Method for preparing lymphocytes for cell therapy

Abstract

The present disclosure relates to methods of making genetically engineered lymphocytes. In addition, methods of using genetically engineered lymphocytes in T cell therapy of cancer are also disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] 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. Technical Field

[0003] The present application relates to a method for preparing one or more lymphocytes (e.g., T cells) for cell therapy. The present application also relates to cells prepared, produced or processed using the methods disclosed herein. In some aspects, the present application relates to a method for improving the efficacy of cell therapy by contacting one or more lymphocytes with anti-CD81 antibodies, exogenous interleukin-7 (IL-7) and exogenous interleukin-21 (IL-21). Background Art

[0004] Human cancers are essentially made up of normal cells that have undergone genetic or epigenetic transformation to become abnormal cancer cells. As a result, cancer cells begin to express proteins and other antigens that are different from those expressed by normal cells. The body's innate immune system can use these abnormal tumor antigens to specifically target and kill cancer cells. However, cancer cells use various mechanisms to prevent immune cells (such as T and B lymphocytes) from successfully targeting cancer cells.

[0005] Human T cell therapy relies on ex vivo enriched or modified human T cells to target and kill cancer cells in a subject (e.g., a patient). A variety of techniques have been developed to enrich the concentration of naturally occurring T cells that can target tumor antigens or genetically modify T cells to specifically target known cancer antigens. These therapies have been shown to have promising effects on tumor size and patient survival. However, it has been shown to be difficult to predict whether a given T cell therapy is effective in each patient.

[0006] The transplantation of mixed populations of T cells is one of the factors that hinder T cell therapy from reaching its full potential. In conventional T cell therapy, donor T cells are collected, optionally modified to target specific antigens (e.g., tumor cells) or selected for anti-tumor characteristics (e.g., tumor infiltrating lymphocytes), amplified in vitro, and administered to subjects in need thereof. Typically, the resulting T cells include a mixed population of a large number of mature cells, many of which are terminally differentiated. As a result, the expected in vivo persistence of these cells may be limited, and the initially observed positive effects may disappear over time because tumors rebound in the absence of transplanted T cells. Therefore, it is still necessary to increase the in vivo persistence of T cells for T cell therapy.

[0007] Current T cell therapies rely on enriched or modified human T cells to target and kill cancer cells in patients. In order to increase the ability of T cells to target and kill specific cancer cells, methods for engineering T cells to express constructs that guide T cells to specific target cancer cells have been developed. Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs) containing binding domains that can interact with specific tumor antigens allow T cells to target and kill cancer cells that express specific tumor antigens.

[0008] There is a need for improved CARs and TCRs for targeting and killing cancer cells and for improved methods for preparing lymphocytes expressing CARs and / or TCRs for cell therapy. Summary of the invention

[0009] Any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein.Other aspects, advantages, and modifications are within the scope of the present application.

[0010] The present disclosure provides a method for producing 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.

[0011] The present disclosure also provides a method for producing genetically engineered lymphocytes, 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 (TILs), myeloid-derived suppressor cells (MDSCs) and dendritic cells.

[0012] 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.

[0013] In certain embodiments, T cells include CD8+T cells and CD4+T cells. In some embodiments, CD8+T cells express CCR7+ and / or CD45RA+. In some embodiments, CD4+T cells express CCR7+ and / or CD45RA+. In certain embodiments, CD8+T cells express CD27+ and / or CD28+. In some embodiments, CD4+T cells express CD27+ and / or CD28+. In certain embodiments, CD8+T cells express CD27+CD28+CCR7+ and / or CD45RA+. In some embodiments, CD4+T cells express CD27+CD28+CCR7+ and / or CD45RA+.

[0014] The present disclosure also provides a method for manufacturing genetically engineered lymphocytes, wherein T cells express chimeric antigen receptors (CAR). In certain embodiments, chimeric antigen receptors (CAR) are bicistronic. In certain embodiments, chimeric antigen receptors (CAR) are bispecific. In some embodiments, chimeric antigen receptors (CAR) are combined with CD19. In certain embodiments, chimeric antigen receptors (CAR) include single-chain variable fragments (scFv) of tumor antigens identified by targeting, and the tumor antigens identified include 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.

[0015] The present disclosure also provides a method for making genetically engineered lymphocytes, wherein the vector is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof. In certain embodiments, the DNA vector is a transposon.

[0016] The present disclosure also provides a method of making genetically engineered lymphocytes, wherein the lymphocytes are not contacted with exogenous interleukin-2 (IL-2).

[0017] In certain embodiments, the donor is a subject in need of T cell therapy.

[0018] The present disclosure also provides a method for making genetically engineered lymphocytes, wherein the lymphocytes are transduced with a viral vector containing a target gene. In certain embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a retroviral vector.

[0019] In certain embodiments, lymphocytes are harvested no more than 24 hours after conversion. In certain embodiments, lymphocytes are harvested no more than 2 days after conversion. In certain embodiments, lymphocytes are harvested no more than 3 days after conversion. In certain embodiments, lymphocytes are harvested no more than 5 days after conversion. In certain embodiments, lymphocytes are harvested no more than 7 days after conversion. In certain embodiments, lymphocytes are harvested no more than 8 days after conversion. In certain embodiments, lymphocytes are harvested no more than 9 days after conversion. In certain embodiments, lymphocytes are harvested no more than 9 days after conversion. In certain embodiments, lymphocytes are harvested no more than 10 days, no more than 11 days, no more than 12 days, no more than 13 days, or no more than 14 days after conversion.

[0020] Another aspect of the present disclosure relates to a genetically engineered lymphocyte produced by a method comprising the following steps: 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 target gene; transforming the contacted lymphocytes with a vector containing a target gene; and collecting 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.

[0021] In certain embodiments, the genetically engineered lymphocytes have a more naive and less differentiated CAR-T phenotype compared to genetically engineered lymphocytes produced by contacting one or more lymphocytes from a subject with exogenous interleukin-2 (IL-2) in vitro.

[0022] In certain embodiments, genetically engineering lymphocytes further comprises transforming the contacted lymphocytes with a vector containing the gene of interest and harvesting the lymphocytes. In some embodiments, the lymphocytes are T cells.

[0023] In some embodiments, the more naive and less differentiated CAR-T phenotypes include CCR7+CD45RA+CD4+T cells. In certain embodiments, the more naive and less differentiated CAR-T phenotypes include CCR7+CD45RA+CD8+T cells.

[0024] In some embodiments, when lymphocytes are harvested no more than 6 days after transformation, a higher juvenile and lower differentiated CAR-T phenotype is generated. In certain embodiments, when lymphocytes are harvested no more than 8 days after transformation, a higher juvenile and lower differentiated CAR-T phenotype is generated. In certain embodiments, when lymphocytes are harvested no more than 9 days, no more than 10 days, no more than 11 days, no more than 12 days, no more than 13 days, or no more than 14 days after transformation, a higher juvenile and lower differentiated CAR-T phenotype is generated.

[0025] In certain embodiments, the genetically engineered lymphocytes secrete lower effector cytokines compared to genetically engineered lymphocytes produced by contacting one or more lymphocytes from a subject with exogenous interleukin-2 (IL-2) in vitro.

[0026] In certain embodiments, the lower effector cytokine is granzyme A. In certain embodiments, the lower effector cytokine is IFN-γ. In certain embodiments, the genetically engineered lymphocytes also secrete higher IL-2.

[0027] In certain embodiments, the genetically engineered lymphocytes exhibit a more naive phenotype compared to genetically engineered lymphocytes produced by contacting one or more lymphocytes from a subject with exogenous interleukin-2 (IL-2) in vitro.

[0028] Another aspect of the present disclosure relates to a composition comprising genetically engineered lymphocytes.

[0029] The present disclosure also 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.

[0030] Another aspect of the present disclosure relates to the use of genetically engineered lymphocytes for the manufacture of a composition for treating cancer, wherein the genetically engineered lymphocytes are T cells.

[0031] In certain embodiments, genetically engineered lymphocytes are used to treat cancer.

[0032] In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, carcinoma, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoma, central nervous system cancer, B cell leukemia, lymphoma, refractory B cell malignancies or other B cell malignancies, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic spinal Myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, embryonal tumors, central nervous system cancer, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, esthesia neuroblastoma, Ewing sarcoma family of tumors, extracranial germ cell tumors, extracranial germ cell tumors, extrahepatic bile duct cancer, eye cancer, bone fibrous histiocytoma, malignant tumors, and osteosarcoma, gallbladder cancer, stomach (gastric) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), soft tissue sarcomas, germ cell tumors, gestational trophoblastic tumors, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors (endocrine pancreas), Kaposi's meat tumors, 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, medullary epithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary midline tract cancer involving the NUT gene, oral cancer, multiple endocrine neoplasms syndrome, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), multiple myeloma, myeloproliferative disorders, nasal cavity and nasal Sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate differentiated pinealoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, transitional cell cancer of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, small cell lung cancer, small intestine cancer,Soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin cancer, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, 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, Waldenstrom's macroglobulinemia, and Wilm's tumor.

[0033] Another aspect of the present disclosure relates to 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).

[0034] The present disclosure also provides a method of reducing or diminishing tumor size or inhibiting tumor growth 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).

[0035] In some embodiments, genetically engineered lymphocytes can be used in autologous cell therapy or allogeneic cell therapy.

[0036] In some embodiments, the genetically engineered lymphocytes produce more cytokines than a method in which the genetically engineered lymphocytes are prepared in the presence of IL-2. In some embodiments, the genetically engineered lymphocytes produce more IL-2 than a method in which the genetically engineered lymphocytes are prepared in the presence of IL-2.

[0037] In some embodiments, the genetically engineered lymphocytes are more juvenile than in methods in which the genetically engineered lymphocytes are prepared in the presence of IL-2. In some embodiments, the genetically engineered lymphocytes are more proliferative or more robust than in methods in which the genetically engineered lymphocytes are prepared in the presence of IL-2. DETAILED DESCRIPTION

[0038] The present disclosure relates to methods for producing genetically engineered lymphocytes for T cell therapy. In particular, the present disclosure relates to methods for producing CAR T cells for T cell therapy.

[0039] definition

[0040] In order to make the present disclosure more easily understood, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0041] 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 the present disclosure relates. For example, "The Concise Dictionary of Biomedicine and Molecular Biology", Juo, Pei-Show, 2nd edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised Edition, 2000, Oxford University Press provide a general dictionary of many of the terms used in the present disclosure for a skilled person.

[0042] Units, prefixes and symbols are expressed in their SI (International System of Units) accepted forms. Numerical ranges include numbers that define the ranges. The headings provided herein do not limit the various aspects of the disclosure, which can be referenced to this specification as a whole. Therefore, the terms defined immediately below are more fully defined by reference to the entire content of the specification.

[0043] As used herein, the indefinite articles "a" and "an" should be understood to mean "one or more" of any stated or listed components.

[0044] As used herein, the term "about" refers to approximately + / - 10% variation from a given value. When specific values ​​or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable error range for that specific value or composition.

[0045] As described herein, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the range, and fractions thereof (such as tenths and hundredths of integers) where appropriate, unless otherwise specified.

[0046] As used herein, the term "and / or" should be considered as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: 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).

[0047] The terms "activation" and "activated" refer to the state of lymphocytes (e.g., 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 primarily to T cells undergoing cell division. T cell activation can be characterized by an increase in 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.

[0048] As used herein, the phrase "administer" refers to any of various methods and delivery systems known to those skilled in the art that medicament is physically introduced into a subject. Exemplary routes of administration for the T cells prepared by the method disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration (e.g., by injection or infusion). As used herein, the phrase "parenteral administration" means the mode of administration (usually by injection) except enteral and topical administration, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion and in vivo electroporation. In some embodiments, the T cells prepared by this method are administered by non-parenteral routes (e.g., oral). Other non-parenteral routes include local, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or topical. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0049] The term "antibody" (Ab) includes, but is not limited to, immunoglobulins that specifically bind to an antigen. In general, an antibody may include 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 may 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 may include one constant domain CL. The VH region and the VL region can be further subdivided into hypervariable regions (called complementary determining regions (CDRs)), with more conserved regions (called framework regions (FRs)) interspersed therebetween. Each VH and VL includes three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains include binding domains that interact with antigens.

[0050] Immunoglobulins can be derived from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those skilled in the art, including but not limited to human IgG1, IgG2, IgG3 and IgG4. "Isotype" refers to the Ab (antibody) class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. For example, the term "antibody" includes both naturally occurring antibodies and non-naturally occurring antibodies; monoclonal antibodies and polyclonal antibodies; chimeric antibodies and humanized antibodies; human antibodies or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. In the absence of explicit instructions, and unless the context otherwise indicates, the term "antibody" also includes an antigen-binding fragment or antigen-binding portion of any of the aforementioned immunoglobulins, and includes monovalent and divalent fragments or portions, as well as single-chain antibodies.

[0051] "Antigen binding molecule" or "antibody fragment" refers to any antibody portion that is smaller than the entire antibody. Antigen binding molecules may include antigen complementary determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen binding molecules.

[0052] The term "autologous" refers to any material that originates from the same individual and is later reintroduced into that individual. For example, the engineered autologous cell therapy (eACT) described herein TM ) methods involve collecting lymphocytes from a donor (e.g., a patient), then engineering them to express, for example, a CAR construct, and subsequently administering them back to the same donor (e.g., patient).

[0053] The term "allogeneic" refers to any material derived from one individual and subsequently introduced into another individual of the same species, such as an allogeneic T cell transplant.

[0054] "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors, which invade adjacent tissues and can also be transferred to distant parts of the body through the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors at various stages. In certain embodiments, the cancer or tumor is in stage 0, so that, for example, the cancer or tumor is in very early development and has not yet metastasized. In some embodiments, the cancer or tumor is in stage I, so that, for example, the size of the cancer or tumor is relatively small, has not spread to nearby tissues, and has not yet metastasized. In other embodiments, the cancer or tumor is in stage II or stage III, so that, for example, the cancer or tumor is greater than stage 0 or stage I, and it has grown into adjacent tissues, but it has not yet metastasized, except potentially lymph nodes. In other embodiments, the cancer or tumor is in stage IV, so that, for example, the cancer or tumor has metastasized. Stage IV can also be referred to as advanced or metastatic cancer.

[0055] As used herein, "anti-tumor effect" refers to a biological effect that can be manifested as a reduction in tumor volume, inhibition of tumor growth, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number of metastases, an extension of overall survival or progression-free survival, an extension of life expectancy, or an improvement in various physiological symptoms associated with tumors. Anti-tumor effect can also refer to the prevention of tumor occurrence, such as vaccines.

[0056] As used herein, the term "progression-free survival" (which may be abbreviated as PFS) refers to the time from the date of treatment to the date of disease progression or death due to any cause according to the revised IWG Response Criteria for Malignant Lymphoma.

[0057] "Disease progression" assessed by measurement of malignant lesions on radiographs or other methods should not be reported as an adverse event. Death due to disease progression in the absence of signs and symptoms should be reported for the primary tumor type (e.g., DLBCL).

[0058] "Duration of response" (which may be abbreviated as DOR) refers to the period of time between the subject's first objective response according to the modified IWG malignant lymphoma response criteria to the date of confirmed disease progression or death.

[0059] The term "overall survival" (which may be abbreviated as OS) is defined as the time from the date of treatment to the date of death.

[0060] As used herein, "cytokine" 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, those cytokines secreted in response to T cell therapy can be a hallmark of effective T cell therapy.

[0061] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of T cells produced by the present method and which protect a subject from the onset of disease or promote disease regression (evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of damage or disability due to disease affliction) when used alone or in combination with another therapeutic agent. The ability of T cells to promote disease regression can be assessed using a variety of methods known to the skilled artisan, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of an agent in an in vitro assay.

[0062] As used herein, the term "lymphocyte" may include natural killer (NK) cells, T cells or B cells. NK cells are a type of cytotoxic (toxic to cells) lymphocytes that represent the main components of the innate immune system. NK cells repel tumors and cells infected by viruses. It works through the process of apoptosis or programmed cell death. They are called "natural killers" because they do not need activation to kill cells. T cells play a major role in cell-mediated immunity (without antibody involvement). Their T cell receptors (TCRs) distinguish themselves from other lymphocyte types. The thymus is a specialized organ of the immune system, primarily 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), bone marrow-derived suppressor cells (MDSC) and dendritic cells. The term also includes precursors of these lymphocytes. Hematopoietic stem cells and / or progenitor cells can be derived from adult peripheral blood after bone marrow, cord blood, cytokine mobilization, etc. by methods known in the art. Some precursor cells are those cells that can differentiate into lymphoid lineages (e.g., hematopoietic stem cells or progenitor cells of lymphoid lineages). Additional examples of lymphocytes that can be used for immunotherapy are described in U.S. Publication No. 20180273601, which is incorporated herein by reference in its entirety.

[0063] There are several types of T cells, namely: helper T cells (e.g., CD4+ cells, effector TEFFs ("effector T cells")), cytotoxic T cells (also known as TCs, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells or killer T cells), memory T cells ((i) stem memory TSC cells (such as naive cells) are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Rα+, but they also express large amounts of CD95+, IL-2Pβ, C XCR3+ and LFA-, and display many functional attributes that are unique to memory cells); (ii) central memory TCM cells express L-selectin and are CCR7+ and CD45RO+, they secrete IL-2, but not IFNγ or IL-4, whereas (iii) effector memory TEM cells do not express L-selectin or CCR7, but do 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 γδT cells. T cells found within tumors are called "tumor infiltrating lymphocytes" or "TILs." On the other hand, B cells play a major role in humoral immunity (with the involvement of antibodies). They make antibodies and antigens and function as antigen presenting cells (APCs), and transform into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow from which they derive their name.

[0064] "Immature" T cells refer to mature T cells that maintain immune undifferentiation. After positive and negative selection in the thymus, T cells appear as CD4+ or CD8+ immature T cells. In their immature state, T cells express L-selectin (CD62L+), IL-7 receptor α (IL-7R-α) and CD132, but they do not express CD25, CD44, CD69 or CD45RO. As used herein, "immature" can also refer to T cells showing phenotypic characteristics of immature T cells or immature T cells (such as TSC M cells or TCM cells). For example, immature T cells can express one or more of L-selectin (CD62L+), IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3 and LFA-1. Immature or immature T cells can be contrasted with terminally differentiated effector T cells (such as TEM cells and TEFF cells).

[0065] After encountering antigen, naive T cells undergo activation and differentiate into TSCM (stem memory T cells), TC M (central memory T cells) and effector T cells. The hallmarks of this differentiation are the expression of various cell surface markers and transcription factors and significant changes in cell metabolic pathways. Generally, T cell activation and differentiation are characterized by increased dependence on glycolysis and mitochondrial membrane potential. These metabolic pathways are critical for mediating the effector functions of T cells in response to infection and cancer. Naive T cells are T cells that have matured but have not encountered antigens. In addition to being CCR7+, CD45RO- and CD95-, other common markers of naive T cells are CD45RA+, IL-2Rβ-, (L-selectin), CD27+, CD28+, IL-7Ra+ and CD62L+. Stem memory T cells (TSCM) have been described in mice, non-human primates and humans, which constitute approximately 2%-4% of total CD4+ and CD8+T cell populations in the periphery. TSCM represents the earliest and long-term developmental stage of memory T cells, exhibits stem cell-like properties, and exhibits a gene spectrum between natural and central memory T cells. In addition to CCR7+, CD45RO- and CD95+, other common markers of stem memory T cells (TSCM) are CD45RA+, IL-2Rβ+, CD62L+, (L-selectin), CD27+, CD28+, IL-7Ra+, IL-2RP+, CXCR3+ and LFA-. Central memory T cells (T CM) express CD45RO+, CCR7+ and CD62L+. This memory subset is usually present in lymph nodes and peripheral circulation. Other common markers of central memory T cells (T CM) are CD95+, IL-2Rβ+, CD3+, CD28+, CD127+ and granzyme B-. Effector memory T cells (T EM) express CD45RO, but lack the expression of CCR7. Because these memory T cells lack CCR7 lymph node homing receptors, they are found in peripheral circulation and tissues. Other common markers of 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 of effector T cells (TEFF) are CD95+, IL-2Rβ+, CD62L-, CD28-, CD62L-, CD 127-, granzyme B+ and perforin+.

[0066] As referred to 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 activity. In some embodiments, T cell function includes cytolytic activity.

[0067] As used herein, cell "proliferation" or "cell expansion" refers to the ability of T cells to grow in quantity by cell division. Proliferation can be measured by staining cells with carboxyfluorescein succinimidyl ester (CFSE). Cell proliferation may occur in vitro (e.g., during T cell culture) or in vivo (e.g., after administering T cell therapy).

[0068] As used herein, "T cell activity" refers to any activity common to healthy T cells. In some embodiments, T cell activity includes cytokine production. In certain embodiments, T cell activity includes the production of one or more cytokines selected from interferon gamma (IFNg), tissue necrosis factor alpha (TNFa), and both.

[0069] As used herein, "cytolytic activity" or "cytotoxicity" refers to the ability of a T cell to destroy a target cell. In some embodiments, the target cell is a cancer cell, such as a tumor cell. In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the target cell expresses a target antigen.

[0070] The terms "genetic engineering", "gene editing" or "engineering" refer to methods of modifying the genome of a cell, including but not limited to deleting a coding region or a non-coding region or a portion thereof, or inserting a coding region or a portion thereof. In some embodiments, the modified cell is a lymphocyte, such as a T cell, which can be obtained from a patient or a donor. The cell can be modified to express an exogenous construct incorporated into the genome of the cell, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0071] The chimeric antigen receptor (CAR or CAR-T) and T cell receptor (TCR) of the present application are genetically engineered receptors. According to techniques known in the art, these engineered receptors can be easily inserted into lymphocytes including T cells and expressed by them. Using CAR, a single receptor can be programmed to recognize specific antigens and activate lymphocytes to attack and destroy cells carrying or expressing the antigen when binding to the antigen. When these antigens are present on tumor cells, lymphocytes expressing CAR can target and kill tumor cells. In one embodiment, the cell prepared according to the present application is a cell having a chimeric antigen receptor (CAR) or T cell receptor comprising an antigen binding molecule, a costimulatory domain and an activation domain. The costimulatory domain may 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.

[0072] "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 produced by any of these cells or the liver (including Abs, cytokines, and complement) that result in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, cells or tissues infected by pathogens, cancer cells or other abnormal cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation from the body of a vertebrate.

[0073] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of developing a disease or recurrence by methods including inducing, enhancing, suppressing or otherwise altering an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy may include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT) TM ) and allogeneic T cell transplantation. Then, those skilled in the art will recognize that the methods for preparing T cells disclosed herein will enhance the efficacy of any transplanted T cell therapy.

[0074] The T cells of immunotherapy can be from any source known in the art. For example, T cells can be differentiated from a hematopoietic stem cell population in vitro, or T cells can be obtained from a donor. The donor can be a subject, such as a subject in need of anticancer treatment. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In addition, T cells can be derived from one or more T cell lines available in the art. A variety of techniques known to technicians (such as FICOLL) can also be used. TM T cells can be obtained from a blood unit collected from a subject by separation and / or apheresis. T cells can also be obtained from an artificial thymic organoid (ATO) cell culture system, which replicates the human thymus environment to support efficient ex vivo differentiation of T cells from primary and reprogrammed pluripotent stem cells.

[0075] The term "engineered autologous cell therapy" (which may be abbreviated as "eACT") is TMAdoptive cell transfer (“ATC”), also known as adoptive cell transfer, is a process by which a patient’s own T cells are collected and subsequently genetically engineered 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 to express, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR). CAR-positive (+) T cells are engineered to express an extracellular single-chain variable region fragment (scFv) specific for a particular tumor antigen, linked to an intracellular signaling moiety comprising a co-stimulatory domain and an activation domain.The "costimulatory domain" can be derived from, 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 co-stimulator (ICOS), 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 molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, 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, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, D1 1b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, 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, a ligand specifically bound to CD83, or any combination thereof. "Activation domain" can be derived from, for example, CD3, such as CD3ζ, ε, δ, γ, etc. In certain embodiments, the CAR is designed to have two, three, four or more co-stimulatory domains.CAR scFv can be designed to target, for example, CD19, which is a transmembrane protein expressed by cells in the B cell lineage (including all normal B cells and B cell malignancies, including but not limited to NHL, CLL, and non-T cell ALL). Exemplary CAR+T cell therapies and constructs are described in U.S. Patent Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, and these references are incorporated by reference in their entirety.

[0076] As used herein, "patient" includes anyone suffering from cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein. The term "donor subject" refers to a subject whose cells are obtained for further in vitro engineering. The donor subject can be a cancer patient (i.e., an autologous donor) who will be treated with a cell colony produced by the methods described herein, or can be an individual (i.e., an allogeneic donor) who donates a lymphocyte sample, which will be used to treat different individuals or cancer patients after producing a cell colony produced by the methods described herein. Those subjects who receive cells prepared by this method can be referred to as "recipient subjects."

[0077] As used herein, "stimulation" refers to a primary response induced by the binding of a stimulatory molecule to its cognate ligand, wherein the binding mediates a signal transduction event. "Stimulatory molecules" are molecules on T cells (e.g., T cell receptor (TCR) / CD3 complexes) that specifically bind to cognate stimulatory ligands presented on antigen presenting cells. "Stimulatory ligands" are ligands that can specifically bind to stimulatory molecules on T cells when presented on antigen presenting cells (e.g., artificial antigen presenting cells (aAPCs), dendritic cells, B cells, etc.), thereby mediating the primary response of T cells (including but not limited to activation, initial immune response, proliferation, etc.). Stimulatory ligands include but are not limited to MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies. As used herein, "activated" or "active" refers to T cells that have been stimulated. Active T cells can be characterized by the expression of one or more markers selected from CD137, CD25, CD71, CD26, CD27, CD28, CD30, CD154, CD40L and CD134.

[0078] The term "exogenous" refers to any substance derived from an external source. For example, exogenous IL-7 or exogenous IL-21 can be commercially obtained or recombinantly produced. When added to one or more T cells or in contact with one or more T cells, "exogenous IL-7" or "exogenous IL-21" means that T cells do not produce IL-7 and / or IL-21. In some embodiments, the T cells before mixing with "exogenous" IL-7 or IL-21 can contain trace IL-7 and / or IL-21 (i.e., endogenous IL-7 or IL-21) produced by T cells or separated from a subject together with 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, including adding separated IL-7 and / or IL-21 to the culture medium, including IL-7 and / or IL-21 in the culture medium, or expressing IL-7 and / or IL-21 by one or more cells in the culture (such as by a feeder layer) except one or more T cells.

[0079] "Treatment" or "treatment" of a subject refers to any type of intervention or process performed on a subject / patient, or the administration of one or more T cells prepared by the present disclosure to a subject, with the purpose of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of symptoms, complications or conditions or biochemical indicators associated with the disease. In one embodiment, "treatment" includes partial remission. In another embodiment, "treatment" includes complete remission.

[0080] As used herein, the term "heterologous" refers to any source other than a naturally occurring sequence. For example, a heterologous sequence that is part of a costimulatory protein having an amino acid sequence of a corresponding human costimulatory protein is an amino acid that does not occur naturally, i.e., is not identical to a wild-type human costimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence that is different from a wild-type human costimulatory protein coding sequence.

[0081] "Antigen" refers to any molecule that causes an immune response or can be bound by an antibody or antigen binding molecule. An immune response may involve antibody production, or the activation of specific immunocompetent cells, or both. It will be readily understood by those skilled in the art that any macromolecule (including nearly all proteins or peptides) can serve as an antigen. Antigens can be endogenously expressed, that is, expressed by genomic DNA, or can be recombinantly expressed. Antigens can be specific for certain tissues (such as cancer cells), or can be widely expressed. In addition, fragments of larger molecules can serve as antigens. In one embodiment, the antigen is a tumor antigen. In a specific embodiment, the antigen is all or a fragment of BCMA, FLT3 or CLL-1.

[0082] The term "transformation" is a specific process in which exogenous genetic material is taken up and incorporated directly by a cell through its cell membrane. This usually occurs when the cell is in a competent state, which is a state in which the cell can take up exogenous material.

[0083] The terms "transduction" and "transduced" refer to the process of introducing exogenous DNA into cells by viral vectors (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 virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0084] The term "transposon" is a segment of DNA that can move to different locations in the genome of a single cell. In the process, they can cause mutations and increase (or decrease) the amount of DNA in the cell's genome, and if the cell is a precursor to gametes, increase (or decrease) the amount of DNA in the genome of any offspring.

[0085] As used herein, the term "in vitro cell" refers to any cell cultured ex vivo. Specifically, the in vitro cell may include a T cell.

[0086] 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.

[0087] As used herein, "costimulatory ligands" include molecules on antigen presenting cells that specifically bind to cognate costimulatory molecules on T cells. The binding of costimulatory ligands provides signals that mediate T cell responses (including but not limited to proliferation, activation, differentiation, etc.). Costimulatory ligands induce signals other than the primary signals provided by stimulatory molecules, for example, by binding of T cell receptor (TCR) / CD3 complexes to major histocompatibility complex (MHC) molecules loaded with peptides. Co-stimulatory ligands may include, but are not limited to, 3 / TR6, 4-1BB ligands, agonists or antibodies that bind to Toll ligand receptors, B7-1 (CD80), B7-2 (CD86), CD30 ligands, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT) 3, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), ligands that specifically bind to B7-H3, lymphotoxin beta 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. 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, CD81, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, a ligand that specifically binds to 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).

[0088] "Co-stimulatory molecules" are cognate binding partners on T cells that specifically bind to co-stimulatory ligands, thereby mediating co-stimulatory responses of T cells, such as but not limited to proliferation. Co-stimulatory molecules include but are not limited to 4-1BB / CD137, B7-H3, BAFF-R, BLAME (SLAMF8), BTLA, CD 33, CD 45. CD100(SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160(BY55), CD18, CD19, CD19 a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (α; β; δ; ε; γ; ζ), CD30, CD37, C D4, 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 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling 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, truncations or combinations thereof.

[0089] As used herein, "juvenile phenotype" or "juvenile cells" refers to less differentiated immune cells, e.g., immature immune cells. In some embodiments, the juvenile phenotype or juvenile cells refers to less differentiated T cells (defined by CCR7+CD45RA+). In some embodiments, the juvenile T cells express CCR7+ and / or CD45RA+. In other embodiments, the juvenile T cells express CCR7+ and / or CD45RA+. In some embodiments, the juvenile T cells comprise a CAR-T phenotype.

[0090] As used herein, the term "bicistronic" refers to a single messenger RNA molecule capable of producing two proteins. In some embodiments, a chimeric antigen receptor (CAR) is a bicistronic. As used herein, the term "bispecific" refers to an artificial protein that can bind to two different types of antigens or two different epitopes on the same antigen at the same time. In some embodiments, a chimeric antigen receptor (CAR) is bispecific.

[0091] Various aspects of the disclosure are described in further detail in the following subsections.

[0092] Method for preparing genetically engineered lymphocytes :

[0093] Methods described herein can enhance the effectiveness of cell therapy. In some aspects, cell therapy can be adoptive T cell therapy, including autologous cell therapy or allogeneic cell therapy. In some other aspects, T cell therapy widely includes any selection, in vitro enrichment, and identification and autologous or allogeneic T cells capable of binding tumor cells are applied to patients. In some aspects, cell therapy is a therapy using lymphocytes that are not T cells, and the lymphocytes include but are 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 suppressor cells (MDSC) and dendritic cells, these cells can be genetically engineered to express at least one CAR, and it can be autologous or allogeneic for patients. Genetically engineered lymphocytes produced in the presence of a combination of anti-CD81, IL7, and IL21 are very useful for autologous as well as allogeneic therapies because the cells produced are more robust or more proliferative and more naive when exposed to tumor antigens and produce more cytokines, such as IL-2, than cells produced in standard manufacturing methods in the presence of IL-2. In contrast, cells produced in the presence of exogenous IL-2 are less active, i.e., more differentiated, produce fewer cytokines, such as IL-2, and expand less when contacted with tumor antigens.

[0094] In some embodiments, the methods described herein may further include enriching a lymphocyte population obtained from a donor. Enriching a lymphocyte (e.g., one or more T cells) population may be accomplished by any suitable separation method, including but not limited to using a separation medium (e.g., FICOLL-PAQUE TM ROSETTESEP TM HLA total lymphocyte enrichment mixture, lymphocyte separation medium (LSA) (MP Biomedical catalog number 0850494X), etc.), cell size, shape or density separation by filtration or panning, immunomagnetic separation (e.g., magnetic activated cell sorting system, MACS), fluorescent separation (e.g., fluorescence activated cell sorting system, FACS) or bead-based column separation.

[0095] Stimulating the lymphocyte population with one or more stimulatory agents to produce a lymphocyte population :

[0096] In some embodiments, the method described herein also includes stimulating lymphocyte colonies with one or more stimulants under suitable conditions to produce activated cell colonies. Any combination of one or more suitable stimulants can be used to produce activated lymphocyte colonies, and the suitable stimulant includes but is not limited to targeting lymphocyte stimulation or the antibody or its functional fragment of costimulatory molecules (for example, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD28 antibodies, anti-CD81 antibodies or their functional fragments) or any other suitable mitogen (for example, tetradecanoylphorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM) or T cell stimulation or the natural ligand of costimulatory molecules.

[0097] In some embodiments, suitable conditions for stimulating a lymphocyte population as described herein may include temperature, amount of time, and / or in the presence of a certain CO2 level. 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°C-38°C. In certain embodiments, the temperature for stimulation is about 35°C-37°C. In certain embodiments, the temperature for stimulation is about 36°C-38°C. In certain embodiments, the temperature for stimulation is about 36°C-37°C or about 37°C.

[0098] In some embodiments, another condition for stimulating lymphocyte colonies as described herein may include the time for stimulation. In some embodiments, the time for stimulation is about 24 hours to 72 hours. In some embodiments, the time for stimulation is about 24 hours to 36 hours, about 30 hours to 42 hours, about 36 hours to 48 hours, about 40 hours to 52 hours, about 42 hours to 54 hours, about 44 hours to 56 hours, about 46 hours to 58 hours, about 48 hours to 60 hours, about 54 hours to 66 hours or about 60 hours to 72 hours. In a specific embodiment, the time for stimulation is about 48 hours or at least about 48 hours. In other embodiments, the time for stimulation is about 44 hours to 52 hours. In certain embodiments, the time for stimulation is about 40 hours to 44 hours, about 40 hours to 48 hours, about 40 hours to 52 hours or about 40 hours to 56 hours.

[0099] In some embodiments, other conditions for stimulating lymphocyte populations as described herein may include CO2 levels. In some embodiments, the CO2 level for stimulation is about 1.0%-10% CO2. In some embodiments, the CO2 level 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 CO2 level for stimulation is about 3%-7% CO2. In other embodiments, the CO2 level for stimulation is about 4%-6% CO2. In still other embodiments, the CO2 level for stimulation is about 4.5%-5.5% CO2. In a specific embodiment, the CO2 level for stimulation is about 5% CO2.

[0100] In one embodiment, the conditions for stimulating a lymphocyte population may include any combination of temperature, amount of stimulation time, and / or in the presence of a certain level of CO2. For example, the step of stimulating a lymphocyte population may include stimulating a lymphocyte population with one or more T cell stimulators at a temperature of about 36°C-38°C, for an amount of time of about 44 hours-52 hours, and in the presence of a CO2 level of about 4.5%-5.5% CO2.

[0101] In some embodiments, the concentration of lymphocytes useful in the methods herein is about 0.5-10.0×10 6 In some embodiments, the concentration of lymphocytes is about 0.5-1.0×10 6 cells / mL, about 1.0-2.0×10 6 cells / mL, about 1.0-3.0×10 6cells / mL, about 1.0-4.0×10 6 cells / mL, about 1.0-5.0×10 6 cells / mL, about 1.0-6.0×10 6 cells / mL, about 1.0-7.0×10 6 cells / mL, about 1.0-8.0×10 6 cells / mL, 1.0-9.0×10 6 cells / mL or about 1.0-10.0×10 6 In some embodiments, the concentration of lymphocytes is about 0.5-1.0×10 6 In some embodiments, the concentration of lymphocytes is about 1.0-2.0×10 6 In some 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 In some 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 6cells / 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.

[0102] In some embodiments, anti-CD81 antibodies (or their functional fragments) can be used according to the step of stimulating lymphocyte populations. Any soluble or immobilized anti-CD81 antibodies or their functional fragments (e.g., clone 5A6; anti-CD81) can be used. In some aspects, antibodies can be purchased commercially from suppliers known in the art, including but not limited to Miltenyi Biotec, BD Biosciences (e.g., 1 mg / mL pure MACS GMP CD3, part number 170-076-116) and eBioscience, Inc. In addition, those skilled in the art will understand how to produce anti-CD81 antibodies by standard methods. In some embodiments, one or more T cell stimulators used according to the step of stimulating lymphocyte populations include antibodies or their functional fragments targeting T cell stimulation or co-stimulatory molecules in the presence of T cell cytokines. In one aspect, one or more T cell stimulators include anti-CD81 antibodies. In certain embodiments, the T cell stimulator includes an anti-CD81 antibody at a concentration of about 100 ng / mL-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 specific embodiment, the concentration of the anti-CD81 antibody is about 1 μg / mL. In a specific embodiment, the concentration of the anti-CD81 antibody is about 2 μg / mL. In a specific embodiment, the concentration of the anti-CD81 antibody is about 3 μg / mL. In a specific embodiment, the concentration of the anti-CD81 antibody is about 4 μg / mL. In a specific embodiment, the concentration of the anti-CD81 antibody is about 5 μg / mL. In a specific embodiment, the concentration of the anti-CD81 antibody is about 6 μg / mL. In a specific embodiment, the concentration of the anti-CD81 antibody is about 7 μg / mL. In a specific embodiment, the concentration of the anti-CD81 antibody is about 8 μg / mL. In a specific embodiment, the concentration of the anti-CD81 antibody is about 9 μg / mL. In a specific embodiment, the concentration of the anti-CD81 antibody is about 10 μg / mL.

[0103] In some embodiments, anti-CD3 antibodies (or functional fragments thereof), anti-CD28 antibodies (or functional fragments thereof), or a combination of anti-CD3 and anti-CD28 antibodies can be used according to the step of stimulating a lymphocyte population. Any soluble or fixed anti-CD2 antibody, anti-CD3 antibody, and / or anti-CD28 antibody or their functional fragments (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 aspects, antibodies can be purchased commercially from suppliers known in the art, including but not limited to Miltenyi Biotec, BD Biosciences (e.g., 1 mg / mL pure MACS GMP CD3, part number 170-076-116) and eBioscience, Inc. In addition, those skilled in the art will understand how to produce anti-CD3 antibodies and / or anti-CD28 antibodies by standard methods. In some embodiments, one or more T cell stimulators used according to the step of stimulating a lymphocyte population include antibodies or functional fragments thereof targeting T cell stimulation or co-stimulatory molecules in the presence of T cell cytokines. In one aspect, one or more T cell stimulators include soluble anti-CD28 antibodies. In certain embodiments, the T cell stimulator includes a soluble anti-CD28 antibody at a concentration of about 1.00 μg / mL-2.00 μg / mL. In certain embodiments, the concentration of anti-CD28 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 specific embodiment, the concentration of anti-CD28 antibody is about 1.00 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.10 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.20 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.30 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.40 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.50 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.60 μg / mL.In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.61 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.62 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.63 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.64 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.65 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.66 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.67 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.68 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.69 μg / mL. In a specific embodiment, the concentration of the anti-CD28 antibody is about 1.70 μg / mL. In one aspect, one or more T cell stimulators include anti-CD3 antibodies. In certain embodiments, the T cell stimulator comprises an anti-CD3 antibody at a concentration of about 0.50 μg / mL-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. In one embodiment, the concentration of the anti-CD3 antibody is about 1.00 μg / mL. In one embodiment, the concentration of the anti-CD3 antibody is about 1.10 μg / mL. In one embodiment, the concentration of the anti-CD3 antibody is about 1.20 μg / mL. In one embodiment, the concentration of the anti-CD3 antibody is about 1.21 μg / mL. In one embodiment, the concentration of the anti-CD3 antibody is about 1.22 μg / mL. In a specific embodiment, the concentration of the anti-CD3 antibody is about 1.23 μg / mL. In a specific embodiment, the concentration of the anti-CD3 antibody is about 1.24 μg / mL. In a specific embodiment, the concentration of the anti-CD3 antibody is about 1.25 μg / mL.In a specific embodiment, the concentration of anti-CD3 antibodies is about 1.26 μg / mL. In a specific embodiment, the concentration of anti-CD3 antibodies is about 1.27 μg / mL. In a specific embodiment, the concentration of anti-CD3 antibodies is about 1.28 μg / mL. In a specific embodiment, the concentration of anti-CD3 antibodies is about 1.29 μg / mL. In a specific embodiment, the concentration of anti-CD3 antibodies is about 1.30 μg / mL. In an alternative embodiment, T cell activation is not required. In such an embodiment, the method omits the step of stimulating a lymphocyte population to produce an activated T cell population, and transduces a lymphocyte population that can be enriched for T lymphocytes according to the following steps.

[0104] Transduction of activated lymphocyte populations with viral vectors :

[0105] In some embodiments, the methods described herein may include using a single transduction cycle to transduce an activated T cell population with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor to produce a transduced T cell population. Several recombinant viruses have been used as viral vectors to deliver genetic material to cells. The viral vector that can be used according to the transduction step can be any symtropic or bidirectional viral vector, including but not limited to recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated virus (AAV) vectors. In some embodiments, the method also includes transducing one or more T cells with a retrovirus. According to one aspect of this embodiment, the viral vector grows in a suspension culture in a culture medium (referred to herein as "viral vector inoculum") that is specific to viral vector manufacturing. According to the methods described herein, any suitable growth medium and / or supplement for growing viral vectors can be used in the viral vector inoculum. According to some aspects, the viral vector inoculum is then added to the serum-free culture medium described below during the transduction step.

[0106] In some embodiments, one or more T cells can be transduced with a retrovirus. In one embodiment, the retrovirus comprises a heterologous gene encoding a cell surface receptor. In a specific embodiment, the cell surface receptor is capable of binding to an antigen on the surface of a target cell (e.g., on a tumor cell surface).

[0107] In some embodiments, the conditions for transducing an activated T cell population as described herein may include a specific time, a specific temperature, and / or a specific CO2 level present. 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°C-38°C. In another embodiment, the temperature for transduction is about 35°C-37°C. In another embodiment, the temperature for transduction is about 36°C-38°C. In still another embodiment, the temperature for transduction is about 36°C-37°C. In a specific embodiment, the temperature for transduction is about 37°C.

[0108] In certain embodiments, the time for transduction is about 12 hours to 120 hours. In some embodiments, the time for transduction is about 12 hours to 16 hours, about 12 hours to 20 hours, about 12 hours to 24 hours, about 12 hours to 28 hours, about 12 hours to 32 hours, about 12 hours to 40 hours, about 12 hours to 50 hours, about 12 hours to 60 hours, about 12 hours to 70 hours, about 12 hours to 80 hours, about 12 hours to 90 hours, about 12 hours to 100 hours, about 12 hours to 110 hours, or about 12 hours to 120 hours. In other embodiments, the time for transduction is about 20 hours or at least about 20 hours. In one embodiment, the time for transduction is about 16 hours to 24 hours. In other embodiments, the time 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.

[0109] In certain embodiments, the CO2 level used for transduction is about 1.0%-10% CO2. In other embodiments, the CO2 level used for transduction 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 CO2 level used for transduction is about 3%-7% CO2. In another embodiment, the CO2 level used for transduction can be about 4%-6% CO2. In another embodiment, the CO2 level used for transduction is about 4.5%-5.5% CO2. In a specific embodiment, the CO2 level used for transduction is about 5% CO2.

[0110] Non-viral vectors can be loosely grouped into plasmid DNA, liposome-DNA complexes (liposome complexes) and polymer-DNA complexes (complexes). Oligonucleotides and analogs thereof, either alone or in complexes, are also examples of gene transfer mediated by non-viral vectors. Transposon vectors based on DNA provide mechanisms for delivering non-viral genes to mammals and human cells. These vectors work by shearing and pasting (cut-and-paste) mechanisms, whereby the transposon DNA containing the target transgene is integrated into the chromosomal DNA by transposase. Transposons have become promising vectors for transfection, which can potentially overcome some limitations of conventional viral vectors. Transposons are stably integrated into the target cell genome, enabling sustained expression of the target gene.

[0111] In some embodiments, transduction of an activated T cell population as described herein can be performed at a specific time, at a specific temperature, and / or in the presence of a specific CO2 level, in any combination: a temperature of about 36°C-38°C, for an amount of time of about 16 hours-24 hours, and in the presence of a CO2 level of about 4.5%-5.5% CO2.

[0112] Expansion of transduced lymphocyte populations :

[0113] In some embodiments, the methods described herein may include amplifying one or more lymphocyte populations of transduction for a specific time to produce engineered lymphocyte populations. The predetermined time of amplification may be any suitable time, and any suitable time allows (i) to produce a sufficient number of cells in engineered lymphocyte populations for at least one dosage applied to a patient, (ii) to produce an engineered lymphocyte population with a favorable ratio of immature cells compared to a typical long process, or (iii) (i) and (ii) both. The time will depend on the cell surface receptors expressed by lymphocytes, the carrier used, the dosage required for therapeutic effect and other variables. Therefore, in some embodiments, the predetermined time of amplification may 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 respects, the amplification time is shorter than amplification methods known in the art. For example, the predetermined expansion time can be shorter by 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% or can be shorter by 75%. In one aspect, the expansion time is about 3 days, and the time from enrichment of the lymphocyte population producing the engineered lymphocytes is about 6 days.

[0114] In some embodiments, the conditions for amplifying the transduced T cell population may include temperature and / or in the presence of a certain CO2 level. 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°C-38°C. In another embodiment, the temperature is about 35°C-37°C. In another embodiment, the temperature is about 36°C-38°C. In yet another embodiment, the temperature is about 36°C-37°C. In a specific embodiment, the temperature is about 37°C. In certain embodiments, the CO2 level is 1.0%-10% CO2. In other embodiments, the CO2 level 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 CO2 level is about 4.5%-5.5% CO2. In another embodiment, the CO2 level is about 5% CO2. In other embodiments, the CO2 level 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 expanding the transduced T cell population include any combination of temperature and / or the CO2 level present. For example, the conditions for expanding the transduced T cell population include a temperature of about 36°C-38°C and in the presence of a CO2 level of about 4.5%-5.5% CO2.

[0115] 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 cell culture bag using any suitable cell culture bag (e.g., Miltenyi Biotec GMP cell differentiation bags, Origen Biomedical permeable life cell culture bags (PermaLife Cell Culturebags)) closed bag culture system. In some embodiments, the cell culture bags used in the closed bag culture system are coated with recombinant human fibronectin fragments during the transduction step. The recombinant human fibronectin fragment can include three functional domains: a central cell binding domain, a heparin binding domain II, and a CS1 sequence. The recombinant human fibronectin fragment can increase the gene transfer efficiency of retroviral transduction of lymphocytes by assisting the co-localization of target cells and viral vectors. In certain embodiments, the recombinant human fibronectin fragment is (Takara Bio, Japan). In certain embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at a concentration of about 1 μg / mL-60 μg / mL or about 1 μg / mL-40 μg / mL. In other embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at a concentration of about 1 μg / mL-20 μg / mL, 20 μg / mL-40 μg / mL, or 40 μg / mL-60 μg / mL. In some embodiments, the cell culture bag is coated with 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 of recombinant human fibronectin fragment. In other embodiments, the cell culture bag is coated with about 2 μg / mL-5 μg / mL, about 2 μg / mL-10 μg / mL, about 2 μg / mL-20 μg / mL, about 2 μg / mL-25 μg / mL, about 2 μg / mL-30 μg / mL, about 2 μg / mL-35 μg / mL, about 2 μg / mL-40 μg / mL, about 2 μg / mL-50 μg / mL or about 2 μg / mL-60 μg / mL recombinant human fibronectin fragment. In certain embodiments, the cell culture bag is coated with 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 recombinant human fibronectin fragment. In a specific embodiment, the cell culture bag is coated with at least about 10 μg / mL recombinant human fibronectin fragment. The cell culture bag used in the closed bag culture system can be optionally blocked with human albumin serum (HSA) during the transduction step. In an alternative embodiment, during the transduction step, the cell culture bag is not blocked with HSA.

[0116] T-cell therapy :

[0117] In some embodiments, for example, but not limited to, the methods described herein can enhance the effectiveness of T cell therapy, which can be an adoptive T cell therapy selected from the group consisting of: tumor infiltrating lymphocytes (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 widely includes any selection, in vitro enrichment, and the administration of autologous or allogeneic T cells that recognize and can bind tumor cells to the patient. TIL immunotherapy is an adoptive T cell therapy type, in which lymphocytes that can infiltrate tumor tissue are separated, enriched in vitro, and administered to the patient. TIL cells can be autologous or allogeneic. Autologous cell therapy is an adoptive T cell therapy that involves separating T cells that can target the patient's tumor cells, enriching T cells in vitro, and administering T cells back to the same patient. Allogeneic T cell transplantation can include the transplantation of naturally occurring T cells or genetically engineered T cells that are expanded ex vivo. Engineered autologous cell therapy, as described in more detail above, is adoptive T cell therapy in which the patient's own lymphocytes are isolated, genetically modified to express tumor targeting molecules, expanded in vitro, and administered back to the patient. Non-T cell transplants can include autologous or allogeneic therapies with non-T cells, such as, but not limited to, natural killer (NK) cells.

[0118] In some embodiments, one or more T cells are transduced with a retrovirus comprising a heterologous gene encoding a cell surface receptor. In a specific embodiment, the cell surface receptor is capable of binding to an antigen on the surface of a target cell (e.g., on a tumor cell surface). In some embodiments, the cell surface receptor is a chimeric antigen receptor or a T cell receptor.

[0119] 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 to 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 fragment or combination thereof.

[0120] In some embodiments, the chimeric antigen receptor may further include a hinge region. The hinge region may be derived from the hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28 or CD8α. In a specific embodiment, the hinge region is derived from the hinge region of IgG4.

[0121] In some embodiments, the chimeric antigen receptor may further comprise a transmembrane domain. The transmembrane domain may be the transmembrane domain of any transmembrane molecule, which is a co-receptor on lymphocytes or a transmembrane domain of an immunoglobulin superfamily member. In certain embodiments, the transmembrane domain is derived from the transmembrane domain of the following substances: CD28, CD8α, CD4 or CD19. In a specific embodiment, the transmembrane domain comprises a domain derived from the CD28 transmembrane domain. In another specific embodiment, the transmembrane domain comprises a domain derived from the CD28 transmembrane domain.

[0122] In some embodiments, the chimeric antigen receptor may also include one or more costimulatory signaling regions. For example, the costimulatory signaling region may be a signaling region of CD28, OX-40, 41BB, CD27, inducible T cell costimulation (ICOS), CD3γ, CD3δ, CD3ε, CD247, Igα (CD79a, CD79b) or Fcγ receptors. In a specific embodiment, the costimulatory signaling region is a CD28 signaling region.

[0123] In one embodiment, the chimeric antigen receptor further comprises a CD3 zeta signaling domain.

[0124] In some embodiments, a chimeric antigen receptor can be engineered to target a specific tumor antigen, ie, a "gene of interest." In some embodiments, the tumor antigen is selected from 707-AP (707 alanine proline), AFP (alpha (a)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; b-catenin / m, b-catenin / mutated), BCMA (B cell maturation antigen), Bcr-abl (cluster of differentiation 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 (antigen recognized by CTL on melanoma), CAP-1 (carcinoembryonic antigen peptide 1), CASP-8 (caspase 8), CDC27m (mutated cell division cycle protein 27), CDK4 / m (mutated cyclin-dependent kinase 4), CEA (carcinoembryonic antigen), CT (carcinoma / testis (antigen)), Cyp-B (cyclophilin B), DAM (differentiation antigen on melanoma), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor variant III), EGP-2 (epidermal glycoprotein 2), EGP-40 (epidermal glycoprotein 40), Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4), ELF2M (mutated elongation factor 2), 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-acetylglucosamine transferase V), Gp100 (glycoprotein 100kD), HAGE (helicase antigen), HER-2 / neu (human epidermal receptor 2 / neural;also known as EGFR2), HLA-A (human leukocyte antigen A), HPV (human papillomavirus), HSP70-2M (mutated heat shock protein 70-2), HST-2 (human cyclic tumor factor 2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), IL-13R-a2 (interleukin 13 receptor subunit alpha-2), KIAA0205, KDR (kinase insert domain receptor), kappa light chain, LAGE (L antigen), LDLR / FUT (low-density lipid receptor / GDP-L-fucose: bD-galactosidase 2-aL Fucosyltransferase), LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma associated antigen 1), mesothelin, murine CMV-infected cells, MART-1 / Melan-A (melanoma antigen recognized by T cells 1 / melanoma antigen A), MC1R (melanocortin 1 receptor), myosin / m (mutated myosin), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitously mutated proteins 1, 2, 3), NA88-A (NA from patient M88) cDNA clone), 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), carcinoembryonic antigen (h5T4), P15 (protein 15), p190 smaller bcr-abl (190KD protein bcr-abl), Pml / RARa (promyelocytic leukemia / retinoic acid receptor a), 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 ubiquitin 1 or 2), SAGE (meat Tumor antigen), SART-1 or SART-3 (squamous antigen for tumor rejection 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 (translocation Ets family leukemia / acute myeloid leukemia 1), TPI / m (mutated triosephosphate isomerase), 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 (Wilm's tumor gene), and any combination thereof. In a specific embodiment, the tumor antigen is CD19. ;

[0125] In some embodiments, T cell therapy comprises administering to a patient an engineered T cell expressing a T cell receptor ("engineered TCR T cell"). The T cell receptor (TCR) may comprise 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, KIA A0205, KDR, kappa light chain, LAGE, LDLR / FUT, LeY, L1CAM, MAGE, MAGE-A1, mesothelin, murine 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, carcinoembryonic antigen, P15, p190 smaller 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.

[0126] In one embodiment, the TCR comprises a binding molecule to a viral oncogene. In a specific embodiment, the viral oncogene is selected from human papillomavirus (HPV), Epstein-Barr virus (Epstein-Barr virus; EBV) and human T-lymphotropic virus (HTLV).

[0127] In yet another embodiment, the TCR comprises a binding molecule to a testicular, placental or fetal tumor antigen. In a specific embodiment, the testicular, placental or fetal tumor antigen is selected from the group consisting of: NY-ESO-1, synovial sarcoma X-spot 2 (SSX2), melanoma antigen (MAGE), and any combination thereof.

[0128] In another embodiment, the TCR comprises a binding molecule to a lineage-specific antigen. In a specific embodiment, the lineage-specific antigen is selected from the group consisting of: melanoma antigen 1 recognized by T cells (MART-1), gp100, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), and any combination thereof.

[0129] In one embodiment, T cell therapy includes administering to the patient an engineered CAR T cell expressing a chimeric antigen receptor bound to CD19 and further comprising a CD28 co-stimulatory domain and a CD3-ζ signaling region. In another embodiment, the engineered CAR T cell comprises a CD81 co-stimulatory domain. In a specific embodiment, T cell therapy includes administering KTE-C19 to the patient.

[0130] In one embodiment, the antigenic portion further includes, but is not limited to, 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, type 2, or type 8 (HSV1, HSV2, or HSV8) virus antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, UL20, UL3 2, US43, UL45, UL49A), cytomegalovirus (CMV) virus antigen (e.g., glycoprotein gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM or other envelope proteins), human immunodeficiency virus (HIV) virus antigen (glycoprotein gp120, gp41 or p24), influenza virus antigen (e.g., hemagglutinin (HA) or neuraminidase (NA)), measles or mumps virus antigen, human papillomavirus (HPV) virus antigen (e.g., L1, L2), parainfluenza virus antigen, rubella virus antigen, respiratory syncytial virus (RSV) virus antigen or varicella-zoster virus antigen. In such an embodiment, the cell surface receptor can be any TCR, or any CAR that recognizes any of the aforementioned viral antigens on a virus-infected target cell.

[0131] In other embodiments, the antigenic portion is associated with cells having immune or inflammatory dysfunction. Such antigenic portions may include, but are not limited to, myelin basic protein (MBP), myelin proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), carcinoembryonic antigen (CEA), proinsulin, glutamine decarboxylase (GAD65, GAD67), heat shock proteins (HSP), or any other tissue-specific antigens involved in or associated with pathogenic autoimmune processes.

[0132] In some embodiments, the methods disclosed herein may involve T cell therapy comprising transferring one or more T cells to a patient. The T cells may be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells (e.g., engineered CAR+T cells or engineered TCR+T cells) may 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 In another embodiment, the therapeutically effective amount of T cells (e.g., 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 In a specific embodiment, the therapeutically effective amount of T cells (e.g., 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, 4×10 4 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, 7×105 cells / kg, 8×10 5 cells / kg, 9×10 5 cells / kg, 1×10 6 cells / kg, about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, about 1×10 7 cells / kg, about 2×10 7 cells / kg, about 3×10 7 cells / kg, about 4×10 7 cells / kg, about 5×10 7 cells / kg, about 6×10 7 cells / kg, about 7×10 7 cells / kg, about 8×10 7 cells / kg or about 9×10 7 cells / kg.

[0133] In some embodiments, the patient is preconditioned before the T cell therapy is administered. The patient can be preconditioned according to any method known in the art, and the known any method includes but is not limited to treatment with one or more chemotherapy drugs and / or radiotherapy. In some embodiments, preconditioning can include reducing the number of endogenous lymphocytes, removing cytokine levels, increasing the serum levels of one or more stable cytokines or proinflammatory factors, enhancing the effector function of the T cell administered after conditioning, enhancing antigen presenting cell activation and / or availability before the T cell therapy, or any treatment of any combination thereof. In one embodiment, preconditioning includes increasing the serum levels of one or more cytokines of the subject.

[0134] Compositions containing lymphocytes :

[0135] In some embodiments, interleukin-7 (IL-7) is a cytokine that promotes lymphocyte homeostasis and is essential 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 T cell subsets, including naive T cells and T CMCells. IL-7 signaling occurs in 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 isolating IL-7 from one or more IL-7 producing cells or obtaining commercially available IL-7.

[0136] In some embodiments, any concentration of IL-7 can be used in the methods described herein. For example, the method may comprise exposing one or more T cells to at least about 0.001 ng / ml IL-7, at least about 0.005 ng / ml IL-7, at least about 0.01 ng / ml IL-7, at least about 0.05 ng / ml IL-7, at least about 0.1 ng / ml IL-7, at least about 0.5 ng / ml IL-7, at least about 1.0 ng / ml IL-7, at least about 1 ng / ml IL-7, at least about 2 ng / ml IL-7, at least about 3 ng / ml IL-7, at least about 4 ng / ml IL-7, at least about 5 ng / ml IL-7, at least about 6 ng / ml IL-7, at least about 7 ng / ml IL-7, at least about 8 ng / ml IL-7, at least about 9 ng / ml IL-7, at least about 10 ng / ml IL-7, at least about 11 ng / ml IL-7, at least about 12 ng / ml 100 ng / ml IL-7, at least about 13 ng / ml IL-7, at least about 14 ng / ml IL-7, at least about 15 ng / ml IL-7, at least about 20 ng / ml IL-7, at least about 25 ng / ml IL-7, at least about 30 ng / ml IL-7, at least about 35 ng / ml IL-7, at least about 40 ng / ml IL-7, at least about 45 ng / ml IL-7, at least about 50 ng / ml IL-7, at least about 100 ng / ml IL-7, at least about 200 ng / ml IL-7, at least about 300 ng / ml IL-7, at least about 400 ng / ml IL-7, at least about 500 ng / ml IL-7, or at least about 1000 ng / ml IL-7. In one embodiment, one or more T cells are contacted with about 0.001 ng / ml to about 500 ng / ml IL-7, about 0.01 ng / ml to about 100 ng / ml IL-7, about 0.1 ng / ml to about 50 ng / ml IL-7, about 1 ng / ml to about 10 ng / ml IL-7, about 1 ng / ml to about 5 ng / ml IL-7, about 5 ng / ml to about 10 ng / ml IL-7, about 3 ng / ml to about 7 ng / ml IL-7, or about 4 ng / ml to about 6 ng / ml IL-7. In a specific embodiment, one or more T cells are contacted with about 5 ng / ml IL-7.

[0137] In some embodiments, interleukin-21 (IL-21) is a cytokine that promotes lymphocyte homeostasis and is necessary for T cell development. IL-21 is produced by T cells and natural killer T cells, and it has pleiotropic effects on a wide range of immune and non-immune cell types, including but not limited to CD4+ and CD8+ T cells, B cells, macrophages, monocytes and dendritic cells (DC). Any exogenous IL-21 can be used in the methods described herein. In some embodiments, exogenous IL-21 is human IL-21. In some embodiments, exogenous IL-21 is wild-type IL-21. In other embodiments, 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 separating IL-21 from one or more IL-21 producing cells or obtaining commercially available IL-21.

[0138] In some embodiments, any concentration of IL-21 can be used in the methods described herein. For example, the method may include culturing one or more T cells with at least about 0.001 ng / ml IL-21, at least about 0.005 ng / ml IL-21, at least about 0.01 ng / ml IL-21, at least about 0.05 ng / ml IL-21, at least about 0.1 ng / ml IL-21, at least about 0.5 ng / ml IL-21, at least about 1.0 ng / ml IL-21, at least about 1 ng / ml IL-21, at least about 2 ng / ml IL-21, at least about 3 ng / ml IL-21, at least about 4 ng / ml IL-21, at least about 5 ng / ml IL-21, at least about 6 ng / ml IL-21, at least about 7 ng / ml IL-21, at least about 8 ng / ml IL-21, at least about 9 ng / ml IL-21, at least about 10 ng / ml ng / ml IL-21, at least about 11 ng / ml IL-21, at least about 12 ng / ml IL-21, at least about 13 ng / ml IL-21, at least about 14 ng / ml IL-21, at least about 15 ng / ml IL-21, at least about 20 ng / ml IL-21, at least about 25 ng / ml IL-21, at least about 30 ng / ml IL-21, at least about 35 ng / ml IL-21, at least about 40 ng / ml IL-21, at least about 45 ng / ml IL-21, at least about 50 ng / ml IL-21, at least about 100 ng / ml IL-21, at least about 200 ng / ml IL-21, at least about 300 ng / ml IL-21, at least about 400 ng / ml IL-21, at least about 500 ng / ml IL-21, or at least about 1000 ng / ml IL-21. In one embodiment, one or more T cells are contacted with about 0.001 ng / ml to about 500 ng / ml IL-21, about 0.01 ng / ml to about 100 ng / ml IL-21, about 0.1 ng / ml to about 50 ng / ml IL-21, about 1 ng / ml to about 10 ng / ml IL-21, about 1 ng / ml to about 5 ng / ml IL-21, about 5 ng / ml to about 10 ng / ml IL-21, about 3 ng / ml to about 7 ng / ml IL-21, or about 4 ng / ml to about 6 ng / ml IL-21. In a specific embodiment, one or more T cells are contacted with about 5 ng / ml IL-21.

[0139] In certain embodiments, the one or more T cells are free of and not contacted with exogenous IL-2.

[0140] In some embodiments, one or more T cells described herein may be obtained from any source, including, for example, human donors. The donor may be a subject in need of anticancer treatment, such as a subject treated with a T cell produced by the methods described herein (i.e., an autologous donor), or may be an individual (i.e., an allogeneic donor) who donates a lymphocyte sample, which will be used to treat different individuals or cancer patients after generating a cell colony produced by the methods described herein. Lymphocyte colonies may be obtained from donors by any suitable method used in the art. For example, lymphocyte colonies may be obtained by any suitable in vitro method, venipuncture, or other blood collection method, by which samples of blood and / or lymphocytes are obtained. In one embodiment, lymphocyte colonies are obtained by apheresis. One or more T cells may be collected from any tissue (including, but not limited to, tumors) comprising one or more T cells. In some embodiments, a tumor or a portion thereof is collected from a subject, and one or more T cells are separated from the tumor tissue. Any T cell may 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 may 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 specific embodiment, the T cells are tumor infiltrating leukocytes. In certain embodiments, one or more T cells express CD8, such as CD8 + In other embodiments, one or more T cells express CD4, such as CD4 + T cells.

[0141] Cancer treatment :

[0142] In some embodiments, the methods of the present disclosure can be used to treat cancer in a subject, reduce the size of a tumor, kill tumor cells, prevent tumor cell proliferation, prevent the growth of a tumor, eliminate a patient's tumor, prevent the recurrence of a tumor, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In certain embodiments, the methods induce a complete response. In other embodiments, the methods induce a partial response.

[0143] In some embodiments, treatable cancers include tumors that are not vascularized, not yet fully vascularized or vascularized. Cancers may also include solid tumors or non-solid tumors. In certain embodiments, cancers may be selected from tumors derived from the following: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, cancer, AIDS-related cancers, anal cancer, appendix cancer, astrocytoma, atypical teratoid / baculoblastoma, central nervous system cancer, B cell leukemia, lymphoma or other B cell malignancies, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (C ML), chronic myeloid proliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, embryonal tumor, central nervous system cancer, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, esthesia neuroblastoma, Ewing sarcoma family of tumors, extracranial germ cell tumor, extracranial germ cell tumor, extrahepatic bile duct cancer, eye cancer, bone fibrous histiocytoma, malignant tumor, 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 (endocrine pancreas), Kaposi's 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, medullary epithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary midline cancer involving the NUT gene, oral cancer, multiple endocrine neoplasms syndrome, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, chronic myeloid leukemia (CML ML), acute myeloid leukemia (AML), multiple myeloma, myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinealoma of intermediate differentiation, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasms / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer,Transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, small cell lung cancer, small intestinal cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin cancer, testicular cancer, laryngeal 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, Waldenstrom's macroglobulinemia, Wilm's tumor.

[0144] In one embodiment, the method can be used to treat tumors, wherein the tumor is lymphoma or leukemia. Lymphoma and leukemia are blood cancers that specifically affect lymphocytes. All white blood cells in the blood are derived from a single type of multipotent hematopoietic stem cell found in the bone marrow. This stem cell produces both bone marrow progenitor cells and lymphocyte progenitor cells, and then the bone marrow progenitor cells and lymphocyte progenitor cells produce various types of white blood cells found in vivo. The white blood cells produced by bone marrow progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells and plasma cells. The white blood cells produced by lymphocyte progenitor cells include megakaryocytes, mast cells, basophils, neutrophils, eosinophils, monocytes and macrophages. Lymphoma and leukemia may affect one or more cell types in these cell types in patients.

[0145] In some embodiments, lymphoma can be divided into at least two subgroups: Hodgkin's lymphoma and non-Hodgkin's lymphoma. Non-Hodgkin's lymphoma (NHL) is a heterogeneous cancer group derived from B lymphocytes, T lymphocytes or natural killer cells. In the United States, B cell lymphoma represents 80-85% of reported cases. In 2013, it was estimated that approximately 69,740 new NHL cases related to the disease and more than 19,000 deaths occurred. Non-Hodgkin's lymphoma is the most common hematological malignancy, and is the seventh major site of new cancer in men and women, and accounts for 4% of all new cancer cases and 3% of cancer-related deaths.

[0146] In some embodiments, diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, accounting for approximately 30% of NHL cases. There are approximately 22,000 newly diagnosed DLBCL in the United States each year. It is classified as an aggressive lymphoma, with most patients cured with conventional chemotherapy (NCCN Guidelines NHL 2014).

[0147] In some embodiments, first-line therapy for DLBCL typically includes an anthracycline-containing regimen with rituximab, such as R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), which has an objective response rate of about 80% and a complete response rate of about 50% (Coiffier 2002), with about one-third of patients having disease refractory to initial therapy or relapsing after R-CHOP (Sehn 2005). For those patients who relapse after responding to first-line therapy, approximately 40%-60% of patients can achieve a second response with additional chemotherapy. The standard of care for second-line therapy for eligible patients for autologous stem cell transplantation (ASCT) includes rituximab and combination chemotherapy, such as R-ICE (rituximab, ifosfamide, carboplatin, and etoposide) and R-DHAP (rituximab, dexamethasone, cytarabine, and cisplatin), each of which has an objective response rate of approximately 63% and a complete response rate of approximately 26% (Gisselbrecht 2010). Patients who respond to second-line therapy and are deemed adequately suitable for transplantation receive a combination 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 no cure options.

[0148] In some embodiments, compared with DLBCL, primary mediastinal large B cell lymphoma (PMBCL) has different clinical, pathological and molecular characteristics.PMBCL is believed to be produced by thymic (medullary) B cells, and represents about 3% of the patients diagnosed with DLBCL.PMBCL is usually identified in the 40th decade of life in younger adult populations, and women are slightly dominant.Gene expression profiles show that PMBCL overlaps with Hodgkin lymphoma deregulation pathways.The initial therapy of PMBCL generally includes an anthracycline-containing regimen with rituximab, such as etoposide, doxorubicin and cyclophosphamide and vincristine, prednisone and rituximab (DA-EPOCH-R) with adjustable doses of infusion, with or without radiation therapy for affected areas.

[0149] In some embodiments, follicular lymphoma (FL), a B-cell lymphoma, is the most common indolent (slow-growing) form of NHL, accounting for approximately 20% to 30% of all NHL. Some patients with FL will histologically transform (TFL) to DLBCL, which is more aggressive and associated with poor outcomes. Histological transformation to DLBCL occurs at an annual rate of approximately 3% for 15 years, with the risk of transformation continuing to decline in subsequent years. The biological mechanism of histological transformation is unknown. The initial treatment of TFL is influenced by previous therapies for follicular lymphoma, but typically includes an anthracycline-containing regimen with rituximab to eliminate the aggressive component of the disease.

[0150] In some embodiments, the treatment options for relapsed / refractory PMBCL and TFL are similar to those in DLBCL. In view of the low prevalence of these diseases, a large number of prospective randomized studies have not been conducted in these patient populations. Patients with chemotherapy-refractory disease have similar or worse prognosis than patients with refractory DLBCL.

[0151] In some embodiments, subjects with refractory, aggressive NHL (e.g., DLBCL, PMBCL, and TFL) have major unmet medical needs, and further investigation with novel treatments is warranted in these populations.

[0152] Thus, in some embodiments, the method can be used to treat lymphoma or leukemia, wherein the lymphoma or leukemia is a B-cell malignancy. 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's 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., Waldenstrom's macroglobulinemia), splenic marginal zone lymphoma, hairy cell leukemia, plasma cell neoplasms (e.g., plasma cell myeloma (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 follicular center lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Epstein-Barr virus-positive DLBCL, lymphocytic granulomatosis, primary mediastinal (thymic) large B-cell lymphoma (PMBCL), intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma leukemia, 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 / Sezary syndrome, primary cutaneous anaplastic large lymphoma In some embodiments, the cancer is refractory to one or more prior treatments, and / or the cancer has relapsed after one or more prior treatments.

[0153] 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 specific embodiment, the cancer is diffuse large B-cell lymphoma.

[0154] In some embodiments, the cancer is refractory to, or has relapsed after, one or more of chemotherapy, radiation therapy, immunotherapy (including T cell therapy and / or treatment with an antibody or antibody-drug conjugate), autologous stem cell transplantation, or any combination thereof. In a specific embodiment, the cancer is refractory diffuse large B-cell lymphoma.

[0155] In some embodiments, cancer is treated by administering one or more T cells to a subject, wherein the one or more T cells have been contacted with anti-CD81 antibodies, exogenous interleukin-7 (IL-7) and exogenous interleukin-21 (IL-21). In some embodiments, one or more T cells include engineered CAR cells or engineered TCR cells. In one embodiment, engineered CAR cells or engineered T cells treat a subject's tumor.

[0156] In some embodiments, the T cell phenotype is assessed by CCR7 and CD45RA expression. In some embodiments, the T cell phenotype is a CAR T cell phenotype. In some embodiments, the blood apheresis material has a more juvenile phenotype (CCR7 + CD45RA + ) is directly correlated with a lower product doubling time. In CD8 T cells, CCR7 + CD45RA + The number of T cells is associated with a durable response. In some embodiments, a higher peak expansion of CAR T cells in peripheral blood (particularly estimated as CAR cells per unit blood volume) is associated with an objective and durable response. The number of CAR T cells in peripheral blood early 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, October 13, 2020, Vol. 4; Issue 19).

[0157] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can also be used in the practice or testing of the present disclosure, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0158] The specific embodiments listed below are merely illustrative and are in no way limiting.

[0159] Example

[0160] Example 1: Effects of duration and conditions of CAR-T activation and expansion on CAR-T phenotype

[0161] CAR-T phenotype during manufacturing can be affected by the duration and exact conditions of CAR-T activation and expansion. A more juvenile and less differentiated CAR-T phenotype is associated with better clinical outcomes. The effects of different CAR-T manufacturing conditions on product phenotype were evaluated using a CD19 / CD20 dual-targeting CAR delivered by a lentiviral vector. The conditions tested in the initial screening using three different healthy donors were as follows:

[0162] 1) Standard manufacture, where cells are activated by contacting plate-bound anti-CD3 and soluble anti-CD28 antibodies and culturing in optimized medium containing IL2. Indicated as "IL2"

[0163] 2) Manufactured in which cells are activated by contacting plate-bound anti-CD3, soluble anti-CD28 and soluble anti-CD81 antibodies and culturing in optimized medium containing IL2. Denoted as "aCD81 / IL2"

[0164] 3) Manufactured in which cells are activated by contacting plate-bound anti-CD3, soluble anti-CD28 antibodies and culturing in an optimized medium containing IL7 and IL21. Denoted as "IL7 / IL21"

[0165] 4) Manufactured in which cells are activated by contacting plate-bound anti-CD3, soluble anti-CD28 and soluble anti-CD81 and culturing in an optimized medium containing IL7 and IL21. Denoted as "aCD81 / IL7 / IL21"

[0166] Use (Prodigy TM ) was obtained from AllCells TM Pan-CD3+ cells or CD4+ cells were isolated in-house from leukocytes of healthy donors (Alameda, CA). + / CD8 + cells, and in Cell cryopreservation medium (Sigma ). Frozen T cells were thawed, 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 rested overnight in IL2 (Prometheus) or IL7 (Peprotech) and IL21 (Peprotech). The next day, cells were transduced with lentiviral vectors and cultured in a 4% culture medium containing expansion supplements, CTS Immune Cell SR, CTS Glutamax (Gibco TM ) T cell culture medium (OpTmizer TM CTS TM The cells were cultured in T cell expansion basal medium (T cell expansion basal medium) supplemented with cytokines appropriate for each of the above conditions for approximately 8 days with feeding every other day. For conditions using anti-CD81, co-stimulatory antibodies were added during the activation step using anti-CD3 and anti-CD28. On day 8, the cells were cultured in CS5 Media (BioLife ) and frozen. Cells were sampled throughout the manufacturing process at days 0, 3, 4, 5, 6, 7, and 8, and cell phenotypes were assessed using flow cytometry. All antibody staining was performed at room temperature in a BD Pharmingen TM The staining was performed in FBS. TM BD FACSDiva TM All flow cytometry data were collected using FlowJo (BD and Company) software and analyzed using FlowJo (BD and Company).

[0167] The viability of T cells during manufacturing is shown in Table 1 below:

[0168]

[0169] The fold expansion of T cells during manufacturing is shown in Table 2 below:

[0170]

[0171] CAR expression by T cells during manufacturing is shown in Table 3 below:

[0172]

[0173] Since cells in all conditions exhibited healthy viability, fold expansion, and CAR transduction, the phenotype of the cells was assessed using different combinations of cell surface markers as shown in the table below.

[0174] The CD4% of T cells during manufacturing are shown in Table 4 below:

[0175]

[0176] The CD8% of T cells during manufacturing are shown in Table 5 below:

[0177]

[0178] Next, multiple cell surface markers were used to assess the memory phenotype of the CD4 and CD8 compartments.

[0179] The CCR7+CD45RA+ % of CD4+ T cells during manufacturing are shown in Table 6 below:

[0180]

[0181] The CCR7+CD45RA+ % of CD8+ T cells during manufacturing are shown in Table 7 below:

[0182]

[0183] As shown in the table above, cells manufactured in CD81 / IL7 / IL21 showed a higher naive phenotype (defined by CCR7+CD45RA+), especially in the later stages of manufacturing. Similar results were observed using alternative cell surface markers as shown in Tables 8, 9, 10 and 11 below.

[0184] The CD27+CD28+ % of CD4+ T cells during manufacturing are shown in Table 8 below:

[0185]

[0186] The CD27+CD28+ % of CD8+ T cells during manufacturing are shown in Table 9 below:

[0187]

[0188] The CD27+CD28+CCR7+CD45RA+% of CD4+ T cells during manufacturing are shown in Table 10 below:

[0189]

[0190]

[0191] The CD27+CD28+CCR7+CD45RA+% of CD8+ T cells during manufacturing are shown in Table 11 below:

[0192]

[0193] Conversely, we also observed a reduction in effector phenotypes in our products, as shown in the table below:

[0194] The CCR7-CD45RA-% of CD4+ T cells during manufacturing are shown in Table 12 below:

[0195]

[0196] The CCR7-CD45RA-% of CD8+ T cells during manufacturing are shown in Table 13 below:

[0197]

[0198]

[0199] Example 2: Functional Characterization of CAR-T Cells

[0200] As shown in Example 1, manufacturing cells in the presence of anti-CD81, IL7 and IL21 resulted in a more juvenile phenotype compared to the IL-2-based manufacturing method. For functional characterization, two different healthy donor T cells were used as starting material and the following conditions were used to manufacture CAR-T cells. The teams to be compared are as follows:

[0201] 1) Standard manufacture, where cells were activated using plate-bound anti-CD3 and soluble anti-CD28 antibodies and cultured in optimized medium containing IL2. Indicated as "IL2"

[0202] 2) Manufactured in which cells are activated using plate-bound anti-CD3, soluble anti-CD28 and soluble anti-CD81 and cultured in IL7 and IL21 containing optimized medium. Denoted as "aCD81 / IL7 / IL21"

[0203] The manufactured cells from the above teams were frozen on manufacturing day 6. These cells were thawed overnight in RPMI medium (Gibco) containing 10% FBS (Gibco), phenotyped the next day, and established in co-culture assays with multiple target lines to evaluate cytotoxicity and cytokines as a readout of CAR-T cell functionality.

[0204] The % cytotoxicity of the manufactured CAR T cells against three different target lines was measured at 24 hours. Four different effector:target ratios were tested and the results are summarized in Tables 14, 15, and 16 below:

[0205] Table 14

[0206]

[0207] Table 15

[0208]

[0209]

[0210] Table 16

[0211]

[0212] As shown above, there was no significant difference in % cytotoxicity between cells made in IL2 and cells made in aCD81 / IL7 / IL21.

[0213] However, cytokine assessment of co-culture supernatants collected at 24 hours using the U-Plex CAR-T cell combo 1 kit (MSD) showed that cells grown in aCD81 / IL7 / IL21 secreted lower effector cytokines (such as granzyme A) and IFN-γ, while also secreting higher IL2. These results are shown in Tables 17 and 18 below:

[0214] Table 17

[0215]

[0216] Table 18

[0217]

[0218]

[0219] Next, the ability of these cells to expand in continuous restimulation assays is evaluated. In short, CAR-T cells and CD19+Nalm6 target cells from American Type Culture Company (ATCC, Manassas, VA) are incubated with 1:1 effector: target ratio. After 2 days, samples are collected, different markers are stained, and flow cytometry is used for phenotyping. The absolute cell counts of both effector cells and target cells are also determined by including counting beads (ThermoFisher Scientific) during flow cytometry. As CAR-T cells expand during the assay, additional target cells are added so that E:T ratios are returned to 1:1 when each cell is phenotyped. The assay lasts for 21 days.

[0220] The fold expansion of the manufactured CAR T cells from the co-cultures is summarized in Table 19 below:

[0221]

[0222] The above results clearly show that CAR-T cells manufactured in aCD81 / IL7 / IL21 are superior in expansion capacity after repeated antigen stimulation compared with CAR-T cells manufactured in IL2.

[0223] Example 3: In vivo efficacy of CAR-T cells produced in aCD81 / IL7 / IL21

[0224] This example describes the evaluation of the efficacy of CAR-T cells manufactured in IL2 relative to CAR-T cells manufactured in aCD81 / IL7 / IL21 tested in vivo in the Nalm6-luc-MHC DKO disseminated mouse model.

[0225] CD19+Nalm6-luc-MHC DKO cells containing bioluminescent reporters are grown in 90% RPMI, 10% FBS, 1% L-glutamine.NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ) from Jackson Laboratory were used for research.On day 0, 5.0×105CD19+Nalm6-luc-MHC DKO cells of 0.1ml were injected intravenously through the caudal vein using a BD U-100 insulin syringe 1 / 2cc, 28G, and 8-week-old mice were transplanted.All CAR-T cells and untransduced (NTD) cells were manufactured as described in Example 1 and frozen on the 3rd day of manufacture.On the 7th day after CD19+Nalm6-luc-MHC DKO implantation, 100ul freshly thawed CAR-T cells were given to mice by intravenous injection. Three different CAR+ doses were tested: 2e5 cells (high), 4e4 cells (medium), and 8e3 cells (low).

[0226] In vivo bioluminescence imaging was performed using IVIS Lumina S5. Under approximately 2%-3% isoflurane gas anesthesia, animals were imaged five times at a time. Each mouse was injected with 150mg / kg D-luciferin IP and imaged in a prone position within 15 minutes after injection. The 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 the metastatic tumors observable in each mouse in the image and to avoid saturation of the CCD chip. BLI images were collected on the 5th, 8th, 12th, 15th, 19th, 22nd, 26th, 29th, 33rd, 36th, 40th, 44th, 48th, 51st, 55th, 58th, 61st and 65th days. Images were analyzed using Living Image 4.5.4 version software. Whole body fixed volume ROIs were placed on the prone images of each animal. Calculate and output the total flux (photons / second) for all ROIs.

[0227] BLI (bioluminescent imaging) values ​​(shown as mean ± SEM) corresponding to CD19+Nalm6-luc-MHC DKO tumor load in mice are given for different treatment groups (Table 20A-Table 20E). Higher values ​​indicate higher tumor loads. Although the cells manufactured by IL2 and aCD81 / IL7 / IL21 show considerable in vivo efficacy at high and medium doses, the CAR-T cells manufactured with aCD81 / IL7 / IL21 show better than the lowest dose of tumor control dynamics during the study. In contrast, mice treated with only vehicle or non-transduced cells (NTD IL2 and NTD aCD81 / IL7 / IL21) did not show any tumor control as expected.

[0228]

[0229]

[0230]

[0231]

[0232]

[0233] ***

[0234] Although multiple embodiments have been described, it is apparent that the present disclosure and embodiments may provide other embodiments utilizing or being encompassed by the compositions and methods described herein. Therefore, it should be understood that the scope of the present disclosure will be defined by what can be understood from the present disclosure and the appended claims, rather than by the embodiments that have been represented by way of example.

Claims

1. A method for producing genetically engineered lymphocytes, the method comprising: contacting one or more lymphocytes from the 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 target gene; and collecting the lymphocytes.

2. The method of 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 (TILs), myeloid-derived suppressor cells (MDSCs), and dendritic cells.

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

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

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

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

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

11. The method of 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 cell expresses a chimeric antigen receptor (CAR), wherein the T cell is transformed with a vector containing the chimeric antigen receptor (CAR).

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

14. The method of claim 12, wherein the chimeric antigen receptor (CAR) comprises CD19.

15. A method according to any one of claims 12 to 14, wherein the chimeric antigen receptor (CAR) comprises a single-chain variable fragment (scFv) targeting an identified tumor antigen, wherein the identified tumor antigen includes 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 adenovirus-associated vector, a lentiviral vector, or any combination thereof.

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

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

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

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

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

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

23. The method of any one of claims 1 to 22, wherein the lymphocytes are harvested no more than 24 hours after transformation.

24. The method of any one of claims 1 to 23, wherein the lymphocytes are harvested no more than 2 days after transformation.

25. The method of any one of claims 1 to 24, wherein the lymphocytes are harvested no more than 3 days after transformation.

26. The method of any one of claims 1 to 25, wherein the lymphocytes are harvested no more than 5 days after transformation.

27. The method of any one of claims 1 to 26, wherein the lymphocytes are harvested no more than 7 days after transformation.

28. The method of any one of claims 1 to 27, wherein the lymphocytes are harvested no more than 8 days after transformation.

29. The method of any one of claims 1 to 28, wherein the lymphocytes are harvested no more than 9 days after transformation.

30. The method of any one of claims 1 to 29, wherein the lymphocytes are harvested no more than 10 days after transformation.

31. The method of any one of claims 1 to 30, wherein the lymphocytes are harvested no more than 11 days after transformation.

32. The method of any one of claims 1 to 31, wherein the lymphocytes are harvested no more than 12 days after transformation.

33. The method of any one of claims 1 to 32, wherein the lymphocytes are harvested no more than 13 days after transformation.

34. The method of any one of claims 1 to 33, wherein the lymphocytes are harvested no more than 14 days after transformation.

35. A genetically engineered lymphocyte produced by a method comprising the steps of: contacting one or more lymphocytes from the 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 target gene; and collecting the lymphocytes.

36. The lymphocyte of 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 (TILs), myeloid-derived suppressor cells (MDSCs), and dendritic cells.

37. A genetically engineered lymphocyte produced by a method comprising the steps of: contacting one or more lymphocytes from the 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, wherein the genetically engineered lymphocytes have a more naive and less differentiated CAR-T phenotype compared to genetically engineered lymphocytes produced by contacting one or more lymphocytes from a subject with exogenous interleukin-2 (IL-2) in vitro.

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

39. The genetically engineered lymphocyte according to any one of claims 37 to 38, wherein the lymphocyte is a T cell.

40. The genetically engineered lymphocyte of any one of claims 37 to 39, wherein the more naive and less differentiated CAR-T phenotype comprises CCR7+CD45RA+CD4+T cells.

41. The genetically engineered lymphocyte of any one of claims 37 to 40, wherein the more naive and less differentiated CAR-T phenotype comprises CCR7+CD45RA+CD8+T cells.

42. The genetically engineered lymphocytes of any one of claims 37 to 41, wherein the more naive and less differentiated CAR-T phenotype is produced when the lymphocytes are harvested no more than 6 days after transformation.

43. The genetically engineered lymphocytes of any one of claims 37 to 42, wherein the more naive and less differentiated CAR-T phenotype is produced when the lymphocytes are harvested no more than 8 days after transformation.

44. The genetically engineered lymphocytes of any one of claims 37 to 43, wherein the more naive and less differentiated CAR-T phenotype is produced when the lymphocytes are harvested no more than 9 days after transformation.

45. The genetically engineered lymphocytes of any one of claims 37 to 44, wherein the more naive and less differentiated CAR-T phenotype is produced when the lymphocytes are harvested no more than 10 days after transformation.

46. ​​The genetically engineered lymphocytes of any one of claims 37 to 45, wherein the more naive and less differentiated CAR-T phenotype is produced when the lymphocytes are harvested no more than 11 days after transformation.

47. The genetically engineered lymphocytes of any one of claims 37 to 46, wherein the more naive and less differentiated CAR-T phenotype is produced when the lymphocytes are harvested no more than 12 days after transformation.

48. The genetically engineered lymphocytes of any one of claims 37 to 47, wherein the more naive and less differentiated CAR-T phenotype is produced when the lymphocytes are harvested no more than 13 days after transformation.

49. The genetically engineered lymphocytes of any one of claims 37 to 48, wherein the more naive and less differentiated CAR-T phenotype is produced when the lymphocytes are harvested no more than 14 days after transformation.

50. A genetically engineered lymphocyte produced by a method comprising the following steps: 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, wherein the genetically engineered lymphocytes secrete lower effector cytokines than genetically engineered lymphocytes produced by contacting one or more lymphocytes from a subject with exogenous interleukin-2 (IL-2) in vitro.

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

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

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

54. A genetically engineered lymphocyte produced by a method comprising the steps of: 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, wherein the genetically engineered lymphocytes exhibit a more juvenile phenotype compared to genetically engineered lymphocytes produced by contacting one or more lymphocytes from a subject with exogenous interleukin-2 (IL-2) in vitro.

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

56. A method of treating cancer, the method comprising administering the composition of 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 treating cancer, wherein the genetically engineered lymphocytes are T cells.

58. The genetically engineered lymphocyte according to any one of claims 1 to 57, wherein the genetically engineered lymphocyte is used to treat cancer.

59. The method of claim 56, wherein the cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, carcinoma, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoma, central nervous system cancer, B cell leukemia, lymphoma, refractory B cell malignancy or other B cell malignancy, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia, blood disease (CML), chronic myeloid proliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, embryonal tumor, central nervous system cancer, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, esthesia neuroblastoma, Ewing sarcoma family of tumors, extracranial germ cell tumor, extracranial germ cell tumor, extrahepatic bile duct cancer, eye cancer, bone fibrous histiocytoma, malignant tumor, and osteosarcoma, gallbladder cancer, gastric (gastric) 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 ( endocrine pancreas), Kaposi's 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, medullary epithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary midline cancer involving the NUT gene, oral cancer, multiple endocrine neoplasms syndrome, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), multiple myeloma, bone marrow Proliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate differentiation pinealoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasms / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, transitional cell cancer of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome,Small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumors, T-cell lymphoma, skin cancer, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, 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, Waldenstrom's macroglobulinemia, and Wilm's tumor.

60. 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 lymphocytes, 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 of reducing or diminishing tumor size or inhibiting tumor growth in a subject in need of T cell therapy, the method comprising administering to the subject one or more lymphocytes, 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 lymphocyte of any one of claims 1 to 61, wherein the lymphocyte can be used for autologous cell therapy or allogeneic cell therapy.

63. The genetically engineered lymphocyte of any one of claims 1 to 62, wherein the lymphocyte produces more cytokines compared to a method of preparing genetically engineered lymphocytes in the presence of IL-2.

64. The genetically engineered lymphocyte of any one of claims 1 to 63, wherein the lymphocyte produces more IL-2 compared to a method of preparing the genetically engineered lymphocyte in the presence of IL-2.

65. The genetically engineered lymphocytes of any one of claims 1 to 64, wherein the lymphocytes are more juvenile compared to a method of preparing genetically engineered lymphocytes in the presence of IL-2.

66. The genetically engineered lymphocytes of any one of claims 1 to 65, wherein the lymphocytes are more proliferative or more robust compared to a method of preparing genetically engineered lymphocytes in the presence of IL-2.

Images