RU2774232C2 - Methods for modulating cart-cells - Google Patents

Abstract

FIELD: medicine; genetic engineering.

SUBSTANCE: invention relates to a method for the expansion of cells modified by genetic engineering methods, which includes one or both of administration of an inhibitor of an immune control point and biopsy of an individual with tumor or malignant tumor. Cells modified by genetic engineering methods were previously administered to the specified individual for the treatment of the specified tumor or malignant tumor, 12 months or less before one or both of administration of the inhibitor of an immune control point and biopsy. Cells modified by genetic engineering methods contain T-cells, and the specified cells modified by genetic engineering methods express a recombinant receptor.

EFFECT: proposed method leads to an increase in the expansion and, in some cases, to more stable and long-lasting response of cells modified by genetic engineering methods after the destruction and/or treatment.

52 cl, 9 dwg, 10 tbl, 6 ex

Description

Cross-reference to related applications

[1] This application claims priority over U.S. Provisional Application No. 62/429740, filed December 3, 2016, entitled "METHODS FOR MODULATION OF GENETICALLY ENGINEERED CELLS", U.S. Provisional Application No. 62/444784, filed January 10, 2017, entitled "METHODS FOR MODULATION OF GENETICALLY ENGINEERED CELLS", U.S. Provisional Application No. 62/492950, filed May 1, 2017, titled "METHODS FOR MODULATION OF GENETICALLY ENGINEERED CELLS", U.S. Provisional Application No. 62/514777, filed June 2, 2017, entitled "METHODS FOR MODULATION OF GENETICALLY ENGINEERED CELLS", US Provisional Application No. 62/515512, filed June 5, 2017, entitled "METHODS FOR MODULATION OF GENETICALLY ENGINEERED CELLS", US Provisional Application No. 62/549391, filed August 23, 2017 2017 titled "METHODS FOR MODULATION OF GENETICALLY ENGINEERED CELLS" and U.S. Provisional Application No. 62/580414 filed November 1, 2017 titled "METHODS FOR MODULATION O F GENETICALLY ENGINEERED CELLS", the contents of which are incorporated herein by reference in their entirety.

Include a sequence listing as a link

[2] This application is filed with the sequence listing in electronic format. The sequence list is provided as a file named 735042009140SeqList.txt, created on November 29, 2017, which is 34.855 kilobytes in size. Sequence listing information in electronic format is incorporated herein by reference in its entirety.

Field of invention

[3] The present invention relates in some aspects to an in vivo modulation method, to cells into which a recombinant receptor, such as a T cell receptor (TCR) or a chimeric antigen receptor (CAR), has been engineered. In some embodiments, the methods include destroying an area in an individual in which cells and/or a lesion are present or likely to be present, such as a tumor, and/or providing treatment that includes one or more of physical or mechanical manipulation of the lesion or portion thereof, irradiation or administration of an immunomodulatory agent. In some embodiments, the implementation of the destruction and/or treatment changes the environment of the lesion, for example, the microenvironment of the tumor. In some embodiments, the destruction and/or treatment is a biopsy. In some aspects, the provided methods lead to increased expansion, and in some cases to a more robust and long lasting response of engineered cells after disruption and/or treatment.

State of the art

[4] Various strategies are available for immunotherapy, for example, adoptive cell therapy methods involving the administration of T cells, such as cells with genetically engineered antigen receptors such as CARs. In some aspects, the available methods may not be completely satisfactory. There is a need for additional strategies for adoptive cell therapy, for example, strategies to increase the survival, activity and/or proliferation of introduced cells and responses, and cell modulation strategies. Methods are provided that satisfy such needs.

The essence of the invention

[5] Within the scope of the present invention, there is provided a method for expanding genetically engineered cells, comprising interfering with an area in an individual in which the modified cells are, or are likely to be, or have been or are likely to be present, and/or providing a treatment that includes one or several of physically or mechanically manipulating a lesion or part thereof, irradiating or administering an immunomodulatory agent, where said individual has previously been administered genetically engineered cells to treat a disease or condition, where the method results in the expansion of the engineered cells in the individual in the area, and /or in a tissue, or in an organ, or in the fluid of the individual and/or to an increase in the number of engineered cells in this area, tissue, or organ, or fluid. In certain embodiments, the region is or contains a lesion or a portion thereof. In some embodiments, the lesion is a tumor. In certain embodiments, the tumor is a primary or secondary tumor. In specific embodiments, the region is or contains bone marrow tissue. In certain embodiments, the region is or contains a lesion or a portion thereof.

[6] In some of any such embodiments, at or shortly before the intervention and/or treatment time, the subject relapsed after remission in response to the administration of genetically engineered cells. In some of any such embodiments, at or just prior to the intervention and/or treatment time point, the individual is in remission; the number of engineered cells detectable in blood is reduced or undetectable; the number of engineered cells detectable in a fluid or tissue or sample, optionally blood, from an individual is reduced compared to a previous point in time after administration of the engineered cells; and/or the number of engineered cells detectable in a fluid or tissue or sample, optionally blood, from an individual is reduced by or more than 1.5 times, 2.0 times, 3.0 times, 4, 0 times, 5.0 times, 10 times or more compared to the peak or maximum number of engineered cells detectable or detectable in the blood of an individual after initiation of the administration of engineered cells and/or compared to the level at a point in time within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or 28 days after cell injection.

[7] In some of any such embodiments, the intervention and/or treatment is carried out after, about after, or more than, or more than about 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months , 1 year or more after the start of the administration of genetically engineered cells or after the last dose of genetically engineered cells. In some of any such embodiments, the intervention and/or treatment comprises one or more of the administration of an immunomodulatory agent, radiation therapy, or physical or mechanical manipulation of the area or lesion.

[8] In some of any such embodiments, the intervention and/or treatment comprises the administration of an immunomodulatory agent. In specific embodiments, the immunomodulatory agent is or contains an immune inhibitory molecule, is or contains an immune checkpoint molecule or a member of the immune checkpoint cascade, and/or is or contains a modulator of the immune checkpoint molecule or cascade. In certain embodiments, the immune control point molecule or cascade is or contains PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM3, VISTA, adenosine receptor, CD73, CD39, adenosine receptor 2A (A2AR ), or adenosine, or a cascade involving any of the above. In certain embodiments, the immunomodulatory agent is thalidomide or is a derivative or analogue of thalidomide. In specific embodiments, the immunomodulatory agent is lenalidomide or pomalidomide, avadomide, a stereoisomer of lenalidomide, pomalidomide, avadomide, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate, or polymorph thereof. In certain embodiments, the immunomodulatory agent is lenalidomide, a stereoisomer of lenalidomide, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate, or polymorph thereof.

[9] In some of any such embodiments, after relapse and before intervention and/or treatment, the subject is not administered an exogenous or recombinant agent to treat the disease or condition or to modulate the activity of the engineered cells. In some of any such embodiments, the intervention and/or treatment comprises radiation therapy. In some of any such embodiments, intervention and/or treatment includes physical or mechanical manipulation of the area or lesion, and optionally includes probing, piercing, or penetrating the area or lesion. In some embodiments, physical or mechanical manipulation includes a biopsy. In specific embodiments, the biopsy is performed using a needle or trocar. In certain embodiments, the biopsy includes an incisional biopsy.

[10] In some of any such embodiments, the methods result in an expansion of the genetically engineered cells or an increase in the number of genetically engineered cells compared to the time immediately before the intervention and/or treatment. In specific embodiments, cell expansion occurs within or approximately within 24 hours, 48 hours, 96 hours, 7 days, 14 days, or 28 days after intervention and/or treatment. In certain embodiments, the expansion provides more than or about more than 1.5 times, 2.0 times, 5.0 times, 10 times, 100 times, 200 times or more engineered cells, amenable to detection in the blood, compared with the time immediately before the intervention and/or treatment; or expansion provides more than or about more than 1.5 times, 2.0 times, 5.0 times, 10 times, 100 times, 200 times or more engineered blood detectable cells , compared to previous peak levels of engineered cells in the blood prior to intervention and/or treatment. In specific embodiments, the number of engineered cells detectable in the blood at some point in time after the intervention and/or treatment: increases (e.g., increases by 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times or more) compared to the number of engineered cells at the previous point in time before intervention and/or treatment; more than 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, 100 times or more the peak or maximum number of engineered cells detectable in the blood of an individual prior to intervention and/or treatment; more than or approximately more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1 % of engineered cells are detectable in the blood at the point in time after detection of the peak of the maximum level of such cells in the blood.

[11] In some of any such embodiments, the engineered cells express a recombinant receptor that specifically binds to an antigen associated with a disease or disorder, or expressed in cells of the environment or site of injury. In some of any such embodiments, the disease or condition is a tumor or cancer. In some of any such embodiments, the disease or condition is leukemia or lymphoma. In some of any such embodiments, the disease or condition is non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL). In some of any such embodiments, the recombinant receptor is a T cell receptor or a functional non-T cell receptor. In some of any such embodiments, the recombinant receptor is a chimeric antigen receptor (CAR).

[12] In some embodiments, the implementation of the CAR contains an extracellular antigen recognition domain that specifically binds to the antigen, and an intracellular signaling domain containing ITAM. In certain embodiments, the antigen is CD19. In specific embodiments, the intracellular signaling domain includes the intracellular domain of the CD3-zeta chain (CD3ζ). In some embodiments, the CAR further comprises a costimulatory signaling region. In certain embodiments, the co-stimulatory signaling domain includes the CD28 or 4-1BB signaling domain. In specific embodiments, the engineered cells are CD4+ or CD8+ T cells. In some of any such embodiments, the engineered cells are autologous to the individual.

[13] In some of any such embodiments, the engineered cells are allogeneic to the individual. In some of any such embodiments, the engineered cells are administered in an amount of from about 0.25×10 ⁶ cells/kg of the individual's body weight to 5×10 ⁶ cells/kg, from 0.5×10 ⁶ cells/kg of the individual's body weight up to 3×10 ⁶ cells/kg, from about 0.75×10 ⁶ cells/kg to 2.5×10 ⁶ cells/kg, or from about 1×10 ⁶ cells/kg to 2×10 ⁶ cells/kg, in each case included. In some of any such embodiments, the engineered cells are administered in a single pharmaceutical composition containing the cells. In some of any such embodiments, the engineered ones are administered in a divided dose, where the cells of the dose are administered in a plurality of compositions, collectively containing the cells of the dose, over a period of no more than three days.

[14] It is within the scope of the present invention to provide methods of treatment comprising administering a treatment regimen to an individual, wherein the individual has previously been administered genetically engineered cells to treat a disease or condition, wherein the method results in expansion of the engineered cells in the individual, into a region and/or in a tissue or organ or fluid of an individual, and/or to an increase in engineered cells in a region, tissue or organ or fluid.

[15] In certain embodiments, the treatment regimen includes intervention in an area in an individual in which the modified cells are, or are suspected to be, or have been or are likely to be present, and/or include one or more physical or mechanical manipulations of the lesion or its part, radiation therapy or the introduction of an immunomodulatory agent. In specific embodiments, the treatment regimen and/or method does not include post-administration of genetically engineered cells or genetically engineered cells, and/or expansion is achieved without such post-administration. In some embodiments, the treatment regimen is administered at a subtherapeutic dose and/or provides its therapeutic effect through the expansion of genetically engineered cells. In certain embodiments, the individual has relapsed after responding to and/or has not responded to prior administration of genetically engineered cells. In some embodiments, the individual responded to the genetically engineered cells and then stopped responding and/or relapsed.

Brief description of the drawings

[16] Figure 1A shows peripheral blood CD3 ⁺ /CAR ⁺ T cell counts measured at specific time points post-infusion for individuals grouped by best overall response.

[17] FIGS. 1B-1D show the levels of CD3 ⁺ /CAR ⁺ T cells, CD4 ⁺ /CAR ⁺ T cells, and CD8 ⁺ /CAR ⁺ T cells in peripheral blood measured at specific time intervals post-infusion for individuals who achieved a response, grouped by long-term response after 3 months.

[18] FIG. 2A shows the number of CD3+/CAR+, CD4+/CAR+, CD8+/CAR+ T cells in the peripheral blood of an individual with a chemorefractory transformed DLBCL as determined at specific time points. Figure 2B is a pre-treatment axial PET-CT image showing an intracranial abnormality in the right middle cranial fossa and a marked abnormality in the subcutaneous tissues of the right posterior auditory region. FIG. 2C is a post-treatment PET-CT image showing resolution of the anomaly shown in FIG. 2B after treatment with anti-CD19 CAR+ T cells. Figure 2D is an MRI image of the brain before treatment (T ₁ -weighted high resolution image using a contrast material; axial view) demonstrating a homogeneous hyperdensity mass in the right middle cranial fossa. FIG. 2E is a post-treatment MRI image demonstrating the almost complete resolution of the hyperdensity mass. FIG. 2F is an axial PET-CT image of relapse showing tumor reappearance in the right posterior auditory region associated with intense uptake of ¹⁸ F-fluorodeoxyglucose (arrow). FIG. 2G is a PET-CT image demonstrating resolution of a posterior auditory tumor after incisional biopsy and re-expansion of CAR+ T cells.

[19] Figure 3 shows the percentage of individuals who experienced laboratory abnormalities and adverse events during treatment (TEAE), which occurred in ≥20% of individuals. *: One Grade 5 AE representing multiple organ failure unrelated to study treatment and resulting from progression of lymphoma; †: One Grade 5 AE representing diffuse alveolar injury assessed by the Investigator to be associated with fludarabine, cyclophosphamide, and CAR T cell therapy occurring on day 23 in an individual who was off mechanical ventilation for progressive respiratory failure with growth factor-induced neutropenia and broad-spectrum antibiotics and antifungals.

[20] Figure 4 is a Kaplan-Meier curve depicting the observed time to onset of CRS and neurotoxicity.

[21] FIG. 5A and FIG. 5B shows response rates among subsets of treated individuals.

[22] FIG. 6A and FIG. 6B shows duration of response (CR/PR, CR or PR) and overall survival in the complete and core cohorts of individuals.

[23] FIG. 7A shows the pharmacokinetics of CAR ⁺ T cells in peripheral blood at various time points after treatment with doses at various levels.

[24] FIG. 7B shows the pharmacokinetics of CAR ⁺ T cells in peripheral blood at various time points after treatment between responders and non-responders.

[25] FIG. 7C shows the pharmacokinetics of CAR ⁺ T cells in peripheral blood at various time points post-treatment in individuals who developed or did not develop any neurotoxicity.

[26] Figure 8 shows the levels of test compounds measured in the serum of individuals prior to administration of CAR+ T cells and the correlation with the development of neurotoxicity.

[27] Figure 9 is a graph showing progression-free time (months) showing overall response and duration of response, and individual clinical outcomes observed over time in individuals in the full group and core cohorts of individuals with NHL who were treated with anti-CD19 cell therapy, including CAR-T cells expressing CD4+ and CD8+.^a: Patients achieved BOR at 1 month unless otherwise noted;^b:Complete elimination of CNS involvement in lymphoma observed in 2 patients;^c: One patient experienced re-expansion after biopsy as the disease progressed.

Detailed description

I. Modulation of Genetically Engineered Cells in Adoptive Cell Therapy

[28] The present invention provides methods for modulating engineered cells in vivo, such as facilitating, enhancing or increasing the expansion, proliferation and/or activation of engineered cells administered to an individual. In some embodiments, upon administration of genetically engineered cells, such as cells expressing a recombinant receptor (e.g., CAR+ T cells) to an individual, the methods provided involve intervention, such as manipulation, on a region in the individual, such as a tissue, organ, mass or a region or portion of a lesion of an individual, or a region or portion thereof, in which the engineered cells are present or likely to be present, and/or the administration of a treatment that includes one or more of physical or mechanical manipulation of the lesion or portion thereof, radiation therapy, or the introduction of an immunomodulatory agent. In some embodiments, a region (eg, tissue, organ, mass, or lesion areas of an individual, or a region or portion thereof) is known or suspected to contain antigen-expressing cells that are recognized by genetically engineered cells. In particular, relative to other areas or regions in an individual, a target area is one that is known or suspected to have a higher or increased concentration or amount of antigen or number of antigen-specific cells relative to or compared to other areas in the individual.

[29] In some embodiments, the treatment and/or intervention changes the environment of the area, eg, changes the environment of the lesion, eg, the tumor microenvironment. In some cases, the change is such as to directly or indirectly modulate the activity of engineered T cells. In some aspects, the change is sufficient to allow reactivation, expansion, and/or proliferation in vivo of previously introduced genetically engineered cells, such as recombinant receptor-expressing cells (eg, CAR+ T cells).

[30] T-cell-based therapies, such as adoptive T-cell therapy (including therapy involving the administration of cells expressing chimeric receptors specific to the disease or disorder of interest, such as chimeric antigen receptors (CAR) and/or other recombinant antigen receptors, as well as other adoptive immune cell therapies and adoptive T cell therapies) may be effective in the treatment of cancer and other diseases and disorders. In certain contexts, the available approaches to adoptive cell therapy may not always be completely satisfactory. In some contexts, optimal efficacy may depend on the ability of the administered cells to recognize and bind to the target, eg, the target antigen, transport, localize, and successfully enter appropriate regions in the individual, tumors, and their environment. In some contexts, optimal efficacy may depend on the ability of the injected cells to activate, expand, to exhibit various effector functions, including cytotoxic destruction and secretion of various factors such as cytokines, to persist, including for a long time, to differentiate, go or be involved in reprogramming into certain phenotypic conditions (such as long-term memory, less differentiated state, and effector state) to avoid or reduce immunosuppressive states in the local microenvironment of the disease, to ensure effective and persistent secondary responses after removal and re-exposure to the target ligand or target antigen, and avoid or reduction of exhaustion, anergy, peripheral tolerance, terminal differentiation and/or differentiation into a suppressive state.

[31] In some aspects, the effectiveness of immunotherapy, such as T-cell therapy, may be limited by immunosuppressive activity or factors present in the local microenvironment of the disease or disorder, such as TME. In some aspects, the TME contains or produces factors or conditions that can inhibit the activity, function, proliferation, survival, and/or persistence of T cells administered for T cell therapy.

[32] In some embodiments, the exposure and persistence of the engineered cells is reduced or reduced after administration to an individual. However, observations indicate that in some cases, increased exposure in an individual to administered cells expressing recombinant receptors (eg, increased cell number or duration over time) may increase the efficacy and improve therapeutic outcomes of adoptive cell therapy. Preliminary analyzes following administration of various CD19-targeted CAR-expressing T cells to individuals with various CD19-expressing cancers in a variety of clinical trials have demonstrated a correlation between greater and/or longer exposure to CAR-expressing cells and treatment outcomes. These outcomes included patient survival and remission, even in individuals with severe or significant tumor burden. In some aspects, the safety profile observed with the provided methods may reduce the risk of undesirable safety issues associated with combination therapy involving the therapeutic T cell composition described herein and another therapy for the treatment of a disease or condition, such as an immunomodulatory agent, such as a control point antagonist.

[33] It has been found within the scope of the present invention that interfering with a lesion and/or providing a treatment that includes one or more of physical or mechanical manipulation of a lesion or a portion thereof, radiation therapy, or administration of an immunomodulatory agent in an individual receiving modified T-cell engineering techniques can result in significant cell expansion in an individual even after the individual has relapsed. It is within the scope of the present invention to intervene, such as manipulate, an area in an individual in which cells are or are likely to be present, and/or provide treatment that includes one or more of physical or mechanical manipulation of a lesion or portion thereof, radiation therapy or administration of an immunomodulatory agent. In some embodiments, the area is a lesion, such as a tumor or cancer. In some embodiments, intervention and/or treatment can be carried out by a mechanical or physical change at or near the lesion, e.g., in or near the tumor, or in or near the microenvironment that is associated with the lesion, e.g., the tumor microenvironment ( TME). In some embodiments, intervention and/or treatment may be by administering a pharmacological agent or therapeutic agent, such as an immunomodulatory agent or other agent capable of modulating the activity of T cells, or a cell or cells associated with the lesion or lesion microenvironment. In some cases, the pharmacological agent is a therapeutic agent that targets the area of the lesion or specifically binds to a cell of the lesion, for example, a cell in the tumor microenvironment. In some embodiments, the intervention and/or treatment, such as mechanical intervention or the administration of a pharmacological agent, such as an immunomodulatory agent, is more than or about more than one week, two months, one month, two months, three months, four months, five months, six months, 1 year, 2 years or more after the start of administration of T cells expressing the recombinant receptor, such as CAR+ T cells.

[34] In some embodiments, at or just before the time point of intervention and/or treatment, such as the administration of a pharmacological agent or therapeutic agent, the subject relapsed after remission after treatment.

A. Lesions

[35] In aspects of the methods provided, the region in an individual in which the engineered cells are, or are likely to be, or were, or were likely to be, is a lesion or part of a lesion. In some aspects, a lesion is a lesion known or suspected to contain antigen-expressing cells recognized by cells expressing the recombinant receptor. The contemplated methods are carried out to intervene in a lesion and/or to treat an individual to modulate engineered cells in vivo.

[36] In some embodiments, the lesion includes any area of an organ or tissue that is damaged as a result of injury or disease. In certain embodiments, a lesion is any area of an organ or tissue that has undergone and/or is undergoing an abnormal structural change due to injury or disease. In some embodiments, the lesion is limited and well defined. In some embodiments, the lesion is non-cancerous. In specific embodiments, the lesion is non-neoplastic. In certain embodiments, the lesion is or is suspected to be malignant. In specific embodiments, the lesion is a tumor.

[37] In some embodiments, the lesion is a lesion located in an organ or tissue. In certain embodiments, the lesion is located in connective tissue, muscle tissue, nervous tissue, or epithelial tissue. In certain embodiments, the lesion is in the heart, blood vessels, salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestines, toast, rectum, hypothalamus, pituitary, epiphysis, thyroid, parathyroid, adrenal, kidney , ureter, bladder, urethra, lymphatic system, skin, mice, brain, spinal cord, nerves, ovaries, uterus, testes, prostate, pharynx, larynx, trachea, bronchi, lungs, diaphragm, bones, cartilage, ligaments or tendons.

[38] In certain embodiments, the lesion is selected from: a soft tissue lesion, eg, a lesion, a Morel-Lavalle lesion, a Bankart lesion, a Perthes lesion, a Stener lesion, or a SLAP lesion; a bone lesion, such as a non-ossifying fibroma, an ALPSA lesion, or a Hill-Sachs lesion; a skin lesion, such as a melanocytic nevus, a segmental lesion, or Osler's nodule; blenorrhagic keratoderma, papular dermatosis of blacks, leukemid, Janeway lesion, Kaposi's sarcoma, melasma or chronic cicatricial keratosis; a gastrointestinal lesion, for example a Dieulafoy lesion or a Cameron lesion; endodermal lesions, for example, melanocytic lesions of the oral cavity, endodermal intraepithelial neoplasia; another lesion such as Gon's lesion, benign lymphoepithelial lesion, multiple sclerosis lesion, tropical ulcer, or herpetic panaritium.

[39] In certain embodiments, the lesion is a tumor or neoplasm. In certain embodiments, the tumor is benign. In specific embodiments, the tumor is premalignant or malignant, or is suspected to be malignant or premalignant. In certain embodiments, the tumor is a primary tumor, i. the tumor is located in the anatomical region in which the lesion originally developed or appeared. In some embodiments, the tumor is a secondary tumor, for example, a cancer derived from a cell in a primary tumor located in another area of the body.

[40] In some embodiments, the lesion is associated with or caused by, or suspected to be associated with or caused by, a cancer or proliferative disease, which is a B-cell cancer or a hematologic cancer. In some embodiments, the cancer or proliferative disease is lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), or chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is CLL. In some embodiments, the lesion is associated with or is caused by, or is suspected to be associated with or is caused by, myeloma, lymphoma, or leukemia. In some embodiments, the lesion is associated with or caused by, or suspected to be associated with or caused by, non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML) or myeloma, such as multiple myeloma (MM). In some embodiments, the lesion is associated with or caused by, or is suspected to be associated with or caused by, MM or DBCBL.

[41] In specific embodiments, the lesion is associated with or caused by, or suspected to be associated with or caused by, a non-hematological malignancy, eg, the lesion is a solid tumor. In some embodiments, the lesion is associated with or caused by, or suspected to be associated with or caused by, cancer of the bladder, lung, brain, melanoma (e.g., small cell lung cancer, melanoma), breast, cervix, ovary, colon, and rectum, pancreas, endometrium, esophagus, kidney, liver, prostate, skin, thyroid gland of the body of the uterus. In some embodiments, the lesion is associated with or caused by pancreatic cancer, bladder cancer, colon and rectal cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, cervical cancer, pancreatic cancer. cancer, rectal cancer, thyroid cancer, endometrial cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine malignancies, CNS malignancies, brain tumors, bone cancer, or soft tissue sarcoma.

[42] In specific embodiments, the lesion is a tumor that contains or is suspected to contain at least one malignant cell. In some embodiments, the lesion is a tumor that contains or is suspected to contain a cancer cell derived from AIDS-associated cancer, breast cancer, digestive/gastrointestinal cancer, anal cancer, appendix cancer, bile duct cancer, colon cancer, colon and rectal cancer, esophageal cancer, gallbladder cancer, islet cell tumors, pancreatic neuroendocrine tumors, liver cancer, pancreatic cancer, rectal cancer, small intestine cancer, gastric (gastric) cancer, endocrine cancer systems, adrenal carcinoma, parathyroid cancer, pheochromocytoma, pituitary tumor, thyroid cancer, malignant eye tumor, intraocular melanoma, retinoblastoma, bladder cancer, kidney cancer (renal cell), penile cancer, prostate cancer, transitional cell renal pelvis cancer ureteral cancer, testicular cancer, urethral cancer, Wilms tumor or other childhood kidney tumor, germinoma, malignant tumor of the central nervous system, extracranial germinoma, extragonadal germinoma, ovarian germinoma, gynecological malignancy, cervical cancer, endometrial cancer, gestational trophoblastic tumors, epithelial malignant tumors of the ovary, uterine sarcoma, vaginal cancer, vulvar cancer, head and neck cancer, hypopharyngeal cancer, laryngeal cancer, lip and mouth cancer, metastatic squamous cell neck cancer, nasopharyngeal cancer, oropharyngeal cancer, cancer of the paranasal sinuses and nasal cavity , pharyngeal cancer, salivary gland cancer, throat cancer, musculoskeletal cancer, bone cancer, Ewing's sarcoma, gastrointestinal stromal tumors (GIST), osteosarcoma, malignant fibrous histiocytoma of bone, rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, neurological malignancy , brain tumors, ac trocytomas, brainstem gliomas, atypical teratoid/rhabdoid tumors of the central nervous system, embryonic tumors of the central nervous system, germinomas of the central nervous system, craniopharyngiomas, ependymomas, medulloblastomas, tumors of the spinal cord, supratentorial primitive neuroectodermal tumors and pineoblastoma, neuroblastoma, cancer of the respiratory tract, thoracic cancer, non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, thymoma, thymic carcinoma, skin cancer, Kaposi's sarcoma, melanoma or Merkel cell carcinoma.

B. Treatment and/or intervention at the site of injury

[43] The present invention provides methods for providing treatment and/or intervention to a lesion, such as a tumor, to modulate engineered cells in vivo, such as facilitating, enhancing, or increasing the expansion of engineered cells administered to an individual. In some embodiments, the treatment and/or intervention includes mechanical intervention, such as a biopsy, treatment and/or intervention through radiation, such as external beam irradiation, and/or pharmacological intervention, such as treatment with an immunomodulatory agent.

[44] In some embodiments, treatment and/or intervention at a lesion is any manipulation, procedure, or treatment that alters engineered T cells, such as recombinant receptor-expressing T cells, e.g., CAR+ T cells, directly or indirectly, for example, by changing the microenvironment associated with the lesion. In some embodiments, the manipulation, procedure, or treatment is a mechanical intervention, such as a biopsy. In specific embodiments, the manipulation, procedure, or treatment is the administration of a pharmacological agent to an individual with a lesion. In specific embodiments, the manipulation, procedure, or treatment is the application of radiation to a lesion. In some embodiments, the implementation of treatment and/or intervention, at least initially or immediately reduces the number of cells in the lesion.

[45] In certain embodiments, treating and/or interfering with a lesion includes interfering with an area in an individual in which engineered cells, eg, cells expressing CAR, are or are likely to be present. In some embodiments, interfering with a lesion includes interfering with an area where the engineered cells were ever present, or an area where the engineered cells were likely to be present.

[46] In specific embodiments, the lesion is subjected to treatment and/or intervention to modulate genetically engineered cells in vivo, where the treatment and/or intervention is or results in a change in the lesion or microenvironment that is associated with the lesion, e.g., with the tumor microenvironment (TME). In some embodiments, the implementation of the change may include modification, change, rearrangement or transformation of at least one component of the microenvironment. In certain embodiments, change refers to the modification, alteration, rearrangement, or transformation of a lesion or microenvironment compared to the lesion or microenvironment prior to treatment and/or intervention. In specific embodiments, change refers to the modification, alteration, rearrangement, or transformation of a lesion or microenvironment compared to a similar lesion, e.g., a lesion of the same type, e.g., a tumor type, or a microenvironment of the same tumor type, in which no treatment and/or intervention. In certain embodiments, a similar lesion is located in another individual. In specific embodiments, the implementation of a similar lesion is in the same individual as the lesion subjected to treatment and/or intervention.

[47] In some embodiments, components of the microenvironment include cells within or surrounding the lesion, such as cancer cells, non-cancerous cells, and molecules, such as signaling molecules, that are secreted, released, and/or expressed by cells in the microenvironment. Non-lesion cells may include cells that are not cells of the lesion but are located in the periphery or at the lesion. Non-lesion cells may include, but are not limited to, immune cells, fibroblasts, adipocytes, vascular endothelial cells, pericytes, and lymphatic endothelial cells. Signaling molecules may include, but are not limited to, cytokines, chemokines, growth factors, and inflammatory and matrix remodeling enzymes.

[48] In specific embodiments, the treatment and/or intervention is a manipulation, procedure or treatment that reduces, at least initially or over a period of time, the number of cells or the number of cells of at least one type in the microenvironment of the lesion. In certain embodiments, the lesion is a tumor and the manipulation, procedure or treatment reduces the number of tumor cells in the lesion. In specific embodiments, the lesion is cancerous and the manipulation, procedure, or treatment reduces the number of malignant cells in the lesion. In some embodiments, the treatment and/or intervention is a manipulation, procedure, or treatment that reduces the number of non-lesion cells that are in the microenvironment of the lesion. Non-lesion cells present in the microenvironment of the lesion include, but are not limited to, immune cells, fibroblasts, adipocytes and vascular endothelial cells, pericytes and/or lymphatic endothelial cells that reside in the microenvironment. In certain embodiments, the treatment and/or intervention is a manipulation, procedure, or treatment that results in an increase in immune cells, fibroblasts, adipocytes, and vascular endothelial cells, pericytes, and/or lymphatic endothelial cells in the tumor microenvironment.

[49] In specific embodiments, the treatment and/or intervention is a manipulation, procedure, or treatment that removes or destroys the cells of the lesion, at least initially or for some period of time. In some embodiments, the lesion is a tumor and the manipulation, procedure or treatment destroys or eliminates cells from the lesion, at least initially or for some period of time. In specific embodiments, the lesion is cancerous and the manipulation, procedure, or treatment destroys or eliminates cells from the lesion. In some embodiments, the treatment and/or intervention eliminates or eliminates at least about 0.001%, at least about 0.01%, at least about 0.1%, at least about 1%, at least about 5% at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.9% of the cells in the lesion.

[50] In specific embodiments, the implementation of the treatment and/or intervention is a manipulation, procedure or treatment that leads to a change in the number of one or more types of immune cells in the microenvironment of the lesion. The types of immune cells found in the microenvironment may include, but are not limited to, T lymphocytes, B lymphocytes, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, e.g. macrophages, myeloid suppressor cells (MDSC), dendritic cells, and neutrophils, such as tumor-associated neutrophils (TANs). In certain embodiments, the treatment and/or intervention is a manipulation, procedure, or treatment that increases the number of CD8 ⁺ cells, cytotoxic CD8 ⁺ memory T cells (CD8 ⁺ CD45RO ⁺ ), CD4 ⁺ T helper 1 (T _H 1), CD4 ⁺ T-helper 2 ( _TH 2), natural killer (NK), natural killer T-cells (NKT) and/or γδ T-lymphocytes in the microenvironment of the lesion. In specific embodiments, the treatment and/or intervention is a manipulation, procedure or treatment that reduces the number of TH2 cells, CD4 ⁺ T helper 17 (T _H 17), immunosuppressive regulatory T cells (Treg), regulatory B cells ( Breg), B10 cells and/or tumor associated macrophages (TAM) in the microenvironment of the lesion.

[51] In some embodiments, the treatment and/or intervention is a manipulation, procedure, or intervention that increases the amount of one or more signaling molecules that are present in the lesion and/or the microenvironment of the lesion. In some embodiments, signaling molecules may include, but are not limited to, cytokines, chemokines, growth factors, and inflammatory and matrix remodeling enzymes. In certain embodiments, the manipulation, procedure, or treatment increases interleukin-2 (IL-2), interleukin-17A (IL-17A), interleukin-17F (IL-17F), interleukin-21 (IL-21), interleukin-22 (IL-22), and/or interferon gamma (IFN-γ). In some embodiments, the treatment and/or intervention is a manipulation, procedure, or treatment that increases the amount of a signaling molecule that is present at the lesion and/or the microenvironment of the lesion by at least about 1%, at least about 5%. , at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least at least about 95%, at least about 1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times , at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 500 times, or at least about 1000 times.

[52] In some embodiments, the treatment and/or intervention is a manipulation, procedure, or treatment that increases the amount of one or more signaling molecules that are present in the lesion and/or the microenvironment of the lesion. In certain embodiments, the manipulation, procedure, or treatment reduces interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-17A (IL-17A), interleukin-17F (IL-17F), interleukin-21 (IL-21), interleukin-22 (IL-22), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF ), endothelin-1, endothelin-2, endothelin-3, endothelial monocyte-activating polypeptide II (EMAP2, also known as AIMP1), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), TGF-β, C-X-C motif chemokine 12 (CXCL12), platelet-derived growth factor (PDGF), matrix metalloproteinase (MMP), and/or cathepsins, e.g., cathepsin L. In some embodiments, treatment and/ or an intervention is a manipulation, procedure, or treatment that reduces the amount of a signaling molecule at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20% , at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.9%, or about 100 %.

1. Mechanical intervention

[53] In some embodiments, treatment and/or intervention involves physical or mechanical manipulation of an area, such as a lesion, for example, by probing, piercing, and/or penetrating the lesion. In some embodiments, treatment and/or intervention is performed by biopsy of the area, such as a biopsy of a lesion (eg, tumor). In some embodiments, the biopsy is performed with a needle. In some embodiments, the biopsy is an incisional biopsy.

[54] In some embodiments, physical or mechanical intervention is performed at the lesion using a biopsy procedure to modulate engineered cells in vivo, for example, to facilitate, enhance, or increase the expansion of genetically engineered cells administered to an individual. In some embodiments, the biopsy procedure is a fine needle aspiration that uses a long thin needle that can be inserted into the lesion and a syringe to collect cells and/or fluid from the lesion. In specific embodiments, the biopsy is a core biopsy in which a larger, cutting-tipped needle is used to remove a tissue column from the lesion. In specific embodiments, the biopsy is a vacuum biopsy that uses a suction device to increase the amount of fluid and/or cells that are removed through the needle. In certain embodiments, the biopsy is an image-guided biopsy in which the lesion is imaged using imaging modalities, including, but not limited to, x-ray, ultrasound, CT scan, or MRI scan, to facilitate visualization by a healthcare professional, such as a physician, the lesion and directing the biopsy device, such as a needle, into the tumor.

[55] In specific embodiments, intervention at the lesion is performed using one or more biopsy devices (eg, needles) to modulate genetically engineered cells in vitro. In some embodiments, the biopsy device is a bovine biopsy needle. In certain embodiments, the biopsy device is a needle that can be used for fine needle aspiration. In certain embodiments, the biopsy device is a trocar.

[56] In specific embodiments, the biopsy device is a bovine biopsy needle. In some embodiments, the core biopsy needle is 10 gauge, 11 gauge, 12 gauge, 13 gauge, 14 gauge, 15 gauge, 16 gauge, 17 gauge, 18 gauge, 19 gauge, 20 gauge, 21 gauge, 22 gauge, 23 gauge , 24 gauge, 25 gauge, or 26 gauge. In certain embodiments, the core biopsy needle has a gauge between 10 and 30, between 10 and 24, or between 14 and 20. In certain embodiments, the needle has a length of about 10 cm, about 11 cm , approximately 12 cm, approximately 13 cm, approximately 14 cm, approximately 15 cm, approximately 16 cm, approximately 17 cm, approximately 18 cm, approximately 19 cm, approximately 20 cm, approximately 21 cm, approximately 22 cm, approximately 23 cm, approximately 24 cm, approximately 25 cm, approximately 26 cm, approximately 27 cm, approximately 28 cm, approximately 29 cm, approximately 30 cm, approximately 31 cm, or approximately 32 cm. The biopsy needle is about 5 cm to about 30 cm long, about 10 cm to about 25 cm long, or about 10 cm to about 20 cm long. In certain embodiments, the core biopsy needle is between 14 and 20 gauge and is about 10 cm to 20 cm. In particular embodiments, the bovine biopsy needle is disposable. In certain embodiments, the bovine biopsy needle is reusable.

[57] In some embodiments, intervention in the lesion is carried out by means of fine needle aspiration. In specific embodiments, the implementation of the needle is a thin needle. In some embodiments, the needle that can be used for fine needle aspiration is 20 gauge, 21 gauge, 22 gauge, 23 gauge, 24 gauge, 25 gauge, 26 gauge, 27 gauge, 28 gauge, 29 gauge, 30 gauge, 31 gauge, or 32 gauge. In certain embodiments, the needle has a gauge between 20 and 30, between 22 and 28, between 20 and 26, or between about 24 and about 28. In certain embodiments, the needle can be used for fine needle aspiration and is about 1 cm long. , approximately 2 cm, approximately 3 cm, approximately 4 cm, approximately 5 cm, approximately 6 cm, approximately 7 cm, approximately 8 cm, approximately 9 cm, approximately 10 cm, approximately 12 cm, approximately 14 cm, approximately 16 cm, approximately 18 cm or about 20 cm. In some embodiments, the needle can be used for fine needle aspiration and is about 1 cm to about 10 cm in size, from approx. 5 cm to about 10 cm, or about 1 cm to about 5 cm. In certain embodiments, the needle can be used for fine needle aspiration and has a gauge between 22 and 28 and a length of 1 cm to 10 cm.

[58] In some embodiments, intervention in the lesion is performed using a trocar or using a trocar to modulate genetically engineered cells in vivo. Trocars are often used for laparoscopic surgical techniques, for example, to gain or provide access to a body cavity, such as the abdominal cavity. A conventional trocar may include, for example, an obturator, a sharp trocar, a cannula, and a protective septum to protect organs after the trocar has entered the abdominal cavity. The baffle is usually designed as a mechanical device that is spring loaded and activated when the trocar tip is inserted into the cannula. The tip of the trocar is protected by a protective septum. The trocar passes through the layers of the abdominal cavity wall, the protective septum is pulled back, exposing the sharp tip of the trocar. When the device finally passes through the last layer of abdominal tissue and just before entering the open space of the abdominal cavity, the protective septum moves forward to close the trocar tip again.

[59] Examples of trocars include bladed trocars, which include a tip with a blade, and blunt trocars. The type of bladed trocar tip that is most commonly used has a three-sided pyramidal design that facilitates entry into tissue through three sharp edges that can cut through tissue, such as abdominal wall tissue. Bladed trocars also include hybrid tip trocars. Hybrid tips have smaller linear guide blades for making incisions, which are then widened with a blunt trocar component. Blunt trocars are designed to enter the cavity without a blade tip. Blunt trocars include radially expanding trocars that are designed to enter tissue after a small incision is made with another instrument, such as a scalpel. In some embodiments, intervention in the lesion is carried out with or with a bladed trocar. In certain embodiments, intervention in the lesion is performed with or by means of a blunt trocar.

[60] In some embodiments, intervention in the lesion is performed using a trocar that has a diameter of at least or about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm , approximately 8 mm, approximately 9 mm, approximately 10 mm, approximately 11 mm, approximately 12 mm, approximately 13 mm, approximately 14 mm, approximately 15 mm, approximately 16 mm, approximately 17 mm, approximately 18 mm, approximately 19 mm, or approximately 20 mm. In certain embodiments, the trocar has a diameter of from about 1 mm to about 20 mm, from about 1 mm to about 15 mm, from about 5 mm to about 15 mm, from about 10 mm to about 15 mm, or from about 5 mm to about 12 mm. In certain embodiments, the implementation of the trocar has a diameter of from about 5 mm to about 12 mm.

[61] In certain embodiments, intervention in the lesion is performed by needle biopsy to modulate engineered cells in vivo. In some embodiments, the needle biopsy is performed with a disc-shaped blade that can be rotated down tissue to collect a cylindrical, columnar tissue sample, such as a skin sample. In certain embodiments, the puncture therapy needle has a diameter of at least or about 0.1 mm, about 0.5 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, approx 7 mm approx 8 mm approx 9 mm approx 10 mm approx 11 mm approx 12 mm approx 13 mm approx 14 mm approx 15 mm approx 16 mm approx 17 mm approx 18 mm approx 19 mm, or approximately 20 mm. In certain embodiments, the puncture therapy needle has a diameter of from about 1 mm to about 8 mm.

[62] In some embodiments, intervention in the lesion is performed by excisional biopsy. In certain embodiments, excisional biopsy involves removal of all or most of the lesion. In specific embodiments, excisional biopsy comprises removing at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99 .9%, or approximately 100% of the lesion.

[63] In specific embodiments, intervention in the lesion is performed using incisional biopsy. In certain embodiments, an incisional biopsy comprises removing at least a portion of a lesion. In certain embodiments, an incisional biopsy comprises extracting at least about 0.01%, at least about 0.1%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85 %, at least about 90%, or at least about 95% of the lesion. In specific embodiments, an incisional biopsy comprises a recovery of less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 1%, less than about 0.5% or less than about 0.1% of the lesion.

[64] In some embodiments, intervention in the lesion is carried out by piercing, piercing, cutting, tearing, dissecting, opening, cutting, shaving and/or cutting the lesion with a surgical instrument, such as a trocar, knife or needle. In specific embodiments, piercing, piercing, cutting, tearing, cutting, opening, cutting, shaving, and/or shearing the lesion results in injury to the lesion. For example, in some embodiments, the injury includes a puncture, puncture, shear, cut, or tear. In some embodiments, tampering with the lesion results in injury to the lesion, which includes a puncture, puncture, shear, cut, or tear that is less than or about 0.001 mm, about 0.01 mm, about 0.1 mm, about 0 .2 mm, approximately 0.3 mm, approximately 0.4 mm, approximately 0.5 mm, approximately 0.6 mm, approximately 0.7 mm, approximately 0.8 mm, approximately 0.9 mm, approximately 1 mm, approx 1.1 mm approx 1.2 mm approx 1.3 mm approx 1.4 mm approx 1.5 mm approx 1.6 mm approx 1.7 mm approx 1.8 mm approx 1 .9 mm, approximately 2 mm, approximately 2.5 mm, approximately 3 mm, approximately 4 mm, approximately 5 mm, approximately 6 mm, approximately 7 mm, approximately 8 mm, approximately 9 mm, approximately 1 cm, approximately 2 cm, approx 3 cm, approx 4 cm, approx 5 cm, approx. 6 cm, about 7 cm, about 8 cm, about 9 cm, or about 10 cm. piercing, piercing, cutting, tearing, dissecting, opening, slicing, shaving, and/or shearing the lesion with a surgical instrument, resulting in a puncture, puncture, shear, cut, or tear of the lesion that is between about 0.001 mm and about 0 .1 mm, from about 0.1 mm to about 1 mm, from about 1 mm to about 1 cm, or from about 1 cm to about 10 cm.

[65] In some embodiments, the implementation of mechanical intervention is carried out by changing the temperature of the lesion, for example, through thermotherapy. In specific embodiments, mechanical intervention is performed using cryoablation. In certain embodiments, the mechanical intervention is hyperthermia therapy.

[66] In certain embodiments, intervention in the lesion is performed using cryoablation. Cryoablation involves freezing the neoplastic mass, which results in the deposition of intracellular and extracellular ice crystals; destruction of cell membranes, proteins and organelles; and inducing a hyperosmotic environment, thereby causing cell death. Methods and devices suitable for cryoablation are described in Murphy et al, Sent. Urol. oncol. 79:133-140 (2001) and US Pat. Nos. 6,383,181; 6,383,180; 5,993,444; 5,654,279;

[67] In specific embodiments, intervention in the lesion is carried out using hyperthermia therapy. Hyperthermia therapy typically involves raising the temperature of the neoplastic mass to a range of about 42°C to about 44°C. The temperature of the lesion can be further increased above this range; however, such temperatures can increase damage to surrounding healthy tissue without causing increased cell death at the site of injury being treated. The tumor can be heated in hyperthermia therapy by any means known to the person skilled in the art. In some embodiments, the lesion may be heated by microwave radiation, high intensity focused ultrasound radiation, ferromagnetic thermal primers, localized current fields, infrared radiation, wet or dry RF albation, laser photocoagulation, laser interstitial thermal therapy, and electrocaustics. Microwaves and radio waves can be generated by waveguide applicators, horn applicators, spiral applicators, current sheet applicators and compact applicators.

[68] Other methods, devices, and compositions for raising the temperature of a lesion, such as a tumor, are discussed in Wust et al, Lancet Oncol. 3:487-97 (2002), and are described in US Pat.

2. Treatment and/or intervention by irradiation

[69] In certain embodiments, the lesion is treated and/or interfered with by radiation or radiation therapy to modulate genetically engineered cells in vivo. In certain embodiments, radiotherapy uses high energy radiation to reduce the size of a lesion, such as a tumor, and kills cells in the lesion, such as cancerous cells. In some embodiments, the treatment and/or intervention in the lesion is carried out using ionizing radiation, i.e. radiation involving particles or photons of sufficient energy, and it can provide sufficient energy through nuclear interactions to provide ionization (gain or loss of electrons). In some embodiments, treatment and/or intervention at the lesion is by exposing the lesion to x-rays, gamma rays, charged particles such as electrons, or any other type of radiation that can be used to treat cancer.

[70] Radiation therapy may include any source of therapeutic radiation used to treat cancer and/or a related disease, including, but not limited to, ionizing radiation therapy, brachytherapy, close focus radiation therapy, systemic radioisotope therapy, open source radiation therapy. , radionuclide therapy, external beam radiation therapy, radiation surgery, charged particle radiation therapy, neutron radiation therapy, x-ray therapy, gamma ray therapy and cobalt therapy.

[71] In certain embodiments, treatment and/or intervention at the lesion is carried out by external beam therapy (EBT), in which an external source of ionizing radiation is applied to the area of the body of the individual that contains the lesion. In some embodiments, the implementation of the EBT includes orthovoltage (ie, surface) beams of radiation for the treatment and/or intervention in the lesion located on the skin. In certain embodiments, EBT includes megavoltage, eg, deep beams of radiation that are used to treat internal lesions, eg, lesions in the bladder, intestines, prostate, lung, or brain. In specific embodiments, treatment and/or intervention at a lesion by EBT includes delivering x-rays, gamma rays, electron beams, proton beams, or ionized nuclear beams to the lesion. In some embodiments, the treatment and/or intervention in the lesion is carried out by EBT, which is carried out using a linear accelerator, a collimator, a cobalt device, a surface radiation therapy (SRT) device, an orthovoltage x-ray device.

[72] In some embodiments, the treatment and/or intervention in the lesion is carried out using internal radiation therapy, i. brachytherapy. In certain embodiments, the implementation of brachytherapy includes the use of radiation sources in or near the area of the lesion. In specific embodiments, the implementation of brachytherapy includes interstitial radiation, where the radiation source is contained in small beads, grains, wires, tubes and/or containers and is placed directly at or near the lesion. In certain embodiments, brachytherapy includes intracavitary radiation, where a container of radioactive material is placed in a body cavity, such as the chest cavity or colon. In some embodiments, ultrasound, X-ray, and/or CT scanning is used to facilitate placement of the radioactive source.

[73] In some embodiments, treatment and/or intervention at the lesion is performed with permanent brachytherapy, which includes placing small containers, eg, containers approximately the size of a grain of rice, into the lesion. In some embodiments, the containers emit radiation for a period of several weeks or months, and they remain in place after radiation ceases.

[74] In specific embodiments, the treatment and/or intervention in the lesion is carried out using temporary brachytherapy, which includes the placement of cylinders, hollow needles, tubes (catheters) and/or fluid-filled balloons in the area being treated, which are then removed after treatment . In some embodiments, radioactive materials are placed in these containers for a short period of time and then removed. In some embodiments, temporary brachytherapy may be high dose rate (HDR) brachytherapy where the radiation source is placed for a few minutes at a time at or near the lesion and then removed. This process can be repeated twice a day for up to a week, or once a week for several weeks. In some embodiments, the temporary brachytherapy is low dose rate (LDR) brachytherapy where the radiation source is left in place for up to 7 days prior to removal.

[75] In some embodiments, the implementation of the treatment and/or intervention in the lesion is carried out through systemic radiation therapy. In some embodiments, systemic radiation therapy includes the administration of radioactive substances, such as radioactive iodine, which travel through the blood to destroy cells at the site of injury. Representative radioisotopes that can be administered in radionuclide therapy include, but are not limited to, phosphorus-32, yttrium-90, dysprosium-165, indium-111, strontium-89, samarium-153, rhenium-186, iodine-131, iodine -125, lutetium-177 and bismuth-213. While all of these radioisotopes can be linked to a biomolecule providing targeting specificity, iodine-131, indium-111, phosphorus-32, samarium-153 and rhenium-186 can be administered systemically without such conjugation. One skilled in the art can select a particular biomolecule to use to target a particular neoplasm for radionuclide therapy based on the cell surface molecules present on that neoplasm. In some embodiments, the radioactive particle is conjugated to a monoclonal antibody, or active fragment or variant thereof, that binds to a cell at the site of injury. Examples of radiopharmaceuticals include, but are not limited to, ibritutomaba tiuxetan, an anti-CD20 monoclonal antibody conjugated to either yttrium-90 or indium-111; and tositumomab, an anti-CD20 monoclonal antibody that is conjugated to iodine-131.

3. Pharmacological treatment and/or intervention

[76] In some embodiments, treatment and/or intervention at a lesion is performed to modulate engineered cells in vivo, e.g., to facilitate, enhance, or increase the expansion of engineered cells administered to an individual by administering an agent to the individual. In specific embodiments, the agent is a pharmaceutical agent. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is an immunomodulatory agent. In specific embodiments, the agent is a chemotherapeutic agent.

[77] In some embodiments, an agent, such as an immunomodulatory agent, is capable of inhibiting or blocking the function of a molecule or a signal transduction cascade involving said molecule. In some embodiments, the molecule is expressed on immune cells or is part of an immune synapse, such as being expressed on a T cell or an antigen presenting cell, or other cell associated with an immune response. In certain such aspects, the molecule is an immune inhibitory molecule or an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule or cascade is PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM3, VISTA, adenosine 2A receptor (A2AR), or adenosine, or a cascade involving any of the above.

[78] In some embodiments, the chemotherapeutic agent is or contains an antibody, which may be an antibody fragment, a single chain antibody, a multispecific antibody, or an immunoconjugate. In some embodiments, the antibody specifically binds to an immune checkpoint molecule, or ligand or receptor thereof. In some embodiments, the antibody is capable of blocking or disrupting the interaction between an immune control point molecule and its ligand or receptor.

[79] In some embodiments, treatment and/or intervention at the site of injury is carried out to modulate genetically engineered cells in vivo by administering an immunomodulatory agent to an individual. In some embodiments, an immunomodulatory agent blocks, inhibits, or antagonizes a component of the immune checkpoint cascade. The immune system has many inhibitory pathways that are involved in maintaining self tolerance and in modulating immune responses. Tumors may use certain immune checkpoint cascades as the main mechanism of immune resistance, particularly against T cells that are specific for tumor antigens, such as those modified by cell engineering techniques such as CAR-expressing cells (Pardoll (2012) Nature Reviews Cancer 12:252-264). Because many of these immune checkpoints are initiated by ligand-receptor interactions, they can be readily blocked by anti-ligand antibodies and/or their receptors. In contrast to most cancer drugs, checkpoint inhibitors do not necessarily target tumor cells directly, but rather target lymphocyte receptors or their ligands to enhance the immune system's endogenous antitumor activity.

[80] In specific embodiments, the implementation of treatment and/or intervention in the lesion is carried out by introducing an inhibitor of the immune control point to the individual. In some embodiments, an immune checkpoint inhibitor is a molecule that wholly or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. In some embodiments, the control point protein is any protein that regulates T cell activation or function and/or is responsible for a costimulatory or inhibitory interaction with a T cell response. Immune control point proteins regulate and maintain self-tolerance and the duration and amplitude of the physiological immune response. Immune checkpoint inhibitors include any agent that blocks, inhibits or reduces the activity or function of inhibitory cascades of the immune system. Such inhibitors may include small molecule inhibitors, or may include antibodies, or antigen-binding fragments thereof, that bind to and block or inhibit immune checkpoint receptors, ligands, and/or receptor-ligand interaction. In some embodiments, modulation, enhancement, and/or stimulation of specific receptors may override components of the immune checkpoint cascade. Exemplary immune control point molecules that can be targeted to block, inhibit, modulate, enhance and/or stimulate include, but are not limited to, PD-1 (CD279), PD-L1 (CD274, B7-H1), PDL2 (CD273, B7-DC), CTLA-4, LAG-3 (CD223), TIM-3, 4-1BB (CD137), 4-1BBL (CD137L), GITR (TNFRSF18, AITR), CD40, OX40 (CD134, TNFRSF4), CXCR2, tumor associated antigens (TAA), B7-H3, B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK cells, T γδ cells and CD8+ memory T cells (αβ)), CD160 (also referred to as BY55), CGEN-15049, CEACAM (e.g. CEACAM-1, CEACAM-3 and/or CEACAM-5), TIGIT, LAIR1, CD160 , 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and transforming growth factor receptor (TGFR) ; for example, TGFR-beta). Immune checkpoint inhibitors include antibodies, or antigen-binding fragments thereof, or other binding proteins that bind and block or inhibit and/or enhance or stimulate one or more of any of the above molecules.

[81] Exemplary immune checkpoint inhibitors include tremelimumab (an antibody that blocks CTLA-4, also known as ticilimumab, CP-675,206), an anti-OX40 antibody, a monoclonal antibody against PD-L1 (an anti-B7-H1 antibody; MEDI4736, also called durvalumab ), MK-3475 (PD-1 blocker), nivolumab (anti-PD-1 antibody), CT-011 (anti-PD-1 antibody), BY55 monoclonal antibody, AMP224 (anti-PD-L1 antibody), BMS-936559 (anti-PD-1 antibody), anti-PD-L1), MPLDL3280A (anti-PD-L1 antibody), MSB0010718C (anti-PD-L1 antibody) and ipilimumab (anti-CTLA-4 antibody also known as Yervoy®, MDX-010 and MDX-101). Illustrative immunomodulatory antibodies include, but are not limited to, daclizumab (Zenapax), Bevacizumab (AVASTIN ®), Basiliximab, Ipilimumab, Nivolumab, pembrolizumab, MPDL3280A, Pidilizumab (CT-011), MK-3475, BMS-936559, MPDL3280A (Atezolizumab) , tremelimumab, IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, MK-4166, dacetuzumab (SGN-40), lucatumumab (HCD122), SEA-CD40, CP-870, CP-893, MEDI6469, MEDI6383 , MOXR0916, AMP-224, MSB0010718C (avelumab), MEDI4736 (durvalumab), PDR001, rHIgM12B7, Ulokuplumab, BKT140, Varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2201), IRG2201 -115, Emactuzumab, CC-90002 and MNRP1685A, or an antigen binding fragment thereof. Other illustrative immunomodulators include, for example, afutuzumab (available from ROCHE®); pegfilgrastim (NEULASTA®); lenalidomide (CC-5013, REVLIMID®); thalidomide (THALOMID®), actimide (CC4047); and IRX-2 (a mixture of human cytokines, including interleukin 1, interleukin 2, and interferon-gamma, CAS 951209-71-5, available from IRX Therapeutics).

[82] In some embodiments, treatment and/or intervention at the site of injury is carried out by administering to the individual an immunomodulatory agent that binds and/or inhibits programmed cell death 1 (PD-1) protein. PD-1 is an immune checkpoint protein that is expressed in B cells, NK cells, and T cells (Shinohara et al., 1995, Genomics 23:704-6; Blank et al., 2007, Cancer Immunol Immunother 56 :739-45; Finger et al., 1997, Gene 197:177-87; Pardoll (2012) Nature Reviews Cancer 12:252-264). The main role of PD-1 is to limit the activity of T cells in peripheral tissues during inflammation in response to infection, as well as to limit autoimmunity. Expression of PD-1 is induced in activated T cells and binding of PD-1 to one of its endogenous ligands inhibits T cell activation by inhibiting stimulatory kinases. PD-1 also inhibits the TCR "stop signal". PD-1 is highly expressed on Treg cells and can increase their proliferation in the presence of a ligand (Pardoll (2012) Nature Reviews Cancer 12:252-264). Anti-PD 1 antibodies are used to treat melanoma, non-small cell lung cancer, bladder cancer, prostate cancer, colon and rectal cancer, head and neck cancer, triple negative breast cancer, leukemia, lymphoma, and renal cell cancer (Topalian et al ., 2012, N Engl J Med 366:2443-54; Lipson et al., 2013, Clin Cancer Res 19:462-8; Berger et al., 2008, Clin Cancer Res 14:3044-51; Gildener-Leapman et al., 2013, Oral Oncol 49:1089-96; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85). In some embodiments, treatment and/or intervention at the lesion is carried out by administering an anti-PD-1 antibody or antigen-binding fragment thereof to an individual. Exemplary anti-PD-1 antibodies include nivolumab (Opdivo from BMS), pembrolizumab (Keytruda from Merck), pidilizumab (CT-011 from Cure Tech), lambrolizumab (MK-3475 from Merck) and AMP-224 (Merck), nivolumab (also referred to as Opdivo, BMS-936558 or MDX1106; Bristol-Myers Squibb), which is a fully human IgG4 monoclonal antibody that specifically blocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are described in US 8008449 and WO2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are described in WO2009/101611. Pembrolizumab (formerly known as lambrolizumab and also called Keytruda, MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are described in US 8354509 and WO2009/114335. Other anti-PD-1 antibodies include AMP 514 (Amplimmune), among others, for example, anti-PD-1 antibodies described in US 8609089, US 2010028330, US 20120114649 and/or US 20150210769. AMP-224 (B7-DCIg; Amplimmune; eg described in WO2010/027827 and WO2011/066342) is a soluble PD-L2 and Fc fusion receptor that blocks the interaction between PD-1 and B7-H1.

[83] In certain embodiments, treatment and/or intervention at the site of injury is carried out by administering to the individual an immunomodulatory agent that binds or inhibits PD-L1 (also known as CD274 and B7-H1) and/or PD-L2 (also known as CD273 and B7-DC). PD-L1 and PD-L2 are PD-1 ligands present on activated T cells, B cells, myeloid cells, macrophages, and some types of tumor cells. Anticancer therapy focuses on antibodies against PD-L1. The complex of PD-1 and PD-L1 inhibits CD8 ⁺ T cell proliferation and reduces the immune response (Topalian et al., 2012, N Engl J Med 366:2443-54; Brahmer et al., 2012, N Eng J Med 366: 2455-65). Anti-PD-L1 antibodies are used to treat non-small cell lung cancer, melanoma, colorectal cancer, renal cell cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, and hematological malignancies (Brahmer et al., 2012, N Eng J Med 366:2455-65; Ott et al., 2013, Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res 19:5541; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Berger et al., 2008, Clin Cancer Res 14:13044-51). In certain embodiments, treatment and/or intervention at the site of injury is carried out by administering to the individual an anti-PD-L1 antibody or an antigen-binding fragment thereof. Exemplary anti-PD-L1 antibodies include MDX-1105 (Medarex), MEDI4736 (durvalumab, Medimmune), MPDL3280A (Genentech), BMS-935559 (Bristol-Myers Squibb), and MSB0010718C.

[84] In some embodiments, the implementation of the immunomodulatory agent is an antibody against PD-L1. An exemplary anti-PD-L1 antibody is MEDI4736 (durvalumab, Medimmune), which is a human monoclonal antibody that binds to PD-L1 and inhibits ligand interaction with PD-1 (see US Pat. No. 8,779,108). In some embodiments, the immunomodulatory agent is MDPL3280A (Genentech/Roche), which is an Fc region optimized human IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are described in US Pat. No. 7,943,743 and US Publication No. 20120039906. Other PD-L1 binding molecules include YW243.55.S70 (see WO2010/077634), MDX-1105 (also referred to as BMS -936559, and, for example, PD-L1 binding molecules described in WO2007/005874), LY3300054 (see US2017/0058033), atezolizumab (see US Patent No. 8217149) and avelumab (US Patent No. 9624298).

[85] In certain embodiments, treatment and/or intervention at the lesion is carried out by administering an inhibitor of cytotoxic T-lymphocyte-associated antigen (CTLA-4), also known as CD152. CTLA-4 is a co-inhibitory molecule that functions to regulate T cell activation. CTLA-4 is a member of the immunoglobulin superfamily that is exclusively expressed on T cells. CTLA-4 inhibits T cell activation and has been reported to inhibit helper T cell activity and enhance the immunosuppressive activity of regulatory T cells. Although the exact mechanism of action of CTLA-4 remains unexplored, it has been suggested that it inhibits T cell activation by displacing CD28 from binding to CD80 and CD86, as well as actively delivering inhibitory signals to the T cell (Pardoll (2012) Nature Reviews Cancer 12:252 -264). Anti-CTLA-4 antibodies are used in clinical trials to treat melanoma, prostate cancer, small cell lung cancer, non-small cell lung cancer (Robert & Ghiringhelli, 2009, Oncologist 14:848-61; Ott et al., 2013, Clin Cancer Res 19: 5300; Weber, 2007, Oncologist 12:864-72; Wada et al., 2013, J Transl Med 11:89). A characteristic feature of the anti-CTLA-4 antibody is the kinetics of the antitumor effect with a delay period of up to 6 months after the initial treatment required for a physiological response. In some cases, tumors may actually increase in size after starting treatment before shrinking is observed (Pardoll (2012) Nature Reviews Cancer 12:252-264). In certain embodiments, treatment and/or intervention at the lesion is carried out by administering an anti-CTLA-4 antibody, or antigen-binding fragment thereof, to the subject. Exemplary anti-CTLA-4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (Pfizer). Ipilimumab has recently been approved by the FDA for the treatment of metastatic melanoma (Wada et al., 2013, J Transl Med 11:89).

[86] In certain embodiments, treatment and/or intervention at the lesion is carried out by administering an immunomodulatory agent that binds and/or inhibits the lymphocyte activation gene-3 (LAG-3), also known as CD223. LAG-3 is an immune checkpoint protein that is associated with inhibition of lymphocyte activity and in some cases induction of lymphocyte anergy. LAG-3 is expressed on various cells of the immune system, including B cells, NK cells, and dendritic cells. LAG-3 is a natural ligand for the class II MHC receptor, which is highly expressed on melanoma-infiltrating T cells, including cells with potent immunosuppressive activity. In certain embodiments, treatment and/or intervention at the lesion is carried out by administering an anti-LAG-3 antibody or an antigen-binding fragment thereof to an individual. Exemplary anti-LAG-3 antibodies include BMS-986016 (Bristol-Myers Squib), which is a monoclonal antibody that targets LAG-3. IMP701 (Immutep) is a LAG-3 antagonist antibody and IMP731 (Immutep and GlaxoSmithKline) is a LAG-3 depleting antibody. Other LAG-3 inhibitors include IMP321 (Immutep), which is a recombinant LAG-3 soluble portion fusion protein, and Ig, which binds to MHC class II molecules and activates antigen presenting cells (APCs). Other antibodies are described, for example, in WO2010/019570 and US 2015/0259420.

[87] In some embodiments, treatment and/or intervention at the lesion is carried out by administering an immunomodulatory agent that binds and/or inhibits T-cell immunoglobulin and mucin domain 3 (TIM-3). TIM-3, originally identified on activated Th1 cells, has been shown to be an inhibitor of the immune response. Blockade of TIM-3 stimulates T-cell mediated antitumor immunity and has antitumor activity in a number of mouse tumor models. Combinations of TIM-3 blockade with other immunotherapeutic agents such as TSR-042, anti-CD137 antibodies, and others may be additive or synergistic in increasing antitumor effects. TIM-3 expression is associated with a number of different tumor types, including melanoma, NSCLC, and renal cancer, and, in addition, intratumoral TIM-3 expression has been shown to correlate with poor prognosis in a number of tumor types, including NSCLC, cervical cancer, and gastric cancer. . Blockade of TIM-3 is also of interest in terms of stimulating increased immunity to a number of chronic viral diseases. TIM-3 has been shown to interact with a number of ligands, including galectin-9, phosphatidylserine, and HMGB1, although it is currently unclear which, if any, of these are involved in the regulation of antitumor responses. In some embodiments, antibodies, antibody fragments, small molecules, or peptide inhibitors that target TIM-3 can bind to the IgV domain of TIM-3, inhibiting interactions with its ligands. In some embodiments, treatment and/or intervention in the lesion is carried out by administering an antibody, or antigen-binding fragment or peptide thereof, that binds and/or inhibits TIM-3. Exemplary antibodies and peptides that inhibit TIM-3 are described in US 2015/0218274, WO2013/006490 and US 2010/0247521. Other anti-TIM-3 antibodies include humanized versions of RMT3-23 (Ngiow et al., 2011, Cancer Res, 71:3540-3551) and clone 8B.2C12 (Monney et al., 2002, Nature, 415:536-541) . Bispecific antibodies that inhibit TIM-3 and PD-1 are described in US 2013/0156774.

[88] In some embodiments, treatment and/or intervention at the lesion is carried out by administering to the individual a CEACAM inhibitor (eg, a CEACAM-1, CEACAM-3, and/or CEACAM-5 inhibitor). In certain embodiments, the CEACAM inhibitor is an anti-CEACAM antibody, or an antigen-binding fragment or variant thereof. Illustrative antibodies against CEACAM-1 are described in WO 2010/125571, WO 2013/082366 WO 2014/059251 and WO 2014/022332, for example, monoclonal antibody 34B1, 26H7 and 5F4; or their recombinant form, as described, for example, in US 2004/0047858, US 7132255 and WO 99/052552. In some embodiments, an anti-CEACAM antibody binds to CEACAM-5 as described, for example, in Zheng et al. PLOS One. (2011) 6(6): e21146) or cross-reacts with CEACAM-1 and CEACAM-5 as described in WO 2013/054331 and US 2014/0271618, for example.

[89] In certain embodiments, treatment and/or intervention at the lesion is carried out by administering an immunomodulatory agent that binds and/or inhibits 4-1BB, also known as CD137. 4-1BB is a transmembrane glycoprotein belonging to the TNFR superfamily. 4-1BB receptors are found on activated T cells and B cells and monocytes. In some embodiments, the anti-4-1BB antibody, or antigen-binding fragment thereof, is administered to an individual for treatment and/or intervention at a lesion. An exemplary anti-4-1BB antibody is urelumab (BMS-663513), which has potential immunostimulatory and antineoplastic activity.

[90] In some embodiments, treatment and/or intervention at the lesion is carried out by administering an immunomodulatory agent that is a structural or functional analog or derivative of thalidomide and/or an inhibitor of E3 ubiquitin ligase. In some embodiments, an immunomodulatory agent binds to cereblon (CRBN). In some embodiments, the immunomodulatory agent binds to the CRBN-ubiquitin ligase E3 complex. In some embodiments, the immunomodulatory agent binds to CRBN and the CRBN-ubiquitin ligase E3 complex. In some embodiments, an immunomodulatory agent activates the expression of a CRBN protein or gene. In some aspects, CRBN is a substrate adapter for CRL4 ^CRBN E3 ubiquitin ligase, and modulates the specificity of this enzyme. In some embodiments, binding to CRB or the CRBN-E3 ubiquitin ligase complex inhibits the activity of the E3 ubiquitin ligase. In some embodiments, the immunomodulatory agent induces ubiquitinylation of KZF1 (Ikaros) and IKZF3 (Aiolos) and/or induces degradation of IKZF1 (Ikaros) and IKZF3 (Aiolos). In some embodiments, the immunomodulatory agent induces ubiquitinylation of casein kinase 1A1 (CK1α) by E3 CRL4 ^CRBN ubiquitin ligase. In some embodiments, CK1α ubiquitinylation results in degradation of CK1α.

[91] In some embodiments, the implementation of the immunomodulatory agent is an inhibitor of the transcription factor Ikaros (IKZF1). In some embodiments, an immunomodulatory agent enhances Ikaros ubiquitinylation. In some embodiments, the implementation of the immunomodulatory agent enhances the degradation of Ikaros. In some embodiments, the implementation of the immunomodulatory agent suppresses the expression of the Ikaros protein or gene. In some embodiments, administration of an immunomodulatory agent causes a decrease in Ikaros protein levels.

[92] In some embodiments, the implementation of the immunomodulatory agent is an inhibitor of the transcription factor Aiolos (IKZF3). In some embodiments, an immunomodulatory agent enhances Aiolos ubiquitinylation. In some embodiments, the implementation of the immunomodulatory agent enhances the degradation of Aiolos. In some embodiments, an immunomodulatory agent suppresses the expression of an Aiolos protein or gene. In some embodiments, administration of an immunomodulatory agent causes a decrease in Aiolos protein levels.

[93] In some embodiments, the immunomodulatory agent is an inhibitor of both Ikaros (IKZF1) and Aiolos (IKZF3) transcription factors. In some embodiments, an immunomodulatory agent increases ubiquitinylation of both Ikaros and Aiolos. In some embodiments, the immunomodulatory agent enhances the degradation of both Ikaros and Aiolos. In some embodiments, the immunomodulatory agent enhances ubiquitinylation and degradation of both Ikaros and Aiolos. In some embodiments, administration of an immunomodulatory agent causes a decrease in the levels of both the Aiolos protein and the Ikaros protein.

[94] In some embodiments, the immunomodulatory agent is a selective cytokine inhibitory drug (SelCID). In some embodiments, the implementation of the immunomodulatory agent inhibits the activity of phosphodiesterase-4 (PDE4). In some embodiments, an immunomodulatory agent inhibits the enzymatic activity of CDC25 phosphatases. In some embodiments, an immunomodulatory agent alters the intracellular transport of CDC25 phosphatases.

[95] In some embodiments, the immunomodulatory agent is thalidomide (2-(2,6-dioxopiperidin-3-yl)-1H-isoindol-1,3(2H)-dione), or an analog or derivative of thalidomide. In certain embodiments, the thalidomide derivative includes structural variants of thalidomide that have similar biological activity. Exemplary thalidomide derivatives include, but are not limited to, lenalidomide (REVLIMMUNOMODULATORY COMPOUND ^™ ; Celgene Corporation), pomalidomide (also known as ACTIMMUNOMODULATORY COMPOUND ^™ or POMALYST ^™ (Celgene Corporation)), CC-1088, CDC-501, and CDC-801, and compounds described in US patent No. 5712291; 7320991; and 8716315; US Application No. 2016/0313300; and PCT Publication Nos. WO 2002/068414 and WO 2008/154252.

[96] In some embodiments, the immunomodulatory agent is 1-oxo- and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindolines substituted with an amino group on the benzo ring as described in US Pat. No. 5,635,517 which is incorporated to the present description by way of reference.

[97] In some embodiments, the immunomodulatory agent is a compound of the following formula:

where one of X and Y is -C(O)- and the other of X and Y is -C(O)- or -CH ₂ - and R ⁵ is hydrogen or lower alkyl, or a pharmaceutically acceptable salt thereof . In some embodiments, X is -C(O)- and Y is -CH ₂ -. In some embodiments, both X and Y are -C(O)-. In some embodiments, R ⁵ is hydrogen. In other embodiments, R ⁵ is methyl.

[98] In some embodiments, the immunomodulatory compound is a compound that belongs to the class of substituted 2-(2,6-dioxopiperidin-3-yl)phthalimnomodulatory compounds and substituted 2-(2,6-dioxopiperidin-3-yl)-1 -oxoisoindoles such as those described in US Pat. No. 6,281,230; 6316471; 6,335,349 and 6,476,052 and International Patent Application No. PCT/US97/13375 (International Publication No. WO 98/03502), each of which is incorporated herein by reference.

[99] In some embodiments, the immunomodulatory agent is a compound of the following formula:

where

one of X and Y is -C(O)- and the other of X and Y is -C(O)- or -CH ₂ -;

(1) each of R ¹ , R ² , R ³ and R ⁴ is independently halogen, alkyl of 1-4 carbon atoms, or alkoxy of 1-4 carbon atoms, or

(2) one of R^one, R³, R^four and R⁵represents -NHR^a, the rest from R^one, R², R³ and R^fourare hydrogen, where R^ais hydrogen or alkyl of 1-8 carbon atoms;

R ⁵ is hydrogen or alkyl of 1-8 carbon atoms, benzyl or halogen;

with the proviso that R ⁵ is other than hydrogen, if X and Y are -C(O)-, and (i) each of R ¹ , R ² , R ³ and R ⁴ is fluorine; or (ii) one of R ¹ , R ² , R ³ and R ⁴ is amino;

or their pharmaceutically acceptable salt thereof.

[100] In some embodiments, the immunostimulatory agent is a compound that belongs to the class of isoindole immunomodulatory compounds described in US Pat. /059106), each of which is included in the present description by reference. For example, in some embodiments, the immunostimulatory agent is [2-(2,6-diox-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl]-amide; (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl)-carbamic acid tert-butyl ester; 4-(aminomethyl)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; N-(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl)acetamide; N-{(2-(2,6-dioxo(3-piperidyl)-1,3-dioxoisoindolin-4-yl)methyl}cyclopropylcarboxamide; 2-chloro-N-{(2-(2,6-dioxo(3 -piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}acetamide; N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-3- pyridylcarboxamide; 3-{1-oxo-4-(benzylamino)isoindolin-2-yl}piperidine-2,6-dione; 2-(2,6-dioxo(3-piperidyl))-4-(benzylamino)isoindoline- 1,3-dione; N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}propanamide;N-{(2-(2,6- dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-3-pyridylcarboxamide; N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindoline- 4-yl)methyl}heptanamide; N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-2-furylcarboxamide; {N-(2- (2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)carbamoyl}methyl acetate;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindoline -4-yl)pentanamide;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-2-thienylcarboxamide;N-{[2-(2,6 -dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(butylamine o) carboxamide; N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(octylamino)carboxamide; or N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(benzylamino)carboxamide.

[101] In some embodiments, the immunomodulatory agent is a compound that belongs to the class of isoindole immunomodulatory compounds described in US Patent Application Publication No. 2002/0045643, International Publication No. WO 98/54170, and US Pat. No. 6,395,754, each of which is included to the present description by way of reference. In some embodiments, the immunomodulatory agent is the tetra-substituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolines described in US Pat. No. 5,798,368, which is incorporated herein by reference. In some embodiments, the immunomodulatory agent is 1-oxo- and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindolines as described in US Pat. No. 6,403,613, which is incorporated herein by reference. In some embodiments, the immunomodulatory agent is 1-oxo- or 1,3-dioxoisoindoline substituted at position 4 or 5 of the indoline ring as described in US Pat. No. 6,380,239 and US Pat. links.

[102] In some embodiments, the immunomodulatory agent is 2-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-4-carbamoylbutyric acid or 4-(4-amino-1-oxo-1 ,3-dihydroisoindol-2-yl)-4-carbamoylbutyric acid. In some embodiments, the immunomodulatory compound is 4-carbamoyl-4-{4-[(furan-2-ylmethyl)amino]-1,3-dioxo-1,3-dihydroisoindol-2-yl}-butyric acid, 4- carbamoyl-2-{4-[(furan-2-ylmethyl)-amino]-1,3-dioxo-1,3-dihydroisoindol-2-yl}-butyric acid, 2-{4-[(furan-2- ylmethyl)amino]-1,3-dioxo-1,3-dihydroisoindol-2-yl}-4-phenylcarbamoylbutyric acid or 2-{4-[(furan-2-ylmethyl)amino]-1,3-dioxo-1 ,3-dihydroisoindol-2-yl}-pentanedioic acid.

[103] In some embodiments, the immunomodulatory agent is isoindolin-1-one or isoindoline-1,3-dione substituted at the 2-position with 2,6-dioxo-3-hydroxypiperidin-5-yl, as described in US Pat. No. 6,458,810 , which is included in the present description by reference. In some embodiments, the immunomodulatory compound is 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidin-2,6-dione, or an enantiomer or mixture of enantiomers; or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph. In some embodiments, the immunomodulatory compound is 3-[4-(4-morpholin-4-ylmethylbenzyloxy)-1-oxo-1,3-dihydroisoindol-2-yl]-piperidine-2,6-dione.

[104] In some embodiments, the immunomodulatory agent is as described in Oshima, K. et al., Nihon Rinsho., 72(6):1130-5 (2014); Millrine, D. et al., Trends Mol Med., 23(4):348-364 (2017); and Collins, et al., Biochem J., 474(7):1127-1147 (2017).

[105] In some embodiments, the immunomodulatory agent is lenalidomide, pomalidomide, avadomide, a stereoisomer of lenalidomide, pomalidomide, avadomide, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate, or polymorph thereof. In some embodiments, the immunomodulatory compound is lenalidomide, a stereoisomer of lenalidomide, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate, or polymorph thereof. In some embodiments, the immunomodulatory compound is lenalidomide or ((RS)-3-(4-amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione).

[106] In certain embodiments, treatment and/or intervention at the site of injury is carried out by administering to the individual a thalidomide derivative lenalidomide ((RS)-3-(4-amino-1-oxo-1,3-dihydro-2H-isoindole-2- yl) piperidine-2,6-dione). Lenalidomide is FDA approved for the treatment of multiple myeloma, 5q deletion myelodysplastic syndrome, and later relapsed/refractory mantle cell lymphoma (MCL). Lenalidomide is typically a synthetic derivative of thalidomide and is currently understood to have a variety of immunomodulatory effects, including increased immune synapse formation between T cells and antigen presenting cells (APCs). For example, in some cases, lenalidomide modulates T-cell responses and leads to an increase in the production of interleukin (IL)-2 in CD4 ⁺ and CD8 ⁺ T-cells, induces a shift in T-helper (Th) responses from Th2 to Th1, and inhibits the expansion of the regulatory subgroup T cells (Treg) and improves immunological synapse function in follicular lymphoma and chronic lymphocytic leukemia (CLL) (Otahal et al., Oncoimmunology (2016) 5(4):e1115940). Lenalidomide also has direct tumoricidal activity in patients with multiple myeloma (MM) and directly and indirectly modulates the survival of CLL tumor cells by influencing supportive cells, such as nurse cells, found in the microenvironment of lymphoid tissues. Lenalidomide can also enhance T cell proliferation and interferon-γ production in response to T cell activation via CD3 binding or dendritic cell-mediated activation. In addition, lenalidomide is believed to decrease the proliferation of pro-inflammatory cytokines including TNF-α, IL-1, IL-6 and IL-12 and enhance antibody-dependent cellular cytotoxicity (ADCC) through increased NK cell activation. Lenalidomide can also induce malignant B cells to express higher levels of immunostimulatory molecules such as CD80, CD86, HLA-DR, CD95 and CD40 (Fecteau et al., Blood (2014) 124(10):1637-1644). Cereblon, an E3 ubiquitin ligase, has been identified as the primary target for thalidomide-induced teratogenesis (Ito et al., T., (2010) Science 327: 1345-1350). Lenalidomide also targets cereblon and this has been shown to result in a decrease in c-Myc and IRF4 expression while also increasing p21 expression, which results in cell cycle arrest in the G1 phase (Lopez-Girona et al., (2012) Leukemia 26 : 2326-2335).

[107] In some embodiments, treatment and/or intervention at the lesion is by administering an agent that modulates adenosine levels and/or modulates the activity or amount of a component of the adenosine cascade. Adenosine may function as an immunomodulatory agent in the body. For example, adenosine and certain adenosine analogs that nonselectively activate adenosine receptor subtypes reduce neutrophil production of inflammatory oxidative products (Cronstein et al., Ann. N.Y. Acad. Sci. 451:291, 1985; Roberts et al., Biochem. J., 227:669, 1985; Schrier et al., J. Immunol. 137:3284, 1986; Cronstein et al., Clinical Immunol. Immunopath. 42:76, 1987). In some cases, concentration levels of extracellular adenosine or adenosine analogues may increase in a particular environment, such as the tumor microenvironment (TME). In some instances, adenosine or adenosine analog signaling is dependent on hypoxia or factors involved in or regulating hypoxia, such as hypoxia inducible factor (HIF). In some embodiments, an increase in adenosine signaling can lead to an increase in intracellular levels of cAMP and cAMP-dependent protein kinase, which leads to inhibition of the production of pro-inflammatory cytokines and can lead to the synthesis of immunosuppressive molecules and the development of Treg (Sitkovsky et al., Cancer Immunol Res (2014) 2(7):598-605). In some embodiments, an additional agent can reduce or reverse the immunosuppressive effects of adenosine, adenosine analogs, and/or adenosine signaling. In some embodiments, an additional agent can reduce or reverse hypoxia-triggered immunosuppression of A2 adenosinergic T cells. In some embodiments, the additional agent is selected from adenosine receptor antagonists, extracellular adenosine-degrading agents, inhibitors of adenosine formation by CD39/CD73 ectoenzymes, and inhibitors of hypoxia-HIF-1α signaling. In some embodiments, the additional agent is an adenosine receptor antagonist or agonist.

[108] In specific embodiments, an agent that inhibits or reduces the level of extracellular adenosine is administered to an individual for treatment and/or intervention at a lesion. In some embodiments, an agent that inhibits the activity and/or amount of the adenosine receptor is administered to an individual to treat and/or interfere with a lesion. Specific embodiments provide that inhibition or reduction of the level of extracellular adenosine or adenosine receptor by an inhibitor of extracellular adenosine (such as an agent that interferes with the formation, degrades, inactivates and/or reduces the level of intracellular adenosine), and/or an adenosine receptor inhibitor (such as an antagonist adenosine receptor) can enhance the immune response, such as macrophage, neutrophil, granulocyte, dendritic cell, T- and/or B-cell mediated response. In addition, inhibitors of the Gs-mediated cAMP-dependent intracellular cascade and inhibitors of the adenosine receptor-triggered Gi-mediated intracellular cascades can also increase acute and chronic inflammation.

[109] In some embodiments, an adenosine receptor antagonist is administered to an individual for treatment and/or intervention at a lesion. In specific embodiments, an adenosine receptor antagonist is administered to an individual for treatment and/or intervention at a lesion. In some embodiments, the adenosine receptor antagonist is an A2a, A2b and/or A3 antagonist. The A2a, A2b and A3 receptors can suppress or reduce the immune response, thus being immunosuppressive adenosine receptor antagonists, which can facilitate, enhance or enhance the immune response. In some embodiments, an agent that inhibits extracellular adenosine production and/or inhibits adenosine-triggered adenosine receptor signaling is administered to an individual for treatment and/or intervention at a lesion. In some embodiments, the immune response to the lesion, tissue inflammation at the lesion, and targeted tissue destruction at the lesion can be enhanced by inhibiting or reducing local tissue hypoxia causing adenosine production; by degradation (or inactivation) of accumulated extracellular adenosine; by preventing or reducing the expression of adenosine receptors on immune cells; and/or by inhibiting signal transduction by adenosine ligands through adenosine receptors.

[110] In specific embodiments, an adenosine receptor antagonist is administered to an individual for treatment and/or intervention at a lesion. In some embodiments, the antagonist is a small molecule adenosine receptor antagonist, such as an A2a, A2b, or A3 receptor antagonist. In some embodiments, the antagonist is a peptide or peptidomimetic that binds to the A2a, A3b and/or A3 adenosine receptor but does not trigger the G _i protein dependent intracellular signaling cascade. Examples of such antagonists are described in US Pat. Nos. 5,565,566; 5545627, 5981524; 5861405; 6066642; 6326390; 5670501; 6117998; 6232297; 5786360; 5424297; 6313131, 5504090 and 6322771.

[111] In some embodiments, an A2 receptor (A2R) antagonist is administered to a subject to treat and/or interfere with a lesion. Exemplary A2R antagonists include, but are not limited to, KW6002 (Istradephylline), SCH58261, caffeine, paraxanthine, 3,7-dimethyl-1-propargylxanthine (DMPX), 8-(m-chlorostyryl)caffeine (CSC), MSX-2, MSX-3, MSX-4, CGS-15943, ZM-241385, SCH-442416, preladenant, vipadenant (BII014), V2006, ST-1535, SYN-115, PSB-1115, ZM241365, FSPTP and inhibitor nucleic acid targeted on the expression of A2R, for example, siRNA or shRNA, or any antibodies or antigen-binding fragments thereof that target A2R. In some embodiments, the agent is an A2R antagonist as described in, for example, Ohta et al., Proc Natl Acad Sci U S A (2006) 103:13132-13137; Jin et al., Cancer Res. (2010) 70(6):2245-2255; Leone et al., Computational and Structural Biotechnology Journal (2015) 13:265-272; Beavis et al., Proc Natl Acad Sci U S A (2013) 110:14711-14716; and Pinna, A., Expert Opin Investig Drug (2009) 18:1619-1631; Sitkovsky et al., Cancer Immunol Res (2014) 2(7):598-605; US 8080554; US 8716301; US20140056922; WO2008/147482; US 8883500; US20140377240; WO02/055083; US 7141575; US 7405219; US 8883500; US 8450329 and US 8987279).

[112] In specific embodiments, an adenosine receptor antagonist that is an antisense molecule, an inhibitory nucleic acid molecule (e.g., a small inhibitory RNA (siRNA)) or a catalytic nucleic acid molecule (e.g., a ribozyme) that specifically binds an mRNA encoding an adenosine receptor , administered to the individual for treatment and/or intervention in the lesion. In some embodiments, an antisense molecule, an inhibitory nucleic acid molecule, or a catalytic nucleic acid molecule binds nucleic acids encoding A2a, A2b, or A3. In some embodiments, the antisense molecule, inhibitory nucleic acid molecule, or catalytic nucleic acid targets biochemical pathways downstream of the adenosine receptor. For example, an antisense molecule or a catalytic nucleic acid can inhibit an enzyme involved in an intracellular cascade dependent on the G _s protein or on the G _i protein. In some embodiments, the additional agent comprises a dominant negative mutant form of an adenosine receptor, such as A2a, A2b, or A3.

[113] In some embodiments, treatment and/or intervention at the site of injury is carried out by administering an agent that inhibits extracellular adenosine in an individual. Agents that inhibit extracellular adenosine include agents that render extracellular adenosine non-functional (eg render extracellular adenosine unable to bind and/or activate the adenosine receptor), such as a substance that modifies the structure of extracellular adenosine. In some embodiments, the additional agent is an enzyme that generates extracellular adenosine or degrades adenosine, a modified form thereof, or a modulator. For example, in some embodiments, the additional agent is an enzyme (e.g., adenosine deaminase) or other catalytic molecule that selectively binds and degrades adenosine, thereby eliminating or reducing the ability of endogenously formed adenosine to signal through adenosine receptors and stop inflammation.

[114] In certain embodiments, treatment and/or intervention at the lesion is by administering adenosine deaminase (ADA) or a modified form thereof, eg, recombinant ADA and/or polyethylene glycol modified ADA (ADA-PEG), to the individual. Adenosine deaminase can inhibit local tissue accumulation of extracellular adenosine. ADA-PEG is used to treat patients with ADA SCID (Hershfield (1995) Hum Mutat. 5:107). In some embodiments, an agent that inhibits extracellular adenosine is administered to the individual, which includes agents that prevent or reduce the formation of extracellular adenosine and/or prevent or reduce the accumulation of extracellular adenosine, thereby eliminating or substantially reducing the immunosuppressive effects of adenosine. In some embodiments, an agent is administered to the individual that specifically inhibits enzymes and proteins that are involved in regulating the synthesis and/or secretion of pro-inflammatory molecules, including modulators of nuclear transcription factors. Suppression of expression of the adenosine receptor or dependent on G _s protein or G _i protein intracellular cascade, or dependent on cAMP intracellular cascade can lead to an increase/strengthening of the immune response.

[115] In some embodiments, an agent targeted by ectoenzymes that generate or produce extracellular adenosine is administered to an individual for treatment and/or intervention at a lesion. In some embodiments, the agent targets the CD39 and CD73 ectoenzymes, which function in tandem to generate extracellular adenosine. CD39 (also called ectonucleoside triphosphate diphosphohydrolase) converts extracellular ATP (or ADP) to 5'AMP. CD73 (also called 5'-nucleotidase) then converts 5'AMΡ to adenosine. CD39 activity is reversible by NDP kinase and adenylate kinase, while CD73 activity is irreversible. CD39 and CD73 are expressed on tumor stromal cells, including endothelial cells and Tregs, as well as on many cancer cells. For example, expression of CD39 and CD73 on endothelial cells is increased under hypoxic conditions of the tumor microenvironment. Tumor hypoxia may be the result of inadequate blood supply and tumor vascular organization that impair oxygen delivery (Carroll and Ashcroft (2005), Expert. Rev. Mol. Med. 7(6):1-16). Hypoxia also inhibits adenylate kinase (AK), which converts adenosine to AMP, resulting in very high extracellular concentrations of adenosine. Thus, in response to hypoxia, which is a condition that often occurs in the tumor microenvironment (TME), in or around solid tumors, adenosine is released. In some embodiments, the additional agent is one or more of an anti-CD39 antibody or antigen-binding fragment thereof, an anti-CD73 antibody or antigen-binding fragment thereof, e.g., MEDI9447 or TY/23, α-β-methylene adenosine diphosphate (ADP), ARL 67156, POM-3, IPH52 (See e.g. Allard et al. Clin Cancer Res (2013) 19(20):5626-5635; Hausler et al., Am J Transl Res (2014) 6(2):129-139 ; Zhang, B., Cancer Res. (2010) 70(16):6407-6411).

[116] In some embodiments, a chemotherapeutic agent (sometimes referred to as a cytotoxic agent) is administered to an individual to treat and/or interfere with a lesion. In certain embodiments, the lesion is a tumor. In specific embodiments, the lesion is malignant. In particular embodiments, the chemotherapeutic agent is any agent known to those skilled in the art to be effective in treating, preventing, or alleviating hyperproliferative disorders such as cancer. Chemotherapeutic agents include, but are not limited to, small molecule compounds, synthetic drugs, peptides, polypeptides, proteins, nucleic acids (e.g., DNA and RNA polynucleotides, including, but not limited to, antisense nucleotide sequences, triple helixes, and nucleotide sequences encoding biologically active proteins, polypeptides or peptides), antibodies, synthetic or natural inorganic molecules, mimetics, and synthetic or natural organic molecules. In specific embodiments, chemotherapeutic drugs include alkylating agents, anthracyclines, cytoskeletal disruptors (taxanes), epothilones, histone deacetylase inhibitors, topoisomerase inhibitors, topoisomerase II inhibitors, kinase inhibitors, nucleotide analogs and precursor analogs, peptide antibiotics, platinum-based agents. and vinca alkaloids and their derivatives.

[117] In certain embodiments, treatment and/or intervention at the site of injury is carried out by administering a chemotherapeutic agent to modulate genetically engineered cells in vivo. Chemotherapeutic agents may include, but are not limited to, abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, BCG live, bevacelizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, camptothecin, capecitocin carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cinacalcet, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, darbepoetin alfa, daunorubicin, denileukin diphthitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone, alfa eliotta solution, estramucin, estramucin, eliott's solution, epietrubi , etoposide, exemestane, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gemcitabine, gemtuzumab ozagomycin, gefitinib, goserelin, hydroxyurea, ibritutomab tiuxetan, idarubicin, ifosfamide, imatinib, interferon alfa-2a, interferon alfa-2b, leukovorin, lectrozolecan , levamisole, lomustine, meclorethamine, magestrol, melphalan, mercaptopurine, mesna, methotrexate, methoxsalen, methylprednisolone, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oblimersen, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pegademazu, pegaspargasu, pegfilgrastim, pemetrexed, pentostatin, pipobroman, plicamycin, polyfeprosan, porfimer, procarbazine, , rituximab, sargramostim, streptozocin, talc, tamoxifen, tarceva, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, and zoledronate.

C. Reexpansion of genetically engineered cells

[118] In some embodiments, the methods provided provide for re-expansion of the engineered cells in an individual, which in some cases may significantly exceed the initial maximum level of expansion prior to treatment and/or intervention. In some embodiments, the provided methods modulate the expansion and/or persistence of engineered T cells at time points when peak levels of engineered cells are reduced or undetectable. In some embodiments, the genetically engineered cell induced to re-expand exhibits superior efficacy in the individual to which it is administered.

[119] Methods for monitoring or detecting CAR+ T cells are known, and exemplary methods are described in section IV.C. In some embodiments, after administration to an individual, the degree or extent of expansion of the injected cells can be detected or quantified. For example, in some aspects, quantitative PCR (qPCR) is used to estimate the number of cells expressing the chimeric receptor (eg, CAR expressing cells) in the blood or serum or organ or tissue (eg, diseased area) of an individual. In some aspects, expansion, including the number of engineered cells, is quantified as copies of DNA or plasmid encoding a receptor, e.g., CAR, per microgram of DNA, or as number of receptor-expressing, e.g., CAR-expressing cells per microliter of sample. , for example, blood or serum, or total peripheral blood mononuclear cells (PBMC), or leukocytes, or T cells, per microliter of sample. In some embodiments, a flow cytometric assay can be performed to detect cells expressing the receptor, typically using antibodies specific for the receptor. Cellular assay methods can also be used to detect the number or percentage of functional cells, such as cells capable of binding and/or neutralizing and/or inducing responses, such as cytotoxic responses, against cells associated with a disease or condition, or expressing an antigen, recognized by the receptor. In any of such embodiments, the extent or level of expression of another marker associated with the recombinant receptor (eg, CAR-expressing cells) can be used to distinguish introduced cells from endogenous cells in an individual.

[120] In some embodiments, intervention at a lesion in accordance with the methods provided provides for activation, re-expansion and/or increased exposure of cells in an individual, for example, T cells introduced for T-cell therapy, for example, by allowing them to expand and/ or persistence over time. In some embodiments, T cell therapy exhibits increased or prolonged expansion and/or persistence in an individual, or an average of a plurality of individuals, treated in this manner, compared to a method in which T cell therapy is administered to the individual(s) at no intervention in the lesion. In some embodiments, contemplated methods involving in vivo intervention at a lesion may increase the maximum, total exposure, and/or duration of exposure of cells, such as T cells administered for T cell therapy, in an individual, or an average of multiple individuals, who are treated in this way, compared with the introduction of T-cells alone in the absence of intervention in the lesion. Such an increase may be, or approximately, or be at least about 1.2 times, 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5.0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, 10.0 times, 20.0 times, 30.0 times, 40.0 times, 50.0 times or more.

[121] In some embodiments, the increased exposure of the administered cells in an individual (eg, increased number of cells or duration of exposure) provided by the provided methods improves the efficacy and therapeutic outcomes of immunotherapy, such as T-cell therapy. In some aspects, the methods are advantageous in that higher exposure and/or longer exposure to recombinant receptor-expressing cells, eg, CAR-expressing cells, improves treatment outcomes over other methods. Such outcomes may include patient survival and remission, even in individuals with severe tumor burden. In some aspects, the increased or prolonged expansion and/or persistence of cell dose in an individual or, on average, across multiple individuals so treated, which is achieved after treatment and/or intervention at a lesion, is associated with benefit in terms of tumor-related outcomes in the individual. (s). In some embodiments, the tumor-related outcome includes a decrease in tumor burden or a decrease in blasts in the bone marrow of the individual(s). In some embodiments, tumor burden is reduced by, or at least by, or about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent after the method is performed. In some embodiments, disease burden, tumor size, tumor volume, tumor mass and/or tumor burden or tumor mass are reduced after a dose of cells by at least or about 50%, 60%, 70%, 80%, 90%, or more than an individual or, on average, a plurality of individuals who are treated in a manner that does not involve intervention at the site of injury.

[122] In some embodiments, the provided methods effectively treat an individual despite the individual becoming resistant to another therapy and/or relapsing after administration of engineered cells, such as cells expressing a recombinant receptor, e.g., CAR+ T cells. In some embodiments, criteria evaluated for effective treatment include overall response rate (ORR), complete response (CR), duration of response (DOR), progression-free survival (PFS), and/or overall survival (OS). In some embodiments, the methods and uses provide or achieve longer lasting responses in an individual or, on average, in a plurality of individuals treated in this way, compared to a method that does not involve intervention at the site of injury. In specific embodiments of any of the methods envisaged, the response, e.g., ORR or CR, lasts for more than 3 months, more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 30 months, more than 36 months or more after the intervention in the site of damage and/or the introduction of a pharmacological or therapeutic agent in accordance with the envisaged methods.

II. Cell therapy and engineered cells

[123] Contemplated therapeutic methods involve administering recombinant receptor-expressing cells and compositions thereof to individuals, eg, patients. In some embodiments, the cells contain or are modified to contain an engineered receptor, for example, an engineered antigen receptor, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR). Cells include populations of such cells, compositions containing such cells and/or enriched with such cells, for example, in which cells of a certain type, such as T cells or CD8 ⁺ or CD4 ⁺ cells, are expanded or selected. Compositions include pharmaceutical compositions and formulations for administration, such as compositions and formulations for adoptive cell therapy.

[124] In some embodiments, cells comprise one or more genetically engineered nucleic acids and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, gene transfer is carried out first by stimulating cells, for example, by combining them with a stimulus that induces a response, such as proliferation, survival, and/or activation, for example, as determined by the expression of a cytokine or an activation marker, followed by transduction of activated cells and an increase in in an amount in culture to amounts sufficient for clinical applications.

[125] Various methods of introducing genetically modified components, for example, antigen receptors, such as CARs, are well known and can be used in conjunction with the provided methods and compositions. Illustrative methods include methods for transferring receptor-encoding nucleic acids, including via viral, eg, retroviral or lentiviral transfer, transduction, transposons, and electroporation.

A. Recombinant receptors

[126] Cells typically express recombinant receptors, such as antigen receptors, including functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs), and other antigen-binding receptors, such as transgenic T cell receptors (TCRs). Receptors also include other chimeric receptors such as chimeric autoantibody receptors (CAARs).

1. Chimeric antigen receptors (CARs)

[127] In some embodiments, the implementation of the recombinant receptor includes a chimeric antigen receptor (CAR). In some embodiments, the CAR is specific for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type. In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, an antigen is selectively expressed or overexpressed on cells associated with a disease or condition, such as tumor or pathogenic cells, over normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on engineered cells.

[128] In specific embodiments, the implementation of the recombinant receptor, such as a chimeric receptor, contains an intracellular signaling region that includes a cytoplasmic signaling domain (also interchangeably referred to as an intracellular signaling domain), such as a cytoplasmic (intracellular) region capable of inducing a primary activation signal in T- cells, e.g., a T-cell receptor (TCR) component cytoplasmic signaling domain (e.g., a CD3 zeta chain (CD3ζ) cytoplasmic signaling domain or a functional variant or signal portion thereof) and/or which contains an immunoreceptor tyrosine-based activation motif (ITAM) .

[129] In some embodiments, the chimeric receptor further comprises an extracellular binding domain that specifically binds to an antigen (or ligand). In some embodiments, the chimeric receptor is a CAR that contains an extracellular antigen recognition domain that specifically binds to an antigen. In some embodiments, the antigen (or ligand) is a protein expressed on the surface of cells. In some embodiments, the CAR is a TCR-like CAR and the antigen is a truncated peptide antigen, such as an extracellular protein peptide antigen that, like TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.

[130] Exemplary antigen receptors, including CARs, and methods for constructing and introducing such receptors into cells, include antigen receptors as described, for example, in International Patent Application Publication Nos. 071154, WO2013/123061, публикациях патентных заявок США номер US2002131960, US2013287748, US20130149337, патентах США № 6451995, 7446190, 8252592, 8339645, 8398282, 7446179, 6410319, 7070995, 7265209, 7354762, 7446191, 8324353 и 8479118, и патентной заявке Европы number EP2537416, and/or antigen receptors described by Sadelain et al., Cancer Discov. April 2013; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, antigen receptors include CAR as described in US Pat. No. 7,446,190 and antigen receptors as described in International Patent Application Publication No. WO/2014055668 A1. Examples of CARs include CARs as described in any of the above publications such as WO2014031687, US 8339645, US 7446179, US 2013/0149337, US Patent No. 7446190, US Patent No. 8389282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8339645, US 7446179, US 2013/0149337, US Pat. No. 7,446,190, and US Pat. No. 8,389,282. chain (VH) and/or variable region (VL) of the light chain of an antibody, for example, an scFv fragment of an antibody.

[131] 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, an antigen is selectively expressed or overexpressed on cells associated with a disease or condition, such as tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on engineered cells.

[132] In some embodiments, a CAR is constructed that has specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, for example, a cancer marker and/or an antigen intended to inducing an inhibition of a response, such as an antigen expressed on a normal or non-disease-associated cell type. Thus, a CAR typically includes, in its extracellular portion, one or more antigen-binding molecules, such as one or more antigen-binding fragments, domains, or portions, or one or more antibody variable domains and/or antibody molecules. In some embodiments, the CAR comprises an antigen-binding portion or portions of an antibody molecule, such as a single chain antibody fragment (scFv) derived from a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody (mAb).

[133] In some embodiments, the antibody, or antigen-binding portion thereof, is expressed on cells as part of a recombinant receptor, such as an antigen receptor. Antigen receptors include functional non-TCR antigen receptors such as chimeric antigen receptors (CARs). Generally, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity against peptide-MHC complexes may also be referred to as a TCR-like CAR. In some embodiments, an extracellular antigen-binding domain specific for the MHC-TCR-like CAR peptide complex is linked to one or more components of intracellular signaling, in some aspects via linkers and/or transmembrane domain(s). In some embodiments, such molecules typically mimic or approximate a signal through a natural antigen receptor such as TCR, and optionally a signal through such a receptor in combination with a co-stimulatory receptor.

[134] In some embodiments, a recombinant receptor, such as a chimeric receptor (eg, CAR), includes a ligand-binding domain that binds, eg, specifically binds, to an antigen (or ligand). Among the antigens targeted by chimeric receptors are antigens expressed in the context of the disease, condition, or cell type to be targeted by adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic and malignant diseases and disorders, including cancer and tumors, including hematological malignancies, immune system malignancies such as lymphomas, leukemias and/or myelomas such as B, T and myeloid leukemias, lymphomas and multiple myelomas.

[135] In some embodiments, the antigen (or ligand) is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen (or ligand) is selectively expressed or overexpressed on cells associated with a disease or condition, such as tumor or pathogenic cells, compared to normal or non-target cells or tissues.

[136] In some embodiments, the CAR comprises an antibody or antigen-binding fragment (eg, scFv) that specifically recognizes an antigen, such as an intact antigen expressed on a cell surface.

[137] In some embodiments, the antigens targeted by the receptors are or include orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, folate 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 antigen, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligands, NY-ESO-1, MART-1, gp100, oncoembryonic 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, antigen Wilms tumor 1 (WT-1), cyclin such as cyclin A1 (CCNA1), G-protein coupled receptor 5D (GPCR5D) and/or biotinylated molecules and/or molecules expressed by HIV, HCV, HBV or other chemical pathogens.

[138] In certain embodiments, the engineered cell expresses a recombinant receptor and/or CAR that binds to an antigen. In specific embodiments, the antigen is αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), testicular cancer antigen, cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, C-C chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), epidermal growth factor truncated protein (tEGFR), epidermal growth factor receptor type III mutant (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), estrogen receptor, Fc-like protein receptor 5 (FCRL5; also known as homologue 5 Fc receptor or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), rec folate eptor alpha, fetal acetylcholine receptor, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), Her2/neu (erbB2 receptor tyrosine kinase), Her3 (erb-B3), Her4 (erb-B4) , erbB dimers, high molecular weight human melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte A1 antigen (HLA-A1), human leukocyte A2 antigen (HLA-A2), IL-22 receptor alpha (IL-22Ra ), IL-13 alpha 2 receptor (IL-13Ra2), kinase insertion domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), L1-CAM CE7 epitope, member of the A family of 8 proteins with leucine- repeat-rich (LRRC8A), Lewis Y antigen, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, representative ligands natural killer group 2 D (NKG2D), melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, preferably exp melanoma repressing antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG, also known as 5T4 ), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor antigen 1 (WT-1), G protein-coupled receptor 5D ( GPCR5D), a pathogen-specific or universal label-associated antigen, and/or biotinylated molecules and/or molecules expressed by HIV, HCV, HBV, or other pathogens. The antigens targeted by the receptors, in some embodiments, include antigens associated with B-cell cancer, such as any of a number of known B-cell markers. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig-kappa, Ig-lambda, CD79a, CD79b, or CD30.

[139] In some embodiments, the CAR binds a pathogen-specific or pathogen-expressed antigen. In some embodiments, the CAR is specific for viral antigens (such as HIV, HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens.

[140] In some embodiments, the CAR comprises a TCR-like antibody, such as an antibody or an antigen-binding fragment (e.g., scFv), that specifically recognizes an intracellular antigen, such as a tumor-associated antigen, presented on the surface as an MHC-peptide complex. In some embodiments, the antibody or antigen-binding portion thereof that recognizes the MHC-peptide complex may be expressed on cells as part of a recombinant receptor, such as an antigen receptor. Antigen receptors include functional non-TCR antigen receptors such as chimeric antigen receptors (CARs). Generally, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity against peptide-MHC complexes may also be referred to as a TCR-like CAR.

[141] A "major histocompatibility complex" (MHC) refers to a protein, principally a glycoprotein, that contains a polymorphic peptide-binding center or binding groove, which in some cases may complex with peptide antigens of polypeptides, including peptide antigens processed cellular apparatus. In some cases, MHC molecules can be exposed or expressed on the cell surface, including as a complex with a peptide, i.e. an MHC-peptide complex to present the antigen in a conformation recognized by an antigen receptor on T cells, such as a TCR or a TCR-like antibody. Typically, class I MHC molecules are heterodimers having a membrane-spanning α-chain, in some cases with three α-domains, and a non-covalently bound β2-microglobulin. Typically, class II MHC molecules are composed of two transmembrane glycoproteins, α and β, both of which tend to pass through the membrane. The MHC molecule may include an effective portion of the MHC that contains the antigen binding site or sites for binding the peptide and sequences required for recognition by the appropriate antigen receptor. In some embodiments, class I MHC molecules deliver cytosol-derived peptides to the cell surface, where the MHC-peptide complex is recognized by T cells, primarily CD8 ⁺ T cells, but in some cases CD4+ T cells. In some embodiments, class II MHC molecules deliver peptides generated in the vesicular system to the cell surface, where they are normally recognized by CD4 ⁺ T cells. In general, MHC molecules are encoded by a group of related loci collectively referred to as H-2 in the mouse and human leukocyte antigen (HLA) in humans. Thus, in general, human MHC may also be referred to as human leukocyte antigen (HLA).

[142] The terms "MHC-peptide complex" or "peptide-MHC complex", or variants thereof, refer to the complex or association of a peptide antigen and an MHC molecule, usually by non-covalent interactions of the peptide in the binding groove or cleft of the MHC molecule. In some embodiments, the MHC-peptide complex is presented or displayed on the surface of cells. In some embodiments, the MHC-peptide complex may be specifically recognized by an antigen receptor, such as a TCR, a TCR-like CAR, or antigen-binding portions thereof.

[143] In some embodiments, a peptide, such as a peptide antigen or an epitope, of a polypeptide can be associated with an MHC molecule, for example, for recognition by the antigen receptor. Typically, a peptide is derived from or based on a fragment of a longer biological molecule, such as a polypeptide or protein. In some embodiments, the implementation of the peptide usually has a length of from about 8 to about 24 amino acids. In some embodiments, the peptide is from or about 9 to 22 amino acids in length for recognition in complex with MHC class II. In some embodiments, the peptide is from or about 8 to 13 amino acids in length for recognition in a class I MHC complex. A TCR-like CAR generates or triggers an activation signal on T cells that induces a T cell response such as T cell proliferation, cytokine production, cytotoxic T cell response, or other response.

[144] In some embodiments, the TCR-like antibody or antigen-binding portion is known or can be obtained by known methods (see, for example, US Published Application No. US 2002/0150914; US 2003/0223994; US 2004/0191260; US 2006/0034850 ; US 2007/00992530; US20090226474; US20090304679; and PCT International Publication No. WO 03/068201).

[145] In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to an MHC-peptide complex can be obtained by immunizing the host with an effective amount of an immunogen containing the particular MHC-peptide complex. In some cases, the peptide of the MHC-peptide complex is an epitope of an antigen capable of binding to MHC, such as a tumor antigen, for example, a universal tumor antigen, myeloma antigen, or other antigen, as described below. In some embodiments, an effective amount of the immunogen is then administered to the host to induce an immune response, wherein the immunogen retains its three-dimensional shape for a period of time sufficient to induce an immune response against the three-dimensional configuration of the peptide in the binding groove of the MHC molecule. Serum obtained from the host is then analyzed to determine if the desired antibodies are being produced that recognize the three-dimensional configuration of the peptide in the binding groove of the MHC molecule. In some embodiments, antibodies produced can be assessed to confirm that the antibody can distinguish between the MHC-peptide complex from the MHC molecule alone, the peptide of interest alone, and the MHC-peptide complex. The desired antibodies can then be isolated.

[146] In some embodiments, an antibody, or an antigen-binding portion thereof, that specifically binds to an MHC-peptide complex can be generated using library display techniques, such as antibody phage libraries. In some embodiments, for example, phage display libraries of mutant Fab, scFv, or other forms of antibodies can be generated in which members of the library are mutated at one or more CDR or multiple CDR residues. See, for example, US Published Application No. US20020150914, US2014/0294841; and Cohen CJ. et al. (2003) J Mol. Recognize. 16:324-332.

[147] the Term "antibody" in the framework of the present invention is used in the broadest sense, and it includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) fragments of antibodies, including antigen-binding (Fab) fragments, F(ab') ₂ - fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable regions (V _H ) capable of specifically binding antigen, single chain antibody fragments, including single chain variable fragments (scFv), and fragments in the form of single domain antibodies (eg, sdAb, sdFv, nanoantibody). The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins such as intraantibodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugated antibodies, multispecific, e.g. scFv, tandem tri-scFv. Unless otherwise indicated, the term "antibody" should be understood to encompass functional fragments of antibodies. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[148] In some embodiments, antigen-binding proteins, antibodies, and antigen-binding fragments thereof specifically recognize the antigen of a full-length antibody. In some embodiments, the antibody heavy and light chains may be full length or may be an antigen binding portion (Fab, F(ab')2, Fv, or single chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, specifically selected from, for example, IgG1, IgG2, IgG3, and IgG4, more specifically , IgG1 (for example, human IgG1). In another embodiment, the antibody light chain constant region is selected from, for example, kappa or lambda, in particular kappa.

[149] Antibodies contemplated include antibody fragments. "Antibody fragment" refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diantibodies; linear antibodies; single chain antibody molecules (eg scFv); and multispecific antibodies formed from antibody fragments. In specific embodiments, the antibodies are single chain antibody fragments containing a heavy chain variable region and/or a light chain variable region, such as scFv.

[150] The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is involved in the binding of an antibody to an antigen. The heavy chain and light chain variable domains (V _H and V _L , respectively) of a native antibody typically have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007) A single V _H or V _L domain may be sufficient to provide antigen binding specificity Moreover, antibodies that bind a particular antigen can be isolated using a V _H domain or V _L from an antibody that binds an antigen to screen for a library of V _L or V _H complementary domains, respectively See, for example, Portolano et al., J. Immunol 150:880-887 (1993); Clarkson et al. Nature 352:624-628 (1991).

[151] Single domain antibodies are antibody fragments containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody. In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds an antigen, such as a cancer marker or cell surface antigen of the cell or disease to be targeted, such as a tumor cell or cancer cell, such as any of those described herein or known target antigens.

[152] Antibody fragments can be obtained by various methods, including, but not limited to, proteolytic cleavage of intact antibodies, as well as production by recombinant host cells. In some embodiments, antibodies are fragments produced by recombinant methods, such as fragments containing an arrangement of elements that does not occur naturally, such as fragments in which two or more regions or chains of an antibody are joined by synthetic linkers, e.g., peptide linkers, and /or which cannot be obtained by enzyme cleavage of a naturally occurring intact antibody. In some aspects, the antibody fragments are scFv.

[153] A "humanized" antibody is an antibody in which all or substantially all of the CDR amino acid residues are from non-human CDRs and all or substantially all of the FR amino acid residues are from human FR. The humanized antibody may optionally include at least a portion of the antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has been humanized, typically to reduce immunogenicity in humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, the antibody from which the CDR residues are derived), for example, to restore or improve the specificity or affinity of the antibody.

[154] Thus, in some embodiments, the chimeric antigen receptor, including TCR-like CARs, includes an extracellular portion containing an antibody or antibody fragment. In some embodiments, the antibody or fragment comprises an scFv. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment and an intracellular signaling region. In some embodiments, the implementation of the intracellular signaling region contains an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain that is capable of inducing primary signal activation in a T cell, a T cell receptor (TCR) component signaling domain, and/or a signaling domain containing an immunoreceptor activating motif. based on tyrosine (ITAM).

[155] In some embodiments, a recombinant receptor, such as CAR, comprising an antibody portion of a recombinant receptor, such as CAR, further comprises at least a portion of an immunoglobulin constant region, such as a hinge region, such as an IgG4 hinge region, and/or C _H 1 /C _L and/or Fc region. In some embodiments, the recombinant receptor, such as CAR, including the antibody moiety, further comprises a spacer, which may be or may include at least a portion of an immunoglobulin constant region or variant, or a variant or modified version thereof, such as a hinge region, e.g. , IgG4 hinge region, and/or C _H 1/C _L and/or Fc region. In some embodiments, the recombinant receptor further comprises a spacer and/or a hinge region. In some embodiments, the constant region or portion is a human IgG, such as IgG4 or IgG1. In some aspects, a portion of the constant region serves as a spacer region between the antigen recognition component, eg scFv, and the transmembrane domain. The spacer may be of a length that provides an increased ability of the cell to respond after antigen binding compared to no spacer. Exemplary spacers, such as hinge regions, include those described in International Patent Application Publication Number WO2014031687. In some examples, the spacer is exactly or about 12 amino acids long, or no more than 12 amino acids long. Exemplary spacers include spacers having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids , about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between final values of any of the listed ranges. In some embodiments, the spacer region is about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include an IgG4 hinge alone, an IgG4 hinge associated with the C _H 2 and C _H 3 domains, or an IgG4 hinge associated with the C _H 3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al., (2013), Clin. Cancer Res., 19:3153, International Patent Publication No. WO2014031687, US Patent No. 8822647, or Publication Application No. US2014/0271635.

[156] In some embodiments, the implementation of the constant region or part is a human IgG, such as IgG4 or IgG1. In some embodiments, the spacer has the sequence ESKYGPPCPPCP (listed in SEQ ID NO: 1) and is encoded by the sequence listed in SEQ ID NO: 2. In some embodiments, the spacer has the sequence listed in SEQ ID NO: 3. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 4. In some embodiments, the implementation of the constant region or part is from IgD. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 5. In some embodiments, the spacer has an amino acid sequence that has at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 1, 3, 4, or 5. In some embodiments, the spacer has the sequence shown in SEQ ID NOs: 26-34. In some embodiments, the spacer has an amino acid sequence that has at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 26-34.

[157] The antigen recognition domain is typically associated with one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as the TCR complex, in the case of CAR, and/or signal through another cell surface receptor. Thus, in some embodiments, an antigen-binding component (eg, an antibody) is associated with one or more transmembrane or intracellular signaling domains or regions. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain is used that is naturally associated with one of the domains in the receptor, such as CAR. In some instances, the transmembrane domain is chosen or modified by amino acid substitution to avoid such domains from binding to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[158] In some embodiments, the transmembrane domain is either from a natural or synthetic source. When the source is natural, in some aspects the domain is derived from any membrane-bound or transmembrane protein. Transmembrane regions include regions derived from (i.e., contain at least the transmembrane region(s) from) T cell receptor alpha, beta, or zeta chain, CD28, CD3-epsilon, CD45, CD4, CD5, CD8, CD9 , CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain contains predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine is located at each end of the synthetic transmembrane domain. In some embodiments, binding is via linkers, spacers, and/or transmembrane domain(s).

[159] Among the intracellular signaling domains or regions are those that mimic or approximately correspond to a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker is present, e.g., a linker between 2 and 10 amino acids in length, such as a linker containing glycine and serine residues, e.g., a glycine-serine doublet, and forms a link between the transmembrane domain and the cytoplasmic signaling domain. or CAR area.

[160] A receptor, such as a CAR, typically includes at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of the TCR complex, such as the CD3 TCR chain that mediates T cell activation and cytotoxicity, such as the CD3 zeta chain. Thus, in some aspects, the antigen-binding portion is associated with one or more signaling modules of cells. In some embodiments, cell signaling modules include a CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the implementation of the receptor, such as CAR, further includes part of one or more additional molecules, such as Fc receptor γ, CD8, CD4, CD25 or CD16. For example, in some aspects, the CAR or other chimeric receptor comprises a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

[161] In some embodiments, upon binding to a CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domains or regions of the receptor activate at least one of the normal effector functions or immune cell responses, e.g., T cells modified to express CAR. For example, in some contexts, CAR induces T cell function such as cytolytic activity or T helper activity such as the secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain or region of the receptor component of an antigen or co-stimulatory molecule is used instead of an intact immunostimulatory chain, for example if it signals an effector function. In some embodiments, the intracellular signaling domain or domains or regions include cytoplasmic T cell receptor (TCR) sequences, and in some aspects also co-receptor sequences, that naturally act in conjunction with such receptors to initiate signal transduction upon antigen receptor binding, and /or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functionality.

[162] In the context of natural TCR, full activation usually requires not only signaling through the TCR, but also a co-stimulatory signal. Thus, in some embodiments, a component for generating a secondary or co-stimulatory signal is also included in the CAR to ensure full activation. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, the additional CAR is expressed in the same cell and provides a component for a secondary or co-stimulatory signal.

[163] T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation via TCRs (primary cytoplasmic signaling sequences), and sequences that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signal sequences). In some aspects, the CAR includes one or both of these signaling components.

[164] In some aspects, CAR includes a primary cytoplasmic signal sequence that regulates the primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs that are known as tyrosine-based immunoreceptor activating motifs, or ITAMs. Examples of ITAMs containing primary cytoplasmic signal sequences include ITAMs derived from TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD8, CD22, CD79a, CD79b and CD66d. In some embodiments, the cytoplasmic signaling molecule(s) in the CAR comprise(s) a cytoplasmic signaling domain or region, portion thereof, or sequence derived from CD3-zeta.

[165] In some embodiments, the CAR includes the signaling domain or region and/or the transmembrane portion of a co-stimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both activating and costimulatory components.

[166] In some embodiments, an activating domain is included in one CAR while a co-stimulatory component is provided to other CARs recognizing a different antigen. In some embodiments, CARs include activating or stimulatory CARs and costimulatory CARs, both of which are expressed on the same cell (see WO2014/055668). In some aspects, the cells include one or more stimulatory or activating CARs and/or costimulatory CARs. In some embodiments, cells further comprise inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December 2013), such as CARs that recognize an antigen other than the antigen associated with and/ or specific to a disease or condition, wherein the activating signal delivered via the disease-targeted CAR is reduced or inhibited by binding the inhibitory CAR to its ligand, for example, to reduce non-specific effects.

[167] In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain associated with an intracellular CD3 domain (eg, CD3-zeta). In some embodiments, the intracellular signaling domain comprises CD28 and CD137 chimeric co-stimulatory domains (4-1BB, TNFRSF9) associated with the CD3-zeta intracellular domain.

[168] In some embodiments, the CAR encompasses one or more, eg, two or more, co-stimulatory domains and an activation domain, eg, a primary activation domain, in the cytoplasmic portion. Exemplary CARs include the intracellular components CD3-zeta, CD28, and 4-1BB.

[169] In some embodiments, the CAR or other antigen receptor further includes a marker, such as a cell surface marker, that can be used to confirm transduction or modification by cell engineering techniques to express a receptor, such as a truncated version of a cell surface receptor, such as EGFR (tEGFR). In some aspects, the marker includes all or part (eg, a truncated form) of CD34, NGFR, or epidermal growth factor receptor (eg, tEGFR). In some embodiments, a nucleic acid encoding a marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, such as T2A. For example, the marker and optionally the linker sequence can be any as described in International Patent Application Publication No. WO2014031687. For example, the marker may be a truncated EGFR (tEGFR) that is optionally linked to a linker sequence, such as a cleavable T2A linker sequence. An exemplary polypeptide for a truncated EGFR (e.g., tEGFR) comprises an amino acid sequence as set forth in SEQ ID NO: 7 or 16, or an amino acid sequence that has at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 7 or 16. An exemplary T2A linker sequence contains an amino acid sequence as set forth in SEQ ID NO: 6 or 17, or an amino acid sequence that has at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 6 or 17.

[170] In some embodiments, the marker is a molecule, such as a cell surface protein, that does not naturally occur on T cells or does not naturally occur on the surface of T cells, or a portion thereof. In some embodiments, the molecule is a non-self molecule, e.g., a non-self protein, i. e. a protein that is not recognized as "self" by the host's immune system, to which cells are introduced via adoptive transfer.

[171] In some embodiments, the implementation of the marker does not perform a therapeutic function and/or does not have an effect different from the effect used for genetic engineering, for example, to select cells that have been successfully modified. In other embodiments, the marker may be a therapeutic molecule or a molecule exhibiting another desired effect, such as a ligand for an in vivo occurring cell, such as a co-stimulatory molecule or an immune checkpoint molecule, to enhance and/or inhibit cell responses to adoptive transfer, and meeting with the ligand.

[172] In some cases, CARs are referred to as first, second and/or third generation CARs. In some aspects, a first generation CAR is a CAR that only provides an inducible CD3 target signal upon antigen binding; in some aspects, a second generation CAR is a CAR that provides such a signal and a costimulatory signal, such as a CAR that includes an intracellular signaling domain from a costimulatory receptor, such as CD28 or CD137; in some aspects, a third generation CAR is a CAR that includes multiple costimulatory domains of various costimulatory receptors.

[173] In some embodiments, the chimeric antigen receptor includes an extracellular portion containing an antibody or antibody fragment. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises scFv and the intracellular domain contains ITAM. In some aspects, the intracellular signaling domain includes the CD3 zeta chain (CD3ζ) signaling domain. In some embodiments, the implementation of the chimeric antigen receptor includes a transmembrane domain that binds an extracellular domain and an intracellular signaling domain. In some aspects, the transmembrane domain contains the transmembrane portion of CD28. In some embodiments, the chimeric antigen receptor contains the intracellular domain of a co-stimulatory molecule for T cells. The extracellular domain and the transmembrane domain may be linked directly or indirectly. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer, such as any spacer described herein. In some embodiments, the implementation of the receptor contains an extracellular part of the molecule from which the transmembrane domain is derived, such as the extracellular part of CD28. In some embodiments, the chimeric antigen receptor comprises an intracellular domain derived from a co-stimulatory molecule for T cells, or a functional variant thereof, for example, between the transmembrane domain and the intracellular signaling domain. In some aspects, the co-stimulatory molecule for T cells is CD28 or 41BB.

[174] In some embodiments, the scFv is derived from FMC63. FMC63 is a mouse IgG1 monoclonal antibody raised against Nalm-1 and -16 cells expressing human CD19 (Ling, NR, et al. (1987). Leucocyte typing III. 302). The FMC63 antibody contains CDRH1 and H2 as listed in SEQ ID NO: 38 and 39, respectively, and CDRH3 as listed in SEQ ID NO: 40 or 54, and CDRL1 as listed in SEQ ID NO: 35, and CDR L2 as listed in SEQ ID NO: 36 or 55 and CDR L3 as set forth in SEQ ID NO: 37 or 56. The FMC63 antibody contains a heavy chain variable region (V _H ) containing the amino acid sequence of SEQ ID NO: 41 and a light chain variable region (V _L ) containing the amino acid sequence of SEQ ID NO: 42. In some embodiments, the implementation of svFv contains a light chain variable region containing CDRL1 specified in SEQ ID NO: 35, CDRL2 specified in SEQ ID NO: 36 or 55, and CDRL3 specified in SEQ ID NO: 37 or 56, and/or a heavy chain variable region comprising CDRH1 as set forth in SEQ ID NO:38, CDRH2 as set forth in SEQ ID NO:39, and CDRH3 as set forth in SEQ ID NO:40 or 54. in some embodiments, the scFv comprises the FMC63 heavy chain variable region set forth in SEQ ID NO:41 and the light chain variable region epi FMC63 as set forth in SEQ ID NO: 42. In some embodiments, the heavy chain variable region and the light chain variable region are connected by a linker. In some embodiments, the implementation of the linker is as indicated in SEQ ID NO:24. In some embodiments, the implementation of the scFv contains VH, linker and VL in this order. In some embodiments, the implementation of the scFv contains VL linker and VH in this order. In some embodiments, the svFc is encoded by the nucleotide sequence shown in SEQ ID NO:25, or by a sequence that has at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:25. In some embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO:43, or a sequence that has at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 43.

[175] In some embodiments, the scFv is derived from SJ25C1. SJ25C1 is a mouse IgG1 monoclonal antibody raised against Nalm-1 and -16 cells expressing human CD19 (Ling, NR, et al. (1987). Leucocyte typing III. 302). The SJ25C1 antibody contains CDRH1, H2 and H3 as shown in SEQ ID NOs: 47-49, respectively, and the CDRL1, L2 and L3 sequences as shown in SEQ ID NOs: 44-46, respectively. The SJ25C1 antibody comprises a heavy chain variable region (V _H ) comprising the amino acid sequence of SEQ ID NO: 50 and a light chain variable region (V _L ) comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the svFv comprises a light chain variable region containing CDRL1 as set forth in SEQ ID NO: 44, CDRL2 as set forth in SEQ ID NO: 45, and CDRL3 as set forth in SEQ ID NO: 46, and/or a heavy chain variable region containing CDRH1 as set forth in SEQ ID NO: 47, CDRH2 as set forth in SEQ ID NO: 48 and CDRH3 as set out in SEQ ID NO: 49. In some embodiments, the scFv comprises the SJ25C1 heavy chain variable region as set forth in SEQ ID NO:50 and the SJ25C1 light chain variable region as set forth in SEQ ID NO:51. In some embodiments, the heavy chain variable region and the light chain variable region are connected by a linker . In some embodiments, the implementation of the linker is as indicated in SEQ ID NO:52. In some embodiments, the implementation of the scFv contains VH, linker and VL in this order. In some embodiments, the implementation of the scFv contains VL, linker and VH in this order. In some embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO:53, or a sequence that has at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:53.

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

[177] In some embodiments, the transmembrane domain of the recombinant receptor, e.g., CAR, is or includes a transmembrane domain of human CD28 (e.g., accession number P01747.1) or a variant thereof, such as a transmembrane domain that contains the amino acid sequence specified in SEQ ID NO: 8, or an amino acid sequence that has at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 8; in some embodiments, the transmembrane domain-containing portion of the recombinant receptor comprises the amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least exactly or about 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or such as the 27 amino acid human CD28 transmembrane domain.

[178] In some embodiments, the chimeric antigen receptor contains the intracellular domain of a costimulatory molecule for T cells. In some aspects, the co-stimulatory molecule for T cells is CD28 or 4-1BB.

[179] In some embodiments, the intracellular signaling domain or region, region or component(s) of a recombinant receptor, e.g., CAR, comprises an intracellular costimulatory signaling domain or region of human CD28, or a functional variant or portion thereof, such as a domain or region with LL replaced by GG at positions 186-187 of the native CD28 protein. For example, in some embodiments, an intracellular signaling domain or region may comprise an amino acid sequence as set forth in SEQ ID NO: 10 or 11, or an amino acid sequence that has at least or at least about 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 10 or 11. In some embodiments embodiment, the intracellular domain or region comprises an intracellular costimulatory signaling domain or region 4-1BB (e.g. Accession No. Q07011.1) or a functional variant or portion thereof, such as the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or more sequence identity to SEQ ID NO: 12, or such as human cytoplasmic domain 4-1BB of 42 am foreign acids.

[180] In some embodiments, the intracellular signaling domain or region of the recombinant receptor, e.g., CAR, comprises the human CD3 zeta chain, optionally its stimulatory signaling domain or region, or a functional variant thereof, such as the cytoplasmic domain or the human CD3ζ isoform 3 region of 112a. to. (Accession No. P20963.2) or CD3-zeta signaling domain or region as described in US Pat. No. 7,446,190 or US Pat. No. 8,911,993. : 13, 14, or 15, or an amino acid sequence that has at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 13, 14 or 15.

[181] In some aspects, the spacer comprises an IgG hinge only, such as an IgG4 or IgG1 hinge only, for example, the hinge only spacer specified in SEQ ID NO: 1. In other embodiments, the spacer is or contains an Ig hinge. , e.g., an IgG4-derived hinge optionally associated with C _H 2 and/or C _H 3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge associated with C _H 2 and C _H 3 domains. as indicated in SEQ ID NO: 4. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, associated only with the C _H 3 domain, e.g., as indicated in SEQ ID NO: 3. In some embodiments, the implementation the spacer is or contains a glycine-serine rich sequence or other flexible linker such as known flexible linkers.

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

[183] In some embodiments, nucleic acid molecules encoding such CAR constructs further comprise a sequence encoding a T2A ribosomal skip element and/or a tEGFR sequence, for example, downstream of the sequence encoding the CAR. In some embodiments, the sequence encodes a T2A ribosomal skip element as set forth in SEQ ID NO: 6 or 17, or an amino acid sequence that has at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:6 or 17. In some embodiments, T antigen receptor (e.g. CAR) expressing cells to express truncated EGFR (EGFRt) as a non-immunogenic selective epitope (e.g. by introducing a construct encoding CAR and EGFRt separated by a T2A ribosomal switch to express two proteins from one construct), which can then be used as a marker to detect such cells (see, for example, US patent No. 8802374). In some embodiments, the sequence encodes a tEGFR sequence as set forth in SEQ ID NO: 7 or 16, or an amino acid sequence that has at least or at least about 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 7 or 16.

[184] Recombinant receptors, such as CARs, expressed by cells introduced into an individual typically recognize or specifically bind a molecule that is expressed in, associated with, and/or specific to the disease or condition being treated, or associated cells. When specifically bound to a molecule, such as an antigen, the receptor typically delivers an immunostimulatory signal, such as an ITAM-transmitted signal, to the cell, thereby stimulating an immune response that targets the disease or condition. For example, in some embodiments, cells express a CAR that specifically binds to an antigen expressed by a cell or tissue affected by, or associated with, a disease or condition.

2. T cell receptors (TCR)

[185] In some embodiments, engineered cells, such as T cells, that express a T cell receptor (TCR) or antigen-binding portion thereof that recognize a peptide epitope or T cell epitope of a target polypeptide, such as a tumor antigen, are provided. , viral or autoimmune protein.

[186] In some embodiments, a "T cell receptor" or "TCR" is a molecule that contains the α and β variable chains (also known as TCRα and TCRβ, respectively) or the γ and δ variable chains (also known as TCRα and TCRβ, respectively), or an antigen-binding portion thereof, and which is capable of specifically binding to a peptide associated with an MHC molecule. In some embodiments, the implementation of the TCR has the form αβ. Typically, TCRs that exist in the αβ and γδ forms are structurally similar, however, T cells expressing them may have different anatomical positions or functions. The TCR may be on the cell surface or may be in a soluble form. Typically, the TCR is found on the surface of T cells (or T lymphocytes), where it is usually responsible for recognizing antigens associated with major histocompatibility complex (MHC) molecules.

[187] Unless otherwise indicated, the term "TCR" should be interpreted to encompass complete TCRs as well as their antigen-binding portions or antigen-binding fragments. In some embodiments, the implementation of the TCR is an intact or full-length TCR, including TCR in the form of αβ or in the form of γδ. In some embodiments, the TCR is an antigen-binding portion that is smaller than the full-length TCR, but that binds to a specific peptide associated with the MHC molecule, ie. binds to the MHC-peptide complex. In some instances, an antigen-binding portion or fragment of a TCR may contain only a portion of the structural domains of a full-length or intact TCR, but is still capable of binding a peptide epitope, such as an MHC-peptide complex, to which the entire TCR binds. In some instances, the antigen-binding portion contains TCR variable domains, such as the TCR α variable chain and the β variable chain of the TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex. Typically, TCR variable chains contain complementarity-determining regions involved in peptide, MHC and/or MHC-peptide recognition.

[188] In some embodiments, the TCR variable domains comprise hypervariable loops or complementarity-determining regions (CDRs), which typically make a major contribution to antigen recognition and binding capacity and specificity. In some embodiments, the TCR CDR or combination thereof forms all or substantially all of the antigen-binding site of a given TCR molecule. The various CDRs in the variable region of the TCR chain are usually separated by framework regions (FRs), which generally exhibit less variability among TCR molecules compared to CDRs (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. U.S.A. 87: 9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; also see Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the primary CDR responsible for antigen binding or specificity, or is the most important of the three CDRs on a given TCR variable region for antigen recognition and/or interaction with the truncated peptide portion of the peptide-MHC complex. In some contexts, the alpha chain CDR1 may interact with the N-terminal portion of certain antigenic peptides. In some contexts, the CDR1 of the beta chain may interact with the C-terminal portion of the peptide. In some contexts, CDR2 contributes the most or is the primary CDR responsible for interacting with or recognizing the MHC portion of the MHC-peptide complex. In some embodiments, the β-chain variable region may contain an additional hypervariable region (CDR4 or HVR4) that is normally involved in superantigen binding and not involved in antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

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

[190] In some embodiments, the implementation of the TCR chain contains one or more constant domains. For example, the extracellular portion of a given TCR chain (eg, α-chain or β-chain) may contain two immunoglobulin-like domains, such as a variable domain (eg, Vα or Vβ; typically amino acids 1 to 116 based on Kabat numbering). , Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) and a constant domain (e.g., α chain constant domain or Cα, usually positions 117 to 259 of the chain based on Kabat numbering, or a β-chain constant domain or Cβ, usually positions 117 to 295 of the chain based on Kabat) adjacent to the cell membrane.For example, in some cases, the extracellular part of the TCR, formed by two chains, contains two constant domains closest to the membrane and two variable domains farthest from the membrane, each of which contains a CDR.The TCR constant domain may contain short connecting sequences in which the cysteine residue is forms a disulfide bond, thereby linking two TCR chains. In some embodiments, the TCR may have an additional cysteine residue in each of the α and β chains such that the TCR contains two disulfide bonds in the constant domains.

[191] In some embodiments, the TCR chains comprise a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules such as CD3 and their subunits. For example, a TCR containing transmembrane constant domains can anchor a protein to the cell membrane and bind to invariant subunits of the CD3 signal transduction apparatus or complex. The intracellular tail portions of the CD3 signaling subunits (eg, the CD3γ, CD3δ, CD3ε, and CD3ζ chains) contain one or more tyrosine- or ITAM-based immunoreceptor activating motifs that are involved in the signaling ability of the TCR complex.

[192] In some embodiments, the TCR may be a heterodimer of two chains α and β (or optionally γ and δ), or it may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β chains or γ and δ chains) that are linked, for example, by a disulfide bond or disulfide bonds.

[193] In some embodiments, TCRs can be derived from known TCR sequence(s), such as Vα,β chain sequences, the entire coding sequence of which is substantially readily available. Methods for obtaining full length TCR sequences, including V chain sequences, from cellular sources are well known. In some embodiments, TCR-encoding nucleic acids can be obtained from a variety of sources, for example, by polymerase chain reaction (PCR) amplification of TCR-encoding nucleic acids residing in or isolated from a given cell or cells, or by synthesizing publicly available TCR DNA sequences.

[194] In some embodiments, the TCR is derived from a biological source, such as a T cell (eg, cytotoxic T cell), T cell hybridoma, or other publicly available source. In some embodiments, T cells can be obtained from cells isolated in vivo. In some embodiments, the TCR is a thymus-selected TCR. In some embodiments, the TCR is neoepitope restricted TCR. In some embodiments, the T cells may be a cultured T cell hybridoma or a clone. In some embodiments, the implementation of the TCR or antigennegative part can be obtained by methods of synthesis, knowing the sequence of the TCR.

[195] In some embodiments, the TCR is derived from a TCR identified or selected by screening a candidate TCR library against the target antigen polypeptide or its target T-cell epitope. TCR libraries can be obtained by amplifying the Vα and Vβ repertoire from isolated T cells from an individual, including cells present in the PBMC, spleen, or other lymphoid organ. In some cases, T cells can be increased in number from tumor infiltrating lymphocytes (TILs). In some embodiments, TCR libraries can be obtained from CD4+ or CD8+ cells. In some embodiments, the TCR may be amplified from a T cell source from a normal healthy individual, i.e. normal TCR libraries. In some embodiments, the TCR may be amplified from a source of T cells from an individual suffering from a disease, i. e. disease-associated TCR libraries. In some embodiments, degenerate primers are used to amplify the Vα and Vβ gene repertoire, for example by RT-PCR, in samples such as human T cells. In some embodiments, scTv libraries can be assembled from naïve Vα and Vβ libraries in which the amplified products are cloned or assembled to be separated by a linker. Depending on the individual and the cell source, the libraries may be specific for the HLA allele. Alternatively, in some embodiments, TCR libraries can be obtained by mutagenesis or diversification of the parent or framework TCR molecule. In some aspects, the TCR is subjected to directed evolution, for example, through mutagenesis, for example, chain α or β. In some aspects, specific residues in the CDR of the TCR are changed. In some embodiments, selected TCRs can be modified by affinity maturation. In some embodiments, antigen-specific T cells can be selected, for example, by screening for CTL activity against a peptide. In some aspects, TCRs, eg, those present on antigen-specific T cells, can be selected, eg, by binding activity, eg, specific affinity or avidity for the antigen.

[196] In some embodiments, the genetically engineered antigen receptors include recombinant T cell receptors (TCRs) and/or TCRs cloned from naturally occurring T cells. In some embodiments, a high affinity T cell clone for an isolated target antigen (eg, cancer antigen) from a patient is identified and introduced into the cells. In some embodiments, a TCR clone for the target antigen is obtained from transgenic mice that have been engineered with human immune system genes (eg, the human leukocyte antigen, or HLA) system. See, for example, tumor antigens (see, for example, Parkhurst et al. (2009) Clin Cancer Res. 15:169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808). In some embodiments, phage display is used to isolate the TCR against the target antigen (see, for example, Varela-Rohena et al. (2008) Nat Med. 14:1390-1395 and Li (2005) Nat Biotechnol. 23:349-354 ).

[197] In some embodiments, the TCR, or an antigen-binding portion thereof, is a TCR, or an antigen-binding portion thereof, that is modified or engineered. In some embodiments, targeted alteration techniques are used to generate TCRs with altered properties, such as higher affinity for a specific MHC-peptide complex. In some embodiments, targeted changes are made 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 U S A, 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, approaching a display involves constructing or modifying a known, original, or reference TCR. For example, in some cases, wild-type TCRs can be used as a template to generate mutant TCRs in which one or more CDR residues are mutated and mutants with a desired altered property, such as higher affinity for the desired target antigen, are selected.

[198] In some embodiments, the target polypeptide peptides for use in the production or production of a TCR of interest are known and can be readily identified by one of skill in the art. In some embodiments, peptides suitable for use in making TCRs or antigen-binding portions can be determined based on the presence of an HLA-restricted motif in a target polypeptide of interest, such as the target polypeptide described below. In some embodiments, peptides are identified using computer predictive models known to those skilled in the art. In some embodiments, for 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 (see 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 about 39-46% of all Europioids and thus is an appropriate choice. an MHC antigen for use in making a TCR or other MHC-peptide binding molecule.

[199] HLA-A0201 binding motifs and cleavage sites for proteasomes and immune proteasomes using computer predictive models are known to those skilled in the art. For prediction of MHC class I binding sites, such models include, but are not limited to, ProPred1 (described in more detail in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17(12):1236-1237 2001), and SYFPEITHI (See Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction. in Immunoinformatics Methods in Molecular Biology, vol 409(1): 75-93 2007).

[200] In some embodiments, the TCR, or an antigen-binding portion thereof, may be a recombinantly produced natural protein, or a mutant form thereof, in which one or more properties, such as binding characteristics, are altered. In some embodiments, the TCR may be from one of a variety of animal species, such as a human, mouse, rat, or other mammal. The TCR may be in cell-bound or soluble form. In some embodiments, for the purposes of the provided methods, the TCR is in cell-bound form, expressed on the cell surface.

[201] 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 have structures as described in WO 03/020763, WO 04/033685, WO2011/044186.

[202] In some embodiments, the TCR contains a sequence corresponding to a transmembrane sequence. In some embodiments, the TCR does not contain a sequence corresponding to cytoplasmic sequences. In some embodiments, the TCR is capable of complexing the TCR with CD3. In some embodiments, any of the TCRs, including dTCR or scTCR, may be associated with signaling domains that provide active TCR on the T cell surface. In some embodiments, the TCR is expressed on the surface of cells.

[203] In some embodiments, the dTCR comprises a first polypeptide, wherein the sequence corresponding to the variable region of the TCR α chain is fused to the N-terminus of the sequence corresponding to the extracellular sequence of the TCR α chain constant region, and a second polypeptide, wherein the sequence corresponding to the sequence of the TCR α chain variable region of the β-chain of TCR, fused to the N-terminus of the sequence corresponding to the extracellular sequence of the constant region of the β-chain of TCR, and the first and second polypeptides are linked by a disulfide bond. In some embodiments, the bond may correspond to a native interchain disulfide bond present in native dimeric αβ TCRs. In some embodiments, interchain disulfide bonds are not present in native TCR. For example, in some embodiments, one or more cysteine residues may be included in the extracellular constant region sequences of a pair of dTCR polypeptides. In some cases, both native and non-native disulfide bonds may be desirable. In some embodiments, the TCR contains a transmembrane sequence for anchoring to the membrane.

[204] In some embodiments, the dTCR comprises a TCR α-chain containing an α-variable domain, a constant α-domain and a first dimerization motif linked to the C-terminus of the constant α-domain, and a TCR β-chain containing a β-variable domain , a constant β-domain, and a first dimerization motif linked to the C-terminus of the constant β-domain, wherein the first and second dimerization motifs readily interact to form a covalent bond between the amino acid in the first dimerization motif and the amino acid in the second dimerization motif binding the α-chain of the TCR and the TCR β-chain together.

[205] In some embodiments, the TCR is scTCR. Typically, scTCRs can be generated using methods known to those of skill in the art, see, for example, Soo Hoo, W. F. et al. PNAS (USA) 89, 4759 (1992); Wülfing, C. and Plückthun, A., J. Mol. Biol. 242, 655 (1994); Kurucz, I. et al. PNAS (USA) 90 3830 (1993); PCT International Publication Nos. WO 96/13593, WO 96/18105, WO99/60120, WO99/18129, WO 03/020763, WO2011/044186; and Schlueter, C. J. et al. J. Mol. Biol. 256, 859 (1996). In some embodiments, the scTCR contains an introduced non-native disulfide interchain bond to facilitate association of the TCR chains (see, for example, PCT International Publication No. WO 03/020763). In some embodiments, the scTCR is a disulfide-linked truncated TCR in which heterogeneous leucine zippers fused to their C-terminus facilitate chain association (see, for example, PCT International Publication No. WO99/60120). In some embodiments, the scTCR comprises a TCRα variable domain covalently linked to a TCRβ variable domain via a peptide linker (see, for example, PCT International Publication No. WO99/18129).

[206] In some embodiments, the scTCR comprises a first segment consisting of an amino acid sequence corresponding to the TCR α chain variable region, a second segment consisting of an amino acid sequence corresponding to the TCR β chain variable region sequence fused to the N-terminus of the amino acid sequence, the corresponding extracellular sequence of the TCR β-chain constant domain, and a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

[207] In some embodiments, the scTCR comprises a first segment consisting of an α-chain variable region sequence fused to the N-terminus of an extracellular α-chain constant domain sequence and a second segment consisting of a β-chain variable region sequence fused to N the end of the extracellular constant and transmembrane sequence of the β-chain, and optionally a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

[208] In some embodiments, the scTCR comprises a first segment consisting of a TCR β-chain variable region sequence fused to the N-terminus of a β-chain extracellular constant domain sequence and a second segment consisting of an α-chain variable region sequence fused to The N-terminus of an extracellular constant and transmembrane α-chain sequence, and optionally a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

[209] In some embodiments, the scTCR linker that binds the first and second segments of the TCR can be any linker capable of forming a single polypeptide chain while retaining TCR binding specificity. In some embodiments, the implementation of the linker sequence may have the formula, for example, -P-AA-P-, where P is a proline and AA is an amino acid sequence, where the amino acids are glycine and 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 is long enough 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 too long to block or reduce binding of the scTCR to the ligand. target. In some embodiments, the implementation of the linker may contain from or from about 10 to 45 amino acids, for example, from 10 to 30 amino acids or from 26 to 41 amino acid residues, for example, 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 (SEQ ID NO: 22). In some embodiments, the implementation of the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO: 23).

[210] In some embodiments, the implementation of the scTCR contains a covalent disulfide bond linking the remainder of the immunoglobulin region of the constant domain of the α-chain to the remainder of the immunoglobulin region of the constant domain of the β-chain. In some embodiments, there is no interchain disulfide bond in the native TCR. For example, in some embodiments, one or more cysteine residues may be included in the extracellular constant region sequences of the first and second segments of the scTCR polypeptide. In some cases, both native and non-native disulfide bonds may be desirable.

[211] In some embodiments, the implementation of the dTCR or scTCR, containing embedded interchain disulfide bonds, native disulfide bonds are not present. In some embodiments, one or more native cysteine residues forming native interchain disulfide bonds are replaced by another residue, such as serine or alanine. In some embodiments, an inserted disulfide bond can be formed by mutating cysteine residues on the first and second segments to cysteine. Exemplary non-native TCR disulfide bonds are described in PCT International Publication No. WO2006/000830.

[212] In some embodiments, the TCR or antigen-binding fragment thereof has an affinity with an equilibrium binding constant for the target antigen of from or about 10-5 to 10-12 M, including all individual values and ranges in between. In some embodiments, the target antigen is an MHC-peptide or ligand complex.

[213] In some embodiments, the TCR-encoding nucleic acid or nucleic acids, such as α- and β-strands, can be amplified by PCR, cloning, or other suitable method and cloned into a suitable expression vector or vectors. The expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include vectors designed for expansion and expansion, or expression, or both, such as plasmids and viruses.

[214] In some embodiments, the vector may be pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden) or the pEX series (Clontech, Palo Alto, Calif.). In some cases, bacteriophage-based vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. In some embodiments, plant expression vectors can be used and include 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, a viral vector is used, such as a retroviral vector.

[215] In some embodiments, recombinant expression vectors can be generated using standard recombinant DNA techniques. In some embodiments, the vectors may contain regulatory sequences, such as transcription and translation initiation and termination codons, that are specific for the type of host (e.g., bacteria, fungi, plants, or animals) to which the vector is administered, as appropriate and given whether the vector is a DNA or RNA based vector. In some embodiments, the vector may contain a non-native promoter operably linked to a nucleotide sequence encoding a TCR or an antigen binding portion (or other MHC peptide binding molecule). In some embodiments, the promoter may be a non-viral promoter or a viral promoter such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and a promoter found in the long terminal repeat of a mouse stem cell virus. Other promoters known to the skilled artisan are also contemplated.

[216] In some embodiments, after a T cell clone has been obtained, the alpha and beta TCR chains are isolated and cloned into a gene expression vector. In some embodiments, the TCR alpha and beta chain genes are linked through a picornavirus 2A ribosomal transmission peptide such that both chains are co-expressed. In some embodiments, TCR gene transfer is via retroviral or lentiviral vectors, or via transposons (see, for example, Baum et al. (2006) Molecular Therapy: The Journal of the American Society of Gene Therapy. 13:1050-1063; Frecha et al (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy 18:1748-1757 and Hackett et al (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy 18:674- 683.

[217] In some embodiments, to obtain a vector encoding a TCR, the α and β chains are PCR amplified with total cDNA isolated from a T cell clone expressing the TCR of interest and cloned into an expression vector. In some embodiments, the α and β chains are cloned into the same vector. In some embodiments, the α and β chains are cloned into different vectors. In some embodiments, the resulting α and β chains are inserted into a retroviral, eg, lentiviral, vector.

3. Chimeric Autoantibody Receptors (CAAR)

[218] In some embodiments, the recombinant receptor is a chimeric autoantibody receptor (CAAR). In some embodiments, the CAAR is specific for an autoantibody. In some embodiments, a cell expressing CAAR, such as a T cell modified to express CAAR, can be used to specifically bind and kill cells expressing autoantibodies, but not normal cells expressing antibodies. In some embodiments, CAAR-expressing cells can be used to treat an autoimmune disease associated with the expression of self antigens, such as autoimmune diseases. In some embodiments, CAAR-expressing cells can be targeted to B cells that eventually produce autoantibodies and display autoantibodies on their cell surfaces, marking these B cells as disease-specific targets for therapeutic intervention. In some embodiments, CAAR-expressing cells can be used to efficiently target and kill pathogenic B cells in autoimmune diseases by targeting disease-causing B cells using an antigen-specific autoantibody receptor chimeric. In some embodiments, the recombinant receptor is a CAAR, such as any CAAR described in US Patent Application Publication No. US 2017/0051035.

[219] In some embodiments, the CAAR comprises an autoantibody binding domain, a transmembrane domain, and an intracellular signaling region. In some embodiments, the implementation of the intracellular signaling region contains an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or contains a primary signaling domain, a signaling domain that is capable of inducing a primary activation signal in a T cell, a T cell receptor (TCR) signaling domain component, and/or a signaling domain containing an immunoreceptor activating motif. based on tyrosine (ITAM). In some embodiments, the intracellular signaling region comprises a secondary or costimulatory signaling region (secondary intracellular signaling regions).

[220] In some embodiments, the implementation of the binding domain of the autoantibody contains a self-antigen or fragment thereof. The choice of autoantigen may depend on the type of autoantibody being targeted. For example, an autoantigen may be selected because it recognizes an autoantibody on a target cell, such as a B cell, associated with a particular disease state, eg, an autoimmune disease, such as an autoantibody-mediated autoimmune disease. In some embodiments, the autoimmune disease includes pemphigus vulgaris (PV). Illustrative autoantigens include desmoglein 1 (Dsg1) and Dsg3.

4. Multiple targeting

[221] In some embodiments, cells and methods include multiple targeting strategies, such as expressing two or more genetically engineered receptors on a cell that recognize the same or different antigens and typically involve different components of intracellular signaling. Such multiple targeting strategies are described, for example, in International Patent Application Publication No. WO 2014055668 A1 (which describes combinations of activating and costimulatory CARs, e.g., targeting two different antigens that are present separately on non-targeted, e.g. on cells associated with the disease or condition being treated) and Fedorov et al., Sci. Transl. Medicine, 5(215) (December 2013) (which describes cells expressing an activating and an inhibitory CAR, such as cells in which an activating CAR binds to a single antigen expressed on both normal or non-disease-associated cells and cells associated with the disease or condition being treated and the inhibitory CAR binds to another antigen expressed only on normal or undesirable cells).

[222] For example, in some embodiments, cells include a receptor that expresses a first engineered antigen receptor (e.g., CAR or TCR) that is capable of inducing an activating signal in the cell, typically upon specific binding to an antigen recognized by the first receptor, e.g. , with the first antigen. In some embodiments, the cell further comprises a second genetically engineered antigen receptor (e.g., CAR or TCR), e.g., a chimeric co-stimulatory receptor, that is capable of inducing a co-stimulatory signal in an immune cell, typically upon specific binding 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.

[223] In some embodiments, the first and/or second engineered antigen receptor (eg, CAR or TCR) is capable of inducing an activating signal in a cell. In some embodiments, the implementation of the receptor includes an intracellular signaling component containing ITAM or ITAM-like motifs. In some embodiments, activation induced by the first receptor involves signal transduction or a change in protein expression in a cell that results in the initiation of an immune response, such as ITAM phosphorylation and/or initiation of an ITAM-mediated signal transduction cascade, formation of an immunological synapse, and/or clustering of molecules near binding receptor (e.g., CD4 or CD8, etc.), activation of one or more transcription factors such as NF-κB and/or AP-1, and/or induction of gene expression of factors such as cytokines, proliferation and/ or survival.

[224] In some embodiments, the first and/or second receptor includes intracellular signaling domains or regions of costimulatory receptors such as CD28, CD137 (41BB), OX40, and/or ICOS. In some embodiments, the first and second receptors comprise intracellular co-stimulatory receptor signaling domains that are different. In one embodiment, the first receptor contains the CD28 costimulatory signaling region and the second receptor contains the 4-1BB costimulatory signaling region, or vice versa.

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

[226] In some embodiments, the first receptor contains an intracellular signaling domain containing ITAM or ITAM-like motifs, and the second receptor contains an intracellular signaling domain of a costimulatory receptor. A costimulatory signal, in combination with an activating signal induced in one cell, is a signal that results in an immune response, such as a sustained and prolonged immune response such as increased gene expression, secretion of cytokines and other factors, and T-cell mediated effector functions. such as killing cells.

[227] In some embodiments, neither binding to the first receptor alone nor binding to the second receptor alone induces a sustained immune response. In some aspects, if only one receptor binds, the cell becomes tolerant or non-responsive to the antigen, or is inhibited and/or not induced to proliferate or secrete factors or perform effector functions. However, in some such embodiments, when multiple receptors bind, for example, when a cell expressing the first and second antigens is encountered, the desired response is achieved, such as complete immune activation or stimulation, as indicated by, for example, secretion of one or more cytokines, proliferation, persistence and/or performance of an immune effector function such as cytotoxic killing of a target cell.

[228] In some embodiments, two receptors induce, respectively, an activating and inhibitory signal in a cell, such that binding of one of the receptors to its antigen activates the cell or induces a response, however, binding of the second inhibitory receptor to its antigen induces a signal that suppresses or inhibits this answer. Examples are combinations of activating CARs and inhibitory CARs, or iCARs. A strategy can be used, for example, in which an activating CAR binds an antigen that is expressed in the disease or condition, but which is also expressed on normal cells, and an inhibitory receptor binds to a separate antigen that is expressed on normal cells, but not on cells associated with the disease. or state.

[229] In some embodiments, a multi-targeting strategy is used when an antigen associated with a particular disease or condition is expressed on a non-disease-associated cell and/or is expressed on the engineered cell itself, either transiently (e.g., upon stimulation in association with genetic engineering), or permanently. In such cases, due to the need to bind two separate and individually specific antigen receptors, specificity, selectivity and/or efficacy may be increased.

[230] In some embodiments, a plurality of antigens, eg, the first and second antigens, are expressed on the cell, tissue, or disease or condition being targeted, eg, on a cancerous cell. In some aspects, the cell, tissue, disease, or condition is multiple myeloma or a multiple myeloma cell. In some embodiments, one or more antigens are typically also expressed on a cell that is undesirable to be targeted by cell therapy, such as a normal or non-disease-associated cell or tissue, and/or the engineered cell itself. In such embodiments, due to the need to bind multiple receptors in order to achieve a cell response, specificity and/or efficiency can be achieved.

C. Cells and Producing Cells for Modification by Genetic Engineering

[231] Among the cells expressing the receptors and administered by the provided methods, cells modified by the methods of engineering are presented. Modification by genetic engineering typically involves introducing a nucleic acid encoding a recombinant or engineered component into a composition containing cells, for example, via retroviral transduction, transfection, or transformation.

[232] In some embodiments, the implementation of the nucleic acids are heterologous, ie. not normally present in a cell or a sample derived from such a cell, e.g., nucleic acids derived from another organism or cells which, for example, are not normally present in the cell being engineered and/or the organism from which the cell is derived . In some embodiments, the nucleic acids are non-naturally occurring, such as non-naturally occurring nucleic acid, including nucleic acid containing chimeric combinations of nucleic acids encoding different domains of many different cell types.

[233] The cells are typically eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph, or lymphoid organs, and are cells of the immune system, such as cells of innate or adaptive immunity, for example, myeloid or lymphoid cells, including lymphocytes, primarily T cells and/ or NK cells. Other exemplary cells include stem cells such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as cells isolated directly from the individual and/or isolated from the individual and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as entire populations of T cells, CD4+ cells, CD8+ cells, and subpopulations thereof, such as subpopulations defined by function, activation state, maturity, potential for differentiation, expansion, recycling, localization and/or persistence, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With regard to the individual being treated, the cells may be allogeneic and/or autologous. The methods include standard methods. In some aspects, such as in the case of standard technologies, the cells are pluripotent and/or multipotent, such as stem cells such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from an individual, preparing, processing, culturing and/or engineering them, and reintroducing them to the same individual, before or after cryopreservation.

[234] Among the subtypes and subpopulations of T cells and/or CD4+ and/or CD8+ T cells are naive T (T _N ) cells, effector T cells (T _EFF ), memory T cells and their subtypes, such as stem Memory T cells (T _SCM ), central memory T cells (T _CM ), effector memory T cells (T _EM ), 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 cells (MAIT), naturally occurring and adaptive regulatory T(Treg) cells, helper T cells such as TH1 cells, cells TH2, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

[235] In some embodiments, the cells are natural killer (NK). In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.

[236] In some embodiments, cells include one or more genetically engineered nucleic acids and thereby express recombinant or genetically engineered products, such as nucleic acids. In some embodiments, the nucleic acids are heterologous, ie. not normally present in a cell or sample derived from a cell, such as nucleic acids derived from another organism or cells which, for example, are not normally found in a cell being modified by engineering techniques and/or the organism from which the cell is derived . In some embodiments, the nucleic acids are not naturally occurring, such as a non-naturally occurring nucleic acid, including nucleic acid containing chimeric combinations of nucleic acids encoding different domains from several different cell types.

[237] In some embodiments, the implementation of obtaining modified methods of engineering includes one or more stages of cultivation and/or obtaining. Cells for the introduction of a nucleic acid encoding a transgenic receptor, such as a CAR, can be isolated from a sample, such as a biological sample, for example, a sample obtained from or derived from an individual. In some embodiments, the individual whose cell is isolated is an individual having a disease or condition, or an individual who is in need of, or will receive, cell therapy. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy for which cells are isolated, processed and/or engineered.

[238] Thus, in some embodiments, the cells are primary cells, eg, human primary cells. Samples include tissue, fluid, and other samples taken directly from the individual, as well as samples resulting from one or more processing steps such as separation, centrifugation, genetic modification (eg, transduction with a viral vector), washing, and/ or incubation. The biological sample may be a sample obtained directly from a biological source or a sample that is being processed. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, organ and tissue samples, including processed samples derived from them.

[239] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Illustrative specimens include whole blood, peripheral blood mononuclear cells (PBMC), leukocytes, bone marrow, thymus, tissue biopsy, tumor cells, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosal-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestines, large intestine, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organ, and/or cells derived from them. Samples include, in the context of cell therapy, such as adoptive cell therapy, samples from autologous and allogeneic sources.

[240] In some embodiments, the cells are derived from cell lines, eg, T cell lines. In some embodiments, the cells are obtained from a xenogeneic source, such as a mouse, a rat, a non-human primate, and a pig.

[241] In some embodiments, cell isolation includes one or more preparative and/or non-affinity cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desirable components, lyse, or remove cells that are sensitive to particular reagents. In some examples, cells are separated based on one or more properties such as density, attachment, size, sensitivity, and/or resistance to particular components.

[242] In some examples, cells from the circulating blood of an individual are obtained, for example, by apheresis or leukapheresis. In some aspects, the samples contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and/or platelets, and in some aspects they contain cells other than erythrocytes and platelets.

[243] In some embodiments, blood cells obtained from an individual are washed, for example, to remove a plasma fraction and to place the cells in a suitable buffer or medium for subsequent processing steps. In some embodiments, cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all of the divalent cations. In some aspects, the washing step is carried out in a semi-automated "flow through" centrifuge (eg, Cobe 2991 Cell Processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is carried out by flow filtration along the flow (TFF) in accordance with the manufacturer's instructions. In some embodiments, after washing, the cells are suspended in various biocompatible buffers, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, the components of the blood sample are removed and the cells directly resuspended in culture medium.

[244] In some embodiments, the methods include methods for density separation, such as obtaining leukocytes from peripheral blood by lysing erythrocytes and centrifuging in a Percoll or Ficoll gradient.

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

[246] Such separation steps may be based on positive selection, in which cells that have bound reagents are retained for further use, and/or negative selection, in which cells that have not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for later use. In some aspects, negative selection may be useful, particularly when there is no available antibody that specifically identifies a cell type in a heterogeneous population, such that separation is best done based on markers expressed by cells other than the desired population.

[247] Separation should not lead to 100% enrichment or elimination of a particular cell population or cells expressing a particular marker. For example, positive selection or an increase in the number of cells of a particular type, such as cells expressing a marker, refers to an increase in the number or percentage of such cells, but should not lead to a complete absence of cells expressing the marker. Similarly, negative selection, removal or depletion of cells of a particular type, such as cells expressing a marker, refers to a reduction in the number or percentage of such cells, but should not lead to the complete elimination of such cells.

[248] In some examples, several rounds of separation are carried out, where the positively or negatively selected fraction is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, for example, by incubating cells with multiple antibodies or binding partners, each of which is specific for the marker targeted by negative selection. Similarly, multiple cell types can be simultaneously positively selected by incubation with multiple antibodies or binding partners expressed on different cell types.

[249] For example, in some aspects, certain subpopulations of T cells are isolated by positive or negative selection methods, such as cells positive or expressing high levels of one or more surface markers, for example, CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+ , CD45RA+, and/or CD45RO+ T cells.

[250] For example, CD3+, CD28+ T cells can be positively selected using anti-CD3/anti-CD28 conjugated magnetic beads (eg, DYNABEADS® M-450 CD3/CD28 T Cell Expander).

[251] In some embodiments, isolation is accomplished by increasing a particular cell population by positive selection or depleting a particular cell population by negative selection. In some embodiments, positive or negative selection is made by incubating cells with one or more antibodies or other binding compounds that specifically bind to one or more surface markers expressed (marker+) or expressed at a relatively higher level (marker ^high ) on the cells. subjected to positive or negative selection, respectively.

[252] In some embodiments, T cells are isolated from a PBMC sample by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection of markers expressed or expressed at a relatively higher level on one or more naive T cell subsets, memory T cell subsets, and/or effector T cell subsets.

[253] In some embodiments, among CD8+ cells, naïve cells, central memory cells, effector memory cells, and/or central memory stem cells are further increased or depleted, e.g., by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, the increase in memory central T cells (T _CM ) is carried out to increase efficiency, for example, to increase long-term survival, expansion and/or engraftment after administration, which in some aspects are 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, the combination of TCM-enriched CD8+ T cells and CD4+ cells further enhances efficacy.

[254] In some embodiments, memory T cells are present in both the CD62L+ and CD62L- subgroups of CD8+ human peripheral blood lymphocytes. The CD62L-CD8+ and/or CD62L+CD8+ fractions can be increased or depleted in PBMC, for example using anti-CD8 and anti-CD62L antibodies.

[255] In some embodiments, the increase in central memory T cells ( _TCM ) is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3 and/or CD127; in some aspects it is based on negative selection of cells expressing or overexpressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched in TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA and positive selection or increase in the content of cells expressing CD62L. In one aspect, central memory TCM (TCM) expansion is performed by starting with a negative cell fraction selected based on CD4 expression, negatively selected based on CD14 and CD45RA expression, and positively selected based on CD62L. Such selection in some aspects is carried out simultaneously and in other aspects it is carried out sequentially, in any order. In some aspects, the same selection step based on CD4 expression that is used to generate a population or subset of CD8+ cells is also used to generate a population or subset of CD4+ cells, such that both positive and negative fractions in the CD4-based separation are retained and used for subsequent method steps, optionally after one or more additional positive or negative selection steps.

[256] In a specific example, a PBMC sample or other leukocyte sample is subjected to CD4+ cell selection, where both negative and positive fractions are retained. The negative fraction is then negatively selected based on CD14 and CD45RA or CD19 expression, and positively selected based on a central memory T cell marker such as CD62L or CCR7, with positive and negative selection in any order.

[257] CD4+ T helper cells are sorted into naive cells, central memory cells, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, the naïve CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, the central CD4+ memory cells are CD62L+ and CD45RO+. In some embodiments, the CD4+ effector cells are CD62L- and CD45RO-.

[258] In one example, to increase the content of CD4+ cells through negative selection, the monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is coupled to a solid support or matrix, such as magnetic beads or paramagnetic beads, to allow cell separation for positive and/or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation methods (discussed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

[259] In some aspects, the sample or cell composition to be separated is incubated with a small, magnetizable or magnetically sensitive material, such as magnetically sensitive particles or microparticles, such as paramagnetic beads (eg, such as Dynalbeads or MACS beads). The magnetically sensitive material, such as particles, is typically associated directly or indirectly with a binding partner, such as an antibody, that specifically binds to a molecule, such as a surface marker, present on the cell(s) or population of cells that it is desired to separate, such as that are intended to be subjected to negative or positive selection.

[260] In some embodiments, the implementation of the magnetic particle or granule contains a magnetically sensitive material associated with a specific binding partner, such as an antibody or other binding partner. There are many magnetically sensitive materials used in separation processes. Suitable magnetic particles include those described in Molday US Pat. No. 4,452,773 and European Patent Specification EP 452342 B, which are incorporated herein by reference. Other examples are colloidal sized particles such as those described in US Pat. No. 4,795,698 to Owen and US Pat. No. 5,200,084 to Liberti et al.

[261] or binding partners, or molecules such as secondary antibodies or other reagents that specifically bind to such antibodies or binding partners that are associated with a magnetic particle or bead, specifically bind to cell surface molecules if they are present on cells in the sample.

[262] In some aspects, the sample is placed in a magnetic field and cells to which magnetically sensitive or magnetizable particles are associated are associated with a magnet and separated from unlabeled cells. For positive selection, cells bound to the magnet are retained; for negative selection, cells that have not bound (unlabeled cells) are retained. In some aspects, the combination of positive and negative selection is carried out during the same selection step, where the positive and negative fractions are retained and further processed or subjected to other separation steps.

[263] In certain embodiments, the magnetically sensitive particles are coated with primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are associated with cells by coating with primary antibodies 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 beads coated with a cell type-specific secondary antibody or other binding partner (eg, streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[264] In some embodiments, magnetically sensitive particles are left associated with cells, which are then incubated, cultured, and/or modified by engineering methods; in some aspects, the particles are left associated with cells for administration to a patient. In some embodiments, magnetizable or magnetically sensitive particles are recovered from cells. Methods for extracting magnetizable particles from cells are known and include, for example, the use of competing unlabeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[265] In some embodiments, affinity-based selection is performed by magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). Magnetically activated cell sorting (MACS) systems are capable of high purity selection of cells to which magnetized particles are bound. In certain embodiments, the MACS operates in a mode in which non-target and target cell types are sequentially eluted following the application of an external magnetic field. In other words, cells bound to the magnetic particles are held in place while unbound cell types are eluted. Then, after this first elution step is complete, the cell types that were trapped in the magnetic field and prevented from eluting are released so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and eliminated from a heterogeneous population of cells.

[266] In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that performs one or more of the steps of isolation, cell preparation, separation, processing, incubation, culture, and/or formulation. In some aspects, a system is used to carry out 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 the system as described in International Patent Application Publication Number WO2009/072003 or US Patent Application Publication Number US 20110003380 A1.

[267] In some embodiments, the system or device performs one or more, for example, all of the steps of isolation, processing, modification, and formulation in an integrated or stand-alone system, and/or in an automated or programmable manner. In some aspects, the system or device includes a computer and/or computer program coupled to the system or device that allows a user to program, control, evaluate, and/or correct various aspects of the processing, extraction, modification, engineering, and composition steps.

[268] In some aspects, the separation and/or other steps are performed using the CliniMACS system (Miltenyi Biotec), for example, to automatically separate cells on a clinical scale in a closed and sterile system. Components may include an integrated microcomputer, a magnetic separation element, a peristaltic pump, and various shut-off valves. In some aspects, the integrated computer controls all components of the device and controls the system to perform repetitive procedures in a standardized sequence. In some aspects, the magnetic separation element includes a movable permanent magnet and a selection column stand. The peristaltic pump controls the flow rate through the tubing and, together with the check valve, provides a controlled flow of buffer through the system and continuous cell suspension.

[269] In some aspects, the CliniMACS system uses magnetizable particles to which the antibody is associated, which are provided in a sterile, non-pyrogenic solution. In some embodiments, after labeling cells with magnetic particles, the cells are washed to remove excess particles. The cell collection bag is then connected to the tubing set, which in turn is connected to the bag containing the buffer and the cell collection bag. The tubing set consists of pre-assembled sterile tubing, including guard column tubing and separation column, and is for single use only. After starting the separation program, the system automatically feeds the cell sample into the separation column. Labeled cells are retained on the column while unlabeled cells are removed through a series of washing steps. In some embodiments, cell populations for use in the methods described herein are unlabeled and not retained on the column. In some embodiments, cell populations for use in the methods described herein are labeled and remain on the column. In some embodiments, cell populations for use in the methods described herein are eluted from the column after the magnetic field has been removed and collected in a cell collection bag.

[270] In certain embodiments, separation and/or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). In some aspects, the CliniMACS Prodigy system is equipped with a cell processing element that allows automatic washing and cell fractionation via centrifugation. The CliniMACS Prodigy system can also include an integrated camera and image recognition software that determines the optimal cell fractionation endpoint by distinguishing macroscopic layers of the original cell product. For example, peripheral blood is automatically divided into layers of erythrocytes, leukocytes, and plasmacytes. The CliniMACS Prodigy system can also include an integrated cell culture chamber that performs cell culture protocols such as cell differentiation and expansion, antigen loading, and long-term cell culture, for example. The inlets may allow sterile removal and replenishment of the medium, and cell monitoring may be performed using an in-line microscope. See, for example, Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[271] In some embodiments, the cell population described herein is harvested and enriched (or depleted) by flow cytometry, in which cells stained for multiple cell surface markers are moved in a fluid stream. In some embodiments, the cell population described herein is harvested and enriched (or depleted) by preparative scale sorting (FACS). In certain embodiments, the cell population described herein is harvested and enriched (or depleted) using microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, for example, International Patent Application Publication number WO 2010/033140 , Cho et al (2010) Lab Chip 10, 1567-1573 and Godin et al (2008) J Biophoton 1(5):355-376). In both cases, cells can be labeled with multiple markers, allowing the isolation of certain subsets of T cells in high purity.

[272] In some embodiments, antibodies or binding partners are 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 fluorescently labeled antibodies. In some examples, cell separation based on the binding of antibodies or other binding partners specific for one or more cell surface markers is carried out in a fluid stream, for example, by fluorescence-activated cell sorting (FACS), including preparative scale (FACS), and/or on microelectromechanical systems (MEMS) chips, for example in combination with a flow cytometric detection system. Such methods allow positive and negative selection based on multiple markers simultaneously.

[273] In some embodiments, methods of preparation include the steps of freezing, eg, cryopreserving, cells either before or after isolation, incubation, and/or engineering modification. In some embodiments, the step of freezing and then thawing eliminates granulocytes and to some extent monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution, for example, after a plasma and platelet wash step. In some aspects, any of a variety of known freezing solutions and parameters may be used. One example involves the use of PBS containing 20% DMSO and 8% human serum albumin (HSA) or other suitable cell freezing medium. It is then diluted 1:1 with medium to a final concentration of DMSO and HSA of 10% and 4%, respectively. Cells are then typically frozen to -80° C. at a rate of 1° per minute and stored in a vapor phase liquid nitrogen storage tank.

[274] In some embodiments, cells are incubated and/or cultured prior to or concomitantly with modification by genetic engineering. Incubation steps may include cultivation, processing, stimulation, activation and/or expansion. The incubation and/or engineering modification can be carried out in a culture vessel such as a cell, chamber, well, column, tube, tubing, valve, container, culture dish, bag or other cell culture or processing container. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulating agent. Such conditions include conditions designed to induce proliferation, expansion, activation and/or survival of cells in a population, to mimic antigen exposure, and/or to prepare cells for modification by genetic engineering, for example, to introduce a recombinant antigen receptor.

[275] Conditions may include one or more of specific environment, temperature, oxygen content, carbon dioxide content, time, means, 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 means designed to activate cells.

[276] In some embodiments, the stimulation conditions or agents include one or more agents, such as a ligand, that are capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent turns on or initiates an intracellular TCR/CD3 signaling cascade in a T cell. Such agents may include antibodies, such as antibodies specific for TCR, for example, antibodies against CD3. In some embodiments, the stimulatory conditions include one or more agents, such as a ligand, that are capable of stimulating a co-stimulatory receptor, such as an anti-CD28 antibody. In some embodiments, such agents and/or ligands may be associated with a solid support, such as beads, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding the anti-CD3 and/or anti-CD28 antibody to the culture medium (eg, at a concentration of at least about 0.5 ng/mL). In some embodiments, stimulants include IL-2, IL-15 and/or IL-7. In some aspects, the concentration of IL-2 is at least about 10 units/ml.

[277] In some aspects, the incubation is carried out in accordance with methods, such as those described in US patent No. 6040177 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.

[278] In some embodiments, T cells are increased in number by adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the composition to initiate culture (e.g., such that the final 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 culture incubation (eg, for a period of time sufficient to increase the number of T cells). In some aspects, non-dividing feeder cells may include gamma irradiated PBMC feeder cells. In some embodiments, the implementation of PBMC irradiated with gamma rays in the range from about 3000 to 3600 rad to prevent cell division. In some aspects, feeder cells are added to the culture medium prior to the addition of T cell populations.

[279] In some embodiments, stimulation conditions include a temperature suitable for growing human T lymphocytes, e.g., at least about 25 degrees Celsius, typically at least about 30 degrees Celsius, and typically at or about 37 degrees Celsius. Optionally, the incubation may further include the addition of non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of approximately 6000 to 10000 rad. In some aspects, LCL feeder cells are provided in any suitable amount, such as a ratio of LCL feeder cells to original T lymphocytes of at least about 10:1.

[280] In some embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naïve or antigen-specific T lymphocytes with an antigen. For example, lines or clones of antigen-specific T cells for cytomegalovirus antigens can be obtained by obtaining T cells from infected individuals and in vitro stimulation of the cells with the same antigen.

B. Genetic engineering vectors and methods

[281] Various methods of introducing genetically modified components, for example, recombinant receptors, for example, CAR or TCR, are well known and can be used in the provided methods and compositions. Illustrative methods include methods for transferring receptor-encoding nucleic acids, including viral, eg, retroviral or lentiviral, transduction, transposons, and electroporation.

[282] In some embodiments, cells are transfected with recombinant nucleic acids using recombinant infectious viral particles, such as, for example, vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors such as retroviral gamma vectors (see, for example, Koste et al. (2014) Gene Therapy 2014 Apr 3. 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 2011 November 29(11): 550-557.

[283] In some embodiments, the retroviral vector has a long terminal repeat (LTR) sequence, e.g., a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse cells (MSCV), spleen necrosis virus (SFFV) or adeno-associated virus (AAV). Most retroviral vectors are derived from mouse retroviruses. In some embodiments, retroviruses include retroviruses derived from any source of avian or mammalian cells. Retroviruses are generally amphoteric, which means that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the gag, pol and/or env retroviral sequences. A number of illustrative retroviral systems have been described (e.g., US Pat. 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.

[284] Methods for lentiviral transduction are known. Illustrative 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.

[285] In some embodiments, recombinant nucleic acids are transferred to T cells 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, recombinant nucleic acids are transferred into T cells via transposition (see, for example, Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74 and Huang et al (2009) Methods of Mol Biol 506: 115-126)). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (eg, as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-assisted microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and coprecipitation of DNA with strontium phosphate (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

[286] Other approaches and vectors for transferring nucleic acids encoding recombinant products are the vectors and approaches described, for example, in International Patent Application Publication No. WO2014055668 and US Patent No. 7446190.

[287] In some embodiments, cells, eg, T cells, can be transfected either during or after expansion, eg, with a T cell receptor (TCR) or a chimeric antigen receptor (CAR). This transfection to introduce the gene of the desired receptor can be carried out, for example, using any suitable retroviral vector. The population of genetically engineered cells can then be released from the initial stimulus (a CD3/CD28 stimulus, for example) and then stimulated with a second type of stimulus, eg, via a de novo injected receptor). This second type of stimulus may include an antigenic stimulus in the form of a peptide/MHC molecule, a genetically engineered native ligand (e.g., a natural CAR ligand), or any ligand (such as an antibody) that directly binds to the novel receptor scaffold (e.g., , through recognition of constant regions in 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 Vol. 65: 333-347).

[288] In some cases, you can use a vector that does not require activation of cells, such as T cells. In some such cases, the cell may be selected and/or transduced prior to activation. Thus, cells can be engineered before or after culturing the cells, and in some cases at the same time or during at least part of the culturing.

[289] In some aspects, the cells are also engineered to express cytokines or other factors. Among the additional nucleic acids, eg genes to be introduced, are genes that increase the effectiveness of therapy, eg by ensuring the viability and/or function of the transferred cells; genes providing a genetic marker for selection and/or evaluation of cells, for example, for evaluation of survival or localization in vivo; safety-enhancing genes, for example, making the cell susceptible to negative selection in vivo, as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); also see PCT/US91/08442 and PCT/US94/05601, Lupton et al., which describe the use of bifunctional selective fusion genes formed by fusion of a dominant positive selectable marker with a negative selectable marker. See, for example, Riddell et al., US Pat. No. 6,040,177 columns 14-17.

[290] In some contexts, overexpression of a stimulatory factor (eg, lymphokine or cytokine) can be toxic to the individual. Thus, in some contexts, engineered cells include gene segments that render cells susceptible to negative selection in vivo, such as when administered in adoptive immunotherapy. For example, in some aspects, the cells are engineered to be removed by changing the in vivo conditions of the individual to which they are administered. A negatively selectable phenotype may result from the insertion of a gene that confers sensitivity to the introduced agent, eg a compound. Negatively selectable genes include the herpes simplex virus type I (HSV-I TK) thymidine kinase gene (Wigler et al., Cell 2:223, 1977), which confers susceptibility to ganciclovir; cellular hypoxanthine phosphoribosyl transferase (HPRT) gene, cellular adenine phosphoribosyl transferase (APRT) gene, bacterial cytosine deaminase gene (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[291] In some embodiments, a single promoter may control the expression of an RNA that contains in one reading frame (ORF) two or three genes (e.g., encoding a molecule involved in modulating the metabolic cascade and encoding a recombinant receptor) separated by sequences encoding a self-cleaving peptide (eg sequence 2A) or a protease recognition site (eg furin). Thus, the ORF encodes a single polypeptide, which is either processed during (in the case of 2A) or after translation into individual proteins. In some cases, a peptide such as T2A can cause a ribosome to skip (ribosomal skip) the synthesis of a peptide bond at the C-terminus of element 2A, resulting in a separation between the end of the 2A sequence and the next peptide (see, for example, de Felipe. Genetic Vaccines and Ther 2:13 (2004) and deFelipe et al Traffic 5:616-626 (2004)). Many elements of 2A are known. Examples of 2A sequences that can be used in the methods and nucleic acids described herein include, but are not limited to, 2A sequences from foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 21), equine rhinitis virus A (E2A, e.g. , SEQ ID NO: 20), Thosea asigna virus (T2A, eg, SEQ ID NO: 6 or 17), and porcine teschovirus 1 (P2A, eg, SEQ ID NO: 18 or 19) as described in US Patent Publication No. 20070116690.

III. Compositions and formulations

[292] In some embodiments, the cell therapy is provided as a composition or formulation, such as a pharmaceutical composition or formulation. Such compositions can be used in accordance with the methods envisaged, for example, for the prevention or treatment of diseases, conditions and disorders, or in methods of detection, diagnosis or prognosis.

[293] The term "pharmaceutical formulation" refers to a formulation that is shaped to ensure the effectiveness of the biological activity of the active ingredient contained therein, and which does not contain additional components that are unacceptably toxic to the individual to whom the composition is intended to be administered.

[294] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to the individual. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[295] In some embodiments, a T cell therapy agent, such as engineered T cells (eg, CAR T cells), is formulated with a pharmaceutically acceptable carrier. In some aspects, the choice of carrier is determined in part by the particular cell and/or route of administration. Thus, there are many suitable formulations. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative, or mixture thereof, is typically present in an amount of from about 0.0001% to about 2% by weight of the entire composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (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 (such as octadecyldimethylbenzyl ammonium 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 polypeptides (less than 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 (eg Zn-protein complexes) and/or non-ionic surfactants such as polyethylene glycol (PEG).

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

[297] the Compositions may include aqueous solutions. The formulation or composition may also contain more than one active ingredient suitable for a particular indication, disease or condition being prevented or treated by cells, including one or more active ingredients whose activity is complementary to cells and/or the respective activities do not adversely affect each other. on a friend. Preferably, such active ingredients are present in combination in amounts that are effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active substances or drugs such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

[298] In some embodiments, the implementation of the pharmaceutical composition contains cells in amounts effective to treat or prevent a disease or condition, such as a therapeutically effective or prophylactically effective amount. In some embodiments, monitoring of therapeutic or prophylactic efficacy is carried out by periodically evaluating the individuals being treated. For multiple administrations over several days or more, depending on the condition, the treatment is repeated until the desired suppression of the symptoms of the disease occurs. However, other dosing regimens may be suitable and may be determined. The desired dosage may be delivered by a single bolus of the composition, by multiple boluses of the composition, or by continuous infusion of the composition.

[299] Cells can be administered using standard administration methods, formulations, and/or devices. Formulations and devices such as syringes and vials are provided for storing and administering the compositions. With respect to cells, administration may be autologous or heterologous. For example, immunoreactive cells or progenitors can be obtained from one individual and administered to the same individual or another compatible individual. Immunoreactive peripheral blood cells or their progeny (eg, occurring in vivo, ex vivo, or in vitro) can be administered by localized injection, including catheter injection, systemic injection, localized injection, intravenous injection, or parenteral administration. When a therapeutic composition (eg, a pharmaceutical composition containing an engineered immunoreactive cell) is administered, it is usually formulated in a single dose injectable form (solution, suspension, emulsion).

[300] Formulations include formulations for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the agent or cell populations are administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration. In some embodiments, the agent or cell populations are administered to an individual using peripheral systemic delivery via intravenous, intraperitoneal, or subcutaneous injection.

[301] In some embodiments, the compositions are provided as sterile liquid preparations, for example, isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects can be buffered for a specific pH. Liquid formulations are generally easier to prepare than gels, other viscous formulations, and solid formulations. In addition, liquid formulations are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within a suitable viscosity range to provide longer periods of contact with certain tissues. Liquid or viscous compositions may include carriers, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, a polyhydric alcohol (eg, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[302] Sterile injectable solutions can be obtained by incorporating cells into a solvent, for example, in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, and the like. The compositions may also be lyophilized. The compositions may contain adjuvants such as wetting agents, dispersing agents or emulsifiers (e.g. methylcellulose), pH buffering agents, gelling agents or viscosity enhancers, preservatives, flavoring agents, coloring agents, etc., depending on the desired route. administration and drug. To obtain suitable drugs in some aspects, you can be guided by standard guides.

[303] You can add various additives that increase the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents and buffers. Prevention of the action of microorganisms can be ensured by using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Sustained absorption of an injectable pharmaceutical form can be achieved using delayed absorption agents such as aluminum monostearate and gelatin.

[304] Compositions for use for in vivo administration are usually sterile. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes.

[305] For the prevention or treatment of a disease, a suitable dosage may depend on the type of disease being treated, the type of agent or agents, the type of cells or recombinant receptors, the severity and course of the disease, the administration of the agent or cells for prophylactic or therapeutic purposes, prior therapy, clinical history individual and response to the agent or cells, and the opinion of the attending physician. In some embodiments, the compositions are preferably administered to an individual once or over a series of administrations.

IV. Treatment and methods

[306] In some embodiments, the provided methods are associated with conducting cell therapy, for example, for the treatment of diseases or conditions, including various tumors. The methods involve introducing genetically engineered cells expressing recombinant receptors designed to recognize and/or specifically bind to molecules associated with a disease or condition and result in a response, such as an immune response, against such molecules when bound to such molecules. Receptors may include chimeric receptors, such as chimeric antigen receptors (CARs), and other transgenic antigen receptors, including transgenic T cell receptors (TCRs), including any of the receptors described herein. In some embodiments, the methods provided are followed by re-expansion of the recombinant immune cells in vivo in the individual, for example, by interfering with a region in the individual in which the engineered cells are, or are likely to be, or were, or are likely to be. In some embodiments, the region may be a region containing antigen-expressing cells recognized by modified cell engineering techniques, such as a lesion site, such as a tumor, or a lesion microenvironment, eg, a tumor microenvironment.

[307] In some embodiments, a dose of cells expressing the recombinant receptor is administered to an individual to treat or prevent diseases, conditions, and disorders, including cancer. In some embodiments, cells, populations, and compositions are administered to an individual or patient having a particular disease or condition being treated, for example, through adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, cells and compositions, such as engineered compositions and ready-to-use compositions, after incubation and/or other processing steps, are administered to an individual, such as an individual who has or is at risk of developing a disease or condition. In some aspects, the methods thus treat, eg, alleviate, one or more symptoms of a disease or condition, eg, by reducing the tumor burden of a cancer expressing an antigen recognized by the engineered T cell.

[308] Methods for introducing the introduction of cells for adoptive cell therapy are known and can be used in conjunction with the envisaged methods and compositions. For example, methods of adoptive T cell therapy are described, for example, in US Patent Application Publication No. 2003/0170238, Gruenberg et al; US patent No. 4690915 issued to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.

[309] The disease or condition being treated can be any disease or condition in which antigen expression is associated with and/or involved in the etiology of the disease, condition, or disorder, e.g., causes, exacerbates, or is otherwise involved in such disease, condition, or violation. Exemplary diseases and conditions may include diseases or conditions associated with cancer or cell transformation (eg, cancer), an autoimmune or inflammatory disease, or an infectious disease, eg, caused by a bacterial, viral, or other pathogen. Illustrative antigens, which include antigens associated with various diseases and conditions that can be treated, are described above. In specific embodiments, the chimeric antigen receptor or transgenic TCR specifically binds to an antigen associated with a disease or condition.

[310] Diseases, conditions, and disorders include tumors, including solid tumors, hematologic malignancies, and melanomas, and including localized and metastatic tumors, infectious diseases such as infection with a virus or other pathogen, e.g., HIV, HCV, HBV, CMV , and parasitic diseases, and 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, leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), 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, lung, liver, breast, prostate, ovary, skin, melanoma, bone and brain cancers, ovarian cancer, epithelial malignancies, renal cell carcinoma, adenocarcinoma pancreas, Hodgkin's lymphoma, cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma and/or mesothelioma. In some embodiments, the individual has acute lymphoblastic leukemia (ALL). In some embodiments, the individual has non-Hodgkin's lymphoma.

[311] 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, immunodeficiency, cytomegalovirus (CMV) infections, Epstein-Barr virus (EBV), adenovirus, polyomavirus BK. In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease glands, Graves' disease, Crohn's disease, multiple sclerosis, asthma, and/or a disease or condition associated with transplantation.

[312] In some embodiments, the antigen associated with the disease or disorder is or includes an antigen selected from αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 ( CA9, also known as CAIX or G250), testicular cancer antigen, testicular cancer antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, chemokine ligand 1 with C-C motif (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), epidermal growth factor truncated protein (tEGFR), mutant epidermal growth factor receptor type III (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), receptor estrogen, Fc receptor like protein 5 (FCRL5; also known as the Fc homologue 5 receptor or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha, fetal acetylcholine receptor, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), conjugated with 5D receptor G protein (GPCR5D), Her2/neu (erbB2 receptor tyrosine kinase), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, high molecular weight human melanoma-associated antigen (HMW-MAA), surface antigen hepatitis B, human leukocyte A1 antigen (HLA-A1), human leukocyte A2 antigen (HLA-A2), IL-22 receptor alpha (IL-22Ra), IL-13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), L1-CAM CE7 epitope, member A of the leucine-rich repeat protein family 8 (LRRC8A), Lewis Y antigen, melanoma-associated antigen (MAGE)- A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, mouse cytomegalovirus (CMV), mucin a 1 (MUC1), MUC16, natural killer group 2 D ligands (NKG2D), melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, preferentially expressed melanoma antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), orphan receptor tyrosine kinase-like 1 (ROR1), survivin, trophoblast glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor antigen 1 (WT-1), pathogen-specific or universal label-associated antigen, and/or biotinylated molecules, and /or molecules expressed by HIV, HCV, HBV or other pathogens. The antigens targeted by the receptors, in some embodiments, include antigens associated with B-cell cancer, such as any of a number of known B-cell markers. In some embodiments, the antigen targeted by the receptor is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig-kappa, Ig-lambda, CD79a, CD79b, or CD30. In some embodiments, the antigen is or includes a pathogen-specific antigen or a pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens.

[313] In some embodiments, cell therapy, e.g., adoptive T cell therapy, is performed via autologous transfer, in which cells are isolated and/or otherwise obtained from the individual to be treated with cell therapy, or from a sample obtained from such an individual. . Thus, in some aspects, cells are obtained from an individual, such as a patient in need of treatment, and after isolation and processing, the cells are administered to the same individual.

[314] In some embodiments, cell therapy, such as adoptive T-cell therapy, is performed via allogeneic transfer, in which cells are isolated and/or otherwise obtained from an individual other than the individual who intends to receive or ultimately receive cell therapy , for example, from the first individual. In such embodiments, the cells are then administered to another individual, such as a second individual, of the same species. In some embodiments, the first and second individuals are genetically identical. In some embodiments, the first and second individuals are genetically similar. In some embodiments, the second individual expresses the same HLA class or supertype as the first individual.

[315] Cells can be administered by any suitable route, e.g., by bolus infusion, injection, e.g. intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection , subconjunctival injection, subconjunctival injection, subtenon injection, retrobulbar injection, peribulbar injection, or posterior parascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal routes, and if desired for topical treatment, via intralesional administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration. In some embodiments, the implementation of this who is administered through a single bolus of cells. In some embodiments, it is administered via multiple bolus injections of cells, such as over a period of no more than 3 days, or via continuous cell infusion.

[316] For the prevention or treatment of a disease, a suitable dosage may depend on the type of disease being treated, the type of cells or recombinant receptors, the severity and course of the disease, the administration of cells for prophylactic or therapeutic purposes, prior therapy, the individual's clinical history and response to cells, and opinion of the attending physician. In some embodiments, the compositions and cells may be administered to an individual once or over a series of administrations.

[317] In some embodiments, the cells are administered as part of a combination treatment, e.g., simultaneously or sequentially in any order with another therapeutic intervention, such as an antibody, or an engineered cell, or a receptor, or an agent, such as a cytotoxic or therapeutic agent. . In some embodiments, the cells are administered in conjunction with one or more additional therapeutic agents or in conjunction with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, cells are co-administered with another therapy close enough in time that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, cells are administered up to one or more additional therapeutic agents. In some embodiments, cells are administered after one or more additional therapeutic agents. In some embodiments, one or more additional agents include a cytokine, such as IL-2, for example, to increase persistence. In some embodiments, the methods include administering a chemotherapeutic agent. In some instances, such a chemotherapeutic agent is not the same agent used in the contemplated methods of interfering with a lesion.

[318] In some embodiments, methods include administering a chemotherapeutic agent, eg, a conditioning chemotherapeutic agent, eg, to reduce tumor burden prior to administration.

[319] In some aspects, pretreatment of individuals with immunodepletion-causing (eg, lymphodepletion) therapies may improve the effects of adoptive cell therapy (ACT).

[320] Thus, in some embodiments, the methods include administering a preconditioning agent, such as a lymphodepletion-inducing or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, to the individual prior to initiation of cell therapy. For example, the subject may be administered the preconditioning agent at least 2 days, such as at least 3, 4, 5, 6, or 7 days prior to the start of cell therapy. In some embodiments, the individual is administered the preconditioning agent no more than 7 days, such as no more than 6, 5, 4, 3, or 2 days prior to initiation of cell therapy.

[321] In some embodiments, the individual is pre-administered with cyclophosphamide at a dose of from or about 20 mg/kg to 100 mg/kg, such as from or about 40 mg/kg to 80 mg/kg. In some aspects, the subject is pre-administered exactly or approximately 60 mg/kg of cyclophosphamide. In some embodiments, cyclophosphamide may be administered as a single dose, or may be administered in multiple doses, such as every day, every two days, or every three days. In some embodiments, the implementation of cyclophosphamide is administered once a day for one or two days.

[322] In some embodiments, when the lymphodepletion-inducing agent comprises fludarabine, the individual is administered fludarabine at a dose of from or about 1 mg/m ² to 100 mg/m ² , for example, from or about 10 mg/m ² to 75 mg /m ² , from 15 mg/m ² to 50 mg/m ² , from 20 mg/m ² to 30 mg/m ² , or from 24 mg/m ² to 26 mg/m ² . In some cases, the subject is administered 25 mg/m ² fludarabine. In some embodiments, fludarabine may be administered as a single dose, or may be administered in multiple doses, such as every day, every other day, or every three days. In some embodiments, fludarabine is administered every day, such as for 1-5 days, such as for 3-5 days.

[323] In some embodiments, the lymphodepletion-inducing agent includes a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents may include cyclophosphamide at any dose or schedule, such as the dose and schedule described above, and fludarabine at any dose and schedule, such as the dose and schedule described above. For example, in some aspects, the individual is administered 60 mg/kg (~2 g/m ² ) of cyclophosphamide and 3 to 5 doses of fludarabine, 25 mg/m ² , prior to the first or subsequent dose.

[324] After the introduction of cells, the biological activity of the engineered cell populations in some embodiments is determined, for example, by any of a number of known methods. Parameters for evaluation include specific binding of engineered or native T cells or other immune cells to the antigen in vivo, eg, by imaging, or ex vivo, eg, by ELISA or flow cytometry. In certain embodiments, the ability of engineered cells to destroy target cells can be determined using any suitable method known in the art, such as the cytotoxicity assay methods described, for example, in Kochenderfer et al., J. Immunotherapy, 32(7) : 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of cells is determined by analyzing the expression and/or secretion of one or more cytokines such as CD107a, IFNγ, IL-2, and TNF. In some aspects, biological activity is determined by assessing a clinical outcome, such as a reduction in tumor mass or burden.

[325] In certain embodiments, the engineered cells are further modified in any number of ways to increase their therapeutic or prophylactic efficacy. For example, an engineered CAR or TCR expressed by a population can be conjugated either directly or indirectly via a linker to a targeting moiety. Methods for conjugating compounds, eg CAR or TCR, with targeting moieties are known in the art. See, for example, Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and US Patent 5,087,616.

A. Dosing

[326] In some embodiments, the implementation of the pharmaceutical composition contains cells in amounts effective to treat or prevent a disease or condition, such as a therapeutically effective or prophylactically effective amount. In some embodiments, the implementation of the composition includes cells in an amount effective to reduce the load of the disease or condition.

[327] In the context of adoptive cell therapy, administering a given "dose" encompasses administering a given amount or number of cells as a single formulation and/or a single continuous administration, e.g., as a single injection or continuous infusion, and also encompasses administering a given amount or number of cells as a divided dose, provided as a plurality of individual compositions or infusions, over a period of time, which is not more than 3 days. Thus, in some contexts, the dose is a single or continuous administration of a certain number of cells, carried out or started at the same time. However, in some contexts, the dose is administered as multiple injections or infusions over a period of no more than three days, for example, once a day for three days or for two days, through multiple infusions over one day.

[328] Thus, in some aspects, the dose cells are administered in a single pharmaceutical composition. In some embodiments, the cells of the dose are administered in a plurality of compositions collectively containing the cells of the first dose.

[329] The term "split dose" refers to a dose that is divided so that it is administered over more than one day. This type of dosing is covered by the methods of the present invention and is considered a single dose.

[330] Thus, in some aspects, the dose can be administered as a divided dose. For example, in some embodiments, a dose may be administered to an individual over 2 days or over 3 days. Exemplary split dosing methods include administering 25% of the dose on the first day and administering the remaining 75% of the dose on the second day. In other embodiments, 33% of the first dose may be administered on the first day and the remaining 67% may be administered on the second day. In some aspects, 10% of the dose is administered on the first day, 30% of the dose is administered on the second day, and 60% of the dose is administered on the third day. In some embodiments, the divided dose is not spread over more than 3 days.

[331] In some embodiments, the dose cells can be administered by administering a plurality of compositions or solutions, for example, the first and second, optionally more, each containing a number of dose cells. In some aspects, multiple compositions, each containing a different population and/or cell subtypes, are administered separately or independently, optionally over a period of time. For example, cell populations or subtypes may include CD8 ⁺ and CD4 ⁺ T cells, respectively, and/or CD8+ and CD4+ rich populations, respectively, e.g., CD4+ and/or CD8+ T cells, which in each case individually include cells modified by genetic engineering to express the recombinant receptor. In some embodiments, administering a dose comprises administering a first composition containing a dose of CD8+ T cells or a dose of CD4+ T cells and administering a second composition containing a different dose of CD4+ T cells and CD8+ T cells.

[332] In some embodiments, administering a composition or dose, for example, administering multiple cell compositions, involves administering the cell compositions individually. In some aspects, a separate administration is carried out simultaneously or sequentially in any order. In some embodiments, the dose comprises a first composition and a second composition, and the first composition and second composition are administered at intervals of 0 to 12 hours, 0 to 6 hours, or 0 to 2 hours. In some embodiments, the start of administration of the first composition and the start of administration of the second composition are at intervals of no more than 2 hours, no more than 1 hour, or no more than 30 minutes, no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes. In some embodiments, the start and/or end of the administration of the first composition and the end and/or start of the administration of the second composition are within no more than 2 hours, no more than 1 hour, or no more than 30 minutes, no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes.

[333] In some compositions, the first composition, for example, the first dose composition, contains CD4+ T cells. In some compositions, the first composition, eg, the first dose composition, contains CD8+ T cells. In some embodiments, the first composition is administered prior to the second composition.

[334] In some embodiments, the dose or cell composition comprises a specific or predetermined ratio of CD4+ cells expressing the recombinant receptor to CD8+ cells expressing the recombinant receptor and/or CD4+ cells to CD8+ cells, which is optionally about 1:1 or from about 1:3 to about 3:1, such as about 1:1. In some aspects, administering a composition or dose at a given or desired ratio of different cell populations (such as a CD4+:CD8+ ratio or a CAR+CD4+:CAR+CD8+ ratio, e.g. 1:1) involves administering a cell composition containing one of the populations and then the introduction of a separate composition of cells containing another of the populations, where the introduction occurs in or approximately in a given or desired ratio.

[335] In some embodiments, one or more subsequent or repeated doses of cells can be administered to an individual. In some embodiments, a subsequent or repeat dose is administered more than or more than about 7 days, 14 days, 21 days, 28 days, or 35 days after the start of the first dose of cells. The subsequent or repeated dose of cells may be greater than, approximately the same as, or less than the first dose. In some embodiments, the implementation of T-cell therapy, such as the introduction of the first and/or second dose, may be repeated.

[336] In some embodiments, the implementation of the dose of cells is administered to individuals in accordance with the methods provided. In some embodiments, the implementation of the size or timing of the introduction of doses is determined depending on the specific disease or condition in the individual. One skilled in the art is able to empirically determine the size or timing of doses for a particular disease. Dosages may vary depending on the indications of the particular disease or disorder and/or the patient and/or other treatments.

[337] In certain embodiments, cells or distinct populations of cell subtypes are administered to an individual in the range of from about 0.1 million to about 100 billion cells and/or the number of cells per kilogram of body weight of the individual, such as from 0.1 million to about 50 billion cells (e.g., approximately 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 30 billion cells, approximately 40 billion cells, or a range determined by any two of the above), 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the above values), for example, from about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, approximately 70 million cells, approximately 80 million cells, approximately 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or a range defined by any two of above values), and in some cases from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells). current, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value between these ranges and/or per kilogram of body weight of the individual. The dosage may vary depending on the characteristics of the disease or disorder and/or the patient and/or other treatments. In some embodiments, such values refer to numbers of cells expressing the recombinant receptor; in other embodiments, they refer to the number of T cells, or PBMCs, or all cells administered.

[338] In some embodiments, for example, when the subject is a human, the dose includes less than about 5×10 ^{8 of} all cells expressing the recombinant receptor (e.g., CAR), T cells, or peripheral blood mononuclear cells (PBMCs), e.g., in the range of approximately 1×10 ⁶ to 5×10 ⁸ such cells, such as 2×10 ⁶ , 5×10 ⁶ , 1×10 ⁷ , 5×10 ⁷ , 1×10 ⁸ , or 5×10 ⁸ all such cells or in the range between any two of the above values.

[339] In some embodiments, cell therapy comprises administering a dose containing a cell count of from or about 1×10 ⁵ to 5×10 ^{8 of} all cells expressing the recombinant receptor, all β-cells, or all peripheral blood mononuclear cells (PBMC), from or from about 5×10 ⁵ to 1×10 ⁷ all cells expressing the recombinant receptor, all T cells, or all peripheral blood mononuclear cells (PBMCs), or from or from about 1×10 ⁶ to 1×10 ⁷ all cells expressing recombinant receptor, all T cells or all peripheral blood mononuclear cells (PBMC), in each case inclusive. In some embodiments, cell therapy comprises administering a dose of cells containing a cell count of at least or at least about 1×10 ^{5 of} all cells expressing the recombinant receptor, all T cells, or all peripheral blood mononuclear cells (PBMC) as for example, at least or at least 1x10 ⁶ , at least or at least about 1x10 ⁷ , at least or at least about 1x10 ⁸ such cells. In some embodiments, this number refers to the total number of CD3+ or CD8+ cells, in some cases also cells expressing the recombinant receptor (eg, CAR+). In some embodiments, cell therapy comprises administering a dose containing a cell count from or from about 1x10 ⁵ to 5x10 ⁸ CD3+ or CD8+ of all T cells, or CD3+ or CD8+ cells expressing the recombinant receptor from or from about 5x10 ⁵ to 1x10 ⁷ CD3+ or CD8+ of all T cells or CD3+ or CD8+ cells expressing the recombinant receptor, or from or about 1x10 ⁶ to 1x10 ⁷ CD3+ or CD8+ of all T cells or CD3+ or CD8+ cells, expressing the recombinant receptor, in each case inclusive. In some embodiments, cell therapy comprises administering a dose containing a number of cells from or from about 1x10 ⁵ to 5x10 ^{8 of} all CD3+/CAR+ or CD8+/CAR+ cells, from or from about 5x10 ⁵ to 1x10 ^{7 of} all CD3+/CAR+ or CD8+/CAR+ cells, or from or from about 1×10 ⁶ to 1×10 ^{7 of} all CD3+/CAR+ or CD8+/CAR+ cells, in each case inclusive.

[340] In some embodiments, the T cells of the dose include CD4+ T cells, CD8+ T cells, or CD4+ and CD8+ T cells.

[341] In some embodiments, for example, when the individual is human, the CD8+ T cell dose, including the dose including CD4+ and CD8+ T cells, comprises from about 1 x 10 ⁶ to 5 x 10 ^{8 of} all CD8+ cells expressing the recombinant receptor (e.g., CAR), e.g., in the range of about 5×10 ⁶ to 1×10 ⁸ such cells, such as 1×10 ⁷ , 2.5×10 ⁷ , 5×10 ⁷ , 7.5×10 ⁷ , 1×10 ⁸ or 5×10 ^{8 of} all such cells, or a range between any of the above two values. In some embodiments, a plurality of doses are administered to a patient, and each of the doses or the total dose may be within any of the above values. In some embodiments, the cell dosage comprises administering from or about 1x10 ⁷ to 0.75x10 ^{8 of} all recombinant CD8+ receptor-expressing T cells, 1x10 ⁷ to 2.5x10 ^{7 of} all recombinant CD8+ T receptor-expressing -cells, from or from about 1×10 ⁷ to 0.75×10 ^{8 of} all expressing the recombinant CD8+ receptor T-cells, in each case, inclusive. In some embodiments, the cell dosage comprises administering exactly or about 1×10 ⁷ , 2.5×10 ⁷ , 5×10 ⁷ 7.5×10 ⁷ , 1×10 ⁸ , or 5×10 ⁸ all expressing the recombinant CD8+ T- receptor cells.

[342] In some embodiments, cell therapy comprises administering a dose containing a cell count of from or about 1×10 ⁵ to 5×10 ^{8 of} all recombinant receptor-expressing cells, all β-cells, or all peripheral blood mononuclear cells (PBMC), from or from about 5×10 ⁵ to 1×10 ⁷ all cells expressing the recombinant receptor, all T cells, or all peripheral blood mononuclear cells (PBMCs), or from or from about 1×10 ⁶ to 1×10 ⁷ all cells expressing recombinant receptor, all T cells or all peripheral blood mononuclear cells (PBMC), in each case inclusive. In some embodiments, cell therapy comprises administering a dose of cells containing a cell count of at least or at least about 1×10 ^{5 of} all cells expressing the recombinant receptor, all T cells, or all peripheral blood mononuclear cells (PBMC) as for example, at least or at least 1x10 ⁶ , at least or at least about 1x10 ⁷ , at least or at least about 1x10 ⁸ such cells. In some embodiments, this number refers to the total number of CD3+ or CD8+ cells, in some cases also cells expressing the recombinant receptor (eg, CAR+). In some embodiments, cell therapy comprises administering a dose containing a cell count from or from about 1x10 ⁵ to 5x10 ⁸ CD3+ or CD8+ of all T cells, or CD3+ or CD8+ cells expressing the recombinant receptor from or from about 5x10 ⁵ to 1x10 ⁷ CD3+ or CD8+ of all T cells or CD3+ or CD8+ cells expressing the recombinant receptor, or from or about 1x10 ⁶ to 1x10 ⁷ CD3+ or CD8+ of all T cells or CD3+ or CD8+ cells, expressing the recombinant receptor, in each case inclusive. In some embodiments, cell therapy comprises administering a dose containing a number of cells from or from about 1x10 ⁵ to 5x10 ^{8 of} all CD3+/CAR+ or CD8+/CAR+ cells, from or from about 5x10 ⁵ to 1x10 ^{7 of} all CD3+/CAR+ or CD8+/CAR+ cells, or from or from about 1×10 ⁶ to 1×10 ^{7 of} all CD3+/CAR+ or CD8+/CAR+ cells, in each case inclusive.

[343] In certain embodiments, cells or distinct populations of cell subtypes are administered to an individual in the range of from about 0.1 million to about 100 billion cells and/or the number of cells per kilogram of body weight of the individual, for example, from 0.1 million to about 50 billion cells (e.g., approximately 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 30 billion cells, approximately 40 billion cells, or a range defined by any two of the above), 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion to cells, about 40 billion cells, or a range defined by any two of the above), e.g., from about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, approximately 70 million cells, approximately 80 million cells, approximately 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or a range defined by any two of the above values), and in some cases from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells to, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value between these ranges and/or per kilogram of body weight of the individual. Dosages may vary depending on the characteristics of the disease or disorder and/or the patient and/or other treatments. In some embodiments, such values refer to numbers of cells expressing the recombinant receptor; in other embodiments, they refer to the number of T cells, or PBMCs, or all of the injected cells.

[344] In some embodiments, cell therapy comprises administering a dose comprising a number of cells that is at least, or at least about, or is or is about 0.1×10 ⁶ cells/kg of body weight of the individual, 0.2 ×10 ⁶ cells/kg, 0.3×10 ⁶ cells/kg, 0.4×10 ⁶ cells/kg, 0.5×10 ⁶ cells/kg, 1×10 ⁶ cells/kg, 2.0×10 ⁶ cells/kg, 3×10 ⁶ cells/kg or 5×10 ⁶ cells/kg.

[345] In some embodiments, the implementation of cell therapy includes the introduction of a dose containing a number of cells, which is from or about 0.1×10 ⁶ cells/kg of body weight of the individual to 1.0×10 ⁷ cells/kg, from or approximately from 0.5x10 ⁶ cells/kg to 5x10 ⁶ cells/kg, from or about 0.5x10 ⁶ cells/kg to 3x10 ⁶ cells/kg, from or about 0.5x10 ⁶ cells/kg up to 2x10 ⁶ cells/kg, from or about 0.5x10 ⁶ cells/kg to 1x10 ⁶ cells/kg, from or about 1.0x10 ⁶ cells/kg body weight of the individual up to 5x10 ⁶ cells/kg, from or about 1.0x10 ⁶ cells/kg to 3x10 ⁶ cells/kg, from or about 1.0x10 ⁶ cells/kg to 2x10 ⁶ cells /kg, from or approximately from 2.0×10 ⁶ cells/kg of body weight of the individual to 5×10 ⁶ cells/kg, from or approximately from 2.0×10 ⁶ cells/kg to 3×10 ⁶ cells/kg, or from or about 3.0×10 ⁶ cells/kg of body weight of the individual to 5×10 ⁶ cells/kg, in each case, inclusive.

[346] In some embodiments, the cell dose comprises from or about 2x10 ⁵ cells/kg to or up to about 2x10 ⁶ cells/kg, such as from or about 4x10 ⁵ cells/kg to or up to about 1×10 ⁶ cells/kg, or from or from about 6×10 ⁵ cells/kg to or up to about 8×10 ⁵ cells/kg. In some embodiments, the cell dose contains no more than 2x10 ⁵ cells (e.g., antigen-expressing cells, such as CAR-expressing cells) per kilogram of the individual's body weight (cells/kg), e.g., no more than or about 3x10 ⁵ cells /kg, not more than or approximately 4×10 ⁵ cells/kg, not more than or approximately 5×10 ⁵ cells/kg, not more than or approximately 6×10 ⁵ cells/kg, not more than or approximately 7×10 ⁵ cells/kg, no more than or about 8x10 ⁵ cells/kg, no more than or about 9x10 ⁵ cells/kg, no more than or about 1x10 ⁶ cells/kg, or no more than or about 2×10 ⁶ cells/kg. In some embodiments, the cell dose contains at least, or at least about, or exactly, or about 2×10 ⁵ cells (e.g., antigen-expressing cells, such as CAR-expressing cells) per kilogram of an individual's body weight (cells/kg), for example, at least, or at least about, or exactly, or about 3x10 ⁵ cells/kg, at least, or at least about, or exactly, or about 4x10 ⁵ cells/kg, at least at least, or at least about, or exactly, or about 5x10 ⁵ cells/kg, at least, or at least about, or exactly, or about 6x10 ⁵ cells/kg, at least, or at least about, or exactly, or about 7x10 ⁵ cells/kg, at least, or at least about, or exactly, or about 8x10 ⁵ cells/kg, at least, or at least about, or exactly, or approximately 9×10 ⁵ cells/kg, p about at least, or at least about, or exactly, or about 1x10 ⁶ cells/kg, or at least, or at least about, or exactly, or about 2x10 ⁶ cells/kg.

[347] In some embodiments, cells are administered at the desired dosage, which in some aspects includes the desired dose or number of cells or cell type(s), and/or the desired ratio of cell types. Thus, in some embodiments, the cell dosage is based on the total number of cells (or number per kg of body weight) and the desired ratio of individual populations or subtypes, such as the ratio of CD4+ to CD8+. In some embodiments, cell dosage is based on the desired total number (or number per kg of body weight) of cells in particular populations or particular cell types. In some embodiments, the implementation of the dosage is based on a combination of such signs, such as the required number of total cells, the desired ratio, and the required total number of cells in individual populations.

[348] In some embodiments, cell populations or subtypes, such as CD8 ⁺ and CD4 ⁺ T cells, are administered at or within a tolerance of the required dose of all cells, such as the required dose of T cells. In some aspects, the desired dose is the desired number of cells or the desired number of cells per unit body weight of the individual to which the cells are administered, for example, the number of cells/kg. In some aspects, the required dose is at or above the minimum number of cells or the minimum number of cells per unit body weight. In some aspects, for all cells administered at the required dose, individual populations or subtypes are present at levels or close to the final ratio (such as the ratio of CD4 ⁺ to CD8 ⁺ ), for example, within a certain acceptable difference or error of such a ratio.

[349] In some embodiments, cells are administered at or within a tolerance range for a desired dose of one or more distinct cell populations or subtypes, such as a desired CD4+ cell dose and/or a desired CD8+ cell dose. In some aspects, the required number of cells is the required number of cells of a subtype or population, or the required number of such cells per unit body weight of the individual to which the cells are administered, for example, the number of cells/kg. In some aspects, the required dose is at or above the minimum cell population or subtype, or the minimum population or subtype per unit body weight.

[350] Thus, in some embodiments, the implementation of the dosage is based on the required fixed dose of all cells and the required ratio and/or based on the required fixed dose of one or more, for example each, of the individual subtypes or subpopulations. Thus, in some embodiments, dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4 ⁺ to CD8 ⁺ cells, and/or is based on a desired fixed or minimum dose of CD4 ⁺ and/or CD8 ⁺ cells.

[351] In some embodiments, cells are administered at or within an acceptable range of the desired final ratio of multiple cell populations or subtypes, such as CD4+ and CD8+ cells or subtypes. In some aspects, the desired ratio may be a specific ratio, or it may be a range of ratios. for example, in some embodiments, the desired ratio (e.g., the ratio of CD4 ⁺ to CD8 ⁺ cells) is from or about 5:1 to or up to about 5:1 (or more than about 1:5 and less than about 5:1) , or from or about 1:3 to or up to about 3:1 (or more than about 1:3 and less than about 3:1), for example, from or about 2:1 to or up to about 1:5 ( or more than about 1:5 and less than about 2:1, for example, exactly or about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2: 1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1: 1.7, 1:1.8, 1:1.9: 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5. In some aspects, the tolerable difference is in the range of about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including anything in between.

[352] In specific embodiments, the numbers and/or concentrations of cells refer to the number of recombinant receptor (eg, CAR) expressing cells. In other embodiments, cell numbers and/or concentrations refer to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.

[353] In some aspects, the amount of dose is determined based on one or more criteria, such as the response of the individual to prior treatment, such as chemotherapy, disease load in the individual, such as tumor burden, volume, size or extent, severity or type of metastases, stage and/or the likelihood or occurrence in the individual of toxic outcomes, eg, CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or host immune response against injected cells and/or recombinant receptors.

B. Intervention and/or treatment

[352] In conjunction with administering a dose of genetically engineered cells, such as cells expressing a recombinant receptor, such as CAR+ T cells, any method of intervening in an area, such as a lesion, in which cells are present or likely to be present, can be used, and/or providing treatment, which includes one or more of physical or mechanical manipulation of the lesion or part thereof, radiation therapy, or administration of an immunomodulatory agent, such as the methods described in section B. In some embodiments, the intervention and/or treatment is carried out after introduction of genetically engineered cells. In some embodiments, the intervention and/or treatment is more than or more than about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years or more after administration of engineered cells (eg, CAR-T cells).

[353] In some embodiments, the treatment and/or intervention is carried out after the individual has experienced a partial response (PR) to the engineered cells, and/or after the individual has not responded to the engineered cells for a certain period of time, e.g., 14 -28 days, to improve the outcome in the form of a response. In certain embodiments, treatment and/or intervention is carried out after the individual has experienced a partial response. In certain embodiments, the intervention is carried out after more than or more than about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years or more after the onset of the individual's PR. In certain embodiments, the intervention is carried out after more than or more than about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years or more after the onset of the individual's PR. In some embodiments, a complete response (CR) to treatment is observed as a result of the provided methods. In some embodiments, the individual has not previously achieved remission, such as CR, on prior therapy or genetically engineered cells, such as cells expressing a recombinant receptor, such as CAR+ T cells.

[354] In some embodiments, at or just before the time point of treatment and/or intervention, the subject relapsed after remission in response to the administration of genetically engineered cells. In some embodiments, relapse occurs after (e.g., within 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or more) after a complete response (CR) or PR to the administration of genetically engineered cells , such as cells expressing the recombinant receptor, such as CAR+ T cells. In some embodiments, treatment and/or intervention is carried out within or approximately within 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or one week after a relapse or after a relapse is detected or observed. In certain embodiments, treatment and/or intervention is carried out within or about 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or one week after the individual is determined or suspected of relapse.

[355] In some embodiments, the treatment and/or intervention is carried out at a point in time when the number of engineered cells found in the individual's blood is reduced compared to a previous point in time after administration of the modified cells to the individual. In some embodiments, treatment and/or intervention is performed at a time point when the number of T cell therapy cells detected in the blood is less than or less than about 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, or 100 times or less than the peak or maximum number of T-cell therapy cells found in the blood of an individual after the start of T-cell therapy; and/or at a point in time after detection of a peak or peak level of T-cell therapy cells in the individual's blood, the amount of T-cell therapy cells detectable in the individual's blood is less than 10%, less than 5%, less than 1%, or less than 0.1% from all peripheral blood mononuclear cells (PBMC) in the blood of an individual. In some embodiments, the treatment and/or intervention is carried out at a point in time when the number of T cell therapy cells detected in the blood is at or about 10%, 20%, 30%, 40%, 50%, 60%, 70% , 80%, 90%, 95%, 98%, 99%, or 99.9% less than the peak or maximum number of T-cell therapy cells found in an individual's blood after initiation of T-cell therapy.

[356] In some embodiments, treatment and/or intervention is performed at a time point where there is (i) less than or about 10 modified cells per microliter, (ii) less than or less than about 20%, 30%, 40%, or 50% of total peripheral blood mononuclear cells (PBMCs), (iii) less than or less than about 1×10 ⁵ modified cells; or (iv) less than or less than about 5000 copies of DNA encoding the recombinant receptor per microgram of DNA.

[357] In some embodiments, the intervention and/or treatment is carried out as part of a treatment regimen involving a single administration, procedure, or manipulation. In specific embodiments, the intervention and/or treatment is carried out as part of a treatment regimen involving more than one administration, procedure, or manipulation. In certain embodiments, the treatment and/or intervention is carried out in accordance with a treatment regimen involving multiple administrations over a treatment period of about an hour, about 6 hours, about 12 hours, about 24 hours, about 1 day, about 2 days, about 3 about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 2 weeks, approximately 3 weeks approximately 4 weeks approximately 5 weeks approximately 6 weeks approximately 7 weeks approximately 8 weeks approximately 9 weeks approximately 10 weeks approximately 11 weeks approximately 12 weeks approximately 1 month approximately 2 months approximately 3 months, approximately 4 months, with about 5 months or about 6 months. In some embodiments, the treatment and/or intervention is carried out through multiple administrations over a treatment period of from about 1 minute to about 1 hour, from 1 hour to 12 hours, from about 12 hours to about 24 hours, from about 1 day to about 2 days, from about 1 day to about 5 days, from about 1 day to about 7 days, from about 1 week to about 4 weeks, from about 1 month to about 2 months. In some embodiments, multiple administration is carried out every hour, every day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, twice a week, three times a day. week, four times a week, five times a week, six times a week, seven times a week, eight times a week, nine times a week, ten times a week, eleven times a week, twelve times a week, thirteen times a week week, fourteen times a week, once a month, twice a month, three times a month, four times a month, five times a month, six times a month, seven times a month, eight times a month, nine times a month, ten times a month, eleven times a month, twelve times a month, thirteen times a month, fourteen times a month, every two months, or once every three months during the treatment period.

[358] In certain embodiments, treatment and/or intervention in an area (e.g., tissue, organ, mass, or lesion in an individual, or a region or portion thereof) is carried out through a treatment regimen involving multiple administration (or procedures or manipulations) over the course of a course. treatment. A course of treatment is a course of treatment that is repeated on a regular basis. In some embodiments, the implementation of the course of treatment may include several days of treatment, followed by several days of rest (ie days off from the drug). For example, treatment may be given every day for three weeks followed by a week without treatment in a course of treatment of 28 days, or treatment may be given five times a week for the first three weeks followed by a week without treatment. In specific embodiments, the implementation of the treatment is carried out for the treatment and/or intervention in the lesion during one or more courses of treatment. The course of treatment may be at least two, at least three, at least four, at least five, at least six, at least seven, at least 14, at least 21, at least 28, at least 48, or at least 96 days or more. In one embodiment, the course of treatment is 28 days. In various embodiments, the course of treatment is determined by a medical professional based on the condition and needs of the individual. In some embodiments, the treatment is carried out through a course of treatment of at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least eight days, at least nine days, at least ten days, at least eleven days, at least twelve days, at least 13 days, at least 14 days, at least 21 days or all 28 days of treatment of 28 days.

[359] In some embodiments, treatment and/or intervention in an area (eg, tissue, organ, mass, or lesion in an individual, or a region or portion thereof) is carried out by mechanical treatment and/or intervention, such as a biopsy. In certain embodiments, the implementation of mechanical treatment and/or intervention in the lesion is carried out through a single introduction, procedure or manipulation. In specific embodiments, the implementation of mechanical treatment and/or intervention includes more than one introduction, procedure or manipulation, such as biopsy. In some embodiments, the individual is treated and/or intervened in more than one lesion.

[360] In specific embodiments, treatment and/or intervention in an area (eg, tissue, organ, mass, or lesion in an individual, or a region or portion thereof) is carried out by thermotherapy, eg, cryotherapy or hyperthermia therapy. Thermotherapy can be administered according to any schedule, dose, or method known to those skilled in the art to be effective in treating and/or interfering with a lesion. In some embodiments, the implementation of the treatment and/or intervention in the lesion is carried out through a single application of thermotherapy. In specific embodiments, the implementation of the treatment and/or intervention in the lesion is carried out by more than a single application of thermotherapy. In certain embodiments, thermotherapy may be cryoablation. In some embodiments, the thermotherapy may be hyperthermia therapy. In some embodiments, thermotherapy is a therapy that raises the temperature of the tumor higher than hyperthermia therapy.

[361] In some embodiments, the implementation of the treatment and/or intervention in the lesion is carried out by radiation and/or radiation therapy. Dosing is based on an international unit known as the Gray (Gy, also expressed as cGy where 100 cGy=1 Gy) and the dose delivered in a single treatment session is known as a fraction. For example, a typical dosing schedule for surface radiotherapy (SRT) might be a total dose of 4500 cGy (45 Gy) delivered in 300 cGy doses over just 15 fractions. Radiation treatment can be carried out over several weeks, and fractions can be given on certain days, for example, from Monday to Friday. Also in clinical practice, alternation schemes are used, which include 2-3 fractions per week (i.e. according to the scheme: Monday, Wednesday, Friday). The dosage for the patient is determined by a qualified clinician, including with the assistance of a qualified technician, such as a radiation physicist, and is based on the size of the lesion, the age and health of the individual.

[362] In certain embodiments, treatment and/or intervention in an area (eg, tissue, organ, mass, or lesion in an individual, or a region or portion thereof) is performed with one or more radiation treatments. In specific embodiments, the treatment and/or intervention at the lesion is carried out with more than one treatment or dose of radiation and the total dose is about 5 Gy, about 10 Gy, about 15 Gy, about 20 Gy, about 25 Gy, about 30 Gy , approximately 35 Gy, approximately 40 Gy, approximately 41 Gy, approximately 42 Gy, approximately 43 Gy, approximately 44 Gy, approximately 45 Gy, approximately 46 Gy, approximately 47 Gy, approximately 48 Gy, 49 Gy, approximately 50 Gy, approximately 51 Gy, approximately 52 Gy, approximately 53 Gy, approximately 54 Gy, approximately 55 Gy, approximately 56 Gy, approximately 57 Gy, approximately 58 Gy, approximately 59 Gy, approximately 60 Gy, approximately 61 Gy, approximately 62 Gy, approximately 63 Gy, about 64 Gy, about 65 Gy, about 70 Gy, about 80 Gy, about 90 Gy, or about 100 Gy. In some embodiments, the total dose is from about 0.01 Gy to about 1 Gy, from about 1 Gy to about 30 Gy, from about 1 Gy to about 15 Gy, from about 15 Gy to about 30 Gy, from about 30 Gy to about 90 Gy, about 30 Gy to about 45 Gy, about 40 Gy to about 70 Gy, or about 45 Gy to about 60 Gy.

[363] In some embodiments, the implementation of the treatment and/or intervention in the lesion is carried out through two or more fractional treatments using radiation. In certain embodiments, the fractional dose is about 100 cGy, about 200 cGy, about 300 cGy, about 400 cGy, about 500 cGy, about 600 cGy, about 700 cGy, about 800 cGy, about 900 cGy, about 1 Gy, about 2 Gy , about 3 Gy, about 4 Gy, or about 5 Gy. In specific embodiments, the fractional dose is from about 10 cGy to about 100 cGy, from about 100 cGy to about 500 cGy, from about 500 cGy to about 1 Gy, or from about 1 Gy to about 5 Gy.

[364] In some embodiments, the implementation of the treatment and/or intervention in the lesion is carried out through a single treatment with radiation. In certain embodiments, a single dose is about 100 cGy, about 200 cGy, about 300 cGy, about 400 cGy, about 500 cGy, about 600 cGy, about 700 cGy, about 800 cGy, about 900 cGy, about 1 Gy, about 2 Gy , about 3 Gy, about 4 Gy, or about 5 Gy. In specific embodiments, a single dose is about 10 cGy to about 100 cGy, about 100 cGy to about 500 cGy, about 500 cGy to about 1 Gy, or about 1 Gy to about 5 Gy.

[365] In certain embodiments, the treatment and/or intervention in the lesion is carried out by external beam radiation therapy (EBT). For treatment and/or intervention at a lesion, EBT can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in treating or alleviating, but not limited to, a hyperproliferative disorder. In some embodiments, treatment and/or intervention at the lesion is carried out by administering EBT at a dose that is less than a dose that one of skill in the art would consider effective in treating or alleviating a hyperproliferative disorder. Typically, external beam radiation therapy involves irradiating a specific volume within an individual with a high energy beam, thereby causing cell death in that volume. In some embodiments, the irradiated volume contains the lesion to be treated and/or intervention, and preferably contains as little healthy and/or unaffected tissue as possible. In certain embodiments, the irradiated volume contains mostly or only the lesion. In some embodiments, administration methods and devices and compositions suitable for external beam radiation therapy can be found in US Pat. Nos. 6,449,336; 6,398,710;

[366] In some embodiments, the implementation of the treatment and/or intervention in the lesion is carried out through brachytherapy. In particular embodiments, brachytherapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in treating and/or intervening in a lesion to treat or mitigate a hyperproliferative disorder. In certain embodiments, the implementation of brachytherapy can be carried out in accordance with the scheme, dose or method, which is known to the person skilled in the art that they are less than those that are effective for treating or alleviating a hyperproliferative disorder. Typically, brachytherapy involves placing radioactive sources into the body of the individual being treated for cancer, eg, inside the tumor itself, so that the tumor is exposed to the maximum exposure from the radioactive source, and minimizing exposure to healthy tissue. Representative radioisotopes that can be administered in brachytherapy include, but are not limited to, phosphorus 32, cobalt 60, palladium 103, ruthenium 106, iodine 125, cesium 137, indium 192, xenon 133, radium 226, californium 252, or gold 198. Methods introduction, and devices and compositions suitable for brachytherapy are described in Mazeron et al, Sem. Rad. one. 12:95-108 (2002), Kovacs J. Contemp. Brachytherapy. 6(4):404-416 (2015) and US Pat. Nos. 6,319,189; 6,179,766; 6,168,777;

[367] In some embodiments, one or more treatments, such as administrations of a pharmaceutical agent, such as a therapeutic agent, are performed to treat and/or interfere with a lesion. In certain embodiments, treatment comprises administering a dose of a pharmaceutical agent, such as any of the agents described, for example, an immunomodulatory agent or compound. In specific embodiments, the implementation of the pharmaceutical agent is administered once for the treatment and/or intervention in the lesion. In certain embodiments, the implementation of the pharmaceutical agent is administered more than once for the treatment and/or intervention in the lesion. In some embodiments, the pharmaceutical agent is administered at a dose ranging from about 0.0001 to about 100 mg/kg body weight, such as from about 0.0005 to about 50 mg/kg body weight, such as from about 0.001 to about 10 mg/kg body weight, for example, from about 0.01 to about 1 mg/kg body weight. In specific embodiments, the pharmaceutical agent is administered to the individual at a dose of about 0.001 mg to about 100 mg, about 0.05 mg to about 50 mg, about 0.01 mg to about 1 mg, about 1 mg to about 20 mg, or from about 5 mg to about 15 mg. In some embodiments, the pharmaceutical agent is administered systemically. In certain embodiments, the agent is administered locally to the lesion. In specific embodiments, the pharmaceutical agent is administered orally, topically, sublingually, intravenously, subcutaneously, enterally, parenterally, by inhalation and/or by injection.

[368] In some embodiments, the pharmaceutical agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agents may be administered at a dose or doses considered by those skilled in the art to be effective in treating a hyperproliferative disorder. In certain embodiments, the implementation of chemotherapeutic agents can be entered in doses that are lower than the doses used in this field, which are considered effective for the treatment of hyperproliferative disorders. In some embodiments, a single dose of a chemotherapeutic agent is administered to the lesion for treatment and/or intervention. In certain embodiments, more than one dose of a chemotherapeutic agent is administered to an individual over a period of treatment, such as one or more courses of treatment, to treat and/or interfere with a lesion. In specific embodiments, the number of treatments or administrations of a chemotherapeutic agent is an amount that is considered by those skilled in the art to be effective in treating a hyperproliferative disorder. In some embodiments, the number of administrations is less than the amount considered effective by those skilled in the art for treating a hyperproliferative disorder.

[369] In certain embodiments, the pharmaceutical agent is an immunomodulatory agent, such as a checkpoint inhibitor. In certain embodiments, the immunomodulatory agents may be administered at a dose or doses that are considered effective for the treatment of a hyperproliferative disorder by those skilled in the art. In certain embodiments, the immunomodulatory agents may be administered at doses that are lower than the doses used in the art that are considered effective for the treatment of a hyperproliferative disorder. In certain embodiments, a single dose of an immunomodulatory agent is administered to the lesion for treatment and/or intervention. In some embodiments, more than one dose of an immunomodulatory agent is administered to an individual over a treatment period, such as one or more courses, to treat and/or interfere with a lesion. In specific embodiments, the number of treatments or administrations of an immunomodulatory agent is an amount considered effective by those skilled in the art to treat a hyperproliferative disorder. In some embodiments, the number of administrations is less than the amount considered effective by those skilled in the art for treating a hyperproliferative disorder.

[370] In certain embodiments, the pharmaceutical agent is lenalidomide or a thalidomide derivative. In specific embodiments, the pharmaceutical agent is lenalidomide. In some embodiments, lenalidomide or a thalidomide derivative is administered at a dosage of about 1 mg to about 20 mg, such as about 1 mg to about 10 mg, about 2.5 mg to about 7.5 mg, about 5 mg to about 15 mg, such as approximately 5 mg, 10 mg, 15 mg, or 20 mg. In some embodiments, lenalidomide is administered at a dose of from about 10 µg/kg to 5 mg/kg, for example, from about 100 µg/kg to about 2 mg/kg, from about 200 µg/kg to about 1 mg/kg, from about 400 µg/kg to about 600 µg/kg, for example about 500 µg/kg. In specific embodiments, the dose of lenalidomide is or is about 10 mg. In certain embodiments, the treatment and/or intervention in the lesion is carried out by administering a single dose of lenalidomide to the individual. In specific embodiments, treatment and/or intervention in the lesion is carried out by administering multiple doses of lenalidomide to the individual. In specific embodiments, multiple doses of lenalidomide are administered over one or more courses of treatment. In some embodiments, the courses of treatment include a day off from the drug. In certain embodiments, the implementation of lenalidomide is administered once a day for 14 days during a course of treatment of 21 days. In certain embodiments, the implementation of lenalidomide is administered once a day for 21 days during a course of treatment of 28 days.

[371] In some embodiments, the implementation of the treatment and/or intervention is repeated one or more times, for example, by providing one or more subsequent treatments and/or interventions after the previous or previous treatment and/or intervention. In some embodiments, the follow-up or re-treatment and/or intervention is carried out after the expansion of the genetically engineered cells in the individual or after the expansion is detected after the previous treatment and/or intervention and before the previous treatment and/or intervention. In some cases, the subsequent treatment and/or intervention is carried out at a point in time just before or just before the subsequent treatment and/or intervention when the individual is in remission. In some examples, the subsequent treatment and/or intervention is carried out at a time point immediately or immediately before the time point when the number of genetically engineered cells found in the blood is reduced or undetectable. In some cases, the subsequent treatment and/or intervention is carried out at a point in time just before or just before the subsequent treatment and/or intervention when the amount of genetically engineered cells found in the fluid or tissue or sample, optionally in the individual's blood, is reduced compared to with a previous point in time after the start of the previous treatment and/or intervention. In some cases, the subsequent treatment and/or intervention is carried out at a point in time immediately or immediately before the subsequent treatment and/or intervention when the number of genetically engineered cells found in the fluid or tissue or sample is optionally at or greater than 1, 5 times, 2.0 times, 3.0 times, 4.0 times, 5.0 times, 10 times or more the peak or maximum number of genetically engineered cells found or detected in the blood of an individual after the start of prior treatment and/ or intervention, and/or compared with a level within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or 28 days after the start of prior treatment and/or intervention.

[372] In some embodiments, the method includes providing a follow-up treatment and/or intervention, optionally after a relapse in an individual after a response from a prior treatment and/or intervention and/or a failure to achieve a complete response after a prior treatment and/or intervention. In some cases, an individual has responded to genetically engineered cells after prior treatment and/or intervention(s) and then ceased responding and/or relapsed prior to subsequent treatment and/or intervention. In some aspects, the genetically modified have been expanded in an individual, or found to have been expanded, after prior treatment and/or intervention(s) and prior to subsequent treatment and/or intervention.

C. Cell Expansion Monitoring

[373] In some embodiments, the implementation of the method includes assessing the exposure, persistence and proliferation of T cells, for example, T cells introduced for therapy based on T cells. In some embodiments, exposure, or long-term expansion and/or residence time of cells, and/or changes in cellular phenotypes or functional activity of cells, e.g., cells administered for immunotherapy, e.g., T-cell therapy, in the methods described herein , can be determined by characterizing T cells in vitro or ex vivo. In some embodiments, such assays can be used to determine or confirm the function of T cells used for immunotherapy, eg, T cell therapy, before or after the cell therapy described herein.

[374] In some embodiments, the presence and/or amount of recombinant receptor-expressing cells (e.g., CAR-expressing cells introduced for T-cell therapy) is detected in an individual after T-cell administration and before, during, and/or after the therapy. In specific embodiments, the presence and/or amount of cells expressing the recombinant receptor in an individual is determined after T cell administration and before, during and/or after the intervention. In some aspects, persistence or number of cells, e.g. to monitor persistence, is quantified as copies of DNA or plasmid encoding a receptor, e.g., CAR, per microgram of DNA, or as number of receptor-expressing, e.g., CAR-expressing cells per microliter of sample, for example, blood or serum, or total peripheral blood mononuclear cells (PBMC) or leukocytes or T cells per microliter of sample. In some cases, detection or monitoring of the presence of engineered cells, e.g., cells expressing a recombinant receptor, can be performed in other biological samples, such as an organ or tissue sample (e.g., a sample from a disease area, e.g., a tumor sample) of an individual.

[375] In some aspects, to assess the number of cells expressing the recombinant receptor (e.g., CAR-expressing cells introduced for T-cell therapy) is evaluated in a sample of blood or serum, or an organ or tissue (e.g., a sample from a region disease, e.g., a sample of a tumor) of an individual. In some cases, methods for assessing the presence or amount of cells expressing a recombinant receptor may include taking peripheral blood (or other biological sample) from individuals who have received the modified cells and determining the amount or ratio of the modified cells in the peripheral blood or biological sample. Cell selection and/or isolation approaches may include the use of antibodies specific for the chimeric antigen receptor (CAR) (e.g., Brentjens et al., Sci. Transl. Med. 2013 Mar; 5(177): 177ra38), protein L (Zheng et al., J. Transl. Med. 2012 Feb; 10:29), epitope tags such as Strep tag sequences that are inserted directly at specific sites in CAR, after which Strep tag binding reagents are used to directly assess CAR (Liu et al. (2016) Nature Biotechnology, 34:430; International Patent Publication No. WO2015095895) and monoclonal antibodies that specifically bind to a CAR polypeptide (see International Patent Publication No. WO2014190273). In some instances, extrinsic marker genes can be used in conjunction with modified cell therapies to allow cell discovery or selection, and in some cells also to allow cell suicide. In some cases, a truncated epidermal growth factor receptor (EGFRt) can be co-expressed with a transgene of interest (CAR or TCR) in transduced cells (see, for example, US Pat. No. 8,802,374). EGFRt may contain an epitope recognized by the cetuximab antibody (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule that can be used to identify or select cells modified with an EGFRt construct and another recombinant receptor, such as a chimeric antigen receptor (CAR), and /or to eliminate or separate cells expressing the receptor. See US Pat. No. 8,802,374 and Liu et al., Nature Biotech. April 2016; 34(4): 430-434).

[376] In some embodiments, cells are detected in an individual at or at least 4, 14, 15, 27, or 28 days after administration of T cells, eg, CAR-expressing T cells. In some aspects, cells are detected after or at least 2, 4, or 6 weeks after, or 3, 6, or 12, 18, or 24, or 30, or 36 months, or 1, 2, 3, 4, 5, or more than years after the introduction of T-cells, for example, CAR-expressing T-cells. In certain embodiments, cells are detected in an individual through or at least through 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days after intervention in an area in an individual, such as an area of a tissue, organ, mass, or lesion of an individual, or a region or part thereof. In some embodiments, cells are detected at or at least 2, 4, or 6 weeks after, or 3, 6, or 12, 18, or 24, or 30, or 36 months, or 1, 2, 3, 4, 5, or more than years after treatment and/or intervention in an area in an individual, such as an area of a tissue, organ, mass or lesion of the individual, or a region or part thereof.

[377] In some embodiments, the presence and/or number of cells is detected after a period of time following treatment or intervention in an area in an individual, such as an area of a tissue, organ, mass, or lesion of an individual, or a region or portion thereof, by more than one administration. , procedure or manipulation over a period of time, and determine the period of time after the start of the first administration, procedure or manipulation. In specific embodiments, the presence and/or number of cells is detected after a certain period of time after treatment and/or intervention in an area in an individual, such as an area of a tissue, organ, mass or lesion in an individual, or a region or part thereof, by more than one administration, procedure or manipulation over a period of time, and determine the period of time from the end of the last administration, procedure and manipulation.

[378] Exposure, e.g., the number of cells, e.g., T cells, introduced for T-cell therapy, indicating expansion and/or persistence, can be indicated in terms of the maximum numbers of cells that affect the individual, the duration of detection of cells on a detectable level or cells above a certain number or percentage, area under the curve for number or concentration of cells over time, and/or a combination of these and their features. Such outcomes can be assessed using known methods such as qPCR to detect the copy number of the nucleic acid encoding the recombinant receptor compared to the total amount of nucleic acid or DNA in a particular sample, e.g. a blood, serum, plasma or tissue sample such as blood, and/or analysis using flow cytometry, detecting cells expressing the receptor, usually using antibodies specific for the receptor. Also, to determine the number or percentage of functional cells, such as cells capable of binding to and/or neutralizing and/or inducing responses, for example, cytotoxic responses, against cells associated with a disease or condition, or expressing an antigen recognized by a receptor, one can use cellular methods of analysis.

[379] In some aspects, increased exposure of an individual to cells includes increased cell expansion. In some embodiments, the expansion of receptor-expressing cells, eg, CAR-expressing cells, occurs in the individual following administration of T cells, eg, CAR-expressing T cells. In specific embodiments, intervention in a region (e.g., region of a tissue, organ, mass, or lesion in an individual, or a region or portion thereof) results in increased exposure of receptor-expressing cells, e.g., CAR-expressing cells, e.g., increased cell expansion in of an individual compared to cell expansion immediately prior to intervention or compared to peak cell expansion in an individual prior to intervention in an area (e.g., an area of a tissue, organ, mass, or lesion in an individual, or a region or portion thereof).

[380] In some respects, progression-free survival (PFS) is described as the period of time during and after treatment for a disease, such as cancer, in which an individual lives with the disease but does not worsen. In some aspects, an objective response (OR) is described as a measurable response. In some aspects, the objective response rate (ORR) is described as the proportion of patients who achieved CR or PR. In some aspects, overall survival (OS) is described as the period of time either from the date of diagnosis or from the start of treatment for a disease, such as cancer, while individuals diagnosed with the disease are still alive. In some aspects, event-free survival (EFS) is described as the period of time after the end of cancer treatment when the individual remains free from certain complications or events that the treatment was supposed to prevent or delay. These events may include the return of the cancer or the onset of certain symptoms, such as bone pain from the cancer that has spread to the bone, or death.

[380] In some aspects, the method, for example, interfering with an area (e.g., a region of a tissue, organ, mass, or lesion in an individual, or a region or portion thereof), results in high in vivo proliferation of the introduced cells, for example, as determined by flow cytometry . In some aspects, high maximum cell ratios are detected. For example, in some embodiments, at the peak or maximum level or concentration following administration of T cells, e.g., CAR expressing T cells, in the blood or diseased area of an individual, or their leukocyte fraction, e.g., PBMC fraction or T cell fraction, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells express the recombinant receptor, eg, CAR.

[381] In some embodiments, the implementation of the method, for example, intervention in the area (for example, areas of tissue, organ, mass or lesion in an individual, or a region or part thereof), leads to a maximum concentration in blood or serum, or other body fluid, or an organ or tissue of an individual comprising at least 100, 500, 1000, 1500, 2000, 5000, 10000 or 15000 copies of a nucleic acid encoding a receptor, e.g. CAR, per microgram of DNA, or at least 0.1, 0, 2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 receptor-expressing, e.g., CAR-expressing cells, per total peripheral blood mononuclear cells (PBMC), the total number of mononuclear cells, the total number of T cells, or the total microliters of blood or serum or other body fluid, or organ, or tissue of an individual. In some embodiments, cells expressing the receptor are detected as at least 10%, 20%, 30%, 40%, 50%, or 60% of the PBMC in an individual's blood, and/or at that level for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, or 52 weeks after administration of T cells, such as CAR-expressing T cells, or within 1, 2, 3, 4 or 5 or more years after such administration.

[382] In some aspects, the method leads to an increase in copies of the nucleic acid encoding the recombinant receptor, for example, CAR, at least 2-fold, at least 4-fold, at least 10-fold, or at least 20 times, per microgram of DNA, for example, in a sample of serum, plasma, blood or tissue, for example, a sample of a tumor, an individual.

[383] In some embodiments, cells expressing the receptor are detected in a serum, plasma, blood, or tissue sample, e.g., a tumor sample, of an individual, e.g., by a specific method such as qPCR or a flow cytometric detection method, at least through 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days or more after administration of T cells, e.g., CAR-expressing T cells, by at least or approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 or more weeks after the introduction of T cells, for example, CAR-expressing T cells. In specific embodiments, cells expressing the receptor are found in a serum, plasma, blood, or tissue sample, e.g., a tumor sample, of an individual at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 or more days after treatment and/or intervention in an area (e.g., areas of a tissue, organ, mass or lesion in an individual, or a region or part thereof), at least through or approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks after treatment and/or intervention in the lesion.

[384] In some aspects, at least about 1x10 ² , at least about 1x10 ³ , at least about 1x10 ⁴ , at least about 1x10 ⁵ , or at least about 1x 10 ⁶ or at least about 5x10 ⁶ or at least about 1x10 ⁷ or at least about 5x10 ⁷ or at least about 1x10 ⁸ recombinant receptor-expressing cells, e.g., CAR-expressing cells, and/or at least 10, 25, 50, 100, 200, 300, 400 or 500 or 1000 receptor expressing cells per microliter, e.g. at least 10 per microliter, is found or present in the individual or in his fluid, plasma, serum, tissue or department, for example, in its peripheral blood, the area affected by the disease, for example, the focus of tumor damage. In some embodiments, such a number or concentration of cells is detected in an individual for at least about 20 days, at least about 40 days, or at least about 60 days, or at least about 3, 4, 5, 6, 7 , 8, 9, 10, 11 or 12 months, or at least 2 or 3 years after administration of T cells, eg CAR expressing T cells. In certain embodiments, such a number or concentration of cells is detectable in an individual for at least about 20 days, at least about 40 days, or at least about 60 days, or at least about 3, 4, 5, 6, 7 , 8, 9, 10, 11 or 12 months, or at least 2 or 3 years after treatment and/or intervention in the lesion. Such cell numbers may be as detected using flow cytometric or quantitative PCR methods and extrapolated to total cell numbers using known methods. See, for example, Brentjens et al., Sci Transl Med. 2013 5(177), Park et al, Molecular Therapy 15(4):825-833 (2007), Savoldo et al., JCI 121(5):1822-1826 (2011), Davila et al., (2013) PLoS ONE 8(4):e61338, Davila et al., Oncoimmunology 1(9):1577-1583 (2012), Lamers, Blood 2011 117:72-82, Jensen et al., Biol Blood Marrow Transplant 2010 September; 16(9): 1245-1256, Brentjens et al., Blood 2011 118(18):4817-4828.

[385] In some aspects, the number of copies of the nucleic acid encoding the recombinant receptor, for example, the number of copies of the vector, per 100 cells, for example, in peripheral blood or bone marrow, or other department, as determined by immunohistochemistry, PCR and/or flow cytometry, is at least 0.01, at least 0.1, at least 1, or at least 10, after about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or at least about 6 weeks, or at least about 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, or 12 months, or at least 2 or 3 years after administration of cells, e.g., CAR-expressing T- cells. In some embodiments, the number of copies of a vector expressing a receptor, such as CAR, per microgram of genomic DNA is at least 100, at least 1000, at least 5000, or at least 10,000, or at least 15,000, or at least 20,000 at a time point of about 1 week, about 2 weeks, about 3 weeks, or at least about 4 weeks after administration of T cells, e.g., CAR-expressing T cells, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or at least 2 or 3 years after such administration.

[386] In certain embodiments, the number of copies of the nucleic acid encoding the recombinant receptor, e.g., the number of copies of the vector, per 100 cells, e.g., in peripheral blood or bone marrow, or other location, as determined by immunohistochemistry, PCR, and/or flow cytometry , is at least 0.01, at least 0.1, at least 1, or at least 10, after about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or at least about 6 weeks, or at least about 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, or 12 months, or at least 2 or 3 years after treatment and/or intervention at the lesion. In some embodiments, the number of copies of a vector expressing a receptor, such as CAR, per microgram of genomic DNA is at least 100, at least 1000, at least 5000, or at least 10,000, or at least 15,000, or at least 20,000 at a time point of about 1 week, about 2 weeks, about 3 weeks, or at least about 4 weeks after administration of T cells, e.g., CAR-expressing T cells, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or at least 2 or 3 years after treatment and/or intervention in the lesion.

[387] In some aspects, a receptor, e.g., CAR, expressed by cells is detected by quantitative PCR (qPCR) or flow cytometry in an individual, in plasma, serum, blood, tissue, and/or a diseased area, e.g., a tumor area, at a time point, which is at least about 3 months, at least about 6 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, or more than 3 years after cell injection , for example, after the start of the introduction of T-cells. In specific embodiments, the cell-expressed receptor is detectable in the individual, in plasma, serum, blood, tissue, and/or diseased area, e.g., in a lesion or tumor area, at a time point that is at least about 3 months. , at least about 6 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, or more than 3 years, after treatment and/or intervention at the lesion . In some embodiments, the area under the curve (AUC) of the concentration of receptor-expressing (e.g., CAR) cells in fluid, plasma, serum, blood, tissue, organ, and/or diseased area, e.g., tumor area, of an individual is measured over time following administration. T cells, for example, CAR expressing T cells.

D. Response, efficacy and survival

[388] In some embodiments, the administration effectively treats the individual despite the individual having become resistant to other therapy and/or relapsed after administration of genetically engineered cells, such as cells expressing a recombinant receptor, e.g., CAR+ T cells . In some embodiments, at least or about at least 50% of individuals, at least or about at least 60% of individuals, at least or about at least 70% of individuals, at least or about at least 80% individuals or at least or approximately at least 90% of individuals treated with the method achieve complete remission (CR) and/or achieve an objective response (OR).

[389] In some embodiments, individuals treated in accordance with the intended method, for example, involving intervention in the area or site of damage after the introduction of genetically modified cells, achieve a longer response or achieve a longer response on average in a population of individuals who are treated in this manner, as compared to methods involving the administration of genetically engineered cells, such as cells expressing the recombinant receptor, but not involving treatment and/or intervention at the site or site of injury as described herein. In some embodiments, the Duration of Response Rate (DOR) includes the time from documentation of tumor response to disease progression. In some embodiments, the parameter for evaluating response may include a sustained response, such as a response that persists after a period of time from the start of therapy or after treatment and/or intervention at an area or site of injury after initiation of the administration of genetically engineered cells. In some embodiments, a sustained response is indicated by a response rate at approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months after initiation of therapy or after treatment and/or intervention. into the area or focus of damage after the start of the introduction of genetically modified cells. In some embodiments, the response lasts for more than 3 months, more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 30 months, more than 36 months, or more. In some specific embodiments, individuals treated with the method achieve a sustained response after the individual has previously relapsed after remission in response to the administration of genetically engineered cells.

[390] In some aspects, response rates in individuals, such as those with chronic lymphocytic leukemia (CLL), are based on the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) response criteria (Hallek, et al., Blood 2008, Jun 15; 111( 12): 5446-5456). In some aspects, these criteria are described as follows: complete remission (CR), which in some aspects requires the absence of clonal lymphocytes in the peripheral blood as determined by immunophenotyping, the absence of lymphadenopathy, the absence of hepatomegaly or splenomegaly, the absence of systemic symptoms, and a satisfactory leukocyte count; complete remission with incomplete bone marrow recovery (CRi), which in some aspects is described as CR above, but without a normal leukocyte count; partial remission (PR), which in some aspects is described as ≥ 50% reduction in lymphocyte count, ≥ 50% reduction in lymphadenopathy, or ≥ 50% reduction in liver or spleen size along with improvement in peripheral blood count; progressive disease (PD), which in some aspects is described as ≥ 50% increase in lymphocyte count to > 5×10 ⁹ /l, ≥ 50% increase in lymphadenopathy, ≥ 50% increase in liver or spleen size, Richter's transformation, or new cytopenias due to CLL; and stable disease, which in some aspects is described as not meeting the criteria for CR, CRi, PR, or PD.

[391] In some embodiments, individuals demonstrate a CR or OR if, within 1 month of cell dosing, the individual's lymph nodes are less than or about 20 mm, less than or about 10 mm or less, or about 10 mm. In specific embodiments, individuals demonstrate CR or OR if, within 1 month of treatment and/or lesion intervention, the individual's lymph nodes are less than or about 20 mm, less than or about 10 mm, or less than or about 10 mm.

[392] In some embodiments, the implementation of the index clone CLL is not detected in the bone marrow of the individual (or in the bone marrow of more than 50%, 60%, 70%, 80%, 90% or more of individuals treated with methods). In some embodiments, the CLL index clone is evaluated by IgH deep sequencing. In some embodiments, no index clone is found at, or about, or at least or about 1, 2, 3, 4, 5, 6, 12, 18, or 24 months after cell injection.

[393] In some aspects, any clinical, hematological and/or molecular methods are used to evaluate the response. In some respects, response is assessed using the Lugano criteria (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338; Cheson, B.D. (2015) Chin Clin Oncol 4(1):5). In some aspects, any of the clinical, hematological and/or molecular methods are used to evaluate the response. In some aspects, response assessed using the Lugano criteria involves the use of positron emission tomography (PET)-computed tomography (CT) and/or CT, as appropriate. Evaluation by PET-CT may additionally include the use of fluorodeoxyglucose (FDG) for FDG avid lymphomas. In some aspects, when PET-CT is used to assess response in FDG-avid histology, a 5-point scale may be used. In some respects, the 5-point scale includes the following criteria: 1, no capture above background level; 2, capture ≤ mediastinum; 3, capture > mediastinal but ≤ liver; 4, seizure moderately > liver; 5, capture well above the liver and/or new lesions; X, new areas of involvement unlikely to be associated with lymphoma.

[394] In some aspects, a complete response, as described using the Lugano criteria, involves a complete metabolic response and a complete radiological response in various measurable areas. In some aspects, these areas include lymph nodes and extralymphatic areas, where CR is described as a score of 1, 2, or 3 with or without residual mass on a 5-point scale when PET-CT is used. In some aspects, in Waldeyer's ring or extranodal areas of high physiological uptake, or when activated in the spleen or bone marrow (eg, through chemotherapy or myeloid colony stimulating factors), uptake may exceed normal mediastinal and/or hepatic uptake. In this case, a complete metabolic response can be established if uptake in areas of initial involvement does not exceed uptake in normal surrounding tissue, even if the tissue has high physiological uptake. In some aspects, the response is assessed in the lymph nodes using CT, where the CR is described as the absence of extralymphatic areas of disease and the size of the given nodes / mass of nodes should be reduced to ≤ 1.5 cm in the largest transverse diameter of the lesion (LDi). The following areas of evaluation include the bone marrow, where PET-CT-based evaluation should indicate no evidence of FDG-avid disease in the bone marrow, and CT-based evaluation should indicate normal morphology, which, if undetectable, should be IHC-negative. The following areas may include an assessment of organ enlargement that should return to normal. In some aspects, unmeasured lesions and new lesions are evaluated, which in the case of CR should be absent (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323- 338 Cheson, B.D. (2015) Chin Clin Oncol 4(1):5).

[395] In some aspects, a partial response (PR), as described using the Lugano criterion, involves a partial metabolic and/or radiological response in various measurable areas. In some aspects, these areas include lymph nodes and extralymphatic areas, where PR is described as a score of 4 or 5 with decreased capture from baseline and residual mass(s) of any size when PET-CT is used. At an interim assessment, such data may indicate a response to the disease. At the end of treatment, such findings may indicate residual disease. In some aspects, the response is assessed in the lymph nodes using CT, where PR is described as ≥50% SPD for up to 6 measurable nodes and extranodal areas. If the lesion is too small to be measured by CT, it is assigned a size of 5 mm×5 mm as the default value; if the focus is no longer visible, the value is 0 mm×0 mm; for a node> 5 mm × 5 mm, but less than the norm, actual indicators are used for calculation. Other areas of evaluation include the bone marrow, where PET-CT-based evaluation should indicate residual uptake greater than that in normal bone marrow, but reduced from baseline (diffuse uptake consistent with reactive changes from chemotherapy is assumed). In some aspects, if there are persistent focal changes in the bone marrow in the context of nodal response, further evaluation by MRI or biopsy or intermediate scan should be considered. In some aspects, additional areas may include an assessment of organ enlargement, where the spleen should be reduced to >50% of the length exceeding the norm. In some aspects, unmeasured lesions and new lesions are evaluated, which in the case of PR should be absent / normal, regress or not increase. Non-response/stable disease (SD) or progressive disease (PD) can also be defined using PET-CT and/or CT-based assessment (Cheson et al., (2014) JCO 32(27):3059-3067; Johnson et al. al., (2015) Radiology 2:323-338 Cheson, B.D. (2015) Chin Clin Oncol 4(1):5).

[396] In some respects, progression-free survival (PFS) is described as the period of time during and after treatment for a disease, such as cancer, when an individual lives with the disease but does not worsen. In some aspects, an objective response (OR) is described as a measurable response. In some aspects, the objective response rate (ORR) is described as the proportion of patients who achieved CR or PR. In some aspects, overall survival (OS) is described as the period of time either from the date of diagnosis or from the start of treatment for a disease, such as cancer, while individuals diagnosed with the disease are still alive. In some aspects, event-free survival (EFS) is described as the period of time after the end of cancer treatment when the individual remains free from certain complications or events that the treatment was supposed to prevent or delay. These events may include the return of the cancer or the onset of certain symptoms, such as bone pain from the cancer that has spread to the bone, or death.

[397] In some aspects, RECIST criteria are used to determine an objective tumor response, in some aspects in solid tumors (Eisenhauer et al., European Journal of Cancer 45 (2009) 228-247). In some aspects, the RECIST criteria are used to determine an objective response to a tumor for given lesions. In some respects, a complete response, as defined using the RECIST criteria, is described as the disappearance of all given lesions and any pathological lymph nodes (both given and unspecified), and should be a decrease in the short axis to <10 mm. In other aspects, a partial response, as determined using the RECIST criteria, is described as at least a 30% reduction in the sum of the diameters of the given lesions, taking the original sum of the diameters as a comparison value. In other aspects, progressive disease (PD) is described as at least a 20% increase in the sum of target lesion diameters, using the smallest study sum as the comparison value (this includes the original sum if it is the smallest study). In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5 mm (in some aspects, the appearance of one or more new lesions is also considered progression). In other aspects, stable disease (SD) is described as neither a sufficient decrease to be defined as a PR, nor a sufficient increase to be defined as a PD, taking as a comparison value the smallest sum of diameters in the study.

[398] In some aspects, the introduction or treatment in accordance with the methods provided, as a rule, reduces or prevents the expansion or load of the disease or condition in the individual. For example, when the disease or condition is a tumor, the methods typically reduce tumor size, volume, metastasis, bone marrow blast rate, or molecularly detectable cancer, and/or improve prognosis or survival or other symptom associated with tumor burden.

[399] The disease burden may encompass the total number of diseased cells in an individual or in an organ, tissue, or body fluid of an individual, such as an organ or tissue of a tumor, or from another area, for example, that may indicate metastasis. For example, tumor cells can be detected and/or quantified in blood or bone marrow in the context of certain hematological malignancies. In some embodiments, the disease burden may include tumor weight, number and extent of metastases, and/or percentage of blast cells present in the bone marrow.

[400] In some embodiments, the individual has leukemia. The degree of disease burden can be determined by assessing residual leukemia in the blood or bone marrow.

[401] In some embodiments, an individual has a morphological disorder if more than or equal to 5% blasts are present in the bone marrow, e.g., as determined by light microscopy, e.g., greater than or equal to 10% of blasts in the bone marrow, greater than or equal to 20 % of bone marrow blasts, greater than or equal to 30% of bone marrow blasts, greater than or equal to 40% of bone marrow blasts, or greater than or equal to 50% of bone marrow blasts. In some embodiments, the individual demonstrates a complete or clinical remission if the number of blasts in the bone marrow is less than 5%.

[402] In some embodiments, the individual may be in complete remission, but there is a small proportion of morphologically undetectable (by light microscopy) residual leukemic cells. An individual is said to have minimal residual disease (MRD) if the individual has less than 5% of blasts in the bone marrow and a molecularly detectable malignancy is determined. In some embodiments, a molecularly detectable cancer can be assessed using any of a variety of molecular methods that allow sensitive detection of a small number of cells. In some aspects, such methods include a PCR assay that can detect unique Ig/T cell receptor gene rearrangements or fusion transcripts resulting from chromosome translocations. In some embodiments, flow cytometry can be used to identify malignant cells based on leukemia-specific immunophenotypes. In some embodiments, molecular cancer detection may detect as early as 1 leukemia or blast cell per 100,000 normal cells, or 1 leukemic cell per 10,000 normal cells. In some embodiments, an MRD is detected in an individual that is molecularly detectable if at least or more than 1 leukemic cell per 100,000 cells is detected, for example, by PCR or flow cytometry. In some embodiments, the individual's disease load is molecularly undetectable, or MRD- ^, such that in some cases it is not possible to detect leukemic cells in an individual using PCR or flow cytometry techniques.

[403] In some aspects, the disease or condition persists after the first dose and/or the first dose is insufficient to eliminate the disease or condition in the individual. In some embodiments, the response to treatment, such as resolution of the disease and/or remission, is observed in accordance with the provided methods after intervention in the area or site of damage, as described.

[404] In some embodiments, the method reduces disease or condition burden, e.g., tumor cell count, tumor size, patient survival, or disease-free survival, to a greater extent and/or for a longer period of time compared to the reduction that would be observed in in the case of a comparable method using an alternative dosing regimen, such as a method in which one or more alternative therapeutic agents are administered to the individual, and/or a method in which the individual does not receive treatment and/or intervention in the area in the individual in which the engineered cells are present, or are likely to be present, or were present, or are likely to be present, in accordance with the means envisaged. In some embodiments, an individual's disease or condition load is detected, assessed, or quantified. In some aspects, disease load detection can be performed by detecting the total number of disease-affected or disease-associated cells, such as tumor cells, in an individual, or in an organ, tissue, or body fluid of the individual, such as blood or serum. In some aspects, an individual's survival, survival over a period of time, degree of survival, presence or duration of event-free or symptom-free survival, or relapse-free survival is assessed. In some embodiments, any symptom of a disease or condition is evaluated. In some embodiments, a disease or condition load is quantified.

[405] In some embodiments, the methods improve event-free survival or overall survival in an individual, compared to other methods, such as methods in which one or more alternative therapeutic agents are administered to the individual, and/or methods in which the individual is not treating and/or interfering with an area of the individual in which the modified cells are, or are likely to be, or were, or were likely to be, in accordance with the methods envisaged. For example, in some embodiments, the event-free survival rate or probability for individuals treated with the methods at 6 months post-dose is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, more than about 80%, more than about 90%, or more than about 95%. In some aspects, the overall survival rate is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some embodiments, the individual being treated with the methods has event-free survival, relapse-free survival, or survival for at least 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, the time to progression is increased, for example, to a time to progression of greater than or about 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

[406] In some embodiments, after treatment with the method, the likelihood of relapse is reduced compared to other methods, for example, methods in which one or more alternative therapeutic agents are administered to the individual, and / or methods in which the individual is not treated and / or intervention to a region in an individual in which the engineered cells are, or are likely to be, or were, or are likely to be, according to the methods envisaged. For example, in some embodiments, the probability of recurrence 6 months after the first dose is less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%. , less than about 20% or less than about 10%.

V Definitions

[407] Unless otherwise defined, all terms of the art, designations and other technical and scientific terms or terminology used in the present description, have the same meaning, which usually implies a specialist in the field to which the claimed invention belongs. In some instances, terms with well-known meanings are defined herein for clarity and/or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as materially different from what is well known in the art.

[408] As used herein, an "individual" is a mammal, such as a human or other animal, and typically is a human. In some embodiments, the subject, eg, the patient, to whom the immunomodulatory polypeptides modified by cell engineering or composition are administered, is a mammal, typically a primate, such as a human. In some embodiments, the primate is a marmoset or great ape. The individual may be male or female, and may be of any suitable age, including infant, child, adolescent, adult, and senior. In some embodiments, the individual is a non-primate mammal, such as a rodent.

[409] As used herein, "treatment" (and its grammatical variants such as "treat" or "treating") refers to the total or partial alleviation or reduction of a disease, or condition, or disorder, or symptom, or adverse effect or outcome, or phenotype associated with it. The desired effects of treatment include, but are not limited to, prevention of the onset or recurrence of the disease, mitigation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, mitigation or alleviation of the disease state, and remission or improvement in prognosis. The terms do not imply complete cure of a disease or complete elimination of any symptom or effect(s) with respect to all symptoms or outcomes.

[410] As used herein, "delaying the development of a disease" means delaying, preventing, slowing down, inhibiting, stabilizing, suppressing and/or delaying the development of a disease (such as cancer). This delay may be of varying duration depending on the medical history and/or the individual being treated. As one of skill in the art would appreciate, a sufficient or significant delay may, in effect, cover prevention such that the individual does not develop the disease. For example, late stage cancer, such as the development of metastases, may be delayed.

[411] "Prevention" as used herein includes providing prophylaxis against the onset or recurrence of a disease in an individual who may be predisposed to the disease but in whom the disease has not yet been diagnosed. In some embodiments, the provided and compositions are used to slow the progression of a disease or to slow the progression of a disease.

[412] As used herein, "suppress" function or activity means to reduce function or activity compared to otherwise the same conditions except for the state or parameter of interest, or alternatively compared to another state. For example, cells that inhibit tumor growth reduce the rate of tumor growth compared to the rate of tumor growth in the absence of cells.

[413] An "effective amount" of an agent, e.g., pharmaceutical formulation, cell, or composition, in the context of administration, refers to an amount effective, in dosages/amounts, and for periods of time necessary to achieve the desired result, such as a therapeutic or prophylactic result.

[414] A "therapeutically effective amount" of an agent, such as a pharmaceutical formulation or engineered cells, refers to an amount effective, in dosages, and for periods of time necessary to achieve a desired therapeutic result, for example, to treat a disease, condition, or disorder. , and/or pharmacokinetic or pharmacodynamic effect of treatment. The therapeutically effective amount may vary depending on such factors as the disease state, age, sex and body weight of the individual, the immunomodulatory polypeptides administered, or the cells modified by techniques. In some embodiments, provided methods involve administering immunomodulatory polypeptides modified by cell engineering techniques or compositions in effective amounts, eg, therapeutically effective amounts.

[415] "Prophylactically effective amount" refers to the amount, effective, in dosages and for periods of time necessary to achieve the desired prophylactic result. Usually, but not necessarily, since a prophylactic dose is used in an individual before or at an early stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.

[416] The term "pharmaceutical formulation" refers to a formulation that is in a form that provides the effectiveness of the biological activity of the active ingredient contained therein, and which does not contain additional components that are unacceptably toxic to the individual to whom the formulation is administered.

[417] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to the individual. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[418] As used herein, an indication that nucleotide or amino acid positions "correspond" to nucleotide or amino acid positions in a described sequence, such as those listed in a sequence listing, refers to the nucleotide or amino acid positions identified when aligned with the described sequence for identity maximization using a standard alignment algorithm such as the GAP algorithm. By aligning sequences, one of skill in the art can identify appropriate residues, for example, using conserved and identical amino acid residues as guides. As a general rule, amino acid sequences are aligned to identify relevant positions to achieve the greatest match (see, for example: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith , D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New. Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carrillo et al. (1988) SIAM J Applied Math 48 : 1073).

[419] The term "vector", as used herein, refers to a nucleic acid molecule capable of increasing in the amount of another nucleic acid to which it is associated. The term includes a vector as a self-replicating nucleic acid structure, as well as a vector that integrates into the genome of the host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors". Vectors include viral vectors, such as retroviral, eg, retroviral gamma and lentiviral vectors.

[420] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid is introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and their progeny, regardless of the number of passages. The progeny may not be completely identical to the original cell in terms of nucleic acid content, and may contain mutations. The term includes mutant progeny that have the same function or biological activity when screened or selected as the originally transformed cell.

[421] As used herein, an indication that a cell or population of cells is "positive" for a particular marker refers to the detectable presence on or in a cell of a particular marker, typically a cell surface marker. When referring to a cell surface marker, the term refers to the presence of surface expression detectable by flow cytometry, for example, by staining with an antibody that specifically binds to the marker, and detecting such an antibody, where the staining is detectable by flow cytometry at a level substantially higher than staining, detectable by performing the same procedure with the same isotype control under otherwise identical conditions and/or at a level substantially similar to that of a cell known to be positive for the marker and/or at a level significantly higher than the level for a cell known to be negative for the marker.

[422] As used herein, an indication that a cell or population of cells is "negative" for a particular marker, to the absence of a significant detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a cell surface marker, the term refers to the absence of surface expression detectable by flow cytometry, for example, by staining with an antibody that specifically binds to the marker, and the detection of said antibody, where the staining is not detected by flow cytometry at a level substantially higher than staining, detectable by performing the same procedure with the same isotype control under otherwise identical conditions and/or at a level substantially lower than that of a cell known to be positive for the marker and/or at a level substantially similar to the level for a cell known to be negative for the marker.

[423] As used herein, "percent identity (%) of amino acid sequences" and "percent identity" when used in relation to an amino acid sequence (reference polypeptide sequence) are defined as the percentage of amino acid residues in a candidate sequence (e.g., the one under consideration). antibody or fragment) that are identical to the amino acid residues in the reference polypeptide sequence after sequence alignment and gaps, if necessary, to achieve maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are known to those skilled in the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithms necessary to ensure maximum alignment over the entire length of the compared sequences.

[424] As used herein, the singular form includes the plural of the entities referred to, unless the context clearly indicates otherwise. For example, the singular form means "at least one" or "one or more". It is understood that aspects and variants described herein include "consisting of" and/or "essentially consisting of" aspects and variants.

[425] Throughout the present description, various aspects of the claimed invention are presented in range format. It should be understood that the description in range format is provided for convenience and brevity only and should not be construed as a strict limitation on the scope of the claimed invention. Thus, a description of a range should be understood as a specific description of all possible subranges as well as individual numerical values within that range. For example, when a range of values is provided, it is understood that each value included in it between the upper and lower limits of this range and any other value specified or included in this specified range is covered by the claimed invention. The upper and lower limits of these smaller ranges can be independently included in the smaller ranges, and are also covered by the claimed invention, with the exception of any specifically excluded limit in the specified range. When the specified range includes one or both of the limits, the claimed invention also includes any or both of these included limits. This applies regardless of the range width.

[426] The term "approximately", as used herein, refers to the usual range of error for the corresponding value, well known to a person skilled in the art. The indication of "approximately" a value or parameter in the present specification includes (and describes) embodiments that refer to that value or parameter itself. For example, a description that mentions "about X" includes a description of "X".

[427] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, suspension, liquid, powder, paste, aqueous composition, non-aqueous composition, or any combination thereof.

[428] All publications, including patent documents, scientific articles and databases referred to in this application, are incorporated herein as references in their entirety for any purpose, to the extent that each individual publication was individually included in as a link. If the definition given in the present description conflicts with or otherwise does not correspond to the definition given in patents, applications, published applications and other publications, which are incorporated in the present description by reference, the definition given in the present description takes precedence over the definition that included in the present description by reference.

[429] The section headings used herein are for organizational purposes only and should not be construed as limiting the invention described.

IX. ILLUSTRATIVE EMBODIMENTS

[430] Embodiments contemplated by the present invention include:

1. A method of expanding genetically engineered cells, comprising intervening in an area in an individual in which the modified cells are present, or likely to be present, or were present or likely to be present, and said individual has previously been administered the genetically modified cells to treat a disease or condition where the method results in the expansion of engineered cells in an individual in a region and/or tissue or organ or fluid of the individual and/or an increase in the number of engineered cells in that region, tissue, or organ, or liquids.

2. The method according to Embodiment 1, wherein the method does not include post-administration of the genetically engineered cells, and/or expansion is achieved without such post-administration of the genetically engineered cells.

3. A method of treatment comprising administering a treatment regimen to an individual, wherein the individual has previously been administered genetically engineered cells to treat a disease or condition, wherein the method results in expansion of the genetically engineered cells in the individual, region, and/or tissue, or organ or fluid of an individual, and/or an increase in the number of genetically engineered cells in that region, tissue, or organ, or fluid.

4. The method according to embodiment 4, wherein the treatment regimen comprises interfering with an area in the subject in which the engineered cells are present, or suspected to be present, or were present, or likely to be present.

5. The method according to embodiment 3 or 4, wherein the treatment regimen and/or method does not include post-administration of genetically engineered cells or genetically engineered cells, and/or expansion is achieved without such post-administration.

6. The method of any one of embodiments 3-5, wherein the treatment regimen is administered at a sub-therapeutic dose and/or provides its therapeutic effect through the expansion of genetically engineered cells.

7. The method according to any one of embodiments 1, 2 and 4-6, wherein the area is or contains a lesion or a portion thereof.

8. The method of Embodiment 7 wherein the lesion is a tumor.

9. The method according to embodiment 8, wherein the tumor is a primary or secondary tumor.

10. The method according to any one of embodiments 1, 2 and 4-6, wherein the region is or contains bone marrow tissue.

11. The method according to any one of embodiments 1, 2 and 4-10, wherein at or just before the intervention time point, the individual has a post-response relapse, optionally post-remission, and/or has failed to respond to the genetically engineered cells.

12. The method according to any one of embodiments 1-11, wherein the subject has relapsed after responding and/or has not responded to prior administration of the genetically engineered cells.

13. The method according to any one of embodiments 1-12, wherein the individual responded to genetically engineered cells and then stopped responding and/or relapsed prior to intervention.

14. The method according to any one of embodiments 1-13, wherein the genetically engineered cells have previously expanded or found to have expanded prior to intervention.

15. The method according to any of embodiments 1-14, where at or just before the time of intervention:

the individual is in remission;

the number of genetically engineered cells detectable in the blood is reduced or undetectable;

the number of genetically engineered cells found in the fluid or tissue or sample, optionally blood, of the individual is reduced compared to a previous point in time after the administration of the genetically engineered cells; and/or

the number of genetically engineered cells found in a fluid or tissue or sample, optionally blood, of an individual is reduced by or more than 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5 .0 times, 10 times or more compared to the peak or maximum number of genetically engineered cells detected or detected in the blood of an individual after the start of administration of genetically engineered cells and / or compared to the level at a time point within 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or 28 days after the introduction of genetically engineered cells.

16. The method according to any one of embodiments 1-15, wherein the intervention is carried out after, about after, or more than, or more than about 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months , 1 year or more after the start of the administration of genetically engineered cells or after the last dose of genetically engineered cells.

17. The method according to any one of embodiments 1-16, wherein the intervention directly or indirectly modulates the activity or function of engineered T cells in vivo in an individual.

18. The method according to any one of embodiments 1-17, wherein the intervention comprises one or more of administration of an immunomodulatory agent, radiation, or physical or mechanical manipulation of an area or lesion.

19. The method according to any one of embodiments 1-18, wherein the intervention comprises administering an immunomodulatory agent.

20. The method of Embodiment 19 wherein the immunomodulatory agent is or contains an immune inhibitory molecule, is or contains an immune checkpoint molecule or a member of the immune checkpoint cascade, and/or is or contains a modulator of the immune checkpoint molecule or cascade.

21. The method according to embodiment 20, wherein the immune checkpoint molecule or cascade is or comprises PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM3, VISTA, adenosine receptor, CD73, CD39, adenosine 2A receptor (A2AR), or adenosine, or a cascade involving any of the above.

22. The method according to any one of embodiments 1-20, wherein the immunomodulatory agent is BY55, MSB0010718C, ipilimumab, daclizumab, bevacizumab, basiliximab, ipilimumab, nivolumab, pembrolizumab, MPDL3280A, pidilizumab, MK-3475, BMS-936559, atezolimumab, tremelimumab , IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, MK-4166, dacetuzumab, lucatumumab, SEA-CD40, CP-870, CP-893, MEDI6469, MEDI6383, MOXR0916, AMP-224, avelumab, MEDI473 , PDR001, rHIgM12B7, ulokuplumab, BKT140, varlilumab, ARGX-110, MGA271, lirilumab, IPH2201, ARGX-115, emaktuzumab, CC-90002 and MNRP1685A, or an antigen-binding fragment thereof.

23. The method according to any one of embodiments 1-22, wherein the immunomodulatory agent is an anti-PD-L1 antibody.

24. The method according to embodiment 1-23, wherein the anti-PD-L1 antibody is MEDI14736, MDPL3280A, BMS-936559, LY3300054, atezolizumab, or avelumab, or an antigen-binding fragment thereof.

25. The method of Embodiment 19 wherein the immunomodulatory agent is thalidomide, or is a derivative or analogue of thalidomide.

26. The method according to embodiment 19 or 25, wherein the immunomodulatory agent is lenalidomide or pomalidomide, avadomide, a stereoisomer of lenalidomide, pomalidomide, avadomide, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate, or polymorph thereof.

27. The method according to any of embodiments 19, 25 and 26, wherein the immunomodulatory agent is lenalidomide, a stereoisomer of lenalidomide, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof.

28. The method according to any one of embodiments 1-27 wherein, after relapse and prior to intervention, the subject was not administered an exogenous or recombinant agent to treat a disease or condition, or to modulate the activity of genetically engineered cells.

29. The method according to any one of embodiments 1-19 and 28, wherein the intervention comprises radiation therapy.

30. The method according to any one of embodiments 1-19 and 28, where the intervention includes physical or mechanical manipulation of the area or lesion, optionally includes probing, piercing or penetrating the area or lesion.

31. The method according to embodiment 30, wherein the physical or mechanical manipulation comprises a biopsy.

32. The method of embodiment 31 wherein the biopsy is performed with a needle or trocar.

33. The method according to embodiment 31 or embodiment 32, wherein the biopsy comprises an incisional biopsy.

34. The method according to any one of embodiments 1-34, wherein the method results in an expansion of the genetically engineered cells or an increase in the number of genetically engineered cells compared to the time immediately before the intervention.

35. The method of any one of embodiments 1-34, wherein the genetically engineered cells expand within or about 24 hours, 48 hours, 96 hours, 7 days, 14 days, or 28 days after the intervention.

36. The method according to any of embodiments 1-35, where:

expansion provides more than or about more than 1.5 times, 2.0 times, 5.0 times, 10 times, 100 times, 200 times or more engineered cells detectable in blood, compared to the time immediately before the intervention; or

expansion provides more than or approximately more than 1.5 times, 2.0 times, 5.0 times, 10 times, 100 times, 200 times or more engineered cells detectable in blood, compared to previous peak levels of engineered cells in blood prior to intervention.

37. The method according to any one of embodiments 1-36, wherein the amount of genetically engineered cells found in the blood at a time point after the intervention is:

increases (for example, increases by 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times or more) compared with the number of cells modified by the methods of engineering at the previous time point before the intervention;

more than 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, 100 times or more the peak or maximum number of engineered cells detectable in the blood of the individual prior to intervention;

more than or approximately more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1 % of engineered cells are detectable in the blood at the point in time after detection of the peak of the maximum level of such cells in the blood.

38. The method according to any one of embodiments 1-37, wherein the engineered cells express a recombinant receptor that specifically binds to an antigen associated with a disease or disorder or expressed in cells of the environment or lesion.

39. The method according to any one of embodiments 1-38, wherein the disease or condition is a tumor or cancer.

40. The method according to any one of embodiments 1-39, wherein the disease or condition is leukemia or lymphoma.

41. The method according to any one of embodiments 1-40, wherein the disease or condition is non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL).

42. The method according to any one of embodiments 1-41, wherein the recombinant receptor is a T cell receptor or a functional non-T cell receptor.

43. The method according to any one of embodiments 1-42, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

44. The method according to embodiment 43, wherein the CAR comprises an extracellular antigen recognition domain that specifically binds to an antigen and an extracellular signaling domain containing ITAM.

45. The method according to any one of embodiments 38-44, wherein the antigen is CD19.

46. The method of Embodiment 44 or Embodiment 45, wherein the intracellular signaling domain comprises an intracellular CD3-zeta (CD3ζ) chain domain.

47. The method according to any one of embodiments 43-46, wherein the CAR further comprises a co-stimulatory signaling region.

48. The method according to embodiment 35, wherein the costimulatory signaling domain comprises a CD28 or 4-1BB signaling domain.

49. The method according to any one of embodiments 1-48, wherein the engineered cells are CD4+ or CD8+ T cells.

50. The method according to any one of embodiments 1-49, wherein the T cell therapy comprises primary cells obtained from an individual.

51. The method according to any one of embodiments 1-49, wherein the engineered cells are autologous to the individual.

52. The method according to any one of embodiments 1-49, wherein the engineered cells are allogeneic to the individual.

53. The method according to any one of embodiments 1-52, wherein the subject is a human.

54. The method according to any one of embodiments 1-53, wherein the dose of genetically engineered cells previously administered is from or about 1×10 ⁵ to 1×10 ^{8 of} all cells expressing the recombinant receptor, all T cells, or all peripheral blood mononuclear cells (PBMC), from or from about 5x10 ⁵ to 1x10 ⁷ all cells expressing the recombinant receptor, all T-cells, or all peripheral blood mononuclear cells (PBMC) or from or from about 1x10 ⁶ to 1×10 ⁷ all cells expressing the recombinant receptor, all T cells, or all peripheral blood mononuclear cells (PBMC), in each case inclusive.

55. The method according to any one of embodiments 1-54, wherein the dose of genetically engineered cells previously administered is no more than 1 x 10 ^{8 of} all cells expressing the recombinant receptor, all T cells, or all peripheral blood mononuclear cells ( PBMC), not more than 1×10 ^{7 of} all cells expressing the recombinant receptor, all T-cells or all peripheral blood mononuclear cells (PBMC), not more than 0.5×10 ^{7 of} all cells expressing the recombinant receptor, all T- cells or all peripheral blood mononuclear cells (PBMC), not more than 1×10 ^{6 of} all cells expressing the recombinant receptor, all T-cells or all peripheral blood mononuclear cells (PBMC), not more than 0.5×10 ^{6 of} all cells expressing the recombinant receptor, all T cells or all peripheral blood mononuclear cells (PBMC).

56. The method according to any one of embodiments 1-53, wherein the dose of genetically engineered cells previously administered is from about 0.25×10 ⁶ cells/kg of body weight of the individual to 5×10 ⁶ cells/kg, from 0 5×10 ⁶ cells/kg of body weight of an individual up to 3×10 ⁶ cells/kg, from about 0.75×10 ⁶ cells/kg to 2.5×10 ⁶ cells/kg, or from about 1×10 ⁶ cells/ kg up to 2×10 ⁶ cells/kg, in each case inclusive.

57. The method according to any one of embodiments 1-56, wherein the dose of genetically engineered cells is administered in a single cell-containing pharmaceutical composition or as multiple cell-containing compositions together.

58. The method according to any one of embodiments 1-57, wherein the engineered cells administered are a divided dose, wherein the dose cells are administered in multiple formulations containing the dose cells collectively over a time period of no more than three days.

59. The method according to any one of embodiments 1-58, wherein the method comprises providing a follow-up intervention, optionally after the individual has relapsed after a response after a prior intervention, and/or has not achieved a complete response after a prior intervention.

60. The method according to any one of embodiments 59, wherein the individual responded to the genetically engineered cells after the prior intervention and then stopped responding and/or relapsed prior to the subsequent intervention.

61. The method of Embodiment 59 or Embodiment 60 wherein the genetically engineered cells have expanded or are found to have expanded after the prior intervention or prior to the subsequent intervention.

62. The method according to any one of embodiments 59-61, wherein at or just before the time of the subsequent intervention:

the individual is in remission;

the number of genetically engineered cells found in the blood is reduced or undetectable;

the number of genetically engineered cells found in a fluid or tissue or sample, optionally in blood, of an individual is reduced compared to a previous point in time after the start of the previous intervention; and/or

the amount of genetically engineered cells found in a fluid or tissue or sample, optionally blood, from an individual is reduced by or more than 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5 .0 times, 10 times or more compared to the peak or maximum number of genetically engineered cells detected or detected in the blood of an individual after the start of the previous intervention and/or compared to the level at a time point within 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or 28 days after the start of the previous intervention.

63. The method according to any one of embodiments 1-62, wherein the genetically engineered cells exhibit increased or prolonged expansion and/or persistence in an individual compared to a method in which the genetically engineered cells are administered to the individual in the absence of intervention.

64. The method according to any one of embodiments 1-63, wherein the method reduces tumor burden to a greater extent and/or for a longer period of time compared to the reduction that would be observed in the case of a comparable method in which genetically engineered cells are administered an individual in the absence of intervention, and/or in which the therapeutic regimen is carried out or the intervention is carried out in the absence of genetically modified cells, optionally with the same dose or the same dosing schedule.

VII. Examples

[431] The following examples are included for illustration only and are not intended to limit the scope of the invention.

Example 1 Administration of Anti-CD19 CAR Expressing Cells to Individuals

[432] Twenty-eight individuals with relapsed or refractory (R/R) non-Hodgkin's lymphoma (NHL) were injected with autologous T cells expressing the chimeric antigen receptor (CAR) against CD19. Individual demographics and baseline characteristics are shown in Table 1. CAR contained an anti-CD19 scFv, mouse antibody-derived, immunoglobulin-derived spacer, CD28-derived transmembrane domain, 4-1BB-derived costimulatory region, and CD3-zeta intracellular signaling domain. . To obtain autologous CAR-expressing T cells, T cells were isolated by immunoaffinity-based enrichment from individual leukapheresis samples, activated, and transduced with a viral vector encoding anti-CD19 CAR, followed by upscaling). Cells were generally administered to individuals at a target ratio of CAR ⁺ CD4 ⁺ T cells and CAR ⁺ CD8 ⁺ T cells of approximately 1:1.

Table 1. Demographics and Baseline Characteristics Characteristic N=28 Median age, years (range) 63(37-79) ≥ 70 years, n (%) 6 (21) Male/Female, n (%) 19/9 (68/32) Subtype B-NHL, n (%) DLBCL, NOS 15 (54) Transformed DLBCL 10 (36) Follicular Grade 3B fourteen) MCL 2 (7) Disease status, n (%) Refractory ^* 24 (86) Chemorefractory ^† 23 (82) Baseline ECOG, n (%) 0 14 (50) one 10 (36) 2 4 (14) Prior Lines of Therapy Median (range) 4 (1-8) ≥ 5, n (%) 7 (25) Prior hematological stem cell transplantation, n (%) Any HSCTs 16 (57) Allogeneic 4 (14) autologous 13 (46) *<CR on last therapy
^† SD or PD on last chemotherapy regimen or relapse <12 months after autologous SCT

[433] Before the introduction of CAR-expressing T cells, (d=0), individuals were treated with 30 mg/m ² fludarabine every day for 3 days and 300 mg/m ² cyclophosphamide every day for 3 days. Compositions of cryopreserved cells were thawed before intravenous administration. A therapeutic dose of T cells was administered as a defined cell composition by administering a pooled population of CD4+ CAR+ cells and a pooled population of CD8+ CAR+ administered in a predetermined ratio of approximately 1:1. At d=0, individuals were started on a single dose or two dose schedule, at one of two dose levels (dose level 1 (DL-1) or dose level 2 (DL-2), by intravenous infusion. Each dose administered included 5 x10 ⁷ (DL-1) or 1 x 10 ⁸ (DL-2) CAR-expressing T cells (specified CD4+:CD8+ ratio 1:1) The results of this example refer to the results observed up to and at the particular time the time of the ongoing study, in a particular group(s) of individuals.

[434] The presence or absence of various treatment-related adverse events was assessed in individuals treated with regimens with various doses of CAR-T cell therapy (Table 2). As shown in Table 3, there was no severe cytokine release syndrome (sCRS) (grade 3-5); cytokine release syndrome (CRS) was observed in 36% (10/28) of individuals. Grade 3-4 neurotoxicity was observed in 14% (4/28) of individuals and 18% (5/28) of individuals had some degree of neurotoxicity. One individual was treated with tocilizumab and four patients were treated with dexamethasone for CRS or early-onset Grade 2 neurotoxicity. Six individuals were administered prophylactic antiepileptic agents.

Table 2. Treatment-related adverse events DL1-S N=22 DL1-D N=3 DL2-S N=3 Total N=28 Any TEAE 21 (96) 3 (100) 3 (100) 27 (96) Any TEAE degree 3-5 ^* 16 (73) 3 (100) 0 19 (68) Any associated TEAE 14 (64) 2 (67) 1 (33) 17 (61) Any associated TEAE grades 3-5 ^* 4 (18) 1 (33) 0 5 (18) Any grade of TEAE reported in ≥15% of patients
preferred term, n (%) Fatigue 7 (32) 2 (67) 2 (67) 11 (39) Cytokine release syndrome 8 (36) 2 (67) 0 10 (36) Decreased appetite 6 (27) 1 (33) 1 (33) 8 (29) Constipation 5 (23) 1 (33) 1 (33) 7 (25) Vomit 5 (23) 1 (33) 1 (33) 7 (25) Diarrhea 5 (23) 1 (33) 0 6 (21) Dizziness 6 (27) 0 0 6 (21) Headache 4 (18) 1 (33) 0 5 (18) hypertension 4 (18) 1 (33) 0 5 (18) Nausea 3 (14) 1 (33) 1 (33) 5 (18) peripheral edema 5 (23) 0 0 5 (18) Laboratory anomalies Anemia 16 (73) 1 (33) 1 (33) 18 (64) Neutropenia 22 (100) 3 (100) 2 (67) 27 (96) Thrombocytopenia 13 (59) 3 (100) 2 (67) 18 (64) *1 Grade 5 respiratory failure assessed as possibly related to CAR-T cell therapy in a patient with MCL who progressed and was started on next therapy

Table 3 Treatment-related adverse events of particular interest Preferred term, n (%) DL1-S N=22 DL1-D N=3 DL2-S N=3 Total N=28 Cytokine release syndrome (CRS), any 8 (36) 2 (67) 0 10 (36) Degrees 3-4 0 0 0 0 Neurotoxicity, any ^* 4 (18) 1 (33) 0 5 (18) Degrees 3-4 3 (14) 1 (33) 0 4 (14) ^* Includes: encephalopathy, confusion, decreased level of consciousness, lethargy or convulsions

[435] Individuals in the group were evaluated for the best overall response observed over a period up to a specific point in time during therapy after the last infusion of a single dose of CAR+ T cells according to DL1. Overall response results are shown in Table 4. Of the 20 individuals who received a single dose of DL1 in the diffuse large B-cell lymphoma (DLBCL) group, an overall response rate (ORR) of 80% (16/20) and 60% (12/20) was observed. individuals showed signs of complete remission (CR). 20% (4/20) of individuals showed signs of a partial response (PR) and 20% (4/20) of individuals showed signs of progressive disease (PD). Among individuals who are chemorefractory (having stable or progressive disease after the last chemotherapy regimen or relapsed less than 12 months after autologous SCT) prior to CAR+ T cell administration, the overall response rate was 83% (10 ORR, 7 CR, 3 PR, 2PD, n=12). Among individuals who are refractory (having less than complete remission after the last treatment but considered chemorefractory), the overall response rate was 77% (13 ORR, 9 CR, 4 PR, 4 PD, n=17).

Table 4. General response DLBCL cohort, DL1 single dose regimen All (n=20) Refractory ^* (n=17) Chemorefractory ^† (n=12) ORR, n (%) [95% CI] 16 (80) [56, 94] 13 (77) [50, 93] 10 (83) [52, 98] CR, n (%) [95% CI] 12 (60) [36, 81] 9 (53) [28, 77] 7 (58) [28, 85] PR 4 (20) 4 (24) 3 (25) PD 4 (20) 4 (24) 2 (17) *<CR on last therapy
^† SD or PD on last chemotherapy regimen or relapse <12 months after autologous SCT

[436] Of the three individuals with DLBCL who were treated with two doses of DL-1 at the time of assessment, two had a partial response (PR) and 1 had progressive disease (PD). Among the 2 DLBCL individuals who were treated with a single dose according to DL-2 at the time of assessment, both individuals were observed to achieve CR. In the MCL group, in which only two individuals were treated with a single dose according to DL-1 at the time of evaluation, 1 PR and 1 PD were observed. Two individuals with double-hit DLBCL, three individuals with triple-hit DLBCL, and four individuals with double-expressor DLBCL were treated and all achieved a response (7 CR, 2 PR).

[437] The number of CAR ⁺ T cells in peripheral blood was determined at certain time points after treatment by incubating the cells with a reagent specific for the transgene. The number of CD3 ⁺ /CAR ⁺ T-cells in peripheral blood, determined at certain time points after infusion, is presented for individuals grouped by the best overall response in figa. Responding individuals (CR/PR) exhibited higher peak CD3 ⁺ /CAR ⁺ T cells than individuals with PD. 1B-1D show the levels of CD3 ⁺ /CAR ⁺ T cells, CD4 ⁺ /CAR ⁺ T cells, and CD8 ⁺ /CAR ⁺ T cells (number of cells/µl of blood; mean ± SEM) in individuals who achieved response grouped by duration of response, either by sustained response (CR/PR) or by PD at 3 months.

[438] C _max (number of CAR ⁺ cells/µl of blood) and area under the curve (AUC) for responding individuals (CR/PR) and for individuals with PD were determined and are presented in Table 5. The results were consistent with the conclusion that long-term responses correlated with higher levels of CD3 ⁺ /CAR ⁺ T-cells in the blood over time and at maximum expansion.

Table 5. Higher _Cmax and AUC _0-28 in patients with CR/PR vs. PD CD3 CD4 CD8 CR/PR
(n=16) PD
(n=4) CR/PR
(n=16) PD
(n=4) CR/PR
(n=16) PD
(n=4) C _max (CAR ⁺ cells/µl of blood) Mean value (SD) 612 (1919) 2(1) 220 (754) 1 (0.6) 426 (1314) 0.5 (0.5) Median (Min, Max) 33 (1, 7726) 1 (1, 3) 8 (1, 3040) 1 (0, 2) 4 (0.5238) 0.3 (0.1) Q1, Q3 7, 123 0.7, 2 2, 46 0.6, 2 0.8, 104 0.1, 0.9 AUC _0-28 Mean value (SD) 5883 (18821) 16 (13) 2369 (8388) 10 (7) 3873 (11963) 6 (6) Median (Min, Max) 196 (11, 75773) 14 (4, 31) 47 (7, 33740) 9 (3, 17) 23(1, 47834) 4 (1, 14) Q1, Q3 52, 781 5, 26 16, 261 4, 16 4,761 1, 10

AUC _0-28= counts per microliter for a specified population of CAR+ cells between days 0 and 28

Example 2: Reexpansion of Cells Expressing Anti-CD19 CAR

[438] For one individual with chemorefractory transformed DLBCL (germinal center subtype with BCL2 rearrangement and multiple copies of MYC and BCL6) treated with CAR+ T cells according to the DL-1 regimen as described in Example 1, the number of CD3+/CAR+ , CD4+/CAR+, CD8+/CAR+ T-cells in peripheral blood, determined after certain periods of time, is presented in figa. Subject previously treated with, and refractory to, five prior line therapies, including dose-adjusted etoposide, doxorubicin and cyclophosphamide with vincristine, and prednisone plus rituximab (DA-EPOCH-R), and intermediate-intensity allogeneic stem cell transplantation from an unrelated matched donor HLA 8/8. Following allogeneic stem cell transplantation and prior to CAR+ T cell administration, the individual demonstrated 100% donor chimerism in all blood outgrowths, discontinued immunosuppressive therapy, and had no graft-versus-host disease (GVHD). Prior to administration of CAR+ T cells, he had a parotid mass and a lesion in the right temporal lobe of the brain observed by positron emission tomography and computed tomography (PET-CT) (Fig. 2B) and confirmed by magnetic resonance imaging (MRI) (Fig. .2D).

[439] Following treatment with anti-CD19 CAR-T cells, the individual achieved CR 28 days post-infusion as shown by PET-CT (Figure 2C) and brain MRI (Figure 2E) with no observable signs of neurotoxicity or CRS. Three months after CAR-T cell infusion, this individual was found to have a recurrent parotid mass (Figure 2F) and underwent an incisional biopsy. As shown in Figure 2A, after the biopsy, the visible tumor resolved without further therapy. Pharmacokinetic analysis demonstrated a marked re-expansion of CAR-T cells in the peripheral blood (to a level above the initially observed expansion, with peak levels observed approximately 113 days after infusion), which coincided with tumor regression. The individual then achieved a second CR, which was confirmed by PET-CT for restaging one month after biopsy (FIG. 2G) and remained in CR 6 months after CAR-T cell infusion. Further evaluation of the individual demonstrated that the CNS response was sustained and the individual remained in the CR state after 12 months.

[440] The results are consistent with the conclusion that re-expansion and activation of CAR+ T cells can be initiated in vivo following a decrease or loss of functional or active CAR+ T cells and/or relapse following an antitumor response to CAR-T cell therapy. In addition, after late re-expansion in vivo after the initial infusion of CAR+ T cells, CAR+ T cells are able to again demonstrate antitumor activity. This result confirms that re-expansion and activation of CAR+ T cells can be triggered in vivo and that methods for reactivating CAR+ T cells can further enhance their effectiveness.

Example 3 Administration of Anti-CD19 CAR Expressing Cells to Individuals with Relapsed and Refractory Non-Hodgkin's Lymphoma (NHL)

A. Individuals and treatment

[441] Therapeutic compositions of CAR+ T cells containing autologous T cells expressing a chimeric antigen receptor (CAR) specific for CD19 were administered to individuals with B-cell malignancies.

Example 3.A.1

[442] Example 3.A.1 describes the results of a point-in-time evaluation (3.A.1) in an ongoing clinical trial of such therapy in patients with B-cell malignancies. Specifically, a cohort (complete cohort) (currently of fifty-five (55) adults with relapsed or refractory (R/R) aggressive non-Hodgkin's lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL), de novo) was studied. or transformed from indolent lymphoma (NOS), primary mediastinal large B-cell lymphoma (PMBCL), and grade 3b follicular lymphoma (FLG3B) after failure of 2 lines of therapy). Subjects included those with Eastern Cooperative Oncology Group (ECOG) scores of 0 to 2 (median follow-up 3.2 months). The complete cohort did not include individuals with mantle cell lymphoma (MCL). Individuals were not excluded based on prior allogeneic stem cell transplantation (SCT), secondary central nervous system (CNS) involvement, and ECOG score 2, and there was no minimum required absolute lymphocyte count (ALC) for apheresis.

[443] Outcomes were assessed separately for the main subgroup from the complete cohort (individuals in the complete cohort excluding those with poor performance (ECOG 2), DLBCL transformed from marginal zone lymphoma (MZL), and/or chronic lymphocytic leukemia (CLL, Richter ) (main cohort)). At the time point assessed in Example 3.A.1, outcomes for 44 individuals in the core cohort were assessed separately.

[444] The demographics and baseline characteristics of individuals in the complete and core cohorts, estimated at the time point in accordance with Example 3.A.1, are presented in Table 6.

Table 6 Demographic and Baseline Characteristics Characteristic Complete
N=55 Main
N=44 Median age, years (range) 61(29-82) 61(29-82) ≥ 65 years, n (%) 22 (40) 17 (39) Male/female, n (%) 38/17 (69/31) 28/16 (64/36) Number of months after diagnosis, median (range) 17 (3-259) 20 (8-259) Subtype B-NHL, n (%) DLBCL, NOS 40 (73) 35 (80) Transformed DLBCL 14 (26) 8 (18) Follicular Grade 3B 12) 12) Molecular subtype, n (%) Subtype "Double/triple hit" 15 (27) 12 (27) Subtype "Double expressor" 6 (11) 4 (9) Patient characteristics, n (%) Chemorefractory ^† 42 (76) 34 (77) ECOG 0-1 48 (87) 44 (100) ECOG2 7 (13) 0 Previous lines of therapy, median (range) 3 (1-11) 3 (1-8) < 5 lines of therapy 44 (80) 37 (84) Any HSCT 27 (49) 22 (50) allogeneic 4(7) 3 (7) autologous 24 (44) 20 (45) ^* SD or PD on last chemotherapy regimen or relapse <12 months after autologous SCT

[445] The administered T cell therapeutic compositions were prepared by a method comprising immunoaffinity enrichment of CD4+ and CD8+ cells from leukapheresis-derived samples from individual treated individuals. The isolated CD4+ and CD8+ T cells were activated and transduced with a viral vector encoding anti-CD19 CAR, followed by expansion and cryopreservation of the modified cell populations. The CAR contained an anti-CD19 scFv, mouse antibody-derived, immunoglobulin-derived spacer, CD28-derived transmembrane domain, 4-1BB-derived costimulatory region, and CD3-zeta intracellular signaling domain.

[446] Cryopreserved cell compositions were thawed prior to intravenous administration. The dose of therapeutic T cells was administered as a formulated cell composition by administering a pooled population of CD4+ CAR+ cells and a pooled population of CD8+ CAR+ cells administered in a predetermined ratio of approximately 1:1. Individuals were administered a single or double dose of CAR-expressing T cells (each single dose via separate infusions of CD4+ CAR-expressing T cells and CD8+ CAR-expressing T cells, respectively) as follows: single dose according to Dose Level 1 (DL -1) containing 5×10 ⁷ total CAR-expressing T cells (n=30 for individuals assessed according to example 3.A.1), a double dose according to DL1, where each dose was administered at an interval of approximately fourteen (14) days (n=6 for subjects evaluated according to Example 3.A.1, including one subject who accidentally received two doses of DL2 in a two-dose regimen due to dosing error), or a single dose according to Dose Level 2 (DL-2) containing 1×10 ⁸ (DL-2) of all CAR-expressing T cells (n=18 for individuals assessed according to Example 3.A.1 ). Beginning three (3) days prior to CAR+ T cell infusion, subjects received lymphodepletion chemotherapy with fludarabine (flu, 30 mg/m ² ) and cyclophosphamide (Cy, 300 mg/m ² ).

Example 3.A.2

[447] For example 3.A.2, an analysis of the results at a subsequent time point in the clinical trial described in this example 3 above was performed. At the time of this analysis, 74 patients (51 men, 23 women) were treated. Subjects included sixty-nine (69) individuals in the complete DLBCL cohort (including 67 DLBCL NOS (45 de novo, 14 cases transformed from FL, 8 cases transformed from CLL or MZL), 1 grade 3B FL, 1 PMBCL); and 5 individuals in the MCL cohort. Among individuals in the complete cohort (DLBCL), median age was 61 years (range 26.82), median number of previous treatments was 3 (range 1.12), 46 (67%) were chemo-refractory, 32 (46%) had any or a previous transplant and at least 16 (23%) patients had double/triple hit lymphoma. Forty-nine (49) individuals in the core cohort were evaluated at this time point in accordance with 3.A.2.

B. Security

[448] Evaluated the presence or absence of post-treatment adverse events (TEAE) during CAR-T cell therapy. Individuals were also assessed and monitored for neurotoxicity (neurological complications including symptoms of confusion, aphasia, encephalopathy, myoclonic seizures, spasms, lethargy, and/or altered mental state), assessed on a scale of 1-5, according to the National Cancer Institute scale. -Common Toxicity Criteria (CTCAE) version 4.03 (NCI-CTCAE v4.03). See Common Terminology for Adverse Events (CTCAE) Version 4, U.S. Department of Health and Human Services, Published: May 28, 2009 (v4.03: June 14, 2010); and Guido Cavaletti & Paola Marmiroli Nature Reviews Neurology 6, 657-666 (December 2010). Also cytokine release syndrome (CRS) was determined and monitored for severity. See Lee et al, Blood. 2014;124(2):188-95.

Example 3.B.1

[449] Example 3.B.1 describes results based on the analysis time point in accordance with Example 3.A.1.

[450] Figure 3 shows the percentage of such individuals who had laboratory abnormalities and TEAE that occurred in ≥20% of individuals. In addition to TEAE shown in Figure 3, the following events were observed in ≥5% of Grade 3-4 patients: decreased white blood cell count (13.6%), encephalopathy (12%), hypertension (7%). The degree of toxicity observed was comparable between dose levels 1 and 2.

[451] 84% of individuals from the complete cohort in the analysis according to 3.B.1 did not experience severe (grade 3 or higher) cytokine release syndrome (CRS) and severe neurotoxicity. In addition, it was found that 60% of individuals in the full cohort did not develop any CRS or neurotoxicity. No differences were observed in the incidence of CRS, neurotoxicity (NT), sCRS, or severe neurotoxicity (sNT) between dose levels. Table 7 summarizes the incidence of adverse events in the form of cytokine release syndrome (CRS) and neurotoxicity in patients 28 days after administration of at least one dose of CAR-T cells. As shown in Table 7, sCRS (grade 3-4) was not observed in any of the individuals who received a single dose of DL2 or a double dose of DL1. Severe neurotoxicity or severe CRS (grade 3-4) was observed in 16% (9/55) of individuals in the complete cohort and in 18% (8/44) of individuals in the main subgroup. 11% (n=6) of individuals received tocilizumab, 24% (n=13) of individuals received dexamethasone. Among individuals in the ECOG2 full cohort, the observed rates of CRS and neurotoxicity were 71% and 29%, respectively.

Table 7. Assessment of the presence or absence of adverse events in the form of CRS and neurotoxicity for example 3.B.1 COMPLETE MAIN All dose levels DL1S DL2S DL1D ^† Safety, N 55 thirty 19 6 44 sCRS or sNT, n (%) 9 (16) 6 (20) 2 (11) 1 (17) 8 (18) CRS or NT, n (%) 22 (40) 12 (40) 7 (37) 3 (50) 15 (34) CRS Grade 1-2, n (%) 18 (33) 10 (33) 5 (26) 3 (50) 12 (27) Grade 3-4, n (%) 12) 13) 0 0 12) Neurotoxicity Grade 1-2, n (%) 3 (6) 13) 2 (11) 0 2(5) Grade 3-4, n (%) 9 (16) 6 (20) 2 (11) 1 (17) 8 (18) ^† includes one patient treated with a 2-dose schedule according to DL2 due to dosing error

[452] Figure 4 is a Kaplan-Meier curve depicting the observed time to onset of CRS and/or neurotoxicity for Example 3.B.1. As shown, the observed median time to onset of CRS and to onset of neurotoxicity was 5 and 11 days, respectively, with only 11% of patients having CRS onset less than 72 hours after starting cell therapy. The median time to resolution of CRS and neurotoxicity of grade 1 or better was 5 and 7 days, respectively. The median time to complete resolution of CRS and neurotoxicity was 5 and 11 days, respectively. The results were consistent with the conclusion that the rate of early onset of any CRS or neurotoxicity in individuals was low.

Example 3.B.2

[453] Example 3.B.2 describes the evaluation at a point in time according to Example 3.B.2. Up to this point in time, data on adverse events (AE) was collected from lymphodepletion (LD) up to 90 days after the introduction of CAR-expressing T cells. At the second time point, 69 individuals in the DLBCL cohort (complete cohort) were assessed for safety: 38 who were administered a single dose according to DL1, 25 who were administered a single dose according to DL2, and 6 who were administered a double dose according to DL1. The most common TEAEs other than CRS or NT included neutropenia (41%, 28/69), fatigue (30%, 21/69), thrombocytopenia (30%, 21/69) and anemia (26%, 18/69) . One grade 5 TEAE was observed, which was a diffuse alveolar lesion.

[454] No acute infusion toxicity was observed in the full cohort and it was observed that the majority of individuals, 64% (44/69), did not have CRS or NT, indicating that outpatient delivery of CAR-expressing T cells may be possible. Levels of CAR T cell-associated toxicity, including CRS and NT, did not differ between dose levels. The safety profile was observed to be similar across cohorts and dose levels. Among the 25 individuals in the full cohort (36%) who had CRS or NT of any degree, 21 (30%) had CRS and 14 (20%) had NT. None of the individuals had CRS grade 3 and only one (1%, 1/69) had CRS grade 4 and required intensive care care; the other 29% (20/69) had grade 1-2 CRS. Of the 20% of individuals with NT, 6% (4/69) had grade 1-2 and 14% (10/69) had grade 3-4; 2 (3%) had seizures. No grade 5 CRS or grade 5 NT was observed. No cases of cerebral edema were observed. All cases of CRS and NT resolved except for one case of grade 1 tremor, which was ongoing at the time of analysis. The median time to onset of first CRS and NT was 5 days (range 2, 12) and 10 days (range 5, 23), respectively. In the first 72 hours after the infusion, no NT was observed in individuals, and only 10% (7/69) experienced CRS (in all cases grade 1); NT preceded CRS in >70% of individuals. Overall, thirteen (13) individuals (19%) required intervention for CRS or NT with anti-cytokine therapy (tocilizumab alone 1 (1%), dexamethasone alone 6 (9%), or both 6 (9%)) and only one required some kind of support with a vasoconstrictor. The median doses of tocilizumab and dexamethasone were 1 and 6, respectively. The median duration of CRS and NT was 5 days and 11 days, respectively. The core cohort analysis (n=49) also showed similar rates of CRS and NT.

[455] In this evaluation, there was a low incidence and late onset of CRS and/or NT at both dose levels, which confirms the feasibility of outpatient infusion, for example, with a hospital visit at the first sign of fever or for fever lasting longer than a certain period of time. No grade 5 CRS or grade 5 NT was observed, and all cases of severe CRS and severe NT resolved. In addition, approximately 2 out of 3 patients did not have CRS or NT, which confirms that the cells can be administered on an outpatient basis. At the time of evaluation in accordance with 3.B.2, four individuals were treated on an outpatient basis. In addition, there were no significant differences in toxicity between individuals administered according to DL1 or DL2, indicating that higher response rates were achieved without increasing the risk of toxicity or safety concerns.

C. Response outcomes after treatment

[456] Individuals were monitored for response, including by assessing tumor burden at 1, 3, 6, 7, 12, 18, and 24 months after administration of CAR+ T cells.

Example 3.C.1

[457] Response rates are shown in Table 8. High sustained response rates were observed in this cohort of individuals, which included individuals with previous treatment failure or poor prognosis and/or relapsed or refractory disease. For individuals across all doses in the main (n=44) cohort, the observed overall response rate (ORR) was 86% and the observed complete response rate (CR) was 59%. At three months, the core cohort had an overall response rate (ORR) of 66%; the CR rate at three months was 50% in the main cohort. In the core cohort, the ORR at 3 months was 58% (11/19) at dose level 1 and 78% at dose level 2; the CR at 3 months was 42% (8/19) for dose level 1 and 56% (5/9) for dose level 2, consistent with the estimated effect of dose response on treatment outcome. In addition, the results were consistent with the relationship between dose and duration of response.

Table 8. Answer Complete Main All dose levels DL1S DL2S DL1D ^c All dose levels DL1S DL2S DL1D ^c Best overall answer, N ^a 54 thirty eighteen 6 44 25 fifteen four ORR, % (95% CI) 76 (62, 87) 80 (61, 92) 72 (47, 90) 67 (23, 96) 86 (73, 95) 84 (64, 95) 87 (60, 98) 100 (40, 100) CR% (95% CI) 52 (38, 66) 53 (34, 72) 50 (26, 74) 50 (12, 88) 59 (43, 74) 56 (35, 76) 60 (32, 84) 75 (19, 99) ≥ 3 months, f/u, ^nb 41 24 eleven 6 32 19 9 four ORR at 3 months,% (95% CI) 51 (35, 67) 46 (26, 67) 64 (31, 89) 50 (12, 88) 66 (47, 81) 58 (34, 80) 78 (40, 97) 75 (19, 99) CR at 3 months, % (95% CI) 39 (24, 56) 33 (16, 55) 46 (17, 77) 50 (12, 88) 50 (32, 68) 42 (20, 67) 56 (21, 86) 75 (19, 99) DL1S: DL1 1-dose regimen; DL2S: DL2 1-dose regimen; DL1D: 2-dose DL1 regimen;
^a Includes patients with PD events, death, or restaging imaging at 28 days. Excluded treated patients <28 days prior to imaging.
^b The denominator is the number of patients who received CAR T cell therapy ≥3 months prior to the date of acquisition of imaging data, with efficacy assessed at 3 months or until assessment of PD or death.
^c Included one patient treated with a 2-dose DL2 schedule due to dosing error

[459] Overall response rates among various subgroups of individuals in the full and core cohort are presented in Figures 5A and 5B, respectively. In low-risk DLBCL subgroups, response rates were mostly high. An ORR greater than 50% was observed after 3 months in patients with the double/triple hit molecular subtype who had primary refractory or chemorefractory DLBCL or who had never previously achieved CR. Complete resolution of CNS involvement in lymphoma was observed in 2 patients.

[460] Among individuals who were treated six months or more prior to a specific time point, of ten (10) patients who had a response after three months, 9 (90%) maintained a response after six months. At the time point of assessment, 97% of the individuals in the main subgroup who responded were alive and at follow-up with a median follow-up time of 3.2 months.

[461] Results for duration of response and overall survival (grouped by best overall response (non-responders, CR/PR, CR and/or PR)) are presented for the complete and core cohorts of individuals in FIGS. 6A and 6B, respectively. As shown, long-term survival was observed in responders with an increase in the duration of response in subjects with CR. All patients responding at three months remained alive at the time of evaluation, although 5/6 of the individuals with low efficacy status (ECOG 2) died.

Example 3.C.2

[462] Example 3.C.2 describes the results based on the analysis time point in accordance with 3.A.2 and 3.B.2.

[463] Up to the time point in accordance with example 3.C.2, 68 individuals in the complete DLBCL cohort were evaluated for response. Overall or objective response (OR), objective response rates at 3 months and 6 months were 75% (51/68), 49% (27/55) and 40% (14/35), respectively. The complete response rate (CR), CR rate at 3 months, and CR rate at 6 months were 56% (38/68), 40% (22/55), and 37% (13/35), respectively. A trend towards an increased response rate at 3 months was observed in individuals treated according to DL2 compared to DL1: 63% (12/19; 95% CI 38, 84) vs. 40% (12/30; 95% CI 23 , 59) for ORR, with p=0.148, and 58% (11/19; 95% CI 34.80) vs. 27% (8/30; 95% CI: 12.46) for CR, with p=0. 0385. Among 16 individuals with double/triple hit lymphoma, the ORR was 81% and the CR at 3 months was 60%.

[464] In the main cohort (n=49 for time point according to Example 1.C.2), OR, OR at 3 months, and OR at 6 months were 84% (41/49), 65% (26/ 40) and 57% (13/23), respectively. CR score, CR score at 3 months, and CR score at 6 months were 61% (30/49), 53% (21/40), and 52% (12/23), respectively. At higher doses, a similar trend was observed for improved long-term ORR and CR at 3 months. Specifically, for patients in the core cohort administered according to DL2, ORR at 3 months was 80% (12/15; 95% CI 52, 96) and CR at 3 months was 73% (11/15; 95% CI 45.92), compared with ORR and CR at 3 months 52% (11/21; 95% CI 30.74) and 33% (7/21; 95% CI 15.57) in individuals in the core cohort treated with DL1, with p=0.159 and p=0.0409, respectively. Among individuals in the core cohort administered according to DL2 and followed up for 3 months (n=15), the ORR at 3 months was 80% and the CR at 3 months was 73%.

[465] The median DOR in the full cohort and core cohort at this time point according to 1.C.2 was 5.0 and 9.2 months, respectively; the median duration of CR was 9.2 months in the entire cohort. This median CR duration was not achieved in the core cohort. The median overall survival (OS) was 13.7 months in the complete cohort and was not achieved in the core cohort. OS at 6 months was 75% in the full cohort with a median follow-up of 5.8 months. OS at 6 months was 88% in the main cohort with a median follow-up of 5.6 months.

D. Assessment of CAR ⁺ T cells in the blood

[466] Based on the data for the time point described in examples 3.A.1, 3.B.1 and 3.C.1, a pharmacokinetic analysis was performed to estimate the amounts of CAR ⁺ T cells in peripheral blood at various time points after treatment. Results were analyzed for fifty-five (55) individuals assessed at time point according to Example 3.A.1 in the DLBCL cohort and four (4) individuals (assessed at the same time point) for mantle cell lymphoma (MCL), described in example 4 below. Pharmacokinetic (PK) determinations were performed using validated flow cytometry to detect a marker expressed in the CAR construct and PCR-based quantification to detect insertion of the CAR construct. B cell aplasia was assessed by flow cytometry using anti-CD19 antibodies. As shown in FIG. 7A, CD4+ and CD8+ CAR-expressing cells, as measured by cell count/µl blood (median ± quartiles) plotted on a logarithmic scale, were detected during evaluation at both dose levels. Individuals administered according to DL2 relative to DL1 had higher median _Cmax and median AUC _0-28 for CD3 ⁺ /CAR ⁺ , CD4 ⁺ /CAR ⁺ , and CD8 ⁺ /CAR ⁺ T cell subgroups in peripheral blood ( AUC _0-28 : for _DL2 vs. DL1 was 1836 vs. 461, 350 vs. 182, and 1628 vs. 114, for CD3 ⁺ , CD4 ⁺ , and CD8 ⁺ , respectively; p<0.05 for CD8 ⁺ ; DL1 was 99.8 vs. 27.9, 15.1 vs. 5.2, and 73.1 vs. 5.5 cells/µl, respectively). The median time to maximum expansion of CD3 ⁺ CAR ⁺ T cells was 15 days (range 8-29) and did not differ between dose levels. CD4 ⁺ and CD8 ⁺ CAR-expressing T cells settled in the bone marrow at relatively similar levels.

[467] Increased median area under the curve (AUC) (the number of CD8+ CAR+ cells in the blood over time) was observed for individuals administered at a higher dose level compared to a lower dose level without an observed increase in toxicity. Higher maximum exposure of CD8 ⁺ /CAR ⁺ T cells was observed in responders (CR/PR) than non-responders (PD); the presence of cells at the time of assessment, including 3 and 6 months, was observed even in individuals in whom the disease had progressed (FIG. 7B). The median C _max and median AUC _0-28 for CD8 ⁺ CAR ⁺ T cells were higher in responders and in those with sustained response at 3 months (median CD8 ⁺ C _max = 20.8 vs 5.5; median CD8 ⁺ AUC _0-28 = 235 vs 55 in CR/PR group at 3 months vs PD at 3 months). Among individuals assessed for CAR T cell persistence, 90% and 93% of 29 individuals had detectable CD8 ⁺ and CD4 ⁺ CAR ⁺ T cells, respectively, at 3 months; 63% and 58% of 19 individuals had detectable CD8 ⁺ and CD4 ⁺ CAR ⁺ T cells, respectively, at 6 months. After 3 and 6 months, there were no statistically significant differences in CAR ⁺ T cell persistence between individuals with sustained response or relapse. CAR ⁺ T cells were detectable at the time of relapse in 89% and 11 individuals with PK, even though B cell aplasia (<1 cell/µl) was demonstrated in virtually all individuals, 97% (34/35) , after 3 months, and in 100% (24/24) after 6 months.

[468] There was no association of higher C _max and AUC _0-28 in the case of DL2 compared to DL1 with an increased frequency of CRS or NT. For any NT or for CRS grade > 2, the median AUC for CD4 ⁺ /CAR ⁺ and CD8 ⁺ /CAR ⁺ T cells was 5-10-fold and 3-5-fold higher, respectively, than the median AUC for DL2. Higher disease burden and baseline inflammatory cytokine levels were associated with higher peak CAR ⁺ T cell levels, higher peak cytokine levels, and higher incidence of CRS and NT. The results were consistent with the conclusion that higher Cmax and median AUC0-28 in the case of DL2 did not increase the incidence of CRS or NT.

[469] The results were consistent with the conclusion that treatment resulted in long-term exposure and persistence of engineered cells, even in individuals with poor response. In some embodiments, combined approaches are used, such as administering an immune checkpoint modulator or other modulating agent, e.g., after disease recurrence or progression, at a point in time when the modified cells are present in the individual, e.g., as determined by peripheral blood cell levels. In some aspects, cells present for an extended period are re-expanded or activated and/or exhibit antitumor function after administration of another agent or other treatment. Generally, higher median counts of CD4+ and CD8+ CAR+ T cells were observed over time in the blood of individuals who developed (FIG. 7C). The results showed that CAR ⁺ T cells showed expansion and persistence, a duration of response after 3 months that was higher at higher dose levels, without increasing toxicity. The results were found to be consistent with the suggestion that high peak levels of CAR ⁺ T cells and cytokines in the blood may be associated with NT and CRS, and may be influenced by baseline factor levels in the individual. It was found that CAR ⁺ T cells were present at the time of relapse, indicating that combination and retreatment approaches may provide certain benefits.

D. Analyzed Blood Molecules and Neurotoxicity, CRS and Response

[470] Various analyzed molecules in the pre-treatment blood, including cytokines, were quantified in the serum of individuals (individuals evaluated at the time point in accordance with example 3.A.1) before the introduction of CAR+ T cells. Quantitative determination of cytokines was performed using multiplex analysis of cytokines. The potential correlation with the risk of developing neurotoxicity was assessed using statistical analysis based on one-way non-parametric criteria.

[471] FIG. 8 shows median levels of assessed analytes in units of (LDH, U/L; ferritin, ng/mL; CRP, mg/L; cytokines, pg/mL) in individuals who did not develop neurotoxicity vs. individuals who developed neurotoxicity after CAR+ T cell therapy. Blood levels of certain analyzed molecules, including LDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1, and MIP-1β, have been found to be associated with risk level. development of neurotoxicity (Wilcoxon p-values <0.05, adjusted for multiple comparisons). In particular, the results were consistent with the conclusion that pre-treatment LDH levels, which in some embodiments replace disease burden, may be useful in assessing the risk of potential neurotoxicity and/or risk-adapted dosing or adjustment of treatment in certain individuals. In addition, tumor burden measured prior to administration of the CAR-T cell composition correlated (Spearman's p-values <0.05) with the risk of developing neurotoxicity. In some aspects, LDH levels may be assessed alone and/or in combination with another pre-treatment parameter, such as another measure or indicator of tumor burden, such as a tumor volumetric measure, such as the sum of products of dimensions (SPD) or other volumetric measures of disease burden based on CT or MRI. In some aspects, one or more parameters are assessed indicative of disease burden, and in some contexts, they may indicate the presence, absence, or risk of developing neurotoxicity following T cell therapy. In some aspects, one or more parameters include LDH and/or tumor volumetric index.

[472] In additional analyses, fifty-five (55) individuals in the DLBCL cohort at time point according to Example 3.A.1 and four (4) individuals with mantle cell lymphoma (MCL) described in Example 4 were included in the analysis to assess correlation with safety. Of the 59 individuals evaluated for safety, CRS was observed in 32% (30% grade 1-2, 0% grade 3, 2% grade 4); NT was observed in 20% (5% grade 1-2, 10% grade 3, 5% grade 4). The dose level did not correlate with CRS or NT (p=0.565 and p=1.00, respectively). Individual factors that correlate with any degree of CRS and NT were a worse predictor of overall health status (e.g., ECOG status 2) (p=0.03) and higher disease burden (p<0.05) when summed products of diameters (SPD) based on imaging results. It was observed that pre-CAR+ T cell infusion clinical laboratory parameters and pre-CAR+ T cell infusion cytokine values associated with the onset of any grade of NT included higher serum levels of LDH, ferritin, and CRP, and higher plasma levels of IL- 6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1 and MIP-1β (p<0.05 for each). Higher pre-infusion plasma levels of CAR+ T cells IL-8, IL-10 and CXCL10 were also associated with grade 3-4 NT (p<0.05 for each).

[473] Of the 54 individuals in the DLBCL cohort assessed for response, higher ECOG and DLBCL transformed from CLL or MZL correlated with lower long-term response at 3 months (p=0.02 for both). Pre-CAR+ T cell infusion parameters associated with superior ORR included lower levels of ferritin, LDH, CXCL10, G-CSF, and IL-10, and parameters associated with sustained response at 3 months included lower levels of ferritin, CRP, LDH, CXCL10, IL-8, IL-10, IL-15, MCP-1, MIP-1β, TNF-α, and higher hemoglobin and albumin before CAR+ T cell infusion (p<0.05 for each).

[474] In some cases, the sample for apheresis and the composition of CAR+ T cells for injection were evaluated and compared with clinical outcomes. The results indicated that memory T cell subsets and T cell functionality may be correlated with certain clinical outcomes.

[475] The results indicated that certain baseline patient characteristics, including inflammatory status and high pretreatment tumor burden, may be useful in identifying patients at risk of increased toxicity following administration of CAR-expressing T cells. It has been found that low tumor burden and low inflammatory status are associated with an improved toxicity profile and a longer duration of response. The results confirm that treating individuals earlier in the course of therapy and/or evaluating a panel of clinical and laboratory risk biomarkers that determine potential earlier intervention in individuals may reduce the risk of toxicity and increase duration of response.

[476] FIG. 9 is a graph plotting time without progression (months) for individuals in the full or core cohorts. Each column corresponds to one patient. Shading indicates the best overall response (in each case, unless otherwise indicated, achieved after 1 month); the texture indicates the dose (solid=dose level 1, single dose; cross-hatched, dose level 2, single dose; vertically hatched=dose level 1, two doses). Horizontal arrows indicate an ongoing response. Some individuals were initially assessed (eg, after 1 month) as having stable disease (SD) or partial response (PR), and were later found to have achieved PR (eg, SD to PR transition) or CR. In such cases, the shading of an individual patient's bar as indicated indicates the best overall response, and the dots (same shading correspondence to achieved response) along each individual's bar indicate when each SD, PR, and/or CR occurred in the individual. Complete resolution of CNS involvement in lymphoma was observed in two patients. It was found that CAR+ cells in one individual increased in number according to the biopsy after relapse.

Example 4: Administration of anti-CD19 CAR expressing cells to an individual with mantle cell lymphoma (MCL)

[477] Therapeutic compositions of CAR+ T cells containing autologous T cells expressing a chimeric antigen receptor (CAR) specific for CD19 prepared as described in Example 1 were administered to four (4) humans with mantle cell lymphoma (MCL ), who failed first-line therapy. Cryopreserved cell compositions were thawed prior to intravenous administration. The therapeutic T cell composition was administered as a formulated cell product, incorporating pooled CD4+ and CD8+ populations of engineered CAR+ T cells originating from the same individual in a predetermined ratio of approximately 1:1. Individuals were dosed with CAR expressing T cells (as a divided dose of CD4+ and CD8+ CAR expressing T cells) in a single dose corresponding to Dose Level 1 (DL1) containing 5×10 ⁷ CAR expressing T cells. Beginning (3) days prior to CAR+ T cell infusion, lymphodepleted individuals received chemotherapy with flurabine (flu, 30 mg/m ² ) and cyclophosphamide (Cy, 300 mg/m ² ).

[478] Individuals were monitored for response and toxicity as described in Example 1. No CRS or neurotoxicity was observed in any of the individuals. Among the 4 treated individuals, two (2) individuals achieved PR (non-long term) and two (2) patients had progressive disease.

Example 5 Evaluation of Biomarkers in Tumor Biopsies from Individuals with Relapsed and Refractory Non-Hodgkin's Lymphoma (NHL) Before and After Administration of Anti-CD19 CAR Expressing Cells

[479] The expression of several biomarkers was assessed in tumor biopsies obtained from individuals before and/or after the introduction of CAR-expressing cells.

A. Tumor biopsies

[480] Tumor biopsies were obtained from selected individuals with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) or mantle cell lymphoma (MCL) who were treated with CAR ⁺ T cell therapeutic compositions containing autologous T cells expressing the CD19-specific chimeric antigen receptor (CAR) described in Examples 3 and 4 above based on the time point of evaluation according to Example 3.A.2. Tumor biopsies were obtained before the introduction of CAR ⁺ T-cells (before treatment) and 7-20 days after administration (after treatment). This example describes the results for the evaluation at the point in time in accordance with example 3.A.2 in the ongoing study. Results were examined for 43 biopsies (26 before treatment; 17 after treatment and 15 matched pairs) from a total of 28 individuals (25 DLBCL and 3 MCL).

B. Evaluation of biomarkers, response outcomes and safety

[481] Infiltration of CAR ⁺ T cells into tumor biopsy was quantified using in situ hybridization (ISH) probes specific for mRNA encoding anti-CD19 CAR. CAR ⁺ T cells, non-CAR T cells and B cells were counted using multiplex immunofluorescence (IF) assays detecting a surrogate cell surface marker for CAR expressing cells, CD4, CD8, CD19, CD20, CD73, FOXP3, CD163 , IDO and PD-L1. Tumor biopsies were stained with hematoxylin and eosin (H&E) and evaluated for tissue quality and tumor identification. Immunofluorescence images were analyzed using image analysis software. Potential correlation with response outcomes was assessed using univariate t-test statistical analysis and p-values were 2-tailed without adjustment for multiplicity.

[482] Individuals were assessed for response and safety outcomes, including by assessing tumor burden at various time points after CAR ⁺ T cell administration, including 3 months post-administration, and determining whether the individual had progressive disease (PD), stable disease (SD), partial response (PR), or complete response (CR). Safety outcomes assessed included neurotoxicity (neurological complications including symptoms of confusion, aphasia, encephalopathy, myoclonic seizures, convulsions, lethargy, and/or altered mental state) scored on a scale of 1-5, according to the National Cancer Institute-Common Scale. Toxicity Criteria (CTCAE) version 4.03 (NCI-CTCAE v4.03).

C. Results

[483] The observed objective response rate (ORR; including CR and PR) was 71% (20/28) in individuals for whom biopsies were evaluated. Grade 1, 2 CRS was observed in 36% (10/28; grade 1, 2) of individuals assessed for biopsies, and NT grades 2-4 were observed in 18% (5/28) of individuals assessed for biopsies.

[484] Pretreatment tumor biopsies were found to have a variable cellular composition: tumor cells (median: 77%; range 5-96%), CD4 ⁺ cells (0.90%; 0.02-15%), and CD8 ⁺ cells (1.5%; 0-23%). The results showed that individuals with CR or PR 3 months after CAR+ T cell administration had a higher percentage of endogenous CD4 ⁺ cells in pre-treatment tumors compared to individuals with PD (CR, PR, median: 7.9%; PD, median: 0.38%, p < 0.0001). The percentages of CD8 ⁺ cells in pre-treatment tumors did not differ between response groups at 3 months (CR, PR, median: 1.9%; PD, median: 0.47%; p=0.6496).

[485] In post-treatment biopsies, infiltration of the tumor with CAR+ T cells was observed, and they accounted for up to 22% of the cells in the biopsy. The rate of tumor infiltration in post-treatment specimens (7 to 20 days after administration) was found to be higher in individuals who then achieved a CR (median: 3.9%) or PR (median: 1.1%) compared to with individuals who then achieved a best overall response (BOR) as SD or PD (median: 0.51%). Although both CD4 ⁺ and CD8 ⁺ CAR T cells were found to infiltrate the tumor area at the time point post-treatment (7 to 20 days post-treatment), individuals who then achieved CR had a higher CD8 ⁺ CAR ⁺ ratio. T cells and CD4 ⁺ CAR ⁺ T cells at this time point post-treatment compared with individuals who then achieved BOR as SD or PD (median CR: 0.83; SD, median PD: 0.14; p= 0.0097).

[486] Comparison of outcomes for matched pre- and post-treatment biopsies from individual individuals showed a trend towards eventually achieving BOR in the form of CR or PR in case of a greater post-treatment increase in CD8 ⁺ cells (CAR ⁺ T and non-CART) in tumors. compared to individuals who achieved BOR as SD or PD (CR, PR, median change: +5.3%; SD, PD, median change: +0.06%; p=0.1225).

[487] Expression of immunosuppressive factors, including CD73, FOXP3, CD163, IDO, and PD-L1, varied among individuals before treatment (CD73 (median: 1.5%; range 0-42%), FOXP3 (0.10%; 0 -1.5%), IDO (0.06%; 0-11%), CD163 (1.2%; 0-24%) and PD-L1 (0.16%; 0-56%)) and after treatment (CD73 (1.6%; 0-53%), FOXP3 (0.09%; 0-4.3%), IDO (0.28%; 0-15%), CD163 (3.6%; 0-22%) and PD-L1 (3.3%; 0-65%)). An increase in post-treatment CD8 ⁺ cells in matched biopsies was observed to be associated with an increase in post-treatment expression of IDO (R ² =0.64) and PD-L1 (R ² =0.61). This result is consistent with the conclusion that infiltration of CD8+ CAR+ cells at the estimated time point may indicate a potential likelihood of achieving a response rate or duration of response, and that the presence and/or activity of such cells may lead to the activation of TME factors.

D. Conclusion

[488] It was found that the duration of response 3 months after the introduction of CAR+ T cells is associated with higher levels of CD4 ⁺ cells in tumors before treatment. In tumor cells, post-treatment infiltration of CAR+ T cells, both CD4 ⁺ and CD8 ⁺ , into the tumor and adjacent tissue was observed. ORR was associated with increased levels of CAR+ T cells in tumor biopsy. An increase in the levels of CD8 ⁺ cells in the tumor biopsy after treatment compared with the levels of CD8 ⁺ in the tumor biopsy before treatment was associated with an increase in the expression of IDO and PD-L1. In some embodiments, therapies targeting these cascades, such as therapies delivered at the time of administration or after administration of CAR-T cells, may improve one or more therapeutic outcomes or their duration after administration of CAR+ T cells.

Example 6: Evaluation of persistence in individuals with relapsed and refractory non-Hodgkin's lymphoma (NHL) after administration of anti-CD19 CAR expressing cells

[489] Persistence and expansion were evaluated in patients at a time point after the evaluation time point in the clinical trial described in Example 3 above.

A. Individuals, response and security

[490] The time point analysis presented in this example is based on the evaluation of a total of 91 individuals in the complete DLBCL cohort (88 (34 from the main cohort) assessed for response and 91 assessed for safety) who were administered cells, expressing CARs against CD19. As shown in Table 9, the Objective Response Rate (ORR) was 74%, including 52% of individuals who demonstrated a complete response (CR). The incidence of cytokine release syndrome (CRS) of any degree was 35% with 1% severe CRS; and the incidence of neurotoxicity (NT) of any degree was 19% with 1% severe NT.

Table 9. Response and safety after administration of CAR+ cells Complete Main All dose levels All dose levels ^a DL1S DL2S Best overall response (BOR), n ^b 88 65 34 27 ORR,% (95% CI) 74 (63.83) 80(68, 89) 77 (59.89) 82 (62, 94) CR% (95% CI) 52 (41.63) 55(43, 68) 47 (30, 65) 63 (42, 81) Security, n ^c 91 67 34 29 Any CRS, % (95% CI) 35 (25, 46) 36 (24, 48) 41 (25, 59) 24 (10, 44) sCRS(grade 3-4),% (95% CI) 1 (0, 6) 1 (0.8) 38 (0.15) 0 Any NTx,% (95% CI) 19 (11.28) 21 (12, 33) 24 (11, 41) 17 (6, 36) sNTx (grade 3-4),% (95% CI) 12 (6, 21) 15 (7, 26) 21 (9, 38) 7 (1, 23)

a Four patients treated according to DL1D (dose level 1, two-dose regimen) with similar outcomes.

b Includes patients with a PD event, death, or restaging results after 28 days. One patient did not have available restaging results.

c Includes all individuals who received at least one dose of the relevant CAR-expressing cell product in the 28 days prior to data collection or who died.

D. Persistence

[491] Persistence of CAR-expressing cells and aplasia of CD19+ B cells (low or no CD19+ B cells) were assessed at various time points in measurable DLBCL individuals injected with CAR+ T cells based on detectable CD3 ⁺ levels. , CD4 ⁺ or CD8 ⁺ CAR-expressing cells and levels of CD19 ⁺ B-cells detected in the blood, respectively. The results are shown in Table 10. Among individuals assessed at progression (n=37), a median of 0.17 CD4+ CAR-expressing cells/µl (range, 0-65.5 cells/µl) and a median of CD8+ CAR+ 0.15 cells were observed. /μl (range, 0-131.8 cells/μl) during progression. Among individuals assessed at relapse (progression after reaching CR/PR) (n=12), a median of 0.17 cells/μl (range, 0-35.1 cells/μl) of CD4+ CAR-expressing cells and a median of 0.20 cells/µl (range, 0-131.8 cells/µl) CD8+ CAR-expressing cells pre-relapsed. Long-term persistence of CAR-expressing cells was observed in 75% of assessed individuals with DLBCL after 12 months. Long-term persistence of B-cell aplasia was also observed in 75% of individuals after 12 months, regardless of relapse status. The results are consistent with the conclusion that cells expressing anti-CD19 CAR showed long-term persistence in most individuals, and show the potential for continued low-level disease control even in relapsed patients.

[492] Among individuals with relapse, 91.7% (11/12) had detectable CAR-expressing cells in the blood at the time of relapse. This result is consistent with the conclusion that a combination therapy or other intervention, in some embodiments, can be used to enhance and/or increase the efficiency of CAR-expressing cells, such as cells that may be depleted.

Table 10 Long-term persistence of CAR+ cells and aplasia of CD19 3 months 6 months 9 months 12 months With progression With a relapse Persistence of CAR T cells in evaluable patients, n fifty thirty eighteen 12 37 12 CD3 ⁺ ,% 100 80.0 77.8 75.0 91.9 91.7 CD4 ⁺ ,% 88.0 63.3 50.0 41.7 83.8 83.3 CD8 ⁺ ,% 90.0 70.0 55.6 50.0 83.8 75.0 CD19 ⁺ B cell aplasia
(< 1 cell/µl),% 96.0 93.3 77.8 75.0 97.3 100

[493] The scope of the present invention is not limited to the specific described embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications of the described compositions and methods will become apparent based on the description and ideas described in the present description. Such variations may be practiced without departing from the scope and spirit of the invention, and are intended to be within the scope of the present invention.

SEQUENCES

SEQID NO SUBSEQUENCE DESCRIPTION one ESKYGPPCPPCP Spacer (IgG4 hinge region) (a.k.)
Homo sapiens 2 GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT Spacer (IgG4 hinge region) (nucleotide)
homo sapiens 3 ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Spacer hinge area-CH3
Homo sapiens four ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Spacer Hinge-CH2-CH3
Homo sapiens 5 RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH IgD-hinge-Fc
Homo sapiens 6 LEGGGEGRGSLTCGDVEENPGPR T2A, artificial 7 MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM tEGFR, artificial eight FWVLVVVGGVLACYSLLVTVAFIFWV CD28 (amino acids 153-179 sequence accession number P10747)
Homo sapiens 9 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWV CD28 (amino acids 114-179 sequence accession number P10747)
Homo sapiens ten RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (amino acids 180-220 P10747)
Homo sapiens eleven RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (LL to GG)
Homo sapiens 12 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB (amino acids 214-255 Q07011.1)
Homo sapiens 13 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3-zeta
Homo sapiens fourteen RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3-zeta
Homo sapiens fifteen RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3-zeta
Homo sapiens 16 RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM tEGFR, artificial 17 EGRGSLLTCGDVEENPGP T2A, artificial eighteen GSGATNFSLLKQAGDVEENPGP P2A 19 ATNFSLLKQAGDVEENPGP P2A twenty QCTNYALLKLAGDVESNPGP E2A 21 VKQTLNFDLLKLAGDVESNPGP F2A 22 PGGG-(SGGGG) ₅ -P- where P is proline, G is glycine and S is serine Linker 23 GSADDAKKDAAKKDGKS Linker 24 GSTSGSGKPGSGEGSTKG Linker 25
gtgaccgtgagcagc scFv coding sequence 26 _{X1PPX2P _}
X ₁ is glycine, cysteine or arginine
X ₂ is cysteine or threonine Hinge area 27 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Hinge area 28 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Hinge area 29 ELKTPLGDTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP Hinge area thirty Glu Ser Lys Tyr Gly Pro Cys Pro Ser Cys Pro Hinge area 31 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge area 32 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge area 33 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge area 34 Glu Val Val Val Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge area 35 RASQDISKYLN CDR L1 FMC63 36 SRLHSGV CDR L2 FMC63 37 GNTLPYTFG CDR L3 FMC63 38 DYGVS CDR H1 FMC63 39 VIWGSETTYYNSALKS CDR H2 FMC63 40 YAMDYWG CDR H3 FMC63 41 EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS VH FMC63 42 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT VL FMC63 43 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS scFv FMC63 44 KASQNVGTNVA CDR L1 SJ25C1 45 SATYRNS CDR L2 SJ25C1 46 QQYNRYPYT CDR L3 SJ25C1 47 SYWMN CDR H1 SJ25C1 48 QIYPGDGDTNYNGKFKG CDR H2 SJ25C1 49 KTISSVVDFYFDY CDR H3 SJ25C1 fifty EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSS VH SJ25C1 51 DIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR VL SJ25C1 52 GGGGSGGGGSGGGGS Linker 53 EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR scFv SJ25C1 54 HYYYGGSYAMDY CDR H3 FMC63 55 HTSRLHS CDR L2 FMC63 56 QQGNTLPYT CDR L3 FMC63

--->

SEQUENCE LIST

<110> JUNO THERAPEUTICS, INC.

ALBERTSON, Tina

<120> METHODS FOR MODULATING CART CELLS

<130> 735042009140

<140> Not yet assigned

<141> Attached hereto

<150> 62/580.414

<151> 2017-11-01

<150> 62/549.391

<151> 2017-08-23

<150> 62/515.512

<151> 2017-06-05

<150> 62/514.777

<151> 2017-06-02

<150> 62/492.950

<151> 2017-05-01

<150> 62/444.784

<151> 2017-01-10

<150> 62/429.740

<151> 2016-12-03

<160> 56

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 12

<212> PROTEIN

<213> Homo sapiens

<220>

<223> Spacer (IgG4 hinge region)

<400> 1

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 2

<211> 36

<212> DNA

<213> Homo sapiens

<220>

<223> Spacer (IgG4 hinge region)

<400> 2

gaatctaagt acggaccgcc ctgcccccct tgccct 36

<210> 3

<211> 119

<212> PROTEIN

<213> Homo sapiens

<220>

<223> Spacer Hinge-CH3

<400> 3

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg

1 5 10 15

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

20 25 30

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

35 40 45

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

50 55 60

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

65 70 75 80

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

85 90 95

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

100 105 110

Leu Ser Leu Ser Leu Gly Lys

115

<210> 4

<211> 229

<212> PROTEIN

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<223> Spacer Hinge CH2-CH3

<400> 4

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 5

<211> 282

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<223> hinge region IgD-Fc

<400> 5

Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala

1 5 10 15

Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala

20 25 30

Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys

35 40 45

Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

50 55 60

Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln

65 70 75 80

Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly

85 90 95

Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val

100 105 110

Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly

115 120 125

Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn

130 135 140

Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro

145 150 155 160

Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys

165 170 175

Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser

180 185 190

Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu

195 200 205

Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro

210 215 220

Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser

225 230 235 240

Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr

245 250 255

Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg

260 265 270

Ser Leu Glu Val Ser Tyr Val Thr Asp His

275 280

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<213> Artificial sequence

<220>

<223> T2A

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Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

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Val Glu Glu Asn Pro Gly Pro Arg

twenty

<210> 7

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<212> PROTEIN

<213> Artificial sequence

<220>

<223>tEGFR

<400> 7

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala

325 330 335

Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly

340 345 350

Ile Gly Leu Phe Met

355

<210> 8

<211> 27

<212> PROTEIN

<213> Homo sapiens

<220>

<223>CD28

<300>

<308> UniProt P10747

<309> 1989-07-01

<400> 8

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 9

<211> 66

<212> PROTEIN

<213> Homo sapiens

<220>

<223>CD28

<300>

<308> UniProt P10747

<309> 1989-07-01

<400> 9

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly

35 40 45

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

50 55 60

Trp Val

65

<210> 10

<211> 41

<212> PROTEIN

<213> Homo sapiens

<220>

<223>CD28

<300>

<308> UniProt P10747

<309> 1989-07-01

<400> 10

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 11

<211> 41

<212> PROTEIN

<213> Homo sapiens

<220>

<223>CD28

<400> 11

Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 12

<211> 42

<212> PROTEIN

<213> Homo sapiens

<220>

<223> 4-1BB

<300>

<308> UniProt Q07011.1

<309> 1995-02-01

<400> 12

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 13

<211> 112

<212> PROTEIN

<213> Homo sapiens

<220>

<223> CD3-zeta

<400> 13

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 14

<211> 112

<212> PROTEIN

<213> Homo sapiens

<220>

<223> CD3-zeta

<400> 14

Arg Val Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 15

<211> 112

<212> PROTEIN

<213> Homo sapiens

<220>

<223> CD3-zeta

<400> 15

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 16

<211> 335

<212> PROTEIN

<213> Artificial sequence

<220>

<223>tEGFR

<400> 16

Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu

1 5 10 15

Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile

20 25 30

Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe

35 40 45

Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr

50 55 60

Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn

65 70 75 80

Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg

85 90 95

Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile

100 105 110

Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val

115 120 125

Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp

130 135 140

Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn

145 150 155 160

Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu

165 170 175

Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser

180 185 190

Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu

195 200 205

Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln

210 215 220

Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly

225 230 235 240

Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro

245 250 255

His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr

260 265 270

Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His

275 280 285

Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro

290 295 300

Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala

305 310 315 320

Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met

325 330 335

<210> 17

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> T2A

<400> 17

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

GlyPro

<210> 18

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<223> P2A

<400> 18

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

twenty

<210> 19

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> P2A

<400> 19

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 20

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223>E2A

<400> 20

Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

1 5 10 15

Asn Pro Gly Pro

twenty

<210> 21

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<223> F2A

<400> 21

Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val

1 5 10 15

Glu Ser Asn Pro Gly Pro

twenty

<210> 22

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<220>

<221> REPEAT

<222> (5)...(9)

<223> SGGGG repeated 5 times

<400> 22

Pro Gly Gly Gly Ser Gly Gly Gly Gly Pro

1 5 10

<210> 23

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 23

Gly Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Gly Lys

1 5 10 15

Ser

<210> 24

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 24

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210> 25

<211> 735

<212> DNA

<213> Artificial sequence

<220>

<223>scFv

<400> 25

gacatccaga tgacccagac cacctccagc ctgagcgcca gcctgggcga ccgggtgacc 60

atcagctgcc gggccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

gacggcaccg tcaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc 180

cggtttagcg gcagcggctc cggcaccgac tacagcctga ccatctccaa cctggaacag 240

gaagatatcg ccacctactt ttgccagcag ggcaacacac tgccctacac ctttggcggc 300

ggaacaaagc tggaaatcac cggcagcacc tccggcagcg gcaagcctgg cagcggcgag 360

ggcagcacca agggcgaggt gaagctgcag gaaagcggcc ctggcctggt ggcccccagc 420

cagagcctga gcgtgacctg caccgtgagc ggcgtgagcc tgcccgacta cggcgtgagc 480

tggatccggc agccccccag gaagggcctg gaatggctgg gcgtgatctg gggcagcgag 540

accacctact acaacagcgc cctgaagagc cggctgacca tcatcaagga caacagcaag 600

agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc 660

gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 720

gtgaccgtga gcagc 735

<210> 26

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge area

<220>

<221> OPTION

<222> (1)...(1)

<223> Xaa1 is glycine, cysteine or arginine

<220>

<221> OPTION

<222> (4)...(4)

<223> Xaa4 is cysteine or threonine

<400> 26

Xaa Pro Pro Xaa Pro

fifteen

<210> 27

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge region

<400> 27

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 28

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge area

<400> 28

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

1 5 10

<210> 29

<211> 61

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge area

<400> 29

Glu Leu Lys Thr Pro Leu Gly Asp Thr His Thr Cys Pro Arg Cys Pro

1 5 10 15

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu

20 25 30

Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro

35 40 45

Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

50 55 60

<210> 30

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge area

<400> 30

Glu Ser Lys Tyr Gly Pro Cys Pro Ser Cys Pro

1 5 10

<210> 31

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge area

<400> 31

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 32

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge area

<400> 32

Tyr Gly Pro Pro Cys Pro Pro Cys Pro

fifteen

<210> 33

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge area

<400> 33

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 34

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge region

<400> 34

Glu Val Val Val Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 35

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR L1 FMC63

<400> 35

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 36

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR L2 FMC63

<400> 36

Ser Arg Leu His Ser Gly Val

fifteen

<210> 37

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR L3 FMC63

<400> 37

Gly Asn Thr Leu Pro Tyr Thr Phe Gly

fifteen

<210> 38

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR H1 FMC63

<400> 38

Asp Tyr Gly Val Ser

fifteen

<210> 39

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR H2 FMC63

<400> 39

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 40

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR H3 FMC63

<400> 40

Tyr Ala Met Asp Tyr Trp Gly

fifteen

<210> 41

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH FMC63

<400> 41

Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 42

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL FMC63

<400> 42

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

100 105

<210> 43

<211> 245

<212> PROTEIN

<213> Artificial sequence

<220>

<223> scFv FMC63

<400> 43

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser

245

<210> 44

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR L1 SJ25C1

<400> 44

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala

1 5 10

<210> 45

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR L2 SJ25C1

<400> 45

Ser Ala Thr Tyr Arg Asn Ser

fifteen

<210> 46

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR L3 SJ25C1

<400> 46

Gln Gln Tyr Asn Arg Tyr Pro Tyr Thr

fifteen

<210> 47

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR H1 SJ25C1

<400> 47

Ser Tyr Trp Met Asn

fifteen

<210> 48

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR H2 SJ25C1

<400> 48

Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys

1 5 10 15

gly

<210> 49

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR H3 SJ25C1

<400> 49

Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr

1 5 10

<210> 50

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH SJ25C1

<400> 50

Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 51

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL SJ25C1

<400> 51

Asp Ile Glu Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Thr Tyr Arg Asn Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser

65 70 75 80

Lys Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr

85 90 95

Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 52

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker

<400> 52

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 53

<211> 245

<212> PROTEIN

<213> Artificial sequence

<220>

<223> scFv SJ25C1

<400> 53

Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp

100 105 110

Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser

130 135 140

Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys

145 150 155 160

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr Ser Ala Thr Tyr Arg Asn

180 185 190

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Thr Asn Val Gln Ser Lys Asp Leu Ala Asp Tyr Phe

210 215 220

Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr Thr Ser Gly Gly Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys Arg

245

<210> 54

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR H3 FMC63

<400> 54

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 55

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR L2 FMC63

<400> 55

His Thr Ser Arg Leu His Ser

fifteen

<210> 56

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR L3 FMC63

<400> 56

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

fifteen

<---

Claims (65)

1. A method for expanding engineered cells comprising one or both of administering an immune checkpoint inhibitor and biopsy to an individual having a tumor or cancer, said individual having previously received the engineered cells to treat said tumor or cancer within 12 months or less before one or both of the administration of the immune checkpoint inhibitor and the biopsy, wherein said engineered cells contain T cells and said engineered cells express the recombinant receptor. 2. The method of claim 1, wherein the method does not include subsequent administration of the genetically engineered cells and/or expansion is achieved without subsequent administration of the genetically engineered cells. 3. The method according to claim 1 or 2, wherein in the lesion or part thereof, the engineered cells are present, or are likely to be present, or were present, or were likely to be present. 4. The method according to any one of claims 1 to 3, wherein the lesion is a tumor. 5. The method of claim 4 wherein the tumor is a primary or secondary tumor. 6. The method according to any one of claims 1 to 5, wherein the lesion is or contains bone marrow tissue. 7. The method according to any one of claims 1, 2 and 4-6, wherein at or just before the time of one or both of the administration of the immune checkpoint inhibitor and the biopsy, the subject relapsed after responding to the genetically engineered cells, optionally after remission, and/or he did not respond to the introduction of genetically engineered cells. 8. The method according to any one of claims 1 to 7, wherein the subject has relapsed after response and/or has not responded to prior administration of the genetically engineered cells. 9. The method of any one of claims 1 to 7, wherein the subject responded to the genetically engineered cells and then stopped responding and/or relapsed prior to either or both of the immune checkpoint inhibitor administration and biopsy. 10. The method of any one of claims 1 to 9, wherein the engineered cells have previously expanded in the subject or found to have expanded prior to either or both of the immune checkpoint inhibitor administration and biopsy. 11. The method according to any one of claims 1 to 10, wherein at or shortly before the time of one or both of the administration of the immune control point inhibitor and the biopsy: the individual is in remission; the number of genetically engineered cells detectable in the blood is reduced or undetectable; the number of genetically engineered cells found in a fluid, or tissue, or sample, optionally blood, of an individual is reduced compared to a previous point in time after the administration of the genetically engineered cells; and/or the number of genetically engineered cells found in a fluid or tissue or sample, optionally blood, of an individual is reduced by or more than 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5 .0 times, 10 times or more compared to the peak or maximum number of genetically engineered cells detected or found in the blood of an individual after the start of administration of genetically engineered cells and / or compared to the level at a time point within 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or 28 days after the introduction of genetically engineered cells. 12. The method according to any one of claims 1 to 11, wherein one or both of the administration of the immune control point inhibitor and the biopsy are carried out after or more than 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months , 1 year or more after the start of the administration of genetically engineered cells or after the last dose of genetically engineered cells. 13. The method of any one of claims 1-12, wherein either or both of administration of the immune checkpoint inhibitor and biopsy directly or indirectly modulate the activity or function of the engineered T cells in vivo in the subject. 14. The method of any one of claims 1-13, wherein said immune checkpoint inhibitor inhibits or blocks an immune checkpoint molecule or cascade that is or includes PD-1, PD-L1, PD-L2, CTLA-4, LAG -3, TIM3, VISTA, adenosine receptor, CD73, CD39, adenosine 2A receptor (A2AR), or adenosine. 15. The method according to any one of claims 1-14, wherein said immune control point inhibitor is selected from the group consisting of BY55, daclizumab, bevacizumab, basiliximab, ipilimumab, nivolumab, pembrolizumab, MPDL3280A, pidilizumab, MK-3475, BMS-936559, atezolizumab, tremelimumab, IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, MK-4166, dacetuzumab, lucatumumab, SEA-CD40, CP-870, CP-893, MEDI6469, MEDI6383, MOXR-2916, AMPR0926 avelumab, MEDI4736, PDR001, rHIgM12B7, ulokuplumab, BKT140, varlilumab, ARGX-110, MGA271, lirilumab, IPH2201, ARGX-115, emaktuzumab, CC-90002 and MNRP1685A, or an antigen-binding fragment thereof. 16. The method of any one of claims 1 to 15, wherein said immune control point inhibitor is an anti-PD-L1 antibody. 17. The method of claim 16 wherein the anti-PD-L1 antibody is MEDI14736, MDPL3280A, BMS-936559, LY3300054, atezolizumab, or avelumab, or an antigen-binding fragment thereof. 18. The method of any one of claims 8-17, wherein, after relapse and prior to either or both of the administration of the immune checkpoint inhibitor and the biopsy, the subject was not administered an exogenous or recombinant agent to treat a disease or condition or to modulate the activity of genetically engineered cells. 19. The method according to any one of claims 1 to 18, wherein the biopsy is performed using a needle or trocar. 20. The method of any one of claims 1-19, wherein the biopsy comprises an incisional biopsy. 21. The method of any one of claims 1 to 20, wherein the method results in an expansion of the genetically engineered cells or an increase in the number of genetically engineered cells compared to a time immediately prior to one or both of the administration of the immune checkpoint inhibitor and the biopsy. 22. The method according to any one of claims 1 to 21, wherein the expansion of the genetically engineered cells occurs within 24 hours, 48 hours, 96 hours, 7 days, 14 days, or 28 days after one or both of the administration of the immune control point inhibitor and biopsy. 23. The method according to any one of claims 1-22, where: expansion provides more than 1.5 times, 2.0 times, 5.0 times, 10 times, 100 times, 200 times or more engineered blood detectable cells compared to the time immediately before one or both of immune control point inhibitor administration and biopsy; or expansion provides more than 1.5 times, 2.0 times, 5.0 times, 10 times, 100 times, 200 times or more engineered cells detectable in blood compared to previous peak levels of engineered cells cells in the blood before one or both of the administration of the immune control point inhibitor and the biopsy. 24. The method according to any one of claims 1 to 23, wherein the amount of genetically engineered cells detectable in blood at a time point after one or both of the administration of the immune checkpoint inhibitor and the biopsy is: increases, optionally increases by 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times or more, compared to the number of engineered cells at the previous time point before one or both of the administration of the immune control point inhibitor and biopsies; greater than 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, 100 times or more the peak or maximum number of engineered cells detectable in an individual's blood to one or both of administration of an immune control point inhibitor and biopsy; more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2% or 0.1% engineered cells are detectable in the blood at a point in time after the detection of the peak of the maximum level of such cells in the blood. 25. The method according to any one of claims 1 to 24, wherein the recombinant receptor specifically binds to an antigen associated with a tumor or malignant tumor or expressed in the cells of the lesion or part thereof. 26. The method of claim 25, wherein the antigen is selected from 5T4, 8H9, avb6 integrin, B7-H6, B cell maturation antigen (BCMA), CA9, testicular cancer 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, dual antigen, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, ephrin B2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, estrogen receptor, Fetal AchR, folate receptor alpha, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, G250/CAIX, GD2, GD3, gp100, Her2/neu (erbB2 receptor tyrosine kinase), HMW-MAA, IL -22R-alpha, IL-13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, Lewis Y antigen, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE )-A1, MAGE-A3, MAGE-A6, MART-1, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, NCAM, NKG2D, NKG2D ligands, NY-ESO-1, O-acetylated GD2 (OGD2), oncoembryonic antigen, preferably expressed melanoma antigen (PRAME), PSCA, progesterone receptor, survivin, ROR1, TAG72, tEGFR, VEGF receptors, VEGF-R2, Wilms tumor antigen 1 (WT-1), pathogen-specific antigen. 27. The method of any one of claims 1 to 26, wherein the tumor or cancer is leukemia or lymphoma. 28. The method of any one of claims 1 to 27, wherein the tumor or cancer is a B-cell cancer. 29. The method according to any one of claims 1 to 28, wherein the tumor or malignant tumor is lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma (NHL ) or diffuse large B-cell lymphoma (DLBCL) or a subtype of any of the above. 30. The method of any one of claims 1-29, wherein the recombinant receptor is a T cell receptor or a functional non-T cell receptor. 31. The method of any one of claims 1-30, wherein the recombinant receptor is a chimeric antigen receptor (CAR). 32. The method of claim 31 wherein the CAR comprises an extracellular antigen recognition domain that specifically binds to an antigen and an intracellular signaling domain containing ITAM. 33. The method according to any one of claims 25-32, wherein the antigen is CD19. 34. The method according to claim 32 or 33, wherein the intracellular signaling domain comprises an intracellular domain of the CD3-zeta chain (CD3ζ). 35. The method of any one of claims 31-34, wherein the CAR further comprises a costimulatory signaling region. 36. The method of claim 35, wherein the co-stimulatory signaling domain comprises a CD28 or 4-1BB signaling domain. 37. The method of any one of claims 1-36, wherein the T cells are CD4+ or CD8+ T cells. 38. The method of any one of claims 1-37, wherein the cells used for therapy with the modified T-cell engineering techniques comprise primary cells obtained from an individual. 39. The method according to any one of claims 1 to 38, wherein the cells of the genetically engineered cells are autologous to the individual. 40. The method of any one of claims 1 to 38, wherein the cells of the genetically engineered cells are allogeneic to the individual. 41. The method of any one of claims 1-40, wherein the subject is a human. 42. The method according to any one of claims 1 to 41, wherein the dose of genetically engineered cells previously administered comprises a dose of 1 x 10 ⁵ to 5 x 10 ^{8 of} all cells expressing the recombinant receptor, all T cells, or all mononuclear peripheral blood cells (PBMC), 1×10 ⁵ to 1×10 ⁸ all cells expressing the recombinant receptor, all T cells, or all peripheral blood mononuclear cells (PBMC), 5×10 ⁵ to 1×10 ⁷ all cells expressing the recombinant receptor, all T-cells or all peripheral blood mononuclear cells (PBMC) or from 1x10 ⁶ to 1x10 ^{7 of} all cells expressing the recombinant receptor, all T-cells or all peripheral blood mononuclear cells (PBMC), in each case inclusive. 43. The method according to any one of claims 1 to 42, wherein the dose of genetically engineered cells previously administered is no more than 5×10 ^{8 of} all cells expressing the recombinant receptor, all T cells, or all peripheral blood mononuclear cells (PBMC ), not more than 1×10 ^{8 of} all cells expressing the recombinant receptor, all T-cells or all peripheral blood mononuclear cells (PBMC), not more than 1×10 ^{7 of} all cells expressing the recombinant receptor, all T-cells or all mononuclear cells blood cells (PBMC), not more than 0.5×10 ⁷ all cells expressing the recombinant receptor, all T-cells or all peripheral blood mononuclear cells (PBMC), not more than 1×10 ⁶ all cells expressing the recombinant receptor, all T -cells or all peripheral blood mononuclear cells (PBMC), not more than 0.5×10 ^{6 of} all cells expressing the recombinant receptor, all T-cells or all peripheral blood mononuclear cells (PBMC). 44. The method according to any one of claims 1 to 43, wherein the dose of genetically engineered cells that was previously administered comprises a dose of from about 0.25×10 ⁶ cells/kg of body weight of the individual to 5×10 ⁶ cells/kg, from 0.5×10 ⁶ cells/kg of individual body weight up to 3×10 ⁶ cells/kg, from about 0.75×10 ⁶ cells/kg to 2.5×10 ⁶ cells/kg, or from about 1×10 ⁶ cells /kg up to 2×10 ⁶ cells/kg, in each case inclusive. 45. The method according to any one of claims 1 to 44, wherein the dose of genetically engineered cells is administered in a single pharmaceutical composition containing cells, or as several compositions together containing cells. 46. The method of any one of claims 1-45, wherein the engineered cells administered are a divided dose, wherein the dose cells are administered in multiple compositions containing the dose cells collectively over a time period of no more than three days. 47. The method of any one of claims 1 to 46, wherein the method comprises providing a follow-up of one or both of the immune check point inhibitor administration and a biopsy, optionally wherein the follow-up of one or both of the immune check point inhibitor administration and the biopsy are performed following a relapse in the post-response individual. after prior one or both of the immune control point inhibitor administration and biopsy and/or after it has not achieved a complete response after prior one or both of the immune control point inhibitor administration and biopsy. 48. The method of claim 47, wherein the subject has responded to the genetically engineered cells following prior one or both of the immune point inhibitor administration and biopsy, and then ceased responding and/or relapsed prior to subsequent one or both of the immune point inhibitor administration. control and biopsy. 49. The method of claim 47 or 48, wherein the genetically engineered cells have expanded, or are found to have expanded, following one or both of the immune checkpoint inhibitor administration and biopsy, or prior to the subsequent one or both of the immune checkpoint inhibitor administration. points of control and biopsy. 50. The method according to any one of claims 47-49, wherein at or immediately before subsequent one or both of the administration of the immune control point inhibitor and the biopsy: the individual is in remission; the number of genetically engineered cells found in the blood is reduced or undetectable; the number of genetically engineered cells found in the fluid or tissue or sample, optionally blood, of the individual is reduced compared to a previous point in time after the start of prior one or both of immune checkpoint inhibitor administration and biopsy; and/or the amount of genetically engineered cells found in a fluid or tissue or sample, optionally blood, from an individual is reduced by or more than 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5.0 times, 10 times, or more than the peak or maximum number of genetically engineered cells detected or detected in the blood of an individual after the start of the previous one or both of the administration of the immune control point inhibitor and biopsy and / or compared with the level in time point within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or 28 days after the start of the preceding one or both of immune control point inhibitor administration and biopsy . 51. The method according to any one of claims 1 to 50, wherein the genetically engineered cells exhibit increased or prolonged expansion and/or persistence in an individual compared to a method in which the genetically engineered cells are administered to the individual in the absence of one or both of the administration immune checkpoint inhibitor and biopsy. 52. The method according to any one of claims 1 to 51, wherein the method reduces the tumor burden to a greater extent and/or for a longer period of time compared to the decrease that would be observed in the case of a comparable method in which genetically modified cells are introduced to an individual in the absence of one or both of the immune checkpoint inhibitor administration and biopsy and/or in which one or both of the immune checkpoint inhibitor administration and biopsy are performed in the absence of genetically engineered cells, optionally at the same dose or on the same dosing schedule .

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