Treatment of B-cell malignancies

Abstract

Aspects of the present disclosure include methods of effectively treating relapsed / refractory B cell malignancies in patients in need thereof who have been previously treated with, and ultimately failed, at least one, two, or at least three previous therapies. In one example, the method comprises administering to the subject one or more doses comprising a therapeutically effective amount of anti-CD20 CAR γδ T cells expressing a chimeric antigen receptor (CAR) comprising a binding domain that specifically binds to CD20 on the malignant B cells. After administration, the subject methods optionally further comprise monitoring one or more biomarkers of cell activation and / or therapeutic efficacy that inform the need for and / or type of subsequent treatment regimen.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 286,086, filed December 5, 2021, the contents of which are incorporated herein for all purposes.

[0002] The present disclosure generally relates to a highly effective salvage therapy for relapsed / refractory B-cell malignancies, comprising administering one or more doses of anti-CD20 CAR γδ T cells to patients previously treated with one or more alternative therapies. [Background technology]

[0003] Non-Hodgkin's lymphoma (NHL) is a type of cancer that affects the lymphatic system. NHL is not a single disease, but rather includes several closely related cancers. The various types of NHL share many common features, but differ in certain characteristics, including their appearance under a microscope, their molecular characteristics and growth patterns, their impact on the body, and how they respond to different types of treatment. NHL is estimated to be the 13th most common cancer and the 11th leading cause of cancer death worldwide (Bray et al. (2018) CA Cancer J Clin., 68(6):394-424, Global Cancer Observatory: Cancer Tomorrow. International Agency for Research on Cancer).

[0004] NHL subtypes are classified according to which lymphocyte subsets are transformed and how fast the lymphoma grows and progresses. NHL arises in B cells, T cells, and natural killer (NK) cells, but the majority (85-90%) of NHL subtypes originate from B cells. NHL subtypes are designated as either asymptomatic (slow growing) or aggressive (rapid growing), with approximately 60% of all cases of NHL being of the aggressive subtype. Relevant examples of aggressive B-cell lymphoma subtypes include diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), and transformed follicular lymphoma (tFL).

[0005] Anti-CD20 monoclonal antibody (mAb) RITUXAN® (rituximab) in combination with chemotherapy is a first-line induction therapy for CD20-positive B-cell NHL. Rituximab engages Fc receptors on NK cells and macrophages, promotes complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity / phagocytosis (ADCC / ADCP), and exerts direct antiproliferative and proapoptotic effects (Tobinai et al. (2017) Adv Ther., 34(2):324-356; Boross and Leusen (2012) Am J Cancer Res., 2(6):676-690). Although the use of rituximab has significantly improved the outcome of some patients with B-cell malignancies (Lim and Levy (2014) J Immunol., 193(4): 1519-1524; Coiffier et al. (2010) Blood, 116(12): 2040-2045; Pfreundschuh et al. (2006) Lancet Oncol., 7(5): 379-391; Hallek et al. (2010) Lancet, 376(9747): 1164-1174), resistance to rituximab is observed in approximately half of patients during the course of long-term treatment. The exact mechanism of rituximab resistance is unknown but is thought to be a function of a complex combination of rituximab's three mechanisms of action (CDC, ADCC / ADCP, and apoptosis) and the patient-specific lymphoma microenvironment (van Meetern and Hagenbeek (2009) The Netherlands Journal of Medicine, 67(7):251-259). Alternative anti-CD20 mAbs have been developed, but their efficacy and safety compared to rituximab remain controversial (Luo et al. (2021) Scientific Reports, 11(3255)).

[0006] Adoptive cell therapy, specifically autologous CAR αβ T cell therapy, is playing an increasingly important role as a salvage treatment for relapsed / refractory B-cell NHL, including the anti-CD19 αβ CAR T cell therapy Yescarta™, which has been approved for the treatment of adult patients with R / R DLBCL. Unfortunately, however, 30% of patients have only a partial response to the treatment, the therapeutic effect tends to wane within 6 months in many other patients, and only 40% of patients achieve a durable response. (With FDA Approval for Advanced Lymphoma, Second CAR T-Cell Therapy Moves to the Clinic; (2017)). In addition, many eligible patients do not have access to the cell product due to manufacturing failures and / or delayed cell infusion, and these cell therapies may also present their own challenging toxicities, namely, graft-versus-host disease (GvHD) and immunogenicity (i.e., host-versus-graft rejection), as well as cytokine release syndrome (CRS). Thus, although autologous CAR αβ T-cell therapy has positively impacted the treatment paradigm for B-cell NHL, a myriad of issues remain, including but not limited to manufacturability challenges, non-responsiveness, severe adverse events, and relapse after achieving remission.

[0007] Thus, outcomes of current salvage therapies remain poor for many patients with relapsed or refractory (R / R) B-cell non-Hodgkin lymphoma (NHL), and more effective treatment options are urgently needed, especially for heavily pretreated patients. Summary of the Invention

[0008] The present disclosure addresses an urgent unmet need in the art for a more effective salvage therapy for patients suffering from relapsed or refractory (R / R) B-cell malignancies by administering at least one dose of anti-CD20 CAR γδ T cells to patients who have been previously treated with at least one, at least two, or at least three prior therapies. As demonstrated for the first time herein, the subject γδ T-cell therapy provides unexpected and truly remarkable results in patients who have failed multiple prior therapies, including prior anti-CD20 antibody and / or autologous αβ CAR T-cell therapy.

[0009] The subject methods will find advantageous use in a wide range of B-cell malignancies, including, for example, non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), acute lymphocytic leukemia (ALL), and acute myeloid leukemia (AML). In embodiments, the B-cell malignancy may be a type of NHL, including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), transformed follicular lymphoma (tFL), primary mediastinal (thymic) large B-cell lymphoma (PMBCL), high-grade B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma (FL), and marginal zone lymphoma (MZL).

[0010] In one aspect, the invention provides a method of treating relapsed / refractory (R / R) B cell malignancies in a patient previously treated with at least one, at least two, or at least three previous therapies, the method comprising administering to the subject a therapeutically effective amount of anti-CD20 CAR γδ T cells expressing a chimeric antigen receptor (CAR) comprising a binding domain that specifically binds to CD20 on the malignant B cell, thereby treating the subject. In embodiments, the patient may have relapsed from or be refractory to a previous therapy, including an anti-CD20 monoclonal antibody (e.g., rituximab or obinutuzumab) or an anti-CD19 CAR αβ T cell therapy (e.g., axicabtagenecilloreucel or tisagenlecleucel).

[0011] In embodiments, the binding domain of the anti-CD20 CAR γδ T cells specifically binds to a CD20 epitope that is distinct from the CD20 epitope recognized by the anti-CD20 monoclonal antibody.

[0012] In embodiments, a therapeutically effective amount of the anti-CD20 CAR γδ T cells is about 3×10 7 ~Approx. 1×10 9 In embodiments, the therapeutically effective amount of the anti-CD20 CAR γδ T cells is about 3×10 7 In embodiments, the therapeutically effective amount of the anti-CD20 CAR γδ T cells is about 1×10 8 In embodiments, the therapeutically effective amount of the anti-CD20 CAR γδ T cells is about 3×10 8 In embodiments, the therapeutically effective amount of the anti-CD20 CAR γδ T cells is about 1×10 9 The anti-CD20 CAR γδ T cells are γδ T cells. The anti-CD20 CAR γδ T cells may comprise, consist essentially of, or consist of γδ1 T cells, γδ2 T cells, γδ3 T cells, or γδ4 T cells, or a combination thereof. In an exemplary embodiment, the anti-CD20 CAR γδ T cells comprise γδ1 T cells.

[0013] In embodiments, the method may further comprise administering to the subject a lymphodepletion (LD) regimen prior to administering to the subject a first dose of a therapeutically effective amount of anti-CD20 CAR γδ T cells. In some embodiments, the LD regimen comprises about 30 mg / m 2 / day fludarabine for 3 days + approx. 500 mg / m 2 / day cyclophosphamide for 3 days, and optionally further comprising an anti-CD52 antibody and / or an anti-CD19 antibody. In some embodiments, the LD regimen comprises about 30 mg / m 2 / day fludarabine for 4 days + approx. 1000 mg / m 2 / day cyclophosphamide for 3 days, and optionally further comprising an anti-CD52 antibody and / or an anti-CD19 antibody.

[0014] In embodiments, the subject method further comprises administering one or more additional doses of anti-CD20 CAR γδ 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 γδ T cells are administered without an additional LD ​​regimen. In embodiments, the one or more additional doses of anti-CD20 CAR γδ T cells are administered after an additional LD ​​regimen. In embodiments, the one or more additional doses comprise an increased amount of anti-CD20 CAR γδ T cells. In embodiments, the one or more additional doses comprise a decreased amount of anti-CD20 CAR γδ T cells. In embodiments, the one or more additional doses comprise the same amount of anti-CD20 CAR γδ T cells. In embodiments, the one or more additional doses comprise anti-CD20 CAR γδ T cells derived from the same donor. In embodiments, one or more additional doses comprise anti-CD20 CAR γδ T cells derived from a different donor.

[0015] In an exemplary embodiment, the method involves administering to the subject a LD regimen on day -5, followed by administration of a therapeutically effective amount of anti-CD20 CAR γδ T cells (e.g., about 3×10 7 ~Approx. 1×10 9 γδ T cells) to the subject on day 1. In embodiments, the therapeutically effective amount of the anti-CD20 CAR γδ T cells is about 3×10 7 In embodiments, the therapeutically effective amount of the anti-CD20 CAR γδ T cells is about 1×10 8 In embodiments, the therapeutically effective amount of the anti-CD20 CAR γδ T cells is about 3×10 8 In embodiments, the therapeutically effective amount of the anti-CD20 CAR γδ T cells is about 1×10 9The anti-CD20 CAR γδ T cells are γδ T cells. The anti-CD20 CAR γδ T cells may comprise, consist essentially of, or consist of γδ1 T cells, γδ2 T cells, γδ3 T cells, or γδ4 T cells, or a combination thereof. In a preferred embodiment, the anti-CD20 CAR γδ T cells comprise, consist essentially of, or consist of γδ1 T cells.

[0016] In further exemplary embodiments, the subject methods may further comprise administering to the subject a second dose comprising a therapeutically effective amount of anti-CD20 CAR γδ T cells on day 7, optionally with or without administration of an additional LD ​​regimen. The second therapeutically effective dose may comprise an increased amount of anti-CD20 CAR γδ T cells, a decreased amount of anti-CD20 CAR γδ T cells, or the same amount of anti-CD20 CAR γδ T cells as the first dose. In embodiments, the subject methods may further comprise administering to the subject a third dose comprising a therapeutically effective amount of anti-CD20 CAR γδ T cells on day 14, optionally with or without administration of an additional LD ​​regimen. The third therapeutic dose may comprise an increased amount of anti-CD20 CAR γδ T cells, a decreased amount of anti-CD20 CAR γδ T cells, or the same amount of anti-CD20 CAR γδ T cells as the first and / or second dose. In embodiments, a therapeutically effective amount of the anti-CD20 CAR γδ T cells is about 3×10 8 The anti-CD20 CAR γδ T cells are γδ T cells. The anti-CD20 CAR γδ T cells may comprise, consist essentially of, or consist of γδ1 T cells, γδ2 T cells, γδ3 T cells, or γδ4 T cells, or a combination thereof. In a preferred embodiment, the anti-CD20 CAR γδ T cells comprise, consist essentially of, or consist of γδ1 T cells.

[0017] In embodiments, the subject methods may further comprise administering to the subject a therapeutically effective amount of IL-2. In some embodiments, the therapeutically effective amount of IL-2 is about 2×10 6 This is IU.

[0018] In another aspect, the method may further comprise monitoring the patient for one or more pharmacodynamic / pharmacokinetic biomarkers as indicators of cell product activation and / or efficacy. In embodiments, the subject method further comprises monitoring the subject for one or more pharmacodynamic / pharmacokinetic biomarkers following administration of the anti-CD20 CAR γδ T cells, wherein the biomarkers comprise or are selected from the group consisting of CAR transgene expression levels, quantitative measurements of CAR+ γδ T cells, serum levels of one or more cytokines and / or serum proteins, and minimal residual disease (MRD). In embodiments, the one or more cytokines / serum proteins include or are selected from the group consisting of INFγ, GM-CSF, IL-2, IL-7, IL-15, TNFα, IL-1β, IL-6, IL-8, IL-10, MIP1α, MIP1β, CRP, ferritin, monocyte chemoattractant protein-1 (MCP-1), 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. In exemplary embodiments, the one or more cytokines are IL-2 and / or IL-8. In embodiments, the subject method may further include measuring CAR transgene expression levels via quantitative polymerase chain reaction (qPCR). In embodiments, the quantitative measurement of CAR+ γδ T cells is determined via flow cytometry. In embodiments, the subject methods further comprise performing analysis of the MRD via immunosequencing methodology.

[0019] In further aspects, the subject method may further comprise administering a secondary therapeutic regimen based at least in part on monitoring one or more of the aforementioned biomarkers. In embodiments, the secondary therapeutic regimen comprises one or more additional doses of anti-CD20 CAR γδ T cells. In embodiments, the one or more additional doses of anti-CD20 CAR γδ T cells are administered without an additional LD ​​regimen. In embodiments, the one or more additional doses of anti-CD20 CAR γδ T cells are administered after an additional LD ​​regimen. In embodiments, the one or more additional doses of anti-CD20 CAR γδ T cells are administered as a consolidation therapy to kill any cancer cells that may be left in the body, preferably the one or more additional doses of anti-CD20 CAR γδ T cells are administered after an additional LD ​​regimen. In embodiments, the one or more additional doses comprise an increased amount of anti-CD20 CAR γδ T cells. In embodiments, the one or more additional doses comprise a decreased amount of anti-CD20 CAR γδ T cells. In embodiments, the one or more additional doses comprise the same amount of anti-CD20 CAR γδ T cells. In embodiments, the one or more additional doses comprise anti-CD20 CAR γδ T cells from the same donor. In embodiments, the one or more additional doses comprise anti-CD20 CAR γδ T cells from a different donor. In embodiments, the subsequent treatment regimen further comprises administration of cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate, and prednisone (CHOP).

[0020] In embodiments, the anti-CD20 CAR may comprise the amino acid sequence of SEQ ID NO: 41. In embodiments, the anti-CD20 γδ T cell may further comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 41. In embodiments, the anti-CD20 γδ T cell may further comprise a nucleic acid sequence encoding a CAR and having the sequence of SEQ ID NO: 46.

[0021] Incorporation by Reference 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]

[0022] [Figure 1] Fluorodeoxyglucose (FDG) positron emission tomography (PET) images obtained in a subject before (A and C) and after (B and D) treatment with 3×10 7 anti-CD20 CAR γδ T cells are shown. [Diagram 2] FDG-PET images obtained in another subject are shown before (A and C) and after (B and D) treatment with 3×10 7 anti-CD20 CAR γδ T cells. [Diagram 3] FDG-PET images obtained in another subject are shown before (A and C) and after (B and D) treatment with 1×10 8 anti-CD20 allogeneic γδ CAR T cells. [Figure 4-1] A and B are graphs showing serum levels of various cytokines in subjects before lymphodepletion, after lymphodepletion but before infusion of anti-CD20 allogeneic γδ CAR T cells, and after infusion of anti-CD20 allogeneic γδ CAR T cells. [Figure 4-2] C is a graph showing serum levels of various cytokines in subjects before lymphodepletion, after lymphodepletion but before infusion of anti-CD20 allogeneic γδ CAR T cells, and after infusion of anti-CD20 allogeneic γδ CAR T cells. [Figure 5A] 1 shows flow cytometry analysis demonstrating in vivo expansion of anti-CD20 allogeneic γδ CAR T cells. [Figure 5B] 1 shows flow cytometry analysis demonstrating in vivo expansion of anti-CD20 allogeneic γδ CAR T cells. [Figure 5C] 1 shows flow cytometry analysis demonstrating in vivo expansion of anti-CD20 allogeneic γδ CAR T cells. [Figure 5D]1 shows flow cytometry analysis demonstrating in vivo expansion of anti-CD20 allogeneic γδ CAR T cells. [Figure 6] We provide a summary of safety findings to date in first-in-human studies of anti-CD20 allogeneic γδ CAR T cells in adults with various B-cell malignancies. [Figure 7] We provide swimmer plots for patients treated to date along with a summary of the independent radiological evaluation of these patients.TH, triple hit;DH, double hit;DLBCL, diffuse large B-cell lymphoma;HGBCL, high-grade B-cell lymphoma;MCL, mantle cell lymphoma;mCR: metabolic complete response (PET negative). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] I. Definition For purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall include the plural and vice versa. In the event that a set forth definition conflicts with any document incorporated herein by reference, the set forth below shall control. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0024] As used herein, "about" when referring to a measurable value, e.g., amount, temporal duration, etc., is meant to encompass variations of ±20% or ±10% from the specified value, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1%, as appropriate for carrying out the disclosed methods.

[0025] As used herein, "w / v" refers to the weight of a component in a given volume of solution.

[0026] "Range": Throughout this disclosure, various aspects of the disclosure may 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. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as each individual numerical value within that range. For example, the description of a range such as 1 to 6 is considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as each individual numerical value within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the broadness of the range. This applies regardless of the broadness of the range.

[0027] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0028] 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., a cell engineered to express a chimeric antigen receptor). An agent may include a therapeutic agent, a diagnostic agent, or a pharmaceutical agent. A therapeutic agent or pharmaceutical agent is one that, alone or together with additional agents, induces a desired response (such as inducing a therapeutic or prophylactic effect when administered to a subject, including treating a subject suffering from cancer, or other disease / condition.

[0029] As used herein, the term "diagnosis" or "diagnosing" refers to the process of identifying a disease, such as cancer, through signs, symptoms, and / or the results of various tests. The conclusion reached through such a process is a diagnosis. Common forms of testing include blood tests, medical imaging, urine tests, biopsies, etc.

[0030] The term "therapeutically effective amount" or simply "effective amount" refers to an amount of an agent or composition (e.g., a composition containing an agent) that elicits the biological or medical response of a tissue, system, or subject that is desired by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of an agent or a composition containing an agent that, when administered, is sufficient to prevent or alleviate to some extent the manifestation of one or more signs or symptoms of the disorder or disease (e.g., hematological or solid tumor) being treated. The therapeutically effective amount will vary depending on the component, the disease and its severity, as well as the age, weight, etc., of the subject to be treated.

[0031] As used herein, the term "treatment" of a disease means to reduce 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 reducing one or more signs or symptoms associated with the disease or condition, e.g., compared to the response in the absence of the therapy.For example, administration of a therapeutic agent can provide an anti-tumor effect that reduces one or more signs or symptoms associated with cancer.

[0032] As used herein, the term "administration" means providing or giving to a subject, by any effective route, one or more agents, such as agents to treat one or more signs or symptoms associated with a condition / disorder or disease, including, but not limited to, cancer (e.g., lymphoma), viral infection, bacterial infection, and the like. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes. Administration "in conjunction with" one or more therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0033] As used herein, the term "pharmaceutical acceptable" refers to a material, including but not limited to, a salt, carrier, or diluent, that does not abrogate the biological activity or properties of a compound and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Pharmaceutically acceptable carriers (vehicles) useful in the present 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, e.g., one or more modulators. In general, the nature of the carrier will depend on the particular mode of administration being used. For example, parenteral formulations may include injectable fluids that include pharmaceutical and physiologically acceptable fluids (e.g., water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol) as vehicles. In addition to biologically neutral carriers, the pharmaceutical agent to be administered may contain minor amounts of non-toxic auxiliary substances (e.g., wetting or emulsifying agents, preservatives, and pH buffering agents, such as, for example, sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate). For example, the invention provides pharmaceutical compositions comprising a pharma- ceutically acceptable excipient and, for example, γδ T cells, preferably γδ T cells engineered to express a CAR directed against CD20, as described herein.

[0034] As used herein, the terms "pharmacodynamic (PD) biomarker" and / or "pharmacokinetic (PK) biomarker" refer to one or more measurable indicators associated with the administration of a therapeutic agent to a subject. Broadly speaking, PK markers relate to how the body affects a therapeutic agent, while PD markers relate to how the therapeutic agent affects the subject.

[0035] As used herein, the term "relapsed / refractory B cell malignancy" encompasses any B cell lymphoma that is ultimately non-responsive to treatment, including, but not limited to, non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), acute lymphocytic leukemia (ALL), and acute myeloid leukemia (AML). Thus, in embodiments, the B cell malignancy may include or be selected from the group consisting of NHL, CLL, ALL, and / or AML. In embodiments, the B cell malignancy may be a form of NHL selected from the group including or consisting of diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), transformed follicular lymphoma (tFL), primary mediastinal (thymic) large B cell lymphoma (PMBCL), high-grade B cell lymphoma, Burkitt lymphoma, follicular lymphoma (FL), and marginal zone lymphoma (MZL).

[0036] As used herein, "activation" refers to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells that are undergoing cell division.

[0037] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immunocompetent cells, or both. One of skill in the art will appreciate that any macromolecule, including a protein or peptide, may function as an antigen.

[0038] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be complete immunoglobulins obtained from natural or recombinant sources, or can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in the present invention may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or complete monoclonal antibodies), antibody compositions with polyepitopic specificity, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), diabodies, single domain antibodies (sdAbs) (so long as they exhibit the desired biological or immunological activity), Fv, Fab, and F(ab), as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY: Harlow et al., 1989, In; Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Nat Acad. Sci. USA 85:5879-5883: Bird et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; ah, 1988, Science 242:423-426).

[0039] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the 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.

[0040] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in antibody molecules in their naturally occurring conformations.

[0041] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0042] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA techniques, such as, for example, an antibody expressed by a bacteriophage, as described herein. The term should also be construed to mean an antibody produced by synthesizing a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein or an amino acid sequence that specifies the antibody, where the DNA or amino acid sequence is available and can be obtained using synthetic DNA or amino acid sequence techniques well known in the art.

[0043] The term "epitope" includes any protein, lipid, or carbohydrate determinant capable of specific binding to an immunoglobulin or receptor (e.g., a T-cell receptor). Epitopic determinants usually consist of active surface groupings of molecules such as amino acids, lipid or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0044] As used herein, the term "specifically binds" refers to a receptor (which may include, but is not limited to, an antibody or antibody fragment) that recognizes a particular molecule / ligand but does not substantially recognize or bind other molecules in a sample. For example, a receptor that specifically binds to a molecule of one kind may also bind to one or more other species of molecules. However, such cross-species reactivity in itself does not change 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 cross-reactivity in itself does not change the classification as specific. In some cases, the terms "specifically bind" or "specifically bind" can be used in reference to the interaction of a protein (or peptide) with a second chemical species, meaning that the interaction is dependent on 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 particular structure, not a general protein. If a receptor is specific for epitope "A", then in a reaction containing labeled "A" and a receptor, the presence of a molecule containing epitope A (or not, unlabeled A) will reduce the amount of labeled A that binds to the receptor.

[0045] In some embodiments, specific binding is at least about 1×10 -8 The binding may be characterized by an equilibrium dissociation constant equal to or less than M (e.g., a smaller K indicates 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.

[0046] As used herein, the term "anti-tumor effect" refers to a biological effect that may be manifested by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the metabolic activity of tumor cells (e.g., PET signal), a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition.

[0047] As used herein, the term "autologous" refers to any material that originates from an individual, which is later to be reintroduced into the same individual.

[0048] As used herein, the term "allogeneic" refers to material derived from an animal that is subsequently introduced into another animal of the same species.

[0049] As used herein, the term "γδ T cells" or "gamma delta T cells" refers to a subset of T cells that express a distinct T cell receptor (TCR) on their surface, i.e., γδ TCR, consisting of one γ chain and one δ chain. The term "γδ T cells" specifically includes all subsets of γδ T cells, including, but not limited to, Vδ1, Vδ2, and Vδ3 γδ T cells, as well as naive, effector memory, central memory, and terminally differentiated γδ T cells. As a further example, the term "γδ T cells" includes Vδ4, Vδ5, Vδ7, and Vδ8 γδ T cells, as well as Vγ2, Vγ3, Vγ5, Vγ8, Vγ9, Vγ10, and Vγ11 γδ T cells.

[0050] "Encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in a biological process, either having a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, which is the nucleotide sequence identical to that of the mRNA and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to code for the protein or other product of that gene or cDNA.

[0051] "Isolated" means changed or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0052] 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 contain introns.

[0053] "Expression cassette" refers to a nucleic acid comprising an expression control sequence operably linked to a nucleic acid encoding a transcript or polypeptide to be expressed. An expression cassette contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. An expression cassette can be a component of a vector, such as a cosmid, a plasmid (e.g., naked in a liposome or contained in a liposome), or a virus (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus). An expression cassette can be present in a host cell, such as a γδ T cell.

[0054] Chimeric antigen receptor constructs Aspects of the invention include nucleic acids encoding CARs, as well as constructs and vectors comprising such nucleic acids. In some cases, the nucleic acid is a component of, e.g., a heterologous expression cassette. In some embodiments, the nucleic acid is a component of, e.g., a heterologous retroviral vector. In some embodiments, the nucleic acid is a component of, e.g., a heterologous γδ T cell. In some embodiments, the nucleic acid is a component of, e.g., a heterologous γ + T cells and / or delta + It is a component of T cells.

[0055] Described herein are nucleic acids encoding a CAR binding domain that specifically binds to a tumor-associated antigen (TAA) expressed on the surface of a hematological tumor cell. In a preferred embodiment, the binding domain is a CD20 binding domain, such as the CD20 binding domain described in US Patent Application Publication No. 2009 / 0035322 and WO2020 / 072536A9, the contents of each of which references are incorporated by reference in their entirety for all purposes, in particular the binding domains, antibodies, antibody fragments, complementarity determining regions, polypeptides comprising the complementarity determining regions, nucleic acids encoding the complementarity determining regions, and epitope specificity and assays for determining epitope specificity described herein. Typically, the region encoding the binding domain is 5' to the linker region (e.g., the region encoding the CD8α hinge domain).

[0056] Exemplary CD20 binding domains include, but are not limited to, 3B9, 3H7, 2B7, 9C11 or 10F2, or a binding domain that selectively binds to an epitope in CD20 that binds with 3B9, 3H7, 2B7 or 9C11 or 3H7, 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 may comprise a complementarity determining region 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 including a complementarity determining region 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 the sequences provided herein.

[0057] By using known methods, it is possible to determine whether a CD20 binding domain binds to the same epitope as a reference antibody or binding domain or competes for binding with the reference antibody or binding domain. For example, to determine whether a test antibody binds to the same epitope as a reference binding domain, the reference binding domain can be bound to CD20 under saturation conditions. The ability of the test binding domain to bind to the CD20 molecule can then be evaluated. If the binding domain being tested is capable of binding to CD20 following saturation binding with the reference binding domain, it is 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 cannot bind to CD20 following saturation binding with the reference binding domain, the test binding domain may bind to the same epitope as the epitope bound by the reference binding domain.

[0058] To determine whether a binding domain competes for binding with a reference binding domain, the above binding methodology is carried out in two orientations. In the first orientation, the reference binding domain is allowed to bind to CD20 under saturating conditions, followed by evaluation of the binding of the test binding domain to the CD20 molecule. In the second orientation, the test binding domain is allowed to bind to CD20 molecules under saturating conditions, followed by evaluation of the binding of the reference binding domain to the CD20 molecule. In both orientations, if only the first (saturating) binding domain is able to bind to the CD20 molecule, it is concluded that the test binding domain and the reference binding domain compete for binding to CD20. As will be understood by those skilled in the art, a binding domain that competes for binding with a reference binding domain may not necessarily be able to bind to the same epitope as the reference binding domain, but may sterically block the binding of the reference binding domain by binding to an overlapping or adjacent epitope.

[0059] Two binding domains bind to the same epitope or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one binding domain inhibits the binding of the other by at least 50%, for example, 75%, 90%, or even 99%, as measured by competitive binding assay (see, for example, 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 the binding of one binding domain also reduce or eliminate the binding of the other. Two binding domains have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one binding domain also reduce or eliminate the binding of the other.

[0060] Further routine experiments (e.g., peptide mutations and binding analysis) can be performed to ascertain whether the observed lack of binding of the test binding domain is indeed due to binding to the same epitope as the reference binding domain, or whether steric hindrance (or other phenomena) is responsible for the observed lack of binding. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative binding assay available in the art.

[0061] In embodiments, the CD20 binding domain disclosed herein binds to a CD20 epitope that is different from the CD20 epitope bound by rituximab. In embodiments, the CD20 binding domain disclosed herein binds to a CD20 epitope that is different from the CD20 epitope bound by ocrelizumab. In some embodiments, the CD20 binding domain disclosed herein binds to the same or overlapping CD20 epitope that is bound by ocrelizumab. In embodiments, the CD20 binding domain disclosed herein binds to a CD20 epitope that is different from the CD20 epitope that is bound by ofatumumab, or obinutuzumab, or veltuzumab. In embodiments, the CD20 binding domain disclosed herein binds to the same or overlapping CD20 epitope that is bound by ofatumumab, or obinutuzumab, or veltuzumab. In embodiments, the CD20 binding domain disclosed herein binds to a different CD20 epitope than one or more anti-CD20 mAbs described by Luo et al. (2021) Scientific Reports, 11 (3255). In embodiments, the CD20 binding domain disclosed herein binds to the 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, the CD20 binding domain disclosed herein binds to a different CD20 epitope than one or more anti-CD20 mAbs described by Casan et al. (2018) Hum Vaccin Immunother, 14 (12): 2820-2841. In embodiments, the CD20 binding domain disclosed herein binds to the same or overlapping CD20 epitopes bound by one or more anti-CD20 mAbs described by Casan et al. (2018) Hum Vaccin Immunother,, 14(12):2820-2841.

[0062] The present disclosure provides antibodies and CARs having "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 a fragment thereof, indicates that when optimally aligned with another nucleic acid (or the complementary strand of another nucleic acid), there is, 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% identity of the nucleotide bases, as measured by any known sequence identity algorithm, such as FASTA, BLAST, or GAP, as described below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, 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.

[0063] When applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share 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% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage 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 skilled in the art. See, for example, Pearson (1994) Methods Mol.Biol.24:307-331, which is incorporated herein by reference. Examples of groups of amino acids with side chains that have 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: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0064] Sequence identity and / or similarity of polypeptides is usually 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 example, GCG software includes 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, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the most overlapping regions between the query sequence and the search sequence (Pearson (2000) supra). Sequences can also be compared using the Smith-Waterman homology search algorithm, using an affine gapped search with a gap open penalty of 12, a gap extension penalty of 2, and a BLOSUM matrix of 62. Another preferred algorithm for comparing the sequences disclosed herein to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly 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 incorporated herein by reference.

[0065] Provided herein are anti-CD20 CARs that include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more substitutions (e.g., conservative substitutions). For example, the disclosure includes anti-CD20 CARs that have HCVR, LCVR, and / or CDR amino acid sequences with, for example, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid substitution compared to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein. For example, an anti-CD20 CAR may include 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions (such as conservative amino acid substitutions) compared to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3) amino acid sequences disclosed herein.

[0066] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain having a heavy chain complementarity determining region 3 (HCDR3) and a light chain CDR3 (LCDR3), wherein the HCDR3 and LCDR3 are selected from the group consisting of SEQ ID NOs: 1 (AKDPSYGSGSYHSYYGMDV) and 2 (QQRFNWPLT), 3 (VKDFHYGSGSNYGMDV) and 4 (QQSNDWPLT), and 5 (TKDGSYGHFYSGLDV) and 6 (QQRYYWPLT).

[0067] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain having a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence, the HCVR and LCVR sequences being set forth in SEQ ID NOs: 7 (EEQLVESGGDLVQPGRSLRLSCAASGFTFHDYTMH WVRQAPGKGLEWVSGISWNSGSLGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDPSYGSGSYHSYYGMDVWGQGTTVTVSS) and 8 (EIVLTQSPATLSLSPGE RATLSCWASQSISRYLVWYQQKCGQAPRLLIYEASKRATGIPVRFSGSGSGTDFTLTISSLESEDFAVYYCQQRFNWPLTFGGGTKVEIK), 9 (EVQLAESGGDLVQSGRSLRLSCAAS GITFHDYAMHWVRQPPGKGLEWVSGISWNSDYIGYADSVKGRFTISRDNAKKSLYLQMNSLRPDDTALYYCVKDFHYGSGSNYGMDVWGQGTTVTVSP) and 10 (EIVMTQSPATL SMSPGERATLSCRASQSVSRNLAWYQQKVGQAPRLLISGASTRATGIPARFSGSGSGTEFTLTINSLQSEDFAVYYCQQSNDWPLTFGQGTRLEIK), and 11 (EVQLVESGGGLVQPGR SLRLLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSDTIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCTKDGSYGHFYSGLDVWGQGTTVTVSS) and 12 (EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYVASNRATGIPARFSGSGSGTDFTLTISSLEPDDFAVYYCQQRYYWPLTFGGGTKVEIK).

[0068] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain having a heavy chain complementarity determining region 3 (HCDR3) domain and a light chain CDR3 (LCDR3) domain, wherein the HCDR3 domain comprises an amino acid sequence of the formula X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19, where X1=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, and X9=G or F. , X10=S or Y, X11=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, and X19=V or absent, and the LCDR3 domain comprises an amino acid sequence of the formula X1-X2-X3-X4-X5-X6-X7-X8-X9, where 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.

[0069] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain having a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence, wherein the HCVR and LCVR sequences are SEQ ID NOs: 13 (EVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSS) and 14 (EIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQKPGQAPR LLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEI).

[0070] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain that binds to, competes with, or is the same epitope as an anti-CD20 binding domain having a heavy chain complementarity determining region (HCDR) and a light chain complementarity determining region (LCDR), wherein the HCDR and LCDR sequences are the HCVR sequence of SEQ ID NO: 13 and the LCVR sequence of SEQ ID NO: 14, respectively.

[0071] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain that binds to, competes with, or is the same epitope as 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 some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain that binds to, competes with, or is the same epitope as 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 some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain that binds to, competes with, or is the same epitope as 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 some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain having an HCDR1 that comprises SEQ ID NO: 15, an HCDR2 that comprises SEQ ID NO: 16, an HCDR3 that comprises SEQ ID NO: 17, an LCDR1 that comprises SEQ ID NO: 18, an LCDR2 that comprises SEQ ID NO: 19, and an LCDR3 that comprises SEQ ID NO: 20.

[0072] Exemplary binding domains described herein typically comprise a heavy chain region followed by a light chain region (VH-VL) in order from amino terminus to carboxy terminus. Where a particular order of VH and VL regions in a binding domain is explicitly or implicitly described, the disclosure is also understood to describe alternative embodiments in which the order of VH and VL regions is reversed, for example in a CAR comprising an scFV or scFv binding domain. Thus, a description of the VH-VL order also describes alternative VL-VH orders, for example in a CAR comprising an scFV or scFv binding domain. Additionally, a description of the VL-VH order also describes alternative VH-VL orders, for example in a CAR comprising an scFV or scFv binding domain.

[0073] Generally, the nucleic acids encoding the CARs described herein include an extracellular linker portion that encodes a peptide linker that connects the binding domain to the transmembrane domain. Exemplary linker portions include, but are not limited to, a linker portion that encodes a CD8 alpha 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., the CD8 alpha hinge domain) is 3' of the region encoding the binding domain and 5' of the region encoding the transmembrane domain.

[0074] The nucleic acids encoding the CARs described herein include a transmembrane domain. The transmembrane domain can link an extracellular antigen binding domain, e.g., a hinge, to one or more intracellular signaling components. For example, the transmembrane domain can link an antigen binding domain, e.g., a hinge, to a CD3ζ signaling domain and, optionally, one or two costimulatory endodomains. Exemplary transmembrane domains include, but are not limited to, a CD8α transmembrane domain, e.g., SEQ ID NO: 23 (IWAPLAGTCGVLLLSLVITLYC). Typically, the region encoding the transmembrane domain (e.g., CD8α transmembrane domain) is 3' to a region encoding a peptide linker (e.g., CD8α hinge domain) and 5' to a region encoding one or more cytoplasmic domains.

[0075] In some embodiments, the isolated nucleic acid encodes a cytoplasmic region comprising one or more cytoplasmic domains. The region encoding the cytoplasmic region is typically 3' of the region encoding the transmembrane domain. The cytoplasmic domain is typically a signaling domain that provides an activation signal for γδ T cell proliferation, cytotoxic activity and / or proinflammatory cytokine expression (e.g., TNF-α or IFNγ). An exemplary cytoplasmic domain is a CD3ζ signaling domain. In some embodiments, the CD3ζ signaling domain is or comprises SEQ ID NO: 24 (RVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP). In some embodiments, the CD3 zeta signaling domain is or comprises SEQ ID NO: 25 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP). In some embodiments, the cytoplasmic region comprises a plurality (e.g., 2, 3, 4, 5, or 6) signaling domains, such as a plurality (e.g., 2, 3, 4, 5, or 6) CD3 zeta signaling domains, each independently selected from SEQ ID NOs: 24 and 25. In some embodiments, the cytoplasmic region comprises a plurality (e.g., 2, 3, 4, 5, or 6) of non-CD3 zeta signaling domains and a CD3 zeta signaling domain. In some embodiments, the cytoplasmic region comprises a non-CD3 zeta signaling domain and a plurality (e.g., 2, 3, 4, 5, or 6) CD3 zeta signaling domains.

[0076] The cytoplasmic region may include one or more costimulatory domains. The region encoding one or more costimulatory domains may be 5' or 3' of the region encoding the signaling domain. In some embodiments, the region encoding one or more costimulatory endodomains is 5' of the region encoding the signaling domain. In some embodiments, the region encoding one or more costimulatory endodomains is 5' of the signaling domain, and the additional region encoding one or more costimulatory endodomains is 3' of the signaling domain. Exemplary costimulatory endodomains include, but are not limited to, CD28, CD137 (4-1BB), CD278 (ICOS), CD27, CD134 (OX40), Dap10, Dap12, DNAm-1, 2B4, SLAM domain, and TLR2 costimulatory endodomain, and combinations thereof.

[0077] In some embodiments, the construct encodes at least one 4-1BB costimulatory endodomain and optionally a second costimulatory endodomain selected from 4-1BB, 2B4, ICOS, CD28, and CD27 costimulatory endodomains. In some embodiments, the construct encodes at least two 4-1BB costimulatory endodomains or two 4-1BB costimulatory endodomains in combination with one, two, three, or four or more costimulatory endodomains selected from 4-1BB, ICOS, CD28, and CD27. In some embodiments, the 4-1BB costimulatory endodomain comprises SEQ ID NO: 26 (KRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL).

[0078] In some embodiments, the construct encodes one CD27 costimulatory endodomain and optionally a second costimulatory endodomain selected from 4-1BB, ICOS, CD28, and a CD27 costimulatory endodomain. In some embodiments, the construct encodes a CD27 costimulatory endodomain and a 4-1BB costimulatory endodomain. In some embodiments, the construct encodes two CD27 costimulatory endodomains. In some embodiments, the CD27 costimulatory endodomain comprises SEQ ID NO: 27 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQED YRKPEPACSP).

[0079] In some embodiments, the construct encodes 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 activation, cytotoxicity, and / or persistence of T cells (e.g., CAR-T cells). In some embodiments, the construct encodes 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 LIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS). Exemplary common gamma chain cytokines include IL-2 and IL-15. In some embodiments, the common gamma chain cytokine is selected from IL-2, IL-7, and IL-15. In some 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 WO2007 / 037780.

[0080] In some embodiments, the construct encodes one or more multicistronic linker regions, for example between the signaling domain and / or the costimulatory endodomain and the operably linked secretion signal to facilitate secretion of the cytokine. A multicistronic linker region is a region of a polypeptide or RNA sequence that facilitates the generation of multiple distinct polypeptides from a single transcription product. In some embodiments, the multicistronic linker region encodes a cleavage sequence. Suitable cleavage sequences include autocleavage sequences, such as P2A, F2A, E2A, or T2A cleavage sequences, and / or sequences that are cleaved by endogenous proteases, such as Furin.

[0081] In some embodiments, the cleavage sequence is a P2A cleavage sequence. In some embodiments, the cleavage sequence is a furin cleavage sequence. In some embodiments, the cleavage sequence is a P2A and furin cleavage sequence. In some embodiments, the cleavage sequence is a P2A cleavage sequence of SEQ ID NO: 30 (SGSGATNFSLLKQAGDVEENPGP). In some embodiments, the cleavage sequence is a furin cleavage sequence of SEQ ID NO: 31 (RAKR). In some embodiments, the cleavage sequence is a P2A + furin cleavage sequence of SEQ ID NO: 32 (RAKRSGSGATNFSLLKQAG DVEENPGP).

[0082] In some embodiments, the cleavage sequence is or comprises a P2A cleavage sequence of SEQ ID NO: 33 (ATNFSLLKQAGDVEENPGP). In some embodiments, the cleavage sequence is or comprises a F2A cleavage sequence of SEQ ID NO: 34 (VKQTLNNFDLLKLAGDVESNPGP). In some embodiments, the cleavage sequence is or comprises an E2A cleavage sequence of SEQ ID NO: 35 (QCTNYALLKLAGDVESNPGP). In some embodiments, the cleavage sequence is or comprises a T2A cleavage sequence of SEQ ID NO: 36 (EGRSLLTCGDVEENPGP). In certain aspects, multiple self-cleavage sequences may be encoded at the carboxy terminus of the signaling and / or costimulatory domain and the amino terminus of the encoded secreted cytokine (e.g., a general gamma chain cytokine such as IL-15), and the multiple self-cleavage sequences are preferably 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 autocleaving sequences and one or more species of sequences cleaved by an endogenous protease are encoded in the constructs described herein, hi certain embodiments, an endogenous protease recognition site is encoded amino-terminal to the autocleaving sequence.

[0083] In some embodiments, the multicistronic linker region encodes an internal ribosome entry site. An exemplary internal ribosome entry site is encoded by SEQ ID NO:37().

[0084] Another exemplary internal ribosome entry site is encoded by SEQ ID NO:38 (AGCAGGTTTCCCCAACTGACACAAAACGTGCAACTTGAAACTCCGCCTGGTCTTTCCAGGTCTAGAGGGGTAACACTTTGTACTGCGTTTGGCTCCACGCTCGATCCACTGGCGAGTGTTAGTAACAGCACTGTTGCTTCGTAGCGGAGCATGACGGCCGTGGGAACTCCTCCTTGGTAACAAGGACCCACGGGGCCAAAAGCCACGCCCACACGGGCCCGTCATGTGTGCAACCCCAGCACGGCGACTTTACTGCGAAACCCACTTTAAAGTGACATTGAAACTGGTACCCACACACTGGTGACAGGCTAAGGATGCCCTTCAGGTACCCCGAGGTAACACGCGACACTCGGGATCTGAGAAGGGGACTGGGGCTTCTATAAAAGCGCTCGGTTTAAAAAGCTTCTATGCCTGAATAGGTGACCGGAGGTCGGCACCTTTCCTTTGCAATTACTGACCAC).

[0085] 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 in WO2018 / 215787, the sequence described in GenBank Accession No. KP019382.1, and the IRES element disclosed in GenBank Accession No. LT727339.1.

[0086] Additional multicistronic linker regions containing cleavable, self-cleavable, and IRES elements are disclosed in US2018 / 0360992 and US8,865,467.

[0087] In some embodiments, the isolated nucleic acid is SEQ ID NO: 39 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTTPAPTIASQ PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCQRRKYRSNKGESPVEPCHYSCPREEEGSTIPIQEDYRKPEPACSPRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), encoding a 3H7-CD8-CD27z polypeptide which, in order, comprises the following domains: a 3H7 binding domain, a CD8α hinge transmembrane domain, a CD27 costimulatory endodomain, and a CD3ζ signaling domain.

[0088] In some embodiments, the isolated nucleic acid is SEQ ID NO: 40 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGTSTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPLTFGGGTKVEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCVASGFTFNDYAMHWVRQAPGKGLEWVSVISWNSDSIGYADSVKGRFTISRDNAKNSLYLQMHSLRAEDTALYYCAKDNHYGSGSYYYYQYGMDVWGQGTTVTVSSTTTPAPRPPTPAPT IASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), which encodes a 3B9-CD8-BBz polypeptide which, in order, comprises the following domains: a 3B9 binding domain, a CD8α hinge and transmembrane domain, a 4-1BB costimulatory endodomain, and a CD3ζ signaling domain.

[0089] In some embodiments, the isolated nucleic acid is SEQ ID NO: 41 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), which encodes a 3H7-CD8-BBz polypeptide which, in order, contains the following domains: a 3H7 binding domain, a CD8α hinge and transmembrane domain, a 4-1BB costimulatory endodomain, and a CD3ζ signaling domain.

[0090] In some embodiments, the isolated nucleic acid is SEQ ID NO: 42 (MSVPTQVLGLLLLWLTDARCEIVLTQSPATLSLSPGERAALSCRASQSVSNYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIRGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFRDYTMHWVRQGPGKGLEWVSGISWNSDYIGYADSVKGRFTISRDNAKNSLYLQMNSLRVEDTALYYCAKLSGTYRDYFYGVDVWGQGTTVTVSSTTTPAPRPPTTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), encoding a 2B7-CD8-BBz polypeptide which, in order, comprises the following domains: a 2B7 binding domain, a CD8α hinge and transmembrane domain, a 4-1BB costimulatory endodomain, and a CD3ζ signaling domain.

[0091] In some embodiments, the isolated nucleic acid is SEQ ID NO: 43 (MSVPTQVLGLLLLWLTDARCEIVVTQSPATLSLSPGERATLSCRTSQTTTSYLAWYRQKPGQAPRLLIYDASNRAAGIPARFSGSGSGTDFTLTINSLEPEDFAVYYCQLRTNWITFGQGTRLEIKGGGGSGGGGSGGGGQVQLVESGGDSVKPGGSLRLSCAASGFTFSDSYMTWIRQAPGKGLEWVSFISSSGSTIYYADSVKGRFTISRDNVKKSLYLQMNRLRAEDTAVYYCAREEPGNYVYYGMDVWGQGTTVTVSSTTTPAPRPPTTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), encoding a 9C11-CD8-BBz polypeptide which comprises, in order, the following domains: a 9C11 binding domain, a CD8α hinge and transmembrane domain, a 4-1BB costimulatory endodomain, and a CD3ζ signaling domain.

[0092] In some embodiments, the isolated nucleic acid is SEQ ID NO: 44 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSYGKFYYG LDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), which encodes a 3H7-CD3z polypeptide which, in order, contains the following domains: a 3H7 binding domain, a CD8α hinge and transmembrane domain, and a CD3ζ signaling domain.

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] In some embodiments, the isolated nucleic acid comprises a codon-optimized sequence encoding a CD8 alpha hinge region. Exemplary codon-optimized CD8 alpha hinge region nucleic acid sequences include, but are not limited to, SEQ ID NO: 50 (ACCACCACCCCTGCACCAAGGCCCCCGACTCCCGCGCCCACCATCGCGTCA CAGCCTCTTAGCCTGCGACCGGAAGCATGCAGACCAGCTGCCGGGGGGGCCGTGCATACGAGAGGTTTGGACTTCGCCTGCGAT). In some embodiments, the CD8 alpha hinge region is encoded by the following sequence SEQ ID NO: 51 (ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT).

[0099] In some embodiments, the isolated nucleic acid encodes the 3B9 binding domain and comprises the following sequence encoding the CD8 alpha hinge domain SEQ ID NO: 50. In some embodiments, the isolated nucleic acid encodes the 2B7 binding domain and comprises the following sequence encoding the CD8 alpha hinge domain SEQ ID NO: 50. In some embodiments, the isolated nucleic acid encodes the 9C11 binding domain and comprises the following sequence encoding the CD8 alpha hinge domain SEQ ID NO: 50. In some embodiments, the isolated nucleic acid encodes the 3H7 binding domain and comprises the following sequence encoding the CD8 alpha hinge domain SEQ ID NO: 51.

[0100] In some embodiments, the isolated nucleic acid encodes an anti-CD20-CAR polypeptide comprising, in order, SEQ ID NO: 52(*), the following domains: a 3H7 binding domain, a CD8α hinge and transmembrane domain, a 4-1BB costimulatory endodomain, a CD3ζ signaling domain, a P2A cleavage domain (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 53), a secretion signal, and a sIL15 domain.

[0101] In some embodiments, the isolated nucleic acid encodes an anti-CD20-CAR polypeptide comprising, in order, SEQ ID NO: 54(*), the following domains: a 3H7 binding domain, a CD8α hinge and transmembrane domain, a 4-1BB costimulatory endodomain, a CD3ζ signaling domain, a P2A cleavage domain of SEQ ID NO: 53, a secretion signal of SEQ ID NO: 55 (MRISKPHLRSISIQCYLCLLLNSHFLTEAG IHVFILGCFSAGLPKTEA), and a sIL15 domain.

[0102]

[0103]

[0104] In some embodiments, the isolated nucleic acid is a sequence similar to SEQ ID NO:58 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL The isolated nucleic acid encodes an anti-CD20-CAR polypeptide comprising the following domains: 3H7 binding domain, CD8α hinge and transmembrane domains, 4-1BB costimulatory endodomain, and CD3ζ signaling domain, and is linked via an internal ribosome entry site (e.g., encoded by SEQ ID NO: 37) 3' to the region encoding SEQ ID NO: 58, the isolated nucleic acid is encoded by SEQ ID NO: 59 (MALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHP SCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*), a secretion signal of SEQ ID NO:28, and further encoding the sIL15 domain.

[0105]

[0106] In some embodiments, the isolated nucleic acid is a linear nucleic acid. In some embodiments, the isolated nucleic acid is a circular nucleic acid. In some embodiments, the isolated nucleic acid is a vector, such as a plasmid vector, an adenoviral vector, an adeno-associated viral vector, a viral vector, a retroviral vector, or a lentiviral vector. In some embodiments, the isolated nucleic acid, or a contiguous portion of the isolated nucleic acid, for example, comprising a binding domain, a transmembrane domain, and one or more signaling and / or costimulatory endodomains, is integrated into the genome of a host cell, such as a host γδ T cell. In an exemplary embodiment, the isolated nucleic acid is a retroviral vector.

[0107] γδ T cells An embodiment of the invention includes a γδ T cell that functionally expresses an isolated nucleic acid described herein, thereby expressing a CAR on the surface of the γδ T cell.

[0108] Embodiments of the invention may alternatively or additionally include γδ T cells having in vitro or in vivo cytotoxic activity against hematological tumor cells exhibiting cell surface expression of a desired tumor-associated antigen (TAA), e.g., CD20. In some cases, the cytotoxic activity is innate activity. In some cases, the cytotoxicity is at least partially, significantly (>about 25%), or entirely due to the presence of a CAR construct having a binding domain that specifically binds to a TAA expressed on the surface of the hematological tumor cells. In some cases, the γδ T cells exhibit hematological tumor cytotoxic activity that is higher than the innate level of in vitro and / or in vivo hematological tumor cytotoxic activity in a control γδ T cell. In some cases, the control γδ T cell does not comprise a CAR construct. In some cases, the control γδ T cell comprises a CAR construct that lacks a binding domain as described herein, a hinge region as described herein, a transmembrane domain as described herein, a signaling domain as described herein, and / or a costimulatory endodomain as described herein.

[0109] In some cases, the cytotoxicity is at least partially, significantly (>about 25%), or completely due to the presence of a CAR construct having a binding domain that specifically binds to CD20 or an epitope within CD20. In some cases, the γδ T cells functionally express a CD20-specific CAR encoded by an isolated nucleic acid described herein.

[0110] In some embodiments, the γδ T cells described herein may exhibit HLA-restricted (e.g., HLA class I restricted) cytotoxicity. In other embodiments, most (>50%), substantially all (>90%), or all cytotoxic activity is not HLA-restricted (e.g., HLA class I restricted). HLA-restricted cytotoxic activity can be measured by in vitro cytotoxicity against HLA (e.g., HLA class I) (null) tumor cell lines and HLA+ (e.g., HLA class I) (null) tumor cell lines. + ) can be assessed by comparing in vitro cytotoxicity against tumor cell lines. In some embodiments, the HLA-restricted cytotoxic activity is at least partially, significantly (>25%), or completely provided by the use of a T cell receptor-like binding domain. A T cell receptor like binding domain is a binding domain that specifically recognizes an antigen when presented on the surface of a cell in a complex with an MHC molecule. T cell receptor-like binding domains are further described, for example, in WO2016 / 199141.

[0111] The γδ T cells described herein can exhibit robust and / or sustained hematologic tumor cytotoxic activity. In some cases, the hematologic tumor cytotoxic activity can persist for at least about 6-120 days, or at least about 6-180 days, from initial contact with hematologic tumor cells. In some cases, the hematologic tumor cytotoxic activity of the γδ T cells described herein or their progeny can persist for at least about 6-120 days, or at least about 6-180 days, from initial contact with hematologic tumor cells or from administration of the γδ T cells described herein. This sustained hematologic tumor cytotoxic activity can be exhibited in vitro, in vivo, or both in vitro and in vivo.

[0112] Aspects of the invention may additionally or alternatively include γδ T cells that proliferate in response to contact with a cell exhibiting cell surface expression or overexpression of a tumor-associated antigen (TAA) (e.g., CD20). The cell exhibiting cell surface expression or overexpression of a tumor-associated antigen (TAA) may be a normal blood cell, such as a normal B cell. The cell exhibiting cell surface expression or overexpression of a tumor-associated antigen (TAA) may be a hematological tumor cell. In some cases, the proliferation is an innate activity. In some cases, the proliferation is at least partially, significantly (>about 20% or >about 25%), or entirely due to the presence of a CAR construct having a binding domain that specifically binds to a TAA expressed on the surface of a blood cell or hematological tumor cell. In some cases, the γδ T cells exhibit a higher level of proliferation in vitro and / or in vivo compared to a control γδ T cell. In some cases, the control γδ T cells do not comprise a CAR construct. In some cases, the control γδ T cell comprises a CAR construct that lacks a binding domain described herein, a hinge region described herein, a transmembrane domain described herein, a signaling domain described herein, and / or a costimulatory endodomain described herein.

[0113] In some cases, the proliferation is at least partially, significantly (>about 20% or >about 25%), or entirely due to the presence of a CAR construct having a binding domain that specifically binds to CD20 or an epitope within CD20. In some cases, γδ T cells that exhibit proliferation in response to contact with blood cells or hematological tumor cells that exhibit cell surface expression of CD20 functionally express a CD20-specific CAR encoded by an isolated nucleic acid described herein.

[0114] The γδ T cells described herein may exhibit robust and / or sustained proliferation in a host organism comprising hematological cells or hematological tumor cells exhibiting cell surface expression or overexpression of a tumor associated antigen (TAA) (e.g., CD20). In some cases, proliferation may persist for at least about 6-120 days, or at least about 6-180 days, from initial contact with hematological tumor cells or from the date of administration of the γδ T cells to the host organism. In some cases, proliferation of the γδ T cells described herein or their progeny in a host organism comprising hematological cells or hematological tumor cells exhibiting cell surface expression or overexpression of a tumor associated antigen (TAA) may persist for at least about 6-120 days, or at least about 6-180 days, from initial contact with hematological cells or from the date of initial administration of the γδ T cells to the host organism. In some cases, proliferation in the host organism is at least partially, significantly (>about 20% or >about 25%), or entirely due to the presence of a CAR construct having a binding domain that specifically binds to CD20 or an epitope within CD20. In some cases, γδ T cells exhibiting proliferation in a host organism that includes blood cells or hematological tumor cells that exhibit cell surface expression of CD20 functionally express a CD20-specific CAR encoded by an isolated nucleic acid described herein.

[0115] In some embodiments, a γδ T cell described herein expresses or persistently expresses a proinflammatory cytokine, such as, but not limited to, tumor necrosis factor alpha or interferon gamma, following contact with blood cells or hematologic tumor cells. In some embodiments, a γδ T cell described herein or its progeny expresses or persistently expresses a proinflammatory cytokine, such as, but not limited to, tumor necrosis factor alpha or interferon gamma, following contact with blood cells or hematologic tumor cells, such as in a host organism that contains the blood cells or hematologic tumor cells.

[0116] In some embodiments, the γδ T cells or pharmaceutical compositions comprising γδ T cells exhibit substantially no or no graft-versus-host response when introduced into an allogeneic host. In some embodiments, the γδ T cells or pharmaceutical compositions comprising γδ T cells exhibit a clinically acceptable level of graft-versus-host response when introduced into an allogeneic host. In some embodiments, a clinically acceptable level is an amount of graft-versus-host response that does not require discontinuation of γδ T cell therapy to achieve a therapeutically effective treatment. In some embodiments, a clinically acceptable level of graft-versus-host response (GvHD) is an acute response less severe than grade C according to the applicable IBMTR grading scale. The severity of acute graft-versus-host reaction is determined by assessing the extent of skin, liver and gastrointestinal involvement. The stages of involvement of individual organs are combined to generate an overall grade that has prognostic significance. Grade I (A) GvHD is characterized as mild disease, grade II (B) GvHD as moderate, grade III (C) as severe, and grade IV (D) as life-threatening. The IBMTR grading system defines the severity of acute GvHD as follows (Rowlings et al., Br J Haematol 1997;97:855): Grade A - No liver or gastrointestinal involvement, only stage 1 skin involvement (maculopapular rash on <25% of the body) Grade B - Stage 2 skin lesions, Stage 1 to 2 intestinal or liver lesions Grade C - Stage 3 disease in any organ system (generalized erythroderma, bilirubin 6.1-15.0 mg / dL, diarrhea 1500-2000 mL / day) • Grade D - Stage 4 involvement of any organ system (generalized erythroderma with blister formation, bilirubin >15 mg / dL, diarrhea >2000 mL / day or pain or ileus). See also, e.g., Tables 1 and 2 in Schoemans et al., Bone Marrow Transplantation volume 53, pages 1401-1415 (2018), which discloses criteria for assessing and grading acute GvHD.

[0117] In some embodiments, the γδ T cells, or pharmaceutical compositions comprising γδ T cells, exhibit a reduced or substantially reduced graft-versus-host response when introduced into an allogeneic host compared to the graft-versus-host response exhibited by a control αβ T cell, or a control pharmaceutical composition comprising a control αβ T cell, administered to the allogeneic host. In some cases, the control αβ T cells are allogeneic, unengineered control αβ T cells. In some cases, the control αβ T cells do not comprise a CAR or do not comprise the same CAR as the reference γδ T cell.

[0118] The γδ T cells described herein can be δ1, δ2, δ3 or δ4 γδ T cells, or a combination thereof. In some cases, the γδ T cells are mostly (>50%), substantially (>90%), essentially all, or entirely δ2 - In some cases, the γδ T cells are mostly (>50%), substantially (>90%), essentially all, or entirely δ1 γδ T cells.

[0119] The γδ T cells can be obtained from allogeneic or autologous donors. The γδ T cells can be partially or completely purified or unpurified and expanded ex vivo. Methods and compositions for ex vivo expansion include, but are not limited to, those described in WO2017 / 197347. Expansion can be performed before or after, or before and after, the CAR construct is introduced into the γδ T cell(s).

[0120] The γδ T cells described herein may be preserved, for example cryopreserved, for use in adoptive cell transfer.

[0121] Biomarkers, detection methods, and uses thereof A biomarker is a biological indicator of disease or treatment efficacy that can be measured in vivo by biomedical / molecular imaging and other in vitro or laboratory methods. As disclosed herein, one or more biomarkers can be advantageously relied upon to inform cell activation, efficacy of treatment, and / or subsequent treatment regimens. As described herein, with respect to administration of anti-CD20 CAR γδ T cells to a subject in need thereof, one or more biomarkers can be relied upon as an indicator(s) of efficacy, potential efficacy, or lack of efficacy, for example, in terms of promoting an anti-tumor effect in the subject. 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 the dosage level (e.g., increase, decrease, or maintain the same dosage of anti-CD20 CAR γδ T cells), whether to include one or more additional or alternative therapies, whether to adjust a previously planned dosing schedule, whether to administer anti-CD20 CAR γδ T cells derived from the same or a different donor, or whether to stop / postpone treatment or discontinue treatment altogether, etc.

[0122] In embodiments, activation and / or expansion of the administered anti-CD20 CAR γδ T cells can be monitored by flow cytometric detection of CAR+ γδ T cells and / or via quantitative polymerase chain reaction (qPCR) detection of the anti-CD20 CAR transgene. Preferably, such methodology(s) is performed at several time points following administration of the anti-CD20 CAR γδ T cells, e.g., daily, every other day, every third day, every fourth day, etc., for up to 14 days, 28 days, 2 months, 3 months, or more, since it is well known that the presence and status of CAR-T cells in peripheral blood can change over time (Shah et al. (2020) Nat Med., 26:1569-75). In the art, PCR results have been reported to correlate with CAR surface expression monitored by flow cytometry, but flow cytometry has the advantage of allowing for the identification and characterization of CAR-T cell subpopulations and immune cells of the patient rapidly and at the single cell level. Furthermore, flow cytometry can detect CARs at a proteomic level and thus provide information regarding CAR cell function. Experimental formats for methods relying on one or more of flow cytometry, qPCR, and / or other detection methodologies to monitor activation and / or expansion of anti-CD20 CAR γδ T cells are readily determined by one of skill in the art, for example, as shown and described in Example 1 below and described, for example, by Hu and Huang (2020) Front. Immunol., 11 (1770).

[0123] In embodiments, the anti-CD20 CAR γδ T cells administered to a subject may induce the release of one or more cytokines. In embodiments, the one or more cytokines are secreted from the anti-CD20 CAR γδ T cells. In further or alternative embodiments, the one or more cytokines are secreted from cells other than the anti-CD20 CAR γδ T cells, including, for example, T cells, NK cells, dendritic cells, and macrophages. In embodiments, the induction of one or more inflammatory cytokines can alleviate immune suppression caused by the tumor microenvironment, which in turn can improve clinical responses to anti-CD20 CAR γδ T cell therapy.

[0124] In embodiments, the one or more cytokines are biomarkers of cell activation and / or therapeutic efficacy of the anti-CD20 CAR γδ T cell therapy disclosed herein. Relevant cytokines may include, but are not limited to, INFγ, GM-CSF, IL-2, IL-7, IL-15, TNFα, IL-1β, IL-6, IL-8, IL-10, MIP1α, MIP1β, CRP, ferritin, monocyte chemoattractant protein-1 (MCP-1), 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. In a preferred embodiment, the one or more cytokines comprise or consist of IL-2 and IL-8.

[0125] In embodiments, induction of cytokines for use as biomarkers of therapeutic efficacy occurs within a time frame of 1 day or less to 28 days after administration of the anti-CD20 CAR γδ T cells. In embodiments, the time frame is 1 day or less to 21 days, or 18 days, or 14 days, or 10 days after administration of the anti-CD20 CAR γδ T cells. In some embodiments, the cytokine biomarker comprises IL-8, and induction of IL-8 occurs 1 day or less to 28 days, such as 1 day or less to 21 days, such as 1 day or less to 14 days after administration of the anti-CD20 CAR γδ T cells. In some further or alternative embodiments, the cytokine biomarker comprises IL-2, and induction of IL-2 occurs 1 day or less to 28 days, such as 1 day or less to 21 days, such as 1 day or less to 14 days after administration of the anti-CD20 CAR γδ T cells. In some further or alternative embodiments, induction of cytokines for use as biomarkers occurs within a time frame of 1 day or less after LD to 28 days after administration of the anti-CD20 CAR γδ T cells. In embodiments, such a time frame is 1 day or less after LD to 21 days, or 18 days, or 14 days, or 10 days after administration of the anti-CD20 CAR γδ T cells. In some embodiments, the cytokine biomarker comprises IL-8, and induction of IL-8 occurs 1 day or less after LD to 28 days after administration of the anti-CD20 CAR γδ T cells, such as 1 day or less after LD to 21 days after administration of the anti-CD20 CAR γδ T cells, such as 1 day or less after LD to 14 days after administration of the anti-CD20 CAR γδ T cells. In some further or alternative embodiments, the cytokine biomarker comprises IL-2 and the induction of IL-2 occurs no more than 1 day after LD to 28 days after administration of the anti-CD20 CAR γδ T cells, such as no more than 1 day after LD to 21 days after administration of the anti-CD20 CAR γδ T cells, such as no more than 1 day after LD to 14 days after administration of the anti-CD20 CAR γδ T cells.

[0126] Measurement of serum levels of a single cytokine is typically performed using enzyme-linked immunosorbent assays (ELISA) and / or chemiluminescence assays, and multiplex bead-based assays can be used to determine serum levels of multiple 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 prior to administration of anti-CD20 CAR γδ T cells, e.g., prior to lymphodepletion and / or after / during lymphodepletion, but prior to anti-CD20 CAR γδ T cells. In additional or alternative embodiments, serum levels of one or more cytokines are measured following administration of anti-CD20 CAR γδ T cells. In embodiments, serum levels of one or more cytokines for use as biomarkers are measured prior to administration of anti-CD20 CAR γδ T cells (e.g., 1-7 days prior to administration) and / or one or more times up to about 28 days following administration of anti-CD20 CAR γδ T cells. In embodiments, multiple measurements of serum levels of one or more cytokines, encompassing a time frame before and / or after administration of anti-CD20 CAR γδ T cells, provide a time course of induction of one or more cytokines. Such a time course can be used to establish peak serum levels of the one or more cytokines, and / or the time course can be used to establish an approximate total level of cytokine induction during the time course. It is within the scope of the present disclosure for the peak levels of one or more cytokines to be used as a biomarker metric. Additionally or alternatively, it is within the scope of the present disclosure for the total release level of one or more cytokines to be used as a biomarker metric.

[0127] In embodiments, the presence of a biomarker (e.g., a cytokine) is confirmed in response to the biomarker being measured above a certain predetermined threshold, for example, after administration of anti-CD20 CAR γδ T cells. In embodiments, the biomarker is IL-8 and the presence of the biomarker is confirmed in response to a serum level of IL-8 reaching or exceeding about 100 pg / mL, or about 125 pg / mL, or about 150 pg / mL, or about 175 pg / mL, or about 200 pg / mL within a predetermined period of time (e.g., 21 days or less) after administration of the anti-CD20 CAR γδ T cells. In further or alternative embodiments, the biomarker is IL-2 and the presence of the biomarker is confirmed in response to a serum level of IL-2 reaching or exceeding about 75 pg / mL, or about 80 pg / mL, or about 85 pg / mL within a predetermined period of time (e.g., 21 days or less) after administration of the anti-CD20 CAR γδ T cells.

[0128] In embodiments, confirmation of the presence of one or more cytokine biomarkers described herein is used to inform subsequent treatment. For example, in response to confirmation of a cytokine biomarker after administration of a first dose of anti-CD20 CAR γδ T cells, a second dose may be optional, or the dosage of the corresponding second dose may be adjusted accordingly (e.g., remain the same as the first dose or be reduced). In additional or alternative embodiments, lack of confirmation of a cytokine biomarker after administration of a first dose of anti-CD20 CAR γδ T cells may indicate the need for a second dose (e.g., with or without another lymphodepletion step), for the cell dosage to be increased for said second dose, and / or for the second dose to include anti-CD20 CAR γδ T cells derived from a different donor compared to the first dose. Similar logic additionally or alternatively applies to indicators of the presence or absence of biomarkers indicative of in vivo activation and / or expansion of the administered anti-CD20 CAR γδ T cells, as described above.

[0129] In additional or alternative embodiments, an indication of minimal residual disease (MRD) may serve as a biomarker to inform subsequent treatment regimens. As discussed herein, MRD refers to a quantity of cancer cells remaining in a subject's body after a course of treatment (e.g., administration of one or more doses of anti-CD20 CAR γδ T cells). In embodiments, MRD analysis is performed for some predefined duration after the last administration of anti-CD20 CAR γδ T cells. In embodiments, the duration is at least 20 days, e.g., at least 25 days, e.g., at least 28 days, e.g., at least 30 days, after the last administration of anti-CD20 CAR γδ T cells. In embodiments, a positive MRD test indicates disease that continues to be detected after treatment, whereas a negative MRD test indicates disease that is not detected after treatment. In embodiments, a positive MRD test may indicate the need for an additional therapeutic regimen, e.g., a second course of treatment comprising administration of another round of anti-CD20 CAR γδ T cells, preferably at a higher cell dose, and preferably including an additional lymphodepletion step. In some embodiments, the first course of treatment comprising administration of anti-CD20 CAR γδ T cells is administered after a standard course of lymphodepletion (e.g., 30 mg / m 2 / day fludarabine + 500mg / m 2 / day cyclosporamide for 3 days), and in response to a positive MRD test, a second course of treatment may be followed by an intensified lymphodepletion step (e.g., 30 mg / m 2 / day fludarabine + 1000mg / m 2 / day of cyclosporamide for three days.

[0130] In embodiments, MRD analysis is performed via one or more of multiparameter flow cytometry and immunosequencing known in the art (see, e.g., Wood et al. (2018) Blood, 131(12):1350-1359).

[0131] Methods for inhibiting or killing tumor cells One or more non-engineered γδ T cell populations, engineered γδ T cell populations and / or mixtures thereof having cytotoxic activity against hematological tumor cells may be administered to a subject in any order or simultaneously. If administered simultaneously, multiple non-engineered γδ T cell populations, engineered γδ T cell populations and / or mixtures thereof of the invention may be provided in a single unified form, such as an intravenous injection, or in multiple forms, such as multiple intravenous infusions, subcutaneous injections or tablets. Non-engineered γδ T cell populations, engineered γδ T cell populations and / or mixtures thereof of the invention may be packaged together or separately in a single package or multiple packages. One or all of the non-engineered γδ T cell populations, engineered γδ T cell populations and / or mixtures thereof of the invention may be given in multiple doses. If not administered simultaneously, the timing between multiple administrations may vary to about one week, one month, two months, three months, four months, five months, six months, or up to about one year. In some cases, the non-engineered enriched γδ T cell populations, engineered enriched γδ T cell populations, and / or mixtures thereof of the present invention may be expanded in vivo in the subject's body after administration to the subject. One or more non-engineered γδ T cell populations, one or more engineered γδ T cell populations, and / or mixtures thereof may be frozen to provide cells for multiple treatments with the same cell preparation. One or more non-engineered γδ T cell populations, one or more engineered γδ T cell populations, and / or mixtures thereof of the present disclosure and pharmaceutical compositions comprising same may be packaged as a kit. The kit may include instructions (such as written instructions) for the use of the non-engineered γδ T cell populations, engineered γδ T cell populations, and / or mixtures thereof and compositions comprising same.

[0132] In some cases, the method of treating a B cell malignancy comprises administering to a subject a therapeutically effective amount of a non-engineered γδ T cell population, an engineered γδ T cell population, and / or a mixture thereof, where the administration treats the B cell malignancy (e.g., expresses CD20 on the cell surface). In some embodiments, the therapeutically effective amount of a non-engineered γδ T cell population, an engineered γδ T cell population, and / or a mixture thereof is 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 some embodiments, the therapeutically effective amount of a non-engineered γδ T cell population, an engineered γδ T cell population, and / or a mixture thereof is administered for at least 1 week. In some embodiments, a therapeutically effective amount of a non-engineered γδ T cell population, an engineered γδ T cell population, and / or a mixture thereof is administered for at least two weeks.

[0133] The unengineered γδ T cell population, engineered γδ T cell population, and / or mixtures thereof described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administration of the pharmaceutical composition comprising the γδ T cell population can vary. For example, the γδ T cell population can be used as a prophylactic and can be administered continuously to a subject with a condition or disease tendency to reduce the likelihood of the disease or condition developing. The initial administration can be via any practical route, such as by any route described herein using any formulation described herein. In some embodiments, administration of the γδ T cell population of the present disclosure is intravenous administration. One or more doses of the γδ T cell population can be administered as soon as practicable after the onset of the hematological cancer and for a time required to treat the immune disease, 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 some embodiments, one or more doses of the γδ T cell population may be administered years after the onset of cancer, and before and after other treatments.

[0134] In some embodiments, the γδ T cell population is administered simultaneously or sequentially with one or more methods of increasing common gamma chain cytokine(s). As used herein, "one or more methods of increasing common gamma chain cytokine(s)" refers to a method or combination of methods that alter the physiological state of a subject such that the level of at least one common gamma chain cytokine is increased in the subject. In some embodiments, the method increases the level of one or more common gamma chain cytokine(s) selected from the group consisting of IL-2, IL-7, and IL-15, preferably wherein the method increases the level of IL-15 in the subject. In some embodiments, the method includes lymphodepletion. In some embodiments, the method includes administering one or more common gamma chain cytokine(s) to the subject. In some cases, IL-2, IL-7, and / or IL-15, preferably IL-15, are administered. In some embodiments, the method includes secreting the common gamma chain cytokine(s), such as from the administered γδ T cells. Optionally, IL-2, IL-7 and / or IL-15, preferably IL-15, is secreted.

[0135] In some embodiments, the one or more administration methods for increasing the general gamma chain cytokine(s) include lymphodepletion prior to the introduction of the γδ T cell(s). In some embodiments, the one or more administration methods for increasing the general gamma chain cytokine(s) include administering an effective amount of a general gamma chain cytokine(s) simultaneously with or consecutively with the introduction of the γδ T cell(s) to increase the proliferation, cytotoxic activity, persistence, or a combination thereof, of the introduced γδ T cell(s), preferably the method includes administering IL-2 or one or more mimetics thereof, more preferably the method includes administering IL-15 or one or more mimetics thereof. The dosage of the general gamma chain cytokine(s) may increase the proliferation, cytotoxic activity, persistence, or a combination thereof, of the introduced γδ T cell(s) prior to and / or after the introduction of the γδ T cell(s). Exemplary amounts of IL-15 include, but are not limited to, 0.01-10 μg / kg / dose every 24 hours for IL-15. An exemplary amount of IL-2 is about 3×10 6 ~Approx. 22×10 6 For example, the dosing regimen for IL2 in RCC is 600,000 international units / kg (0.037 mg / kg) IV every 8 hours infused over 15 minutes for a maximum of 14 doses.

[0136] In some embodiments, the one or more administration methods to elevate the general gamma chain cytokine(s) comprise lymphodepletion prior to administration of the γδ T cell(s), prior to simultaneous administration with the introduced γδ T cell(s), or prior to sequential administration of the general gamma chain cytokine(s) effective to increase the proliferation, cytotoxic activity, persistence, or a combination thereof, of the introduced γδ T cell(s).

[0137] It is understood that other suitable lymphodepletion methods can be utilized in the methods of the present disclosure. Exemplary lymphodepletion methods are disclosed, for example, in 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. EXAMPLES

[0138] The following examples are presented 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 present 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, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0139] Example 1. Phase I anti-CD20 γδ CAR T cells in humans This example demonstrates the safety profile, evidence of cell expansion, and pharmacodynamic implications of anti-CD20 allogeneic γδ CAR T cell therapy.

[0140] A first-in-human (FIH) trial was conducted on a set of NHL patients who had relapsed from at least three prior lines of therapy. Six patients were enrolled. Five of the patients had a ≥3 × 10 7Although patients were administered 1 × 10 CAR+ cells, two of the five patients enrolled had evaluable non-dose-limiting toxicities and were therefore excluded from further analysis. Of the six patients initially enrolled, one patient received 1 × 10 8 Thus, in the study setting, the safety subset included 6 patients (all patients who received anti-CD20 allogeneic γδ CAR T-cell therapy) and the efficacy subset included 4 patients who completed at least one response evaluation.

[0141] The study design consisted of four phases. The first phase (day -5 to day 0) included a lymphodepletion (LD) step, followed by treatment in the second phase (day 0 to day 28). The second phase included a dose escalation study with anti-CD20 allogeneic γδ CAR T-cell therapy. At the end of the second phase, patient response and safety evaluations were performed. The third phase (day 28 to month 12) included an early follow-up phase (response and safety evaluations at months 3, 6, 9, and 12). The fourth phase included a long-term follow-up study. The LD in the first phase included either standard LD (sLD) or enhanced LD (eLD). sLD was 30 mg / m 2 / day fludarabine + 500mg / m 2 The eLD included 30 mg / m2 of cyclosporamide per day for 4 days. 2 / day fludarabine + 1000 mg / m for 3 days 2 / day of cyclosporamide.

[0142] Patient characteristics Subject 101-104-001 is a 62-year-old female with transformed DLBCL (derived from CLL). This subject has been previously treated with 5 prior lines of therapy, with the best response being progressive disease (PD), including: 1) R-CHOP, 2) rituximab-abbs, gemcitabine, and CDDP, 3) rituximab-abbs, gemcitabine, carboplatin, 4) polatuzumab + BR x 2, and 5) obinutuzumab-hypercyclophosphamide and dexamethasone. For the study, this subject received sLD, 3 x 10 7 Each patient received anti-CD20 allogeneic γδ CAR T cells.

[0143] Subject 101-108-009 is a 66 year old female with transformed high grade B cell neoplasm (FL origin). This subject was previously treated with 4 prior lines of therapy including 1) R-CHOP, 2) ibrutinib, 3) bendamustine / rituximab, and 4) rituximab. For the study, this subject received sLD and 3×10 7 Each patient received anti-CD20 allogeneic γδ CAR T cells.

[0144] Subject 101-108-010 is a 75 year old male with DLBCL. This subject was previously treated with 5 prior lines of therapy including: 1) R-CHOP;IT MTX, 2) liso-cel, 3) liso-cel (reinfusion), 4) Revlimid, and 5) tafasitamab-cxix. For the study, this subject received eLD and 3×10 7 Each patient received anti-CD20 allogeneic γδ CAR T cells.

[0145] Subject 101-108-012 is a 62 year old male with MCL. This subject has been previously treated with 5 prior lines of therapy including: 1) bendamustine / rituximab, 2) zanubrutinib, 3) bendamustine / obinutuzumab, 4) bendamustine / rituximab, and 5) rituximab / gemcitabine / dex / carboplatin. For the study, this subject received eLD and 1×10 8Each patient received anti-CD20 allogeneic γδ CAR T cells.

[0146] All of the above subjects completed the DLT protocol. The following two patients did not complete the DLT period but are included herein for reference:

[0147] Subject 101-104-002 is a 29 year old male with primary refractory Burkitt lymphoma. This subject has been previously treated with three prior lines of therapy including: 1) R-CODOC-M / R-IVAC, 2) cy / flu / rituximab + NK cell trial (FK516), and 3) R-EPOCH. For the study, this subject received sLD and 3×10 7 Each patient received anti-CD20 allogeneic γδ CAR T cells.

[0148] Subject 101-102-004 is a 52 year old male with double hit DLBCL. This subject was previously treated with 5 prior lines of therapy including: 1) DA-EPOCH R with IT MTX / ARA-C, 2) R-ICE, 3) polatuzumab / rituximab, 4) tisagenlecleucel, and 5) gemcitabine / oxyliplatin. For the study, this subject received sLD and 3×10 7 Each patient received anti-CD20 allogeneic γδ CAR T cells.

[0149] Table 1 below shows the safety profile data corresponding to the study. [Table 1]

[0150] For the data shown in Table 1, the data correspond to an N of 6 (all enrolled patients). No DLTs were observed, no ICANs were observed, no GvHDs were observed, and no grade 3+ CRSs were observed. Regarding infection subcategories, one subject had COVID-19 and pneumonia, and one subject had Candida.

[0151] Table 2 below shows the responses obtained in efficacy-evaluable patients. [Table 2]

[0152] Thus, the data presented in Table 2 show an overall response rate (ORR) of 75% (3 / 4 patients) and a CR of 50% (2 / 4 patients).

[0153] Antitumor Response Fluorodeoxyglucose (FDG) positron emission tomography (PET) was used to monitor cancerous lesions before and after treatment in subjects enrolled in a study with anti-CD20 allogeneic γδ CAR T cells. Figures 1A-1D show data obtained from subject 101-108-009. Figures 1A and 1C show baseline FDG uptake by tumor lesions imaged from the frontal and lateral views, respectively, and Figures 1B and 1D show the site of tumor response obtained 28 days after administration of anti-CD20 allogeneic γδ CAR T cells from the frontal and lateral views, respectively. Each of Figures 1A-1D also shows FDG uptake by normal tissues. Tumor response was evaluated as immune-related response (PR) according to Lugano 2014. This subject had 3×10 7 There was a near complete response from administration of anti-CD20 allogeneic γδ CAR T cells (see Table 2 above).

[0154] Figures 2A-2D show FDG-PET data obtained from subject 101-108-010. Figures 2A and 2C show baseline FDG uptake by tumor lesions shown from sagittal plane of the right leg and transverse view of the pelvis, respectively. Figures 2B and 2D show the site of tumor response obtained 28 days after administration of anti-CD20 allogeneic γδ CAR T cells from sagittal plane of the right leg and transverse view of the pelvis, respectively. This subject received 3×10 7 had a complete response from administration of anti-CD20 allogeneic γδ CAR T cells (see Table 2 above).

[0155] Figures 3A-3D show FDG-PET data obtained from subject 101-108-012. Figures 3A and 3C show baseline FDG uptake due to tumor lesions, as shown from the frontal and pelvic cross-sectional views, respectively. Figures 3B and 3D show the sites of tumor response obtained on day 28 after administration of anti-CD20 allogeneic γδ CAR T cells, from the frontal and pelvic cross-sectional views, respectively. Each of Figures 3A-3B also shows FDG uptake by normal tissue. This subject had a complete response following administration of 1×10 8 cells of anti-CD20 allogeneic γδ CAR T cells (see Table 2 above).

[0156] In Vivo Expansion and Pharmacodynamic Biomarkers Pharmacodynamic Biomarkers Blood samples for serum cytokine analysis were collected on day -5 before lymphodepletion, day -1 after lymphodepletion, day 1 before infusion, and at 2 and 6 hours (+ / -15 minutes) after infusion, and on days 2, 3, 5, 7, 10, 11, 14, 21, 28, and 3 months (+ / -1 day) after administration of anti-CD20 allogeneic γδ CAR T cells. The samples collected were shipped to the central laboratory for analysis.

[0157] The results are shown graphically in Figures 4A-4C for subjects 101-104-002, 101-104-001, 101-102-004, and 101-108-009. The data points in the shape of an "X" were below the lower limit of quantification (<LLOQ).

[0158] In Vivo Expansion Anti-CD20 allogeneic γδ CAR T cell levels in peripheral blood were measured by flow cytometry on days -1, 1, 3, 5, 10, and 21. Figure 5A shows flow cytometry data gated on CD3+ cells, and Figure 5B shows a negative control using PBMCs. Figure 5C shows flow cytometry data gated on CD3+ and Vδ1+ cells, and Figure 5D shows a positive control using CAR+ Jurkat cells spiked into PBMCs (gated on CD3+ cells). This data shows the in vivo expansion of CAR+ γδ T cells.

[0159] Clinical Trial Updates Methods - This multicenter Phase I clinical trial is evaluating ADI-001 in adults with relapsed / refractory B-cell lymphoma. Eligibility criteria included the presence of measurable disease, expression of CD20 on tumor cells, and ≥2 prior systemic therapies. All patients received conditioning therapy with fludarabine and cyclophosphamide. ADI-001 can be administered at four dose levels (DL) (DL1: 3E7, DL2: 1E8, DL3: 3E8, and DL4: 1E9 CAR+ cells) in a 3+3 dose escalation scheme. Patients were considered evaluable if they completed the 28-day DLT period. In DL3, patients could receive a second course of conditioning therapy and be re-administered with ADI-001 if they were free of DLTs during the first 28 days, free of progressive disease by PET / CT assessment on day 28, and had resolved from cytopenias. Treatment-emergent adverse events were classified according to CTCAE v5.0, and immune effector cell-associated neurological syndrome (ICANS) and cytokine release syndrome (CRS) assessments were performed according to the ASTCT criteria. Objective response rates (ORR) were assessed by independent radiological review according to the Lugano 2014 criteria.

[0160] Results - As of July 15, 2022, 11 patients were enrolled and 9 were evaluable. Of these 9 patients, 6 (67%) were male and the median age was 62 years (range 45-75 years). Eight patients had large B-cell lymphoma (LBCL) and 1 had mantle cell lymphoma (MCL). Of the 8 patients with LBCL, 5 had diffuse large B-cell lymphoma (DLBCL), 2 had high-grade B-cell lymphoma with double / triple hit (HGBCL), and 1 had HGBCL not otherwise specified. At baseline, the median tumor burden was 2,974 (150-7,919) mm 2 of whom 89% (8 / 9) had stage III / IV disease. Median number of prior therapies was 4 (range 2-5). Four patients had received prior anti-CD19 CAR T-cell therapy (2 Liso-cel and 2 Axi-cel). Of the 9 evaluable patients, 3 patients were treated in each of DL1, DL2, and DL3. Two patients in DL3 were re-administered a second course of ADI-001.

[0161] As shown in Figure 6, two patients developed CRS: one grade 1 and one grade 2. One patient developed grade 1 ICANS, which resolved within 24 hours. There was no CRS or ICANS ≥ grade 3. The only relevant SAEs were grade 2 CRS, grade 1 ICANS, and grade 3 adenoviremia. No GvHD or protocol-defined DLT events were reported. As shown in Figure 7, the best ORR was 78% (7 / 9), with a complete response (CR) rate of 78% (7 / 9). In the four patients who received prior CD19 CAR T therapy, the ORR was 100% (4 / 4), with a CR rate of 100%. As of the data cutoff date, of the seven patients who achieved CR, two patients progressed, one died during complete remission, and four were in CR and active follow-up, with follow-up durations ranging from 1.2 to 8.8 months. Based on flow cytometry, CAR+ γδ T cell kinetics was improved in a dose-dependent manner, with peak cell expansion occurring between days 7 and 10 in DL3.

[0162] Conclusions - ADI-001 γδ CAR T cells maintained a favorable safety profile. Preliminary efficacy showed encouraging CR rates and sustained durability in patients, including those with prior exposure to CAR T therapy.

[0163] The sequence information disclosed herein is summarized in Table 3 below. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

[0164] The above embodiments and examples are intended to be merely illustrative and non-limiting. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific compounds, materials, and procedures. All such equivalents are considered to be within the scope of the invention and are covered by the appended claims.

Claims

1. A pharmaceutical for treating relapsed / refractory (R / R) B-cell malignancies in a subject in need of treatment, comprising a therapeutically effective amount of anti-CD20 CAR γδ T cells expressing a chimeric antigen receptor (CAR) comprising a binding domain that specifically binds to CD20 on malignant B cells, wherein the subject has previously been treated with at least one, at least two, or at least three prior therapies.

2. 2. The method of claim 1, wherein the subject has relapsed from and / or is refractory to a previous therapy comprising an anti-CD20 monoclonal antibody, and optionally the anti-CD20 monoclonal antibody is rituximab.

3. 2. The medicament of claim 1, wherein the subject has relapsed from and / or is refractory to a previous therapy comprising an anti-CD19 CAR αβ T cell therapy, and optionally the anti-CD19 CAR αβ T therapy is selected from axicabtagenecilloreucel and tisagenlecleucel.

4. The pharmaceutical according to claim 2 , wherein the binding domain specifically binds to a CD20 epitope different from the CD20 epitope recognized by the anti-CD20 monoclonal antibody.

5. the therapeutically effective amount of the anti-CD20 CAR γδ T cells is about 3×10 ７ ~Approx. 1×10 ９ The medicament according to any one of claims 1 to 4, wherein the γδ T cells are γδ T cells.

6. The medicament of claim 1, wherein a lymphodepleting (LD) regimen is further administered to the subject prior to administering the first dose of the therapeutically effective amount of anti-CD20 CAR γδ T cells to the subject.

7. The LD regimen is about 30 mg / m ２ / day fludarabine + approximately 500 mg / m ２ 7. The method of claim 6, comprising administering cyclophosphamide at a dose of 0.1 mg / day for three days.

8. The LD regimen is about 30 mg / m ２ / day fludarabine for 4 days + approximately 1000 mg / m ２ 7. The method of claim 6, comprising administering cyclophosphamide at a dose of 0.1 mg / day for three days.

9. The pharmaceutical described in claim 7 or 8, wherein the LD regimen further comprises an anti-CD52 antibody and / or an anti-CD19 antibody.

10. The pharmaceutical agent of any one of claims 1 to 4, wherein one or more additional doses of anti-CD20 CAR γδ T cells are further administered to the subject 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 1 month after the first dose of the therapeutically effective amount of anti-CD20 CAR γδ T cells.

11. The method of claim 10, wherein the one or more additional doses of anti-CD20 CAR γδ T cells are administered without or after an additional LD ​​regimen.

12. The medicament of claim 10, wherein the one or more additional doses comprise an increased amount of anti-CD20 CAR γδ T cells, a decreased amount of anti-CD20 CAR γδ T cells, or the same amount of anti-CD20 CAR γδ T cells.

13. The method of claim 10, wherein the one or more additional doses comprise anti-CD20 CAR γδ T cells from the same donor or a different donor.

14. The medicament of claim 1, wherein the anti-CD20 CAR γδ T cells are γδ1 T cells, γδ2 T cells, γδ3 T cells, or γδ4 T cells, and preferably the γδ T cells are γδ1 T cells.

15. 2. The medicament of claim 1, wherein after administration of the therapeutically effective amount of anti-CD20 CAR γδ T cells, the subject is monitored for one or more pharmacodynamic / pharmacokinetic biomarkers, wherein the biomarkers comprise or are selected from the group consisting of CAR transgene expression levels, quantitative measurements of CAR+ γδ T cells, serum levels of one or more cytokines and / or serum proteins, and minimal residual disease (MRD).

16. 16. The pharmaceutical composition of claim 15, wherein the one or more cytokines / serum proteins comprise or are selected from the group consisting of INFγ, GM-CSF, IL-2, IL-7, IL-15, TNFα, IL-1β, IL-6, IL-8, IL-10, MIP1α, MIP1β, CRP, ferritin, monocyte chemoattractant protein-1 (MCP-1), 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.

17. The pharmaceutical according to claim 16, wherein the one or more cytokines are IL-2 and / or IL-8.

18. The pharmaceutical composition of claim 15, wherein the CAR transgene expression level is measured via quantitative polymerase chain reaction (qPCR).

19. The method of claim 15, wherein the quantitative measurement of CAR+ γδ T cells is determined via flow cytometry.

20. The method of claim 15, further comprising analyzing for MRD via immunosequencing methodology.

21. The method of any one of claims 15 to 20, further comprising administering a secondary treatment regimen based at least in part on monitoring one or more of said biomarkers.

22. The method of claim 21 , wherein the second-line treatment regimen comprises one or more additional doses of anti-CD20 CAR γδ T cells.

23. The method of claim 22, wherein the one or more additional doses of anti-CD20 CAR γδ T cells are administered without or after an additional LD ​​regimen.

24. The medicament of claim 22, wherein the one or more additional doses comprise an increased amount of anti-CD20 CAR γδ T cells, a decreased amount of anti-CD20 CAR γδ T cells, or the same amount of anti-CD20 CAR γδ T cells.

25. 23. The medicament of claim 22, wherein the one or more additional doses comprise anti-CD20 CAR γδ T cells from a different donor.

26. 22. The method of claim 21, wherein the subsequent treatment regimen further comprises administration of cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate, and prednisone (CHOP).

27. 2. The pharmaceutical composition of claim 1, wherein the B-cell malignant tumor comprises or is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), and transformed follicular lymphoma (tFL).

28. A pharmaceutical for treating relapsed / refractory (R / R) B-cell malignancy in a subject in need thereof, comprising a therapeutically effective amount of anti-CD20 CAR γδ T cells expressing a chimeric antigen receptor (CAR) comprising a binding domain that specifically binds to CD20 on malignant B cells, wherein a lymphodepletion (LD) regimen is administered to the subject at least 5 days prior to administering a first dose of anti-CD20 CAR γδ T cells to the subject, followed by administering to the subject a therapeutically effective amount of anti-CD20 CAR γδ T cells expressing a chimeric antigen receptor (CAR) comprising a binding domain that specifically binds to CD20 on malignant B cells, thereby treating the subject, and wherein the subject has been previously treated with at least one, at least two, or at least three prior therapies.

29. 29. The medicament of claim 28, wherein the subject is further administered a second dose comprising a therapeutically effective dose of anti-CD20 CAR γδ 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 1 month after the first dose, with or without administration of an additional LD ​​regimen.

30. 30. The medicament of claim 29, wherein the second therapeutically effective dose comprises an increased amount of anti-CD20 CAR γδ T cells, a decreased amount of anti-CD20 CAR γδ T cells, or the same amount of anti-CD20 CAR γδ T cells as in the first dose.

31. the second dose is administered 7 days after the first dose, and the therapeutically effective amount of the anti-CD20 CAR γδ T cells in the first and second doses is about 3×10 ８ γδ T cells, and the second dose is administered without administering an additional LD ​​regimen.

32. The medicament of any one of claims 29 to 31, wherein a third dose comprising a therapeutically effective amount of anti-CD20 CAR γδ T cells is further administered to the subject 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 1 month after the second dose, with or without administration of an additional LD ​​regimen.

33. 33. The medicament of claim 32, wherein the third therapeutically effective dose comprises an increased amount of anti-CD20 CAR γδ T cells, a decreased amount of anti-CD20 CAR γδ T cells, or the same amount of anti-CD20 CAR γδ T cells as in the first and / or second dose.

34. the third dose is administered 7 days after the second dose, and the therapeutically effective amount of the anti-CD20 CAR γδ T cells in the first, second, and third doses is about 3×10 ８ γδ T cells, and the second and third doses are administered without administering an additional LD ​​regimen.

35. The pharmaceutical agent of claim 1 or 28, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

41.

36. The method of claim 35, wherein the anti-CD20 γδ T cells further comprise an isolated nucleic acid encoding the CAR.

37. The medicament of claim 1 or 28, wherein the anti-CD20 CAR γδ T cells further comprise an isolated nucleic acid encoding the CAR having the sequence of SEQ ID NO: 46.

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