Method for preparing lymphocytes for cell therapy

Abstract

The present disclosure relates to a method for preparing genetically engineered lymphocytes. Further disclosed is a method of using genetically engineered lymphocytes in T cell therapy for cancer.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 350,645, filed on June 9, 2022, which is hereby incorporated by reference in its entirety.

[0002] This application relates to methods for preparing one or more lymphocytes, such as T cells, for cell therapy. This application also relates to cells prepared, generated, or processed using the methods disclosed herein. In certain embodiments, this application relates to methods for improving the efficacy of cell therapy by contacting one or more lymphocytes with anti - CD81 antibody, exogenous interleukin - 7 (IL - 7), and exogenous interleukin - 21 (IL - 21).

Background Art

[0003] Human cancers are essentially composed of normal cells that have undergone genetic or epigenetic transformation to become abnormal cancer cells. By doing so, cancer cells begin to express proteins and other antigens that are different from those expressed by normal cells. These abnormal tumor antigens can be used by the body's natural immune system to specifically target and kill cancer cells. However, cancer cells use various mechanisms to prevent immune cells, such as T lymphocytes and B lymphocytes, from normally targeting cancer cells.

[0004] Human T cell therapy relies on ex vivo - enriched or modified human T cells to target and kill cancer cells in a subject, such as a patient. Various techniques have been developed to enrich the concentration of naturally occurring T cells that can target tumor antigens or to genetically modify T cells to specifically target known cancer antigens. These therapies have proven to have promising effects on tumor size and patient survival. However, it has been demonstrated that it is difficult to predict whether a given T cell therapy will be effective in each patient.

[0005] The transplantation of a mixed population of T cells is one of the factors that prevent T cell therapy from reaching its maximum potential. In conventional T cell therapy, donor T cells are collected and optionally modified to target specific antigens (e.g., tumor cells) or selected for anti - tumor properties (e.g., tumor - infiltrating lymphocytes), expanded in vitro, and administered to the subject in need. Typically, the resulting T cells contain a mixed population of mostly mature cells, many of which are terminally differentiated. As a result, the expected in vivo persistence of these cells can be limited, and the initially observed positive effects can be reversed over time as the tumor rebounds in the absence of the transplanted T cells. Thus, there remains a need to increase the in vivo persistence of T cells for use in T cell therapy.

[0006] Current T cell therapy is based on human T cells that are enriched or modified to target and kill a patient's cancer cells. To increase the ability of T cells to target and kill specific cancer cells, methods have been developed to genetically engineer T cells to express constructs that direct the T cells to specific target cancer cells. Chimeric antigen receptors (CARs) that contain binding domains capable of interacting with specific tumor antigens and engineered T cell receptors (TCRs) enable T cells to target and kill cancer cells that express specific tumor antigens.

[0007] There is a need for improved chimeric antigen receptors (CARs) and T cell receptors (TCRs) that target and kill cancer cells, and for improved methods of preparing lymphocytes that express CARs and / or TCRs for use in cell therapy. SUMMARY OF THE INVENTION

[0008] Any aspect or embodiment described herein can be combined with any other aspect or embodiment disclosed herein. Other aspects, advantages, and modifications are within the scope of this application.

[0009] The present disclosure provides a method of manufacturing genetically engineered lymphocytes, the method comprising contacting one or more lymphocytes from a subject with an anti-CD81 antibody, exogenous interleukin-7 (IL-7), and exogenous interleukin-21 (IL-21) in vitro, transforming the contacted lymphocytes with a vector containing a gene of interest, and harvesting the lymphocytes.

[0010] The present disclosure further provides a method of manufacturing genetically engineered lymphocytes, wherein the lymphocytes are selected from the group consisting of macrophages, neutrophils, basophils, eosinophils, granulocytes, natural killer (NK) cells, B cells, T cells, NK-T cells, mast cells, tumor infiltrating lymphocytes (TIL), myeloid derived suppressor cells (MDSC), and dendritic cells.

[0011] In certain embodiments, the lymphocytes are T cells. In some embodiments, the lymphocytes are contacted with an anti-CD3 antibody and an anti-CD28 antibody.

[0012] In certain embodiments, the T cells include CD8+ T cells and CD4+ T cells. In some embodiments, the CD8+ T cells express CCR7+ and / or CD45RA+. In some embodiments, the CD4+ T cells express CCR7+ and / or CD45RA+. In certain embodiments, the CD8+ T cells express CD27+ and / or CD28+. In some embodiments, the CD4+ T cells express CD27+ and / or CD28+. In certain embodiments, the CD8+ T cells express CD27+CD28+CCR7+ and / or CD45RA+. In some embodiments, the CD4+ T cells express CD27+CD28+CCR7+ and / or CD45RA+.

[0013] The present disclosure further provides a method for producing genetically engineered lymphocytes, wherein the T cells express a chimeric antigen receptor (CAR). In certain embodiments, the chimeric antigen receptor (CAR) is bicistronic. In certain embodiments, the chimeric antigen receptor (CAR) is bispecific. In some embodiments, the chimeric antigen receptor (CAR) binds to CD19. In certain embodiments, the chimeric antigen receptor (CAR) targets an identified tumor antigen including CD20, BCMA, CLL-1, CTLA4, CD30, CD40, NKp44, NKp30, GPC-3, CD79a, CD79b, BAFF-R, CS-1, PSMA, NKG2D, CLL-1, CD33, CD22 or NKp46 and includes a single chain variable fragment (scFv).

[0014] The present disclosure also provides a method for producing genetically engineered lymphocytes, wherein the vector is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adeno-associated vector, a lentiviral vector, or any combination thereof. In certain embodiments, the DNA inhibitor is a transposon.

[0015] The present disclosure further provides a method for producing genetically engineered lymphocytes, wherein the lymphocytes have not been contacted with exogenous interleukin-2 (IL-2).

[0016] In certain embodiments, the donor is a subject in need of T cell therapy.

[0017] The present disclosure also provides a method for producing genetically engineered lymphocytes, wherein the lymphocytes are transduced with a viral vector containing a gene of interest. In certain embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a retroviral vector.

[0018] In certain embodiments, the lymphocytes are harvested within 24 hours after transformation. In some embodiments, the lymphocytes are harvested within 2 days after transformation. In certain embodiments, the lymphocytes are harvested within 3 days after transformation. In some embodiments, the lymphocytes are harvested within 5 days after transformation. In certain embodiments, the lymphocytes are harvested within 7 days after transformation. In some embodiments, the lymphocytes are harvested within 8 days after transformation. In certain embodiments, the lymphocytes are harvested within 9 days after transformation. In certain embodiments, the lymphocytes are harvested within 9 days after transformation. In certain embodiments, the lymphocytes are harvested within 10, 11, 12, 13, or 14 days after transformation.

[0019] Another aspect of the present disclosure relates to genetically engineered lymphocytes produced by a method comprising contacting one or more lymphocytes derived from a subject with an anti-CD81 antibody, exogenous interleukin-7 (IL-7), and exogenous interleukin-21 (IL-21) in vitro, transforming the contacted lymphocytes with a vector containing a gene of interest, and harvesting the lymphocytes. In certain embodiments, the lymphocytes are selected from the group consisting of macrophages, neutrophils, basophils, eosinophils, granulocytes, natural killer cells (NK cells), B cells, T cells, NK-T cells, mast cells, tumor-infiltrating lymphocytes (TIL), myeloid-derived suppressor cells (MDSC), and dendritic cells.

[0020] In certain embodiments, the genetically engineered lymphocytes have a more naive and less differentiated CAR-T phenotype compared to genetically engineered lymphocytes produced when one or more lymphocytes derived from a subject are contacted with exogenous interleukin-2 (IL-2) in vitro.

[0021] In certain embodiments, the genetically engineered lymphocytes further comprise transforming the contacted lymphocytes with a vector containing a gene of interest and harvesting the lymphocytes. In some embodiments, the lymphocytes are T cells.

[0022] In some embodiments, the more naive and less differentiated CAR-T phenotype comprises CCR7+CD45RA+CD4+ T cells. In certain embodiments, the more naive and less differentiated CAR-T phenotype comprises CCR7+CD45RA+CD8+ T cells.

[0023] In some embodiments, when lymphocytes are collected within 6 days after transformation, a more juvenile and less differentiated CAR-T phenotype is produced. In certain embodiments, when lymphocytes are collected within 8 days after transformation, a more juvenile and less differentiated CAR-T phenotype is produced. In certain embodiments, when lymphocytes are collected within 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after transformation, a more juvenile and less differentiated CAR-T phenotype is produced.

[0024] In certain embodiments, the genetically engineered lymphocytes secrete lower amounts of effector cytokines compared to genetically engineered lymphocytes produced when one or more lymphocytes from a subject are contacted with exogenous interleukin-2 (IL-2) in vitro.

[0025] In certain embodiments, the lower amount of effector cytokine is granzyme A. In some embodiments, the lower amount of effector cytokine is IFN-γ. In certain embodiments, the genetically engineered lymphocytes further secrete higher amounts of IL-2.

[0026] In certain embodiments, the genetically engineered lymphocytes exhibit a more juvenile phenotype compared to genetically engineered lymphocytes produced when one or more lymphocytes from a subject are contacted with exogenous interleukin-2 (IL-2) in vitro.

[0027] Another aspect of the disclosure relates to a composition comprising genetically engineered lymphocytes.

[0028] The disclosure further provides a method of treating cancer, the method comprising administering the composition to a subject in need of treatment and monitoring the subject to determine the progress of the treatment.

[0029] Another aspect of the present disclosure relates to the use of genetically engineered lymphocytes for the manufacture of a composition for the treatment of cancer, wherein the genetically engineered lymphocytes are T cells.

[0030] In certain embodiments, the genetically engineered lymphocytes are used for the treatment of cancer.

[0031] In some embodiments, the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic cancer, adrenocortical cancer, AIDS-related cancer, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, central nervous system, B-cell leukemia, lymphoma, refractory B-cell malignancy or other B-cell malignancies, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brainstem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, germinoma, central nervous system, endometrial cancer, epithelioblastoma, epithelioma, esophageal cancer,esthesioneuroblastoma, Ewing sarcoma family of tumors, extracranial primitive neuroectodermal tumor, extragonadal primitive neuroectodermal tumor, extrahepatic bile duct cancer, eye cancer, malignant fibrous histiocytoma of bone, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), soft tissue sarcoma, germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (pancreas), Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer (primary), non-invasive lobular carcinoma inselected from the group consisting of situ, LCIS, lung cancer, lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma with potential primary midline cancer associated with the NUT gene, oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell tumor, fungating polyposis, myelodysplastic syndrome, myelodysplasia / myeloproliferative neoplasm, myeloid leukemia, chronic (CML), myeloid leukemia, acute (AML), myeloma, multiple, myeloproliferative disorder, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell tumor / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the neck, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, T cell lymphoma, skin, testicular cancer, pharyngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, villous tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor.

[0032] Another aspect of the disclosure is a method of treating a tumor in a subject in need of T cell therapy, the method comprising administering to the subject one or more T cells, wherein the one or more T cells have been contacted with an anti-CD81 antibody, exogenous interleukin-7 (IL-7), and exogenous interleukin-21 (IL-21).

[0033] The present disclosure further provides a method of reducing or decreasing the size of a tumor or inhibiting the growth of a tumor in a subject in need of T cell therapy, the method comprising administering to the subject one or more T cells that have been contacted with an anti-CD81 antibody, exogenous interleukin-7 (IL-7), and exogenous interleukin-21 (IL-21).

[0034] In some embodiments, the genetically engineered lymphocytes can be used in autologous cell therapy or allogeneic cell therapy.

[0035] In some embodiments, the genetically engineered lymphocytes produce more cytokines as compared to the process of producing genetically engineered lymphocytes in the presence of IL-2. In some embodiments, the genetically engineered lymphocytes produce more IL-2 as compared to the process of producing genetically engineered lymphocytes in the presence of IL-2.

[0036] In some embodiments, the genetically engineered lymphocytes are more naive as compared to the process of producing genetically engineered lymphocytes in the presence of IL-2. In some embodiments, the genetically engineered lymphocytes are more proliferative or robust as compared to the process of producing genetically engineered lymphocytes in the presence of IL-2.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The present disclosure relates to a method for manufacturing genetically engineered lymphocytes for use in T cell therapy. In particular, the present disclosure relates to a method for manufacturing CAR T cells for use in T cell therapy.

[0038] Definitions To make the present disclosure more readily understandable, certain terms are first defined below. As used in this application, unless expressly provided otherwise herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0039] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, the Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide many common dictionaries of terms used in this disclosure to those of ordinary skill in the art.

[0040] Units, prefixes, and symbols are presented in the form accepted in the Systeme International de Unites (SI). Numerical ranges include the numbers defining the range. Headings provided herein are not limitations of the various aspects of the disclosure and may be by reference to the entire specification. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification.

[0041] As used herein, it is to be understood that the articles “a” or “an” refer to “one or more” of any recited or enumerated component.

[0042] As used herein, the term "about" refers to a variation of approximately + / - 10% from a given value. When a specific value or composition is presented in this application and the claims, unless otherwise specified, the meaning of "about" should be assumed to be within the allowable error range of that specific value or composition.

[0043] As described herein, any range of concentrations, percentage ranges, ratio ranges, or integer ranges should be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of an integer), unless otherwise specified.

[0044] The term "and / or" as used herein should be construed as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. Thus, as used herein, the term "and / or" when used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" when used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0045] The term "activated" or "activation" refers to the state of lymphocytes, such as T cells, that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector functions. The term "activated T cells" refers, inter alia, to T cells that have undergone cell differentiation. T cell activation can be characterized by an increase in the T cell expression of one or more biomarkers including, but not limited to, CD57, PD1, CD107a, CD25, CD137, CD69, TIM-3, LAG-3, CD39, and / or CD71.

[0046] As used herein, the term "administering" refers to the physical introduction of an agent to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration of T cells prepared by the methods disclosed herein include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, or other parenteral routes of administration by injection or infusion. As used herein, the term "parenteral administration" generally means a mode of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the T cells prepared by the methods are administered orally, for example, by a non-intravenous route. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, for example, intranasal, intravaginal, rectal, sublingual, or topical. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0047] The term "antibody" (Ab) includes, but is not limited to, immunoglobulins that specifically bind to an antigen. Generally, an antibody can comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region can include three or four constant domains, CH1, CH2, CH3, and / or CH4. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region can include one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) that incorporate more conserved regions called framework regions (FRs). Each VH and VL includes three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus towards the carboxy terminus. The variable regions of the heavy and light chains include a binding domain that interacts with an antigen.

[0048] The immunoglobulin can be derived from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to an Ab (antibody) class or subclass (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. The term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human or non-human antibodies, fully synthetic antibodies, and single-chain antibodies. Non-human Abs can be humanized by recombinant methods that reduce their immunogenicity in humans. Unless explicitly stated otherwise or the context otherwise indicates, the term "antibody" also includes any antigen-binding fragment or antigen-binding portion of any of the aforementioned immunoglobulins, including monovalent and bivalent fragments or portions, and single-chain antibodies.

[0049] "Antigen-binding molecule" or "antibody fragment" refers to any part of an antibody that is smaller than the whole. The antigen-binding molecule may contain antigen complementarity-determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules.

[0050] The term "autologous" refers to any material derived from the same individual that is re-introduced later. For example, the method of engineered autologous cell therapy (eACT™) described herein involves the collection of lymphocytes from a donor, e.g., a patient, which are then engineered to express, e.g., a CAR construct, and then administered to the same donor, e.g., the patient.

[0051] The term "allogeneic" refers to any material that is derived from one individual and then introduced into another individual of the same species, e.g., allogeneic T cell transplantation.

[0052] "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth lead to the formation of malignant tumors that can invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" can include tumors at various stages. In certain embodiments, the cancer or tumor is at stage 0. For example, the cancer or tumor is at a very early stage of development and has not metastasized. In some embodiments, the cancer or tumor is at stage I. For example, the cancer or tumor is relatively small in size, has not spread to nearby tissues, and has not metastasized. In other embodiments, the cancer or tumor is at stage II or stage III. For example, the cancer or tumor is larger than stage 0 or stage I, has grown within adjacent tissues, but has not metastasized except possibly to lymph nodes. In other embodiments, the cancer or tumor is at stage IV. For example, the cancer or tumor has metastasized. Stage IV can also be referred to as advanced cancer or metastatic cancer.

[0053] As used herein, the term "anti-tumor effect" can refer to a biological effect presented as a decrease in tumor volume, inhibition of tumor growth, decrease in the number of tumor cells, decrease in tumor cell proliferation, decrease in the number of metastases, increase in overall survival or progression-free survival, extension of mean lifespan, or improvement of various physiological symptoms related to the tumor. The anti-tumor effect can also refer to the prevention of tumor development, for example, a vaccine.

[0054] The term "progression-free survival," which can be abbreviated as PFS, as used herein, refers to the period from the date of treatment according to the Revised IWG Response Criteria for malignant lymphoma to the date of disease progression or death from any cause.

[0055] "Disease progression" is evaluated by measurement of malignant lesions in radiographs or other methods and should not be reported as an adverse event. Death due to disease progression in the absence of signs and symptoms should be reported as the primary tumor type (e.g., DLBCL).

[0056] The term "duration of response", which may be abbreviated as DOR, as used herein, refers to the period from the first objective response of the subject according to the Revised IWG Response Criteria for malignant lymphoma until the date of disease progression or death is confirmed.

[0057] The term "overall survival", which may be abbreviated as OS, is defined as the period from the treatment date to the date of death.

[0058] "Cytokine", as used herein, refers to a non-antibody protein that can be released by lymphocytes, including macrophages, B cells, T cells, and mast cells, to propagate an immune response. In some embodiments, one or more cytokines are released in response to T cell therapy. In certain embodiments, these cytokines secreted in response to T cell therapy may be indicative of an effective T cell therapy.

[0059] "Therapeutically effective amount" or "therapeutically effective dose", as used herein, refers to the amount of T cells produced by the method, which, when used alone or in combination with another therapeutic agent, protects the subject against the development of a disease or promotes the regression of a disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of periods without disease symptoms, or the prevention of disorders or disabilities resulting from the disease. The ability of T cells to promote the regression of a disease can be evaluated using various methods known to those of skill in the art, such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0060] As used herein, the term "lymphocyte" can include natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cytotoxic) lymphocyte that is a major component of the innate immune system. NK cells reject tumor and virus-infected cells. It acts through the process of apoptosis or programmed cell death. They are called "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without the involvement of antibodies). Its T cell receptor (TCR) differentiates from other lymphocyte types. The thymus, a differentiated organ of the immune system, is mainly responsible for the maturation of T cells. The terms "immune cell" and "lymphocyte" are used interchangeably herein. There are several types of "lymphocytes", including but not limited to macrophages (e.g., tumor-associated macrophages), neutrophils, basophils, eosinophils, granulocytes, natural killer cells (NK cells), B cells, T cells, NK-T cells, mast cells, tumor-infiltrating lymphocytes (TIL), myeloid-derived suppressor cells (MDSC), and dendritic cells. This term also includes the precursors of these lymphocytes. Hematopoietic stem cells and / or progenitor cells can be derived from bone marrow, umbilical cord blood, adult peripheral blood after cytokine mobilization, etc. by methods known in the art. Some progenitor cells can differentiate into the lymphoid lineage, e.g., lymphoid hematopoietic stem cells or progenitor cells. Additional examples of lymphocytes that can be used in immunotherapy are described in U.S. Patent Application Publication No. 2018 / 0273601, which is hereby incorporated by reference in its entirety.

[0061] There are also several types of T cells, namely, helper T cells (e.g., CD4+ cells, effector TEFF (''effector T cells''), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells or killer T cells), memory T cells ((i) Stem memory TTSCM cells, like naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Rα+, but also express large amounts of CD95+, IL-2Pβ, CXCR3+ and LFA, and exhibit a number of functional attributes characteristic of memory cells), (ii) Central memory TCM cells express L-selectin, are CCR7+ and CD45RO+, secrete IL-2 but do not secrete IFNγ or IL-4, (iii) However, effector memory TTEM cells do not express L-selectin or CCR7, but express CD45RO and produce effector cytokines such as IPNγ and IL-4), regulatory T cells (Treg, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and γδ T cells. T cells found within tumors are referred to as ''tumor infiltrating lymphocytes'' or ''TIL''. On the other hand, B cells play a major role in humoral immunity (involving antibodies). B cells produce antibodies and antigens, act as antigen presenting cells (APC), and turn into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow and derive their name from the bone marrow.

[0062] "Naive" T cells refer to mature T cells that remain immunologically undifferentiated. Following positive and negative selection in the thymus, T cells emerge as either CD4+ or CD8+ naive T cells. In their naive state, T cells express L-selectin (CD62L+), IL-7 receptor-α (IL-7R-α), and CD132, but do not express CD25, CD44, CD69, or CD45RO. As used herein, "immature" may also refer to T cells that exhibit phenotypic characteristics of either naive T cells or immature T cells, such as TSCM cells or TCM cells. For example, immature T cells may express one or more of L-selectin (CD62L+), IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, and LFA-1. Naive T cells or immature T cells can be contrasted with terminally differentiated effector T cells, such as TEM cells and TEFF cells.

[0063] Naive T cells, upon encountering an antigen, become activated and differentiate into TSCM (stem memory T cells), TCM (central memory T cells), and effector T cells. This differentiation is characterized by the expression of various cell surface markers and transcription factors, as well as significant changes in cellular metabolic pathways. Generally, T cell activation and differentiation are characterized by an increased dependence on glycolysis and mitochondrial membrane potential. These metabolic pathways are important for mediating the effector functions of T cells in response to infection and cancer. Naive T cells are mature T cells that have not encountered an antigen. In addition to being CCR7+, CD45RO-, and CD95-, other common markers for naive T cells are CD45RA+, IL-2Rβ-, (L-selectin), CD27+, CD28+, IL-7Rα+, and CD62L+. Stem memory T cells (TSCM) have been described in mice, non-human primates, and humans and constitute approximately 2 - 4% of the total CD4+ and CD8+ T cell populations in the periphery. TSCM represents the earliest long-term developmental stage of memory T cells, exhibits stem cell-like properties, and shows a gene profile intermediate between naive T cells and central memory T cells. In addition to being CCR7+, CD45RO-, and CD95+, other common markers for stem memory T cells (TSCM) are CD45RA+, IL-2Rβ+, CD62L+, (L-selectin), CD27+, CD28+, IL-7Rα+, IL-2RP+, CXCR3+, and LFA-. Central memory T cells (TCM) express CD45RO+, CCR7+, and CD62L+. This memory subpopulation is commonly found in lymph nodes and the peripheral circulation. Other common markers for central memory T cells (TCM) are CD95+, IL-2Rβ+, CD3+, CD28+, CD127+, and granzyme B-. Effector memory T cells (TEM) express CD45RO but lack the expression of CCR7. Since these memory T cells lack the CCR7 lymph node homing receptor, they are found in the peripheral circulation and tissues. Other common markers for effector memory T cells (TEM) are CD95+, IL-2Rβ+, CD45RA-, and CD62L-.Effector T cells (TEFF) include cytotoxic T cells, helper T cells, and regulatory T cells. In addition to being CCR7- and CD45RO-, other common markers for effector T cells (TEFF) are CD95+, IL-2Rβ+, CD62L-, CD28-, CD62L-, CD127-, granzyme B+, and perforin+.

[0064] As used herein, "T cell function" refers to the normal characteristics of healthy T cells. In some embodiments, T cell function includes T cell proliferation. In some embodiments, T cell function includes T cell activation. In some embodiments, T cell function includes cytolytic activity.

[0065] Cell "proliferation" or "cell increase", as used herein, refers to the ability of T cells to increase in number through cell division. Proliferation can be measured by staining cells with carboxy fluorescein succinimidyl ester (CFSE). Cell proliferation can occur in vitro, for example, during T cell culture, or in vivo, for example, after administration of T cell therapy.

[0066] "T cell activation", as used herein, refers to any activity common to healthy T cells. In some embodiments, T cell activation includes cytokine production. In certain embodiments, T cell activation includes the production of one or more cytokines selected from interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and both.

[0067] "Cytolytic activity" or "cytotoxicity", as used herein, refers to the ability of T cells to destroy target cells. In some embodiments, the target cells are cancer cells, such as tumor cells. In some embodiments, the T cells express a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the target cells express a target antigen.

[0068] The terms "genetically engineered," "gene edited," or "engineered," while not limiting, refer to methods of modifying the genome of a cell, such as deleting a coding region or non-coding region or a part thereof, or inserting a coding region or a part thereof. In some embodiments, the modified cell is a lymphocyte, such as a T cell, and can be obtained from either a patient or a donor. The cell may be modified to express an exogenous construct, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR), that is integrated into the genome of the cell.

[0069] The chimeric antigen receptors (CARs or CAR-Ts) and T cell receptors (TCRs) of the present application are genetically engineered receptors. These engineered receptors can be readily inserted into lymphocytes, including T cells, according to techniques known in the art and can be expressed by the lymphocytes. In a CAR, a single receptor can be programmed to recognize a specific antigen and, when bound to that antigen, activate the lymphocyte to attack and destroy cells that have or express that antigen. When these antigens are present on tumor cells, lymphocytes expressing the CAR can target and kill the tumor cells. In one embodiment, the cells prepared according to the present application are cells having a chimeric antigen receptor (CAR) or a T cell receptor that includes an antigen-binding molecule, a co-stimulatory domain, and an activation domain. The co-stimulatory domain can include an extracellular domain, a transmembrane domain, and an intracellular domain. In one embodiment, the extracellular domain includes a hinge or a truncated hinge domain.

[0070] "Immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including Abs, cytokines, and complement) produced by any of these cells or the liver, which results in the selective targeting, binding, damage, destruction, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0071] The term "immunotherapy" refers to the treatment of a subject suffering from, or at risk of developing or relapsing with, a disease by a method that includes inducing, enhancing, suppressing, or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy can include adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT™), and allogeneic T cell transplantation. However, one of ordinary skill in the art will understand that the methods of preparing T cells disclosed herein enhance the effectiveness of any adoptive T cell therapy.

[0072] The T cells for immunotherapy can be derived from any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population or T cells can be obtained from a donor. The donor can be the subject, for example, a subject in need of anti-cancer treatment. T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. Further, the T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a blood unit collected from a subject using various techniques known to those of skill in the art, such as FICOLL™ separation and / or apheresis. T cells can also be obtained from an artificial thymic organoid (ATO) cell culture system in which the human thymic environment is replicated to support efficient ex vivo differentiation of T cells derived from primary and reprogrammed pluripotent stem cells.

[0073] The term "engineered autologous cell therapy", also known as adoptive cell transfer and which may be abbreviated as "eACT™", is a process in which a patient's own T cells are harvested and subsequently genetically modified to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. T cells can be engineered, for example, to express a chimeric antigen receptor (CAR) or a T cell receptor (TCR). CAR-positive (+) T cells are engineered to express an extracellular single-chain variable fragment (scFv) specific for a particular tumor antigen linked to an intracellular signaling moiety that includes a co-stimulatory domain and an activation domain. Co-stimulatory domains include, for example, CD81, CD28, CTLA4, CD16, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), programmed death ligand-1 (PD-L1), inducible T cell costimulator (ICOS), ICOS ligand (ICOSL), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18)), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Igα (CD79a, CD79b), DAP-10, Fcγ receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CD5, ICAM-1, GITR, BAFF-R, CS-1, GPC-3, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or a ligand that specifically binds to any combination thereof, and may be derived from. The activation domain can be derived from, for example, CD3 such as CD3ζ, ε, δ, γ. In certain embodiments, the CAR is designed to have two, three, four, or more co-stimulatory domains. The CAR scFv is a transmembrane protein expressed by cells of the B cell lineage, including but not limited to all normal B cells and B cell malignancies, including NHL, CLL, and non-T cell ALL, and can be designed to target, for example, CD19. Exemplary CAR T cell therapies and constructs are described in U.S. Patent Application Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated herein by reference in their entirety.

[0074] "Patient", as used herein, includes any human suffering from cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein. The term "donor subject" refers herein to a subject from whom cells are being harvested for further in vitro manipulation. The donor subject can be a cancer patient (i.e., an autologous donor) to be treated with a population of cells generated by the methods described herein, or can be an individual (i.e., an allogeneic donor) who provides a lymphocyte sample that is used to generate a population of cells for treating another individual or cancer patient at the time of generation of the cell population by the methods described herein. These subjects who receive the cells prepared by the method can be referred to as "recipient subjects".

[0075] "Stimulation", as used herein, refers to the primary response induced by the binding of a stimulatory molecule to its cognate ligand, which binding mediates signal transduction events. A "stimulatory molecule" is a molecule on a T cell, such as the T cell receptor (TCR) / CD3 complex that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen presenting cell (e.g., an artificial antigen presenting cell (aAPC), dendritic cell, B cell, etc.), specifically binds to a stimulatory molecule on a T cell and thereby can mediate, but is not limited to, primary responses by the T cell such as activation, initiation of an immune response, proliferation, etc. Examples of stimulatory ligands include, but are not limited to, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies. "Activation" or "active" refers herein to a T cell that has been stimulated. An activated T cell can be characterized by the expression of one or more markers selected from CD137, CD25, CD71, CD26, CD27, CD28, CD30, CD154, CD40L, and CD134.

[0076] The term "exogenous" refers to any substance that is derived from an external source. For example, exogenous IL-7 or exogenous IL-21 can be commercially available or recombinantly produced. When added to or contacting one or more T cells, "exogenous IL-7" or "exogenous IL-21" indicates that IL-7 and / or IL-21 is not produced by the T cells. In some embodiments, the T cells prior to being mixed with exogenous IL-7 or IL-21 may contain trace amounts of IL-7 and / or IL-21 (i.e., endogenous IL-7 or IL-21) that were produced by the T cells or isolated from a subject having the T cells. One or more T cells described herein can be contacted with exogenous IL-7 and / or exogenous IL-21 by any means known in the art, such as addition to a culture of isolated IL-7 and / or IL-21, inclusion of IL-7 and / or IL-21 in a culture medium, or expression of IL-7 and / or IL-21 by one or more cells other than the T cells in a culture, such as by a feeder layer.

[0077] "Treatment" or "treating" of a subject refers to any type of intervention or process performed on the subject, or administration of one or more T cells prepared according to the present disclosure to the subject, for the purpose of restoring, alleviating, improving, inhibiting, slowing down or preventing the onset, exacerbation, development, severity or recurrence of a symptom, complication or condition, or a biochemical sign associated with a disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission.

[0078] As used herein, the term "heterologous" means derived from any source other than a naturally occurring sequence. For example, a heterologous sequence that is included as part of a co-stimulatory protein having the amino acid sequence of the corresponding human co-stimulatory protein is an amino acid that does not naturally occur as a wild-type human co-stimulatory protein, i.e., an amino acid that does not align with the wild-type human co-stimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence other than the nucleotide sequence of the wild-type human co-stimulatory protein coding sequence.

[0079] "Antigen" refers to any molecule that can induce an immune response or can be bound by an antibody or antigen-binding molecule. The immune response can involve either antibody production or activation of specific immunocompetent cells, or both. One of ordinary skill in the art will readily understand that virtually any macromolecule, including all proteins or peptides, can function as an antigen. An antigen may be expressed endogenously, i.e., by genomic DNA, or recombinantly. An antigen may be specific for a particular tissue, such as a cancer cell, or may be widely expressed. Further, fragments of larger molecules can act as antigens. In one embodiment, the antigen is a tumor antigen. In a particular embodiment, the antigen is all or a fragment of BCMA, FLT3, or CLL-1.

[0080] The term "transformation" is a specific process by which exogenous genetic material is directly taken up and incorporated by a cell through its cell membrane. This usually occurs when the cell is in a state of competence, a state in which the cell can take up exogenous substances.

[0081] The terms "transfection" and "transfected" refer to the process by which foreign DNA is introduced into a cell by a viral vector (see Jones et al., "Genetics: principles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adeno-associated vector, a lentiviral vector, or any combination thereof.

[0082] The term "transposon" is a segment of DNA that can move to different positions within the genome of a single cell. In this process, the transposon can cause mutations and increase (or decrease) the amount of DNA in the cell's genome, and if the cell is a precursor to a gamete, it can increase (or decrease) the amount of DNA in the genome of any progeny.

[0083] As used herein, the term "in vitro cell" refers to any cell cultured ex vivo. In particular, in vitro cells can include T cells.

[0084] As used herein, "substantially" refers to a difference of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more compared to a control.

[0085] As used herein, "costimulatory ligand" includes molecules on antigen-presenting cells that specifically bind to cognate costimulatory molecules on T cells. Binding of a costimulatory ligand provides signals that mediate T cell responses such as, but not limited to, proliferation, activation, differentiation, etc. A costimulatory ligand, in addition to the primary signal provided by a stimulatory molecule, induces a signal, for example, by binding of the T cell receptor (TCR) / CD3 complex with a peptide-loaded major histocompatibility complex (MHC) molecule. Examples of costimulatory ligands include, but are not limited to, 3 / TR6, 4-1BB ligand, an agonist or antibody that binds to the Toll ligand receptor, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT) 3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), a ligand that specifically binds to B7-H3, lymphotoxin β receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or programmed death (PD)L1.Examples of co-stimulatory ligands include, but are not limited to, antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, ligands that specifically bind to CD81, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

[0086] "Costimulatory molecule" specifically binds to a costimulatory ligand and thereby is a cognate binding partner on a T cell that mediates a costimulatory response by the T cell, such as, but not limited to, proliferation.Co-stimulatory molecules include 4-1BB / CD137, B7-H3, BAFF-R, BLAME (SLAMF8), BTLA, CD33, CD45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (α, β, δ, ε, γ, ζ), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD81, CD83 ligand, CD84, CD86, CD8α, CD8β, CD9, CD96 (Tactile), CD1-1a, CD1-1b, CD1-1c, CD1-1d, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Igα (CD79a, CD79b), IL2Rβ, IL2Rγ, IL7Rα, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14, TNFSF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signal transduction lymphocyte activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleavage forms, or combinations thereof.

[0087] The "naive phenotype" or "naive cells", as used herein, refers to more poorly differentiated immune cells, such as immature immune cells. In some embodiments, the naive phenotype or naive cells refer to more poorly differentiated T cells (defined by CCR7+CD45RA+). In some embodiments, naive T cells express CCR7+ and / or CD45RA+. In other embodiments, naive T cells express CCR7+ and / or CD45RA+. In some embodiments, naive T cells include a CAR-T phenotype.

[0088] As used herein, the term "bicistronic" refers to a single messenger RNA molecule capable of producing two types of proteins. In some embodiments, a chimeric antigen receptor (CAR) is bicistronic. As used herein, the term "bispecific" refers to an artificial protein capable of simultaneously binding two different types of antigens or two different epitopes on the same antigen. In some embodiments, a chimeric antigen receptor (CAR) is bispecific.

[0089] Various aspects of the present disclosure are described in more detail in the following subsections.

[0090] Methods for preparing genetically engineered lymphocytes: The methods described herein can enhance the effectiveness of cell therapy. In certain embodiments, the cell therapy can be adoptive T cell therapy, including autologous cell therapy or allogeneic cell therapy. In certain further embodiments, T cell therapy broadly includes any method of selection, in vitro enrichment, and administration of autologous or allogeneic T cells of a patient that can recognize and bind to tumor cells. In certain embodiments, the cell therapy is a therapy that utilizes lymphocytes other than T cells, including but not limited to macrophages, neutrophils, basophils, eosinophils, granulocytes, natural killer cells (NK cells), B cells, NK-T cells, mast cells, tumor infiltrating lymphocytes (TIL), myeloid-derived suppressor cells (MDSC), and dendritic cells, and these cells may be genetically engineered to express at least one CAR and may be autologous or allogeneic to the patient. Genetically engineered lymphocytes produced in the presence of a combination of anti-CD81, IL7, and IL21 are more robust or proliferative when the cells produced are exposed to tumor antigens, as compared to cells produced under standard manufacturing processes in the presence of IL-2, and are also more juvenile and produce more cytokines, such as IL-2 itself, and are thus also very useful for autologous and allogeneic therapies. In comparison, cells produced in the presence of exogenous IL-2 are less active, i.e., more differentiated, produce less cytokines, such as IL-2 itself, and show less increase when contacted with tumor antigens.

[0091] In some embodiments, the methods described herein may further include enriching a population of lymphocytes obtained from a donor. Enrichment of a population of lymphocytes, such as a population of one or more T cells, can be achieved by separation media (e.g., FICOLL-PAQUE™, ROSETTESEP™ HLA Total Lymphocyte Enrichment cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical catalog number 0850494X), etc.), cell size, shape or density separation by filtration or elution, immunomagnetic separation (e.g., magnetic-activated cell sorting system, MACS), fluorescence separation (e.g., fluorescence-activated cell sorting system, FACS), or bead-based column separation.

[0092] Stimulation of a population of lymphocytes with one or more stimulants to produce a population of lymphocytes In some embodiments, the methods described herein can further include stimulating a population of lymphocytes with one or more stimulants to produce a population of activated cells under suitable conditions. Any suitable combination of one or more stimulants can be used to produce a population of activated lymphocytes, including, but not limited to, antibodies or functional fragments thereof that target lymphocyte-stimulating or co-stimulating molecules (e.g., anti-CD2 antibody, anti-CD3 antibody, anti-CD28 antibody, anti-CD81 antibody or functional fragments thereof), or any other suitable mitogen (e.g., tetradecanoyl phorbol acetate (TPA), phytohaemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)), or natural ligands for T cell-stimulating or co-stimulating molecules.

[0093] In some embodiments, suitable conditions for stimulating the population of lymphocytes described herein may include temperature, duration, and / or the presence of a certain level of CO2. In certain embodiments, the temperature for stimulation is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C. In certain embodiments, the temperature for stimulation is about 34 - 38°C. In certain embodiments, the temperature for stimulation is about 35 - 37°C. In certain embodiments, the temperature for stimulation is about 36 - 38°C. In certain embodiments, the temperature for stimulation is about 36 - 37°C or about 37°C.

[0094] In some embodiments, another condition for stimulating the population of lymphocytes described herein may include the duration of stimulation. In some embodiments, the duration of stimulation is about 24 - 72 hours. In some embodiments, the duration of stimulation is about 24 - 36 hours, about 30 - 42 hours, about 36 - 48 hours, about 40 - 52 hours, about 42 - 54 hours, about 44 - 56 hours, about 46 - 58 hours, about 48 - 60 hours, about 54 - 66 hours, about 60 - 72 hours. In a particular embodiment, the duration of stimulation is about 48 hours or at least about 48 hours. In other embodiments, the duration of stimulation is about 44 - 52 hours. In certain embodiments, the duration of stimulation is about 40 - 44 hours, about 40 - 48 hours, about 40 - 52 hours, or about 40 - 56 hours.

[0095] In some embodiments, another condition for stimulating the population of lymphocytes described herein may include the CO2 level. In some embodiments, the level of CO2 for stimulation is about 1.0 to 10% CO2. In some embodiments, the level of CO2 for stimulation is about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In one embodiment, the level of CO2 for stimulation is about 3 to 7% CO2. In other embodiments, the level of CO2 for stimulation is about 4 to 6% CO2. In still other embodiments, the level of CO2 for stimulation is about 4.5 to 5.5% CO2. In a particular embodiment, the level of CO2 for stimulation is about 5% CO2.

[0096] In one embodiment, the conditions for stimulating the population of lymphocytes may further include temperature, the period for stimulation, and / or any combination in the presence of a certain level of CO2. For example, the step of stimulating the population of lymphocytes may include stimulating the population of lymphocytes with one or more T cell stimulants at a temperature of about 36 to 38 °C, over a period of about 44 to 52 hours, in the presence of CO2 at a level of about 4.5 to 5.5% CO2.

[0097] In some embodiments, the concentration of lymphocytes useful in the methods herein is about 0.5 to 10.0×10 6 cells / mL. In certain embodiments, the concentration of lymphocytes is about 0.5 to 1.0×10 6 cells / mL, about 1.0 to 2.0×10 6 cells / mL, about 1.0 to 3.0×10 6 cells / mL, about 1.0 to 4.0×10 6 cells / mL, about 1.0 to 5.0×10 6 cells / mL, about 1.0 to 6.0×10 6 cells / mL, about 1.0 to 7.0×10 6 cells / mL, about 1.0 to 8.0×10 6 cells / mL, 1.0 to 9.0×10 6 cells / mL, or about 1.0 to 10.0×10 6cells / mL. In certain embodiments, the concentration of lymphocytes is about 0.5 - 1.0×10 6 cells / mL. In certain embodiments, the concentration of lymphocytes is about 1.0 - 2.0×10 6 cells / mL. In certain embodiments, the concentration of lymphocytes is about 1.0 - 1.2×10 6 cells / mL, about 1.0 - 1.4×10 6 cells / mL, about 1.0 - 1.6×10 6 cells / mL, about 1.0 - 1.8×10 6 cells / mL, or about 1.0 - 2.0×10 6 cells / mL. In certain embodiments, the concentration of lymphocytes is at least about 0.5×10 6 cells / mL, at least about 0.6×10 6 cells / mL, at least about 0.7×10 6 cells / mL, at least about 0.8×10 6 cells / mL, at least about 0.9×10 6 cells / mL, at least about 1.0×10 6 cells / mL, at least about 1.1×10 6 cells / mL, at least about 1.2×10 6 cells / mL, at least about 1.3×10 6 cells / mL, at least about 1.4×10 6 cells / mL, at least about 1.5×10 6 cells / mL, at least about 1.6×10 6 cells / mL, at least about 1.7×10 6 cells / mL, at least about 1.8×10 6 cells / mL, at least about 1.9×10 6 cells / mL, at least about 2.0×10 6 cells / mL, at least about 4.0×10 6 cells / mL, at least about 6.0×10 6 cells / mL, at least about 8.0×10 6 cells / mL, or at least about 10.0×10 6 cells / mL.

[0098] In some embodiments, an anti-CD81 antibody (or a functional fragment thereof) can be used according to the step of stimulating a population of lymphocytes. Any soluble or immobilized anti-CD81 antibody or a functional fragment thereof can be used (e.g., clone 5A6; anti-CD81). In some aspects, the antibody can be commercially purchased from vendors known in the art including, but not limited to, Miltenyi Biotec, BD Biosciences (e.g., MACS GMP CD3 pure 1 mg / mL, Part No. 170-076-116), and eBioscience, Inc. Further, those skilled in the art will understand methods for producing anti-CD81 antibodies by standard methods. In some embodiments, one or more T cell stimulants used according to the step of stimulating a population of lymphocytes include an antibody or a functional fragment thereof that targets a T cell stimulatory molecule or a co-stimulatory molecule in the presence of a T cell cytokine. In one aspect, one or more T cell stimulants include an anti-CD81 antibody. In certain embodiments, the T cell stimulant includes an anti-CD81 antibody at a concentration of about 100 ng / mL to 10 μg / mL. In certain embodiments, the concentration of the anti-CD81 antibody is about 100 ng / mL, about 500 ng / mL, about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, or about 10 μg / mL. In a particular embodiment, the concentration of the anti-CD81 antibody is about 1 μg / mL. In a particular embodiment, the concentration of the anti-CD81 antibody is about 2 μg / mL. In a particular embodiment, the concentration of the anti-CD81 antibody is about 3 μg / mL. In a particular embodiment, the concentration of the anti-CD81 antibody is about 4 μg / mL. In a particular embodiment, the concentration of the anti-CD81 antibody is about 5 μg / mL. In a particular embodiment, the concentration of the anti-CD81 antibody is about 6 μg / mL. In a particular embodiment, the concentration of the anti-CD81 antibody is about 7 μg / mL. In a particular embodiment, the concentration of the anti-CD81 antibody is about 8 μg / mL. In a particular embodiment, the concentration of the anti-CD81 antibody is about 9 μg / mL. In a particular embodiment, the concentration of the anti-CD81 antibody is about 10 μg / mL.

[0099] In some embodiments, an anti-CD3 antibody (or a functional fragment thereof), an anti-CD28 antibody (or a functional fragment thereof), or a combination of an anti-CD3 antibody and an anti-CD28 antibody can be used according to the step of stimulating a population of lymphocytes. Any soluble or immobilized anti-CD2, anti-CD3, and / or anti-CD28 antibody or a functional fragment thereof (e.g., clone OKT3 (anti-CD3), clone 145-2C11 (anti-CD3), clone UCHT1 (anti-CD3), clone L293 (anti-CD28), clone 15E8 (anti-CD28)) can be used. In some embodiments, the antibodies can be commercially purchased from vendors known in the art including, but not limited to, Miltenyi Biotec, BD Biosciences (e.g., MACS GMP CD3 pure 1mg / mL, Part No.170-076-116), and eBioscience, Inc. Further, those skilled in the art will understand methods for producing anti-CD3 antibodies and / or anti-CD28 antibodies by standard methods. In some embodiments, one or more T cell stimulants used according to the step of stimulating a population of lymphocytes include an antibody or a functional fragment thereof that targets a T cell stimulatory molecule or a costimulatory molecule in the presence of a T cell cytokine. In one embodiment, one or more T cell stimulants include a soluble anti-CD28 antibody. In certain embodiments, the T cell stimulant includes a soluble anti-CD28 antibody at a concentration of about 1.00 μg / mL to 2.00 μg / mL. In certain embodiments, the concentration of the anti-CD28 antibody is about 1.00 μg / mL, about 1.10 μg / mL, about 1.20 μg / mL, about 1.30 μg / mL, about 1.40 μg / mL, about 1.50 μg / mL, about 1.60 μg / mL, about 1.61 μg / mL, about 1.62 μg / mL, about 1.63 μg / mL, about 1.64 μg / mL, about 1.65 μg / mL, about 1.66 μg / mL, about 1.67 μg / mL, about 1.68 μg / mL, about 1.69 μg / mL, about 1.70 μg / mL, about 1.80 μg / mL, about 1.90 μg / mL, or about 2.00 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.00 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.10 μg / mL.In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.20 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.30 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.40 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.50 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.60 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.61 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.62 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.63 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.64 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.65 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.66 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.67 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.68 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.69 μg / mL. In a particular embodiment, the concentration of the anti-CD28 antibody is about 1.70 μg / mL. In one aspect, one or more T cell stimulants comprise an anti-CD3 antibody. In certain embodiments, the T cell stimulant comprises an anti-CD3 antibody at a concentration of about 0.50 μg / mL to 2.00 μg / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 0.50 μg / mL, about 0.60 μg / mL, about 0.70 μg / mL, about 0.80 μg / mL, about 0.90 μg / mL, about 1.00 μg / mL, about 1.10 μg / mL, about 1.20 μg / mL, about 1.21 μg / mL, about 1.22 μg / mL, about 1.23 μg / mL, about 1.24 μg / mL, about 1.25 μg / mL, about 1.26 μg / mL, about 1.27 μg / mL, about 1.28 μg / mL, about 1.29 μg / mL, about 1.30 μg / mL, about 1.40 μg / mL, about 1.50 μg / mL, about 1.60 μg / mL, about 1.70 μg / mL, about 1.80 μg / mL, about 1.90 μg / mL, or about 2.00 μg / mL. In a particular embodiment, the concentration of the anti-CD3 antibody is about 1.00 μg / mL.In certain embodiments, the concentration of the anti-CD3 antibody is about 1.10 μg / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 1.20 μg / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 1.21 μg / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 1.22 μg / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 1.23 μg / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 1.24 μg / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 1.25 μg / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 1.26 μg / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 1.27 μg / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 1.28 μg / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 1.29 μg / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 1.30 μg / mL. In alternative embodiments, T cell activation is not required. In such embodiments, the step of stimulating the population of lymphocytes to produce a population of activated T cells is omitted from this method, and the population of lymphocytes from which T lymphocytes can be enriched is transduced according to the following steps.

[0100] Transduction of a population of activated lymphocytes with a viral vector: In some embodiments, the methods described herein may involve transducing a population of activated T cells with a viral vector containing a nucleic acid molecule encoding a cell surface receptor using single-cycle transduction to produce a population of transduced T cells. Several recombinant viruses have been used as viral vectors to deliver genetic material into cells. Viral vectors that may be used according to the transduction process include, but are not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated viral (AAV) vectors, and can be any ecotropic or amphotropic viral vector. In some embodiments, the method further comprises transducing one or more T cells with a retrovirus. According to one aspect of this embodiment, the viral vector is grown in suspension culture in a medium specific for the production of viral vectors, referred to herein as a "viral vector inoculum." Any suitable growth medium and / or supplement for growing the viral vector can be used with the viral vector inoculum according to the methods described herein. According to some aspects, the viral vector inoculum is then added to the serum-free culture medium described below during the transduction process.

[0101] In some embodiments, one or more T cells can be transduced with a retrovirus. In one embodiment, the retrovirus contains a heterologous gene encoding a cell surface receptor. In a particular embodiment, the cell surface receptor is capable of binding to an antigen on the surface of a target cell, such as a tumor cell.

[0102] In some embodiments, the conditions for transducing the population of activated T cells described herein may include being in the presence of a specific period, a specific temperature, and / or a specific level of CO2. In certain embodiments, the temperature for transduction is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C. In one embodiment, the temperature for transduction is about 34 - 38°C. In another embodiment, the temperature for transduction is about 35 - 37°C. In another embodiment, the temperature for transduction is about 36 - 38°C. In yet another embodiment, the temperature for transduction is about 36 - 37°C. In a particular embodiment, the temperature for transduction is about 37°C.

[0103] In certain embodiments, the period for transduction is about 12 - 120 hours. In some embodiments, the period for transduction is about 12 - 16 hours, about 12 - 20 hours, about 12 - 24 hours, about 12 - 28 hours, about 12 - 32 hours, about 12 - 40 hours, about 12 - 50 hours, about 12 - 60 hours, about 12 - 70 hours, about 12 - 80 hours, about 12 - 90 hours, about 12 - 100 hours, about 12 - 110 hours, or about 12 - 120 hours. In other embodiments, the period for transduction is about 20 hours or at least about 20 hours. In one embodiment, the period for transduction is about 16 - 24 hours. In other embodiments, the period for transduction is at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 26 hours, at least about 28 hours, at least about 32 hours, at least about 40 hours, at least about 50 hours, at least about 60 hours, at least about 70 hours, at least about 80 hours, at least about 90 hours, at least about 100 hours, at least about 110 hours, or at least about 120 hours.

[0104] In certain embodiments, the level of CO2 for transfection is from about 1.0 to 10% CO2. In other embodiments, the level of CO2 for transfection is about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In one embodiment, the level of CO2 for transfection is from about 3 to 7% CO2. In another embodiment, the level of CO2 for transfection can be from about 4 to 6% CO2. In another embodiment, the level of CO2 for transfection is from about 4.5 to 5.5% CO2. In a particular embodiment, the level of CO2 for transfection is about 5% CO2.

[0105] Non-viral vectors can be broadly classified as plasmid DNA, liposome-DNA complexes (lipoplexes), and polymer-DNA complexes (polyplexes). Oligonucleotides and their analogs are also examples of non-viral vector-mediated gene transfer, either alone or in complexes. DNA-based transposon vectors provide a mechanism for non-viral gene delivery into mammalian and human cells. These vectors function via a cut-and-paste mechanism in which transposon DNA containing the transgene(s) of interest is integrated into chromosomal DNA by a transposase enzyme. Transposons have emerged as promising vectors for transfection that can potentially overcome some of the limitations of commonly used viral vectors. Transposons are stably integrated into the target cell genome, enabling sustained expression of the gene of interest.

[0106] In some embodiments, transfection of the population of activated T cells described herein can be performed in any combination of a specific period, a specific temperature, and / or the presence of a specific level of CO2: at a temperature of about 36 to 38 °C, for about 16 to 24 hours, in the presence of CO2 at a level of about 4.5 to 5.5% CO2.

[0107] Increase in the population of transfected lymphocytes: In some embodiments, the methods described herein can include expanding a population of one or more transduced lymphocytes over a specified period of time to produce a population of engineered lymphocytes. The specified period for expansion can be (i) a sufficient number of cells in at least one dose of the population of engineered lymphocytes for administration to a patient, (ii) a population of engineered lymphocytes having a preferred percentage of naïve cells as compared to a typical longer process, or (iii) any suitable period that enables the production of both (i) and (ii). This period depends on cell surface receptors expressed by the lymphocytes, the vectors used, the dose required to have a therapeutic effect, and other variables. Thus, in some embodiments, the specified period for expansion can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more than 21 days. In some aspects, the period for expansion is shorter than expansion methods known in the art. For example, the specified period for expansion can be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% shorter, or more than 75% shorter. In one aspect, the period of expansion is about 3 days, and the period from enrichment of the population of lymphocytes to production of the engineered lymphocytes is about 6 days.

[0108] In some embodiments, the conditions for increasing the population of transduced T cells can include temperature and / or the presence of a level of CO2. In certain embodiments, the temperature is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C. In one embodiment, the temperature is about 34 - 38°C. In another embodiment, the temperature is about 35 - 37°C. In another embodiment, the temperature is about 36 - 38°C. In yet another embodiment, the temperature is about 36 - 37°C. In a particular one embodiment, the temperature is about 37°C. In certain embodiments, the level of CO2 is 1.0 - 10% CO2. In other embodiments, the level of CO2 is about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In one embodiment, the level of CO2 is about 4.5 - 5.5% CO2. In another embodiment, the level of CO2 is about 5% CO2. In other embodiments, the level of CO2 is about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.5% CO2. In some embodiments, the conditions for increasing the population of transduced T cells can include any combination in the presence of temperature and / or a level of CO2. For example, the conditions for increasing the population of transduced T cells include the presence of a temperature of about 36 - 38°C and a level of CO2 of about 4.5 - 5.5% CO2.

[0109] In some embodiments, each step of the methods described herein can be performed in a closed system. In certain embodiments, the closed system is a closed bag culture system using any suitable cell culture bag (e.g., Miltenyi Biotec MACS® GMP Cell Differentiation Bags, Origen Biomedical PermaLife Cell Culture bags). In some embodiments, the cell culture bag used in the closed bag culture system is coated with a recombinant human fibronectin fragment during the transduction step. The recombinant human fibronectin fragment can include three functional domains, namely, a central cell-binding domain, a heparin-binding domain II, and a CS1 sequence. By using the recombinant human fibronectin fragment to assist in the co-localization of target cells and viral vectors, the gene efficiency of retroviral transduction of lymphocytes can be increased. In certain embodiments, the recombinant human fibronectin fragment is RETRONECTIN® (Takara Bio, Japan). In certain embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at a concentration of about 1 - 60 μg / mL or about 1 - 40 μg / mL. In other embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at a concentration of about 1 - 20 μg / mL, 20 - 40 μg / mL, or 40 - 60 μg / mL. In some embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 11 μg / mL, about 12 μg / mL, about 13 μg / mL, about 14 μg / mL, about 15 μg / mL, about 16 μg / mL, about 17 μg / mL, about 18 μg / mL, about 19 μg / mL, or about 20 μg / mL.In other embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at about 2 - 5 μg / mL, about 2 - 10 μg / mL, about 2 - 20 μg / mL, about 2 - 25 μg / mL, about 2 - 30 μg / mL, about 2 - 35 μg / mL, about 2 - 40 μg / mL, about 2 - 50 μg / mL, or about 2 - 60 μg / mL. In certain embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at at least about 2 μg / mL, at least about 5 μg / mL, at least about 10 μg / mL, at least about 15 μg / mL, at least about 20 μg / mL, at least about 25 μg / mL, at least about 30 μg / mL, at least about 40 μg / mL, at least about 50 μg / mL, or at least about 60 μg / mL. In one particular embodiment, the cell culture bag is coated with a recombinant human fibronectin fragment at at least about 10 μg / mL. The cell culture bag used in the closed bag culture system can be optionally blocked with human serum albumin (HSA) during the transduction process. In alternative embodiments, the cell culture bag is not blocked with HSA during the transduction process.

[0110] T cell therapy: In some embodiments, for example, without limitation, the methods described herein can improve the effectiveness of T cell therapy, which can be adoptive T cell therapy selected from the group consisting of tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT™), allogeneic T cell transplantation, non-T cell transplantation, and any combination thereof. Adoptive T cell therapy broadly includes any method of selection, in vitro enrichment, and administration of a patient's autologous T cells or allogeneic T cells that can recognize and bind to tumor cells. TIL immunotherapy is a type of adoptive T cell therapy in which lymphocytes that can infiltrate tumor tissue are isolated, enriched in vitro, and administered to a patient. TIL cells can be either autologous or allogeneic. Autologous cell therapy is an adoptive T cell therapy that includes isolating T cells from a patient that can target tumor cells, enriching the T cells in vitro, and administering the T cells back to the same patient. Allogeneic T cell transplantation can include transplantation of ex vivo-expanded natural-occurring T cells or genetically engineered T cells. As described in more detail above, engineered autologous cell therapy is an adoptive T cell therapy in which a patient's own lymphocytes are isolated, genetically modified to express a tumor-targeting molecule, expanded in vitro, and administered back to the patient. Non-T cell transplantation can include autologous or allogeneic therapies using non-T cells such as, but not limited to, natural killer (NK) cells.

[0111] In some embodiments, one or more T cells are transduced using a retrovirus containing a heterologous gene encoding a cell surface receptor. In a particular embodiment, the cell surface receptor is capable of binding to an antigen on the surface of a target cell, such as on the surface of a tumor cell. In some embodiments, the cell surface receptor is a chimeric antigen receptor or a T cell receptor.

[0112] In some embodiments, one or more T cells can be engineered to express a chimeric antigen receptor. The chimeric antigen receptor can include a binding molecule for a tumor antigen. The binding molecule can be an antibody or an antigen-binding molecule thereof. For example, the antigen-binding molecule can be selected from scFv, Fab, Fab’, Fv, F(ab’)2, and dAb, and any fragments or combinations thereof.

[0113] In some embodiments, the chimeric antigen receptor can further include a hinge region. The hinge region can be derived from the hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8α. In a particular embodiment, the hinge region is derived from the hinge region of IgG4.

[0114] In some embodiments, the chimeric antigen receptor can also include a transmembrane domain. The transmembrane domain can be the transmembrane domain of any transmembrane molecule that is the transmembrane domain of a coreceptor on a lymphocyte or a member of the immunoglobulin superfamily. In certain embodiments, the transmembrane domain is derived from the transmembrane domain of CD28, CD8α, CD4 or CD19. In a particular embodiment, the transmembrane domain includes a domain derived from the CD28 transmembrane domain. In another particular embodiment, the transmembrane domain includes a domain derived from the CD28 transmembrane domain.

[0115] In some embodiments, the chimeric antigen receptor can further include one or more co-stimulatory signaling regions. For example, the co-stimulatory signaling region can be the signaling region of CD28, OX-40, 4-1BB, CD27, inducible T cell co-stimulator (ICOS), CD3γ, CD3δ, CD3ε, CD247, Igα (CD79a, CD79b), or Fcγ receptor. In a particular embodiment, the co-stimulatory signaling region is the CD28 signaling region.

[0116] In one embodiment, the chimeric antigen receptor further includes a CD3ζ signaling domain.

[0117] In some embodiments, the chimeric antigen receptor can be engineered to target a specific tumor antigen, the "gene of interest". In some embodiments, the tumor antigen is 707-AP (707 alanine proline), AFP (α(a)-fetoprotein), ART-4 (a cancer antigen recognized by T4 cells), BAGE (B antigen, b-catenin / m, b-catenin / variant), BCMA (B cell maturation antigen), Bcr-abl (breakpoint cluster region-Abelson), CAIX (carbonic anhydrase IX), CD19 (cluster of differentiation 19), CD20 (cluster of differentiation 20), CD22 (cluster of differentiation 22), CD30 (cluster of differentiation 30), CD33 (cluster of differentiation 33), CD44v7 / 8 (cluster of differentiation 44, exon 7 / 8), CAMEL (a CTL-recognized antigen on melanoma), CAP-1 (cancer fetal antigen peptide-1), CASP-8 (caspase-8), CDC27m (cell division cycle 27, variant), CDK4 / m (cyclin-dependent kinase 4, variant), CEA (carcinoembryonic antigen), CT (cancer / testis (antigen)), Cyp-B (cyclophilin B), DAM (differentiation antigen, melanoma), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor, variant III), EGP-2 (epithelial glycoprotein 2), EGP-40 (epithelial glycoprotein 40), Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4), ELF2M (elongation factor 2, variant), ETV6-AML1 (Ets variant gene 6 / acute myeloid leukemia 1 gene ETS), FBP (folate-binding protein), fAchR (fetal acetylcholine receptor), G250 (glycoprotein 250), GAGE (G antigen), GD2 (disialoganglioside 2), GD3 (disialoganglioside 3), GnT-V (N-acetylglucosaminyltransferase V), Gp100 (glycoprotein 100kD), HAGE (helicos antigen), HER-2 / neu (human epidermal receptor-2 / neural; also known as EGFR2), HLA-A (human leukocyte antigen-A), HPV (human papillomavirus), HSP70-2M (heat shock protein 70-2, variant), HST-2 (human ring finger tumor-2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase),IL-13R-a2 (Interleukin-13 receptor subunit α-2), KIAA0205, KDR (Kinase Insert Domain Receptor), κ-light chain, LAGE (L antigen), LDLR / FUT (Low density lipoprotein receptor / GDP-L-fucose:β-D-galactosidase 2-α-L-fucosyltransferase), LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (Melanoma antigen), MAGE-A1 (Melanoma-associated antigen 1), mesothelin, mouse CMV-infected cells, MART-1 / Melan-A (Melanoma antigen recognized by T cells-1 / Melanoma antigen A), MC1R (Melanocortin 1 receptor), Myosin / m (Myosin, mutant), MUC1 (Mucin 1), MUM-1, -2, -3 (Melanoma ubiquitous, mutant 1, 2, 3), NA88-A (NA cDNA clone of patient M88), NKG2D (Natural killer group 2, member D) ligand, NY-BR-1 (New York breast differentiation antigen 1), NY-ESO-1 (New York esophageal squamous cell carcinoma-1), cancer fetal antigen (h5T4), P15 (Protein 15), p190 minor bcr-abl (190KD bcr-abl protein), Pml / RARa (Promyelocytic leukemia / Retinoic acid receptor α), PRAME (Preferentially expressed antigen in melanoma), PSA (Prostate specific antigen), PSCA (Prostate stem cell antigen), PSMA (Prostate specific membrane antigen), RAGE (Renal antigen), RU1 or RU2 (Renal ubiquitous 1 or 2), SAGE (Sarcoma antigen), SART-1 or SART-3 (Flat antigen that rejects tumors 1 or 3), SSX1, -2, -3, 4 (Synovial sarcoma X1, -2, -3, -4), TAA (Tumor-associated antigen), TAG-72 (Tumor-associated glycoprotein 72), TEL / AML1 (Translocated Ets-family leukemia / Acute myeloid leukemia 1), TPI / m (Triose phosphate isomerase, mutant), TRP-1 (Tyrosinase-related protein 1, or gp75), TRP-2 (Tyrosinase-related protein 2), TRP-2 / INT2 (TRP-2 / Intron 2), VEGF-R2 (Vascular endothelial growth factor receptor 2), WT1 (Wilms tumor gene), and is selected from any combination thereof. In a specific embodiment, the tumor antigen is CD19.,

[0118] In some embodiments, the T cell therapy involves administering to a patient engineered T cells that express a T cell receptor (the “engineered TCR T cells”). The T cell receptor (TCR) can include a binding molecule to a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of 707-AP, AFP, ART-4, BAGE, BCMA, Bcr-abl, CAIX, CD19, CD20, CD22, CD30, CD33, CD44v7 / 8, CAMEL, CAP-1, CASP-8, CDC27m, CDK4 / m, CEA, CT, Cyp-B, DAM, EGFR, EGFRvIII, EGP-2, EGP-40, Erbb2, 3, 4, ELF2M, ETV6-AML1, FBP, fAchR, G250, GAGE, GD2, GD3, GnT-V, Gp100, HAGE, HER-2 / neu, HLA-A, HPV, HSP70-2M, HST-2, hTERT or hTRT, iCE, IL-13R-a2, KIAA0205, KDR, κ-light chain, LAGE, LDLR / FUT, LeY, L1CAM, MAGE, MAGE-A1, mesothelin, mouse CMV-infected cells, MART-1 / Melan-A, MC1R, Myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NKG2D ligand, NY-BR-1, NY-ESO-1, cancer fetal antigen, P15, p190 minor bcr-abl, Pml / RARa, PRAME, PSA, PSCA, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SSX1, -2, -3, 4, TAA, TAG-72, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, VEGF-R2, WT1, and any combination thereof.

[0119] In one embodiment, the TCR includes a binding molecule to a viral oncogene. In a particular embodiment, the viral oncogene is selected from human papilloma virus (HPV), Epstein-Barr virus (EBV), and human T-lymphotropic virus (HTLV).

[0120] In yet another embodiment, the TCR comprises a binding molecule to a testicular, placental, or fetal tumor antigen. In a particular embodiment, the testicular, placental, or fetal tumor antigen is selected from the group consisting of NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), and any combination thereof.

[0121] In another embodiment, the TCR comprises a binding molecule to a lineage-specific antigen. In a particular embodiment, it is selected from the group consisting of melanoma antigen recognized by T cells 1 (MART-1), gp100, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), and any combination thereof.

[0122] In one embodiment, the T cell therapy comprises administering to a patient engineered CAR T cells that express a chimeric antigen receptor that binds to CD19 and further comprises a CD28 co-stimulatory domain and a CD3ζ signaling region. In another embodiment, the engineered CAR T cells comprise a CD81 co-stimulatory domain. In a particular embodiment, the T cell therapy comprises administering KTE-C19 to a patient.

[0123] In one embodiment, the antigenic moiety further includes Epstein - Barr virus (EBV) antigens (e.g., EBNA - 1, EBNA - 2, EBNA - 3, LMP - 1, LMP - 2), hepatitis A virus antigens (e.g., VP1, VP2, VP3), hepatitis B virus antigens (e.g., HBsAg, HBcAg, HBeAg), hepatitis C virus antigens (e.g., envelope glycoproteins E1 and E2), herpes simplex virus type 1, 2, or 8 (HSV1, HSV2, or HSV8) virus antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK,gL, gM, UL20, UL32, US43, UL45, UL49A), cytomegalovirus (CMV) virus antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK,gL, gM or other envelope proteins), human immunodeficiency virus (HIV) virus antigens (glycoproteins gp120, gp41, or p24), influenza virus antigens (e.g., hemagglutinin (HA) or neuraminidase (NA)), measles or mumps virus antigens, human papillomavirus (HPV) virus antigens (e.g., L1, L2), parainfluenza virus antigens, rubella virus antigens, respiratory syncytial virus (RSV) antigens, or varicella - zoster virus antigens, but are not limited thereto. In such an embodiment, the cell - surface receptor can be any TCR or any CAR that recognizes any of the aforementioned viral antigens on the target virus - infected cells.

[0124] In other embodiments, the antigenic portion is associated with cells having immunodeficiency or inflammatory dysfunction. Such antigenic portions include, but are not limited to, myelin basic protein (MBP), myelin proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), carcinoembryonic antigen (CEA), proinsulin, glutamic acid decarboxylase (GAD65, GAD67), heat shock protein (HSP), or any other tissue-specific antigen involved in or associated with a pathogenic autoimmune process.

[0125] In some embodiments, the methods disclosed herein can include T cell therapy involving transplantation of one or more T cells into a patient. The T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells, such as engineered CAR+ T cells or engineered TCR+ T cells, can be at least about 10 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 cells, at least about 10 8 cells, at least about 10 9 , or at least about 10 10 and can be. In another embodiment, a therapeutically effective amount of T cells, such as engineered CAR+ T cells or engineered TCR+ T cells, is about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, or about 10 8 cells. In a particular embodiment, a therapeutically effective amount of T cells, such as engineered CAR+ T cells or engineered TCR+ T cells, is about 1×10 4 cells / kg, 2×10 4 cells / kg, 3×10 4 cells / kg, 4×104 cells / kg, 5×10 4 cells / kg, 6×10 4 cells / kg, 7×10 4 cells / kg, 8×10 4 cells / kg, 9×10 4 cells / kg, 1×10 5 cells / kg, 2×10 5 cells / kg, 3×10 5 cells / kg, 4×10 5 cells / kg, 5×10 5 cells / kg, 6×10 5 cells / kg, 7×10 5 cells / kg, 8×10 5 cells / kg, 9×10 5 cells / kg, 1×10 6 cells / kg, approximately 2×10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 number of cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 cells / kg, or approximately 9×10 7 is cells / kg.

[0126] In some embodiments, the patient is preconditioned prior to administration of the T cell therapy. The patient can be preconditioned according to any method known in the art, including but not limited to treatment with one or more chemotherapeutic agents and / or radiation therapy. In some embodiments, the preconditioning includes any treatment that decreases the number of endogenous lymphocytes, eliminates cytokine sinks, increases the serum levels of one or more homeostatic cytokines or pro-inflammatory factors, enhances the effector function of the T cells administered after treatment, enhances the activation and / or availability of antigen-presenting cells, or any combination of these prior to the T cell therapy. In one embodiment, the preconditioning includes increasing the serum levels of one or more cytokines in the subject.

[0127] Composition comprising lymphocytes: In some embodiments, interleukin-7 (IL-7) is a cytokine that promotes lymphocyte homeostasis and is required for T cell development. Endogenous IL-7 is produced by epithelial cells in the thymus and bone marrow, and its receptor, IL-7 receptor-α (IL-7R-α), is expressed by naive T cells and a subset of T CM cells, including T cells. IL-7 signaling occurs through various tyrosine kinases, including the Janus kinase / signal transducer and activator of transcription (Jak / STAT) pathway, PI3K, and Src family tyrosine kinases. Any exogenous IL-7 can be used in the methods described herein. In some embodiments, the exogenous IL-7 is human IL-7. In some embodiments, the exogenous IL-7 is wild-type IL-7. In other embodiments, the exogenous IL-7 is recombinant IL-7. IL-7 can be produced and obtained by any method known in the art, including but not limited to isolation of IL-7 from one or more IL-7-producing cells or obtaining commercially available IL-7.

[0128] In some embodiments, IL-7 at any concentration can be used in the methods described herein. For example, the methods can include contacting one or more T cells with at least about 0.001 ng / mL of IL-7, at least about 0.005 ng / mL of IL-7, at least about 0.01 ng / mL of IL-7, at least about 0.05 ng / mL of IL-7, at least about 0.1 ng / mL of IL-7, at least about 0.5 ng / mL of IL-7, at least about 1.0 ng / mL of IL-7, at least about 1 ng / mL of IL-7, at least about 2 ng / mL of IL-7, at least about 3 ng / mL of IL-7, at least about 4 ng / mL of IL-7, at least about 5 ng / mL of IL-7, at least about 6 ng / mL of IL-7, at least about 7 ng / mL of IL-7, at least about 8 ng / mL of IL-7, at least about 9 ng / mL of IL-7, at least about 10 ng / mL of IL-7, at least about 11 ng / mL of IL-7, at least about 12 ng / mL of IL-7, at least about 13 ng / mL of IL-7, at least about 14 ng / mL of IL-7, at least about 15 ng / mL of IL-7, at least about 20 ng / mL of IL-7, at least about 25 ng / mL of IL-7, at least about 30 ng / mL of IL-7, at least about 35 ng / mL of IL-7, at least about 40 ng / mL of IL-7, at least about 45 ng / mL of IL-7, at least about 50 ng / mL of IL-7, at least about 100 ng / mL of IL-7, at least about 200 ng / mL of IL-7, at least about 300 ng / mL of IL-7, at least about 400 ng / mL of IL-7, at least about 500 ng / mL of IL-7, or at least about 1000 ng / mL of IL-7. In one embodiment, one or more T cells are contacted with IL-7 in the range of about 0.001 to about 500 ng / mL, about 0.01 to about 100 ng / mL, about 0.1 to about 50 ng / mL, about 1 to about 10 ng / mL, about 1 to about 5 ng / mL, about 5 to about 10 ng / mL, about 3 to about 7 ng / mL, or about 4 to about 6 ng / mL. In a particular embodiment, one or more T cells are contacted with about 5 ng / mL of IL-7.

[0129] In some embodiments, interleukin-21 (IL-21) is a cytokine that promotes lymphocyte homeostasis and is required for T cell development. IL-21 has pleiotropic effects on a wide range of immune cell types and non-immune cell types including, but not limited to, CD4+ and CD8+ T cells, B cells, macrophages, monocytes, and dendritic cells (DC), and is produced by T cells and natural killer T cells. Any exogenous IL-21 can be used in the methods described herein. In some embodiments, the exogenous IL-21 is human IL-21. In some embodiments, the exogenous IL-21 is wild-type IL-21. In other embodiments, the exogenous IL-21 is recombinant IL-21. IL-21 can be produced and obtained by any method known in the art, including, but not limited to, isolation of IL-21 from one or more IL-21-producing cells or obtaining commercially available IL-21.

[0130] In some embodiments, IL-21 at any concentration can be used in the methods described herein. For example, the methods can include contacting one or more T cells with IL-21 at least about 0.001 ng / mL, at least about 0.005 ng / mL, at least about 0.01 ng / mL, at least about 0.05 ng / mL, at least about 0.1 ng / mL, at least about 0.5 ng / mL, at least about 1.0 ng / mL, at least about 1 ng / mL, at least about 2 ng / mL, at least about 3 ng / mL, at least about 4 ng / mL, at least about 5 ng / mL, at least about 6 ng / mL, at least about 7 ng / mL, at least about 8 ng / mL, at least about 9 ng / mL, at least about 10 ng / mL, at least about 11 ng / mL, at least about 12 ng / mL, at least about 13 ng / mL, at least about 14 ng / mL, at least about 15 ng / mL, at least about 20 ng / mL, at least about 25 ng / mL, at least about 30 ng / mL, at least about 35 ng / mL, at least about 40 ng / mL, at least about 45 ng / mL, at least about 50 ng / mL, at least about 100 ng / mL, at least about 200 ng / mL, at least about 300 ng / mL, at least about 400 ng / mL, at least about 500 ng / mL, or at least about 1000 ng / mL. In one embodiment, one or more T cells are contacted with IL-21 at about 0.001 to about 500 ng / mL, about 0.01 to about 100 ng / mL, about 0.1 to about 50 ng / mL, about 1 to about 10 ng / mL, about 1 to about 5 ng / mL, about 5 to about 10 ng / mL, about 3 to about 7 ng / mL, or about 4 to about 6 ng / mL.In certain embodiments, one or more T cells are contacted with about 5 ng / mL of IL-21.

[0131] In certain embodiments, one or more T cells have not been and are not being contacted with exogenous IL-2.

[0132] In some embodiments, one or more T cells described herein can be obtained from any source, including, for example, a human donor. The donor can be a subject in need of cancer treatment, e.g., treatment with one T cell generated by the methods described herein (i.e., an autologous donor), or an individual who provides a lymphocyte sample used to treat another individual or cancer patient during the generation of a cell population generated by the methods described herein (i.e., an allogeneic donor). The population of lymphocytes can be obtained from the donor by any suitable method used in the art. For example, the population of lymphocytes can be obtained by any suitable ex vivo method, venipuncture, or other blood collection method from which a sample of blood and / or lymphocytes can be obtained. In one embodiment, the population of lymphocytes is obtained by apheresis. One or more T cells can be harvested from any tissue containing one or more T cells, including but not limited to tumors. In some embodiments, a tumor or a portion thereof is harvested from the subject and one or more T cells are isolated from the tumor tissue. Any T cell can be used in the methods disclosed herein, including any T cell suitable for T cell therapy. For example, one or more cells useful in the present disclosure can be selected from the group consisting of tumor infiltrating lymphocytes (TILs), cytotoxic T cells, CAR T cells, engineered TCR T cells, natural killer T cells, dendritic cells, and peripheral blood lymphocytes. In a particular embodiment, the T cell is a tumor infiltrating leukocyte. In certain embodiments, one or more T cells express CD8, e.g., CD8 + T cells. In other embodiments, one or more T cells express CD4, e.g., CD4 + T cells.

[0133] Cancer treatment: In some embodiments, the methods of the present disclosure can be used to treat cancer in a subject, to reduce the size of a tumor, to kill tumor cells, to prevent tumor cell proliferation, to prevent tumor growth, to remove a tumor from a patient, to prevent tumor recurrence, to prevent tumor metastasis, to induce remission in a patient, or for any combination thereof. In certain embodiments, the method induces a complete response. In other embodiments, the method induces a partial response.

[0134] In some embodiments, cancers that can be treated include tumors that are not angiogenesis, tumors that are not yet substantially angiogenesis, or tumors that are angiogenesis. Cancer can also include solid tumors or non-solid tumors. In certain embodiments, the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic cancer, adrenocortical cancer, AIDS-related cancer, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, central nervous system, B-cell leukemia, lymphoma or other B-cell malignancies, basal cell cancer, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brainstem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, germinoma, central nervous system, endometrial cancer, epithelioblastoma, epithelioma, esophageal cancer, sensory neuroblastoma, Ewing sarcoma family of tumors extracranial primitive neuroectodermal tumor, extragonadal primitive neuroectodermal tumor extrahepatic bile duct cancer, eye cancer malignant fibrous histiocytoma of bone, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), soft tissue sarcoma, germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (pancreas), Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medulloepithelioma, melanoma, Merkel cell cancer, mesothelioma, metastatic squamous cell carcinoma associated with potential primary midline cancer related to the NUT gene, oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell tumor, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, myeloid leukemia, chronic (CML), myeloid leukemia, acute (AML), myeloma, multiple, myeloproliferative disorder, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papilloma, paraganglioma,Tumors that can be selected from nasal cavity and paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell tumor / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the neck, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, T cell lymphoma, skin, testicular cancer, pharyngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, tumors derived from Wilms tumor.

[0135] In one embodiment, the method can be used to treat a tumor, which is lymphoma or leukemia. Lymphomas and leukemias are cancers of the blood that specifically affect lymphocytes. All white blood cells in the blood are derived from a single type of pluripotent hematopoietic stem cell in the bone marrow. This stem cell produces both myeloid progenitor cells and lymphoid progenitor cells, which then give rise to the various types of white blood cells found in the body. White blood cells arising from myeloid progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells, and plasma cells. White blood cells arising from lymphoid progenitor cells include megakaryocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, and macrophages. Lymphomas and leukemias can affect one or more of these cell types in a patient.

[0136] In some embodiments, generally, lymphomas can be divided into at least two subgroups: Hodgkin lymphoma and non-Hodgkin lymphoma. Non-Hodgkin lymphoma (NHL) is a heterogeneous group of cancers that originate from B lymphocytes, T lymphocytes, or natural killer cells. In the United States, B-cell lymphoma accounts for 80-85% of reported cases. In 2013, it was estimated that there were approximately 69,740 new cases of NHL and more than 19,000 disease-related deaths. Non-Hodgkin lymphoma is the most common hematologic malignancy and the seventh most common site of new cancer in both men and women, accounting for 4% of new cancer cases and 3% of cancer-related deaths.

[0137] In some embodiments, diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, accounting for approximately 30% of NHL cases. In the United States, approximately 22,000 people are newly diagnosed with DLBCL each year. This is classified as an intermediate-grade lymphoma in which the majority of patients are cured with conventional chemotherapy (NCCN guidelines, NHL 2014).

[0138] In some embodiments, the first-line therapy for DLBCL typically includes an anthracycline-containing regimen that includes rituximab, such as R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), which has an objective response rate of approximately 80% and a complete response rate of approximately 50% (Coiffier 2002), and approximately one-third of patients are resistant to initial therapy or relapse after R-CHOP (Sehn 2005). For patients who relapse after responding to first-line therapy, approximately 40 - 60% of patients can achieve a second response with additional chemotherapy. The standard therapy as second-line therapy for autologous stem cell transplant (ASCT)-eligible patients includes rituximab, as well as combination chemotherapy, such as R-ICE (rituximab, ifosfamide, carboplatin, and etoposide), and R-DHAP (rituximab, dexamethasone, cytarabine, and cisplatin), which have an objective response rate of approximately 63% and a complete response rate of approximately 26%, respectively (Gisselbrecht 2010). Patients who respond to second-line therapy and are considered suitable for transplantation receive consolidation therapy with high-dose chemotherapy and ASCT, which is curative in approximately half of transplanted patients (Gisselbrecht 2010). Patients who fail ASCT have a very poor prognosis and have no curable treatment options.

[0139] In some embodiments, primary mediastinal large B-cell lymphoma (PMBCL) has different clinical, pathological, and molecular characteristics compared to DLBCL. PMBCL is thought to arise from thymic (myeloid) B cells and accounts for approximately 3% of patients diagnosed with DLBCL. PMBCL typically presents in a younger adult population in their 30s and is slightly more common in women. Gene expression profiling has suggested deregulated pathways in PMBCL that overlap with Hodgkin lymphoma. Initial therapy for PMBCL generally includes anthracycline-containing regimens with or without rituximab, such as etoposide, doxorubicin, and cyclophosphamide with adjusted infusion volumes, vincristine, prednisone, and rituximab (DA-EPOCH-R), with or without regional radiotherapy.

[0140] In some embodiments, follicular lymphoma (FL), a B-cell lymphoma, is the most common low-grade (slow-growing) type of NHL and accounts for approximately 20% - 30% of all NHLs. Some patients with FL histologically transform (TFL) into DLBCL, which is more aggressive and associated with a poor outcome. Histological transformation to DLBCL occurs at an annual rate of approximately 3% over 15 years, after which the risk of transformation continues to decline. The biological mechanism of histological transformation is unknown. Initial treatment of TFL is influenced by prior treatment for follicular lymphoma but generally includes anthracycline-containing regimens with rituximab to eliminate the intermediate-grade component of this disease.

[0141] In some embodiments, treatment options for relapsed / refractory PMBCL and TFL are similar to those for DLBCL. Given the low prevalence of these diseases, large-scale prospective randomized trials have not been conducted in these patient populations. Patients with chemotherapy-resistant disease are similar to those with refractory DLBCL or have a worse prognosis.

[0142] In some embodiments, by way of example, subjects having refractory intermediate-grade NHL (e.g., DLBCL, PMBCL, and TFL) have unmet high medical needs and further research with new therapies is needed in these populations.

[0143] Thus, in some embodiments, the method can be used to treat lymphoma or leukemia, which are B-cell malignancies. Examples of B-cell malignancies include, but are not limited to, non-Hodgkin lymphoma (NHL), small lymphocytic lymphoma (SLL / CLL), mantle cell lymphoma (MCL), FL, marginal zone lymphoma (MZL), extranodal (MALT lymphoma), nodal (monocytoid B-cell lymphoma), splenic, diffuse large cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, Burkitt lymphoma, and lymphoblastic lymphoma. In some embodiments, the lymphoma or leukemia is selected from B-cell chronic lymphocytic leukemia / small cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., Waldenström macroglobulinemia), splenic marginal zone lymphoma, hairy cell leukemia, plasma cell tumor (e.g., plasmacytoma (i.e., multiple myeloma), or plasmacytoma), extranodal marginal zone B-cell lymphoma (e.g., MALT lymphoma), nodal marginal zone B-cell lymphoma, follicular lymphoma (FL), transformed follicular lymphoma (TFL), primary cutaneous follicle center lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Epstein-Barr virus-positive DLBCL, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma (PMBCL), intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, Burkitt lymphoma / leukemia, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides / Sézary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, B-lymphoblastic leukemia / lymphoma, B-lymphoblastic leukemia / lymphoma with recurrent genetic abnormalities, T-lymphoblastic leukemia / lymphoma, and Hodgkin lymphoma.In some embodiments, the cancer is resistant to one or more prior treatments and / or the cancer has recurred after one or more prior treatments.

[0144] In certain embodiments, the cancer is selected from follicular lymphoma, transformed follicular lymphoma, diffuse large B-cell lymphoma, and primary mediastinal (thymic) large B-cell lymphoma. In a particular embodiment, the cancer is diffuse large B-cell lymphoma.

[0145] In some embodiments, the cancer is resistant to one or more of chemotherapy, radiation therapy, immunotherapy (including treatment with T-cell therapy and / or an antibody or antibody-drug conjugate), autologous stem cell transplantation, or any combination thereof, or the cancer has recurred thereafter. In a particular embodiment, the cancer is refractory diffuse large B-cell lymphoma.

[0146] In some embodiments, the cancer is treated by administering one or more T cells that have been contacted with an anti-CD81 antibody, exogenous interleukin-7 (IL-7), and exogenous interleukin-21 (IL-21). In some embodiments, the one or more T cells include engineered CAR cells or engineered TCR cells. In one embodiment, the engineered CAR cells or engineered T cells treat the tumor in a subject.

[0147] In some embodiments, the T-cell phenotype is evaluated by the expression of CCR7 and CD45RA. In some embodiments, the T-cell phenotype is a CAR T-cell phenotype. In some embodiments, the proportion of T cells (CCR7 + CD45RA + ) with a more naive phenotype in the apheresis product is directly related to a shorter product doubling time. Among CD8 T cells, CCR7 + CD45RA +The number of T cells is associated with sustained efficacy. In some embodiments, a higher peak increase in CAR T cells in peripheral blood, specifically estimated as CAR cells per unit blood volume, is associated with both objective and sustained responses. The number of CAR T cells in the initial peripheral blood immediately after infusion is associated with clinical efficacy. (Locke et.al., Tumor burden, inflammation, and product attributes determine outcomes of axicabtagene ciloleucel in large B-cell lymphoma; Blood Advances, 13 October 2020; Volume 4; Number 19).

[0148] 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 belongs. Any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, but the preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing the methods and / or materials in connection with which the publication is cited.

[0149] The specific examples listed below are illustrative only and are in no way limiting.

Example

[0150] Example 1: Influence of the Period and Conditions of CAR-T Activation and Increase on CAR-T Phenotype The CAR-T phenotypes during manufacturing can be affected by the period and exact conditions of CAR-T activation and expansion. Younger and less differentiated CAR-T phenotypes have been shown to correlate with better clinical outcomes. Using CD19 / CD20 dual-targeting CAR delivered by a lentiviral vector, the effects of different CAR-T manufacturing conditions on the product phenotypes were evaluated. The conditions tested in the first screening using three different healthy donors were as follows. 1) Standard manufacturing in which cells were activated by contacting plate-bound anti-CD3 antibody and soluble anti-CD28 antibody and culturing in an IL2-containing optimized medium. Represented as "IL2". 2) Manufacturing in which cells were activated by contacting plate-bound anti-CD3 antibody, soluble anti-CD28 antibody and soluble anti-CD81 antibody and culturing in an IL2-containing optimized medium. Represented as "aCD81 / IL2". 3) Manufacturing in which cells were activated by contacting plate-bound anti-CD3, soluble anti-CD28 antibody and culturing in an IL7- and IL21-containing optimized medium. Represented as "IL7 / IL21". 4) Manufacturing in which cells were activated by contacting plate-bound anti-CD3, soluble anti-CD28 and soluble anti-CD81 and culturing in an IL7- and IL21-containing optimized medium. Represented as "aCD81 / IL7 / IL21". AllCells™ (Alameda, CA) was used from leukopacks obtained from healthy donors (Prodigy™) to generate pan-CD3+ cells or CD4 + / CD8 +Cells were isolated and frozen in CryoStor® cell freezing medium (Sigma Aldrich®). The frozen T cells were thawed and activated with plate-bound MACS GMP CD3 pure (OKT3) (Miltenyl Biotec) and soluble human anti-CD28 (BD Biosciences) according to the manufacturer's recommendations, and left standing overnight in IL2 (Prometheus), or with IL7 (Peprotech) and IL21 (Peprotech). The next day, the cells were transduced with a lentiviral vector and cultured for approximately 8 days in T cell medium (OpTmizer® CTS® T cell expansion basal medium) containing an increasing supplement, CTS Immune Cell SR, CTS Glutamax (Gibco®), with cytokine replenishment appropriate for each of the above conditions and supplied every other day. For the condition using anti-CD81, the co-stimulatory antibody was added during the activation step using anti-CD3 and anti-CD28. On day 8, the cells were centrifuged and frozen in CryoStor® CS5 medium (BioLife Solutions®). Cells were sampled throughout the manufacturing process on days 0, 3, 4, 5, 6, 7, and 8, and cell phenotype was evaluated using flow cytometry. All antibody staining was performed at room temperature in BD Pharmingen® Azide containing staining buffer (FBS). All flow cytometry data were collected on a BD FACSymphony® A5 Cell Analyzer (BD and Company) using BD FACSDiva® software (BD and Company), and the data were analyzed using FlowJo (BD and Company).

[0151] The viability of T cells during production is shown in Table 1 below. TIFF2025519447000001.tif89166

[0152] The fold increase of T cells during production is shown in Table 2 below. TIFF2025519447000002.tif79166

[0153] The CAR expression of T cells during production is shown in Table 3 below. TIFF2025519447000003.tif56167Since the cells showed healthy survival rates, fold increases, and CAR transduction under all conditions, the phenotypes of the cells were evaluated using combinations of different cell surface markers as shown in the table below.

[0154] The %CD4 of T cells during production is shown in Table 4 below. TIFF2025519447000004.tif59167

[0155] The %CD8 of T cells during production is shown in Table 5 below. TIFF2025519447000005.tif59165Next, the memory phenotypes of the CD4 and CD8 compartments were evaluated using multiple cell surface markers.

[0156] The %CCR7+CD45RA+ of CD4+ T cells during production is shown in Table 6 below. TIFF2025519447000006.tif59167

[0157] The %CCR7+CD45RA+ of CD8+ T cells during production is shown in Table 7 below. TIFF2025519447000007.tif63167

[0158] As shown in the table above, the cells produced in aCD81 / IL7 / IL21 showed a higher naive phenotype (defined by CCR7+CD45RA+) especially during the later stages of production. Similar results were observed using alternative cell surface markers shown in Tables 8, 9, 10, and 11 below.

[0159] The %CD27+CD28+ of CD4+ T cells during production is shown in Table 8 below. TIFF2025519447000008.tif59167

[0160] The percentage of CD27+CD28+ in CD8+ T cells during production is shown in Table 9 below. TIFF2025519447000009.tif59167

[0161] The percentage of CD27+CD28+CCR7+CD45RA+ in CD4+ T cells during production is shown in Table 10 below. TIFF2025519447000010.tif63168

[0162] The percentage of CD27+CD28+CCR7+CD45RA+ in CD8+ T cells during production is shown in Table 11 below. TIFF2025519447000011.tif59169 Conversely, the inventors also observed a decrease in the effector phenotype in the inventors' products, as shown in the following table.

[0163] The percentage of CCR7-CD45RA- in CD4+ T cells during production is shown in Table 12 below. TIFF2025519447000012.tif59167

[0164] The percentage of CCR7-CD45RA- in CD8+ T cells during production is shown in Table 13 below. TIFF2025519447000013.tif59166

[0165] Example 2: Functional Characterization of CAR-T Cells As shown in Example 1, the production of cells in the presence of anti-CD81, IL7, and IL21 resulted in a more naive phenotype compared to the IL-2-based production process. To perform functional characterization, two different healthy donor T cells were used as starting materials, and CAR-T cells were produced using the following conditions. The groups to be compared are as follows. 1) Standard production using plate-bound anti-CD3 and soluble anti-CD28 antibodies to activate the cells and culturing them in an IL2-containing optimized medium. Represented as "IL2". 2) Manufactured by activating cells using anti-CD3, soluble anti-CD28, and soluble anti-CD81 conjugated to a plate and culturing them in an optimized medium containing IL7 and IL21. Represented as "aCD81 / IL7 / IL21". The cells produced from the above groups were frozen on day 6 of production. These cells were thawed overnight in RPMI medium (Gibco) containing 10% FBS (Gibco), phenotyped the next day, and set up in a co-culture assay with multiple target strains to evaluate cytotoxicity and cytokines as a readout of the functionality of CAR-T cells. The % cytotoxicity of the produced CAR T cells against three different target strains was measured at 24 hours. Four different effector:target ratios were tested, and the results are summarized in Tables 14, 15, and 16 below.

[0166] Table 14 TIFF2025519447000014.tif56170

[0167] Table 15 TIFF2025519447000015.tif57166

[0168] Table 16 TIFF2025519447000016.tif56170

[0169] As shown above, there was no significant difference in % cytotoxicity between the cells produced in IL2 and the cells produced in aCD81 / IL7 / IL21. However, cytokine evaluation of the co-culture supernatant collected at 24 hours using the U-Plex CAR-T cell combo 1 kit (MSD) revealed that the cells expanded in aCD81 / IL7 / IL21 secreted lower amounts of effector cytokines such as granzyme A and IFN-γ while also secreting higher amounts of IL2. The results are shown in Tables 17 and 18.

[0170] Table 17 TIFF2025519447000017.tif89169

[0171] Table 18 TIFF2025519447000018.tif102166

[0172] Next, the ability of these cells to increase in a continuous restimulation assay was evaluated. Briefly, CAR-T cells and CD19+ Nalm6 target cells from the American Type Culture Company (ATCC, Manassas, VA) were incubated together at an effector:target ratio of 1:1. After 2 days, samples were taken, stained for different markers, and phenotyped using flow cytometry. Absolute cell counts of both effector and target cells were also determined by including bead counting (ThermoFisher Scientific) during flow cytometry. Since the CAR-T cells increased during the assay, additional target cells were added to return the E:T ratio to 1:1 each time the cells were phenotyped. The assay was continued for 21 days.

[0173] The fold increase in CAR T cells produced from co-culture is summarized in Table 19 below. TIFF2025519447000019.tif70168The above results clearly show that CAR-T cells produced in aCD81 / IL7 / IL21 are superior in their ability to increase upon repeated antigen stimulation compared to CAR-T cells produced in IL2.

[0174] Example 3: In vivo efficacy of CAR-T cells produced in aCD81 / IL7 / IL21 This example describes the evaluation of the efficacy of CAR-T cells produced in IL2 versus CAR-T cells produced in aCD81 / IL7 / IL21 as tested in vivo in a Nalm6-luc-MHC DKO seeded mouse model.

[0175] CD19+ Nalm6-luc-MHC DKO cells containing a bioluminescent reporter were grown in 90% RPMI, 10% FBS, 1% L-glutamine. NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ) from The Jackson Laboratory were used in the study. 8-week-old mice were transplanted on day 0 by intravenous injection via the lateral tail vein with 5.0 x 105 CD19+ Nalm6-luc-MHC DKO cells in 0.1 mL using a BD U-100 insulin syringe 1 / 2 cc, 28G. All CAR-T cells and non-transduced (NTD) cells were manufactured as described in Example 1 and frozen on day 3 of manufacture. 100 uL of freshly thawed CAR-T cells were administered to the mice by intravenous injection 7 days after CD19+ Nalm6-luc-MHC DKO transplantation. Three different CAR+ doses, 2e5 cells (high), 4e4 cells (medium) and 8e3 cells (low) were tested.

[0176] In vivo bioluminescence imaging was performed using an IVIS Lumina S5. Animals were imaged 5 times at once under approximately 2 - 3% isoflurane gas anesthesia. 150 mg / kg of D-luciferin was injected IP into each mouse and the animals were imaged in the prone position 15 minutes after injection. A large binning of the CCD chip was used and the exposure time was adjusted to 15 seconds to obtain at least several hundred counts from metastatic tumors observable in each mouse in the image and avoid saturation of the CCD chip. BLI images were collected on days 5, 8, 12, 15, 19, 22, 26, 29, 33, 36, 40, 44, 48, 51, 55, 58, 61 and 65. Images were analyzed using Living Image version 4.5.4 software. A whole body fixed volume ROI was placed on the prone position image for each individual animal. The total light flux (photons / second) was calculated and exported for all ROIs.

[0177] BLI (bioluminescence imaging) values (shown as mean ± SEM) corresponding to CD19+ Nalm6-luc-MHC DKO tumor burden in mice are shown for different treatment groups (Tables 20A to 20E). Higher values indicate higher tumor burden. Both IL2-producing cells and aCD81 / IL7 / IL21-producing cells demonstrated comparable in vivo efficacy at high and medium doses, but CAR-T cells produced using aCD81 / IL7 / IL21 demonstrated superior tumor control kinetics at the lowest dose over the course of the study. In comparison, mice treated with vehicle only or non-transduced cells (NTD IL2 and NTD aCD81 / IL7 / IL21) showed no tumor control as expected.

[0178] Table 20A TIFF2025519447000020.tif22793

[0179] Table 20B TIFF2025519447000021.tif22797

[0180] Table 20C TIFF2025519447000022.tif22795

[0181] Table 20D TIFF2025519447000023.tif22795

[0182] Table 20E TIFF2025519447000024.tif190145* * *

[0183] Although many embodiments have been described, it will be apparent that the present disclosure and examples may provide other embodiments that utilize the compositions and methods described herein, or other embodiments subsumed within such compositions and methods. Accordingly, it will be understood that the scope of the present invention should be defined not by the embodiments presented as examples, but by what can be understood from the present disclosure and the appended claims.

Claims

1. contacting one or more lymphocytes derived from a subject with an anti-CD81 antibody, exogenous interleukin-7 (IL-7), and exogenous interleukin-21 (IL-21) in vitro; transforming the contacted lymphocytes with a vector containing a gene of interest; and collecting the lymphocytes, a method for producing genetically engineered lymphocytes.

2. The method according to claim 1, wherein the lymphocytes are selected from the group consisting of macrophages, neutrophils, basophils, eosinophils, granulocytes, natural killer cells (NK cells), B cells, T cells, NK-T cells, mast cells, tumor-infiltrating lymphocytes (TIL), myeloid-derived suppressor cells (MDSC), and dendritic cells.

3. The method according to claim 2, wherein the lymphocytes are T cells.

4. The method according to any one of claims 1 to 3, wherein the lymphocytes are contacted with an anti-CD3 antibody and an anti-CD28 antibody.

5. The method according to any one of claims 1 to 4, wherein the T cells include CD8+ T cells and CD4+ T cells.

6. The method according to claim 5, wherein the CD8+ T cells express CCR7+CD45RA+.

7. The method according to claim 5, wherein the CD4+ T cells express CCR7+CD45RA+.

8. The method according to claim 5, wherein the CD8+ T cells express CD27+CD28+.

9. The method according to claim 5, wherein the CD4+ T cells express CD27+CD28+.

10. The method according to claim 5, wherein the CD8+ T cells express CD27+CD28+CCR7+CD45RA+.

11. The method according to claim 5, wherein the CD4+ T cells express CD27+CD28+CCR7+CD45RA+.

12. The method according to any one of claims 1 to 11, wherein the T cells express a chimeric antigen receptor (CAR), and the T cells are transformed with a vector containing the chimeric antigen receptor (CAR).

13. The method according to claim 12, wherein the chimeric antigen receptor (CAR) is bicistronic or bispecific.

14. The method according to claim 12, wherein the chimeric antigen receptor (CAR) includes CD19.

15. The chimeric antigen receptor (CAR) in the method according to any one of claims 12 to 14 comprises a single-chain variable fragment (scFv) targeting an identified tumor antigen including CD20, BCMA, CLL-1, CTLA4, CD30, CD40, NKp44, NKp30, GPC-3, CD79a, CD79b, BAFF-R, CS-1, PSMA, NKG2D, CLL-1, CD33, CD22 or NKp46.

16. The method according to any one of claims 1 to 15, wherein the vector is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adeno-associated vector, a lentiviral vector, or any combination thereof.

17. The method according to claim 16, wherein the DNA vector is a transposon.

18. The method according to any one of claims 1 to 17, wherein the lymphocytes have not been contacted with exogenous interleukin-2 (IL-2).

19. The method according to any one of claims 1 to 18, wherein the donor is a subject in need of T cell therapy.

20. The method according to claim 16, wherein the lymphocytes are transduced with a vector containing the gene of interest.

21. The method according to claim 20, wherein the vector is a lentiviral vector.

22. The method according to claim 20, wherein the vector is a retroviral vector.

23. The method according to any one of claims 1 to 22, wherein the lymphocytes are collected within 24 hours after transformation.

24. The method according to any one of claims 1 to 23, wherein the lymphocytes are collected within 2 days after transformation.

25. The method according to any one of claims 1 to 24, wherein the lymphocytes are collected within 3 days after transformation.

26. The method according to any one of claims 1 to 25, wherein the lymphocytes are collected within 5 days after transformation.

27. The method according to any one of claims 1 to 26, wherein the lymphocytes are collected within 7 days after transformation.

28. The method according to any one of claims 1 to 27, wherein the lymphocytes are collected within 8 days after transformation.

29. The method according to any one of claims 1 to 28, wherein the lymphocytes are collected within 9 days after transformation.

30. The method according to any one of claims 1 to 29, wherein the lymphocytes are collected within 10 days after transformation.

31. The method according to any one of claims 1 to 30, wherein the lymphocytes are collected within 11 days after transformation.

32. The method according to any one of claims 1 to 31, wherein the lymphocytes are collected within 12 days after transformation.

33. The method according to any one of claims 1 to 32, wherein the lymphocytes are collected within 13 days after transformation.

34. The method according to any one of claims 1 to 33, wherein the lymphocytes are collected within 14 days after transformation.

35. Contacting one or more lymphocytes derived from a subject with an anti-CD81 antibody, exogenous interleukin-7 (IL-7), and exogenous interleukin-21 (IL-21) in vitro; Transforming the contacted lymphocytes with a vector containing a gene of interest; and collecting the lymphocytes. A genetically engineered lymphocyte produced by a method comprising:

36. The lymphocyte according to claim 35, wherein the lymphocyte is selected from the group consisting of macrophages, neutrophils, basophils, eosinophils, granulocytes, natural killer cells (NK cells), B cells, T cells, NK-T cells, mast cells, tumor-infiltrating lymphocytes (TIL), myeloid-derived suppressor cells (MDSC), and dendritic cells.

37. Contacting one or more lymphocytes derived from a subject with an anti-CD81 antibody, exogenous interleukin-7 (IL-7), and exogenous interleukin-21 (IL-21) in vitro; Transforming the contacted lymphocytes with a vector containing a gene of interest; and collecting the lymphocytes. A genetically engineered lymphocyte produced by a method comprising: The genetically engineered lymphocyte has a higher juvenility and a more undifferentiated CAR-T phenotype compared to a genetically engineered lymphocyte produced by contacting one or more lymphocytes derived from a subject with exogenous interleukin-2 (IL-2) in vitro.

38. The genetically engineered lymphocyte according to claim 37, wherein the lymphocyte is contacted with an anti-CD3 antibody and an anti-CD28 antibody.

39. The genetically engineered lymphocyte according to claim 37 or 38, wherein the lymphocyte is a T cell.

40. The CAR-T phenotype with higher juvenility and more low differentiation includes CCR7+CD45RA+CD4+ T cells, and is the genetically engineered lymphocyte according to any one of claims 37 to 39.

41. The CAR-T phenotype with higher juvenility and more low differentiation includes CCR7+CD45RA+CD8+ T cells, and is the genetically engineered lymphocyte according to any one of claims 37 to 40.

42. The CAR-T phenotype with higher juvenility and more low differentiation is produced when the lymphocyte is collected within 6 days after the transformation, and is the genetically engineered lymphocyte according to any one of claims 37 to 41.

43. The CAR-T phenotype with higher juvenility and more low differentiation is produced when the lymphocyte is collected within 8 days after the transformation, and is the genetically engineered lymphocyte according to any one of claims 37 to 42.

44. The CAR-T phenotype with higher juvenility and more low differentiation is produced when the lymphocyte is collected within 9 days after the transformation, and is the genetically engineered lymphocyte according to any one of claims 37 to 43.

45. The CAR-T phenotype with higher juvenility and more low differentiation is produced when the lymphocyte is collected within 10 days after the transformation, and is the genetically engineered lymphocyte according to any one of claims 37 to 44.

46. The CAR-T phenotype with higher juvenility and more low differentiation is produced when the lymphocyte is collected within 11 days after the transformation, and is the genetically engineered lymphocyte according to any one of claims 37 to 45.

47. The CAR-T phenotype with higher juvenility and more low differentiation is produced when the lymphocyte is collected within 12 days after the transformation, and is the genetically engineered lymphocyte according to any one of claims 37 to 46.

48. The CAR-T phenotype with higher juvenility and more low differentiation is produced when the lymphocyte is collected within 13 days after the transformation, and is the genetically engineered lymphocyte according to any one of claims 37 to 47.

49. The CAR-T phenotype with higher juvenility and more low differentiation is produced when the lymphocyte is collected within 14 days after the transformation, and is the genetically engineered lymphocyte according to any one of claims 37 to 48.

50. One or more lymphocytes derived from a subject are contacted in vitro with an anti-CD81 antibody, exogenous interleukin-7 (IL-7), and exogenous interleukin-21 (IL-21), the contacted lymphocytes are transformed with a vector containing a gene of interest, and the lymphocytes are collected, and the genetically engineered lymphocytes produced by the method comprising: the genetically engineered lymphocytes secrete a lower amount of effector cytokines compared to genetically engineered lymphocytes produced by contacting one or more lymphocytes derived from a subject with exogenous interleukin-2 (IL-2) in vitro.

51. The genetically engineered lymphocyte according to claim 50, wherein the lower amount of effector cytokine is granzyme A.

52. The genetically engineered lymphocyte according to claim 50, wherein the lower amount of effector cytokine is IFN-γ.

53. The genetically engineered lymphocyte according to any one of claims 50 to 52, wherein the lymphocyte further secretes a higher amount of IL2.

54. One or more lymphocytes derived from a subject are contacted in vitro with an anti-CD81 antibody, exogenous interleukin-7 (IL-7), and exogenous interleukin-21 (IL-21), the contacted lymphocytes are transformed with a vector containing a gene of interest, and the lymphocytes are collected, and the genetically engineered lymphocytes produced by the method comprising: the genetically engineered lymphocytes exhibit a more juvenile phenotype compared to genetically engineered lymphocytes produced by contacting one or more lymphocytes derived from a subject with exogenous interleukin-2 (IL-2) in vitro.

55. A composition comprising the genetically engineered lymphocyte according to any one of claims 35, 37, 50 or 54.

56. A method for treating cancer, comprising administering the composition according to claim 55 to a subject in need of treatment and monitoring the subject to determine the progress of the treatment.

57. Use of the genetically engineered lymphocytes according to any one of claims 35, 37, 50 or 54 for the manufacture of a composition for the treatment of cancer, wherein the genetically engineered lymphocytes are T cells.

58. The genetically engineered lymphocytes according to any one of claims 1 to 57, wherein the genetically engineered lymphocytes are used for the treatment of cancer.

59. The cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic cancer, adrenocortical cancer, AIDS-related cancer, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, central nervous system, B-cell leukemia, lymphoma, refractory B-cell malignancy or other B-cell malignancies, basal cell cancer, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brainstem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, germ cell tumor, central nervous system, endometrial cancer, epithelioblastoma, epithelioma, esophageal cancer,esthesioneuroblastoma, Ewing sarcoma family of tumors, extracranial primitive neuroectodermal tumor, extragonadal primitive neuroectodermal tumor, extrahepatic bile duct cancer, eye cancer, malignant fibrous histiocytoma of bone, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), soft tissue sarcoma, germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (pancreas), Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medulloepithelioma, melanoma, Merkel cell cancer, mesothelioma, metastatic squamous cell carcinoma with potential primary midline cancer associated with the NUT gene, oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell tumor, mycosis fungoides, myelodysplastic syndrome, myelodysplasia / myeloproliferative neoplasm, myeloid leukemia, chronic (CML), myeloid leukemia, acute (AML), myeloma, multiple, myeloproliferative disorder, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell tumor / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer,The method according to claim 56, selected from the group consisting of primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, cervical squamous cell carcinoma, gastric (stomach) cancer, primitive neuroectodermal tumor above the tentorium, T-cell lymphoma, skin, testicular cancer, pharyngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, choriocarcinoma, cancer of the ureter and renal pelvis, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor.

60. A method for treating a tumor in a subject in need of T cell therapy, comprising administering one or more lymphocytes to the subject, wherein the one or more lymphocytes have been contacted with an anti-CD81 antibody, exogenous interleukin-7 (IL-7) and exogenous interleukin-21 (IL-21).

61. A method for reducing or decreasing the size of a tumor or inhibiting the growth of a tumor in a subject in need of T cell therapy, comprising administering one or more lymphocytes to the subject, wherein the one or more lymphocytes have been contacted with an anti-CD81 antibody, exogenous interleukin-7 (IL-7) and exogenous interleukin-21 (IL-21).

62. The genetically engineered lymphocytes according to any one of claims 1 to 61, wherein the lymphocytes can be used in autologous cell therapy or allogeneic cell therapy.

63. The genetically engineered lymphocytes according to any one of claims 1 to 62, which produce more cytokines as compared to the process of producing genetically engineered lymphocytes in the presence of IL-2.

64. The genetically engineered lymphocytes according to any one of claims 1 to 63, which produce more IL-2 as compared to the process of producing genetically engineered lymphocytes in the presence of IL-2.

65. The genetically engineered lymphocytes according to any one of claims 1 to 64, which are more immature as compared to the process of producing genetically engineered lymphocytes in the presence of IL-2.

66. The genetically engineered lymphocytes according to any one of claims 1 to 65, which are more proliferative or robust as compared to the process of producing genetically engineered lymphocytes in the presence of IL-2.

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