MTD or MAD of ADI-001 for each cohort determined in part 1, as follows: Cohort 1: Cohort la: up to approximately 20 subjects with LN; Cohort lb: up to approximately 20 subjects with SLE with extrarenal involvement; Cohort 2: up to approximately 20 subjects with SSc; Cohort 3: up to approximately 20 subjects with AAV; Cohort 4: up to approximately 20 subjects with autoimmune muscle diseases. Results from Part 2 are used to determine the recommended phase 2 dose (RP2D) to be used in subsequent studies.

[0195] Lymphodepletion is given prior to the administration of the y5 T cells. In general, the subjects receive an intravenous lymphodepletion regimen as follows: Cyclophosphamide (Cy) 500 mg / m2on Day -5, -4 and -3; and Fludarabine (Flu) 30 mg / m2on Day -5, -4 and -3. A different regimen may be used for individual subjects, e.g., Cy at 300 mg / m2and Flu at 20 mg / m2both daily for 3 days. Mesna may be used to prevent hemorrhagic cystitis associated with Cy (e.g., at of dose of 15 mg / kg and final concentration 20 mg / mL). Subjects for whom Flu is contraindicated may receive single agent bendamustine 90 mg / m2IV daily for 2 days (Day -4 to Day -3).

[0196] One or more of the following biomarker analyses is performed: (1) high resolution HLA ty ping performed at screening on whole blood to determine; the degree of shared HLA alleles between subject and donor; (2) changes in serum cytokine, chemokine and complementlevels; (3) cytokines and serum proteins involved in CRS and neurotoxicity (e.g., IL-5, IL-6, IL-10, IL-IRA, IL-8, IL-18, MCP-1, MIPla. MIP10, CRP, ferritin); (4) changes in urinary cytokine and chemokine levels (for cohorts 1, 2, and 3 only); (5) evaluate immune cell phenotype and immune cell recovery (e.g., B and T cells) in the peripheral blood before and after transfer of the yb T cells following lymphodepletion; (6) evaluate B cell repertoire by BCR sequencing; (7) serum immunoglobulin (e.g., IgM, IgG, IgA) levels; (8) antibody titers associated with response to treatment, namely dsDNA and antinuclear antibodies (ANA).

[0197] Primary endpoints may include incidence of treatment-emergent adverse events (TEAEs), including severity, seriousness, and relatedness, as well as incidence of DLTs at each dose. Secondary' and exploratory endpoints may include pharmacodynamic endpoints (e.g. dynamics of B cell depletion and reconstitution, immune reset, dynamics of host immune cell recovery in peripheral blood, autoantibody titers, and cellular kinetics) as well as appropriate efficacy endpoints for each indication. For patients with LN, endpoints may include complete / partial response based on kidney function. For patients with SLE, endpoints may include the systemic lupus erythematosus disease activity index 2000 (SLEDAI-2K) and / or definition of remission in systemic lupus erythematosus (DORIS) remission. For patients with SSc, endpoints may include the American College of Rheumatology Composite Response Index in Systemic Sclerosis (CRISS) score, modified Rodnan Skin Score (mRSS) in diffuse cutaneous, and FVC% predicted in ILD (percent predicted forced vital capacity). For patients with IIM, endpoints may include changes in Manual Muscle Test-8 (MMT-8) and muscle enzymes, and Total Improvement Score. For patients with DM, endpoints may include Cutaneous Dermatomyositis Disease Area and Severity Index (CDASI). For patients with SPS, endpoints may include Distribution of Stiffness Index. Timed 25- foot walk, and Rankin scale. For patients with AAV, endpoints may include complete response per Birmingham Vasculitis Activity Score (BVAS).

[0198] The anti-CD20 CAR-expressing yb T cells described in Example 1 are prepared from PBMCs that are obtained via a standard leukapheresis procedure from qualified donors. After activation, transduction, expansion, and a0 T cell depletion, the enriched yb T cells are washed, formulated in a suspension and cryopreserved in vials. The anti-CD20 CAR-expressing yb T cells are supplied in frozen vial(s) containing 5 mL nominal extractable volume of a cryopreserved cell suspension. Each vial of the cells contains > 1.00E7 viable cells / mL, and the product is stored in liquid nitrogen vapor phase (< -120°C). The product will be thawed in a dry -thawing device or water bath just prior to intravenous (IV) infusion. The thawed cell suspension is translucent to opaque and colorless to slightly yellow in appearance. The anti-CD20 CAR-expressing y5 T cells are administered intravenously. The number of CD20 CAR+ V51 T cells per milliliter of cell suspension, number of vials to thaw, and the volume of drug product to administer is determined for each subject depending on the dose cohort.Example 3. Clinical responses and biomarker data on subjects with Lupus Nephritis (LN) and Lupus with extra-renal involvement (SLE) after ADI-001 administration

[0199] This Example provides initial data for three patients enrolled in the clinical study described in Example 2, with follow-up ranging from 3 to 9 months. In particular, the results for two patients with Lupus Nephritis (LN-1 (203-001) and LN-4 (350-006)) and one having Lupus with extra-renal involvement (SLE-2 (209-013)) are shown in more detail below. Remarkably, improved kidney function was seen in the two LN patients, including complete renal responses (and DORIS remissions), as well as rapid and sustained reductions in SLEDAI and PGA in the LN and SLE patients. The protocol was well tolerated, with no CRS (cytokine release syndrome) > Gr2 and no Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS). Additionally, and importantly, there was clear evidence of immune reset with subsequent emergence of a naive B cell repertoire following single treatment, and patients were able to discontinue immunosuppressants and taper corticosteroids to zero or to physiological levels.

[0200] Patient characteristics are provided in Table 2 below.203-001 350-006 209-013LN Non-Renal (Class IV) SLEantidsDNA+ antidsDNA+14 12Baseline UPCR# of pnor therapies 3 4 4 Glucocorticoid dose at baseline 10 20 15 Ong Pred equivday)ANA = antinuclear antibodies; GC = glucocorticoid; UPCR = Urine Protein-to-Creatinine Ratio

[0201] The LN patients achieved renal responses within six months, including complete responses & DORIS remissions, with responses ongoing. UPCR dropped to 0.05 for patient LN-1 (203-001) at last follow up, and to 0.33 for patient LN-4 (350-006). Additionally, as shown in FIG. 6A-6C, there was a significant decline in SLEDAI across all of the patients, highlighting the durable effect of ADI-001 on a broad range of lupus symptoms. As shown in FIG. 7, there were also rapid and sustained reductions in the PGA (physician global assessment) score across all patients as well, again highlighting ADI-001's significant impact on overall disease activity.

[0202] Remarkably, the administration of ADI-001 also demonstrated clear evidence of immune reset, including deep and broad B cell depletion, reconstitution driven by naive, nonclass switched B cells, elimination of clonally dominant and potentially pathogenic clones, and diversification of the B cell repertoire. In all patients, CD 19+ B cells became undetectable post-treatment for an extended period (FIG. 8), and patient cell counts based on individual B cell subsets (FIG. 9A-9E) further illustrate that reconstitution was driven by naive, non-class switched B cells. Importantly, there was complete elimination of circulating B cells and plasmablasts, and reconstitution of naive B cells was observed by month 2. As shown in FIG.10, dominant and potentially pathogenic B cell clones were eliminated, and did not persist or return post-treatment. Finally, as shown in FIG. 11. there was a highly desirable immune reset of clonally dominant and potentially pathogenic clones with the emergence of a new, naïve B cell repertoire.

[0203] Sequence information as herein disclosed is summarized below in Table 3.Table 3: Sequence InformationSEQIDNo. Name Sequence1 HCDR3 AKDPSYGSGSYHSYYGMDVpolypeptide2 LCDR3 QQRFNWPLTpolypeptide3 HCDR3 VKDFHYGSGSNYGMDVpolypeptideLCDR3 QQSNDWPLTpolypeptideHCDR3 TKDGSYGHFYSGLDVpolypeptideLCDR3 QQRYYWPLTpolypeptideHCVR EEQLVESGGDLVQPGRSLRLSCAASGFTFHDYTMHWVRQpolypeptide APGKGLEWVSGISWNSGSLGYADSVKGRFTISRDNAKKSL YLQMNSLRAEDTALYYCAKDPSYGSGSYHSYYGMDVWG QGTTVTVSS LCVR EIVLTQSPATLSLSPGERATLSCWASQSISRYLVWYQQKCGpolypeptide QAPRLLIYEASKRATGIPVRFSGSGSGTDFTLTISSLESEDFA VYYCQQRFNWPLTFGGGTKVEIK HCVR EVQLAESGGDLVQSGRSLRLSCAASGITFHDYAMHWVRQPpolypeptide PGKGLEWVSGISWNSDYIGYADSVKGRFTISRDNAKKSLYL QMNSLRPDDTALYYCVKDFHYGSGSNYGMDVWGQGTTV TVSP LCVR EIVMTQSPATLSMSPGERATLSCRASQSVSRNLAWYQQKVpolypeptide GQAPRLLISGASTRATGIPARFSGSGSGTEFTLTINSLQSEDF AVYYCQQSNDWPLTFGQGTRLEIK HCVR EVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQpolypeptide APGKGLEWVSGISWNSDHGYADSVKGRFTISRDNAKNSLY LQMNSLRAEDTALYYCTKDGSYGHFYSGLDVWGQGTTVTvssLCVR EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGpolypeptide QAPRLLIYVASNRATGIPARFSGSGSGTDFTLTISSLEPDDFA VYYCQQRYYWPLTFGGGTKVEIK HCVR EVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQpolypeptide APGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSL YLQMNSLRAEDTALYYCAKDNSYGKFYYGLDVWGQGTT VTVSS LCVR EIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQKPGpolypeptide QAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAV YYCQQYNNWPITFGQGTRLEIK HCDR1 GFTFYDYApolypeptideHCDR2 ISWNSGYIpolypeptideHCDR3 AKDNSYGKFYYGLDVpolypeptideLCDR1 QSVSSNpolypeptideLCDR2 GASpolypeptideLCDR3 QQYNNWPITpolypeptideCD8a hinge PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY domainpolypeptideCD8a hinge TTFPAPRPdomain PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY polypeptideCD8a IWAPLAGTCGVLLLSLVFFLYCtransmembranedomainpolypeptideCD3£ signaling RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR domain GRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMK polypeptide GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3ζ signaling RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR domain GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG polypeptide ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE costimulatory LdomainpolypeptideCD27 QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKP costimulatory EPACSPdomainpolypeptidesecretion signal MALPVTALLLPLALLLHAARPIL-15 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMK polypeptide CFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTSP2A cleavage SGSGATNFSLLKQAGDVEENPGPsequencepolvpeptidefiirin cleavage RAKRsequencepolypeptideP2A+furin RAKRSGSGATNFSLLKQAG DVEENPGPcleavagesequencepolypeptideP2A cleavage ATNFSLLKQAGDVEENPGPsequencepolvpeptideF2A cleavage VKQTLNNFDLLKLAGDVESNPGPsequencepolypeptideE2A cleavage QCTNYALLKLAGDVESNPGPsequencepolvpeptideT2A cleavage EGRSLLTCGDVEENPGPsequencepolypeptideIRES CTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTT CTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAA AGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAG TTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAG CGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACA GGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACAC CTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTT GGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCG TATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCC CATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGC TTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGC CCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACA CGATGATA IRES AGCAGGTTTCCCCAACTGACACAAAACGTGCAACTTGAA ACTCCGCCTGGTCTTTCCAGGTCTAGAGGGGTAACACTT TGTACTGCGTTTGGCTCCACGCTCGATCCACTGGCGAGT GTTAGTAACAGCACTGTTGCTTCGTAGCGGAGCATGACG GCCGTGGGAACTCCTCCTTGGTAACAAGGACCCACGGG GCCAAAAGCCACGCCCACACGGGCCCGTCATGTGTGCA ACCCCAGCACGGCGACTTTACTGCGAAACCCACTTTAAA GTGACATTGAAACTGGTACCCACACACTGGTGACAGGCT AAGGATGCCCTTCAGGTACCCCGAGGTAACACGCGACA CTCGGGATCTGAGAAGGGGACTGGGGCTTCTATAAAAG CGCTCGGTTTAAAAAGCTTCTATGCCTGAATAGG TGACC GGAGGTCGGCACCTTTCCTTTGCAATTACTGACCAC 3H7-CD8- MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLCD27z SCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARF polypeptide SGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRL EIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCA ASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSY GKFYYGLDVWGQGTTVTVSSTITPAPRPPTPAPTIASQPLSL RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL VFFLYCQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQE DYRKPEPACSPRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR3B9-CD8-BBz MSVPTQVLGLLLLWLTDARCEIVM TQSPATLSVSPGERATLpolypeptide SCRASQSVSSNLAWYQQKPGQAPRLLIYGTSTRATGIPARF SGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPLTFGGGIK VEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSC VASGFTFNDYAMHWVRQAPGKGLEWVSVISWNSDSIGYA DSVKGRFTISRDNAKNSLYLQMHSLRAEDTALYYCAKDNH YGSGSYYYYQYGMDVWGQGTTVTVSSTTTPAPRPPTPAPT IASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT CGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDG CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR3H7-CD8-BBz MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTrL polypeptide SCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARF SGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRL EIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCA ASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSY GKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSL RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVrFLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR2B7-CD8-BBz MSVPTQVLGLLLLWLTDARCEIVM TQSPATLSLSPGERAAL polypeptide SCRASQSVSNYLAWYQQKPGQAPRLLIYDASNRATGIPARF SGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTK VEIRGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSC AASGFTFRDYTMHWVRQGPGKGLEWVSGISWNSDYIGYA DSVKGRFTISRDNAKNSLYLQMNSLRVEDTALYYCAKLSG TYRDYFYGVDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR9C11-CD8- MSVPTQVLGLLLLWLTDARCEIVM TQSPATLSLSPGERATL BBz SCRTSQTTFSYLAWYRQKPGQAPRLLIYDASNRAAGIPARF polypeptide SGSGSGTDFTLTTNSLEP EDFAVYYCQLRTNWITFGQGTRLE IKGGGGSGGGGSGGGGQVQLVESGGDSVKPGGSLRLSCAA SGFTFSDSYMTWIRQAPGKGLEWVSFISSSGSTIYYADSVK GRFTISRDNVKKSLYLQMNRLRAEDTAVYYCAREEPGNYV YYGMDVWGQGTFVTVSSTITPAPRPPTPAPTIASQPLSLRP EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVI TLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR3H7-CD3z MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLpolypeptide SCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARF SGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRL EIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCA ASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSY GKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSL RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVTTLYCRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR3H7-CD8-27z ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTG nucleotide TGGCTGACCGACGCCAGATGCGAAATAGTGATGACGCA GTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAAC CACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAA CTTAGCCTGGTACCTTCAGAAACCTGGCCAGGCTCCCAG GCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTAT CCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTT CATTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGC AGTTTATTACTGTCAGCAGTATAATAACTGGCCGATCAC CTTCGGCCAAGGGACACGGCTGGAGATTAAAGGTGGAG GTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGAA GTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCT GGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC ACCTTTTATGATTATGCCATGCACTGGGTCCGGCAAGCT CCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTG GAATAGTGGTTACATAGGCTATGCGGACTCTGTGAAGGG CCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCT GTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGG CCTTGTATTACTGTGCAAAAGATAACAGCTATGGAAAGT TCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGG TCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCAC CAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCC TGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCA GTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTAC ATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTC CTGTCACTGGTTATCACCCTTTACTGCCAACGACGCAAG TACCGCTCCAATAAAGGAGAGTCACCAGTAGAACCCGC CGAACCTTGTCACTATTCATGTCCACGCGAAGAGGAGGGTTCAACGATCCCTATTCAGGAAGATTACAGAAAGCCGGA ACCTGCTTGTAGCCCCAGAGTGAAGTTCAGCCGCAGCGC CGACGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGT ATAACGAGCTGAACCTGGGCAGGCGGGAGGAATACGAC GTGCTGGACAAGCGCAGAGGCCGGGACCCTGAGATGGG CGGCAAGCCCC AGAGGCGGAAGAACCCCCAGGAAGGCC TGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCC TACAGCGAGATCGGCATGAAGGGCGAGCGGCGACGCGG CAAGGGCCACGACGGCCTGTACCAGGGCCTGTCCACCG CCACCAAGGACACCTACGACGCCCTGCACATGCAGGCC CTGCCTCCCCGTTAG3H7-CD8-BBz ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTG nucleotide TGGCTGACCGACGCCAGATGCGAAATAGTGATGACGCA GTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAAC CACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAA CTTAGCCTGGTACCTTCAGAAACCTGGCCAGGCTCCCAG GCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTAT CCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCATTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCGATCAC CTTCGGCCAAGGGACACGGCTGGAGATTAAAGGTGGAG GTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGAA GTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCT GGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC ACCTTTTATGATTATGCCATGCACTGGGTCCGGCAAGCT CCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTG GAATAGTGGTTACATAGGCTATGCGGACTCTGTGAAGGG CCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCT GTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGG CCTTGTATTACTGTGCAAAAGATAACAGCTATGGAAAGT TCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGG TCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCAC CAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCC TGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCA GTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTAC ATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTC CTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGA AAGAAACTCCTGTATATATTCAAACAACCATTTATGAGA CCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTG CCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGA GAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTAC CAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGAC GTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGA AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA GAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGA TGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGC CTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTAC GACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA3B9-CD8-BBz ATGAGCGTTCCAACCCAAGTTCTGGGACTGCTTCTGCTC nucleotide TGGTTGACTGACGCTAGGTGCGAAATAGTAATGACCCAA TCCCCAGCCACTCTCTCCGTTAGCCCAGGTGAAAGAGCC ACTCTTAGTTGCAGGGCTAGTCAATCCGTATCTAGCAAC CTGGCCTGGTACCAGCAAAAGCCCGGACAAGCGCCGCG GTTGTTGATCTATGGGACGAGCACACGAGCTACGGGTAT TCCGGCCAGGTTCTC AGGGTCTGGCTCCGGAACCGAATT TACATTGACGATCAGTAGTCTGCAATCAGAGGATTTCGC CGTTTACTATTGCCAACAGTACAATAATTGGCCGCTCAC ATTCGGGGGAGGAACCAAGGTCGAGATTAAGGGAGGTG GGGGTAGTGGGGGCGGGGGGTCAGGAGGTGGAGGAGA GGTACAGTTGGTAGAAAGCGGCGGGGGGTTGGTTCAAC CTGGACGGAGTCTGAGATTGTCTTGCGTGGCTTCCGGCTTTACTTTCAATGATTACGCCATGCACTGGGTACGCCAGG CGCCTGGAAAGGGTCTGGAGTGGGTTTCCGTGATATCCT GGAATAGTGATAGTATAGGCTATGCCGATAGTGTAAAA GGAAGGTTTACAATCTCTAGGGATAACGCTAAGAACAG CCTGTACCTTCAAATGCATAGTCTCCGGGCTGAGGACAC AGCCTTGTACTATTGTGCTAAGGACAATCATTATGGAAGCGGGTCATATTATTACTATCAATATGGGATGGATGTGTGGGGTCAGGGAACGACCGTTACGGTATCCTCAACCACCAC CCCTGCACCAAGGCCCCCGACTCCCGCGCCCACCATCGC GTCACAGCCTCTTAGCCTGCGACCGGAAGCATGCAGACC AGCTGCCGGGGGGGCCGTGCATACGAGAGGTTTGGACT TCGCCTGCGATATCTACATCTGGGCGCCCTTGGCCGGGA CTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTA CTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCA AACAACCATTTATGAGACCAGTACAAACTACTCAAGAG GAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGA AGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCG CAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTC TATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGA TG 1 H l GGAC AAGAGACGTGGCCGGGACCCTGAGATGG GGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGG CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGG CCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGG GGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTAC AGCCACCAAGGACACCTACGACGCCCTTCACATGCAGG CCCTGCCCCCTCGCTAA2B7-CD8-BBz ATGTCCGTACCTACCCAGGTGCTGGGCCTGCTGCTGCTG nucleotide TGGCTGACCGACGCCAGATGCGAAATTGTGTTGACACAG TCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCC GCCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAACTAC TTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGG CTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATC CCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTC ACTCTC ACC ATC AGC AGCCTAGAGCCTGAAGA 1111 GCA GTTTATTACTGTCAGCAGCGTAGCAACTGGCCGCTCACT TTCGGCGGAGGGACCAAGGTGGAGATCAGAGGTGGAGG TGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGAAG TGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTG GCAGGTCCCTGCGACTCTCCTGTGCAGCCTCTGGATTCA CCTTTCGAGATTATACCATGCACTGGGTCCGGCAAGGTC CAGGGAAGGGCCTGGAATGGGTCTCAGGTATTAGTTGG AATAGTGATTACATAGGCTATGCGGACTCTGTGAAGGGC CGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTG TATCTGCAAATGAACAGTCTGAGAGTTGAGGACACGGC CTTGTATTACTGTGCAAAGCTCAGTGGGACCTACAGGGA CTACTTCTACGGAGTGGACGTCTGGGGCCAAGGGACCAC GGTCACCGTCTCCTCAACCACCACCCCTGCACCAAGGCC CCCGACTCCCGCGCCCACCATCGCGTCACAGCCTCTTAG CCTGCGACCGGAAGCATGCAGACCAGCTGCCGGGGGGG CCGTGCATACGAGAGGTTTGGACTTCGCCTGCGATATCT ACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTC TCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCA GAAAGAAACTCCTGTATATATTCAAACAACCATTTATGA GACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGC TGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACT GAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAG ACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGA GAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGG GATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGAT GGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA9C11-CD8- ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTG BBz nucleotide TGGCTGACCGACGCCAGATGCGAAATTGTGGTGACACA GTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGC CACCCTCTCCTGCAGGACCAGTCAGACTACTACCAGCTA CTTAGCCTGGTACCGACAGAAACCTGGCCAGGCTCCCAG GCTCCTCATCTATGATGCATCCAACAGGGCCGCTGGCAT CCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACTCTCACCATCAACAGCCTGGAGCCTGAAGATTTTGC AGTTTATTACTGTCAGCTGCGTACCAACTGGATCACCTTC GGCCAAGGGACACGACTGGAGATTAAAGGTGGAGGTGG ATCTGGAGGAGGAGGATCCGGTGGAGGAGGTCAGGTGC AGCTGGTGGAGTCTGGGGGAGACTCGGTCAAGCCTGGA GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACC TTCAGTGACTCCTACATGACTTGGATCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTTTCATTCATTAGTAGTAGT GGAAGTACCATATATTATGCAGACTCTGTGAAGGGCCGA TTCACCATTTCCAGGGACAACGTCAAGAAGTCATTGTAT CTGCAGATGAACAGACTGAGAGCCGAGGACACGGCCGT GTATTACTGTGCGAGAGAAGAACCAGGAAACTACGTCT ATTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCCTCAACCACCACCCCTGCACCAAGGCCCCCG ACTCCCGCGCCCACCATCGCGTCACAGCCTCTTAGCCTG CGACCGGAAGCATGCAGACCAGCTGCCGGGGGGGCCGT GCATACGAGAGGTTTGGACTTCGCCTGCGATATCTACAT CTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCT GTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAA GAAACTCCTGTATATATTCAAACAACCATTTATGAGACC AGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCC GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGA GTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCA GCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAG GACGAAGAGAGGAGTACGATGTTTTTGGAC AAGAGACGT GGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAG GAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGA AAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATG AAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCT TTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGA CGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAACodon- ACCACCACCCCTGCACCAAGGCCCCCGACTCCCGCGCCC optimized ACCATCGCGTCACAGCCTCTTAGCCTGCGACCGGAAGCA CD8a hinge TGCAGACCAGCTGCCGGGGGGGCCGTGCATACGAGAGG region TTTGGACTTCGCCTGCGATnucleotideCodon- ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCC optimized CACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGC CD8a hinge GTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGG region GGCTGGACTTCGCCTGTGATnucleotideanti-CD20- MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTL CAR SCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARFpolypeptide SGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRL EIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCA ASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSY GKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSL RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPRGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLL HAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK VTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNG NVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS* P2A cleavage GSGATNFSLLKQAGDVEENPGPdomainpolypeptideanti-CD20- MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTrL CAR SCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARFpolypeptide SGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRL EIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCA ASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSY GKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSL RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVrrLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPRGSGATNFSLLKQAGDVEENPGPMRISKPHLRSISIQCYL CLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLE SGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNI KEFLQSFVHIVQMFINTS*Secretion signal MRISKPHLRSISIQCYLCLLLNSHFLTEAGpolypeptide 1HVFILGCFSAGLPKTEAanti-CD20 ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTG CAR + sIL15 TGGCTGACCGACGCCAGATGCGAAATAGTGATGACGCA nucleotide GTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAAC CACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAA CTTAGCCTGGTACCTTCAGAAACCTGGCCAGGCTCCCAG GCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCATTCTC ACC ATC AGCAGCCTGC AGTCTGAAGATTTTGC AGTTTATTACTGTCAGCAGTATAATAACTGGCCGATCAC CTTCGGCCAAGGGACACGGCTGGAGATTAAAGGTGGAG GTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGAA GTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCT GGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC ACCTTTTATGATTATGCC ATGC ACTGGGTCCGGC AAGCT CCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTG GAATAGTGGTTACATAGGCTATGCGGACTCTGTGAAGGG CCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCT GTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGG CCTTGTATTACTGTGCAAAAGATAACAGCTATGGAAAGT TCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGG TCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCAC CAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCC TGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCA GTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTAC ATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTC CTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGA AAGAAACTCCTGTATATATTCAAACAACCATTTATGAGA CCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTG CCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGA GAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTAC CAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGTACGATGTTTTTGGACAAGAGAC GTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGA AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA GAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGA TGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGC CTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTAC GACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGTAGC GGGGCTACGAACTTCTCCCTTCTTAAACAAGC'GGGAGAC GTGGAAGAAAATCCCGGACCTATGGCCTTACCAGTGACC GCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCA GGCCGAACTGGGTGAATGTAATAAGTGATTTGAAAAAA ATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTT TATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAA CAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTC ACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGA AAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAA TGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGG AACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGT TTTGTACATATTGTCCAAATGTTCATCAACACTTCTTGAanti-CD20 ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTG CAR + S1L15 TGGCTGACCGACGCCAGATGCGAAATAGTGATGACGCA nucleotide GTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAAC CACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAA CTTAGCCTGGTACCTTCAGAAACCTGGCCAGGCTCCCAG GCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCATTCTC ACC ATC AGCAGCCTGC AGTCTGAAGATTTTGC AGTTTATTACTGTCAGCAGTATAATAACTGGCCGATCAC CTTCGGCCAAGGGACACGGCTGGAGATTAAAGGTGGAG GTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGAA GTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCT GGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC ACCTTTTATGATTATGCC ATGC ACTGGGTCCGGC AAGCT CCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTG GAATAGTGGTTACATAGGCTATGCGGACTCTGTGAAGGG CCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCT GTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGG CCTTGTATTACTGTGCAAAAGATAACAGCTATGGAAAGT TCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGG TCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCAC CAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCC TGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCA GTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTAC ATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTC CTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGA AAGAAACTCCTGTATATATTCAAACAACCATTTATGAGA CCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTG CCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGA GAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTAC CAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGTACGATGTTTTTGGACAAGAGAC GTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGA AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA GAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGA TGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGC CTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTAC GACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGTAGC GGGGCTACGAACTTCTCCCTTCTTAAACAAGC'GGGAGAC GTGGAAGAAAATCCCGGACCTATGAGAATTTCGAAACC ACATTTGAGAAGTATTTCCATCCAGTGCTACTTGTGTTTA CTTCTAAAC AGTCATTTCTAACTGAAGCTGGC ATTC ATG TCTTCATTTGGGCTGTTTC AGTGCAGGGCTTCCTAAAAC AGAAGCCAACTGGGTGAATGTAATAAGTGATITGAAAA AAATTGAAGATCTTATTCAATCTATGCATATTGATGCTA CTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAG TAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTA TTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAG TAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTT CTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGT GAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCA GAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT TGAanti-CD20- MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTL CAR SCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARFpolypeptide SGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSY GKFYYGLDVWGQGITVTVSSnTPAPRPPTPAPTIASQPLSL RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL VETLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR*Secretion signal MALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMH and sIL-15 IDATLYTESDVHPdomain SCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSS polypeptide NGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS* anti-CD20 ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTG CAR + sIL15 TGGCTGACCGACGCCAGATGCGAAATAGTGATGACGCA nucleotide GTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAAC CACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAA CTTAGCCTGGTACCTTCAGAAACCTGGCCAGGCTCCCAG GCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTAT CCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTT C ATTCTC ACC ATC AGC AGCCTGC AGTCTGAAGATTTTGC AGTTTATTACTGTCAGCAGTATAATAACTGGCCGATCAC CTTCGGCCAAGGGACACGGCTGGAGATTAAAGGTGGAG GTGGATCTGGAGGAGGAGGATCCGGTGGAGGAGGTGAA GTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCT GGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC ACCTTTTATGATTATGCCATGCACTGGGTCCGGCAAGCT CCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTG GAATAGTGGTTACATAGGCTATGCGGACTCTGTGAAGGG CCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCT GTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGG CCTTGTATTACTGTGCAAAAGATAACAGCTATGGAAAGT TCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGG TCACCGTCTCCTCAACCACGACGCCAGCGCCGCGACCAC CAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCC TGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCA GTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTAC ATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTC CTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGA AAGAAACTCCTGTATATATTCAAACAACCATTTATGAGA CCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTG CCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGA GAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTAC CAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGT ACGATGTTTTTGGACAAGAGAC GTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGA AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA GAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGA TGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAGAGT ACTGCGGCCGCTACGTAAATTCCGCCCCTCTCCCTCCCCC CCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGC CGGTGTGCGTTTGTCTATATGTTA 11’11 CC ACC ATATTGC CGTCTTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTG TCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGC CAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAG CAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTG TAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCG ACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGAT ACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGT GAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTC AAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGG TACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCA CATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTC TAGGCCCCCCGAACCACGGGGACGTGGTTTTTCCTTTGAA AAACACGATGATATTAATTAAGCCACCGCCATGGCCTTA CCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC ACGCCGCCAGGCCGAACTGGGTGAATGTAATAAGTGAT TTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATT GATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGT TGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTA CAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCAT GATACAGTAGAAAATCTGATCATCCTAGCAAACAACAG TTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAA AGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTGA

[0204] The embodiments and examples described above are intended to be merely illustrative and non-limiting. Those skilled in the art will recognize or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials and procedures. All such equivalents are considered to be within the scope and are encompassed by the appended claims.

Claims

CLAIMS:

1. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of anti-CD20 yd T cells to the subject, wherein the anti-CD20 yd T cells express a chimeric antigen receptor (CAR) comprising a binding domain that specifically binds to CD20.

2. A method for reducing and / or eliminating a need for chronic immunosuppression in a human subject with an autoimmune disease, comprising administering a therapeutically effective amount of anti-CD20 yd T cells to the subject, wherein the anti-CD20 yd T cells express a CAR comprising a binding domain that specifically binds to CD203. A method for effectuating immune reset in a subject with an autoimmune disease, comprising administering a therapeutically effective amount of anti-CD20 yd T cells to the subject, wherein the anti-CD20 yd T cells express a CAR comprising a binding domain that specifically binds to CD20.

4. A method for producing a naive B cell repertoire in a subject in need thereof, comprising administering a therapeutically effective amount of anti-CD20 yd T cells to the subject, wherein the anti-CD20 yd T cells express a CAR comprising a binding domain that specifically binds to CD20.

5. The method of any one of claims 1 to 4, wherein the autoimmune disease is selected from the group comprising or consisting of lupus, lupus nephritis, systemic sclerosis, antineutrophil cytoplasmic autoantibody-associated vasculitis, and autoimmune muscle disease.

6. The method of claim 5, wherein the lupus is systemic lupus erythematosus.

7. The method of claim 6, wherein the systemic lupus erythematosus is systemic lupus erythematosus with extrarenal involvement.

8. The method of claim 5, wherein the autoimmune muscle disease is idiopathic inflammatory myopathies, dermatomyositis, or stiff person syndrome.

9. The method of any one of claims 1-8, wherein the therapeutically effective amount of the anti-CD20 CAR yd T cells is from about 1 x 108to about 1 x 109yd T cells.

10. The method of any one of claims 1-8, wherein the therapeutically effective amount of the anti-CD20 CAR y8 T cells is about 1 x 108y8 T cells, about 3 x 108y8 T cells, or about 1 x 109y8 T cells.

11. The method of any one of the preceding claims, wherein the anti-CD20 CAR y6 T cells are 61 y8 T cells, 82 y8 T cells, 83 y8 T cells, or 64 y8 T cells, preferably wherein the y8 T cells are 81 y8 T cells.

12. The method of any one of the preceding claims, further comprising administering to the subject a lymphodepletion (LD) regimen prior to administering to the subject a first dose of the therapeutically effective amount of anti-CD20 CAR y8 T cells.

13. The method of claim 12, wherein the LD regimen comprises administration of fludarabine at about 30 mg / m2 / day or about 20 mg / m2 / day, and cyclophosphamide at about 500 mg / m2 / day for three days or about 300 mg / m2 / day for three days.

14. The method of claim 12 or 13, wherein the LD regiment further comprises administration of mesna at about 15 mg / kg with the cyclophosphamide.

15. The method of claim 12, wherein the LD regimen comprises administration of bendamustine at about 90 mg / m2 / day for 2 days.

16. The method of any one of the preceding claims, further comprising administering one or more additional doses of anti-CD20 CAR y8 T cells at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least 28 days, or at least one month after the first dose.

17. The method of claim 16, wherein the one or more additional doses of anti-CD20 CAR y6 T cells are administered without an additional LD regimen.

18. The method of claim 16, wherein the one or more additional doses of anti-CD20 CAR y8 T cells are administered following an additional LD regimen.

19. The method of any one of claims 16-18, wherein the one or more additional doses of anti-CD20 CAR y6 T cells comprise an increased amount of anti-CD20 CAR y8 T cells, a decreased amount of anti-CD20 CAR y8 T cells, or the same amount of anti-CD20 CAR y8 T cells.

20. The method of any one of claims 16-19, wherein the one or more additional doses of anti-CD20 CAR y5 T cells comprise anti-CD20 CAR y5 T cells derived from the same donor.

21. The method of any one of claims 16-19, wherein the one or more additional doses of anti-CD20 CAR y6 T cells comprise anti-CD20 CAR y6 T cells derived from a different donor.

22. The method of any one of claims 1-21, further comprising monitoring the subject for one or more pharmacodynamics / pharmacokinetics biomarkers following administration of the therapeutically effective amount of anti-CD20 CAR y5 T cells, wherein the biomarkers are selected from the group comprising or consisting of CAR transgene expression level, quantitative measurement of CAR+ y3 T cells, and serum level of one or more cytokines and / or serum proteins.

23. The method of claim 22, wherein the one or more cytokines and / or serum proteins are selected from the group comprising or consisting of IL-6, TNF-a, IL-8, IL-1, IL-2, GM-CSF, IL-15, IL-17a, IFNy, IL-12, granzyme A, granzyme B, perforin, sFasL, MIP-la, or MCP-1.

24. The method of claim 22 or 23, further comprising administering a secondary treatment regimen based at least in part on monitoring one or more of the biomarkers.

25. The method of claim 24, wherein the secondary treatment regimen comprises one or more additional doses of anti-CD20 CAR y5 T cells.

26. The method of claim 25, wherein the one or more additional doses of anti-CD20 CAR y5 T cells are administered without an additional LD regimen.

27. The method of claim 25, wherein the one or more additional doses of anti-CD20 CAR y5 T cells are administered following an additional LD regimen.

28. The method of any one of claims 25-27, wherein the one or more additional doses comprise an increased amount of anti-CD20 CAR y5 T cells, a decreased amount of anti-CD20 CAR y5 T cells, or the same amount of anti-CD20 CAR y5 T cells.

29. The method of any one of claims 25-28, wherein the one or more additional doses comprise anti-CD20 CAR y8 T cells derived from the same donor.

30. The method of any one of claims 25-28, wherein the one or more additional doses comprise anti-CD20 CAR y5 T cells derived from a different donor.

31. The method of any one of the preceding claims, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 41.

32. The method of any one of the preceding claims, wherein the anti-CD20 y8 T cells further comprise an isolated nucleic acid encoding the CAR.

33. The method of claim 32, wherein isolated nucleic acid encoding the CAR comprises the sequence of SEQ ID NO: 46.

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