Method for cryogenic storage

Abstract

Methods, systems, and compositions, and articles of manufacture are provided for the cryogenic storage of cells and cell compositions, and / or their manipulation and / or administration to a subject, e.g., a recipient, in cell therapy.SOLUTION: A method comprising cryogenically storing cells from a biological sample from a donor, wherein the cells, at a time after the donor is diagnosed with or considered to have or be suspected of having a disease or condition and before the donor receives one or more treatments for the disease or condition: The method wherein the sample is obtained from a donor and the cells are frozen in a controlled rate freezer using a step-wise freezing profile comprising at least one step wherein the sample and / or chamber is cooled at a rate of greater than 1 °C per minute.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Application Information This application claims priority to U.S. Provisional Patent Application No. 62 / 471,343, filed March 14, 2017, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] overview Cell therapy is a technique in which cells are administered to a recipient to achieve a therapeutic goal. For any given recipient, the administered cells may originate from another person or from the recipient himself / herself. The latter case may be referred to as autologous cell therapy, i.e., the cells are administered back to the recipient from whom they were collected. An advantage of autologous cell therapy may be that the recipient is the donor from whom the cells were collected, thereby reducing the likelihood that the recipient's body will reject the administered cells.

[0003] With respect to cell therapy, when and how the cells are collected from a donor, as well as how the cells are treated after collection and before administration, can affect the effectiveness and availability of the therapy, e.g., how quickly the cells can be administered to a recipient when needed. Summary of the Invention [Means for solving the problem]

[0004] To these ends, methods, systems, and compositions, and articles of manufacture are provided for the cryogenic storage of cells and cell compositions, and / or their manipulation and / or administration to a subject, e.g., a recipient. In some aspects, advantages of these embodiments include, among other things, enhancing the availability, efficacy, and / or other aspects of cell therapy. The methods may also, or alternatively, benefit other medical or research processes that use cells collected from a donor.

[0005] In some aspects, the present disclosure relates to methods of cryogenic storage, processing, manipulation, and administration of cells, as well as related articles, compositions, and systems involving apheresis, where cells are collected before a patient requires cell therapy and cryogenically stored for future use.

[0006] In some aspects, the cells and compositions and products of the present disclosure can be used for subsequent therapeutic treatment of a disease or condition, e.g., in the donor and / or another recipient. In some embodiments, the methods involve cryogenically storing cells derived from the donor's blood. The cryogenically stored cells, in some embodiments, can then be used in cell therapy to treat the disease or condition.

[0007] In some embodiments, the cells are collected after the donor is diagnosed with a disease or condition and before the donor receives one or more of the following: any initial treatment for the disease or condition, any targeted or directed treatment for the treatment of the disease or condition, or any treatment other than radiation and / or chemotherapy. In some embodiments, the cells are collected after the first recurrence of the disease after the initial treatment for the disease and before the donor or subject receives a subsequent treatment for the disease. The initial and / or subsequent treatment may be a therapy other than cell therapy, according to certain embodiments. In some embodiments, The collected cells may be used in cell therapy after initial and / or subsequent treatments.

[0008] In some embodiments, the cells are collected after a second recurrence of the disease after a second course of treatment and before the donor or subject receives subsequent treatment for the disease. In some embodiments, the patient is identified as likely to relapse after the second course of treatment, for example, by assessing certain risk factors. In some embodiments, the risk factors are based on the type and / or genetics of the disease, for example, double-hit lymphoma, primary refractory cancer, or activated B-cell lymphoma. In some embodiments, the risk factors are based on clinical symptoms, for example, early recurrence after the first course of treatment, or other indications of poor prognosis after treatment (e.g., IPI greater than 2).

[0009] In some embodiments, the cells are collected before the donor or subject is diagnosed with the disease. In some aspects, the donor or subject may be determined to be at risk for developing the disease, or may not be considered at risk for developing the disease or diagnosed with the disease, but may choose to store or bank the cells in case cell therapy is needed later in life. In some embodiments, the donor or subject may be considered at risk for developing the disease based on factors such as gene mutations, gene abnormalities, gene disruptions, family medical history, protein abnormalities (e.g., defects in protein production and / or processing), and lifestyle choices that may increase the risk of developing the disease. In some embodiments, the cells are collected prophylactically.

[0010] In some embodiments, the cells are stored or preserved for a period of time greater than or equal to the following: 12 hours, 24 hours, 36 hours, or 48 hours. In some embodiments, the cells are stored or preserved for a period of time greater than or equal to the following: 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the cells are preserved or preserved for a long period of time. In some aspects, the cells are preserved for a period of time greater than or equal to the following: 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, or more.

[0011] The present disclosure also relates, in some aspects, to methods for processing apheresis samples. In some embodiments, the methods involve transporting an apheresis sample obtained from a donor to a storage facility in a cryogenic environment and storing the apheresis sample at the storage facility at cryogenic temperatures. In some embodiments, prior to transport, the sample is subjected to cryogenic storage of, for example, T cells, e.g., CD4 + T cells and / or CD8 + In some embodiments, the T cells are treated by selecting for T cells. In some embodiments, such treatment occurs after transporting the sample and before cryogenic storage of the sample. In some embodiments, treatment occurs after thawing the sample after cryogenic storage.

[0012] In some embodiments, advantages of the methods according to the described embodiments include improved efficiency and / or effectiveness of cell therapy. By allowing a donor, and thus the donor's cells, to store cells at a stage when the donor has not undergone extensive treatment for a disease and / or before a disease or condition or its diagnosis has occurred, such cells may have certain advantages for use in cell therapy compared to cells harvested after one or more treatments. For example, cells harvested before one or more treatments may be healthier, may exhibit higher levels of certain cellular activities, may grow more rapidly, and / or may be more genetically stable than cells after multiple treatments. They may be more receptive to genetic manipulation. Another example of an advantage of the embodiments described herein may include convenience. For example, by collecting, optionally processing, and storing a donor's cells before they are needed for cell therapy, the cells may be readily available to a recipient if and when they are needed at a later time. This may increase apheresis lab capacity and provide technicians with greater flexibility in scheduling the apheresis collection process.

[0013] In some embodiments, the cells and / or compositions and / or products, e.g., containers (e.g., cell vials or bags) containing cells, are marked with one or more codes or other identifiers, e.g., for sorting of cells and samples during processing, cryopreservation, and / or storage, e.g., long-term storage. In some embodiments, systems and articles include multiple containers, each containing a cryopreserved cell composition, e.g., one produced according to an embodiment of the provided methods, where each of the multiple containers contains a cryopreserved sample obtained from a different donor. In some embodiments, the containers are marked with one or more identifiers, e.g., barcodes, radio frequency identification (RFID) tags, or other identifiers, corresponding to or indicating the identity of one or more of the donor, sample, composition, vial, container, condition, disease, collection facility, hospital, and / or recipient. In some aspects, additional information contained in or affixed to the container includes the date of apheresis collection and / or cryopreservation, and / or expiration date, and / or information regarding location within a repository or storage facility. In some embodiments, the code corresponds to a code that appears on a patient identification bracelet or in a hospital or medical facility or collection facility system or documentation, for example, on the donor or associated facility.

[0014] Suitable coding or marking methods or systems include, but are not limited to, coding using printed, magnetic, or electronic tags that can be read using light, electronics, or magnetism, such as barcodes, QR codes, RFID, or transponders, such as light-activated microtransponders, low-cost silicon devices that store a unique 30-bit read-only identification code, are powered by a light-emitting reader device, and emit the code as a radio frequency signal upon removal. In some embodiments, all processing components (such as sample collection tubes, cell purification components, cell culture and expansion components, etc.) are pre-registered in a facility's component registry, where each component's function and intended use stage in the processing workflow are recorded against the component's unique identifier code. In some embodiments, transponders are used, and in some aspects, transponder refers to any method or article for encoding a unique sample identification that can be read.

[0015] In some embodiments, at various stages of the method, e.g., at each stage, e.g., processing workflow and / or prior to or at the time of administration to a recipient, one or more identifier codes are read into a record, e.g., a unique patient-specific record in a central database, and / or used to confirm the identity of the sample and / or the patient from whom the sample originated or to whom the sample is to be administered and / or other information regarding the sample and / or its collection or processing, and / or to confirm the correct chain of custody. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description The following detailed description and examples illustrate certain specific embodiments of the present disclosure. Those skilled in the art will recognize that numerous variations and modifications of the present disclosure exist and are within the scope of the present disclosure. Accordingly, the description of certain specific embodiments should not be construed as limiting. There is no.

[0017] As used herein, the terms "cryogenic storage" or "cryogenic storage" generally refer to the storage of a sample, e.g., a cell-containing sample, at a temperature between -210°C and -80°C under conditions such that the cells can be thawed after such storage period, and such that upon thawing or after thawing, at least a portion or a majority of the cells in the sample remain viable and / or retain at least a portion of their biological function. In one embodiment, a cell sample can be thawed such that at least a certain percentage of the cells in the sample, e.g., at, about, or greater than the following, remain viable and / or remain negative for apoptotic markers or indicators thereof, e.g., cleaved caspase and / or Annexin V staining: 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0018] As used herein, the term cryogenically freezing means reducing the temperature of a sample, for example a sample containing cells, to a temperature of -210 to -80°C.

[0019] In some embodiments, the terms enrich or enrichment, as used herein in the context of a cell-containing sample, refer to separating, selecting, or purifying one or more types of cells from a sample so that the cells are obtained in higher concentrations. The term "enriching" does not necessarily include achieving absolute or near-absolute cell purity, although in some embodiments it may.

[0020] As used herein, a subject or donor is a mammal, e.g., a human or other animal, typically a human. In some embodiments, the subject, e.g., a patient, to whom a cell, cell population, or composition is administered is a mammal, typically a primate, e.g., a human. In some embodiments, the primate is a monkey or ape. The subject may be male or female and of any suitable age, including infant, juvenile, adolescent, adult, and / or geriatric subjects. In some embodiments, the subject is a non-primate mammal, e.g., a rodent.

[0021] In some embodiments, the term "freezing solution" refers to a solution that, when combined with a cell-containing sample, e.g., an apheresis sample, helps preserve one or more biological functions of the cells during the process of cooling, cryogenic freezing, and / or cryogenic storage of the sample or cells. In some embodiments, the terms freezing solution and cryogenic medium are interchangeable.

[0022] In some embodiments, the terms "post-cryogenic modification" or "post-cryogenic modification," when used herein in the context of a cryogenically stored sample containing cells, refer to a process applied to the sample after thawing the cells.

[0023] In some embodiments, the term "relapse," as used herein, refers to the return of signs or symptoms of a disease, generally after a period of improvement.

[0024] Apheresis generally refers to the process of collecting blood from a donor or subject. This process can include the process of collecting cells from the donor's blood. Leukapheresis is used to refer to such a process of collecting white blood cells from the donor's blood. In some embodiments, provided embodiments and compositions relate to the collection of a blood sample from a donor, for example, by apheresis, and in some embodiments, the methods and compositions include: The present invention relates to administering a composition, e.g., a cell therapy composition, to a recipient. In some embodiments, the donor and recipient are the same individual. In some embodiments, cells derived from the donor are administered to a recipient that is a different subject.

[0025] In some embodiments, the method involves cryogenically storing cells derived from the donor's blood. In some embodiments, the cryogenically stored cells are subsequently administered to a recipient to treat a disease. The cells can be used as part of a cell therapy treatment, e.g., T cell therapy, as described, for example, in U.S. Patent Application Publication Nos. 2016 / 0158359 and 2016 / 0206656 and PCT International Application Nos. 2016 / 064929 and 2016 / 033570, which are incorporated herein in their entireties.

[0026] In some embodiments, the donor is a subject, e.g., a person who later receives the collected cells, i.e., a recipient. In such embodiments, the therapy is referred to as autologous cell therapy. As discussed herein, advantages of autologous cell therapy can include a reduced likelihood that the recipient's body will reject the administered cells because the donor who collected the cells is the recipient. In some embodiments, the donor and recipient are different individuals. In such embodiments, the therapy can be referred to as allogeneic cell therapy. Advantages of allogeneic therapy can include uniformity and consistency across a cell sample. Other advantages can include, in some aspects, a higher availability of cells compared to autologous cell therapy, e.g., in situations where donor cells are available at a time when recipient-derived cells may not be available, e.g., in situations where the recipient is unable to donate such cells and / or undergo apheresis, e.g., if the recipient is too ill.

[0027] In some embodiments, cells are collected by apheresis, e.g., any of a number of known apheresis techniques. An exemplary apheresis collection method involves drawing blood from a donor using commonly accepted routine procedures performed by a medical professional. For example, the medical professional may select a site on the donor's body, typically the arm, sterilize the site, perform venipuncture, and collect the blood into a suitable container for storing the blood, e.g., a sterile blood bag containing an anticoagulant. For example, the medical professional may perform routine procedures described in the World Health Organization ("WHO"), WHO guidelines on drawing blood: best practices in phlebotomy (2010). The medical professional may or may not be the same person who diagnoses disease in the donor, as described below. After blood collection, blood components, e.g., plasma and various blood cells, may be separated using centrifugation.

[0028] In some embodiments, the cells are collected after the donor is diagnosed with a disease and before the donor receives any treatment for the disease and / or before the donor receives a targeted treatment, such as a treatment that specifically recognizes or specifically binds to an antigen or other ligand associated with the disease or condition. In some embodiments, the cells are collected at a time before the donor is diagnosed with a disease or condition. Advantages of such embodiments can include improved cell viability, activity, and amenability to genetic manipulation compared to cells collected after the donor receives treatment for the disease. In some embodiments, the cells are collected from the donor after the first recurrence of the disease after the initial treatment for the disease and before the donor receives a subsequent treatment for the disease. Advantages of such embodiments can include improved cell viability, activity, and amenability to genetic manipulation compared to cells collected after the donor receives two or more treatments for the disease. In other embodiments, the cells is collected from the donor after the second recurrence of the disease and before the donor undergoes subsequent treatment for the disease.

[0029] Diseases, conditions, and disorders of the donor and / or recipient, and / or that the donor and / or recipient herein have or are suspected to have, and / or that are targeted by the recombinant receptor, include tumors, including solid tumors, hematopoietic malignancies, and melanoma, including localized tumors and metastatic tumors. Diseases, conditions, and disorders also include infectious diseases, such as infections by viruses or other pathogens, e.g., HIV, HCV, HBV, CMV, HPV, and parasitic diseases. Diseases, conditions, and disorders also include autoimmune and inflammatory diseases. In some embodiments, the disease or condition is a tumor, cancer, malignancy, neoplasm, or other proliferative disease or disorder. Such diseases include, but are not limited to, leukemias, lymphomas such as chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, B-cell malignancies, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, and cancer of the brain, ovarian cancer, epithelial carcinoma, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and / or mesothelioma. In some embodiments, the disease or condition is DLBCL, not otherwise specified (including NOS, transformed DLBCL derived from follicular lymphoma), high-grade B-cell lymphoma with MYC and BCL2 and / or BCL6 rearrangements with DLBCL histology.

[0030] In some embodiments, the subject presents with CLL with indications for treatment under the iwCLL guidelines and SLL, which is clinically measurable disease or biopsy-proven SLL. In some aspects, the subject has received and failed Bruton's tyrosine kinase inhibitor (BTKi) treatment or is deemed ineligible for BTKi therapy.

[0031] In some embodiments, subjects with CLL or SLL and high-risk features, such as complex chromosomal abnormalities (three or more chromosomal abnormalities), 17p deletion, TP53 mutation, or unmutated immunoglobulin heavy chain variable region (IGHV), have failed at least two courses of prior therapy, including BTKi. In some embodiments, subjects with CLL or SLL and standard-risk features have failed at least three courses of prior therapy, including BTKi. In some embodiments, subjects with CLL or SLL who are BTKi intolerant and have not received at least six months of BTKi therapy or are BTKi ineligible have failed at least one (high-risk) or two (standard-risk) courses of non-BTKi therapy.

[0032] In some embodiments, the subject is not eligible for one or more clinical trials and / or approved engineered cell immunotherapies. In some embodiments, the subject is not yet eligible for one or more clinical trials and / or approved engineered cell immunotherapies, but is at risk of becoming eligible or may become eligible. In some embodiments, the subject is not eligible for one or more clinical trials and / or approved engineered cell immunotherapies due to a predicted or actual response to one or more courses of prior therapy or after autologous HSCT. In some embodiments, the subject is not eligible for one or more clinical trials and / or approved engineered cell immunotherapies if they have not relapsed and / or are not refractory to one or more courses of prior therapy (e.g., two or more, three or more, or four or more courses of prior therapy) or after autologous HSCT. In some embodiments, the subject is eligible for one or more clinical trials and / or approved engineered cell immunotherapies due to the absence of high-risk chromosomal abnormalities. Not a rank.

[0033] In some aspects, the subject has multiple metastases and / or widespread metastases. In some aspects, the subject's tumor burden is low, and the subject has few metastases. In some embodiments, the size or timing of the administration is determined by the initial disease burden in the subject. For example, in some aspects, the subject may be administered a relatively low number of cells in the initial dose, while in situations of lower disease burden, the dose may be higher.

[0034] In some embodiments, the disease or condition is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus, etc.

[0035] In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition, for example, arthritis, e.g., rheumatoid arthritis (RA), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, asthma, an immunodeficiency, and / or a disease or condition associated with transplantation.

[0036] In some embodiments, the disease or condition is graft-versus-host disease (GVHD), e.g., GVHD in a subject undergoing or who has undergone a transplant, e.g., an allogeneic organ transplant and / or a bone marrow and / or hematopoietic stem cell transplant. + CD25 + The addition of T cells, e.g., in vitro expanded Treg cells, can delay and / or prevent graft-versus-host disease in some situations. In some embodiments, the provided Treg compositions and methods prevent and / or reduce the risk of GVHD or its symptoms or signs. In some embodiments, the disease or condition is or is the risk of organ transplant rejection, such as a heart, liver, cornea, kidney, lung, pancreas, or other organ transplant.

[0037] In some embodiments, the autoimmune or inflammatory disease is a chronic and / or acute inflammatory disease. In some aspects, the disease or disorder is or includes systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), polymyositis, multiple sclerosis (MS), diabetes, inflammatory bowel disease (IBD), type I diabetes mellitus, or autoimmune insulitis, autoimmune thyroiditis, autoimmune uveitis or uveoretinitis, autoimmune orchitis, autoimmune oophoritis, psoriasis, vitiligo, autoimmune prostatitis, any unwanted immune response or other inflammatory or autoimmune disease or condition, e.g., a condition characterized by an unwanted immune response and / or virus-induced immunopathology. In some aspects, the antigen, e.g., the antigen specifically bound by the T cell and / or recombinant receptor, is a self antigen or auto-antigen, e.g., a human antigen expressed in normal or non-diseased tissue. In some embodiments, the antigen is not an antigen expressed in a cancer or is not expressed in a cancer in the subject. In some embodiments, the subject is not known to have and / or is not suspected of having cancer.

[0038] In some embodiments, the antigen recognized by the cell, chimeric antigen receptor (CAR) or T cell receptor (TCR), or other recombinant receptor is or includes an autoantigen or an antigen that cross-reacts with an autoantigen, e.g., a pathogenic antigen in the pathophysiology of an autoimmune disease. In some embodiments, e.g., when the disease or condition is inflammatory bowel disease (IBD), the antigen is one expressed in the affected colon or ileum. In some embodiments, e.g., in the context of RA, the antigen or ligand is an epitope of collagen or or an antigen present in a joint. In some embodiments, e.g., for treating or preventing type 1 diabetes mellitus or autoimmune insulitis, the antigen is a pancreatic beta cell antigen. In some embodiments, e.g., for MS, the antigen is myelin basic protein antigen, MOG-1, MOG-2, or another neuronal antigen. In some embodiments, e.g., when the disease or condition is autoimmune thyroiditis, the antigen or ligand is a thyroid antigen. In some embodiments, e.g., when the disease or condition is autoimmune gastritis, the antigen is a gastric antigen. In some embodiments, e.g., when treating autoimmune uveitis or uveoretinitis, the antigen is S antigen or another uveal or retinal antigen. In some embodiments, e.g., when the disease or condition is orchitis, the antigen is a testis antigen. In some embodiments, e.g., when treating or preventing autoimmune oophoritis, the antigen is an ovarian antigen. In some embodiments, e.g., when treating or preventing psoriasis, the antigen is a keratinocyte antigen or another dermal or epithelial antigen. In some embodiments, e.g., for the treatment or prevention of vitiligo, the antigen is a melanocyte antigen. In some embodiments, e.g., for the treatment or prevention of autoimmune prostatitis, the antigen is a prostate antigen. In some embodiments, the antigen may include an activation antigen expressed on effector T cells present at the site of an unwanted immune response.

[0039] In some embodiments, the antigen is citrullinated vimentin.

[0040] In some embodiments, for example, when the disease or condition is or involves tissue or organ rejection, the antigen may include an MHC molecule or portion thereof having the haplotype of the transplanted tissue.

[0041] In some embodiments, the antigen associated with the disease or disorder is selected from the group consisting of GPRC5D, glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), B-cell maturation antigen (BCMA, BCM), B-cell activating factor receptor (BAFFR, BR3), transmembrane activating factor and CAML interactor (TACI), Fc receptor-like 5 (FCRL5, FcRH5), orphan tyrosine kinase receptor ROR1, Her2, LI-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine receptor (FETA), and / or acetylcholine receptor (EGFR). receptor), GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms' tumor 1 (WT-1), cyclins, e.g., cyclin A1 (CCNA1), and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV, or other pathogens.

[0042] In some embodiments, the disease is cancer. The cells can be used as part of cancer therapy treatment, for example, T cell therapy, as described, for example, in U.S. Patent Application Publication Nos. 2016 / 0158359 and 2016 / 0206656, and PCT Patent Application Publication No. 2016 / 064929, which are incorporated herein in their entireties.

[0043] Cancer can be, for example, benign or malignant. Cancer can include, for example, primary or metastatic cancer. In some embodiments, cancer can be at any stage, e.g., It may be stage TX, stage T0, stage T1, stage T1a, stage T1b, stage T2, stage T2a, stage T2b, stage T3, stage T3a, stage T3b, stage T4, stage T4a, stage T4b, stage NX, stage N0, stage N1, stage N1a, stage N1b, stage N2, stage N2a, stage N2b, stage N2c, stage N3, stage MX, stage M0, stage M1, stage M1a, stage M1b, stage M1c, stage M2, stage M3, stage M3V, stage M4, stage M4E, stage M5, stage M6, or stage M7.

[0044] In some embodiments, the cancer is selected from acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, Kaposi's sarcoma, astrocytoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma, brain cancer, breast cancer, bronchial carcinoma, Burkitt's lymphoma, carcinoid cancer, cardiac cancer, atypical teratoid or rhabdomyosarcoma-like tumor, embryonal tumor, germ cell tumor, primary central nervous system lymphoma, cervical cancer, cholangiocarcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasm, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, nasal neuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, intraocular melanoma, retinoblastoma, thyroid cancer ... Cell tumors, fallopian tube cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, glioblastoma, ovarian germ cell tumors, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, intraocular melanoma, pancreatic islet cell tumors, pancreatic neuroendocrine tumors, kidney cancer, lung cancer (non-small cell and small cell), malignant fibrous histiocytoma, Merkel cell carcinoma, mesothelioma, midline tract carcinoma carcinoma), oral cavity cancer, multiple endocrine neoplasms, mycosis fungoides, myelodysplastic or myeloproliferative neoplasms, chronic myeloid leukemia, acute myeloid leukemia, chronic myeloproliferative neoplasms, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumors, phenotype carcinoma, multiple myeloma, pleuropulmonary blastoma, peritoneal cancer, prostate cancer, rectal cancer, retinoblastoma, salivary gland cancer, rhabdomyosarcoma, Sézary syndrome, small intestine cancer, small lymphocytic leukemia, squamous cell carcinoma, squamous cell cervical cancer, testicular cancer, throat cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thymoma, thymic carcinoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0045] In some embodiments, the cancer is chronic lymphocytic leukemia, small lymphocytic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia, null acute lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, multiple myeloma, follicular lymphoma, splenic marginal zone lymphoma, mantle cell lymphoma, late-onset B-cell lymphoma, or acute myeloid leukemia.

[0046] In some embodiments, the cancer is caused by orphan tyrosine kinase receptor ROR1, EGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light receptor, IL-13R-alpha2, IL-13R-alpha3, IL-13R-alpha4, IL-13R-alpha5, IL-13R-alpha6, IL-13R-alpha7, IL-13R-alpha8, IL-13R-alpha9, IL-13R-alpha10, IL-13R-alpha11, IL-13R-alpha2, IL-13R-alpha12, IL-13R-alpha2, IL-13R-alpha13, IL-13R-alpha2, IL-13R-alpha14, IL-13R-alpha15, IL-13R-alpha2, IL-13R-alpha16, IL-13R-alpha17, IL-13R-alpha18, IL-13R-alpha19, IL-13R-alpha2, IL-13R-alpha2, IL-13R-alpha19 ... chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, B cell maturation antigen (BCMA), FCRL5 / FCRH5, GPRC5D, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms' tumor 1 (WT-1), cyclins, e.g., cyclin A1 (CCNA1), and / or bile ductase inhibitors. In some embodiments, the cancer comprises cells that express at least one or more of the following: a cytosine-3-phosphate dehydrogenase (CDR) molecule, and / or a molecule expressed by HIV, HCV, HBV, or other pathogens. In some embodiments, the cancer comprises cells that express CD19. In some embodiments, the cancer comprises cells that express BCMA.

[0047] In some embodiments, the disease is diagnosed by a medical professional (e.g., a person licensed by a medical regulatory agency in a country, state, province, county, municipality, or township) who confirms the presence of the disease in the donor by examining the donor and observing a structural or functional disorder in the donor. A medical professional can include, for example, a physician, e.g., a hematologist, immunologist, oncologist, or nurse practitioner.

[0048] In some embodiments, diagnosis excludes self-diagnosis by a donor and / or excludes diagnosis by a genetic testing service.

[0049] In some embodiments, the initial and subsequent treatments may each, independently of one another, include cancer therapies such as chemotherapy, radiation therapy, immunotherapy, hormone therapy, and / or surgery. Chemotherapy may include, for example, administering at least one of cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, mustine, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, cisplatin, epirubicin, capecitabine, folinic acid, oxaliplatin, and other small molecule kinase inhibitors. Immunotherapy may include, for example, administering at least one of antibodies and immune cells such as natural killer cells, lymphokine-activated killer cells, cytotoxic T cells, and dendritic cells. In some embodiments, treatments (either initial or subsequent) may include radiation therapy (e.g., 4000 cGy irradiation), autologous stem cell rescue, or other chemotherapy. In some embodiments, the treatment may include any or all of the following: CAR (carcinoma cell rescue), stem cell transplant, bone marrow transplant, and hematopoietic stem cell transplant (HSCT). T cell therapy may be included. In some embodiments, treatment may include tisagenlecleucel (Kymriah). In some embodiments, treatment may include axicabtagene ciloleucel (Yescarta). In some embodiments, initial and / or subsequent therapy may include any or all of cytarabine (ara-C, including high-dose cytarabine), daunorubicin (daunomycin), idarubicin, or cladribine (leustatin, 2-CdA), alone or in combination. In some embodiments, initial and / or subsequent therapy may include any or all of bortezomib, carfilzomib, thalidomide, lenalidomide, pomalidomide, and corticosteroids, such as prednisone and dexamethasone. In some embodiments, initial and / or subsequent therapies may include any or all of the following: alkylating agents, such as cyclophosphamide, chlorambucil, bendamustine, and ifosfamide; platinum drugs, such as cisplatin, carboplatin, and oxaliplatin; purine analogs, such as fludarabine, pentostatin, cladribine, and cytarabine; antimetabolites, such as gemcitabine, methotrexate, and pralatrexate; and other drugs, such as vincristine, doxorubicin, mitoxantrone, etoposide, and bleomycin. In some embodiments, initial and / or subsequent therapies may include any or all of the following: proteasome inhibitors, such as bortezomib; histone deacetylase inhibitors, such as romidepsin and belinstat; and kinase inhibitors, such as ibrutinib and idelalisib. In some embodiments, the initial and / or subsequent therapy includes antibodies targeting CD20, e.g., rituximab, obinutuzumab, ofatumumab, and ibritumomab tiuxetan; antibodies targeting CD52, e.g., alemtuzumab; CD30 Antibodies targeting EGFR-1, such as brentuximab vedotin; interferon; and immunomodulatory agents such as thalidomide and lenalidomide. In some embodiments, the initial and / or subsequent treatment may be combination therapy, such as CHOP, CHOP+R (or R-CHOP), CVP, EPOCH, EPOCH+R, DHAP, and DHAP+R (or R-DHAP). CHOP includes the drugs cyclophosphamide, doxorubicin, vincristine, and prednisone. R-CHOP (or CHOP+R) further includes treatment with rituximab. CVP includes cyclophosphamide, vincristine, and prednisone. CVP may also be administered in combination with rituximab. EPOCH includes the drugs etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin. EPOCH-R further comprises the treatment with rituximab.DHAP comprises the drugs of dexamethasone, high-dose cytarabine and cisplatin.DHAP+R (or R-DHAP) further comprises the treatment with rituximab.Additional combination regimens that can be used according to the methods described herein include any one or more of the following: bendamustine plus rituximab (BR); rituximab, cyclophosphamide, etoposide, procarbazine and prednisone (R-CEPP); rituximab, cyclophosphamide, epirubicin and prednisone (R-CEOP); rituximab, gemcitabine, cisplatin and dexamethasone (R-GDP); rituximab and lenalidomide. Additional anti-cancer therapies that can be used in accordance with the methods described herein include any one or more, or combination, of chlorambucil, bendamustine, cyclophosphamide, fludarabine, ofatumumab, obinutuzumab, rituximab, idelalisib, venetoclax, lenalidomide, and methylprednisolone.

[0050] In some embodiments, the donor may experience a first relapse after initial treatment and a period of improvement of the disease. In some embodiments, the period of improvement is indicated by the complete absence of signs and symptoms of the disease. In some embodiments, during the period of improvement, the signs and symptoms of the disease are alleviated or reduced, but not completely absent. In some embodiments, the complete absence of signs and symptoms of the disease, or the alleviation or reduction of signs and symptoms, is the result of the initial treatment.

[0051] In some embodiments, the donor may experience a second relapse after one or more previous treatments and one or more periods of improvement of the disease. In some embodiments, the period of improvement is indicated by the complete absence of signs and symptoms of the disease. In some embodiments, during the period of improvement, the signs and symptoms of the disease are alleviated or reduced, but not completely absent. In some embodiments, the complete absence of signs and symptoms of the disease, or the alleviation or reduction of signs and symptoms, is the result of previous treatments.

[0052] In some embodiments, recurrence is diagnosed by a medical professional who examines the donor and confirms the return of signs and symptoms of the disease in the donor. In some embodiments, the medical professional is a person licensed by a medical regulatory agency in a country, state, province, county, municipality, or township. A medical professional can include, for example, a physician, e.g., a hematologist, immunologist, or oncologist, or a nurse practitioner. The medical professional who diagnoses the disease and the medical professional who diagnoses the recurrence may or may not be the same person.

[0053] In some embodiments, cells derived from the donor's blood are obtained by apheresis or leukapheresis. In some embodiments, the number of cells when collected from the donor and / or in the total apheresis sample is at or about, or does not exceed, 500 x 10 6 piece, 1000 x 106 pieces, 2000×10 6 pieces, 3000×10 6 pieces, 4000×10 6 pieces, or 5000 x 10 6 In some embodiments, the sample upon administration to the subject contains 10 or more total cells or nucleated cells per kilogram of donor body weight. 5 ~10 pieces 6 pieces or about 10 5 pieces ~ about 10 6 cells or T cells or engineered cells, and / or 5 x 10 6 Pieces or 10 x 10 6 pieces or approximately 5 x 10 6 pieces or approximately 10 x 10 6 In some embodiments, the volume of blood collected from the donor is 0.5 to 5 milliliters per kilogram of donor body weight.

[0054] In some embodiments, the cells comprise and / or are enriched for T cells and / or populations thereof. + T cells and / or CD8 + T cells, either separately or in combination. T cells and / or CD4 + T cells and / or CD8 +T cell subtypes and subpopulations include naive T (TN) cells, effector T cells (TEFF), memory T cells, and their subtypes, such as stem central memory T cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, natural and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. In some embodiments, the T cells include or are bulk T cells, e.g., those selected based on CD3 expression, CD4 or CD8 expression, or negativity for non-T cell markers found on blood cells.

[0055] In some embodiments, the cells are T cells, e.g., CD8 + T cells (e.g., CD8 + naive T cells, central memory T cells, or effector memory T cells), CD4 +The cells may be or include T cells, natural killer T cells (NKT cells), regulatory T cells (Tregs), stem cell memory T cells, lymphoid progenitor cells, hematopoietic stem cells, natural killer cells (NK cells), or dendritic cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. In certain embodiments, the cells are induced pluripotent stem (iPS) cells or cells derived from iPS cells, e.g., generated from a subject and engineered to modify (e.g., induce mutations in) or manipulate the expression of one or more target genes, e.g., T cells, e.g., CD8 + T cells (e.g., CD8 + naive T cells, central memory T cells, or effector memory T cells), CD4 + These are iPS cells that have differentiated into T cells, stem cell memory T cells, lymphoid progenitor cells, or hematopoietic stem cells.

[0056] In some embodiments, the cells are one or more subsets of T cells or other cell types, e.g., the total T cell population, CD4 + cells, CD8 + Cells, and subpopulations thereof, including those defined by, for example, function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, type of antigen receptor, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation.

[0057] In some embodiments, T cells and / or CD4 + T cells and / or CD8 + T cell subtypes and subpopulations include naive T (TN) cells, effector T cells ( T cells include T cells, T cells, and their subtypes, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, natural and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. In some embodiments, cells are cryogenically frozen and / or cryogenically stored after collection from a donor without further processing. In some embodiments, cells are enriched one or more times before cryogenically freezing and / or storing. In some embodiments, cells are enriched one or more times after cryogenic storage. In some cases, not concentrating or further processing the cells before cryogenic freezing and / or storage can provide cost-saving and / or time-saving benefits. In some cases, not concentrating or further processing the cells before cryogenic freezing and / or storage can also allow for a wider selection of collection facilities for donors who do not have access to facilities that can concentrate and / or process the cells. Concentration can be, for example, as described in PCT Application Publication No. 2015 / 164675, which is incorporated herein in its entirety. In some embodiments, the cells are processed before cryogenic freezing and / or storage.

[0058] In certain embodiments, the cells are frozen, e.g., after washing steps to remove plasma and platelets, for example. In some embodiments, the cells are frozen prior to, following, and / or during any of the steps associated with manufacturing and / or generating cells, e.g., CD4+ T cells and / or CD8+ T cells expressing a recombinant receptor, e.g., a CAR. In certain embodiments, such steps may include any step associated with generating engineered cells, including, but not limited to, selection and / or isolation of a subset of cells, e.g., CD4+ T cells and / or CD8+ T cells, stimulation and / or expansion of cells, e.g., T cells or a subset thereof, or transfection or transduction of cells. In some embodiments, the cells are cells of an apheresis sample collected from a subject prior to selection and / or isolation of cells, stimulation and / or expansion of cells, or transfection or transduction of cells.

[0059] Cell processing method

[0060] In some embodiments, cells collected from a subject are washed, e.g., to remove the plasma fraction and place the cells in a buffer or medium suitable for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution does not contain calcium and / or magnesium, and / or many or all divalent cations. In some aspects, the wash step is accomplished using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the wash step is performed in a centrifuge chamber, e.g., those manufactured and sold by Biosafe SA, including the A-200 / F and A-200 centrifuge chambers, including those for use with the Sepax® and Sepax® 2 systems, according to the manufacturer's instructions. In some aspects, the wash step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are washed after washing, e.g., to remove Ca. ++ / Mg ++ The cells are resuspended in various biocompatible buffers, such as free PBS. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture media.

[0061] In some embodiments, the method includes a density-based cell separation method, for example, preparation of white blood cells from peripheral blood by lysing red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0062] In some embodiments, isolation methods include separating different cell types based on the expression or presence of one or more specific molecules, e.g., surface markers, e.g., surface proteins, intracellular markers, or nucleic acids, in cells. In some embodiments, any known separation method based on such markers can be used. In some embodiments, the separation is affinity- or immunoaffinity-based. For example, in some aspects, isolation includes separating cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, by, for example, incubation with an antibody or binding partner that specifically binds to such markers, typically followed by a washing step and separation of cells bound to the antibody or binding partner from cells that are not bound to the antibody or binding partner.

[0063] Such separation steps can be based on positive selection, where cells that bind to the reagent are retained for further use, and / or negative selection, where cells that do not bind to the antibody or binding partner are retained. In some cases, both fractions are retained for further use. In some aspects, negative selection can be particularly useful when antibodies that specifically identify cell types in a heterogeneous population are not available, such that separation is best performed based on markers expressed by cells other than the desired population.

[0064] Separation does not necessarily result in enrichment or removal of 100% of a particular cell population or cells expressing a particular marker. In some embodiments, an enriched population comprises at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the population. For example, positive selection or enrichment of a particular type of cell, e.g., one expressing a marker, refers to an increase in the number or proportion of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, e.g., one expressing a marker, refers to a decrease in the number or proportion of such cells, but does not necessarily result in the complete removal of all such cells.

[0065] In some cases, multiple separation steps are performed, where a positively or negatively selected fraction from one step is subjected to another separation step, for example, a subsequent positive or negative selection. In some cases, for example, by incubating cells with multiple antibodies or binding partners specific to markers targeted by negative selection, cells expressing multiple markers can be simultaneously depleted in a single separation step. Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed by different cell types.

[0066] For example, in some embodiments, specific subpopulations of T cells, e.g., cells that are positive for or express high levels of one or more surface markers, e.g., CD28 + , CD62L + , CCR7 + , CD27 + , CD127 + , CD4 + , CD8 + , CD45RA + , and / or CD45RO + T cells are isolated by positive or negative selection techniques.

[0067] For example, CD3 + , CD28 + T cells were isolated using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 TC Positive selection can be performed using a cellular expander.

[0068] In some embodiments, isolation is achieved by enriching for a particular cell population by positive selection, or by depleting a particular cell population by negative selection. In some embodiments, positive or negative selection is performed by isolating cells from a marker expressed in the positively or negatively selected cells, respectively. + This is achieved by incubating with one or more antibodies or other binding agents that specifically bind to one or more surface markers that are low in the marker population (low) or expressed at relatively high levels (marker high).

[0069] In some embodiments, T cells are isolated from the PBMC sample by negative selection of markers expressed on non-T cells, e.g., B cells, monocytes, or other leukocytes, e.g., CD14. + or CD8 + Using a selection step, CD4 + Helper T cells and CD8 + Isolate cytotoxic T cells. Such CD4 + and CD8 + The population can be further divided into subpopulations by positive or negative selection of markers that are expressed or expressed to a relatively high degree in one or more naive, memory, and / or effector T cell subpopulations.

[0070] In some embodiments, CD8 +The cells may be further enriched or depleted for naive, central memory, effector memory, and / or stem central memory cells, e.g., by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (TCM) cells is performed to increase efficacy, e.g., to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such subpopulations. See Terakura et al. (2012), Blood. 1:72-82; Wang et al. (2012), J Immunother. 35(9):689-701. In some embodiments, TCM-enriched CD8 + T cells and CD4 + Combining T cells further enhances efficacy.

[0071] In some embodiments, the memory T cells are CD8 + CD62L on peripheral blood lymphocytes + and CD62L - PBMCs are present in both CD62L and CD8 subsets, for example, using anti-CD8 and anti-CD62L antibodies. - CD8 + and / or CD62L + CD8 + The fraction can be enriched or depleted.

[0072] In some embodiments, the enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127, and in some aspects, this is based on negative selection of cells that express or highly express CD45RA and / or granzyme B. In some aspects, the enriched CD8 TCM cells are +The population is isolated by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of cells expressing CD62L. In one embodiment, enrichment of central memory T (TCM) cells is performed by starting with a negative cell fraction selected based on CD4 expression, which is then subjected to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. Such selections are performed simultaneously in some embodiments, and sequentially in either order in other embodiments. In some embodiments, the enrichment of central memory T (TCM) cells is performed by depleting cells expressing CD4, CD14, CD45RA, and positive selection based on CD62L. + The same CD4 expression-based selection step used to prepare the cell population or subpopulation may be used to select for CD4 expression, such that both the positive and negative fractions obtained from the CD4-based separation are retained and used in subsequent steps of the method, optionally after one or more further positive or negative selection steps. + It may also be used to generate cell populations or subpopulations.

[0073] In certain instances, a sample of PBMCs or other white blood cell sample may be subjected to CD4 + Subjecting the cells to selection, Here, both the negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on expression of CD14 and CD45RA or ROR1, and positive selection based on markers characteristic of central memory T cells, such as CD62L or CCR7, where positive and negative selection are performed in either order.

[0074] CD4 + Helper T cells are classified into naive, central memory, and effector cells by identifying cell populations that possess cell surface antigens. + Lymphocytes can be obtained by standard methods. In some embodiments, naive CD4 + T lymphocytes are CD45RO - , CD45RA + , CD62L + , CD4 + In some embodiments, central memory CD4+ The cells are CD62L + and CD45RO + In some embodiments, the effector CD4 + The cells are CD62L - and CD45RO - is.

[0075] In one example, negative selection leads to the deletion of CD4 + To enrich for cells, monoclonal antibody cocktails typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are coupled to a solid support or substrate, e.g., magnetic or paramagnetic beads, to allow for cell separation by positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (e.g., affinity magnetic) separation techniques (discussed in Methods in Molecular Medicine, Vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, pp. 17-25: S.A. Brooks and U. Schumacher, eds., Humana Press Inc., Totowa, NJ).

[0076] In some embodiments, two or more selection steps may be performed sequentially. For example, a sample or composition of cells to be separated may be selected from CD8 + The CD8 negative fraction is further subjected to selection of cells, where both the negative and positive fractions are retained. + In some embodiments, the sample or composition of cells to be separated may be subjected to selection of CD4 + The cells are subjected to selection, where both the negative and positive fractions are retained, and the CD4 negative fraction is selected for CD8 +The cells can be subjected to cell selection. Exemplary methods for cell selection are described in PCT Patent Application Publication Nos. 2015 / 157384 and / or 2015 / 164675, which are incorporated by reference in their entirety, and all or part of which can be used in conjunction with the methods described herein.

[0077] In some embodiments, a sample or composition of cells to be separated is incubated with a small magnetizable or magnetically responsive material, e.g., magnetically responsive particles or microparticles, e.g., paramagnetic beads (e.g., Dynalbeads or MACS beads, etc.). The magnetically responsive material, e.g., particles, are generally coupled, directly or indirectly, to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., a surface marker, present on the cell, cells, or cell population desired to be separated, e.g., negatively or positively selected.

[0078] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material bound to a specific binding member, e.g., an antibody or other binding partner. There are many well-known magnetically responsive materials for use in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and European Patent Specification No. 452342B, which are incorporated herein by reference. Colloidal-sized particles, such as those described in Owen, U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084, are other examples. , which is incorporated herein by reference.

[0079] Incubation is generally carried out under conditions such that the antibody or binding partner, or a molecule attached to a magnetic particle or bead that specifically binds to such an antibody or binding partner, e.g., a secondary antibody or other reagent, specifically binds to the cell surface molecule if present on cells in the sample.

[0080] In some embodiments, the sample is placed in a magnetic field, and cells with magnetically responsive or magnetizable particles attached are attracted to the magnet and separated from unlabeled cells. For positive selection, cells attracted to the magnet are retained, and for negative selection, cells that are not attracted (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subjected to additional separation steps.

[0081] In certain embodiments, magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, magnetic particles bind to cells via coating with a primary antibody specific for one or more markers. In certain embodiments, cells, rather than beads, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a cell-type-specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[0082] In some embodiments, the magnetically responsive particles remain bound to the cells to be subsequently incubated, cultured, and / or manipulated, and in some aspects, the particles remain bound to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competing unlabeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, and the like. In some embodiments, the magnetizable particles are biodegradable.

[0083] In some embodiments, affinity-based selection is by magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). The magnetic-activated cell sorting (MACS) system allows for high-purity selection of cells bound to magnetized particles. In certain embodiments, MACS operates in a manner in which non-target and target species are sequentially eluted after application of an external magnetic field. That is, cells bound to magnetized particles are retained in place, while unbound species are eluted. Then, after this first elution step is completed, the species captured by the magnetic field and those prevented from elution are released in some manner so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from a heterogeneous cell population.

[0084] In certain embodiments, the isolation or separation is performed using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the method. In some aspects, a system is used to perform each of these steps in a closed or sterile environment, for example, to minimize error, user handling, and / or contamination. In one example, the system is a system described in PCT Patent Application Publication No. 2009 / 072003 or U.S. Patent Application Publication No. 2011 / 0003380 A1, which are incorporated herein by reference. In some aspects, a system is used to perform each of these steps in a closed or sterile environment, for example, to minimize error, user handling, and / or contamination. In one example, the system is a system described in PCT Patent Application Publication No. 2016 / 073602 or U.S. Patent Application Publication No. 2016 / 0122782. The described systems, devices, apparatus, and / or methods are used to process and / or isolate or select apheresis or leukapheresis products, or samples derived therefrom, the contents of which are incorporated by reference in their entirety. In some embodiments, the isolation or separation is performed according to the methods described in PCT Patent Application Publication No. 2015 / 164675, the contents of which are incorporated by reference in their entirety.

[0085] In some embodiments, the system or device performs one or more, e.g., all, of the isolation, processing, manipulation, and formulation steps in an integrated or self-contained system and / or in an automated or programmable manner. In some aspects, the system or device includes a computer and / or computer program in communication with the system or device that allows a user to program, control, evaluate the results of, and / or adjust various aspects of the processing, isolation, manipulation, and formulation steps.

[0086] In some embodiments, the separation and / or other steps are performed using, for example, the CliniMACS system (Miltenyi Biotic) for automated separation of cells at a clinical-scale level in a closed and sterile system. Components may include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. The integrated computer, in some embodiments, controls all components of the instrument and directs the system to perform repetitive procedures in a standard sequence. The magnetic separation unit, in some embodiments, includes a movable permanent magnet and a holder for the selected column. The peristaltic pump controls the flow rate of the entire tubing set and, together with the pinch valves, ensures controlled flow of buffer through the system and continuous suspension of the cells.

[0087] The CliniMACS system, in some embodiments, uses magnetizable particles coupled to antibodies, which are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labeling the cells with the magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tubing set, which is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and is for single-use only. After starting the separation program, the system automatically applies the cell sample to the separation column. Labeled cells are retained in the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, cell populations for use in the methods described herein are unlabeled and not retained in the column. In some embodiments, cell populations for use in the methods described herein are labeled and retained in the column. In some embodiments, cell populations for use in the methods described herein are eluted from the column after removing the magnetic field and collected in a cell collection bag.

[0088] In certain embodiments, separation and / or other steps are performed using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system, in some aspects, is equipped with a cell processing unity that allows for automated cell washing and centrifugal fractionation. The CliniMACS Prodigy system may also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by recognizing the layers of the source cell product visible to the naked eye. For example, peripheral blood may be automatically separated into red blood cell, white blood cell, and plasma layers. The CliniMACS Prodigy system may also include an integrated cell culture chamber to accomplish cell culture protocols, such as cell differentiation and expansion, antigen loading, and long-term cell culture. An input port may allow for sterile removal and replenishment of media, and cells may be monitored using an integrated microscope. For example, Klebanoff et al. (2012), J See Immunother., 35(9):651-660; Terakura et al. (2012), Blood., 1:72-82; and Wang et al. (2012), J Immunother., 35(9):689-701.

[0089] In some embodiments, the cell populations described herein are collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are passed through a fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) via preparative-scale (FACS) sorting. In certain embodiments, the cell populations described herein are collected and enriched (or depleted) using a microelectromechanical systems (MEMS) chip in combination with a FACS-based detection system. See, e.g., WO 2010 / 033140; Cho et al. (2010), Lab Chip, 10, 1567-1573; and Godin et al. (2008), J Biophoton., 1(5):355-376. In either case, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets with high purity.

[0090] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation for positive and / or negative selection. For example, separation may be based on binding to a fluorescently labeled antibody. In some examples, cell separation based on binding of an antibody or other binding partner specific to one or more cell surface markers is performed in a fluid stream, for example, by fluorescence-activated cell sorting (FACS), including preparative-scale FACS, and / or by a microelectromechanical system (MEMS) chip combined with, for example, a flow cytometry detection system. Such methods allow for simultaneous positive and negative selection based on multiple markers.

[0091] In some embodiments, the preparation method includes a step for freezing, e.g., cryogenically storing, the cells either before or after isolation, incubation, and / or manipulation. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and, to some extent, monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., after a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters can be used in some aspects. One example includes using PBS containing approximately 20% dimethyl sulfoxide (DMSO) and approximately 8% human serum albumin (HSA), or other suitable cell freezing medium. In some aspects, the solution is then diluted 1:1 with medium to achieve final DMSO and HSA concentrations of 10% and 4%, respectively. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0092] Any of a variety of known freezing solutions and parameters can be used in some aspects. In some embodiments, the cell sample may contain a cryopreservation or vitrification medium or solution containing a cryoprotectant. Suitable cryoprotectants include, but are not limited to, DMSO, glycerol, glycol, propylene glycol, ethylene glycol, propanediol, polyethylene glycol (PEG), 1,2-propanediol (PROH), or mixtures thereof. In some examples, the cryopreservation solution may contain one or more non-cell-permeable cryopreservatives, including, but not limited to, polyvinylpyrrolidone, hydroxyethyl starch, polysaccharides, monosaccharides, alginate, trehalose, raffmose, dextran, human serum albumin, Ficoll, lipoproteins, polyvinylpyrrolidone, hydroxyethyl starch, autologous plasma, or mixtures thereof. In some embodiments, the cells are present in a concentration of about 1% to about 1% by volume. The cryoprotectant is suspended in a freezing solution having a final concentration of about 20%, about 3% to about 9%, or about 6% to about 9% by volume. In certain embodiments, the final concentration of the cryoprotectant in the freezing solution is about 3%, about 4%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% by volume.

[0093] In certain embodiments, cells are suspended in a freezing solution having a final concentration of DMSO of about 1% to about 20%, about 3% to about 9%, or about 6% to about 9% by volume. In certain embodiments, the final concentration of DMSO in the freezing solution is about 3%, 4%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% by volume.

[0094] In some embodiments, the composition is packaged in one or more bags suitable for cryogenic storage (e.g., CryoMacs® Freezing Bags, Miltenyi Biotec). In some embodiments, the composition is packaged in one or more vials suitable for cryogenic storage (e.g., CellSeal® Vials, Cook Regentec).

[0095] In some embodiments, provided methods include a cultivation, incubation, culture, and / or genetic manipulation step either before or after the cryopreservation step. In some embodiments, at least a genetic manipulation step is performed after the cryopreservation step. For example, in some embodiments, methods are provided for incubating and / or manipulating a cryopreserved cell population.

[0096] Thus, in some embodiments, the cell population is incubated in a culture starter composition. The incubation and / or manipulation may be performed in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other vessel for culture or cultivating cells.

[0097] In some embodiments, cells are incubated and / or cultured prior to or in conjunction with genetic manipulation. Incubation steps can include culturing, cultivating, stimulating, activating, and / or expanding. In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in a population, mimic antigen exposure, and / or prime cells for genetic manipulation, e.g., introduction of a recombinant antigen receptor.

[0098] The conditions may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells. In some aspects, cells are incubated in the presence of one or more cytokines, and in some embodiments, cytokine cocktails, such as those described in PCT Patent Application Publication No. 2015 / 157384, which is incorporated herein by reference. In some embodiments, cells are incubated with one or more cytokines and / or cytokine cocktails before, simultaneously with, or after transduction. They are also incubated.

[0099] In some embodiments, the stimulatory conditions or agents include one or more agents, e.g., ligands, capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agents activate or initiate the TCR / CD3 intracellular signaling cascade in the T cell. Such agents can include antibodies, e.g., those specific for the TCR, e.g., anti-CD3. In some embodiments, the stimulatory conditions include one or more agents, e.g., ligands, e.g., anti-CD28, capable of stimulating a costimulatory receptor. In some embodiments, such agents and / or ligands may be bound to a solid support, e.g., beads, and / or one or more cytokines. Optionally, the expansion method can further include adding an anti-CD3 antibody and / or an anti-CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulatory agent includes IL-2, IL-15, and / or IL-7. In some aspects, the concentration of IL-2 is at least about 10 units / mL.

[0100] In some embodiments, the incubation is carried out according to techniques such as those described in U.S. Patent No. 6,040,177 to Riddell et al., Klebanoff et al. (2012), J Immunother., 35(9):651-660; Terakura et al. (2012), Blood., 1:72-82; and / or Wang et al. (2012), J Immunother., 35(9):689-701. In some embodiments, the incubation is carried out using the systems, devices, apparatus, and / or methods described in PCT Patent Application Publication No. 2016 / 073602 or U.S. Publication No. 2016 / 0122782, the contents of which are incorporated by reference in their entireties. In some embodiments, the incubation and / or culturing is carried out according to the methods described in PCT Patent Application Publication No. 2015 / 164675, the contents of which are incorporated by reference in their entirety.

[0101] In some embodiments, T cells are expanded by adding feeder cells, e.g., non-dividing peripheral blood mononuclear cells (PBMCs), to the culture starter composition (e.g., so that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded) and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, PBMCs are irradiated with gamma rays in the range of about 3000-3600 rads to prevent cell division. In some aspects, the feeder cells are added to the culture medium prior to the addition of the T cell population.

[0102] In some embodiments, stimulatory conditions include temperatures suitable for the growth of human T lymphocytes, e.g., at least about 25° C., typically at least about 30° C., and typically at or about 37° C. Optionally, incubation can further include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL may be gamma-irradiated in the range of about 6000-10,000 rads. LCL feeder cells are provided in any suitable amount, in some aspects, e.g., at a ratio of LCL feeder cells to primary T lymphocytes of at least about 10:1.

[0103] In some embodiments, antigen-specific T cells, e.g., antigen-specific CD4 + T cells and / or CD8 + T cells can be obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, antigen-specific T cell lines or clones against cytomegalovirus antigens can be obtained by isolating T cells from an infected subject and incubating the cells in vitro. These can be generated by stimulating the cells with the same antigen.

[0104] In some embodiments, the cells are enriched before being cryogenically frozen and / or stored. Advantages of enriching the cells before being cryogenically frozen and / or stored can include time savings. For example, when a recipient needs the cells as part of a cell replacement therapy, the cells can be thawed from cryogenic storage and administered to the recipient without further manipulation. In some embodiments, the method includes enriching for one or more types of cells. In some embodiments, the enriched cells are T cells. In some embodiments, the enriched cells are CD4 + T cells are enriched. In some embodiments, CD8 + T cells are enriched. In some embodiments, CD4 + T cells and CD8 + In some embodiments, both CD4 and T cells are enriched. + T cells and CD8 + T cells are enriched in a separate process. In some embodiments, CD4 + T cells and CD8 + T cells are enriched in a single process: CD4 + T cells and / or CD8 + Enrichment of T cells may be, for example, as described in PCT Application Publication No. 2015 / 164675, which is incorporated herein in its entirety.

[0105] In some embodiments, the cells are analyzed before cryogenic storage. In some embodiments, the cells may be analyzed to measure the activity of the cells. In some embodiments, the activity is a biological function of the cells. In some embodiments, the activity is the ability of the cells to support immunological processes, including maturation of B cells into plasma cells and / or memory B cells, activation of cytotoxic T cells and / or macrophages, etc. In some embodiments, the activity is the ability of the cells to bind to a specific ligand or antigen using a receptor, receptor-like molecule, antibody, or antibody-like molecule. In some embodiments, the activity is the ability of the cells to recognize and destroy virally infected cells and tumor cells. In some embodiments, the cells are analyzed to measure another biological function of the cells that is related to or affects the activity of the cells.

[0106] Cell selection and / or processing steps may also be as described, for example, in WO 2017214207, the contents of which are incorporated herein by reference in their entirety, and / or as described in WO 2016073602, the contents of which are incorporated herein by reference in their entirety.

[0107] Cryogenic freezing method

[0108] In some embodiments, cells are frozen, e.g., at a particular cell density, e.g., a known or controlled cell density. In certain embodiments, the cell density during the freezing process can affect cell death and / or cell damage that occurs during and / or results from the freezing process.

[0109] For example, in certain embodiments, cell density affects equilibrium, e.g., osmotic equilibrium with the surroundings during the freezing process. In some embodiments, this equilibrium is, includes, and / or results in dehydration. In certain embodiments, dehydration is or includes dehydration of cells caused by contact, combination, and / or incubation with a freezing solution, e.g., DMSO and / or a DMSO-containing solution. In certain embodiments, dehydration is or includes dehydration caused by nucleation and growth of ice crystals in the extracellular space, e.g., by reducing the effective liquid water concentration exposed to the cells. In some embodiments, cells are frozen at a cell density that results in slower and / or more gradual dehydration than cells frozen at a different cell density, e.g., a higher or lower cell density. In some embodiments, cells freeze at a cell density that results in approximately faster dehydration than cells frozen at a different cell density, e.g., a higher or lower cell density, under the same or similar conditions. Freeze at a cell density that results in dehydration that is at least as slow as, or by the following values: 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 1x, 2x, 3x, 4x, 5x, 10x, 50x, or 100x.

[0110] In certain embodiments, the cells are 1×10, inclusive. 6 cells / mL ~ 1 x 10 8 cells / mL or approximately 1 x 10 6 cells / mL ~ approx. 1 x 10 8 cells / mL, approximately 1 x 10 6 cells / mL ~ approx. 2 x 10 7 cells / mL, approximately 1 x 10 7 cells / mL ~ approx. 5 x 10 7 cells / mL, or approximately 1 x 10 7 cells / mL ~ 5 x 10 7 In certain embodiments, the cells are suspended in freezing solution at a density of about 1 x 10 cells / mL, inclusive. 6 cells / mL, approximately 2 x 106 cells / mL, approximately 5 x 10 6 cells / mL, approximately 1 x 10 7 cells / mL, approximately 1.5 x 10 7 cells / mL, approximately 2 x 10 7 cells / mL, approximately 2.5 x 10 7 cells / mL, approximately 2.5 x 10 7 cells / mL, approximately 2.5 x 10 7 cells / mL, approximately 3 x 10 7 cells / mL, approximately 3.5 x 10 7 cells / mL, approximately 4 x 10 7 cells / mL, approximately 4.5 x 10 7 cells / mL, or approximately 5 x 10 7 In certain embodiments, the cells are suspended in freezing solution at a density of about 1.5 x 10 cells / mL, inclusive. 7 cells / mL ~ approx. 6 x 10 7 In certain embodiments, the cells are suspended in the freezing solution at a density of at least about 1 x 10 cells / mL. 7 In certain embodiments, the cells are suspended in freezing solution at a density of about 5 x 10 cells / mL, inclusive. 6 cells / mL ~ approx. 150 x 10 6 In certain embodiments, the cells are suspended in the freezing solution at a density of at least about 1.5 x 10 cells / mL. 7 The cells are suspended in a freezing solution at a density of cells / mL. In some embodiments, the cells are viable cells. In some embodiments, the cell density is determined by the diameter of the T cells.

[0111] In some embodiments, the cells are frozen in one or more containers. In certain embodiments, the container is a freezing container and / or a cryoprotectant container. Containers suitable for cryogenic freezing include, but are not limited to, vials, bags, such as plastic bags, and canes. In certain embodiments, cells, for example, cells in the same cell composition, for example, a cell composition containing cells expressing a CAR, are frozen in one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate containers. For example, in some embodiments, the cells and / or cell compositions are suspended in, for example, a solution, freezing solution, and / or cryoprotectant, etc., in a volume larger than the volume suitable for the container, such that the volume is placed in two or more containers. In some embodiments, the volume is, is approximately, or is less than the following values: 100 mL, 50 mL, 25 mL, 20 mL, 15 mL, 10 mL, 5 mL, or less than 5 mL. The cells are frozen in 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 separate vials. In certain embodiments, the same volume of cells is placed in each vial. In some embodiments, the vials are identical, e.g., from the same manufacturer, model, and / or manufacturing lot. In certain embodiments, the volume is at, about, or greater than: 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 120 mL, 150 mL, 200 mL, or greater than 200 mL. The cells are frozen in 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 separate bags. In certain embodiments, the same volume of cells is placed in each bag. In some embodiments, the bags are identical bags, for example bags from the same manufacturer, model, and / or production lot.

[0112] In some embodiments, the container is a vial. The vial has a fill volume of, approximately, or at least 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, or 50 mL. In some embodiments, the vial has a fill volume of 1 mL to 120 mL, 1 mL to 20 mL, 1 mL to 5 mL, 1 mL to 10 mL, 1 mL to 40 mL, or 20 mL to 40 mL, inclusive. In some embodiments, the vial is a cryovial, a cryoprotectant vial, and / or a cryogenic vial. Suitable vials are known and include, but are not limited to, CellSeal® Vials (Cook Regentec) and the vials described in U.S. Pat. Nos. 8,936,905, 9,565,854, and 8,709,797, which are incorporated herein by reference in their entireties.

[0113] In certain embodiments, the container is a bag. In certain embodiments, the container is a bag having a fill volume of, approximately, or at least the following values: 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, or 50 mL. In some embodiments, the bag has a fill volume of 1 mL to 120 mL, 1 mL to 20 mL, 1 mL to 5 mL, 1 mL to 40 mL, 20 mL to 40 mL, 1 mL to 70 mL, or 50 mL to 70 mL, inclusive. In some embodiments, the bags are filled to volumes that are at, approximately, or less than the following values: 100 mL, 75 mL, 70 mL, 50 mL, 25 mL, 20 mL, or 10 mL. Suitable bags are known and include, but are not limited to, CryoMacs® Freezing Bags (Miltenyi Biotec). In certain embodiments, the volume is the volume at room temperature. In some embodiments, the volume is the volume at 37°C to 4°C, 16°C to 27°C, inclusive, or at, approximately, or at least the following temperatures: 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C. In some embodiments, the volume is the volume at 25°C.

[0114] In some embodiments, cells in a volume of 1 mL to 20 mL, inclusive, of a medium or solution, e.g., a freezing solution, are frozen in one or more vials. In some embodiments, the one or more vials have a fill volume of 1 mL to 5 mL, inclusive. In certain embodiments, cells in a volume of 20 mL to 120 mL, inclusive, of a medium or solution, e.g., a freezing solution, are frozen in one or more bags. In certain embodiments, the one or more bags have a fill volume of 20 mL to 40 mL, inclusive. In some embodiments, cells in a volume of 120 mL or more, of a medium or solution, e.g., a freezing solution, are frozen in one or more bags. In certain embodiments, the one or more bags have a fill volume of 50 mL to 70 mL, inclusive.

[0115] In certain embodiments, the cells are frozen in a solution, e.g., a freezing solution, contained in a container, e.g., a bag or vial, with a surface area to volume ratio that is at or about the following, inclusive: 0.1 cm -1 ~100cm -1 , 1cm -1 ~50cm -1 , 1cm -1 ~20cm -1 , 1cm -1 ~10cm -1 , 2cm -1 ~10cm -1 , 3cm -1 ~7cm -1 , or 3 cm -1 ~6cm -1 In certain embodiments, the surface area to volume ratio is 3 cm -1 ~6cm -1 or about 3 cm -1 ~approx. 6cm -1 Some implementations In the form, the surface area to volume ratio is, is approximately, or is at least 3 cm -1 , 4cm -1 , 5cm-1 , 6cm -1 , or 7 cm -1 .

[0116] In some embodiments, cells are frozen to -80°C at a rate of at or about 1°C per minute. In some embodiments, cells are actively and / or effectively cooled at a rate of at or about 1°C per minute using a controlled rate freezer. In some embodiments, cells can be frozen using a controlled rate freezer. In some aspects, a controlled rate freezer is used to freeze cells with a programmed cooling profile, e.g., a profile having multiple cooling and / or heating rates. Such a freezing profile can be programmed to control nucleation, e.g., ice formation, e.g., to reduce intracellular ice formation. In some embodiments, the temperatures selected to initiate and end the rapid cooling profile are related to the type of container and the volume to be frozen. In some embodiments, if the volume is too small or the container has too high a surface area-to-volume ratio, the sample will respond too quickly to the decrease in temperature and freeze too rapidly, risking intracellular ice formation. In other embodiments, if the volume is too large or the diameter of the container has too low a surface area to volume ratio, the sample will not respond to the decrease in temperature, freezing will occur too slowly, the sample will be at risk of uncontrolled nucleation later in the profile, and the solution will be damaged by prolonged exposure to cryogenic preservatives, e.g., DMSO, before ice crystals form.

[0117] In some embodiments, cells are frozen using the following profile: a hold step at 4.0°C, followed by a cooling step of 1.2°C per minute until the sample reaches a temperature of -6°C. In some aspects, the sample is then cooled at a rate of 25°C per minute until the chamber containing the sample reaches -65°C. In some aspects, the sample is then heated at a rate of 15°C per minute until the chamber containing the sample reaches -30°C. In some aspects, the sample is then cooled at a rate of 1°C per minute until the chamber containing the sample reaches -40°C. In some aspects, the sample is then cooled at a rate of 1°C per minute until the chamber containing the sample reaches -90°C. In some aspects, the sample is then held at -90°C until removal from the controlled rate freezer.

[0118] In some embodiments, cells are frozen using the following profile: a hold step at 4.0°C, followed by a cooling step of 1.2°C per minute until the sample reaches a temperature of -6°C. In some aspects, the sample is then cooled at a rate of 25°C per minute until the chamber containing the sample reaches -65°C. In some aspects, the sample is then heated at a rate of 15°C per minute until the chamber containing the sample reaches -30°C. In some aspects, the sample is then cooled at a rate of 1°C per minute until the chamber containing the sample reaches -40°C. In some aspects, the sample is then cooled at a rate of 10°C per minute until the chamber containing the sample reaches -90°C. In some aspects, the sample is then held at -90°C until removal from the controlled rate freezer.

[0119] In some embodiments, the cells are cooled to a temperature of above -80° C. to 0° C. before being cryogenically frozen and / or stored. For example, the cells may be cooled to -20° C., or to a temperature above -80° C. or below -20° C.

[0120] In some embodiments, the cells are cryogenically frozen to a temperature of between −210° C. and −80° C. before being cryogenically stored. For example, the cells are cryogenically frozen to a temperature of −210° C., or −196° C., or − It can be cryogenically frozen to 80°C.

[0121] In some embodiments, cells are cooled and / or cryogenically frozen at a rate of 0.1°C to 5°C per minute. In some embodiments, cells are cooled and / or cryogenically frozen at a rate of 0.2°C to 4°C per minute. In some embodiments, cells are cooled and / or cryogenically frozen at a rate of 0.5°C to 3°C per minute. In some embodiments, cells are cooled and / or cryogenically frozen at a rate of 0.5°C to 2°C per minute. In some embodiments, cells are cooled and / or cryogenically frozen at a rate of 1°C per minute. For example, one method of cooling and / or cryogenically freezing cells at the above rates includes placing the cells in a programmable refrigerator that lowers the temperature therein at such a rate. Another method of doing so includes placing a vial of cells in a container, surrounding the vial with isopropyl alcohol, and placing the container in a cryogenic or cryogenic environment. In some embodiments, the cells are stored at temperatures lower than the temperatures at which the cells would be frozen using a stepwise approach. For example, in some embodiments, storage occurs at temperatures below -80°C, e.g., below -100, -110, -120, -130, -140, -150, -160°C, or lower. In some aspects, such storage results in the cells or the biological activity of the cells being maintained to a greater extent and / or for a longer period of time.

[0122] In some embodiments, before being cooled or cryogenically frozen, the cells are washed to remove certain components of the sample in which the cells are present. For example, the cells can be washed to remove plasma and / or platelets. The cells can be washed, for example, as described in PCT Application Publication No. 2015 / 164675, which is incorporated herein by reference in its entirety.

[0123] In some embodiments, the cells are combined with a freezing solution prior to cooling, cryogenic freezing, and / or cryogenic storage. In some embodiments, the freezing solution results in greater retention of one or more biological functions of the cells after cooling, cryogenic freezing, or cryogenic storage, and after thawing the cells, compared to cells that were cooled, cryogenically frozen, or cryogenically stored without the freezing solution.

[0124] In some embodiments, the freezing solution comprises 0.1% to 50% by volume DMSO and 0.1% to 20% by weight HSA. In some embodiments, the freezing solution comprises 0.5% to 40% by volume DMSO and 0.2% to 15% by weight HSA. In some embodiments, the freezing solution comprises 1% to 30% by volume DMSO and 0.5% to 10% by weight HSA. In some embodiments, the freezing solution comprises 1% to 20% by volume DMSO and 2% to 7.5% by weight HSA. In some embodiments, the freezing solution comprises 5% to 20% by volume DMSO and 1% to 5% by weight HSA. In some embodiments, the freezing solution comprises 10% by volume DMSO, or 7, 7.5, or 8% by volume or approximately these percentages of DMSO, and 4% by weight HSA. In some embodiments, the above concentrations are the concentrations of DMSO and HSA before the freezing solution is combined with the cells. In some embodiments, the concentrations mentioned above are the concentrations of DMSO and HSA after the freezing solution is combined with the cells.

[0125] In some embodiments, the cells are cryogenically stored at a temperature between -210° C. and -80° C. In some embodiments, the cells are cryogenically stored at a temperature between -210° C. and -196° C. In some embodiments, the cells are cryogenically stored at a temperature between -196° C. and -80° C. In some embodiments, the cells are cryogenically stored in the vapor phase of a liquid nitrogen storage tank.

[0126] In some embodiments, cells are cryogenically stored for a period of 1 day to 12 years. For example, cells may be stored for a period of time before they lose viability for use in cell therapy and until the recipient requires treatment. By storing cells in this manner until the recipient requires treatment, in certain embodiments, the disclosed methods provide the advantage that the cells are readily available when the recipient requires cells for cell therapy. In some embodiments, cells are stored or preserved for a period of time greater than or equal to the following: 12 hours, 24 hours, 36 hours, or 48 hours. In some embodiments, cells are stored or preserved for a period of time greater than or equal to the following: 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, cells are "long-term stored" or "long-term preserved." In some embodiments, the cells are stored for a period of time greater than or equal to the following: 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, or more.

[0127] In some embodiments, after a storage period, the cells are thawed. In some embodiments, the cells are thawed by raising the temperature of the cells to 0° C. or above 0° C. to restore at least a portion of the biological function of the cells. In some embodiments, the cells are thawed by raising the temperature of the cells to 37° C. to restore at least a portion of the biological function of the cells. According to certain embodiments, thawing comprises placing the cells in a container and placing them in a 37° C. water bath for 60-90 seconds.

[0128] In some embodiments, the cells are thawed. In certain embodiments, the cells are thawed rapidly, e.g., as rapidly as possible without overheating the cells or exposing them to high temperatures, such as above 37°C. In some embodiments, rapid thawing reduces and / or prevents the cells from being exposed to high concentrations of cryoprotectants and / or DMSO. In certain embodiments, the rate at which thawing occurs can be influenced by the nature of the container, e.g., the vial and / or bag in which the cells are frozen and thawed.

[0129] In certain embodiments, the cells are thawed at, at about, or below the following temperatures: 37°C, 35°C, 32°C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21°C, 20°C, or 15°C, or between 15°C and 30°C, between 23°C and 28°C, or between 24°C and 26°C, inclusive.

[0130] In some embodiments, the cells are thawed on a heat block, a dry thaw apparatus, or a water bath. In certain embodiments, the cells are not thawed on a heat block, a dry thaw apparatus, or a water bath. In some embodiments, the cells are thawed at room temperature.

[0131] In some embodiments, the thickness of the container wall affects the rate of cell thawing; for example, cells in a container with thick walls may thaw more slowly than in a container with thinner walls. In some embodiments, containers with a low surface area to volume ratio may thaw at a slower and / or uneven rate. In some embodiments, cryogenically frozen cells thaw rapidly in containers with a surface area to volume ratio of, approximately, or at least: 1 cm -1 , 2cm -1 , 3cm -1 , 4cm -1 , 5cm -1 , 6cm -1 , or 7 cm -1 , 8cm -1 , 9cm -1 , or 10cm -1 In certain embodiments, the cells are thawed within, within about, or within less than 120 minutes, 90 minutes, 60 minutes, 45 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, or 10 minutes. The cells are thawed for 10 to 60 minutes, 15 to 45 minutes, or 15 to 25 minutes, inclusive. In certain embodiments, the cells are thawed within 20 minutes, within about 20 minutes, or within less than 20 minutes.

[0132] In certain embodiments, the thawed cells are allowed to rest, e.g., incubated or cultured, prior to administration or prior to any subsequent manipulation and / or processing steps. In some embodiments, the cells are allowed to rest under low and / or undetectable amounts of cryoprotectant or in the absence of a cryoprotectant, e.g., DMSO. In certain embodiments, the thawed cells are allowed to rest after or immediately after a wash step, e.g., to remove the cryoprotectant and / or DMSO. In some embodiments, the allowing is or includes culturing and / or incubation at or about 37°C. In some embodiments, the allowing is performed in the absence of any reagents, e.g., stimulatory reagents, bead reagents, or recombinant cytokines, used in and / or associated with any processing or manipulation steps. In some embodiments, the cells are allowed to rest for, about, or at least 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 18 hours, or 24 hours. In certain embodiments, the cells are allowed to rest for 2 hours, about 2 hours, or at least 2 hours.

[0133] In some embodiments, after the storage period, the percentage of viable cells is between 24% and 100%. The percentage of viable cells can be determined, for example, using the trypan blue dye exclusion technique described in, for example, Schulz et al., "Towards a xeno-free and fully chemically defined cryopreservation medium for maintaining viability, recovery, and antigen-specific functionality of PBMCs during long-term storage," Vol. 382, ​​J. Immu. Methods, pp. 24-26, which discloses using a ViCell™ cell viability analyzer (Beckman Coulter, Krefeld, Germany) to perform trypan blue exclusion. Under the trypan blue dye exclusion technique, for example, dead cells appear blue and can therefore be distinguished from viable cells. The percentage of viable cells can also be determined, for example, by using a flow cytometer or another technique or instrument.

[0134] During the process of cooling, cryogenic freezing, and / or cryogenic storage of a sample or cells, one or more biological functions of the cells are preserved. The use of a freezing solution helps preserve these biological functions. Upon thawing the cells, these biological functions are restored. In addition to viability, other biological functions may include the cell's ability to replicate, its receptivity to genetic modification, and its ability to support immunological processes, including the maturation of B cells into plasma cells and / or memory B cells, and the activation of cytotoxic T cells and / or macrophages.

[0135] In some embodiments, characteristics of the frozen cells, including any of the described cells and compositions, e.g., cell compositions at a particular concentration or cell density, frozen in the presence of a cryoprotectant, and / or packed into a container at a particular volume or surface area to volume ratio, include improved, increased, and / or faster expansion after thawing than cells frozen by alternative means; improved, increased, and / or enhanced cell survival and reduced cell death, e.g., necrosis, programmed cell death, and / or apoptotic events; improved, enhanced, and / or increased activity, e.g., cytolytic activity; and / or reduced senescence or quiescence events.

[0136] In certain embodiments, cells are frozen at the cell densities and / or surface area to volume ratios provided herein and have reduced cell death, e.g., necrosis and / or apoptosis, during and / or resulting from freezing, cryogenic freezing, and / or cryopreservation, compared to cells frozen at a different cell density and / or different surface area to volume ratio under the same or similar conditions. In certain embodiments, cells are frozen at the cell densities and / or surface area to volume ratios provided herein and have reduced delayed cell death, e.g., a reduced amount of cells that die by necrosis, programmed cell death, or apoptosis, within 48 hours after freezing, cryogenic freezing, and / or cryopreservation, e.g., after thawing the frozen cells. In certain embodiments, at least or about the following fewer cells die during and / or as a result of freezing and / or cryogenic storage compared to cells frozen at a different cell density and / or surface area to volume ratio under the same or similar conditions: 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99%. In certain embodiments, less than 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.1%, or 0.01% of cells frozen at a provided cell density and / or surface area to volume ratio die during or as a result of freezing, cryogenic freezing, and / or cryogenic storage.

[0137] In some embodiments, cells are frozen at the cell densities and / or surface area to volume ratios provided herein to reduce senescence or quiescence events due to and / or resulting from freezing, cryogenic freezing, and / or cryopreservation compared to cells frozen at a different cell density and / or different surface area to volume ratio under the same or similar conditions. In certain embodiments, compared to cells frozen at a different cell density and / or different surface area to volume ratio under the same or similar conditions, at least or about the following number of cells are senescent and / or quiescent: 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% fewer cells. In certain embodiments, cells are frozen at a provided cell density and / or surface area to volume ratio such that less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of the cells are senescent and / or quiescent as a result of freezing, cryogenic freezing, and / or cryogenic storage.

[0138] In certain embodiments, cells are frozen, e.g., cryogenically frozen, at a cell density and / or surface area to volume ratio provided herein and have improved, faster, and / or more rapid expansion after the cells are thawed under stimulatory conditions, such as by incubation with a stimulatory reagent described herein, compared to cells frozen at a different cell density and / or surface area to volume ratio under the same or similar conditions. In certain embodiments, the cells expand at a faster and / or more rapid rate by, about, or at least by 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 1x, 1.5x, 2x, 3x, 4x, 5x, or 10x compared to cells frozen at a different cell density and / or surface area to volume ratio under the same or similar conditions. For example, in some embodiments, thawed cells reach a threshold increase, e.g., a predetermined cell number, density, or factor, e.g., a 2-fold increase, in a time that is, approximately, or at least as short as 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% shorter than thawed cells frozen under the same or similar conditions at a different cell density and / or different surface area to volume ratio.

[0139] In some embodiments, the cells are frozen, e.g., cryogenically frozen, at that cell density and have improved, increased, and / or higher cytolytic activity, e.g., as measured by any assay for measuring cytolytic activity described herein, after thawing the cells, compared to cells frozen at a different cell density, e.g., a higher or lower density, under the same or similar conditions. In certain embodiments, the cytolytic activity is increased by, about, or at least: 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold, compared to cells frozen at a different density under the same or similar conditions.

[0140] Cellular modification

[0141] In some embodiments, cells can be modified to, for example, confer one or more of the following: new, enhanced, altered, increased, or decreased activities on the cells. In some embodiments, cells are modified after collection and before cryogenic freezing and / or storage. In some embodiments, cells are modified after thawing following cryogenic storage. Exemplary cell modification methods are described in PCT Application Publication Nos. 2016 / 033570 and 2016 / 115559, which are incorporated herein by reference in their entireties. Exemplary cell modification methods are also described in WO 2017214207 and / or WO 2016073602, the contents of which are incorporated herein by reference in their entireties.

[0142] In some embodiments, the activity is a biological function of the cell, e.g., the ability of the cell to support immunological processes, including, by way of example, maturation of B cells into plasma cells and / or memory B cells, activation of cytotoxic T cells and / or macrophages, etc. In some embodiments, the activity is the ability of the cell to bind to a specific ligand or antigen using a receptor, receptor-like molecule, antibody, or antibody-like molecule. In some embodiments, the activity is the ability of the cell to recognize and destroy virally infected cells and tumor cells.

[0143] Genetic modification of cells

[0144] In some embodiments, modifying cells comprises genetically modifying cells.For example, genetic modification can be as described in PCT Application Publication No. 2016 / 033570 and PCT Application Publication No. 2016 / 115559, which are incorporated herein in their entirety.Exemplary genetic modification methods are also described in International Publication No. 2017214207 and / or International Publication No. 2016073602, which are incorporated herein by reference in their entirety.

[0145] In some embodiments, the genetic modification includes genetically modifying the cells in a manner that allows the cells to express a chimeric molecule comprising a single-chain variable fragment ("scFv") that recognizes a protein. In some embodiments, the scFv binds to a specific protein. In some embodiments, the scFv is derived from a portion of an antibody that binds to a specific protein. In some embodiments, when the scFv is expressed by a cell, the cell can recognize cancer cells and activate itself. In some embodiments, the scFv is selected from the group consisting of orphan tyrosine kinase receptor RORI, tEGFR, Her2, LI-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha, and IL-14R-alpha. and / or a biotinylated molecule, and / or a molecule expressed by HIV, HCV, HBV, or other pathogens. In some embodiments, the scFv binds to CD 19. In some embodiments, the scFv binds to BCMA.

[0146] CAR

[0147] In some embodiments, the genetic modification includes genetically modifying the cells to express one or more chimeric antigen receptors (CARs). Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells are described, for example, in PCT Patent Application Publication Nos. 2000 / 14257, 2013 / 126726, 2012 / 129514, 2014 / 031687, 2013 / 166321, 2013 / 071154, 2013 / 123061, U.S. Patent Application Publication Nos. 2002 / 131960, 2013 / 287748, 2013 / 0149337, U.S. Patent Application Publication Nos. 2003 ... Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent No. 2537416, and / or those described in Sadelain et al., Cancer Discov., 3(4):388-398 (2013); Davila et al., PLoS ONE, 8(4):e61338 (2013); Turtle et al., Curr. Opin. Immunol., 24(5):633-39 (2012); Wu et al., Cancer, 18(2):160-75 (2012). In some embodiments, antigen receptors include CARs described in U.S. Patent No. 7,446,190 and those described in PCT Patent Application Publication No. 2014 / 055668 A1.Examples of CARs include those disclosed in any of the aforementioned publications, e.g., WO 2014 / 031687, U.S. Pat. Nos. 8,339,645, 7,446,179, U.S. Pat. Nos. 2013 / 0149337, 7,446,190, 8,389,282, Kochenderfer et al., Nature Reviews Clinical Oncology, 10:267-276 (2013); Wang et al., J. Immunother., 35(9):689-701 (2012); and Brentjens et al., Sci Transl Med., 5(177) (2013). See also International Publication No. WO 2014 / 031687, U.S. Patent Nos. 8,339,645, 7,446,179, U.S. Publication No. 2013 / 0149337, U.S. Patent No. 7,446,190, and 8,389,282. Chimeric receptors, e.g., CARs, generally comprise an extracellular antigen-binding domain, e.g., a portion of an antibody molecule, generally the heavy chain variable (VH) region and / or light chain variable (VL) region of an antibody, e.g., an scFv antibody fragment. In some embodiments, the chimeric receptor comprises an extracellular antigen-binding domain, e.g., a ligand or other binding moiety, that is not derived from an antibody molecule.

[0148] In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed in disease or condition cells, e.g., tumor or pathogenic cells, compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed in normal cells and / or expressed in engineered cells.

[0149] Antigens targeted by receptors include, in some embodiments, orphan tyrosine kinase receptor RORI, tEGFR, Her2, LI-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R. -alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, and MAGE A3, CE7, Wilms' tumor 1 (WT-1), cyclins, e.g., cyclin A1 (CCNA1), and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV, or other pathogens.

[0150] In some embodiments, the CAR binds to a pathogen-specific antigen, hi some embodiments, the CAR is specific for a viral antigen (e.g., HIV, HCV, HBV, etc.), a bacterial antigen, and / or a parasitic antigen.

[0151] In some embodiments, the recombinant receptor, e.g., the antibody portion of the CAR, further comprises at least a portion of an immunoglobulin constant region, e.g., a hinge region, e.g., an IgG4 hinge region, and / or a CH1 / CL and / or Fc region. In some embodiments, the constant region or portion is that of a human IgG, e.g., IgG4 or IgG1. In some aspects, the portion of the constant region serves as a spacer region between the antigen recognition element, e.g., an scFv, and the transmembrane domain. The spacer can be of a length that results in increased cellular responsiveness after antigen binding compared to the absence of the spacer. Exemplary spacers, e.g., hinge regions, include those described in International Patent Application Publication No. 2014 / 031687. In some examples, the spacer is 12 or about 12 amino acids in length, or no more than 12 amino acids in length. Exemplary spacers include those having at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, including any integer between any of the listed ranges. In some embodiments, the spacer region has about 12 or fewer amino acids, about 119 or fewer amino acids, or about 229 or fewer amino acids. Exemplary spacers include an IgG4 hinge alone, an IgG4 hinge linked to the CH2 and CH3 domains, or an IgG4 hinge linked to the CH3 domain. Exemplary spacers include those described in Hudecek et al., Clin. Cancer Res., 19:3153 (2013), International Patent Application Publication No. 2014031687, U.S. Patent No. 8,822,647, or U.S. Patent Application Publication No. 2014 / 0271635.

[0152] In some embodiments, the constant region or portion is that of a human IgG, e.g., IgG4 or IgG1. In some embodiments, the spacer has the sequence ESKYGPPCPPCP. In some embodiments, the constant region or portion is that of an IgD.

[0153] The antigen recognition domain is generally linked to one or more intracellular signaling elements, e.g., in the case of a CAR, a signaling element that mimics activation through an antigen receptor complex, e.g., a TCR complex, and / or signals through another cell surface receptor. Thus, in some embodiments, the antigen binding element (e.g., an antibody) is linked to one or more transmembrane domains and an intracellular signaling domain. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in a receptor, e.g., a CAR, is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding to the transmembrane domain of the same or a different surface membrane protein, in order to minimize interaction with other members of the receptor complex.

[0154] In some embodiments, the transmembrane domain is derived from either a natural or synthetic source. If the source is natural, the domain is derived from any membrane-bound or transmembrane protein in some aspects. Transmembrane regions include those derived from (i.e., at least the transmembrane regions of) the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain is synthetic in some embodiments. In some aspects, synthetic transmembrane domains contain primarily hydrophobic residues, e.g., leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by a linker, spacer, and / or transmembrane domain.

[0155] The intracellular signaling domain can mimic or approximate the signaling by a natural antigen receptor, by such a receptor in combination with a costimulatory receptor, and / or by a costimulatory receptor alone. In some embodiments, a short oligopeptide or polypeptide linker, e.g., a linker 2-10 amino acids in length, e.g., one containing glycine and serine, e.g., a glycine-serine doublet, is present and forms the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0156] Receptors, e.g., CARs, generally comprise at least one intracellular signaling element. In some embodiments, the receptor comprises an intracellular component of the TCR complex, e.g., the TCR CD3 chain, e.g., the CD3 zeta chain, which mediates T cell activation and cytotoxicity. Thus, in some aspects, the antigen-binding moiety is linked to one or more cell signaling modules. In some embodiments, the cell signaling module comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further comprises a portion of one or more additional molecules, e.g., Fc receptor gamma, CD8, CD4, CD25, or CD16. For example, in some aspects, a CAR or other chimeric receptor comprises a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor gamma and CD8, CD4, CD25, or CD16.

[0157] In some embodiments, upon ligation of a CAR or other chimeric receptor, The cytoplasmic domain or intracellular signaling domain activates at least one of the normal effector functions or responses of an immune cell, e.g., a T cell engineered to express the CAR. For example, in some situations, the CAR induces a T cell function, e.g., cytolytic activity or T helper activity, e.g., secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor element or costimulatory molecule is used in place of an intact immunostimulatory chain, e.g., when transmitting an effector function signal. In some embodiments, the one or more intracellular signaling domains comprise the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects also include that of a co-receptor that acts with such receptor in its natural context to initiate signal transduction following antigen receptor binding.

[0158] In the context of natural TCR, full activation generally requires not only signal transduction through TCR but also costimulatory signals.Therefore, in some embodiments, the CAR also contains components for generating secondary or costimulatory signals to promote full activation.In other embodiments, the CAR does not contain components for generating costimulatory signals.In some aspects, another CAR is expressed in the same cell and provides components for generating secondary or costimulatory signals.

[0159] T cell activation, in some embodiments, is described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act to provide secondary or costimulatory signals in an antigen-independent manner (secondary cytoplasmic signaling sequences). In some embodiments, a CAR comprises one or both of such signaling elements.

[0160] In some embodiments, CAR comprises a primary cytoplasmic signaling sequence that regulates the primary activation of TCR complex.The primary cytoplasmic signaling sequence that acts in a stimulatory manner can comprise a signaling motif known as immunoreceptor tyrosine-based activation motif or ITAM.Examples of primary cytoplasmic signaling sequences that comprise ITAM include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta, and CD3 epsilon.In some embodiments, the cytoplasmic signaling molecule in CAR comprises a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3 zeta.

[0161] In some embodiments, the CAR comprises the signaling domain and / or transmembrane portion of a costimulatory receptor, e.g., CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR comprises both an activating component and a costimulatory component.

[0162] In some embodiments, the activation domain is included in one CAR, and the costimulatory component is provided by another CAR that recognizes a different antigen. In some embodiments, the CAR includes an activating or stimulatory CAR and a costimulatory CAR, both of which are expressed on the same cell (see WO 2014 / 055668). In some aspects, the cell includes one or more stimulatory or activating CARs and / or costimulatory CARs. In some embodiments, the cell further includes an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (2013)), e.g., a CAR that recognizes an antigen that is not associated with and / or specific for a disease or condition, such that the activation signal delivered through the disease-targeting CAR is reduced or inhibited by the inhibitory CAR binding to its ligand, e.g., reducing off-target effects.

[0163] In certain embodiments, the intracellular signaling domain is a CD3 (e.g., CD3 In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domain linked to a CD3 zeta intracellular domain.

[0164] In some embodiments, the CAR comprises one or more, e.g., two or more, costimulatory domains and activation domains, e.g., primary activation domains, in the cytoplasmic portion. Exemplary CARs include the intracellular components of CD3-zeta, CD28, and 4-1BB.

[0165] In some embodiments, the CAR or other antigen receptor further comprises a marker, e.g., a cell surface marker, which can be used to confirm transduction or engineering of cells expressing the receptor, e.g., a truncated version of the cell surface receptor, e.g., truncated EGFR (tEGFR). In some aspects, the marker includes all or a portion (e.g., a truncated form) of PSMA, Her2, CD34, NGFR, or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a linker sequence, e.g., a cleavable linker sequence, e.g., a polynucleotide encoding T2A. For example, the sequence of the marker, and optionally the linker, may be any of those disclosed in PCT Patent Application Publication No. 2014031687, which is incorporated herein by reference. In some embodiments, the marker may be as described in PCT Patent Application Publication No. 2011 / 056894, the contents of which are incorporated herein in their entirety. For example, the marker can be a truncated EGFR (tEGFR), optionally linked to a linker sequence, eg, a T2A cleavable linker sequence.

[0166] In some embodiments, the marker is a molecule, e.g., a cell surface protein, or portion thereof, that is not naturally found on or on the surface of T cells. In some embodiments, the molecule is a non-self molecule, e.g., a non-self protein, i.e., one that is not recognized as "self" by the immune system of the host into which the cells are adoptively transferred.

[0167] In some embodiments, the marker serves no therapeutic function and / or has no effect other than being used as a marker for genetic manipulation, e.g., to select successfully manipulated cells. In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise exerts some desired effect, e.g., a ligand for cells encountered in vivo, e.g., a costimulatory or immune checkpoint molecule that enhances and / or dampens the response of cells upon adoptive transfer and encounter with the ligand.

[0168] In some cases, CARs are referred to as first-, second-, and / or third-generation CARs. In some embodiments, first-generation CARs simply provide a signal induced by the CD3 chain upon antigen binding, in some embodiments, second-generation CARs provide such a signal as well as a costimulatory signal, e.g., include intracellular signaling domains from costimulatory receptors such as CD28 or CD137, and in some embodiments, third-generation CARs include multiple costimulatory domains from different costimulatory receptors.

[0169] In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or antibody fragment. In some aspects, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment, and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv, and the intracellular domain comprises an ITAM. In some aspects, the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3-zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain connecting the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain comprises the transmembrane portion of CD28. In some embodiments, the chimeric antigen receptor comprises the intracellular domain of a T cell costimulatory molecule. The extracellular domain and the transmembrane domain can be linked directly or indirectly. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer, e.g., any of those described herein. In some embodiments, the receptor comprises the extracellular portion of the molecule from which the transmembrane domain is derived, e.g., the extracellular portion of CD28. In some embodiments, the chimeric antigen receptor comprises an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, e.g., between the transmembrane domain and the intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.

[0170] For example, in some embodiments, the CAR comprises an antibody, e.g., an antibody fragment, a transmembrane domain that is or includes a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that includes a signaling portion of CD28 or a functional variant thereof and a signaling portion of CD3-zeta or a functional variant thereof. In some embodiments, the CAR comprises an antibody, e.g., an antibody fragment, a transmembrane domain that is or includes a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that includes a signaling portion of 4-1BB or a functional variant thereof and a signaling portion of CD3-zeta or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer that includes a portion of an Ig molecule, e.g., a human Ig molecule, e.g., an Ig hinge, e.g., an IgG4 hinge, e.g., a hinge-only spacer.

[0171] In some embodiments, the transmembrane domain of the recombinant receptor, e.g., CAR, is or comprises the transmembrane domain of human CD28 (e.g., Accession No. P01747.1) or a variant thereof.

[0172] In some embodiments, the intracellular signaling element of the recombinant receptor, e.g., CAR, comprises the intracellular costimulatory signaling domain of human CD28, or a functional variant or portion thereof, e.g., a domain with an LL to GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular domain comprises the intracellular costimulatory signaling domain of 4-1BB (e.g., Accession No. Q07011.1), or a functional variant or portion thereof.

[0173] In some embodiments, the intracellular signaling domain of the recombinant receptor, e.g., a CAR, comprises a human CD3 zeta stimulatory signaling domain, or a functional variant thereof, e.g., the 112 AA cytoplasmic domain of isoform 3 of human CD3ζ (accession number P20963.2), or a CD3 zeta signaling domain described in U.S. Pat. No. 7,446,190 or U.S. Pat. No. 8,911,993.

[0174] In some aspects, the spacer comprises only the hinge region of an IgG, e.g., only an IgG4 or IgG1 hinge. In other embodiments, the spacer is or comprises an Ig hinge, e.g., a hinge from IgG4, optionally linked to the CH2 and / or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to the CH2 and CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to the CH3 domain only. In some embodiments, the spacer is or comprises a glycine-serine-rich sequence or other flexible linker, e.g., a known flexible linker.

[0175] For example, in some embodiments, a CAR comprises an antibody, e.g., an antibody fragment, including an scFv, a spacer, e.g., a portion of an immunoglobulin molecule, e.g., a spacer comprising the hinge region and / or one or more constant regions of a heavy chain molecule, e.g., a spacer comprising an Ig hinge, a transmembrane domain comprising all or a portion of the transmembrane domain from CD28, an intracellular signaling domain from CD28, and a CD3 zeta signaling domain. In some embodiments, a CAR comprises any of an antibody or fragment, e.g., an scFv, a spacer, e.g., a spacer comprising an Ig hinge, a transmembrane domain from CD28, an intracellular signaling domain from 4-1BB, and a signaling domain from CD3 zeta.

[0176] In some embodiments, the nucleic acid molecule encoding such a CAR construct further comprises a sequence encoding a T2A ribosomal skip element and / or a tEGFR sequence, e.g., downstream of the sequence encoding the CAR. In some embodiments, T cells expressing an antigen receptor (e.g., a CAR) can also be generated to express a truncated EGFR (EGFRt) as a non-immunogenic selection epitope (e.g., by introducing a construct encoding the CAR and EGFRt separated by a T2A ribosomal switch such that the two proteins are expressed from the same construct), which can then be used as a marker to detect such cells (see, e.g., U.S. Patent No. 8,802,374).

[0177] The recombinant receptor (e.g., CAR) expressed by the cells administered to the subject generally recognizes or specifically binds to a molecule that is associated with, and / or specific to, the disease or condition being treated or expressed in the cells thereof. Upon specific binding to a molecule, e.g., an antigen, the receptor generally delivers an immunostimulatory signal, e.g., a signal transmitted by an ITAM, to the cell, thereby promoting an immune response that targets the disease or condition. For example, in some embodiments, the cells express a CAR that specifically binds to an antigen expressed by cells or tissues of the disease or condition or associated with the disease or condition.

[0178] TCR

[0179] In some embodiments, the genetic modification includes genetically modifying the cells to express one or more T cell receptors (TCRs) or antigen-binding portions thereof that recognize peptide epitopes or T cell epitopes of a target polypeptide, e.g., a tumor, viral, or autoimmune protein antigen.

[0180] In some embodiments, a "T cell receptor" or "TCR" is a molecule comprising variable α and β chains (known as TCRα and TCRβ, respectively) or variable γ and δ chains (known as TCRγ and TCRδ, respectively), or an antigen-binding portion thereof, which can specifically bind to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the αβ form. TCRs, which typically exist in the αβ and γδ forms, are generally similar in structure, although the T cells that express them may have different anatomical locations or functions. TCRs can be found on the surface of a cell or in a soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes), where they are generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0181] Unless otherwise stated, the term "TCR" shall be understood to encompass complete TCRs as well as antigen-binding portions or fragments thereof. In some embodiments, The TCR may be an intact or full-length TCR, including an αβ or γδ form of the TCR. In some embodiments, the TCR is an antigen-binding portion that is shorter than the full-length TCR but binds to a specific peptide bound to an MHC molecule, e.g., binds to an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of a TCR may contain only a portion of the structural domain of a full-length or intact TCR, but still be able to bind to a peptide epitope, e.g., an MHC-peptide complex, that the complete TCR binds. In some cases, the antigen-binding portion includes the variable domain of the TCR, e.g., the variable α chain and variable β chain of the TCR, which are sufficient to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chain of the TCR contains the complementarity-determining regions involved in recognizing peptides, MHC, and / or MHC-peptide complexes.

[0182] In some embodiments, the variable domain of a TCR comprises hypervariable loops, or complementarity-determining regions (CDRs), which are generally the major contributors to antigen recognition and binding capacity and specificity. In some embodiments, the CDRs of a TCR, or a combination thereof, form all or substantially all of the antigen-binding site of a given TCR molecule. The various CDRs within the variable region of a TCR chain are generally separated by framework regions (FRs), which generally exhibit lower variability between TCR molecules compared to the CDRs (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. USA, 87:9138, 1990; Chothia et al., EMBO J., 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol., 27:55, 2003). In some embodiments, CDR3 is the primary CDR responsible for antigen binding or specificity, or the one of the three CDRs in a given TCR variable region that is most important for antigen recognition and / or interaction with the processed peptide portion of a peptide-MHC complex. In some circumstances, CDR1 of the alpha chain may interact with the N-terminal portion of a particular antigenic peptide. In some circumstances, CDR1 of the beta chain may interact with the C-terminal portion of a peptide. In some circumstances, CDR2 is the primary CDR that most strongly contributes to or is responsible for interaction with or recognition of the MHC portion of an MHC-peptide complex. In some embodiments, the variable region of the beta chain may further comprise a hypervariable region (CDR4 or HVR4), which is generally involved in superantigen binding but not antigen recognition (Kotb (1995) Clinical Microbiology Reviews 8:411-426).

[0183] In some embodiments, the TCR may also comprise a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd ed., Current Biology Publications, p. 4:33, 1997). In some aspects, each chain of a TCR may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short C-terminal cytoplasmic tail. In some embodiments, the TCR is associated with invariant proteins of the CD3 complex, which are involved in mediating signal transduction.

[0184] In some embodiments, a TCR chain comprises one or more constant domains. For example, the extracellular portion of a given TCR chain (e.g., an α chain or a β chain) comprises two immunoglobulin-like domains, e.g., variable domains (e.g., Vα or Vβ, typically amino acids 1-116, based on Kabat numbering (Kabat et al., "Sequences of Proteins of Immunological Interest," U.S. Pat. No. 6,111,169). Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.)) and constant domains adjacent to the cell membrane (e.g., The constant domains of the TCR may include an α chain constant domain or Cα, typically from positions 117 to 259 of the chain according to Kabat numbering, or a β chain constant domain or Cβ, typically from positions 117 to 295 of the chain according to Kabat numbering. For example, in some cases, the extracellular portion of the TCR formed by the two chains includes two membrane-proximal constant domains and two membrane-distal variable domains, each of which includes a CDR. The constant domains of the TCR may include a short connective sequence in which cysteine ​​residues form disulfide bonds, thereby linking the two chains of the TCR. In some embodiments, the TCR may have an additional cysteine ​​residue in each of the α and β chains such that the TCR includes two disulfide bonds in the constant domains.

[0185] In some embodiments, the TCR chain comprises a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain comprises a cytoplasmic tail. In some cases, this structure allows the TCR to associate with other molecules, such as CD3 and its subunits. For example, a TCR that comprises a constant domain with a transmembrane region can anchor the protein to the cell membrane and associate with an invariant subunit of the CD3 signaling apparatus or complex. The intracellular tails of CD3 signaling subunits (e.g., CD3γ, CD3δ, CD3ε, and CD3ζ chains) contain one or more immunoreceptor tyrosine-based activation motifs or ITAMs that are involved in the signaling capacity of the TCR complex.

[0186] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer, e.g., comprising two separate chains (α and β or γ and δ chains) linked by one or more disulfide bonds.

[0187] In some embodiments, TCRs can be generated from known TCR sequences, e.g., sequences of the Vα and β chains, and substantially full-length coding sequences for these chains are readily available. Methods for obtaining full-length TCR sequences, including V chain sequences, from cellular sources are well known. In some embodiments, nucleic acids encoding TCRs can be obtained from a variety of sources, for example, by polymerase chain reaction (PCR) amplification of nucleic acids encoding TCRs within or isolated from a given cell or cells, or by synthesis of publicly available TCR DNA sequences.

[0188] In some embodiments, the TCR is obtained from a biological source, e.g., from a cell, e.g., a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available source. In some embodiments, the T cell can be obtained from a cell isolated in vivo. In some embodiments, the TCR is a thymic-selected TCR. In some embodiments, the TCR is a neoepitope-restricted TCR. In some embodiments, the T cell can be a cultured T cell hybridoma or clone. In some embodiments, the TCR, or an antigen-binding portion thereof, can be synthetically generated from knowledge of the sequence of the TCR.

[0189] In some embodiments, TCRs are generated from TCRs identified or selected by screening a library of candidate TCRs against a target polypeptide antigen or its target T cell epitope. TCR libraries can be generated by expanding Vα and Vβ repertoires from T cells isolated from a subject, including cells present in PBMCs, spleen, or other lymphoid organs. In some cases, T cells can be expanded from tumor-infiltrating lymphocytes (TILs). In some embodiments, TCR libraries are generated from CD4 + cells or CD8 + In some embodiments, the TC can be produced from cells. Rs can be amplified from a T cell source of a normal or healthy subject, i.e., from a normal TCR library. In some embodiments, TCRs can be amplified from a T cell source of a diseased subject, i.e., from a diseased TCR library. In some embodiments, degenerate primers are used to amplify the Vα and Vβ gene repertoire, for example, by RT-PCR in a sample, e.g., T cells, obtained from a human. In some embodiments, scTv libraries can be assembled from naive Vα and Vβ libraries, where the amplified products are cloned or assembled so as to be separated by a linker. Depending on the subject and cell source, libraries can be HLA allele-specific. Alternatively, in some embodiments, TCR libraries can be generated by mutagenesis or diversification of parent or scaffold TCR molecules. In some aspects, TCRs are subjected to directed evolution, such as by mutagenesis of the α or β chain. In some aspects, specific residues within the CDRs of the TCR are altered. In some embodiments, selected TCRs can be modified by affinity maturation. In some embodiments, antigen-specific T cells can be selected, e.g., by screening to assess CTL activity against a peptide. In some aspects, TCRs, e.g., present on antigen-specific T cells, can be selected by avidity, e.g., a particular affinity or avidity for an antigen.

[0190] In some embodiments, the TCR or antigen-binding portion thereof has been modified or engineered. In some embodiments, directed evolution methods are used to generate TCRs with altered properties, e.g., higher affinity for particular MHC-peptide complexes. In some embodiments, directed evolution is achieved by display methods, including, but not limited to, yeast display (Holler et al. (2003) Nat Immunol 4:55-62; Holler et al. (2000) Proc Natl Acad Sci USA 97:5387-92), phage display (Li et al. (2005) Nat Biotechnol 23:349-54), or T cell display (Chervin et al. (2008) J Immunol Methods 339:175-84). In some embodiments, the display approach involves the manipulation or modification of a known parent or reference TCR. For example, in some cases, a wild-type TCR can be used as a template to produce a mutagenized TCR, in which one or more residues in the CDRs are mutated, and variants with desired altered properties, e.g., higher affinity for a desired target antigen, are selected.

[0191] In some embodiments, peptides of target polypeptides used in producing or generating a TCR of interest are known to or can be readily identified by those of skill in the art. In some embodiments, peptides suitable for use in generating a TCR or antigen-binding portion can be determined based on the presence of HLA restriction motifs in the target polypeptide of interest, such as the target polypeptides described below. In some embodiments, HLA-A0201 binding motifs, proteasome and immunoproteasome cleavage sites, and peptides are identified using computer prediction models known to those of skill in the art. In some embodiments, with respect to predicting MHC class I binding sites, such models include, but are not limited to, ProPred1 (Singh and Raghava (2001) Bioinformatics, 17(12):1236-1237) and SYFPEITHI (Schuler et al. (2007) Immunoinformatics Methods in Molecular Biology, 409(1):75-93, 2007). In some embodiments, the MHC-restricted epitope is HLA-A0201, which is expressed in approximately 39-46% of all Caucasian humans and therefore represents a suitable MHC antigen choice for use in preparing TCRs or other MHC-peptide binding molecules.

[0192] In some embodiments, the TCR or antigen-binding portion thereof may be a recombinantly produced native protein or a mutant form thereof, in which one or more properties, e.g., binding properties, are modified. In some embodiments, the TCR may be derived from one of a variety of animal species, e.g., human, mouse, rat, or other mammal. The TCR may be in a cell-associated or soluble form. In some embodiments, for purposes of the provided methods, the TCR is in a cell-associated form that is expressed on the surface of a cell.

[0193] In some embodiments, the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (sc-TCR). In some embodiments, the dTCR or scTCR has a structure described in WO 03 / 020763, WO 04 / 033685, and WO 2011 / 044186, which are incorporated herein by reference.

[0194] In some embodiments, the TCR comprises a sequence corresponding to a transmembrane sequence. In some embodiments, the TCR comprises a sequence corresponding to a cytoplasmic sequence. In some embodiments, the TCR can form a TCR complex with CD3. In some embodiments, any TCR, including dTCR or scTCR, can be linked to a signaling domain that provides an active TCR on the surface of a T cell. In some embodiments, the TCR is expressed on the surface of a cell.

[0195] In some embodiments, a dTCR comprises a first polypeptide in which a sequence corresponding to the variable region sequence of a TCR α chain is fused to the N-terminus of a sequence corresponding to the extracellular sequence of the constant region of the TCR α chain, and a second polypeptide in which a sequence corresponding to the variable region sequence of a TCR β chain is fused to the N-terminus of a sequence corresponding to the extracellular sequence of the constant region of the TCR β chain, the first and second polypeptides being linked by a disulfide bond. In some embodiments, the bond may correspond to a native interchain disulfide bond present in naturally occurring dimeric αβ TCRs. In some embodiments, the interchain disulfide bond is not present in naturally occurring TCRs. For example, in some embodiments, one or more cysteines may be incorporated into the constant region extracellular sequences of the dTCR polypeptide pair. In some cases, both native and non-native disulfide bonds may be desired. In some embodiments, the TCR comprises a transmembrane sequence for membrane anchoring.

[0196] In some embodiments, the dTCR comprises a TCR alpha chain comprising a variable alpha domain, a constant alpha domain, and a first dimerization motif attached to the C-terminus of the constant alpha domain, and a TCR beta chain comprising a variable beta domain, a constant beta domain, and a first dimerization motif attached to the C-terminus of the constant beta domain, wherein the first and second dimerization motifs readily interact to form a covalent bond between an amino acid in the first dimerization motif and an amino acid in the second dimerization motif, linking the TCR alpha chain and the TCR beta chain together.

[0197] In some embodiments, the TCR is an scTCR. Typically, scTCRs can be generated using methods known to those skilled in the art, such as those described in Soo Hoo, WF et al., PNAS (USA), 89, 4759 (1992); Wulfing, C. and Pluckthun, A., J. Mol. Biol., 242, 655 (1994); Kurucz, I. et al., PNAS (USA), 90, 3830 (1993); PCT Application Publication Nos. 96 / 13593, 96 / 18105, 99 / 60120, 99 / 18129, 03 / 020763, 2011 / 044186, and Schlueter, CJ et al., J. Mol. Biol., 256, 859 (1996). In some embodiments, the scTCR contains an introduced non-native interchain disulfide bond to facilitate TCR chain association (see, e.g., PCT Application Publication No. 03 / 020763, which (See, e.g., PCT Publication No. 99 / 60120, which is incorporated herein by reference). In some embodiments, the scTCR is a non-disulfide-bonded truncated TCR in which chain association is facilitated by a heterologous leucine zipper fused to its C-terminus (see, e.g., PCT Publication No. 99 / 60120, which is incorporated herein by reference). In some embodiments, the scTCR comprises a TCR alpha variable domain covalently linked to a TCR beta variable domain via a peptide linker (see, e.g., PCT Publication No. 99 / 18129, which is incorporated herein by reference).

[0198] In some embodiments, the scTCR comprises a first segment constructed from an amino acid sequence corresponding to a TCR alpha chain variable region, a second segment constructed from an amino acid sequence corresponding to a TCR beta chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to a TCR beta chain constant domain extracellular sequence, and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0199] In some embodiments, the scTCR comprises a first segment constructed by an alpha chain variable region sequence fused to the N-terminus of an alpha chain extracellular constant domain sequence, and a second segment constructed by a beta chain variable region sequence fused to the N-terminus of a beta chain extracellular constant sequence and a transmembrane sequence, and optionally a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0200] In some embodiments, the scTCR comprises a first segment constructed from a TCR beta chain variable region sequence fused to the N-terminus of a beta chain extracellular constant domain sequence, and a second segment constructed from an alpha chain variable region sequence fused to the N-terminus of an alpha chain extracellular constant sequence and a transmembrane sequence, and optionally a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0201] In some embodiments, the linker of the scTCR connecting the first and second TCR segments can be any linker capable of forming a single polypeptide chain while retaining TCR binding specificity. In some embodiments, the linker sequence can have the formula -P-AA-P-, for example, where P is proline and AA represents an amino acid sequence, where the amino acids are glycine and / or serine. In some embodiments, the first and second segments are paired such that their variable region sequences are oriented for such binding. Thus, in some cases, the linker has a length sufficient to span the distance between the C-terminus of the first segment and the N-terminus of the second segment, or vice versa, but not so long as to block or reduce binding of the scTCR to its target ligand. In some embodiments, the linker can comprise 10 to 45 or about 10 to about 45 amino acids, e.g., 10 to 30 amino acids, or 26 to 41 amino acid residues, e.g., 29, 30, 31, or 32 amino acids. In some embodiments, the linker has the formula -PGGG-(SGGGG)5-P-, where P is proline, G is glycine, and S is serine. In some embodiments, the linker has the sequence GSADDAKKDAAKKDGKS.

[0202] In some embodiments, the scTCR comprises a covalent disulfide bond linking residues of the immunoglobulin region of the constant domain of the α chain to residues of the immunoglobulin region of the constant domain of the β chain. In some embodiments, interchain disulfide bonds are absent in native TCRs. For example, in some embodiments, one or more cysteines may be incorporated into the constant region extracellular sequences of the first and second segments of the scTCR polypeptide. In some cases, both natural and non-natural disulfide bonds may be desired.

[0203] In some embodiments of dTCRs or scTCRs that contain an introduced interchain disulfide bond In some embodiments, no native disulfide bonds are present. In some embodiments, one or more of the native cysteines forming the native interchain disulfide bonds are substituted with another residue, e.g., serine or alanine. In some embodiments, the introduced disulfide bond can be formed by mutating non-cysteine ​​residues in the first and second segments to cysteines. Exemplary non-native disulfide bonds in TCRs are described in PCT Application Publication No. 2006 / 000830, which is incorporated herein by reference.

[0204] In some embodiments, the TCR or antigen-binding fragment thereof exhibits an affinity for the target antigen with an equilibrium binding constant of 10 M to 10 M, or about 10 M to about 10 M, and all individual values ​​and ranges therein. In some embodiments, the target antigen is an MHC-peptide complex or a ligand.

[0205] In some embodiments, one or more nucleic acids encoding the TCR, e.g., the α and β chains, can be amplified by PCR or other suitable means and cloned into one or more suitable expression vectors. The expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification, or expression, or both, e.g., plasmids and viruses.

[0206] In some embodiments, the vector may be a pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), or pEX series (Clontech, Palo Alto, Calif.) vector. In some cases, bacteriophage vectors, such as λG10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, may also be used. In some embodiments, plant expression vectors may be used, including pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, viral vectors, such as retroviral vectors, are used.

[0207] In some embodiments, recombinant expression vectors can be prepared using standard recombinant DNA techniques. In some embodiments, vectors can include regulatory sequences, e.g., transcriptional and translational initiation and termination codons, which are specific to the type of host into which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), as appropriate, taking into account whether the vector is DNA- or RNA-based. In some embodiments, vectors can include a non-native promoter operably linked to a nucleotide sequence encoding a TCR or antigen-binding portion (or other MHC-peptide binding molecule). In some embodiments, the promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long terminal repeat of murine stem cell virus. Other promoters known to those of skill in the art are also contemplated.

[0208] In some embodiments, to generate a vector encoding a TCR, the alpha and beta chains are PCR amplified from total cDNA isolated from a T cell clone expressing the TCR of interest and cloned into an expression vector. In some embodiments, the α chain and β chain are cloned into different vectors. In some embodiments, the generated α chain and β chain are incorporated into a retroviral vector, e.g., a lentiviral vector.

[0209] multiple targeting

[0210] In some embodiments, the genetic modification includes genetically modifying the cell to express two or more engineered receptors, each of which recognizes the same or different antigens and, in some embodiments, each of which contains a different intracellular signaling element. Such multiple targeting strategies are described, for example, in PCT Patent Application Publication No. 2014 / 055668 A1 and Fedorov et al., Sci. Transl. Medicine, 5 (215) (2013).

[0211] For example, in some embodiments, the cells generally comprise a receptor expressing a first engineered antigen receptor (e.g., a CAR or TCR) that can induce an activating signal in the cell when specifically bound to an antigen recognized by the first receptor, e.g., the first antigen. In some embodiments, the cells generally further comprise a second engineered antigen receptor (e.g., a CAR or TCR), e.g., a chimeric costimulatory receptor, that can induce a costimulatory signal in the immune cell when specifically bound to a second antigen recognized by the second receptor. In some embodiments, the first antigen and the second antigen are the same. In some embodiments, the first antigen and the second antigen are different.

[0212] In some embodiments, the first and / or second engineered antigen receptor (e.g., CAR or TCR) can induce an activation signal in a cell. In some embodiments, the receptor comprises an intracellular signaling element comprising an ITAM or ITAM-like motif. In some embodiments, activation induced by the first receptor comprises signal transduction or changes in protein expression in the cell that result in the initiation of an immune response, such as ITAM phosphorylation and / or initiation of an ITAM-mediated signaling cascade, formation of an immune synapse and / or clustering of molecules (e.g., CD4 or CD8) near the bound receptor, activation of one or more transcription factors, e.g., NF-κB and / or AP-1, and / or induction of gene expression of factors such as cytokines, proliferation, and / or survival.

[0213] In some embodiments, the first and / or second receptor comprises the intracellular signaling domain of a costimulatory receptor, such as CD28, CD137 (4-1BB), OX40, and / or ICOS. In some embodiments, the first and second receptor comprise the intracellular signaling domains of different costimulatory receptors. In some embodiments, the first receptor comprises the CD28 costimulatory signaling region and the second receptor comprises the 4-1BB costimulatory signaling region, or vice versa.

[0214] In some embodiments, the first and / or second receptor comprises both an intracellular signaling domain containing an ITAM or ITAM-like motif and an intracellular signaling domain of a costimulatory receptor.

[0215] In some embodiments, the first receptor comprises an intracellular signaling domain comprising an ITAM or ITAM-like motif, and the second receptor comprises an intracellular signaling domain of a costimulatory receptor. A costimulatory signal in combination with an activating signal induced in the same cell can result in an immune response, e.g., a strong and sustained immune response, e.g., a T cell-mediated response, such as increased gene expression, secretion of cytokines and other factors, and cell killing. These provide effector functions.

[0216] In some embodiments, ligation of neither the first receptor alone nor the second receptor alone induces a strong immune response. In some aspects, when only one receptor is ligated, the cells become tolerant or unresponsive to the antigen, or become inhibited, and / or are not induced to proliferate, secrete factors, or perform effector functions. In some such embodiments, however, when multiple receptors are ligated, a desired response, e.g., complete immune activation or stimulation, is achieved, as indicated by, for example, secretion of one or more cytokines, proliferation, persistence, and / or performance of immune effector functions, such as cytotoxic killing of target cells, upon encounter between cells expressing the first and second antigens.

[0217] In some embodiments, the two receptors induce an activating signal and an inhibitory signal in the cell, respectively, so that when one of the receptors binds to the antigen, the cell is activated or a response is induced, while when the second inhibitory receptor binds to the antigen, a signal that suppresses or reduces the response is induced. An example is the combination of an activating CAR and an inhibitory CAR or iCAR. For example, a strategy can be used in which the activating CAR binds to an antigen that is expressed in a disease or condition but also expressed in normal cells, and the inhibitory receptor binds to another antigen that is expressed in normal cells but not expressed in cells of the disease or condition.

[0218] In some embodiments, multiple targeting strategies are utilized when antigens associated with a particular disease or condition are expressed on non-diseased cells and / or expressed transiently (e.g., upon stimulation associated with genetic engineering) or permanently on the engineered cells themselves. In such cases, specificity, selectivity, and / or efficacy may be improved by requiring ligation of two separate and individual specific antigen receptors.

[0219] In some embodiments, multiple antigens, e.g., first and second antigens, are expressed on targeted cells, tissues, or disease or condition, e.g., cancer cells. In some aspects, the cells, tissues, disease, or condition are multiple myeloma or multiple myeloma cells. In some embodiments, one or more of the multiple antigens are also expressed on cells that are generally not desired to be targeted by cell therapy, e.g., normal or non-diseased cells or tissues, and / or the engineered cells themselves. In such embodiments, specificity and / or efficacy are achieved by requiring ligation of multiple receptors to achieve a cellular response.

[0220] In some embodiments, the modification of the cells is performed based on analysis of the cells after collection and before cryogenic freezing and / or storage. The cells may be modified based on analysis before and / or after cryogenic storage. In some embodiments, the modification of the cells is performed based on analysis of the cells after thawing after cryogenic storage. In some embodiments, the analysis is performed based on analysis of the CD4 + Cellular CD8 + In some embodiments, post-cryogenic modification conditions, such as the time for incubating the cells, the temperature for incubating the cells, the use and concentration of cell stimuli, and the steps for genetically modifying the cells, can be selected based on the analysis or can be used to determine the ratio of CD4 + Cellular CD8 + The selection may be based on the ratio to the cells.

[0221] Vectors for manipulating cells

[0222] Polynucleotides (nucleic acid molecules) encoding recombinant receptors and / or TCRs can be included in vectors for genetically engineering cells to express such receptors. In some embodiments, the vector or construct comprises one or more promoters operably linked to the nucleotides encoding the polypeptides or receptors to drive expression. In some embodiments, the promoter is operably linked to one or more nucleic acid molecules. In some cases, the vector is a viral vector, e.g., a retroviral vector, e.g., a lentiviral vector or a gamma retroviral vector. In some embodiments, a polynucleotide, e.g., a vector, encoding the recombinant receptor is introduced into a composition comprising cultured cells, e.g., by retroviral transduction, transfection, or transformation.

[0223] Various methods for introducing genetically engineered components, for example, recombinant receptors, for example, CAR or TCR, are well known and can be used in the provided methods and compositions.Exemplary methods include those for transferring the nucleic acid encoding polypeptide or receptor, including those by viral vectors, for example, retroviruses or lentiviruses, non-viral vectors or transposons, for example, Sleeping Beauty transposon system.Gene transfer methods can include transduction, electroporation or other methods that result in the transfer of gene into cells.

[0224] In some embodiments, gene transfer is achieved by first stimulating the cells, e.g., by combining the cells with a stimulus that induces a proliferation, survival, and / or activation response, as measured, e.g., by expression of cytokines or activation markers, followed by transduction of the activated cells and expansion in culture to numbers sufficient for clinical application.

[0225] In some situations, it may be desirable to protect against the possibility that overexpression of a stimulatory factor (e.g., a lymphokine or cytokine), e.g., a factor associated with toxicity in a subject, could potentially result in an unfavorable prognosis or reduced efficacy in the subject. Thus, in some situations, the engineered cells contain a gene segment that renders the cells susceptible to negative selection in vivo, e.g., upon administration in adoptive immunotherapy. For example, in some embodiments, the cells are engineered such that the cells can be eliminated as a result of a change in the in vivo conditions of the patient to whom the cells are administered. A negatively selectable phenotype can result from the insertion of a gene that confers selectivity for an administered agent, e.g., a compound. Negative selectable genes include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene, which confers sensitivity to ganciclovir (Wigler et al., Cell 11:223, 1977), the intracellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the intracellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA 89:33 (1992)).

[0226] In some embodiments, the recombinant nucleic acid is transferred into cells using a vector derived from a recombinant infectious viral particle, e.g., Simian Virus 40 (SV40), adenovirus, adeno-associated virus (AAV), etc. In some embodiments, the recombinant nucleic acid is transferred into T cells using a recombinant lentiviral or retroviral vector, e.g., a gamma retroviral vector (see, e.g., Koste et al. (2014), Gene Therapy, April 3, 2014, doi: 10.1038 / gt.2014.25; Carlens et al. (2000), Exp Hematol, 28(10):1137-46; Alonso-Camino et al. (2013), Mol Ther Nucl Acids, 2, e93; Park et al., Trends Biotechnol., November 29, 2011 (11):550-557). stomach).

[0227] In some embodiments, retroviral vectors, such as those derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus-forming virus (SFFV), or adeno-associated virus (AAV), have long terminal repeats (LTRs). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically amphotropic, i.e., they can infect host cells of multiple species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and / or env sequences. Several exemplary retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740, 6,207,453, 5,219,740; Miller and Rosman (1989), BioTechniques 7:980-990; Miller, AD (1990), Human Gene Therapy 1:5-14; Scarpa et al. (1991), Virology 180:849-852; Burns et al. (1993), Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993), Cur. Opin. Genet. Develop. 3:102-109).

[0228] Lentiviral transduction methods are known.Exemplary methods are described, for example, in Wang et al. (2012), J. Immunother., 35(9):689-701; Cooper et al. (2003), Blood., 101:1637-1644; Verhoeyen et al. (2009), Methods Mol Biol., 506:97-114; and Cavalieri et al. (2003), Blood., 102(2):497-505.

[0229] In some embodiments, the recombinant nucleic acid is transferred into the T cell by electroporation (see, e.g., Chicaybam et al. (2013) PLoS ONE 8(3):e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16):1431-1437). In some embodiments, the recombinant nucleic acid is transferred into the T cell by transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4):427-437; Sharma et al. (2013) Molec (See Ther Nucl Acids, 2, e74; and Huang et al. (2009), Methods Mol Biol, 506:115-126.) Other methods for introducing genetic material into and expressing it in immune cells include calcium phosphate transfection (as described, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-assisted microparticle bombardment; particle-facilitated microparticle bombardment) (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)). In some embodiments, the wash step is performed in a centrifugation chamber, e.g., those manufactured and sold by Biosafe SA, including the A-200 / F and A-200 centrifugation chambers, including those for use in the Sepax® and Sepax® 2 systems, according to the manufacturer's instructions.

[0230] Other approaches and vectors for the transfer of nucleic acids encoding recombinant products are described, for example, in PCT Patent Application Publication No. 2014055668 and U.S. Patent No. 7,446,190, which are incorporated herein by reference.

[0231] In some embodiments, cells, e.g., T cells, can be transfected with, e.g., a T cell receptor (TCR) or a chimeric antigen receptor (CAR) either during or after expansion. This transfection for the introduction of the gene for the desired polypeptide or receptor can be performed, for example, using any suitable retroviral vector. The genetically modified cell population can then be released from the initial stimulation (e.g., CD3 / CD28 stimulation) and subsequently stimulated with a second type of stimulation (e.g., via the de novo introduced receptor). This second type of stimulation can include antigenic stimulation in the form of a peptide / MHC molecule, a cognate (cross-linking) ligand of the transfected receptor (e.g., the natural ligand of the CAR), or any ligand (e.g., an antibody) that directly binds to the framework of the new receptor (e.g., by recognizing a constant region within the receptor). See, for example, Cheadle et al., "Chimeric antigen receptors for T-cell based therapy," Methods Mol Biol., 2012;907:645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine, 65:333-347 (2014).

[0232] Additional nucleic acids, e.g., genes for transfer, include those that improve the efficacy of the therapy, e.g., by promoting the survival and / or function of the transferred cells; genes that provide genetic markers for cell selection and / or evaluation, e.g., for assessing in vivo survival or localization; and genes that improve safety, e.g., by making cells susceptible to in vivo negative selection, as described in Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy, 3:319-338 (1992). See also PCT / US91 / 08442 and PCT / US94 / 05601 publications by Lupton et al., which describe the use of bifunctional selectable fusion genes derived by fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., U.S. Pat. No. 6,040,177, columns 14-17, by Riddell et al.

[0233] In some embodiments, cells are incubated and / or cultured prior to or in conjunction with genetic manipulation. Incubation steps may include culturing, cultivating, stimulating, activating, and / or expanding. Incubation and / or manipulation may occur in a culture vessel, e.g., a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other vessel for culturing or cultivating cells. In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in a population, mimic antigen exposure, and / or prime cells for genetic manipulation, e.g., introduction of a recombinant antigen receptor. In some embodiments, one or more of the incubation steps may be performed using a rocking bioreactor, e.g., a WAVE™ Bioreactor (GE Healthcare) or a BIOSTAT® RM (Sartorius). In some embodiments, one or more of the incubation steps may be carried out using a static bioreactor or incubation chamber. In certain embodiments, when a rocking bioreactor is used for one or more incubation steps, an anti-shear agent, e.g., poloxamer, may be added to the composition.

[0234] The conditions may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells. In some aspects, cells are incubated in the presence of one or more cytokines, and in some embodiments, cytokine cocktails, such as those described in PCT Patent Application Publication No. 2015 / 157384, which is incorporated by reference, may be utilized. In some embodiments, cells are incubated with one or more cytokines and / or cytokine cocktails before, simultaneously with, or after transduction.

[0235] In some embodiments, the stimulatory conditions or agents include one or more agents, e.g., ligands, capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agents activate or initiate the TCR / CD3 intracellular signaling cascade in T cells. Such agents can include antibodies, e.g., those specific for TCR components, e.g., anti-CD3. In some embodiments, the stimulatory conditions include one or more agents, e.g., ligands, e.g., anti-CD28, capable of stimulating a costimulatory receptor. In some embodiments, such agents and / or ligands may be bound to a solid support, e.g., beads, and / or one or more cytokines. Optionally, the expansion method can further include adding an anti-CD3 antibody and / or an anti-CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulatory agent includes IL-2 and / or IL-15, e.g., IL-2 at a concentration of at least about 10 units / mL.

[0236] In some aspects, incubation is carried out according to techniques such as those described in U.S. Patent No. 6,040,177 to Riddell et al.; Klebanoff et al. (2012), J. Immunother., 35(9):651-660; Terakura et al. (2012), Blood., 1:72-82; and / or Wang et al. (2012), J. Immunother., 35(9):689-701. In some aspects, transduction is carried out using the systems, devices, apparatus, and / or methods described in PCT Patent Application Publication No. 2016 / 073602 or U.S. Publication No. 2016 / 0122782, the contents of which are incorporated by reference in their entireties. In some embodiments, transduction is carried out according to the methods described in PCT Patent Application Publication No. 2015 / 164675, the contents of which are incorporated by reference in their entireties.

[0237] In some embodiments, T cells are expanded by adding feeder cells, e.g., non-dividing peripheral blood mononuclear cells (PBMCs), to the culture starter composition (e.g., such that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded) and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells can include gamma-irradiated PBMC feeder cells. In some embodiments, PBMCs are added to the culture starter composition at a concentration of about 3000-360 The cells are irradiated with gamma rays in the 0 rad range to prevent cell division. In some embodiments, feeder cells are added to the culture medium prior to the addition of the T cell population.

[0238] In some embodiments, stimulatory conditions include temperatures suitable for the growth of human T lymphocytes, e.g., at least about 25° C., typically at least about 30° C., and typically at or about 37° C. Optionally, incubation can further include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL may be gamma-irradiated in the range of about 6000-10,000 rads. LCL feeder cells are provided in any suitable amount, in some aspects, e.g., at a ratio of LCL feeder cells to primary T lymphocytes of at least about 10:1.

[0239] Methods for processing samples

[0240] In some embodiments, the method includes a method for processing an apheresis sample, the method comprising: (a) transporting an apheresis sample obtained from a donor to a storage facility in a cryogenic environment; and (b) storing the apheresis sample at the storage facility at cryogenic temperatures. The method, according to certain embodiments, may further comprise processing a plurality of apheresis samples, the plurality of apheresis samples each obtained from the same or different donors and transported at either the same or different times, to a storage facility in a cryogenic environment; and (b) storing each of the apheresis samples at cryogenic temperatures at the storage facility.

[0241] In some embodiments, the apheresis sample is blood collected from a donor according to the embodiments described above.

[0242] In some embodiments, the temperature of the refrigerated shipping environment is greater than -80°C to 0°C. In some embodiments, the temperature of the refrigerated shipping environment is greater than -80°C to -20°C. In some embodiments, the temperature of the refrigerated shipping environment is between -20°C and 0°C.

[0243] In some embodiments, the facility where a donor's apheresis sample is collected and the storage facility are affiliated with each other, although this is not required in all embodiments. In some embodiments, the facilities are affiliated with each other by a donor or another entity selecting to collect the apheresis sample at a collection facility and store the apheresis sample at a storage facility. In some embodiments, the collection facility and the storage facility may share the same physical location. In some embodiments, the collection facility and the storage facility may be located in different locations, for example, in different countries or different states.

[0244] In some embodiments, the storage facility is a central or common repository storage facility where apheresis samples from various patients obtained at different collection facilities are stored. In some embodiments, the central or common repository storage facility stores apheresis samples at cryogenic temperatures before sending these samples to one or more manufacturing facilities. In some embodiments, the central or common repository facility and the manufacturing facility are affiliated with each other. In some embodiments, the central or common repository facility and the manufacturing facility are not affiliated with each other. In some embodiments, all of the samples obtained from a donor are sent from the central or common repository facility to a manufacturing facility. In other embodiments, some of the samples obtained from a donor are sent to a manufacturing facility, and other samples are held at the central or common repository facility. In some embodiments, all of the samples obtained from a donor are sent from the central or common repository facility to the same manufacturing facility. In other embodiments, some of the samples obtained from a donor are sent from the central or common repository facility to one manufacturing facility, and other samples obtained from a donor are sent to another manufacturing facility.

[0245] In some embodiments, one or more types of cells are enriched and / or isolated from the apheresis sample prior to transport. In other cases, cells may be enriched and / or isolated from the apheresis sample after transport. For example, cells may be enriched and / or isolated according to the embodiments described above.

[0246] In some embodiments, the apheresis sample or enriched and / or isolated cells are analyzed before being transported. In some embodiments, the apheresis sample or enriched and / or isolated cells are analyzed after being transported and before being cryogenically stored. The apheresis sample or enriched and / or isolated cells may be analyzed according to the embodiments described above.

[0247] In some embodiments, a portion or portions of the apheresis, or enriched and / or isolated cell population, or engineered T cell population or composition, are removed prior to apheresis, or cryogenic freezing of the enriched and / or isolated cell population, or engineered T cell population or composition. In some embodiments, the removed portion or portions are analyzed at any time, including, for example, before or after apheresis, or cryogenic freezing of the enriched and / or isolated cell population, or engineered T cell population or composition.

[0248] In some embodiments, the apheresis sample or cells are combined with a freezing solution before being transported. In some embodiments, the apheresis sample or cells are combined with a freezing solution after being transported and before being cryogenically stored. The freezing solution may be the same as the freezing solution in the above-described embodiments.

[0249] In some embodiments, the apheresis sample is divided into 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 separate containers before or after combining with the freezing solution to be cryogenically frozen. In some embodiments, the apheresis sample is divided into 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 separate containers before being shipped. In some embodiments, the apheresis sample is divided into 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 separate containers after being shipped. In some embodiments, any number of separate containers containing the divided apheresis samples are cryogenically frozen before or after being shipped.

[0250] In some embodiments, the apheresis sample is divided into 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 separate containers, which are stored at cryogenic temperatures at an archiving facility. In some embodiments, the archiving facility is a central or common repository archiving facility. In some embodiments, the archiving facility sends any number of separate containers containing the divided apheresis to one or more manufacturing facilities.

[0251] In some embodiments, one or more containers in which the apheresis sample is cryogenically stored are removed from cryogenic storage, while the remaining containers are maintained in cryogenic storage. In some embodiments, the cells in the one or more containers removed from cryogenic storage are thawed. In some embodiments, the thawed cells are manipulated. In some embodiments, the thawed cells are manipulated to express a CAR molecule. In some embodiments, one or more subsequent containers in which the apheresis sample is cryogenically stored are removed from cryogenic storage, while the remaining containers are maintained in cryogenic storage. In some embodiments, the cells in the one or more subsequent containers removed from cryogenic storage are thawed. In some embodiments, the thawed cells are manipulated. In some embodiments, the thawed cells are manipulated. The thawed cells are engineered to produce cells that express a similar or different CAR molecule than the previously thawed cells. In some embodiments, the containers in which the apheresis samples are cryogenically stored are kept in cryogenic storage for different lengths of time.

[0252] In some embodiments, the apheresis sample or cells are cooled to a temperature of greater than -80°C to 0°C before transport. The apheresis sample or cells may be cooled in a manner according to the embodiments described above. In some embodiments, prior to cooling the cells, the cells are washed in a manner according to the embodiments described above.

[0253] In some embodiments, the apheresis sample or cells are cryogenically frozen to a temperature of -210°C to -80°C before transport. In some embodiments, the apheresis sample or cells are cryogenically frozen after transport. The apheresis sample or cells may be cryogenically frozen in a manner consistent with the embodiments described above. In some embodiments, prior to cryogenic freezing of the cells, the cells are washed in a manner consistent with the embodiments described above.

[0254] In some embodiments, the apheresis sample or cells are cryogenically stored at temperatures between -210°C and -80°C. For example, the apheresis sample or cells may be cryogenically stored in a manner according to the above-described embodiments, e.g., in the vapor phase of a liquid nitrogen storage tank, for a storage period of, e.g., 1 day to 12 years. In some embodiments, the cells are stored or preserved for a period greater than or equal to the following: 12 hours, 24 hours, 36 hours, or 48 hours. In some embodiments, the cells are stored or preserved for a period greater than or equal to the following: 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the cells are stored or preserved for a long-term period. In some embodiments, the cells are stored for a period of time greater than or equal to the following: 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, or more.

[0255] In some embodiments, the apheresis sample or cells are cryogenically stored at temperatures between −210° C. and −80° C. In some embodiments, the cells are stored at temperatures no greater than about −100° C., or −95° C., or −90° C., or −85° C., or −80° C., or −75° C., or −70° C., or −65° C., or −60° C.

[0256] In some embodiments, after the storage period, the apheresis sample or cells are thawed. For example, the apheresis sample or cells may be thawed in a manner consistent with the embodiments described above. Also, according to certain embodiments, after the storage period, the percentage of viable cells is between 24% and 100%. The percentage of viable cells may be determined, for example, in accordance with the embodiments described above.

[0257] In some embodiments, the apheresis sample or enriched cells are analyzed after collection and before transport. In some embodiments, the apheresis sample or enriched cells are analyzed after transport and before cryogenic storage. In some embodiments, the apheresis sample or enriched cells are analyzed after a storage period. In some embodiments, after analysis, the apheresis sample or cells may be modified. In some embodiments, the modification occurs before transport. In some embodiments, the modification occurs after transport and before cryogenic storage. In some embodiments, the modification occurs after cryogenic storage. In such embodiments, the modification is referred to as "post-cryogenic modification." Analysis and / or modification of the apheresis sample or cells may be performed according to the embodiments described above.

[0258] Compositions and Formulations

[0259] Compositions comprising cells are also provided, including pharmaceutical compositions and formulations, for example, compositions in unit dose form containing a number of cells for administration at a given dose or fraction thereof. Pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one additional therapeutic agent.

[0260] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation will be administered.

[0261] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0262] In some embodiments, the choice of carrier is determined, in part, by the particular cell and / or method of administration. Accordingly, a variety of suitable formulations exist. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixture thereof is typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences, 16th Edition, edited by Osol, A. (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (about 10 residues) proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG).

[0263] In some embodiments, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st Edition (May 1, 2005). .

[0264] The formulation may include an aqueous solution. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with complementary activities, where each activity does not adversely affect the other. Such active ingredients are preferably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine.

[0265] In some embodiments, the composition comprises cells in an amount effective to reduce the burden of the disease or condition and / or in an amount that does not result in CRS or severe CRS in the subject and / or does not affect any of the other outcomes of the methods described herein.

[0266] The pharmaceutical composition, in some embodiments, contains the cells in an amount effective to treat or prevent a disease or condition, e.g., a therapeutically or prophylactically effective amount. Therapeutic or prophylactic effectiveness is, in some embodiments, monitored by periodic evaluation of the treated subject. The desired dosage can be delivered by a single bolus of cells, by multiple boluses of cells, or by continuous infusion of cells.

[0267] Cells and compositions can be administered using standard administration techniques, formulations, and / or devices. Cell administration can be autologous or xenogeneic. For example, immunoresponsive cells or precursors can be obtained from one subject and administered to the same subject or another compatible subject. Peripheral blood-derived immunoresponsive cells or their progeny (e.g., derived in vivo, ex vivo, or in vitro) can be administered by local injection, systemic injection, regional injection, intravenous injection, or parenteral administration, including catheter administration. When a therapeutic composition (e.g., a pharmaceutical composition containing genetically modified immunoresponsive cells) is administered, it will generally be formulated in a unit-dosage injectable form (solution, suspension, emulsion).

[0268] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, intrapulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0269] In some embodiments, the compositions are provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may, in some embodiments, be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0270] Sterile injectable solutions may be prepared by dissolving the cells in a solvent, e.g., a suitable carrier, diluent, or excipient, For example, they can be prepared by incorporating them as a mixture with sterile water, saline, glucose, dextrose, etc. Depending on the desired route of administration and preparation, the compositions may contain auxiliary substances, such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavoring agents, and / or coloring agents. Standard texts may be consulted in some embodiments to prepare suitable preparations.

[0271] Various additives that enhance the stability and sterility of the compositions can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0272] Formulations to be used for in vivo administration are generally sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0273] In some embodiments, the therapeutic T cell composition comprises between about 10 million cells per ml and about 70 million cells per ml, or between about 10 million viable cells per ml and about 70 million viable cells per ml. In some embodiments, the therapeutic T cell composition comprises between about 15 million cells or viable cells per ml and about 60 million cells or viable cells per ml. In some embodiments, the T cell composition comprises greater than 10 million cells or viable cells per ml. In some embodiments, the therapeutic T cell composition comprises greater than 15 million cells or greater than 15 million cells per ml.

[0274] In some embodiments, the application provides an article of manufacture comprising a container containing a therapeutic T cell composition. In some embodiments, the article of manufacture further comprises information indicating that the container contains a target number of units of the therapeutic T cell composition. In some embodiments, the article of manufacture comprises multiple containers, wherein each container contains a unit dose comprising a target number of units of the T cell composition. In some embodiments, the container comprises between about 10 million cells or viable cells per mL and about 70 million cells or viable cells per mL, between about 15 million cells or viable cells per mL and about 60 million cells or viable cells per mL, greater than 10 million cells or viable cells per mL, greater than 15 million cells or viable cells per mL, or a combination thereof. In some embodiments, the composition further comprises a cryoprotectant and / or the article of manufacture further comprises instructions for thawing the composition prior to administration to a subject.

[0275] In some embodiments, cells are suspended in a freezing solution, for example, after a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters can be used in some aspects. One example includes using PBS containing 20% ​​DMSO and 8% HSA, or other suitable cell freezing medium. This is then diluted 1:1 with the medium so that the final concentrations of DMSO and HSA are 10% and 4%, respectively.

[0276] Any of a variety of known freezing solutions and parameters can be used in some aspects. In some embodiments, the cell sample may contain a cryopreservation or vitrification medium or solution containing a cryoprotectant. Suitable cryoprotectants include, but are not limited to, DMSO, glycerol, glycol, propylene glycol, ethylene glycol, propanediol, polyethylene glycol (PEG), 1,2-propanediol (PROH), or mixtures thereof. In some examples, the cryopreservation solution may be polyvinylpyrrolidone, hydroxyethyl starch, polysaccharides, monosaccharides, alginates, or the like. The cryoprotectant may contain one or more non-cell-permeable cryopreservatives, including, but not limited to, trehalose, raffinose, dextran, human serum albumin, Ficoll, lipoproteins, polyvinylpyrrolidone, hydroxyethyl starch, autologous plasma, or mixtures thereof. In some embodiments, the cells are suspended in a freezing solution having a final concentration of cryoprotectant of about 1% to about 20%, about 3% to about 9%, or about 6% to about 9% by volume. In certain embodiments, the final concentration of cryoprotectant in the freezing solution is about 3%, 4%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% by volume.

[0277] In some embodiments, the cryoprotectant is DMSO. In certain embodiments, the cells are suspended in a freezing solution having a final concentration of DMSO of about 1% to about 20%, about 3% to about 9%, or about 6% to about 9% by volume. In certain embodiments, the final concentration of DMSO in the freezing solution is about 3%, about 4%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% by volume.

[0278] In certain embodiments, the cells are about 1 x 10 6 cells / ml ~ approx. 1 x 10 8 cells / mL, approximately 1 x 10 6 cells / mL ~ approx. 2 x 10 7 cells / mL, approximately 1 x 10 7 cells / mL ~ approx. 5 x 10 7 cells / mL, or approximately 1 x 10 7 cells / mL ~ 5 x 10 7 The cells are suspended in the freezing solution at a density of about 1 x 10 cells / mL. In certain embodiments, the cells are 6 cells / mL, approximately 2 x 10 6 cells / mL, approximately 5 x 10 6 cells / mL, approximately 1 x 10 7 cells / mL, approximately 1.5 x 10 7 cells / mL, approximately 2 x 10 7 cells / mL, approximately 2.5 x 10 7 cells / mL, approximately 2.5 x 10 7 cells / mL, approximately 2.5 x 10 7 cells / mL, approximately 3 x 10 7 cells / mL, approximately 3.5 x 10 7 cells / mL, approximately 4 x 10 7 cells / mL, approximately 4.5 x 10 7 cells / mL, or approximately 5 x 10 7 In certain embodiments, the cells are suspended in the freezing solution at a density of about 1.5 x 10 cells / mL. 7 cells / mL ~ approx. 6 x 10 7In certain embodiments, the cells are suspended in freezing solution at a density of about 5 x 10 cells / mL. 6 cells / mL ~ approx. 150 x 10 6 In certain embodiments, the cells are suspended in the freezing solution at a density of at least about 1 x 10 cells / mL. 7 In certain embodiments, the cells are suspended in the freezing solution at a density of at least about 1.5 x 10 cells / mL. 7 The cells are suspended in a freezing solution at a density of cells / mL. In some embodiments, the cells are viable cells.

[0279] In certain embodiments, the cells are 0.1 x 10, inclusive. 6 cells / mL ~ 5,000 x 10 6 cells / mL or approximately 0.1 x 10 6 cells / mL ~ approx. 5,000 x 10 6 cells / mL, 1 x 10 6 cells / mL ~ 500 x 10 6 cells / mL or approximately 1 x 10 6 cells / mL ~ approx. 500 x 10 6 cells / mL, 5 x 10 6 cells / mL ~ 150 x 10 6 cells / mL or approximately 5 x 10 6 cells / mL ~ approx. 150 x 10 6 cells / mL, 10 x 10 6 cells / mL ~ 70 x 10 6 cells / mL or approximately 10 x 10 6 cells / mL ~ approx. 70 x 10 6 cells / mL, or 15 x 10 6 cells / mL ~ 60 x 10 6 cells / mL or approximately 15 x 10 6 cells / mL ~ approx. 60 x 10 6 In certain embodiments, the cells are suspended in freezing solution at a density of about 1 x 10 cells / mL, inclusive. 6 cells / mL ~ approx. 1 x 10 8 cells / mL, approximately 1 x 10 6cells / mL ~ approx. 2 x 10 7 cells / mL, approximately 1 x 10 7 cells / mL ~ approx. 5 x 10 7 cells / mL, or approximately 1 x 10 7 cells / mL ~ 5 x 10 7 The cells are suspended in the freezing solution at a density of about 1 x 10 cells / mL. In certain embodiments, the cells are 6 cells / mL, approximately 2 x 10 6 cells / mL, approximately 5 x 10 6 cells / mL, approximately 1 x 10 7 cells / mL, approximately 1.5 x 10 7 cells / mL, approximately 2 x 10 7 cells / mL, approximately 2.5 x 10 7 cells / mL, approximately 2. 5×10 7 cells / mL, approximately 2.5 x 10 7 cells / mL, approximately 3 x 10 7 cells / mL, approximately 3.5 x 10 7 cells / mL, approximately 4 x 10 7 cells / mL, approximately 4.5 x 10 7 cells / mL, or approximately 5 x 10 7 In certain embodiments, the cells are suspended in freezing solution at a density of about 1.5 x 10 cells / mL, inclusive. 7 cells / mL ~ approx. 6 x 10 7 In certain embodiments, the cells are suspended in the freezing solution at a density of at least about 1 x 10 cells / mL. 7 In certain embodiments, the cells are suspended in the freezing solution at a density of at least about 1.5 x 10 cells / mL. 7 The cells are suspended in a freezing solution at a density of cells / mL. In some embodiments, the cells are viable cells.

[0280] In some embodiments, transfer to cryopreservation medium is accompanied by one or more processing steps, which may involve, for example, washing the sample, e.g., cells and / or manipulated cell compositions, to remove the medium and / or replacing the cells in an appropriate cryopreservation buffer or medium for subsequent freezing. In certain embodiments, transfer to cryopreservation medium is fully automated at a clinical scale in a closed and sterile system. In certain embodiments, transfer to cryopreservation medium was performed using a CliniMACS system (Miltenyi Biotec).

[0281] In some embodiments, the cells are frozen, e.g., cryogenically preserved, either before, during, or after the method for processing and / or manipulating the cells. In some embodiments, the freezing and subsequent thawing steps remove granulocytes, and to some extent monocytes, from the cell population. The cells may be frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. In some embodiments, the composition is packaged in a bag suitable for cryogenic storage (e.g., CryoMacs® Freezing Bags, Miltenyi Biotec). In some embodiments, the composition is packaged in a vial suitable for cryogenic storage (e.g., CellSeal® Vials, Cook Regentec).

[0282] Suitable containers include, for example, bottles, vials, syringes, and flexible bags, e.g., infusion bags. In certain embodiments, the container is a bag, e.g., a flexible bag, e.g., one suitable for infusion of cells into a subject, e.g., a flexible plastic or PVC bag, and / or an IV solution bag. The bag, in some embodiments, is sealable and / or sterilizable to provide a sterile solution and delivery of cells and compositions. In some embodiments, the container, e.g., bag, has a volume of, or about, or at least about: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 mL, e.g., a volume of 10 or about 10 to 100 or about 100 mL, or 10 or about 10 to 500 or about 500 mL, inclusive, respectively. In some embodiments, the container, e.g., bag, is and / or is made of a material that is stable and / or provides for stable storage and / or maintenance of the cells at various temperatures, e.g., low temperatures, e.g., temperatures below or at or about the following: -20°C, -80°C, -120°C, 135°C, and / or temperatures suitable for cryogenic storage, and / or other temperatures, e.g., temperatures suitable for thawing the cells and one or more of body temperature, e.g., 37°C or about 37°C, to allow thawing at the subject's location or treatment site, e.g., bedside, immediately prior to treatment.

[0283] The container can be formed from a variety of materials, such as glass or plastic. In some embodiments, the container is suitable for, e.g., connecting tubing or cannulating one or more tubes, e.g., for intravenous or other infusion, and / or or other container, e.g., cell culture and / or storage bag or other container, have one or more ports, e.g., sterile access ports, for connection for purposes of transfer to and from them. Exemplary containers include infusion bags, intravenous solution bags, and vials, including those having stoppers pierceable by an injection needle.

[0284] The present invention is not limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention; all functional equivalents are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and teachings and are likewise intended to be included within the scope of the invention. Such modifications and other embodiments can be made without departing from the true scope and spirit of the invention.

[0285] Irritant agents

[0286] In some embodiments, incubating the enriched cell composition under stimulatory conditions is or includes incubating and / or contacting the enriched cell composition with a stimulatory reagent capable of activating and / or expanding T cells. In some embodiments, the stimulatory reagent can stimulate and / or activate one or more signals in the cells. In some embodiments, the one or more signals are mediated by a receptor. In certain embodiments, the one or more signals are or are associated with changes in signal transduction and / or second messenger levels or amounts, e.g., cAMP and / or intracellular calcium levels or amounts, changes in the amount, subcellular localization, confirmation, phosphorylation, ubiquitination, and / or truncation of one or more intracellular proteins, and / or changes in cellular activity, e.g., transcription, translation, proteolysis, cytogenetics, activation state, and / or cell division. In certain embodiments, the stimulatory reagent activates and / or is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules.

[0287] In certain embodiments, the stimulatory reagent comprises one or more agents, e.g., particles, e.g., beads, conjugated or linked to biomolecules, that can activate and / or expand cells, e.g., T cells. In some embodiments, the one or more agents are bound to the beads. In some embodiments, the beads are composed of a material that is biocompatible, i.e., suitable for biological use. In some embodiments, the beads are non-toxic to cultured cells, e.g., cultured T cells. In some embodiments, the beads can be any particle that can bind to an agent in a manner that allows interaction between the agent and the cell.

[0288] In some embodiments, the stimulatory reagent contains one or more agents capable of activating and / or expanding cells, e.g., T cells, bound to or otherwise attached to the surface of the beads, e.g., beads. In certain embodiments, the beads are non-cellular particles. In certain embodiments, the beads may include colloidal particles, microspheres, nanoparticles, magnetic beads, etc. In some embodiments, the beads are agarose beads. In certain embodiments, the beads are sepharose beads.

[0289] In certain embodiments, the stimulating reagent contains monodisperse beads. In certain embodiments, monodisperse beads have a size distribution with diameters that have a standard deviation of less than 5% from one another. include.

[0290] In some embodiments, the beads comprise one or more agents, e.g., agents coupled, conjugated, or linked (directly or indirectly) to the surface of the beads. In some embodiments, agents contemplated herein may include, but are not limited to, RNA, DNA, proteins (e.g., enzymes), antigens, polyclonal antibodies, monoclonal antibodies, antibody fragments, carbohydrates, lipids, lectins, or any other biomolecules with affinity for a desired target. In some embodiments, the desired target is a T cell receptor and / or a component of a T cell receptor. In certain embodiments, the desired target is CD3. In certain embodiments, the desired target is a T cell costimulatory molecule, e.g., CD28, CD137 (4-1-BB), OX40, or ICOS. The one or more agents can be directly or indirectly bound to the beads by a variety of methods known and available in the art. Binding can be covalent, non-covalent, electrostatic, or hydrophobic and can be achieved by a variety of binding means, including, for example, chemical, mechanical, or enzymatic means. In some embodiments, a biomolecule (e.g., a biotinylated anti-CD3 antibody) can be indirectly bound to a bead via another biomolecule (e.g., an anti-biotin antibody) that is directly bound to the bead.

[0291] In some embodiments, the stimulatory reagent contains beads and one or more agents that directly interact with macromolecules on the surface of cells. In certain embodiments, the beads (e.g., paramagnetic beads) interact with cells via one or more agents (e.g., antibodies) specific to one or more macromolecules (e.g., one or more cell surface proteins) on the cells. In certain embodiments, the beads (e.g., paramagnetic beads) are labeled with a first agent described herein, such as a primary antibody (e.g., an anti-biotin antibody) or other biomolecule, and then a second agent, such as a secondary antibody (e.g., a biotinylated anti-CD3 antibody) or other biomolecule (e.g., streptavidin), is added, whereby the secondary antibody or other biomolecule specifically binds to the primary antibody or other biomolecule on the particle.

[0292] In some embodiments, the stimulatory reagent is coupled to beads (e.g., paramagnetic beads) and contains one or more agents (e.g., antibodies) that specifically bind to one or more of the following macromolecules on cells (e.g., T cells): CD2, CD3, CD4, CD5, CD8, CD25, CD27, CD28, CD29, CD31, CD44, CD45RA, CD45RO, CD54 (ICAM-1), CD127, MHC1, MHCII, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BB (CD137), 4-1BBL, CD30L, LIGHT, IL-2R, IL-12R, IL-1R, IL-15R; IFN-gamma R, TNF-alpha R, IL-4R, IL-10R, CD18 / CD1 Ia (LFA-1), CD62L (L-selectin), CD29 / CD49d (VLA-4), Notch ligands (e.g., Delta-like 1 / 4, Jagged 1 / 2, etc.), CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, and CXCR3, or fragments thereof (including ligands corresponding to these macromolecules or fragments thereof). In some embodiments, the agent (e.g., an antibody) bound to the bead specifically binds to one or more of the following macromolecules on a cell (e.g., a T cell): CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and / or CD45RO.

[0293] In some embodiments, one or more of the agents bound to the beads is an antibody, including polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies, and the like. In some embodiments, the stimulatory reagent is an antibody fragment (including an antigen-binding fragment), e.g., a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. It is understood that constant regions of any isotype may be used in the antibodies contemplated herein, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions may be derived from any human or animal species (e.g., It is understood that the beads can be obtained from various species, e.g., mouse species. In some embodiments, the agent is an antibody that binds to and / or recognizes one or more components of the T cell receptor. In certain embodiments, the agent is an anti-CD3 antibody. In certain embodiments, the agent is an antibody that binds to and / or recognizes a co-receptor. In some embodiments, the costimulatory reagent comprises an anti-CD28 antibody. In some embodiments, the beads are greater than about 0.001 μm, greater than about 0.01 μm, greater than about 0.1 μm, or greater than about 1 μm. In some embodiments, the beads have a diameter of about 1.0 μm to about 500 μm, about 1.0 μm to about 150 μm, about 1.0 μm to about 30 μm, about 1.0 μm to about 10 μm, about 1.0 μm to about 5.0 μm, about 2.0 μm to about 5.0 μm, or about 3.0 μm to about 5.0 μm. In some embodiments, the beads have a diameter of about 3 μm to about 5 μm. In some embodiments, the beads have a diameter of at least The beads have a diameter of at least, or at least about, the following: 0.001 μm, 0.01 μm, 0.1 μm, 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm, 2.5 μm, 3.0 μm, 3.5 μm, 4.0 μm, 4.5 μm, 5.0 μm, 5.5 μm, 6.0 μm, 6.5 μm, 7.0 μm, 7.5 μm, 8.0 μm, 8.5 μm, 9.0 μm, 9.5 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, or 20 μm. In certain embodiments, the beads have a diameter of 4.5 μm or about 4.5 μm.In certain embodiments, the beads have a diameter of 2.8 μm or about 2.8 μm.

[0294] In some embodiments, the beads have a density of 0.001 g / cm 3 Exceeding 0.01g / cm 3 Exceeds 0.05g / cm 3 Exceeding 0.1g / cm 3 Exceeds 0.5g / cm 3 Exceeding 0.6g / cm 3 Exceeding 0.7g / cm 3 Exceeding 0.8g / cm 3 Exceeding 0.9g / cm 3 Exceeds 1g / cm 3 Exceeding 1.1g / cm 3 Exceeding 1.2g / cm 3 Exceeding 1.3g / cm 3 Exceeding 1.4g / cm 3 Exceeds 1.5g / cm 3 Exceeds 2g / cm 3 Exceeds 3g / cm 3 Exceeding 4g / cm 3 or above 5g / cm 3 In some embodiments, the beads have a density greater than about 0.001 g / cm 3 ~About 100g / cm 3 , about 0.01g / cm 3 ~about 50g / cm 3 , about 0.1g / cm 3 ~Approx. 10g / cm 3 , about 0.1g / cm 3 ~Approx. .5g / cm 3 , about 0.5g / cm 3 ~Approx. 1g / cm 3 , about 0.5g / cm 3 ~Approx. 1.5g / cm 3 , about 1g / cm 3 ~Approx. 1.5g / cm 3 , about 1g / cm 3 ~About 2g / cm 3 , or about 1 g / cm 3 ~about 5g / cm 3In some embodiments, the beads have a density of about 0.5 g / cm 3 , about 0.5g / cm 3 , about 0.6g / cm 3 , about 0.7g / cm 3 , about 0.8g / cm 3 , about 0.9g / cm 3 , about 1.0g / cm 3 , about 1.1g / cm 3 , about 1.2g / cm 3 , about 1.3g / cm 3 , approximately 1.4 g / cm 3 , about 1.5g / cm 3 , about 1.6g / cm 3 , about 1.7g / cm 3 , about 1.8g / cm 3 , about 1.9g / cm 3 , or approximately 2.0 g / cm 3 In one particular embodiment, the beads have a density of about 1.6 g / cm 3 In certain embodiments, the beads or particles have a density of about 1.5 g / cm 3 In certain embodiments, the particles have a density of about 1.3 g / cm 3 It has a density of

[0295] In certain embodiments, the plurality of beads has a uniform density, hi certain embodiments, the uniform density comprises a density standard deviation of less than 10%, less than 5%, or less than 1% of the average bead density.

[0296] In some embodiments, the beads have a density of about 0.001 m per gram of particles. 2 (m 2 / g) ~ approx. 1,000m 2 / g, approx. .010m 2 / g~about 100m 2 / g, approx. 0.1m 2 / g~about 10m 2 / g, approx. 0.1m 2 / g ~ approx. 1m 2 / g, approx. 1m 2 / g~about 10m 2 / g, approx. 10m 2 / g~about 100m 2 / g, approx. 0.5m 2 / g~about 20m 2 / g, approx. 0.5m 2 / g~about 5m 2 / g, or approximately 1 m 2 / g ~ approx. 4m 2 In some embodiments, the particles or beads have a surface area of ​​about 1 m 2 / g ~ approx. 4m 2 / g of surface area.

[0297] In some embodiments, the beads comprise at least one material at or near their surface that can be coupled, linked, or conjugated to an agent. In some embodiments, the beads are surface-functionalized, i.e., comprise functional groups that can form covalent bonds with binding molecules, e.g., polynucleotides or polypeptides. In certain embodiments, the beads comprise surface-exposed carboxyl, amino, hydroxyl, tosyl, epoxy, and / or chloromethyl groups. In certain embodiments, the beads comprise surface-exposed agarose and / or sepharose. In certain embodiments, the surface of the beads comprises a bound stimulatory reagent that can bind or attach to a binding molecule. In certain embodiments, the biomolecule is a polypeptide. In some embodiments, the beads comprise surface-exposed protein A, protein G, or biotin.

[0298] In some embodiments, the beads respond to a magnetic field. In some embodiments, the beads are magnetic beads. In some embodiments, the magnetic beads are paramagnetic. In certain embodiments, the magnetic beads are superparamagnetic. In certain embodiments, the beads do not exhibit any magnetic properties unless exposed to a magnetic field.

[0299] In certain embodiments, the beads include a magnetic core, a paramagnetic core, or a superparamagnetic core. In some embodiments, the magnetic core contains a metal. In some embodiments, the metal may be, but is not limited to, iron, nickel, copper, cobalt, gadolinium, manganese, tantalum, zinc, zirconium, or any combination thereof. In certain embodiments, the magnetic core includes a metal oxide (e.g., iron oxide), a ferrite (e.g., manganese ferrite, cobalt ferrite, nickel ferrite, etc.), hematite, and a metal alloy (e.g., CoTaZn). In some embodiments, the magnetic core includes one or more of ferrite, a metal, a metal alloy, iron oxide, or chromium dioxide. In some embodiments, the magnetic core includes elemental iron or a compound thereof. In some embodiments, the magnetic core includes one or more of magnetite (FeO), maghemite (γFeO), or greigite (FeS). In some embodiments, the inner core includes iron oxide (e.g., FeO).

[0300] In certain embodiments, the beads contain a magnetic, paramagnetic, and / or superparamagnetic core coated with a surface-functionalized coat or coating. In some embodiments, the coat may contain a material that may include, but is not limited to, a polymer, a polysaccharide, silica, a fatty acid, a protein, carbon, agarose, sepharose, or a combination thereof. In some embodiments, the polymer may be polyethylene glycol, poly(lactic-co-glycolic acid), polyglutaraldehyde, polyurethane, polystyrene, or polyvinyl alcohol. In certain embodiments, the outer coat or coating comprises polystyrene. In certain embodiments, the outer coating is surface-functionalized.

[0301] In some embodiments, the stimulating reagent comprises a bead containing a metal oxide core (e.g., an iron oxide core) and a coat, wherein the metal oxide core comprises at least one polysaccharide (e.g., dextran) and the coat comprises at least one polysaccharide (e.g., amino nodextran), at least one polymer (e.g., polyurethane), and silica. In some embodiments, the metal oxide core is a colloidal iron oxide core. In certain embodiments, the one or more agents comprise an antibody or antigen-binding fragment thereof. In certain embodiments, the one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the stimulatory reagent comprises an anti-CD3 antibody, an anti-CD28 antibody, and an anti-biotin antibody. In some embodiments, the stimulatory reagent comprises an anti-biotin antibody. In some embodiments, the beads have a diameter of about 3 μm to about 10 μm. In some embodiments, the beads have a diameter of about 3 μm to about 5 μm. In certain embodiments, the beads have a diameter of about 3.5 μm.

[0302] In some embodiments, the stimulatory reagent comprises one or more agents bound to beads comprising a metal oxide core (e.g., an iron oxide inner core) and a coat (e.g., a protective coat), where the coat comprises polystyrene. In certain embodiments, the beads are monodisperse paramagnetic (e.g., superparamagnetic) beads comprising a paramagnetic (e.g., superparamagnetic) iron core, e.g., a core comprising magnetite (Fe3O4) and / or maghemite (γFe2O3)c, and a polystyrene coat or coating. In some embodiments, the beads are non-porous. In some embodiments, the beads comprise a functionalized surface to which one or more agents are attached. In certain embodiments, the one or more agents are covalently bound to the beads at the surface. In some embodiments, the one or more agents comprise an antibody or antigen-binding fragment thereof. In some embodiments, the one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the one or more agents include an anti-CD3 antibody and / or an anti-CD28 antibody, and a labeled antibody (e.g., a biotinylated antibody), e.g., an antibody or antigen fragment thereof that can bind to the labeled anti-CD3 antibody or anti-CD28 antibody. In certain embodiments, the beads have a mass of about 1.5 g / cm 3 density and approximately 1 m 2 / g ~ approx. 4m 2 In certain embodiments, the beads have a diameter of about 4.5 μm and a surface area of ​​about 1.5 g / cm 3 In some embodiments, the beads are monodisperse superparamagnetic beads having an average diameter of about 2.8 μm and a density of about 1.3 g / cm 3 The beads are monodisperse superparamagnetic beads with a density of 0.015 μm.

[0303] In some embodiments, the enriched T cell composition is incubated with a stimulatory reagent at a bead to cell ratio at or about the following ratios: 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1, or 0.2:1. In certain embodiments, the bead to cell ratio is between 2.5:1 and 0.2:1, 2:1 and 0.5:1, 1.5:1 and 0.75:1, 1.25:1 and 0.8:1, or 1.1:1 and 0.9:1. In certain embodiments, the stimulatory reagent to cell ratio is about or is 1:1.

[0304] Removal of irritating agents from cells

[0305] In certain embodiments, the stimulatory reagent is removed and / or separated from the cells. Without wishing to be bound by theory, in certain embodiments, it is contemplated that binding and / or association between the stimulatory reagent and the cells may, in some circumstances, be reduced over time during incubation. In certain embodiments, one or more agents may be added to reduce binding and / or association between the stimulatory reagent and the cells. In certain embodiments, changes in cell culture conditions, e.g., temperature or pH of the medium, may reduce binding and / or association between the stimulatory reagent and the cells. Thus, in some embodiments, the stimulatory reagent may similarly be removed and / or separated from the cells during incubation, e.g., without removing the cells from the incubation, cell culture system, and / or solution. The composition may be removed from the application, cell culture system, and / or solution.

[0306] Methods for removing stimulatory reagents (e.g., stimulatory reagents that are or include particles, such as bead particles or magnetizable particles) from cells are known. In some embodiments, for example, the use of a competing antibody, e.g., an unlabeled antibody, can be used, which binds to the primary antibody of the stimulatory reagent and alters its affinity for its antigen on the cell, thereby allowing for slow desorption. In some cases, after desorption, the competing antibody can remain associated with the particle (e.g., bead particle), while unreacted antibody can be washed away or washed away, and the cells are free of the isolated, selected, enriched, and / or activated antibody. An exemplary such reagent is DETACaBEAD (Friedl et al., 1995; Entschladen et al., 1997). In some embodiments, particles (e.g., bead particles) can be removed in the presence of a cleavable linker (e.g., a DNA linker), whereby the particle-bound antibody is conjugated to the linker (e.g., CELLection, Dynal). In some cases, the linker region provides a cleavable site for removing the particle (e.g., bead particle) from the cell after isolation, for example, by addition of DNase or other detachment buffer. In some embodiments, other enzymatic methods can also be used to detach the particle (e.g., bead particle) from the cell. In some embodiments, the particle (e.g., bead particle or magnetizable particle) is biodegradable.

[0307] In some embodiments, the stimulatory reagent is magnetic, paramagnetic, and / or superparamagnetic, and / or comprises magnetic, paramagnetic, and / or superparamagnetic beads, and the stimulatory reagent can be removed from the cells by exposing the cells to a magnetic field. An example of a suitable instrument that includes a magnet to generate a magnetic field is the DynaMag CTS (Thermo Fisher), Magnetic Separator (Takara), and EasySepMagnet (Stem Cell Technologies).

[0308] In certain embodiments, the stimulatory reagent is removed or separated from the cells prior to harvesting, collecting, and / or formulating the engineered cells produced by the methods provided herein. In some embodiments, the stimulatory reagent is removed and / or separated from the cells prior to manipulating, e.g., transducing or transfecting, the cells. In certain embodiments, the stimulatory reagent is removed and / or separated from the cells after manipulating the cells. In certain embodiments, the stimulatory reagent is removed prior to culturing the cells, e.g., culturing engineered, e.g., transfected or transduced, cells under conditions that promote growth and / or expansion. [Example]

[0309] To evaluate the effect of cryogenic storage of apheresis prior to selection or isolation of a desired cell population, apheresis samples were obtained from various steps of a process designed to produce engineered T cells. Samples were evaluated at various time points for cell viability, cell yield, cell phenotype, and cell activity. These studies were designed to determine whether cryogenic storage of apheresis material affected (1) the phenotypic ratios of relevant CD4+ and CD8+ T cell populations, (2) the ability to sort and select relevant T cell populations after thawing, and / or (3) the health and / or functionality of the cells.

[0310] In the following examples, apheresis refers to apheresis collected from a donor. Cryopreserved apheresis refers to a cell product obtained by cryopreservation of an apheresis sample after collection but prior to selection of any cell populations of interest within the sample. Apheresis refers to a cell product obtained by thawing cryopreserved apheresis and then allowing it to rest for a predetermined period of time before any further processing steps. Cryopreserved selected material refers to a cell product obtained by isolating the cells of interest (in these examples, CD4+ T cells and CD8+ T cells) and then subjecting them to a post-isolation cryopreservation step.

[0311] Example 1 Process for producing therapeutic compositions of CD4+ and CD8+ cells expressing anti-CD19 CAR

[0312] Engineered CD4+ T cells and engineered CD8+ T cells, each expressing the same anti-CD19 chimeric antigen receptor (CAR), were produced by the process outlined herein. As described in Example 2 below, the cells were produced by a process in which separate compositions of CD4+ and CD8+ cells were selected from PBMCs isolated from human leukapheresis samples and cryogenically frozen. The selected CD4+ and CD8+ compositions were then thawed and separately subjected to stimulation, transduction, and expansion steps. A second exemplary process included an additional cryogenic storage step before the selection step.

[0313] Isolated CD4+ and CD8+ cells were separately stimulated in the presence of paramagnetic polystyrene-coated beads conjugated with anti-CD3 and anti-CD28 antibodies at a 1:1 bead-to-cell ratio. Cells were stimulated in a medium containing IL-2, IL-15, and N-acetylcysteine ​​(NAC). The CD4+ cell medium also contained IL-7.

[0314] After bead transduction, CD4+ and CD8+ cells were separately transduced with lentiviral vectors encoding the same anti-CD19 CAR, which contained an anti-CD19 scFv derived from a mouse antibody, an immunoglobulin spacer, a transmembrane domain derived from CD28, a costimulatory region derived from 4-1BB, and a CD3-zeta intracellular signaling domain.

[0315] After transduction, the beads were removed from the cell composition by exposure to a magnetic field. The CD4+ and CD8+ cells were then cultured separately for expansion with continuous mixing and oxygen transfer in a bioreactor (Xuri W25 Bioreactor). Poloxamer was added to the medium. Both cell compositions were cultured in the presence of IL-2 and IL-15. The CD4+ cell medium also contained IL-7. The CD4+ and CD8+ cells were cultured to the desired cell number and / or concentration, respectively, before harvesting. One day after reaching the threshold, cells were harvested separately from each composition, formulated, and cryogenically frozen.

[0316] A controlled rate freezing device utilizing a step-wise freezing profile was used for the cryopreservation steps described in the examples below.

[0317] Example 2 Study design

[0318] Two healthy donors (i.e., Donor 1 and Donor 2) were used in this study, and the initial apheresis (APH) material was divided into five different arms for each donor. One-fifth of the initial apheresis volume (control arm or arms 5 and 10) was washed and subjected to an isolation step to isolate CD4+ T cells and CD8+ T cells. At this point, the selected cells were cryopreserved for two weeks. The remaining apheresis from each donor was divided into four samples and then cryopreserved (Arms 1-4 and 6-9). Each cryopreserved sample was either thawed, washed, and placed at 37°C for two hours before selection, or subjected to the selection step immediately after thawing and washing. Half of the arms were frozen after selection, and the other half were proceeded directly to activation.

[0319] Samples in arms 1, 2, 6, and 7 were cryogenically stored for two weeks before thawing the cells, isolating CD4+ and CD8+ T cell populations, and subjecting them to the cell activation method. Arms 1 and 6 included an additional step in which the cells were allowed to rest in an incubator for two hours after thawing before any further processing. Samples in arms 3, 4, 8, and 9 were cryogenically stored for two to four days before thawing and subjecting them to selection of CD4+ and CD8+ T cell populations, at which point the selected populations were cryogenically stored for one week before thawing the cells and subsequently subjecting them to stimulation. Arms 3 and 8 included an additional step in which the cells were allowed to rest in an incubator for two hours after thawing before any further processing.

[0320] The cells underwent various processing steps, including a selection step to isolate CD4+ T cells and CD8+ T cells. During this selection step, each arm was divided into sub-arms (i.e., CD4+ T cell and CD8+ T cell sub-arms), at which point the selected cells proceeded to the remaining processing steps. Table 1 shows the study design, including the cryopreservation steps each arm underwent.

[0321] [Table 1]

[0322] Example 3 Cryogenic storage of apheresis material does not significantly affect cell phenotype

[0323] To assess the effect of freezing on the distribution of cells with different phenotypes, flow analysis was performed on apheresis samples before and after cryopreservation. A custom flow panel was developed to assess the phenotypic distribution of T cells, B cells, NK cells, NK-T cells, monocytes, dendritic cells, and memory T cells. Results suggest that the distribution of cells with different phenotypes was comparable between pre- and post-cryopreservation samples.

[0324] Both cryopreserved and fresh apheresis samples were analyzed for the presence of CD4 and CD8 molecules on the cell surface using flow cytometry. The results of this assay showed that the levels of surface CD4 and CD8 molecules were unaffected by cryopreservation. These results also suggest that cryopreservation of apheresis samples did not affect the relative proportions of CD4+ and CD8+ T cells in the samples, as the percentages of these cells were similar for both donors before and after cryopreservation.

[0325] Example 4 The effect of cryopreservation on the isolation of CD4+ and / or CD8+ T cell populations

[0326] Furthermore, to assess whether cryopreservation of apheresis affects the processing of CD4+ and CD8+ T cells, viability assays were performed at various steps leading up to the selection of the desired cells. Cell viability was assessed at various steps of the process for cells subjected to cryopreservation before the selection step without a post-cryopreservation rest period (arms 2, 4, 7, and 9), cells subjected to cryopreservation before the selection step with a post-cryopreservation rest period (arms 1, 3, 6, and 8), and cells subjected to cryopreservation after the isolation step (arms 5 and 10, or the control arm). Specifically, viability was assessed after apheresis collection, formulation of the apheresis for cryogenic storage, cryogenic storage of the apheresis for a designated period, thawing and dilution, washing of the thawed and diluted apheresis, placing the washed apheresis in an incubator for 2 hours, adding antibody-coated beads to the sample, and isolating CD8+ and / or CD4+ T cells. Cell viability values ​​across all arms were comparable to those in the control arm at each processing step.

[0327] Total nucleated cell counts (TNC) were also determined for all samples during various steps leading to the isolation of CD4+ T cells and / or CD8+ T cells. Cell loss was found to occur predominantly during the formulation step. Cell yield ratios, obtained by normalizing post-isolation cell count values ​​to pre-isolation cell count values, indicated that cell loss in cryopreserved apheresis samples occurred before the isolation of CD4+ and CD8+ T cell populations, and that cell yields at each step within the isolation process were unaffected. However, in this experiment, the final TNC values ​​corresponding to selected cells were found to vary slightly between the cryopreserved apheresis and control arms for each cell type for each donor, likely due to cell loss occurring before isolation. However, the CD4+ T cell yields for a single donor were found to be comparable between the cryopreserved apheresis and control arms.

[0328] Example 5 Assessment of cell phenotype and viability after isolation and freezing steps

[0329] After the isolation step, cells that required a cryopreservation step (arms 3, 4, 5, 8, 9, and 10) were cryopreserved and then thawed for further analysis. Cells from arms 3, 4, 8, and 9 were cryopreserved for 1.5–2 weeks before being thawed for further analysis and processing. Cells from arms 5 and 10 (or the control arm) were cryopreserved for 2 weeks before being thawed for further analysis and processing. At this point, all isolated T cell populations from all arms of each donor were assayed before any cell activation steps. The TMEM assay assessed the presence of various T cell markers across selected cell populations from different arms of each donor. Cell phenotype distribution (based on detection of selected markers) did not significantly differ between the cryopreserved apheresis arm and the control arm for each cell type for each donor. Cells from the arm that did not undergo a post-isolation freezing step showed a strong tendency toward naive-like cells (CD45RA+ / CCR7+, CD27+ / CD28+) and fewer final effector cells (CD45RA+, CCR7-). CD62L expression was slightly reduced in samples that were subjected to a post-isolation freezing step.

[0330] Cell viability was assessed for all arms of each cell type derived from each donor prior to cell activation. Cell viability did not differ significantly between the cryopreserved apheresis arms and the control arms of each cell type derived from each donor. In addition, to assess the effect of a post-isolation freezing step, a post-isolation freezing step was performed. Cell yield ratios were obtained by normalizing the cell numbers obtained after isolation to the cell numbers obtained immediately after isolation, before freezing. Cell yield ratios were similar between the cryopreserved apheresis arm and the control arm.

[0331] Caspase 3 levels were found to be low (less than or about 5%) across arms.

[0332] Example 6 Assessment of cell viability and cell yield during activation, transduction, and expansion

[0333] As previously discussed, after the isolation step, the study arm that required a post-isolation freezing step was cryogenically stored for a predetermined period of time, then thawed and subsequently subjected to the activation, transduction, and expansion steps. Cell viability and TNC values ​​were determined after thawing the cryopreserved material, stimulating the thawed material in the presence of paramagnetic polystyrene-coated beads conjugated with anti-CD3 and anti-CD28 antibodies, transducing the activated cells, removing the beads from the cells, and expanding the cells for 2 or 3 days. Cell viability was found to be comparable between the cryopreserved apheresis arm and the control arm for each cell type for each donor. During the stimulation step, the cell viability values ​​for the cryopreserved apheresis arm showed a difference of less than 20% compared with the corresponding control arm. This percentage difference was less than 10% for all other steps. Additionally, the TNC values ​​obtained during each of these steps were comparable between the cryopreserved apheresis arms and their corresponding control arms. Furthermore, the fold increase calculated at each step was also found to be comparable between the cryopreserved apheresis arms and their corresponding control arms.

[0334] These results showed that in this experiment, apheresis samples cryopreserved without settling or an additional cryopreservation step immediately after isolation, and apheresis samples cryopreserved with a settling step but without an additional cryopreservation step immediately after isolation, had similar or higher final cell yields compared to their corresponding control arms.

[0335] Example 7 Assessment of cell viability, cell yield, and cell activity during formulation of cryopreserved compositions

[0336] Cells from each arm obtained after the expansion step were formulated in cryopreservation medium and frozen. Samples were then thawed for further analysis. Cell viability and cell yield values ​​were determined for cells in all arms of the study at this step. The average viability and cell yield were comparable between the cryopreserved apheresis arms and their corresponding control arms at this step.

[0337] Additionally, assays were performed to assess the cell phenotype distribution in all arms. Cell phenotypes were found to be statistically equivalent between the cryopreserved apheresis arms and their corresponding control arms. The arms that did not undergo a post-isolation freezing step tended to contain higher percentages of CD45RA+ / CCR7+ and CD27+ / CD28+ cells, and fewer CD45RA+ and CCR7- cells. Additionally, in this experiment, caspase 3 levels were found to be slightly higher in the CD8+ T cell arms compared with the CD4+ T cell arms, and the arms that included a post-isolation freezing step exhibited higher caspase levels than the arms that did not.

[0338] Interferon gamma (IFNγ) secretion was used to assess T cell functionality after treatment. T cells from each arm were stimulated to produce IFNγ. After stimulation, supernatants were collected and the secreted IFNγ in the supernatants was measured. For all experimental conditions, values ​​were consistent with those of their corresponding controls, indicating that the cellular activity of the final cell product was not affected by the initial cryopreservation step.

[0339] In addition, a cytolytic assay was also performed to evaluate the cytolytic activity of the generated CD8+ T cells. Cytolytic activity was measured at various effector cell:target cell ratios to determine the EC50 (the ratio required to kill 50% of the target cells). The fold difference in cytolytic EC50 of the cryopreserved apheresis arms compared to their corresponding control arms was found to be less than two-fold, suggesting that the different arm conditions did not significantly alter the cytolytic EC50 of the resulting cells. Illustrative Embodiments

[0340] 1. A method comprising cryogenically storing cells from a biological sample derived from a donor, wherein the cells were obtained from the donor (i) after the donor has been diagnosed with a disease or condition and before the donor has received one or more of the following: any initial treatment for the disease or condition, any targeted treatment for or directed at the treatment of the disease or condition, or any treatment other than radiation and / or chemotherapy; (ii) after the donor's first recurrence of the disease or condition after initial treatment for the disease or condition and before the donor has received any treatment after the recurrence of the disease or condition; or (iii) when the donor has not been diagnosed with or known to have or is not suspected of having the disease or condition.

[0341] 2. The method of embodiment 1, wherein the biological sample is or is derived from a donor blood sample.

[0342] 3. The method of embodiment 1 or embodiment 2, wherein the cells have not been subjected to a selection step for and / or enriched for blood cell populations and / or T cell populations and / or T cell subsets before being cryogenically stored.

[0343] 4. The method of embodiment 1 or embodiment 2, wherein the cells have been subjected to a selection step and / or enrichment of blood cell and / or T cell populations prior to cryogenic storage, and optionally further comprising selecting or enriching cell populations from the biological sample prior to said cryogenic storage.

[0344] 5. The method of embodiment 4, wherein the selection step and / or enrichment comprises immunoaffinity-based selection and / or comprises positive or negative selection.

[0345] 6. Selection Step and / or Enrichment for CD4 + and / or enrichment and / or isolation of CD4 cells or subsets thereof and / or CD8+ cells or subsets thereof. + Enrichment or isolation of cells or subsets thereof is performed using CD8 + The method may be performed either separately or in combination with the selection and / or isolation of cells or subsets thereof, optionally including the selection and / or isolation of CD8 + Cell subsets and / or CD4 + A subset of cells, when needed, can be identified as memory cells, central memory T (T CM ) cells, effector memory cells (T EM ), Stem Central Memory (T SCM ) cells, effector T (T E ) cells, effector memory RA T(T EMRA ) cells, naive T (T N ) cells, and / or regulatory T(T REG 6. The method of any one of embodiments 2 to 5, wherein the cell is selected from the group consisting of:

[0346] 7. The method of any one of embodiments 1 to 6, wherein the cells comprise T cells or are enriched for T cells.

[0347] 8. T cells are CD4 + T cells or their subsets, CD8 +containing or enriched in T cells or a subset thereof, or a mixture thereof, and + Cell subsets and / or CD4 + A subset of cells, when needed, can be identified as memory cells, central memory T (T CM ) cells, effector memory cells (T EM ), Stem Central Memory (T SCM ) cells, effector T (T E ) cells, effector memory RA T(T EMRA ) cells, naive T (T N ) cells, and / or regulatory T (T REG 8. The method of embodiment 7, wherein the cell is selected from the group consisting of:

[0348] 9. The method of any one of embodiments 1 to 8, further comprising cooling the cells to a temperature below or equal to 0°C before cryogenically storing the cells.

[0349] 10. The method of embodiment 8, further comprising combining the cells with a freezing solution before storing and / or cooling the cells.

[0350] 11. The method of embodiment 10, wherein the freezing solution comprises about 10% dimethyl sulfoxide (DMSO) and serum proteins, optionally human serum albumin, optionally about 4% human serum albumin, and / or the freezing solution, and / or the composition in which the cells are cryopreserved and stored at a final concentration, comprises about 1% to about 20%, about 3% to about 9%, or about 6% to about 9% DMSO by volume, and / or comprises about 3%, about 4%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% DMSO by volume.

[0351] 12. The method of any one of embodiments 9-11, wherein cooling the cells comprises decreasing the temperature at a rate of 1°C or about 1°C per minute, as needed, until the temperature reaches -80°C or about -80°C.

[0352] 13. The method of any one of embodiments 1 to 11, wherein the cells are cryogenically stored in a container in the vapor phase of liquid nitrogen, the container optionally being a bag or vial suitable for cryogenic storage.

[0353] 14. The method of any one of embodiments 1-13, wherein the cells are cryogenically stored for a period of more than or equal to the following: 12 hours, 24 hours, 36 hours, 48 ​​hours, 1 week, 2 weeks, 3 weeks, or 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, or 40 years.

[0354] 15. The method of any of embodiments 1-14, wherein the cells are stored for a period of time, after which the percentage of viable cells or viable T cells or subtypes or subsets thereof in the composition is between about 24% and about 100%, or is at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90%.

[0355] 16. The method of any one of embodiments 1-15, wherein the disease is cancer, an inflammatory disease or condition, an autoimmune disease or condition, or an infectious disease or condition.

[0356] 17. The method of embodiment 16, wherein the cancer is chronic lymphocytic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia, null acute lymphoblastic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, multiple myeloma, follicular lymphoma, splenic marginal zone lymphoma, mantle cell lymphoma, late-onset B-cell lymphoma, or acute myeloid leukemia.

[0357] 18. The cancer expresses ROR1, EGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y , L1-cell adhesion molecule, MAGE-A1, MUC1, MUC16, B-cell maturation antigen (BCMA), FCRL5 / FCRH5, GPRC5D, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, oncofetal antigen, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms' tumor 1 (WT-1), and a cyclin, e.g., cyclin A1 (CCNA1).

[0358] 19. The method according to any one of embodiments 1 to 18, wherein the initial or subsequent treatment is chemotherapy, radiation, and / or surgery, and / or a debulking treatment.

[0359] 20. The method of embodiment 19, wherein the initial treatment or subsequent treatment is or comprises a combination of chemotherapy.

[0360] 21. The method of any one of embodiments 1 to 20, wherein the donor is a human.

[0361] 22. The method of any one of embodiments 1 to 21, further comprising analyzing the cells prior to cryogenic storage, optionally by assessing the surface expression of the cells for one or more phenotypic markers.

[0362] 23. The method of any one of embodiments 1 to 21, further comprising thawing cryogenically stored cells.

[0363] 24. The method of embodiment 23, further comprising performing post-cryogenic modification to increase the activity of the cells.

[0364] 25. The method of embodiment 24, wherein the post-cryogenic modification is based on analyzing the cells before cryogenic storage.

[0365] 26. An embodiment further comprising, after cryogenic storage and / or thawing of the cells, engineering the cells to express a recombinant or exogenous molecule, which optionally is a recombinant protein, optionally a recombinant receptor, which optionally is or includes a T cell receptor (TCR), a chimeric receptor, and / or a chimeric antigen receptor. 26. The method according to any one of aspects 23 to 25.

[0366] 27. The method of embodiment 26, wherein the recombinant molecule is a recombinant receptor that specifically recognizes or specifically binds to an antigen expressed by, specifically expressed by, or associated with the disease or condition.

[0367] 28. The method of any one of embodiments 1 to 26, wherein the number of cells at or about the time of collection from the donor and / or in the total apheresis sample is, or does not exceed, 500 x 10 6 pieces, 1000×10 6pieces, 2000×10 6 pieces, 3000×10 6 pieces, 4000×10 6 pieces, or 5000 x 10 6 or more total cells or total nucleated cells.

[0368] 29. A method for processing an apheresis sample, comprising: (a) transporting an apheresis sample obtained from a donor to a storage facility in a cryogenic environment; and (b) optionally storing the apheresis sample at cryogenic temperatures in the storage facility.

[0369] 30. The method of embodiment 29, further comprising enriching T cells from the apheresis sample before transporting the sample and / or storing the sample at cryogenic temperatures.

[0370] 31. T cells are CD4 + T cells or their subsets, CD8 + T cells or a subset thereof, or a mixture thereof, or comprising or enriched in T cells, and optionally + Cell subsets and / or CD4 + A subset of cells, when needed, can be identified as memory cells, central memory T (T CM ) cells, effector memory cells (T EM ), Stem Central Memory (T SCM ) cells, effector T (T E ) cells, effector memory RA T(T EMRA ) cells, naive T (T N ) cells, and / or regulatory T (T REG 31. The method of embodiment 30, wherein the sample is selected from the group consisting of: ) cells, and / or the sample is enriched for bulk T cells.

[0371] 32. The method of any one of embodiments 29-31, further comprising analyzing the apheresis sample before transporting.

[0372] 33. The method of any one of embodiments 29-32, further comprising adding a freezing solution to the apheresis sample before transport.

[0373] 34. The method of embodiment 32, further comprising adding a freezing solution to the apheresis sample prior to transport, wherein the freezing solution is selected based on analyzing the apheresis sample prior to transport.

[0374] 35. The method of any one of embodiments 29-34, further comprising cryogenically freezing the apheresis sample prior to transport.

[0375] 36. The method of embodiment 35, further comprising enriching T cells from the apheresis sample after transport and before cryogenic storage of the cells.

[0376] 37. T cells are CD4 + T cells or their subsets, CD8 + T cells or a subset thereof, or a mixture thereof, or comprising or enriched in T cells, and optionally + Cell subsets and / or CD4 + A subset of cells, when needed, can be identified as memory cells, central memory T (T CM )cell, Effector memory cells (T EM ), Stem Central Memory (T SCM ) cells, effector T (T E ) cells, effector memory RA T(T EMRA ) cells, naive T (T N ) cells, and / or regulatory T (T REG 37. The method of embodiment 36, wherein the T cells are selected from the group consisting of: ) cells, and / or bulk T cells.

[0377] 38. The method of any one of embodiments 36-37, further comprising analyzing the apheresis sample or T cells after transport and before cryogenic storage of the cells.

[0378] 39. The method of any one of embodiments 36 to 38, further comprising adding a freezing solution to the apheresis sample or T cells after transport and before cryogenic storage of the cells.

[0379] 40. The method of embodiment 38, further comprising adding a freezing solution to the apheresis sample or T cells after transport and before cryogenic storage of the cells, wherein the freezing solution is optionally selected based on analyzing the apheresis sample or T cells after transport and before cryogenic storage of the cells.

[0380] 41. The method of any one of embodiments 29-40, further comprising thawing cryogenically stored cells.

[0381] 42. The method of embodiment 41, further comprising analyzing the cells after thawing.

[0382] 43. The method of embodiment 42, further comprising selecting conditions for further modification of the cells based on analysis after thawing.

[0383] 44. A method of treatment comprising obtaining and optionally thawing a sample of cryogenically frozen cells, optionally including T cells, from a subject, wherein prior to said obtaining, the cells have been cryogenically frozen for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years; modifying the cells to express a recombinant antigen receptor; and administering the cells to the subject.

[0384] 45. The method of embodiment 44, wherein the sample has been frozen and / or stored according to the method of any of embodiments 1 to 43. Further Exemplary Embodiment I

[0385] 1. A method for producing a composition of engineered cells, comprising: (a) incubating an input composition comprising T cells enriched for CD4+ primary human T cells under stimulatory conditions comprising the presence of (i) a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules, and (ii) one or more cytokines, thereby producing a stimulated composition; and (b) introducing a recombinant receptor into the stimulated composition, thereby producing an engineered composition comprising the engineered T cells, wherein the input composition is, or is derived from, a sample that has been cryogenically stored for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

[0386] 2. The method of embodiment 1, wherein the stimulatory reagent comprises a primary agent that specifically binds to a member of the TCR complex, and optionally specifically binds to CD3.

[0387] 3. The method of embodiment 2, wherein the stimulatory reagent further comprises a secondary agent that specifically binds to a T cell costimulatory molecule, optionally wherein the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS.

[0388] 4. The method of embodiment 2 or embodiment 3, wherein the primary and / or secondary agents comprise antibodies, and optionally the stimulatory reagent comprises incubation with an anti-CD3 antibody and an anti-CD28 antibody or antigen-binding fragment thereof.

[0389] 5. The method of any one of embodiments 2-4, wherein the primary agent and / or the secondary agent are present on the surface of a solid support.

[0390] 6. The method of embodiment 5, wherein the solid support is or comprises beads.

[0391] 7. The method of embodiment 6, wherein the beads comprise a diameter greater than or greater than about 3.5 μm, but not greater than about 9 μm, or not greater than about 8 μm, or not greater than about 7 μm, or not greater than about 6 μm, or not greater than about 5 μm.

[0392] 8. The method of embodiment 6 or embodiment 7, wherein the beads comprise a diameter of 4.5 μm or about 4.5 μm.

[0393] 9. The method of any one of embodiments 6 to 8, wherein the beads are inert.

[0394] 10. The method of any one of embodiments 6 to 9, wherein the beads are or comprise a polystyrene surface.

[0395] 11. The method of any one of embodiments 6 to 10, wherein the beads are magnetic or superparamagnetic.

[0396] 12. The method of any one of embodiments 6 to 11, wherein the ratio of beads to cells is less than 3:1.

[0397] 13. The method of any one of embodiments 6 to 12, wherein the ratio of beads to cells is 2:1 or about 2:1 to 0.5:1.

[0398] 14. The method of any one of embodiments 6 to 13, wherein the ratio of beads to cells is 1:1 or about 1:1.

[0399] 15. The method of any one of embodiments 1 to 14, wherein introducing comprises transducing the cells of the stimulated composition with a viral vector comprising a polynucleotide encoding the recombinant receptor.

[0400] 16. The method of embodiment 15, wherein the viral vector is a retroviral vector.

[0401] 17. The method of embodiment 15 or embodiment 16, wherein the viral vector is a lentiviral vector or a gammaretroviral vector.

[0402] 18. The method of any one of embodiments 15-17, wherein the introducing is carried out in the presence of a transduction adjuvant.

[0403] 19. The method of any one of embodiments 1 to 18, wherein introducing comprises transfecting the cells of the stimulated composition with a vector comprising a polynucleotide encoding the recombinant receptor.

[0404] 20. The method of embodiment 19, wherein the vector is a transposon, optionally a Sleeping Beauty (SB) transposon or a piggyBac transposon.

[0405] 21. The method of any one of embodiments 1-20, further comprising culturing the engineered composition under conditions that promote the growth or expansion of the engineered cells, thereby producing an output composition comprising the engineered T cells.

[0406] 22. The method of embodiment 21, wherein the stimulatory agent is removed from the operating composition before culturing.

[0407] 23. The method of embodiment 22, wherein removing the beads comprises exposing the cells of the manipulating composition to a magnetic field.

[0408] 24. The method of any one of embodiments 21-23, wherein at least a portion of the culturing is performed by continuous mixing and / or perfusion.

[0409] 25. A method for producing an engineered cell composition, comprising: (a) incubating an input composition comprising primary T cells, enriched for one or both of CD4+ and CD8+ primary human T cells, under stimulatory conditions comprising the presence of (i) a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules, and (ii) one or more cytokines, thereby producing a stimulated composition; and (b) introducing a recombinant receptor into the stimulated composition, thereby producing an engineered composition comprising the engineered T cells, wherein the input composition is, or is derived from, a sample that has been cryogenically stored for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

[0410] 26. The method of embodiment 24 or embodiment 35, wherein the stimulatory reagent comprises a primary agent that specifically binds to a member of the TCR complex, and optionally specifically binds to CD3.

[0411] 27. The method of embodiment 26, wherein the stimulatory reagent further comprises a secondary agent that specifically binds to a T cell costimulatory molecule, optionally wherein the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS.

[0412] 28. The method of embodiment 26 or embodiment 27, wherein the primary and / or secondary agents comprise antibodies, and optionally the stimulatory reagent comprises incubation with an anti-CD3 antibody and an anti-CD28 antibody or antigen-binding fragment thereof.

[0413] 29. The method of any one of embodiments 26-28, wherein the primary agent and / or the secondary agent are present on the surface of a solid support.

[0414] 30. The method of embodiment 29, wherein the solid support is or comprises beads. Law.

[0415] 31. The method of embodiment 30, wherein the beads comprise a diameter greater than or greater than about 3.5 μm, but not greater than about 9 μm, or not greater than about 8 μm, or not greater than about 7 μm, or not greater than about 6 μm, or not greater than about 5 μm.

[0416] 32. The method of embodiment 30 or embodiment 31, wherein the beads have a diameter of 4.5 μm or about 4.5 μm.

[0417] 33. The method of any one of embodiments 30-32, wherein the beads are inert.

[0418] 34. The method of any one of embodiments 30-33, wherein the beads are or comprise a polystyrene surface.

[0419] 35. The method of any one of embodiments 30 to 34, wherein the beads are magnetic or superparamagnetic.

[0420] 36. The method of any one of embodiments 30-35, wherein the ratio of beads to cells is less than 3:1.

[0421] 37. The method of any one of embodiments 30 to 36, wherein the ratio of beads to cells is 2:1 or about 2:1 to 0.5:1.

[0422] 38. The method of any one of embodiments 30-37, wherein the ratio of beads to cells is 1:1 or about 1:1.

[0423] 39. The method of any one of embodiments 24 to 38, wherein introducing comprises transducing the cells of the stimulated composition with a viral vector comprising a polynucleotide encoding the recombinant receptor.

[0424] 40. The method of embodiment 39, wherein the viral vector is a retroviral vector.

[0425] 41. The method of embodiment 39 or embodiment 40, wherein the viral vector is a lentiviral vector or a gammaretroviral vector.

[0426] 42. The method of any one of embodiments 24 to 41, wherein the introducing is carried out in the presence of a transduction adjuvant.

[0427] 43. The method of any one of embodiments 24 to 38, wherein introducing comprises transfecting the cells of the stimulated composition with a vector comprising a polynucleotide encoding the recombinant receptor.

[0428] 44. The method of embodiment 43, wherein the vector is a transposon, optionally a Sleeping Beauty (SB) transposon or a piggyBac transposon.

[0429] 45. The engineered cell composition does not contain a stimulatory reagent, and / or the stimulatory reagent has been substantially removed from the composition prior to culturing, and the stimulatory reagent inhibits one or more intracellular signaling domains and / or one or more components of a TCR complex. 26. The method of embodiment 24 or embodiment 25, comprising a reagent capable of activating one or more intracellular signaling domains of one or more costimulatory molecules.

[0430] 46. ​​The method of any one of embodiments 21-45, wherein the culturing is carried out at least until the output composition comprises a threshold number of T cells.

[0431] 47. The method of embodiment 46, wherein the culturing is continued for at least 1 day after a threshold number of T cells is achieved.

[0432] 48. The method of any one of embodiments 21-47, wherein following culturing, the cells of the output composition are harvested.

[0433] 49. The method of any of embodiments 21-48, further comprising formulating the cells of the output composition for cryogenic storage and / or administration to a subject, optionally in the presence of a pharmaceutically acceptable excipient.

[0434] 50. The method of embodiment 49, wherein the cells of the output composition are formulated in the presence of a cryoprotectant.

[0435] 51. The method of embodiment 50, wherein the cryoprotectant comprises DMSO.

[0436] 52. The method of any of embodiments 49-51, wherein the cells of the output composition are formulated in a container, optionally a vial or bag.

[0437] 53. The method of any one of embodiments 1-38, further comprising isolating CD4+ T cells and / or CD8+ T cells from the biological sample before incubating.

[0438] 54. The method of embodiment 53, wherein isolating comprises selecting cells based on surface expression of CD4 and / or CD8, optionally by positive or negative selection.

[0439] 55. The method of embodiment 53 or embodiment 54, wherein isolating comprises performing immunoaffinity-based selection.

[0440] 56. The method of any one of embodiments 53-55, wherein the biological sample comprises primary T cells obtained from the subject.

[0441] 57. The method of embodiment 56, wherein the subject is a human subject.

[0442] 58. The method of any one of embodiments 53 to 55, wherein the biological sample is or comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukocyte apheresis product.

[0443] 59. The method of any one of embodiments 53 to 55, wherein the biological sample is or comprises a cryopreserved apheresis product or a cryopreserved leukapheresis product.

[0444] 60. The recombinant receptor is associated with cells or tissues of a disease, disorder, or condition, 60. The method of any one of embodiments 1 to 59, which is specific for and / or capable of binding to a target antigen expressed therein.

[0445] 61. The method of embodiment 60, wherein the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer.

[0446] 62. The method of embodiment 60 or embodiment 61, wherein the target antigen is a tumor antigen.

[0447] 63. Target antigens include 5T4, 8H9, avb6 integrin, B7-H6, B cell maturation antigen (BCMA), CA9, cancer-testis antigen, carbonic anhydrase 9 (CAIX), CCL-1, CD19, CD20, CD22, CEA, hepatitis B surface antigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD171, carcinoembryonic antigen (CEA), CE7, cyclin, cyclin A2, c-Met, bilayer antigen, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, ephrin B2, erb-B2, erb-B3, erb-B4, erbB dimer, and EGFR. vIII, estrogen receptor, fetal AchR, folate receptor alpha, folate-binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, G250 / CAIX, GD2, GD3, gp100, G protein-coupled receptor 5D (GPCR5D), Her2 / neu (receptor tyrosine kinase erbB2), HMW-MAA, IL-22R-alpha, IL-13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1 , MAGE-A3, MAGE-A6, MART-1, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, NCAM, NKG2D, NKG2D ligand, NY-ESO-1, O-acetylated GD2 (OGD2), carcinoembryonic antigen, preferentially expressed antigen in melanoma (PRAME), PSCA, progesterone receptor, survivin, ROR1, TAG72, tEGFR, VEGF receptor, VEGF-R2, Wilms' tumor 1 (WT-1), pathogen-specific antigens, and antigens associated with universal tags.

[0448] 64. The method of any one of embodiments 1 to 63, wherein the recombinant receptor is or comprises a functional non-TCR antigen receptor, or a TCR, or an antigen-binding fragment thereof.

[0449] 65. The method of any one of embodiments 1 to 64, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

[0450] 66. The method of any one of embodiments 1 to 65, wherein the recombinant receptor is an anti-CD19 CAR.

[0451] 67. The method of embodiment 65, wherein the chimeric antigen receptor comprises an extracellular domain comprising an antigen-binding domain.

[0452] 68. The method of embodiment 67, wherein the antigen-binding domain is or comprises an antibody or an antibody fragment thereof, the fragment optionally being a single-chain fragment.

[0453] 69. The method of embodiment 68, wherein the fragment comprises antibody variable regions joined by a flexible linker.

[0454] 70. The method of embodiment 68 or embodiment 69, wherein the fragment comprises an scFv.

[0455] 71. The method of any one of embodiments 67-70, wherein the chimeric antigen receptor further comprises a spacer and / or hinge region.

[0456] 72. The method of any of embodiments 67-71, wherein the chimeric antigen receptor comprises an intracellular signaling region.

[0457] 73. The method of embodiment 72, wherein the intracellular signaling region comprises an intracellular signaling domain.

[0458] 74. The method of embodiment 73, wherein the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM).

[0459] 75. The method of embodiment 74, wherein the intracellular signaling domain is or comprises the intracellular signaling domain of a CD3 chain, optionally the CD3-zeta (CD3ζ) chain, or a signaling portion thereof.

[0460] 76. The method of any one of embodiments 72 to 75, wherein the chimeric antigen receptor further comprises a transmembrane domain located between the extracellular domain and the intracellular signaling region.

[0461] 77. The method of any one of embodiments 72 to 76, wherein the intracellular signaling region further comprises a costimulatory signaling region.

[0462] 78. The method of embodiment 77, wherein the costimulatory signaling region comprises the intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof.

[0463] 79. The method of embodiment 77 or claim 78, wherein the costimulatory signaling region comprises the intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof.

[0464] 80. The method of any one of embodiments 77-79, wherein the costimulatory signaling region is between the transmembrane domain and the intracellular signaling region.

[0465] 81. The method of any one of embodiments 46-47, wherein an output composition comprising at or above the threshold number of cells is produced in greater than or about 85% of the iterations of the method, greater than or about 90%, or greater than or about 95% of the iterations of the method.

[0466] 82. A composition comprising engineered cells produced by the method of any one of embodiments 1 to 79.

[0467] 83. The composition of embodiment 82, further comprising a pharmaceutically acceptable carrier.

[0468] 84. The composition of embodiment 82 or embodiment 83, comprising a cryoprotectant, optionally DMSO.

[0469] 85. The composition according to any one of embodiments 80 to 82 and the output composition The product includes instructions for administration.

[0470] 86. The product of embodiment 85, wherein the subject has a disease or condition, and optionally the recombinant receptor specifically recognizes or specifically binds to an antigen associated with the disease or condition, or expressed or present on cells of the disease or condition.

[0471] 87. The product of embodiment 85 or embodiment 86, wherein the output composition is a composition of engineered CD4+ T cells.

[0472] 88. The product of embodiment 85 or embodiment 86, wherein the output composition is a CD8+ T cell manipulation composition.

[0473] 89. An article of manufacture comprising a composition of engineered CD4+ T cells produced by the method of any one of embodiments 1-25 or 26-81, and a composition of engineered CD8+ T cells produced by the method of any one of claims 2-23, 25, or 26-81, and instructions for administering the engineered CD4+ T cells and the engineered CD8+ T cells to a subject.

[0474] 90. The product of embodiment 89, wherein the instructions specify that the CD4+ T cells and CD8+ T cells are administered separately to the subject.

[0475] 91. The product of embodiment 89 or embodiment 90, wherein the instructions specify administering CD4+ T cells and CD8+ T cells to the subject in a desired ratio. Further Exemplary Embodiment II

[0476] 1. A method of storing a biological sample, the method comprising obtaining a biological sample from a subject, dividing the biological sample into two or more separate containers, cryogenically storing the biological sample, and storing the cryogenically stored biological sample.

[0477] 2. The method of embodiment 1, wherein the subject is a human subject.

[0478] 3. The method of any one of embodiments 1-2, wherein the biological sample is an apheresis product or a leukapheresis product.

[0479] 4. The method of any one of embodiments 1-3, wherein the two or more separate containers are each selected from the group consisting of a cryogenic bag and / or a cryogenic vial.

[0480] 5. The method of any one of embodiments 1-4, wherein two or more separate containers contain unique identifiers thereon.

[0481] 6. The method of embodiment 5, wherein the unique identifier comprises any one or more of text information, an RFID tag, a QR code, and / or a barcode.

[0482] 7. The method of any one of embodiments 5-6, wherein the unique identifier information includes information regarding any one or more of the following categories: subject identification, sample storage location, storage and / or handling instructions, date of receipt, date of cryogenic storage, expiration date, and intended use.

[0483] 8. The biological sample is stored for a period of more than or equal to the following: 12 hours, 24 hours, 36 hours, 48 ​​hours, 1 week, 2 weeks, 3 weeks, or 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, or 10 months. 8. The method of any one of embodiments 1 to 7, wherein the storage period is 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, or 40 years.

[0484] 9. A method of storing a biological sample, comprising: (a) obtaining a biological sample from a subject; (b) cryogenically storing the biological sample in one or more containers; and (c) storing the cryogenically stored biological sample for a period of time greater than or equal to the following: 12 hours, 24 hours, 36 hours, 48 ​​hours, 1 week, 2 weeks, 3 weeks, or 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, or 40 years.

[0485] 10. The method of embodiment 9, wherein the subject is a human subject.

[0486] 11. The method of any one of embodiments 9 to 10, wherein the biological sample is an apheresis product or a leukapheresis product.

[0487] 12. The method of any one of embodiments 9-11, wherein the one or more containers are each selected from the group consisting of a cryogenic bag and / or a cryogenic vial.

[0488] 13. The method of any one of embodiments 9-12, wherein one or more separate containers contain a unique identifier thereon.

[0489] 14. The method of embodiment 13, wherein the unique identifier comprises any one or more of text information, an RFID tag, a QR code, and / or a barcode.

[0490] 15. The method of any one of embodiments 13-14, wherein the unique identifier information includes information regarding any one or more of the following categories: subject identification, sample storage location, storage and / or handling instructions, date of receipt, date of cryogenic storage, expiration date, and intended use.

[0491] 16. A method for obtaining a biological sample corresponding to a subject, the method comprising: (a) locating a cryogenically preserved sample at a central facility based on a unique identifier associating the sample with the subject; and (b) obtaining the cryogenically preserved sample.

[0492] 17. The method of embodiment 16, wherein the biological sample is genetically matched to the subject, suitable for producing an autologous product for the subject, and / or contains cells of the subject. Further Exemplary Embodiment III

[0493] 1. A method comprising cryogenically storing cells from a biological sample derived from a donor, wherein the cells are obtained from the donor at a time after the donor has been diagnosed with or is deemed to have or be suspected of having a disease or condition and before the donor has undergone one or more treatments for the disease or condition, and the cells are frozen in a controlled rate freezer using a step-wise freezing profile including at least one step in which the sample and / or chamber is cooled at a rate greater than 1°C per minute.

[0494] 2. A method comprising cryogenically storing cells from a biological sample derived from a donor, wherein the cells were obtained from the donor at a time after the donor was deemed refractory to a treatment regimen for a disease or condition or experienced a relapse following a treatment regimen, and before the donor underwent subsequent treatment for the disease or condition.

[0495] 3. A method comprising cryogenically storing cells from a biological sample derived from a donor, wherein the cells were obtained from the donor at a time when the donor had not been diagnosed with, known to have, or suspected to have a disease or condition, and the cells were frozen in a controlled rate freezer using a step-wise freezing profile including at least one step in which the sample and / or chamber is cooled at a rate greater than 1°C per minute.

[0496] 4. A method comprising: (a) cryogenically freezing cells from a biological sample derived from a donor; and (b) storing the cryogenically frozen cells for a period of time, wherein the cells are obtained or were obtained from the donor (i) after the donor has been diagnosed with or is deemed to have or be suspected of having a disease or condition and before the donor has received treatment for the disease or condition, or (ii) after the donor has been deemed refractory to a treatment regimen for the disease or condition or has experienced a relapse following a treatment regimen and before the donor has received subsequent treatment for the disease or condition, and during the period of storage, the donor has received or has received at least one treatment for the disease or condition.

[0497] 5. A method comprising: (a) cryogenically freezing cells from a biological sample derived from a donor; and (b) storing the cryogenically frozen cells for a period of time, wherein the cells are obtained from the donor (i) after the donor has been diagnosed with or is deemed to have or be suspected of having a disease or condition and before the donor has undergone treatment for the disease or condition, or (ii) after the donor has been deemed refractory to a treatment regimen for the disease or condition or has experienced a relapse following a treatment regimen and before the donor has undergone subsequent treatment for the disease or condition. or obtained from a donor, wherein the cells are cryogenically stored for a period of greater than or equal to 12 hours, 24 hours, 36 hours, 48 ​​hours, 1 week, 2 weeks, 3 weeks, or 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, or 40 years, or until the donor needs the cells.

[0498] 6. A method comprising: (a) cryogenically freezing cells from a biological sample derived from a donor; and (b) administering to a subject in need thereof a therapeutically effective amount of a composition comprising engineered T cells generated from the cryogenically frozen cells, wherein the cells are obtained or were obtained from the donor (i) after the donor has been diagnosed with or is deemed to have or be suspected of having a disease or condition and before the donor has received treatment for the disease or condition, or (ii) after the donor has been deemed refractory to a treatment regimen for the disease or condition or has experienced a relapse after a treatment regimen and before the donor has received subsequent treatment for the disease or condition, and wherein between the freezing and the administering, the donor has received or has received at least one treatment for the disease or condition.

[0499] 7. (a) cryogenically freezing cells from a biological sample derived from a donor, thereby producing a cryogenically frozen cell composition; and (b) cryogenically freezing the cells. and manipulating the cells of the composition to produce a composition comprising the engineered T cells, wherein the cells are obtained or have been obtained from a donor (i) after the donor has been diagnosed with or is deemed to have or be suspected of having a disease or condition and before the donor has received treatment for the disease or condition, or (ii) after the donor has been deemed refractory to a treatment regimen for the disease or condition or has experienced a relapse after a treatment regimen and before the donor has received subsequent treatment for the disease or condition, and wherein between the freezing and the manipulation, the donor has received or has received at least one treatment for the disease or condition.

[0500] 8. A method of treatment comprising administering a therapeutically effective amount of engineered T cells to a subject in need thereof, wherein the cells are or have been obtained from a subject (i) after the subject has been diagnosed with or is deemed to have or be suspected of having a disease or condition and before the subject has received treatment for the disease or condition, or (ii) after the subject has been deemed refractory to a treatment regimen for the disease or condition or has experienced a relapse following a treatment regimen and before the subject has received subsequent treatment for the disease or condition, and wherein the subject has received or has received at least one treatment for the disease or condition after the cells are or have been obtained from the subject and before administration of the engineered T cells.

[0501] 9. A method for producing a composition of engineered cells, comprising: (a) obtaining and, if necessary, thawing cryogenically stored cells; and (b) introducing a recombinant receptor into the cryogenically stored cells, thereby producing an engineered composition comprising engineered T cells, wherein the cells were cryogenically stored after being harvested from the donor (i) after the donor has been diagnosed with or is deemed to have or be suspected of having a disease or condition and before the donor has received treatment for the disease or condition, or (ii) after the donor has been deemed refractory to a treatment regimen for the disease or condition or has experienced a relapse after a treatment regimen and before the donor has received subsequent treatment for the disease or condition, and wherein the donor has received or has received at least one treatment for the disease or condition after cryogenic storage and before obtaining the cryogenically stored cells.

[0502] 10. The method of any one of embodiments 1 to 9, wherein the biological sample is or is derived from an apheresis sample, optionally a leukapheresis sample, and / or the sample contains leukocytes and / or lymphocytes, and / or the cells or blood cells in the sample consist essentially of leukocytes, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells in the sample, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the blood cells in the sample are leukocytes.

[0503] 11. The method of any one of embodiments 1 to 10, wherein the cells have not been subjected to a step of immunoaffinity-based and / or target-specific selection and / or enrichment of blood cell populations and / or T cell populations and / or T cell subsets before being stored at cryogenic temperatures.

[0504] 12. The method of any one of embodiments 1 to 10, wherein the cells have been subjected to an immunoaffinity-based and / or target-specific selection and / or enrichment step of blood cell and / or T cell populations prior to cryogenic storage, and optionally further comprising performing said selection or enrichment prior to said cryogenic storage.

[0505] 13. The method of embodiment 12, wherein the selection step and / or enrichment comprises immunoaffinity-based selection and / or comprises positive or negative selection.

[0506] 14. The method of any one of embodiments 12-13, wherein the selection step and / or enrichment comprises enrichment and / or isolation of CD4+ cells or a subset thereof and / or CD8+ cells or a subset thereof, wherein the enrichment or isolation of CD4+ cells or a subset thereof is performed either separately from or in combination with the selection and / or isolation of CD8+ cells or a subset thereof, and wherein, optionally, the subset of CD8+ cells and / or the subset of CD4+ cells is selected from the group consisting of memory cells, central memory T (TCM) cells, effector memory cells (TEM), stem central memory (TSCM) cells, effector T (TE) cells, effector memory RA T (TEMRA) cells, naive T (TN) cells, and / or regulatory T (TREG) cells.

[0507] 15. The method of any one of embodiments 1 to 14, wherein the cells comprise T cells or are enriched for T cells.

[0508] 16. The method of embodiment 15, wherein the T cells comprise or are enriched for CD4+ T cells or a subset thereof, CD8+ T cells or a subset thereof, or a mixture thereof, and the subset of CD8+ cells and / or the subset of CD4+ cells is optionally selected from the group consisting of memory cells, central memory T (TCM) cells, effector memory cells (TEM), stem central memory (TSCM) cells, effector T (TE) cells, effector memory RA T (TEMRA) cells, naive T (TN) cells, and / or regulatory T (TREG) cells.

[0509] 17. The method of any one of embodiments 1-16, further comprising combining the cells with a cryogenic storage medium prior to cryogenically storing the cells.

[0510] 18. The method of embodiment 17, wherein the cryogenic storage medium comprises about 10% dimethyl sulfoxide (DMSO) and serum proteins, optionally human serum albumin, optionally about 4% human serum albumin, and / or the freezing solution and / or final concentration of the biological sample comprises about 1% to about 20%, about 3% to about 9%, or about 6% to about 9% DMSO by volume, and / or about 3%, about 4%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% DMSO by volume.

[0511] 19. The method of any one of embodiments 2 or 4-18, wherein the cryogenic storage comprises decreasing the temperature at a rate of 1°C or about 1°C per minute, as needed, until the temperature reaches -80°C or about -80°C.

[0512] 20. The method of any one of embodiments 1 to 19, wherein the cells are cryogenically stored in a container in the vapor phase of liquid nitrogen, the container being optionally a bag or a vial.

[0513] 21. The method of any one of embodiments 1-20, wherein the cells are cryogenically stored for a period of more than or equal to the following: 12 hours, 24 hours, 36 hours, 48 ​​hours, 1 week, 2 weeks, 3 weeks, or 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, or 40 years.

[0514] 22. The method of any one of embodiments 1 to 21, wherein the cells are stored for a period of time, and after the period, the percentage of viable cells or viable T cells or subtypes or subsets thereof in the composition is between about 24% and about 100%, or is at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90%.

[0515] 23. The method of any one of embodiments 1-22, wherein the disease is cancer, an inflammatory disease or condition, an autoimmune disease or condition, or an infectious disease or condition.

[0516] 24. The method of embodiment 23, wherein the cancer is chronic lymphocytic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia, null acute lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, multiple myeloma, follicular lymphoma, splenic marginal zone lymphoma, mantle cell lymphoma, late-onset B-cell lymphoma, or acute myeloid leukemia.

[0517] 25. The cancer expresses ROR1, EGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y , L1-cell adhesion molecule, MAGE-A1, MUC1, MUC16, B-cell maturation antigen (BCMA), FCRL5 / FCRH5, GPRC5D, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, oncofetal antigen, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms' tumor 1 (WT-1), and a cyclin, e.g., cyclin A1 (CCNA1).

[0518] 26. The method according to any one of embodiments 1 to 25, wherein the treatment is chemotherapy, radiation, surgery, cell therapy, and / or a debulking treatment.

[0519] 27. Treatment includes the following: cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, mustine, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, cisplatin, epirubicin, capecitabine, folinic acid, oxaliplatin, small molecule inhibitors, immune cells, natural killer cells, lymphokine-activated killer cells, cytotoxic T cells, dendritic cells, 4000cGy irradiation, autologous stem cell rescue, stem cell transplant, bone marrow transplant, hematopoietic stem cell transplant (HSCT), CAR T-cell therapy, tisagenlecleucel, axicabtagene ciloleucel, cytarabine, high-dose cytarabine, daunorubicin (daunomycin), idarubicin, cladribine, bortezomib, carfilzomib, thalidomide, lenalidomide, pomalidomide, corticosteroids, prednisone, dexamethasone, alkylating agents, chlorambucil, bendamustine, ifosfamide, platinum drugs, cisplatin, carboplatin, oxaliplatin, purine analogs, fludarabine, pemetrexed ibuprofen antostatin, cladribine, antimetabolites, gemcitabine, methotrexate, pralatrexate, vincristine, doxorubicin, mitoxantrone, bleomycin, proteasome inhibitors, histone deacetylase inhibitors, romidepsin, belinstat, kinase inhibitors, ibrutinib, idelalisib, antibodies, anti-CD20 antibodies, rituximab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, anti-CD52 antibodies, alemtuzumab, anti-CD30 antibodies, brentuximab 27. The method of embodiment 26, comprising one or more of the following, alone or in combination: cimab, vedotin, interferon, an immunomodulatory agent, thalidomide, CHOP, CHOP+R (or R-CHOP), CVP, EPOCH, EPOCH+R, DHAP, DHAP+R (or R-DHAP), venetoclax, methylprednisolone, or a Bruton's tyrosine kinase inhibitor (BTKi).

[0520] 28. The method of any one of embodiments 1 to 27, wherein the donor or subject is a human.

[0521] 29. The method of any one of embodiments 1 to 28, further comprising analyzing the cells, if desired, for one or more phenotypic markers by assessing their surface expression prior to cryogenic storage.

[0522] 30. The method of any one of embodiments 1 to 29, further comprising thawing cryogenically stored cells.

[0523] 31. The method of any of embodiments 1-30, further comprising, after cryogenic storage and / or thawing of the cells, engineering the cells to express a recombinant or exogenous molecule, which is optionally a recombinant protein, optionally a recombinant receptor, which optionally is or comprises a T cell receptor (TCR), a chimeric receptor, and / or a chimeric antigen receptor.

[0524] 32. The method of embodiment 31, wherein the recombinant molecule is a recombinant receptor that specifically recognizes or specifically binds to an antigen expressed by or specifically expressed by a cell associated with the disease or condition.

[0525] 33. The method of any one of embodiments 1 to 32, wherein the number of cells when collected from the donor or subject and / or in the total apheresis sample is at or about, or does not exceed, 500 x 10 6 pieces, 1000×10 6 pieces, 2000×10 6 pieces, 3000×10 6 pieces, 4000×10 6 pieces, or 5000 x 10 6 or more total cells or total nucleated cells.

[0526] 34. The method of any one of embodiments 1-33, further comprising enriching T cells from the sample before storing the sample at cryogenic temperatures.

[0527] 35. The method of embodiment 34, wherein the T cells are, comprise, or are enriched for CD4+ T cells or a subset thereof, CD8+ T cells or a subset thereof, or a mixture thereof, and optionally the subset of CD8+ cells and / or the subset of CD4+ cells are optionally selected from the group consisting of memory cells, central memory T (TCM) cells, effector memory cells (TEM), stem central memory (TSCM) cells, effector T (TE) cells, effector memory RA T (TEMRA) cells, naive T (TN) cells, and / or regulatory T (TREG) cells, and / or the sample is enriched for bulk T cells.

[0528] 36. The method of any one of embodiments 1 to 35, further comprising formulating the sample in a cryogenic medium before storing the sample at cryogenic temperatures.

[0529] 37. Cells may be transported to a storage facility either before or after cryogenic freezing. 37. The method of any one of embodiments 1 to 36, further comprising:

[0530] 38. The method of embodiment 37, wherein the storage facility is a central or common repository storage facility.

[0531] 39. The method of embodiment 37 or 38, wherein the sample is transported to a storage facility in a cryogenic environment.

[0532] 40. The method of any one of embodiments 36-39, further comprising enriching the sample for T cells after transport and before cryogenically storing the cells.

[0533] 41. The method of embodiment 40, wherein the T cells are, comprise, or are enriched for CD4+ T cells or a subset thereof, CD8+ T cells or a subset thereof, or a mixture thereof, and optionally the subset of CD8+ cells and / or the subset of CD4+ cells are optionally selected from the group consisting of memory cells, central memory T (TCM) cells, effector memory cells (TEM), stem central memory (TSCM) cells, effector T (TE) cells, effector memory RA T (TEMRA) cells, naive T (TN) cells, and / or regulatory T (TREG) cells, and / or comprise bulk T cells.

[0534] 42. The method of embodiment 40 or embodiment 41, further comprising formulating the sample and / or T cells in a cryogenic medium after transport and before cryogenic storage of the cells.

[0535] 43. The method of any one of embodiments 1 to 42, further comprising thawing cryogenically stored cells.

[0536] 44. The method of any one of embodiments 1 to 43, wherein the sample is placed in a container marked with o...

Claims

1. 1. A method comprising cryogenically storing cells from a biological sample derived from a donor, the cells are obtained from the donor after the donor has been diagnosed with, or is considered to have, or is suspected of having, a disease or condition, and before the donor has undergone one or more treatments for the disease or condition; A method wherein the cells are frozen in a controlled rate freezer using a stepwise freezing profile including at least one step in which the sample and / or chamber is cooled at a rate greater than 1° C. per minute.

2. 1. A method comprising storing cells from a biological sample derived from a donor at cryogenic temperatures, wherein the cells were obtained from the donor after the donor was deemed refractory to a treatment regimen for a disease or condition or experienced a relapse after said treatment regimen, and before the donor underwent subsequent treatment for said disease or condition.

3. 1. A method comprising cryogenically storing cells from a biological sample derived from a donor, wherein the cells were obtained from the donor at a time when the donor was not diagnosed with, known to have, or suspected to have a disease or condition; A method wherein the cells are frozen in a controlled rate freezer using a stepwise freezing profile including at least one step in which the sample and / or chamber is cooled at a rate greater than 1° C. per minute.

4. - cryogenically freezing cells from a biological sample derived from a donor; storing the cryogenically frozen cells for a period of time; A method comprising: the cells are obtained or were obtained from the donor (i) after the donor has been diagnosed with or is considered to have or be suspected of having a disease or condition and before the donor has received treatment for the disease or condition, or (ii) after the donor has been considered refractory to a treatment regimen for a disease or condition or has experienced a relapse following said treatment regimen and before the donor has received subsequent treatment for the disease or condition; During said period of storage, said donor undergoes or has undergone treatment for at least one of said diseases or conditions.

5. - cryogenically freezing cells from a biological sample derived from a donor; storing the cryogenically frozen cells for a period of time; A method comprising: the cells are obtained or were obtained from the donor (i) after the donor has been diagnosed with or is considered to have or be suspected of having a disease or condition and before the donor has received treatment for the disease or condition, or (ii) after the donor has been considered refractory to a treatment regimen for a disease or condition or has experienced a relapse following said treatment regimen and before the donor has received subsequent treatment for the disease or condition; The cells have been in culture for a period of time greater than or equal to the following: 12 hours, 24 hours, 36 hours, 48 ​​hours, 1 week, 2 weeks, 3 weeks, or 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, wherein the cells are cryogenically stored for 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 years, or until needed by the donor.

6. - cryogenically freezing cells from a biological sample derived from a donor; administering to a subject in need thereof a therapeutically effective amount of a composition comprising engineered T cells generated from said cryogenically frozen cells; A method comprising: the cells are obtained or were obtained from the donor (i) after the donor has been diagnosed with or is considered to have or be suspected of having a disease or condition and before the donor has received treatment for the disease or condition, or (ii) after the donor has been considered refractory to a treatment regimen for a disease or condition or has experienced a relapse following said treatment regimen and before the donor has received subsequent treatment for the disease or condition; The method, wherein between said freezing and administering, said donor undergoes or has undergone treatment for at least one of said diseases or conditions.

7. - cryogenically freezing cells from a biological sample derived from a donor, thereby producing a cryogenically frozen cell composition; - manipulating cells of said cryogenically frozen cell composition to produce a composition comprising engineered T cells; A method comprising: the cells are obtained or were obtained from the donor (i) after the donor has been diagnosed with or is considered to have or be suspected of having a disease or condition and before the donor has received treatment for the disease or condition, or (ii) after the donor has been considered refractory to a treatment regimen for a disease or condition or has experienced a relapse following said treatment regimen and before the donor has received subsequent treatment for the disease or condition; A method wherein between said freezing and manipulation, said donor undergoes or has undergone treatment for at least one of said diseases or conditions.

8. 1. A method of treatment comprising administering a therapeutically effective amount of engineered T cells to a subject in need thereof, the cells are obtained or were obtained from the subject (i) after the subject has been diagnosed with or is considered to have or be suspected of having a disease or condition and before the subject has received treatment for the disease or condition, or (ii) after the subject has been considered refractory to a treatment regimen for the disease or condition or has experienced a relapse following said treatment regimen and before the subject has received subsequent treatment for the disease or condition; The method, wherein after the cells are obtained or obtained from the subject and prior to the administration of the engineered T cells, the subject is undergoing or has undergone treatment for at least one of the diseases or conditions.

9. 1. A method for producing a composition of engineered cells, comprising: - obtaining cryogenically stored cells and thawing them as needed; - introducing a recombinant receptor into said cryogenically stored cells, thereby producing an engineered composition comprising engineered T cells; A method comprising: The cells are collected (i) after the donor has been diagnosed with, or is considered to have, or is suspected of having, a disease or condition, and or (ii) after the donor has been deemed refractory to a treatment regimen for a disease or condition or has experienced a relapse following said treatment regimen and before the donor has received subsequent treatment for the disease or condition; and The method, wherein after cryogenic storage and prior to obtaining said cryogenically stored cells, said donor undergoes or has undergone treatment for at least one of said diseases or conditions.

10. 10. The method of any one of claims 1 to 9, wherein the biological sample is or is derived from an apheresis sample, optionally a leukapheresis sample, and / or the sample contains leukocytes and / or lymphocytes, and / or the cells or blood cells in the sample consist essentially of leukocytes, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells in the sample, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the blood cells in the sample are leukocytes.

11. 11. The method of any one of claims 1 to 10, wherein the cells have not been subjected to a step of immunoaffinity-based and / or target-specific selection and / or enrichment of blood cell populations and / or T cell populations and / or T cell subsets prior to cryogenic storage.

12. 11. The method of any one of claims 1 to 10, wherein the cells have been subjected to an immunoaffinity-based and / or target-specific selection and / or enrichment step of blood cell and / or T cell populations prior to cryogenic storage, optionally further comprising carrying out said selection or enrichment prior to said cryogenic storage.

13. 13. The method of claim 12, wherein the selection step and / or enrichment comprises immunoaffinity-based selection and / or comprises positive or negative selection.

14. The selection step and / or enrichment is ＋ and / or enrichment and / or isolation of CD4+ cells or subsets thereof and / or CD8+ cells or subsets thereof, ＋ The enrichment or isolation of cells or subsets thereof may be carried out by the method of claim 1, further comprising: ＋ performed either separately or in combination with said selection and / or isolation of cells or subsets thereof, Optionally, the CD8 ＋ Cell subsets and / or the CD4 ＋ A subset of cells may optionally be designated as memory cells, central memory T (T ＣＭ ) cells, effector memory cells (T ＥＭ ), Stem Central Memory (T ＳＣＭ ) cells, effector T (T Ｅ ) cells, effector memory RA T(T ＥＭＲＡ ) cells, naive T (T Ｎ ) cells, and / or regulatory T (T ＲＥＧ The method according to any one of claims 12 to 13, wherein the cell is selected from the group consisting of:

15. The method of any one of claims 1 to 14, wherein the cells comprise or are enriched for T cells.

16. The T cells are CD4 ＋ T cells or their subsets, CD8 ＋ T cells or a subset thereof, or a mixture thereof, or enriched in such T cells, ＋ Cell subsets and / or the CD4 ＋ A subset of cells may optionally be designated as memory cells, central memory T (T ＣＭ ) cells, effector memory cells (T ＥＭ ), Stem Central Memory (T ＳＣＭ ) cells, effector T (T Ｅ ) cells, effector memory RA T(T ＥＭＲＡ ) cells, naive T (T Ｎ ) cells, and / or is the regulatory T (T ＲＥＧ 16. The method of claim 15, wherein the cell is selected from the group consisting of:

17. 17. The method of any one of claims 1 to 16, further comprising combining the cells with a cryogenic storage medium prior to cryogenically storing the cells.

18. 18. The method of claim 17, wherein the cryogenic storage medium comprises about 10% dimethyl sulfoxide (DMSO) and serum proteins, optionally human serum albumin, optionally about 4% human serum albumin, and / or the freezing solution and / or the final concentration of the biological sample comprises about 1% to about 20%, about 3% to about 9%, or about 6% to about 9% DMSO by volume, and / or about 3%, about 4%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% DMSO by volume.

19. 19. The method of any one of claims 2 or 4-18, wherein the cryogenic storage comprises decreasing the temperature at a rate of at or about 1°C per minute, as needed, until the temperature reaches at or about -80°C.

20. 20. The method of any one of claims 1 to 19, wherein the cells are cryogenically stored in a container in the vapor phase of liquid nitrogen, the container being optionally a bag or a vial.

21. 21. The method of any one of claims 1 to 20, wherein the cells are cryogenically stored for a period of time greater than or equal to 12 hours, 24 hours, 36 hours, 48 ​​hours, 1 week, 2 weeks, 3 weeks, or 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, or 40 years.

22. 22. The method of any one of claims 1 to 21, wherein the cells are stored for a period of time, after which the percentage of viable cells or viable T cells or subtypes or subsets thereof in the composition is from about 24% to about 100%, or is at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90%.

23. 23. The method of any one of claims 1 to 22, wherein the disease is cancer, an inflammatory disease or condition, an autoimmune disease or condition, or an infectious disease or condition.

24. 24. The method of claim 23, wherein the cancer is chronic lymphocytic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia, null acute lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, multiple myeloma, follicular lymphoma, splenic marginal zone lymphoma, mantle cell lymphoma, late-onset B-cell lymphoma, or acute myeloid leukemia.

25. The cancer is characterized by the expression of ROR1, EGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kappa-like protein (KAP), IL-13R-alpha2, kappa-like protein (KAP), IL-13R-alpha3, kappa-like protein (KAP), IL-13R-alpha4, kappa-like protein (KAP), IL-13R-alpha5, kappa-like protein (KAP), IL-13R-alpha6, kappa-like protein (KAP), IL-13R-alpha7, kappa-like protein (KAP), IL-13R-alpha8, kappa-like protein (KAP), IL-13R-alpha9, kappa-like protein (KAP), IL-13R-alpha1, kappa-like protein (KAP), IL-13R-alpha1, kappa-like protein (KAP), IL-13R-alpha2 ... dr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, MUC1, MUC16, B-cell maturation antigen (BCMA), FCRL5 / FCRH5, GPRC5D, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, oncofetal antigen, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms' tumor 1 (WT-1), and a cyclin, e.g., cyclin A1 (CCNA1).

26. The method of any one of claims 1 to 25, wherein the treatment is chemotherapy, radiation, surgery, cell therapy, and / or a debulking treatment.

27. The treatment may be one of the following: cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, mustine, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, cisplatin, epirubicin, capecitabine, folinic acid, oxaliplatin, small molecule inhibitors, immune cells, natural killer cells, lymphokine-activated killer cells, cytotoxic T cells, dendritic cells, 4000 cGy irradiation, autologous stem cell rescue, stem cell transplant, bone marrow transplant, hematopoietic stem cell transplant (HSCT), CAR T-cell therapy, tisagenlecleucel, axicabtagene ciloleucel, cytarabine, high-dose cytarabine, daunorubicin (daunomycin), idarubicin, cladribine, bortezomib, carfilzomib, thalidomide, lenalidomide, pomalidomide, corticosteroids, prednisone, dexamethasone, alkylating agents, chlorambucil, bendamustine, ifosfamide, platinum drugs, cisplatin, carboplatin, oxaliplatin, purine analogs, fludarabine, pentostatin, cladribine, antimetabolites, gemcitabine, methotrexate, pralatrexate, vincristine, doxorubicin, mitoxantrone, bleomycin, proteasome inhibitors, rheumatoid arthritis 27. The method of claim 26, comprising one or more of the following, alone or in combination: a stromal deacetylase inhibitor, romidepsin, belinstat, a kinase inhibitor, ibrutinib, idelalisib, an antibody, an anti-CD20 antibody, rituximab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, an anti-CD52 antibody, alemtuzumab, an anti-CD30 antibody, brentuximab, vedotin, an interferon, an immunomodulatory agent, thalidomide, CHOP, CHOP+R (or R-CHOP), CVP, EPOCH, EPOCH+R, DHAP, DHAP+R (or R-DHAP), venetoclax, methylprednisolone, or a Bruton's tyrosine kinase inhibitor (BTKi).

28. The method of any one of claims 1 to 27, wherein the donor or subject is a human.

29. 29. The method of any one of claims 1 to 28, further comprising analyzing the cells prior to cryogenic storage, optionally by assessing the surface expression of the cells for one or more phenotypic markers.

30. 30. The method of any one of claims 1 to 29, further comprising thawing the cryogenically stored cells.

31. 31. The method of any preceding claim, further comprising, after cryogenic storage and / or thawing of the cells, engineering the cells to express a recombinant or exogenous molecule, optionally a recombinant protein, optionally a recombinant receptor, optionally which is or comprises a T cell receptor (TCR), a chimeric receptor, and / or a chimeric antigen receptor.

32. 32. The method of claim 31 , wherein the recombinant molecule is a recombinant receptor that specifically recognizes or specifically binds to an antigen expressed by or specifically expressed by a cell associated with the disease or condition.

33. 33. The method of any one of claims 1 to 32, wherein the number of cells when collected from the donor or subject and / or in the apheresis sample total is at or about, or does not exceed, 500 x 10 ６ pieces, 1000×10 ６ pieces, 2000×10 ６ pieces, 3000×10 ６ pieces, 4000×10 ６ pieces, or 5000 x 10 ６ or more total cells or total nucleated cells.

34. 34. The method of any one of claims 1 to 33, further comprising enriching the sample for T cells prior to cryogenic storage of the sample.

35. The T cells are CD4 ＋ T cells or their subsets, CD8 ＋ T cells or a subset thereof, or a mixture thereof, or comprising or enriched therein, and optionally ＋ Cell subsets and / or the CD4 ＋ A subset of cells may optionally be designated as memory cells, central memory T (T ＣＭ ) cells, effector memory cells (T ＥＭ ), Stem Central Memory (T ＳＣＭ ) cells, effector T (T Ｅ ) cells, effector memory RA T(T ＥＭＲＡ ) cells, naive T (T Ｎ ) cells, and / or regulatory T (T ＲＥＧ 35. The method of claim 34, wherein the sample is selected from the group consisting of: T cells;

36. 36. The method of any one of claims 1 to 35, further comprising formulating the sample in a cryogenic medium prior to storing the sample at cryogenic temperatures.

37. 37. The method of any one of claims 1 to 36, further comprising transporting the cells to an archival facility either before or after cryogenic freezing.

38. 38. The method of claim 37, wherein the storage facility is a central or common repository storage facility.

39. 39. The method of claim 37 or 38, wherein the sample is transported to the storage facility in a cryogenic environment.

40. 40. The method of any one of claims 36 to 39, further comprising enriching the sample for T cells after transport and before cryogenically storing the cells.

41. The T cells are CD4 ＋ T cells or their subsets, CD8 ＋ T cells or a subset thereof, or a mixture thereof, or comprising or enriched therein, and optionally ＋ Cell subsets and / or the CD4 ＋ A subset of cells may optionally be designated as memory cells, central memory T (T ＣＭ ) cells, effector memory cells (T ＥＭ ), Stem Central Memory (T ＳＣＭ ) cells, effector T (T Ｅ ) cells, effector memory RA T(T ＥＭＲＡ ) cells, naive T (T Ｎ ) cells, and / or regulatory T (T ＲＥＧ 41. The method of claim 40, wherein the T cells are selected from the group consisting of: T cells;

42. 42. The method of claim 40 or claim 41, further comprising formulating the sample and / or the T cells in a cryogenic medium after transport and before cryogenic storage of the cells. The method described.

43. 43. The method of any one of claims 1 to 42, further comprising thawing the cryogenically stored cells.

44. 44. The method of any one of claims 1 to 43, wherein the sample is placed in a container marked with one or more codes or identifiers for categorizing the cells during processing, cryopreservation, and / or storage.

45. 45. The method of claim 44, wherein the one or more codes or identifiers include a text identifier, a barcode, a QR code, an RFID, or a transponder.

46. 46. ​​The method of claim 44 or claim 45, wherein the one or more codes or identifiers correspond to or indicate the identity of one or more of the donor, the sample, the vial, the container, the disease, and / or the storage facility.

47. 47. A method according to any one of claims 44 to 46, wherein the one or more codes or identifiers correspond to a patient identification bracelet or a code appearing on a hospital or medical facility or collection facility system or document.

48. 1. A method of treatment comprising:

48. Obtaining and, if necessary, thawing cryogenically stored cells by the method of any one of claims 1 to 47, wherein prior to said obtaining, the cells have been cryogenically stored for a period of at least 12 hours, 24 hours, 36 hours, 48 ​​hours, 1 week, 2 weeks, 3 weeks, or 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, or 40 years; introducing a recombinant receptor into said stimulated composition, thereby producing an engineered composition comprising engineered T cells; administering the cells to a subject; A method comprising:

49. 49. The method of any one of claims 1-48, wherein said treatment does not include said engineered T cells or cells of said cryogenically frozen composition.

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