Methods of conditioning patients for t cell therapy

Abstract

To provide a method for increasing the efficacy of a T cell therapy in a patient in need thereof.SOLUTION: Provided is a method for conditioning a patient prior to a T cell therapy, wherein the conditioning comprises the step of administering a combination of cyclophosphamide and fludarabine.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Government Interest Statement This invention was developed by an agency of the U.S. Department of Health and Human Services. This invention was made in fulfillment of a Cooperative Research and Development Agreement with the National Cancer Institute (NCI). The United States Government has certain rights in this invention.

[0002] FIELD OF THE INVENTION The present invention provides a method for preconditioning a patient in need of tumor treatment, e.g., T cell therapy. In particular, the present invention relates to a method for improving the efficacy of T cell therapy, including engineered CAR T cell therapy, comprising administering to a patient in need of T cell therapy a combination of cyclophosphamide and fludarabine. by first administering a conditioning chemotherapy regimen comprising: [Background technology]

[0003] Background of the Invention Human cancers, by their nature, consist of normal cells that undergo genetic or epigenetic transformation to become abnormal cancer cells. In doing so, the cancer cells begin to express proteins and other antigens that differ from those expressed by normal cells. The body's innate immune system can exploit these abnormal tumor antigens to specifically target and kill cancer cells. However, cancer cells employ various mechanisms to prevent immune cells, such as T and B lymphocytes, from successfully targeting them.

[0004] Human T cell therapy is a method for targeting and killing cancer cells in patients by enriching or modifying them. These rely on modified human T cells to enrich for the concentration of natural T cells capable of targeting tumor antigens, or to genetically modify T cells to specifically target known cancer antigens. Various techniques have been developed to date. These treatments have proven to have promising but modest effects on tumor size and patient survival. On the other hand, It has proven difficult to predict whether a therapy will be effective in each patient.

[0005] Cyclophosphamide can be given alone or in combination with other drugs, including carmustine (BCNU) and etoposide (VP-16). , 10-20 mg / kg / day for 2-5 days, 40-50 mg / kg (1.5-1.8 g / m 2 ) can be administered IV Cut.

[0006] Recent studies have shown that preconditioning patients with one or more immunosuppressive chemotherapy regimens before T cell infusion can increase the efficacy of the transplanted T cells. (Rosenberg et al., Clin. Cancer. Res. (2011)). However, the current method However, these approaches rely on high doses of toxic and nonspecific drugs, which can cause painful and sometimes fatal adverse events. As a result, improved T cell therapy requires effective preconditioning. There remains a need to identify the method. Summary of the Invention

[0007] The present disclosure provides that patients receive 200 mg / m 2 / day~2000mg / m 2 / day dose of cyclophosphamide and 20 mg / m 2 / day~900mg / m 2

[0013] The present invention relates to a method for treating a patient in need of T cell therapy, the method comprising administering fludarabine at a dose of 100 mg / day to a patient in need of T cell therapy. The present invention provides a method for conditioning a patient.

[0008] The present disclosure provides that patients receive 200 mg / m 2 / day~2000mg / m 2 / day dose of cyclophosphamide and 20 mg / m 2 / day~900mg / m 2

[0013] The present invention relates to a method for treating a patient in need of T cell therapy, the method comprising administering fludarabine at a dose of 100 mg / day to a patient in need of T cell therapy. Further provided is a method for reducing endogenous lymphocytes in a patient suffering from leukemia.

[0009] The present disclosure provides that patients receive 200 mg / m 2 / day~2000mg / m 2 / day dose of cyclophosphamide and 20 mg / m 2 / day~900mg / m 2

[0013] The present invention relates to a method for treating a patient in need of T cell therapy, the method comprising administering fludarabine at a dose of 100 mg / day to a patient in need of T cell therapy. Also provided are methods for increasing serum levels of homeostatic cytokines in a patient suffering from the disease.

[0010] In certain embodiments, the homeostatic cytokine is interleukin 7 (IL-7), interleukin 1 (IL-1), or Interleukin 15 (IL-15), interleukin 10 (IL-10), interleukin 5 (IL-5), gamma induction Protein 10 (IP-10), interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1), placental growth factor (PLGF), C-reactive protein (CRP), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), or any combination thereof.

[0011] The present disclosure provides that patients receive 200 mg / m 2 / day~2000mg / m 2 / day dose of cyclophosphamide and 20 mg / m 2 / day~900mg / m 2

[0013] The present invention relates to a method for treating a patient in need of T cell therapy, the method comprising administering fludarabine at a dose of 100 mg / day to a patient in need of T cell therapy. Also provided are methods for enhancing effector function of administered T cells in a patient.

[0012] The present disclosure provides that patients receive 200 mg / m 2 / day~2000mg / m 2 / day dose of cyclophosphamide and 20 mg / m 2 / day~900mg / m 2

[0013] The present invention relates to a method for treating a patient in need of T cell therapy, the method comprising administering fludarabine at a dose of 100 mg / day to a patient in need of T cell therapy. Also provided are methods for enhancing the activation and / or availability of antigen-presenting cells in a patient.

[0013] In certain embodiments, the T cell therapy is selected from tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation.

[0014] The present disclosure also provides a method of treating a patient with lymphoma, comprising: Prior to administering a therapeutically effective amount of the engineered CAR T cells to the patient, the patient is administered approximately 500 mg / m 2 / day cyclophosphamide and approximately 60 mg / m 2 / day of fludarabine daily for 3 days, The T cell expresses a chimeric antigen receptor, which binds to CD19 and further contains a CD28 costimulatory domain and a CD3-zeta signaling region.

[0015] The present disclosure also provides a method of treating a patient with lymphoma, comprising: (i) approximately 200 mg / m 2 / day cyclophosphamide and approximately 20 mg / m 2 / day fludarabine, and (ii) administering to the patient a therapeutically effective amount of engineered CAR T cells, Engineered CAR T cells express a chimeric antigen receptor, which binds to CD19. and further comprising a CD28 costimulatory domain and a CD3-zeta signaling region.

[0016] The present disclosure also provides a method of treating a patient with lymphoma, comprising: (i) approximately 300 mg / m 2 / day cyclophosphamide and approximately 30 mg / m 2 / day fludarabine, and (ii) administering to the patient a therapeutically effective amount of engineered CAR T cells, Engineered CAR T cells express a chimeric antigen receptor, which binds to CD19. and further comprising a CD28 costimulatory domain and a CD3-zeta signaling region.

[0017] The present disclosure also provides a method of treating a patient with lymphoma, comprising: (i) Approximately 300 mg / m 2 / day cyclophosphamide and approximately 60 mg / m 2 / day fludarabine, and (ii) administering to the patient a therapeutically effective amount of engineered CAR T cells, Engineered CAR T cells express a chimeric antigen receptor, which binds to CD19. and further comprising a CD28 costimulatory domain and a CD3-zeta signaling region.

[0018] The present disclosure also provides a method of treating a patient with lymphoma, comprising: (i) approximately 500 mg / m 2 / day cyclophosphamide and approximately 60 mg / m 2 / day of fludarabine and (ii) administering to the patient a therapeutically effective amount of the engineered CAR T cells, Engineered CAR T cells express a chimeric antigen receptor, which binds to CD19. and further comprising a CD28 costimulatory domain and a CD3-zeta signaling region.

[0019] The present disclosure provides a method for treating a patient with lymphoma, comprising administering to the patient a therapeutically effective amount of engineered CAR T cells. Also provided is a method of treating a patient with steroids, wherein the patient receives about 500 mg / m 2 / day cyclophosphamide and approximately 60 mg / m 2 The engineered CAR T cells were conditioned with fludarabine / day and expressed a chimeric antigen receptor (CAR), which binds to CD19. , and further comprising a CD28 costimulatory domain and a CD3-zeta signaling region.

[0020] The present disclosure provides a method for treating rheumatoid arthritis with (i) cyclophosphamide, (ii) fludarabine, and (iii) engineered CAR T cell therapy. 200 mg / m before treatment for patients requiring 2 / day~2000mg / m 2 / day dose of cyclophosphamide and 20 mg / m 2 / day~900mg / m 2 Also provided is a kit containing instructions for administering fludarabine at a dose of 1 / day daily for three days. [Brief explanation of the drawings]

[0021] [Figure 1] Illustrates an example of CAR-engineered T cells and a schematic diagram of their configuration.In this exemplary CAR-engineered T cells, the target binding domain comprises an antibody-derived scFv domain, the costimulatory domain is derived from CD28, and the essential activation domain is derived from CD3ζ (zeta).A viral vector carries the CAR vector construct, which can then be integrated into T cell genome.T cells can then express the CAR construct as a transmembrane protein. [Figure 2A]Figure 2 shows patient disease response after treatment with anti-CD-19 CAR+ T cells. Figure 2A shows the best response as a percentage change in disease for patients with B-cell malignancies. Hatched bars indicate complete response (CR). Shaded bars indicate partial response. Open bars indicate stable disease (SD). Filled bars indicate progressive disease (PD). [Figure 2B] Figure 2B shows the patient's disease response after treatment with anti-CD-19 CAR+ T cells. Figure 2B shows the patient's disease response versus months after CAR+ T cell infusion. Solid bars indicate partial responses (PR), and gray bars indicate complete responses (CR). Breaks in the bars marked "PD" indicate patients experienced progressive disease. Inverted triangles mark the time of T cell infusion. Solid circles indicate the time of B cell recovery. Open circles indicate the time of CAR+ T cell clearance from the patient's blood. Horizontal arrows indicate that the patient's response is ongoing. [Figure 3] A sample diagram is provided of a Phase 1 clinical trial aimed at determining the safety, efficacy, and dose-limiting toxicities of treating patients with 500 mg / m2 / day cyclophosphamide, 30 mg / m2 / day fludarabine, and 2 x 106 / kg anti-CD19 CAR+ T cells. [Figure 4] Serum levels of selected cytokine analytes before and after conditioning with 300 mg / m² / day of cyclophosphamide and 30 mg / m² / day of fludarabine are shown. Serum levels of interleukin-15 (IL-15; Figure 4A), monocyte chemoattractant protein 1 (MCP-1; Figure 4B), gamma-inducible protein 10 (IP-10; Figure 4C), placental growth factor (PLGF; Figure 4D), soluble intercellular adhesion molecule 1 (sICAM-1; Figure 4E), C-reactive protein (CRP; Figure 4F), vascular endothelial growth factor D (VEGF-D; Figure 4G), and macrophage inflammatory protein 1β (MIP-1b; Figure 4H) are shown before and after administration of 300 mg / m² of cyclophosphamide and 30 mg / m² of fludarabine. Pre-dose serum was collected on days -12 to -5, and post-dose serum was collected on day 0, before administration of T cell therapy (Figures 4A to 4H). [Figure 5]Figure 5 shows the fold changes in serum levels of selected cytokine analytes after conditioning with 300 mg / m² / day cyclophosphamide and 30 mg / m² / day fludarabine in patients who either responded or did not respond to subsequent T cell therapy. Fold changes in serum levels of IL-15 (Figure 5A), MCP-1 (Figure 5B), IP-10 (Figure 5C), PLGF (Figure 5D), sICAM-1 (Figure 5E), CRP (Figure 5F), VEGF (Figure 5G), and MIP-1b (Figure 5H) are shown for responders and non-responders. Horizontal lines indicate the mean (Figures 5A-H). IL-15 changes for individual patients are shown in Figure 5A, and each patient's disease response is indicated next to each data point as partial response (PR), complete response (CR), stable disease (SD), or progressive disease (PD). [Figure 6-1] Serum concentrations of selected cytokine analytes measured at various time points from days -10 to 18 for patients receiving 300 mg / m² / day cyclophosphamide and 30 mg / m² / day fludarabine prior to T-cell therapy on day 0 are shown. Serum concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF; Figure 6A), IL-2 (Figure 6B), MCP-1 (Figure 6C), IL-6 (Figure 6D), IL-10 (Figure 6E), MCP-4 (Figure 6F), CRP (Figure 6G), interferon gamma (IFNγ; Figure 6H), granzyme A (Figure 6I), IL-15 (Figure 6J), IL-5 (Figure 6K), and granzyme B (Figure 6L) are shown. [Figure 6-2] Serum concentrations of selected cytokine analytes measured at various time points from days -10 to 18 for patients receiving 300 mg / m2 / day cyclophosphamide and 30 mg / m2 / day fludarabine prior to T cell therapy on day 0 are shown. Serum concentrations of IL-8 (Figure 6M), IP-10 (Figure 6N), MIP-1b (Figure 6O), and PLGF (Figure 6P) are shown. [Figure 6-3]Serum concentrations of selected cytokine analytes measured at various time points from days -10 to 18 for patients receiving 300 mg / m2 / day cyclophosphamide and 30 mg / m2 / day fludarabine prior to T-cell therapy on day 0 are shown. Serum concentrations of IL-16 (Figure 6Q), thymus and activation-regulated chemokine (TARC; Figure 6R), eotaxin-3 (Figure 6S), and sICAM-1 (Figure 6T) are shown. [Figure 6-4] Serum concentrations of selected cytokine analytes measured at various time points from days -10 to 18 are shown for patients receiving 300 mg / m² / day cyclophosphamide and 30 mg / m² / day fludarabine prior to T-cell therapy on day 0. Serum concentrations of soluble vascular adhesion molecule 1 (sVCAM; Figure 6U), (SAA; Figure 6V) are shown. [Figure 7] Serum concentrations of selected cytokine analytes measured before and after administration of 300 mg / m² / day cyclophosphamide and 30 mg / m² / day fludarabine are shown. Post-administration serum was collected immediately prior to T cell infusion. Serum concentrations of IL-15 (Figure 7A), IL-7 (Figure 7B), PLGF (Figure 7C), CRP (Figure 7D), IL-5 (Figure 7E), IL-10 (Figure 7F), MCP-1 (Figure 7G), IP-10 (Figure 7H), and sICAM-1 (Figure 7I) are shown. Each data point represents a single patient. Horizontal bars indicate the mean (Figures 7A-7I). P values ​​from the Wilcoxon matched-pairs signed-rank test were applied to analytes measured before and after conditioning, and the corresponding P values ​​are shown (Figures 7A-7I). Some IL-7 values ​​exceeded the upper limit of quantification (ULOQ; Figure 7B). [Figure 8]In vitro production of various cytokine analytes by anti-CD19 CAR+ T cells (K562-CD19) after stimulation with K562 cells is shown compared to the negative control (K562-NGFR). Concentrations of GM-CSF (Figure 8A), IL-2 (Figure 8B), IFNγ (Figure 8C), IL-5 (Figure 8D), IL-4 (Figure 8E), IL-13 (Figure 8F), tumor necrosis factor alpha (TNFα; Figure 8G), IL-6 (Figure 8H), granzyme B (Figure 8I), MIP-1β (Figure 8J), MIP-1α (Figure 8K), and soluble CD137 (Figure 8L) are shown for control T cells and anti-CD19 CAR+ T cells. T1, T2, and immune homeostatic cytokines (Figures 8A-8F) as well as pro-inflammatory cytokines and chemokines (Figures 8G-8L) are labeled accordingly. Data were collected prior to infusion by co-incubating product T cells with K562-CD19 cells or control K562-NGFR cells and measuring the media concentrations of the listed analytes (Figures 8A-8L). [Figure 9] The percentage of anti-CD19 CAR+ T cells (K562-CD19) expressing various cytokines after engagement with target antigens is shown compared to the negative control (K562-NGFR). The percentage of cells expressing CD107α (Figure 9A), 4-1BB (Figure 9B), and programmed death 1 (PD-1; Figure 9C) is shown. Pre-infusion data were collected by co-incubating product T cells with K562-CD19 cells or control K562-NGFR cells and measuring the media concentrations of selected activation markers (Figures 9A-9C). The indicated P values ​​represent the results of a paired t-test comparing K562-CD19 test cells with K562-NGFR negative control cells (Figures 9A-9C). [Figure 10]Various characteristics of product T cells and peripheral blood lymphocytes (PBLs) are described in terms of production time (days). Data include the percent of anti-CD-19 CAR+ T cells detected in the product compared to PBLs; the CD8 to CD4 ratio in the product compared to PBLs; the relative abundance of naive, central memory (Tcm), effector memory (Tem), and effector (Teff) T cells within the anti-CD19 CAR+ CD8+ T cell population; and the relative abundance of naive, central memory (Tcm), effector memory (Tem), and effector (Teff) T cells within the anti-CD19 CAR+ CD4+ T cell population (Figure 10). Phenotypic analysis of product T cells prior to infusion and PBLs at peak proliferation in the blood was performed with respect to anti-CD19 CAR+ T cells (Figure 10). p-values ​​represent the results of a rank test for association between production time and T cell subset composition. [Figure 11] Figure 1 shows the expression profile of cytokines, chemokines and other markers observed after conditioning of NHL patients according to the present invention. CRP: C-reactive protein. PLGF: placental growth factor. MCP-1: monocyte chemotactic protein-1. [Figure 12] Quantification of changes observed in cytokines, chemokines and other markers after conditioning with cyclophosphamide and fludarabine according to the present invention. [Figure 13] Magnitude of changes in circulating IL-15 and perforin after conditioning chemotherapy associated with objective response is shown. P values ​​were not multiplicity adjusted. Analysis was performed on markers measured before CAR T-cell infusion. [Figure 14]Biomarker analysis of cytokines, chemokines, and effector molecules is shown. Markers were ranked from lowest to highest p-value within each biomarker category using the Wilcoxon signed-rank test. Biomarkers that were altered in the majority of patients with a p-value <0.05 are displayed. Only 7 of the 41 markers measured showed changes in the majority of patients with a p<0.05. Analysis was performed on markers measured before CAR T-cell infusion. [Figure 15] The sequential induction and elimination of immune homeostatic, inflammatory, and modulating cytokines, chemokines, and immune effector molecules is shown. Representative markers are shown. A total of 22 of the 41 markers measured: IL-15, IL-7, IL-2, granzyme B, granzyme A, CRP, IL-6, GM-CSF, IL-5, IFNg, IL-10, MCP-1, MCP-4, IP-10, IL-8, TARC, MIP1a, MIP1b, PLGF, VEGF-D, sICAM-1, and FGF-2, showed an increase of at least 2-fold above baseline levels after CAR T cell treatment in at least 50% of patients. Peak formation was observed for immune homeostatic cytokines and chemokines on days 3-4. [Figure 16] The sequential induction and elimination of immune homeostatic, inflammatory, and regulatory cytokines, chemokines, and immune effector molecules is shown. Representative markers are shown. A total of 22 of the 41 markers measured: IL-15, IL-7, IL-2, Granzyme B, Granzyme A, CRP, IL-6, GM-CSF, IL-5, IFNg, IL-10, MCP-1, MCP-4, IP-10, IL-8, TARC, MIP1a, MIP1b, PLGF, VEGF-D, sICAM-1, and FGF-2, showed an increase of at least 2-fold above baseline values ​​after CAR T cell treatment in at least 50% of patients. Peak formation was observed between days 5 and 7 for immunoregulatory cytokines and chemokines. "ULOQ": upper limit of quantitation. [Figure 17]Figure 1 shows changes in treatment-related biomarkers and clinical responses induced by anti-CD19 CAR T cells according to the present invention. Maximum fold change in marker levels after CAR T cell treatment compared to baseline (before conditioning). Each row represents an individual subject. Wilcoxon rank-sum tests were used to compare maximum fold change values ​​between responder and non-responder groups for a total of 41 biomarkers evaluated. P values ​​were not multiplicity adjusted, and only biomarkers with p<0.10 are shown. P values ​​for IL-7 and sICAM-1 were <0.05. This association also applied to changes in absolute levels of IL-7 (p=0.0165), IL-15 (p=0.0314), and IL-15 (p=0.041). [Figure 18-1] Figures 18A-18F show changes in analyte levels before and after conditioning with cyclophosphamide and fludarabine. Pre- and post-levels of IL-15 (Figure 18A), IP-10 (Figure 18B), CRP (Figure 18C), IL-7 (Figure 18D), MCP-1 (Figure 18E), and perforin (Figure 18F) are shown. [Figure 18-2] Figure 18G shows the changes in analyte levels before and after conditioning with cyclophosphamide and fludarabine. Figure 18G summarizes the changes in serum levels of various analytes and the corresponding p-values. [Figure 19] Correlations between changes in analyte levels after conditioning and objective response to CAR T cell therapy are shown for IL-15 (Figure 19A), IP-10 (Figure 19B), and perforin (Figure 19C). Figure 19D provides a summary of the statistical significance of the data provided in each of Figures 19A-19C. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description of the Invention The present invention relates to a method for treating a patient with T cell therapy, comprising administering cyclophosphamide and fludarabine prior to administering T cell therapy. The present invention relates to a method of conditioning a patient in need of T cell therapy, e.g., engineered CAR T cell therapy, e.g., autologous cell therapy (eACT™), including administering a dose of these prior to T cell therapy. Preconditioning patients with cyclophosphamide and fludarabine reduces the number of endogenous lymphocytes and increases the homeostatic cytokines and / or cytokines present in the patient. or enhance the efficacy of T cell therapy by increasing serum levels of pro-immune factors. This improves the efficacy of the transplanted T cells, which are then expanded after being administered to the patient. Preconditioning at the doses described herein surprisingly reduced the number of endogenous lymphocytes while minimizing the toxicity associated with cyclophosphamide and fludarabine treatment. The present invention provides a method for preconditioning prior to T cell therapy. The administration of specific doses of cyclophosphamide and fludarabine may result in optimal levels of transplanted T cells. Induce cytokine availability in the T cell therapy while resulting in lower toxicity in patients overall. Provides sex.

[0023] definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning set forth below, unless otherwise expressly provided herein. Additional definitions are set forth throughout this application.

[0024] The term "and / or" as used herein should be considered to specifically disclose each of the two specified features or components together with or without the other. Thus, phrases such as "A and / or B" herein should not be construed as meaning "A and / or B" unless specifically disclosed otherwise. The term "and / or," as used, is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, expressions such as "A, B, and / or C" are also intended to include "A and B," "A or B," "A" (alone), and "B" (alone). The term "and / or" as used in such phrases includes each of the following aspects: It is intended that: A, B, and C; A, B, or C; A or C; A or B; B or C; A and and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0025] Whenever an aspect is described herein using the word "comprising," other similar aspects described with the terms "consisting of" and / or "consisting essentially of" are also provided. It is understood that

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide skilled artisans with a basic understanding of the terms used in this disclosure. It provides a dictionary of many common words.

[0027] Units, prefixes, and symbols are denoted in the form recognized by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of this disclosure that may be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0028] The term "activated" refers to the state of an immune cell, e.g., a T cell, that has been stimulated sufficiently to induce detectable cell proliferation. Activation is characterized by induced cytokine production and detectable The term "activated T cells" can also refer to, among other things, cells that are activated by T cell differentiation. Represents T cells undergoing cleavage.

[0029] "Administering" refers to the introduction of an agent into a subject's body using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary administration routes for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, for example, orally. Other non-parenteral routes include topical, epidermal, or mucosal administration routes, such as intranasal, intravaginal, rectal, sublingual, or topical. Administration can be, for example, one time, multiple times, and / or over one or more extended periods of time. .

[0030] As used herein, an "adverse event" (AE) is any untoward, generally unintended or undesirable sign (including abnormal laboratory findings), symptom, medical occurrence, or disease associated with the use of a medical procedure. The definition of an adverse event includes a worsening of an existing medical condition. A worsening is an increase in severity, frequency, and / or duration of an existing medical condition, or a worsening of the condition. demonstrated that the treatment was associated with better outcomes.

[0031] The term "antibody" (Ab) includes, but is not limited to, immunoglobulins, which are glycoproteins that specifically bind to antigens. Generally, antibodies can comprise at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, inter-connected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains, CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region contains one The VH and VL regions contain highly flexible domains called complementarity-determining regions (CDRs). The variable regions of the heavy and light chains can be further subdivided into variable regions and larger, conserved regions called framework regions (FRs) interspersed between them. Each VH and VL contains three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with antigen. The constant regions of Abs may mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0032] Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. Subclasses of IgG are also well known to those skilled in the art and include: "Isotype" refers to the class or subclass of Abs (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. The term "antibody" includes, by way of example, both natural and non-natural Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or non-human Abs; fully synthetic Abs; and single-chain Abs. Non-human Abs may be humanized by recombinant methods to reduce their immunogenicity in humans. Unless expressly stated and the context indicates otherwise, the term "antibody" also includes antigen-binding fragments or portions of any of the foregoing immunoglobulins, including monovalent and bivalent fragments or portions, and single-chain Abs.

[0033] An "antigen-binding molecule" or "antibody fragment" refers to any portion of an antibody that is less than the entire antibody. An antigen-binding molecule can include an antigen complementarity-determining region (CDR). An antibody fragment Examples of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAbs, linear antibodies. , scFv antibodies, and multispecific antibodies formed from antigen-binding molecules.

[0034] "Antigen" refers to any molecule capable of eliciting an immune response or being bound by an antibody. The immune response may involve either or both antibody production or activation of specific immunocompetent cells. Those skilled in the art will readily appreciate that virtually any macromolecule, including any protein or peptide, can serve as an antigen. Antigens can be endogenously expressed, i.e., expressed by genomic DNA, or recombinantly expressed. Antigens can be expressed by cells that express antigens, such as cancer cells. Antigens may be specific to a particular tissue, such as a tumor, or may be widely expressed. In addition, fragments of relatively large molecules may act as antigens. In one aspect, the antigen is a tumor antigen.

[0035] The term "autologous" refers to any material derived from the same individual that is later reintroduced into that individual. For example, the engineered autologous cell therapy (eACT™) described herein involves collecting lymphocytes from a patient, which are then engineered to express, for example, a CAR construct, and then reintroduced into the same patient. This includes administering and returning.

[0036] The term "allogeneic" refers to any material derived from one individual that is then introduced into another individual of the same species; an example is allogeneic T cell transplantation.

[0037] "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth can lead to the formation of malignant tumors that invade neighboring tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors. Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cancers of the immune system, including lymphoma, leukemia, and other leukemic malignancies. In some embodiments, the methods of the present invention are useful for the treatment of cancers of various cancer types, including, for example, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma, and ovarian cancer. Lymphoma (SMZL), esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's leukemia tumors, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, and asbestos-induced cancers tumors derived from environmentally induced cancers, including B-cell malignancies, other B-cell malignancies, and combinations of the aforementioned cancers It can be used to reduce tumor size. Certain cancers can be responsive to chemotherapy or radiation therapy, or the cancer can be refractory. A refractory cancer refers to a cancer that cannot be corrected with surgical intervention; the cancer is either unresponsive to chemotherapy or radiation therapy from the beginning or becomes unresponsive over time.

[0038] As used herein, "anti-tumor effect" refers to a biological effect that can be manifested as a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall survival or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with tumors.Anti-tumor effect can also refer to the prevention of tumor development, for example, vaccination.

[0039] As used herein, the term "progression-free survival," which can be abbreviated as PFS, refers to the time between treatment From the date of placement to the date of disease progression according to the revised IWG Response Criteria for Malignant Lymphoma or death from any cause Indicates the time until the date.

[0040] "Disease progression" is determined by measuring malignant lesions using radiographic or other methods and should not be reported as an adverse event. Death due to disease progression in the absence of signs and symptoms should be reported as the primary tumor type (e.g., DLBCL).

[0041] As used herein, "duration of response," which may be abbreviated as DOR, refers to the time from a subject's first Objective response was confirmed according to the revised IWG response criteria for malignant lymphoma Represents the time to date of disease progression or death.

[0042] The term "overall survival," which can be abbreviated as OS, is defined as the time from the date of treatment to the date of death.

[0043] As used herein, a "cytokine" refers to a compound produced by a cytokine in response to contact with a specific antigen. Cytokines refer to non-antibody proteins released by a cell, where the cytokines are released by a second cell. Cytokines interact with the immune system to mediate responses in second cells. Cytokines can be expressed intracellularly or administered to a subject. Cytokines can be released by immune cells, including macrophages, B cells, T cells, and mast cells, to propagate immune responses. Cytokines can induce various responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, can promote the survival and proliferation of immune cells, while pro-inflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of pro-inflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF)2, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placenta growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, These include granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0044] A "chemokine" is a type of cytokine that mediates cell chemotaxis or directional migration. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, These include macrophage-derived chemokine (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1α, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC or CCL17).

[0045] Other examples of analytes and cytokines of the present invention include, but are not limited to, chemokine (CC motif) ligand (CCL) 1, CCL5, monocyte-specific chemokine 3 (MCP3 or CCL7), monocyte chemoattractant protein 2 (MCP-2 or CCL8), CCL13, IL-1, IL-3, IL-9, IL-11, IL-12, IL-14, IL-17, IL-20, IL-21, granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), oncostatin M (OSM), CD154, lymphotoxin (LT) beta, 4-1BB ligand (4-1BBL), proliferation-inducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-related ligand (GITRL), tumor Necrosis factor superfamily member 14 (TNFSF14), OX40L, TNF, and ApoL-related leukocyte proliferation These include tumor necrosis factor (TNF)-associated apoptosis-inducing ligand 1 (TALL-1), or TNF-related apoptosis-inducing ligand (TRAIL).

[0046] As used herein, the terms "serum level" and "serum concentration" are used interchangeably and refer to the amount of an analyte in a subject's serum. The serum level of a given analyte can be measured using any method known in the art. For example, the serum level of a cytokine can be measured using enzyme-linked immunosorbent assay (ELISA). In one particular embodiment, Serum levels of cytokines can be measured using EMD Millipore LUMINEX® xMAP® multiplex assays.

[0047] As used herein, "dosing interval" refers to the amount of time that elapses between administration of multiple doses of the formulations disclosed herein to a subject. Thus, the dosing interval can be expressed as a range.

[0048] The doses described herein are "weight-based doses" or "body surface area (BSA)-based doses." A weight-based dose is a dose administered to a patient calculated based on the patient's weight, e.g., mg / kg. A BSA-based dose is a dose administered to a patient calculated based on the patient's surface area, e.g., mg / m 2 For human administration, the weight The two forms of dose measurement can be converted by multiplying the BSA-based dose by 37 or dividing the BSA-based dose by 37. For example, a dose of 60 mg / kg administered to a human subject would be converted to 37 for the same dose. The dose of the same drug administered to elephants is 2220 mg / m 2 Corresponds to.

[0049] As used herein, the term "dosage frequency" refers to the number of times a dose of a formulation disclosed herein is administered within a given time period. The administration frequency can be expressed as the number of doses per given time period. For example, cyclophosphamide can be administered once daily for each of five consecutive days. Once a day for each of four consecutive days, once a day for each of three consecutive days, once a day for each of two consecutive days In certain embodiments, cyclophosphamide is administered once daily for 3 consecutive days or once daily for 2 consecutive days. Fludarabine is administered once daily for each of 8 consecutive days, once daily for each of 7 consecutive days, once daily for each of 6 consecutive days. In other embodiments, fludarabine is administered once daily for five consecutive days or once daily for three consecutive days.

[0050] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject from developing disease or promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free symptom-free periods, or prevention of functional impairment or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to the skilled practitioner, for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0051] As used herein, the term "lymphocytes" includes natural killer (NK) cells, T cells, NK cells are cytotoxic T cells that represent a major component of the innate immune system. NK cells are a type of harmful (cytotoxic) lymphocyte. NK cells reject tumor and virus-infected cells. They work through the process of apoptosis, or programmed cell death. NK cells are named "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without the involvement of antibodies). Their T cell receptors (TCRs) distinguish self from other lymphocyte types. The thymus, a specialized organ of the immune system, is responsible for the majority of There are six types of T cells: helper T cells (e.g., CD4+ cells), Cytotoxic T cells (also known as TCs, cytotoxic T lymphocytes, CTLs, T-killer cells, cytolytic T cells, CD8+ T cells or killer T cells), memory T cells ((i) memory T cells such as naive cells) SCM Stem cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and and IL-7Rα, but these cells also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1. (ii) central memory T cells CM (iii) effector memory T cells express L-selectin and CCR7, and these cells secrete IL-2 but not IFNγ or IL-4; EM These include T cells (which do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Treg, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. B cells, on the other hand, are involved in humoral immunity (antibody-mediated and It plays a major role in the immune system (immune system) by producing antibodies and antigens, acting as an antigen-presenting cell (APC), and transforming into a memory B cell after activation by antigen interaction. , immature B cells are formed in the bone marrow, from which it takes its name.

[0052] The term "genetic engineering" or "engineering" refers to a method of modifying the genome of a cell, including, but not limited to, deleting a coding or non-coding region or portion thereof, or inserting a coding region or portion thereof. In some embodiments, the modified cell is a lymphocyte, e.g., a T cell, which can be obtained from either a patient or a donor. For example, Chimeric antigen receptors (CARs) or T cell receptors (TCRs) that are integrated into the cell's genome The cells can be engineered to express the exogenous construct that is placed in them.

[0053] An "immune response" refers to the activity of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (Abs, cytokines, and and complement) from the vertebrate body to remove invading pathogens, pathogen-infected cells, or tissue, cancerous or other abnormal cells, or in the case of autoimmune or pathological inflammation, normal human cells or tissues, resulting in the selective targeting of, binding to, damaging, destroying, and / or eliminating normal human cells or tissues.

[0054] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of developing or experiencing a recurrence of a disease by a method that involves inducing, enhancing, suppressing, or otherwise modifying the immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy can include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation. However, those skilled in the art will appreciate that the present invention is not limited to the use of immunotherapy. The conditioning method disclosed herein is recognized to enhance the efficacy of any transplanted T cell therapy. Examples of T cell therapies are described in U.S. Patent Publication Nos. 2014 / 0154228 and 2002 / 0006409, U.S. Patent No. 5,728,388, and WO 2008 / 081035. There are.

[0055] Immunotherapeutic T cells can be derived from any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be isolated from, for example, peripheral blood mononuclear cells, bone marrow, lymphocytes, or other organs. T cells can be obtained from nodal tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In addition, T cells can be derived from one or more T cell lines available in the art. T cells can be obtained from blood units collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL™ separation and / or apheresis. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0056] The term "engineered autologous cell therapy," also known as adoptive cell transfer, which may be abbreviated as "eACT™," refers to a therapy in which a patient's own T cells are collected and subsequently engineered to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. It is a process in which T cells are genetically modified. T cells can be engineered to express, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR). CAR-positive (+) T cells are: They are engineered to express an extracellular single-chain variable fragment (scFv) with specificity for a particular tumor antigen linked to an intracellular signaling moiety containing a costimulatory domain and an activation domain. The costimulatory domain can be derived, for example, from CD28, and the activation domain can be derived, for example, from CD3-zeta (Figure 1). In certain embodiments, CARs are designed to have two, three, four, or more costimulatory domains. CAR scFvs are, for example, For example, targeting CD19, a transmembrane protein expressed by cells of the B-cell lineage, including all normal B cells and B-cell malignancies, including, but not limited to, NHL, CLL, and non-T-cell ALL, Examples of CAR+ T cell therapies and constructs are described in U.S. Patent Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which references are incorporated by reference in their entireties.

[0057] As used herein, a "patient" includes any human suffering from cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0058] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids and There is no limit to the maximum number of amino acids that can make up a protein or peptide sequence. A polypeptide contains two or more amino acids joined together by peptide bonds. " Polypeptide " includes any peptide or protein.As used herein, this term refers to both short chains, which are generally referred to in the art as peptides, oligopeptides and oligomers, and longer chains, which are generally referred to in the art as proteins, and there are many types. " Polypeptide " includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0059] As used herein, "stimulation" refers to a primary response induced by the binding of a stimulatory molecule to its cognate ligand, where the binding mediates a signal transduction event. A "stimulatory molecule" refers to a molecule on a T cell, e.g., a cognate stimulatory ligand present on an antigen-presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), can specifically bind to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. A stimulatory ligand is a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), can specifically bind to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. These include, but are not limited to, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonists, These include anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0060] As used herein, a "co-stimulatory signal" refers to a signal that stimulates a cell to produce a signal, such as TCR / CD3 ligation. In combination with primary signals, including but not limited to proliferation and / or upregulation of key molecules Represents signals that lead to T cell responses such as upregulation or downregulation .

[0061] As used herein, a "costimulatory ligand" includes a cognate costimulatory molecule on a T cell. Co-stimulatory ligands include molecules on antigen-presenting cells that specifically bind to T cells. Binding of a co-stimulatory ligand provides a signal that mediates T cell responses, including, but not limited to, proliferation, activation, differentiation, etc. The stimulatory ligand induces a signal that is then transmitted by a stimulatory molecule, e.g., a T cell. Co-stimulatory ligands are additional to the primary signal provided by the binding of the TCR / CD3 complex to peptide-loaded major histocompatibility complex (MHC) molecules. Co-stimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), programmed death (PD) L1, PD-L2, 4-1BB ligand, OX40 ligand, and the like. Gand, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30 ligand, CD40, CD70, CD83, human leukocyte antigen G (HLA-G), MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), herpesvirus entry mediator (HVEM), lymphotoxin B Toll receptor, 3 / TR6, immunoglobulin-like transcript (ILT) 3, ILT4, Toll ligand receptor and a ligand that specifically binds to B7-H3. Costimulatory ligands include, but are not limited to, costimulatory molecules present on T cells, such as non- Exclusively, antibodies that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT), natural killer cell receptor C (NKG2C), B7-H3, and CD83 The present invention also includes a ligand that specifically binds to the

[0062] "Costimulatory molecules" specifically bind to costimulatory ligands to stimulate T cell co-activation. Stimulatory responses, including but not limited to, cognate binding partners on T cells that mediate proliferation Costimulatory molecules include, but are not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, CD83, PD-1, ICOS, LFA-1, CD2, CD7, TNFSF14 (LIGHT), NKG2C, B7-H3, MHC class 1 molecules, B and T lymphocyte attenuator (BTLA), and Toll ligand receptor.

[0063] The terms "conditioning" and "preconditioning" are used interchangeably herein to refer to preparing the appropriate conditions for a patient in need of T cell therapy. As used herein, conditioning includes, but is not limited to, administering the following prior to T cell therapy: Decreasing the number of endogenous lymphocytes, clearing cytokine sinks, and increases serum levels of multiple homeostatic cytokines or pro-inflammatory factors, enhances the effector function of T cells administered after conditioning, and stimulates antigen-presenting cells. or any combination thereof. In one aspect, "conditioning" refers to the administration of one or more cytokines, e.g., Interleukin 7 (IL-7), interleukin 15 (IL-15), interleukin 10 (IL-10), Interleukin 5 (IL-5), gamma-inducible protein 10 (IP-10), interleukin 8 (IL-8) In another embodiment, "conditioning" comprises increasing the serum levels of IL-7, IL-15, IP-10, MCP-1, PLGF, CRP, or any combination thereof.

[0064] The terms "reduce" and "lower" are used interchangeably herein and refer to any change that is less than the original. "Reduce" and "lower" are relative terms that require a comparison between before and after measurement. "Reduce" and "lower" include total depletion.

[0065] "Treatment" of a subject or "treating" a subject refers to any type of intervention or process performed on a subject or administration of an active agent to a subject for the purpose of reversing, alleviating, reversing, inhibiting, slowing, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, or condition, or the biochemical characteristics associated with a disease. In one aspect, "treatment" or "treating" includes a partial response. In another aspect, "treatment" or "treating" includes a complete response.

[0066] The use of alternatives (e.g., "or") means one, both, or any combination of the alternatives. The indefinite article "a" or "b" as used herein should be understood to mean either "A" or "an" means "one or more" of any described or listed components. It should be understood to mean "plural."

[0067] The terms "about" or "consisting essentially of" refer to a value or composition that is within an acceptable range of error for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of" can mean within one standard deviation or more than one standard deviation pertaining to practice in the art. Alternatively, "about" or "consisting essentially of" can mean a range of up to 10% (i.e., ±10%). For example, about 3 mg includes any number between 2.7 mg and 3.3 mg (about 10%). Additionally, particularly with respect to biological systems or processes, these terms can mean up to an order of magnitude or up to five times the value. When specific values ​​or compositions are provided in the applications and claims, unless otherwise stated, the meaning of "about" or "consisting essentially of" should be assumed to be within an acceptable error range for that particular value or composition.

[0068] Any concentration range, percentage range, ratio range, or integer range set forth herein includes any integer within the stated range, and, where appropriate, fractions thereof (e.g., 1 / 2, 1 / 4 ...2, 1 / 4, 1 / 2, 1 / 2, 1 / 2, 1 / 2, 1 / 2, 1 / 2, 1 / 2, 1 / 2, 1 / 2, 1 / 2, 1 / 2, 1 / 2, 1 / 2, 1 / 2, 1 / 2, 1 / 2, 1 This should be understood to include values ​​such as integer tenths and hundredths.

[0069] Various aspects of the invention are described in further detail in the subsections below.

[0070] Method of Invention The present invention provides a method for treating T-cell proliferation, comprising administering to a patient cyclophosphamide and fludarabine. The present invention is directed to a method of conditioning a patient in need of cell therapy. 2 / day~about 2000mg / m 2 / day cyclophosphamide and approximately 20 mg / m 2 / day~900mg / m 2 Conditioning the patient with fludarabine / day is followed by administering T Enhance the efficacy of cell therapy while avoiding higher doses of cyclophosphamide and / or fluticasone It has been shown to reduce the incidence and / or severity of adverse events associated with ludarabine.

[0071] The present invention demonstrates that administration of cyclophosphamide and fludarabine prior to T cell therapy can improve The endogenous lymphocytes that are reduced include, but are not limited to, endogenous regulatory T cells, B cells, natural killer cells, CD4+ T cells, CD8+ T cells, or any combination thereof, which may inhibit the anti-tumor effect of adoptively transferred T cells. Endogenous lymphocytes may compete with adoptively transferred T cells for access to antigens and supportive cytokines. The conditioning treatment used eliminates this competition, leading to increased levels of endogenous cytokines. After the adoptively transferred T cells are administered to the patient, they respond to increased levels of endogenous homeostatic cytokines. In addition, cyclophosphamide and fludarabine treatment can cause tumor cell death and lead to an increase in tumor antigens in the patient's serum. This can enhance the activation and / or availability of antigen-presenting cells in patients prior to receiving T cell therapy. Without being bound by any theory, conditioning with cyclophosphamide and fludarabine may promote homeostatic proliferation, activity, and proliferation of T cells. The immune environment is modified through the induction of molecules that can favor the synthesis and transport of the immune system.

[0072] Previous studies have used high doses of cyclophosphamide and fludarabine to reduce the number of endogenous lymphocytes. However, these harsh conditioning regimens are associated with severe and potentially fatal adverse events. Surprisingly, this method does not significantly reduce the number of adoptively transferred T cells. It has been found that this increases the efficacy of the drug while reducing the incidence and severity of adverse events.

[0073] In some embodiments, administration of cyclophosphamide and fludarabine reduces endogenous lymphocytes. In some embodiments, administration of cyclophosphamide and fludarabine increases the availability of homeostatic cytokines. In some embodiments, administration of cyclophosphamide and fludarabine reduces the effector T cells administered after conditioning. Enhances function. In some embodiments, administration of cyclophosphamide and fludarabine enhances the activation and / or availability of antigen-presenting cells.

[0074] In one aspect, the present invention provides a method for administering a dose of about 200 mg / m 2 / day~about 2000mg / m 2 / day dose of cyclophosphamide amide and approximately 20 mg / m 2 / day~about 900mg / m2 / day dose of fludarabine In another aspect, the present invention provides a method for conditioning a patient in need of T cell therapy. The invention provides patients with approximately 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day) and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day) and a method for conditioning a patient in need of T cell therapy, comprising administering In that case, the patient had increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, and sVCAM-1 after administration of cyclophosphamide and fludarabine. or any combination thereof, e.g., IL-15, IP-10, and / or IL-7, and In one embodiment, the present invention provides a method for treating MIP-1b-associated leukemia, the method comprising administering to a subject a subject a therapeutically effective amount of MIP-1b. Ming gave patients approximately 1110 mg / m 2 / day~about 2000mg / m 2 / day dose of cyclophosphamide and approximately 20 mg / m 2 / day~about 900mg / m 2 / day, e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 In another embodiment, the present invention provides a method of conditioning a patient in need of T cell therapy comprising administering fludarabine at a dose of about 1110 mg / m 2 / day~about 2000mg / m 2 / day dose of cyclophosphamide and approximately 20 mg / m 2 / day~about 900mg / m 2 / day, e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day dose of fludarabine and a method for conditioning a patient in need of T cell therapy, comprising: Subjects reported increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any of its components after administration of cyclophosphamide and fludarabine. Any combination of these, for example, IL-15, IP-10, and / or IL-7, or reduced blood In one aspect, the present invention provides a method for treating a patient with HIV-1-associated leukemia, the method comprising administering to the patient a ... Cyclophosphamide at a dose of about 30 mg / kg / day to less than 60 mg / kg / day and about 20 mg / m 2 / day~about 900mg / m 2 / day, e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day dose Conditioning a patient in need of T-cell therapy, including administering fludarabine This includes a method for

[0075] In another embodiment, the present invention provides a method for administering a dose of about 200 mg / m 2 / day~about 2000mg / m 2 / day dose of cyclophosphamide Famid and approximately 20 mg / m 2 / day~about 900mg / m 2 1 / day dose of fludarabine. and methods for reducing or depleting endogenous lymphocytes in patients in need of T cell therapy. In another embodiment, the present invention provides a method for administering about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day) and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day) 2. The method of claim 1, further comprising administering fludarabine at a dose of and a method for reducing or depleting immune lymphocytes, wherein the patient experiences increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, and / or IL-16 following administration of cyclophosphamide and fludarabine. , IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof, e.g., IL-15, IP-10, and / or IL-7, or reduced serum levels of perforin and In one aspect, the present invention provides a method for administering to a patient a dose of about 1110 mg / m 2 / day~about 2000mg / m 2 / day dose of cyclophosphamide and approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day) of fludarabine. Methods for reducing or depleting endogenous lymphocytes in patients requiring T cell therapy, including In one embodiment, the method comprises administering cyclophosphamide and fludarabine to a patient, wherein the patient exhibits increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof, e.g., IL-15, IP-10, and / or IL-7, or decreased serum levels of perforin and / or MIP-1b. The present invention provides a method for administering cyclophosphamide to a patient at a dose of at least 30 mg / kg / day but less than 60 mg / kg / day and about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day) of fludarabine, wherein the patient exhibits increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof, after administration of cyclophosphamide and fludarabine. For example, decreased serum levels of IL-15, IP-10, and / or IL-7, or perforin MIP-1b and / or MIP-1b.

[0076] In another embodiment, the present invention provides a method for administering a dose of about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day) and cyclophosphamide at a dose of approximately 20 mg / m 2 / day ~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m2 / day, or 60 mg / m 2 / day) Homeostasis in a patient requiring T cell therapy, including administering a dose of fludarabine In another embodiment, the present invention provides a method for increasing cytokine availability in a patient by administering about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day) and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2

[0013] The present invention also includes a method of increasing homeostatic cytokine availability in a patient in need of T cell therapy, comprising administering fludarabine at a dose of 0.05 mg / day (0.05 mg / day), wherein the patient exhibits increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, and / or IL-15 after administration of cyclophosphamide and fludarabine. or any combination thereof, e.g., IL-15, IP-10, and / or IL-7, and In one embodiment, the present invention provides a method for treating MIP-1b-associated leukemia, the method comprising administering to a subject a subject a therapeutically effective amount of MIP-1b. Ming gave patients approximately 1110 mg / m 2 / day~about 2000mg / m 2 / day dose of cyclophosphamide and approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day) of fludarabine, wherein the patient experiences increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof, e.g., after administration of cyclophosphamide and fludarabine. For example, IL-15, IP-10, and / or IL-7, or decreased serum levels of perforin In one aspect, the present invention provides a method for administering to a patient cyclophosphamide at a dose of about 30 mg / kg / day to less than 60 mg / kg / day and about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 and increasing homeostatic cytokine availability in a patient in need of T cell therapy, the method comprising administering fludarabine at a dose of 100 mg / day. wherein the patient exhibits increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof, e.g., IL-15, IP-10, and / or IL-7, or decreased serum levels of perforin and / or MIP-1b, after administration of cyclophosphamide and fludarabine.

[0077] In one particular embodiment, the present invention provides a patient with a dose of about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day) and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day) 2. Administration of fludarabine in a patient in need of T cell therapy, comprising administering a dose of In another aspect, the present invention provides a method for enhancing the effector function of T cells in a patient. Approximately 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day) and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day) of fludarabine. and / or IL-7, wherein the patient exhibits increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof, e.g., IL-15, IP-10, and / or IL-7, or decreased serum levels of perforin and / or MIP-1b, following administration of cyclophosphamide and fludarabine. In such a case, the present invention provides a patient with approximately 1110 mg / m 2 / day~about 2000mg / m 2 / day dose of cyclophosphamide and approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2

[0013] The present invention also includes a method for enhancing effector function of administered T cells in a patient in need of T cell therapy, comprising administering fludarabine at a dose of 100 mg / day (100 mg / day) of fludarabine, wherein the patient After administration of cyclophosphamide and fludarabine, increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination of IL-15, IP-10, and / or IL-7, or reduced serum levels In one aspect, the present invention provides a method for administering to a patient a dose of cyclophosphamide at a dose of about 30 mg / kg / day to less than 60 mg / kg / day and a dose of about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 and / or IL-7, or decreased serum levels of perforin and / or fludarabine. indicates MIP-1b.

[0078] In some embodiments, the present invention provides a method for administering a dose of about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day) and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 In another aspect, the present invention provides a method for enhancing the activation and / or availability of antigen-presenting cells in a patient in need of T cell therapy, comprising administering fludarabine at a dose of 100 mg / day. , patients, approximately 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day) and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day) and / or a method for enhancing the activation and / or availability of antigen-presenting cells in a patient in need of T cell therapy, comprising administering to the patient increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof, e.g., IL-15, IP-10, or a ... , and / or IL-7, or reduced serum levels of perforin and / or MIP-1b. In one aspect, the present invention provides a method for treating a patient with steroid therapy comprising administering a dose of about 1110 mg / m 2 / day~about 2000mg / m 2 / day dose of cyclophosphamide and approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 210. A method for enhancing the activation and / or availability of antigen-presenting cells in a patient in need of T cell therapy, comprising administering fludarabine at a dose of 100 mg / day. wherein the patient exhibits increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof, e.g., IL-15, IP-10, and / or IL-7, or decreased serum levels of perforin and / or MIP-1b, following administration of cyclophosphamide and fludarabine. The invention provides a method for administering cyclophosphamide to a patient at a dose of about 30 mg / kg / day to less than 60 mg / kg / day and about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2

[0013] The present invention also includes a method for enhancing the activation and / or availability of antigen-presenting cells in a patient in need of T cell therapy, comprising administering fludarabine at a dose of 0.1 mg / day, wherein the patient is Increased serum levels of IL-7, IL-15, and I after administration of clofosfamide and fludarabine Il-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any of these any combination thereof, e.g., IL-15, IP-10, and / or IL-7, or reduced serum levels of perforin and / or MIP-1b.

[0079] The methods of the present invention include administering cyclophosphamide and fludarabine prior to T cell therapy. The timing of administration of each component can be adjusted to maximize efficacy. As described herein, the day on which T cell therapy is administered is referred to as day 0. Cyclophosphamide and fludarabine can be administered any time before administration of T cell therapy. In some embodiments, the administration of cyclophosphamide and fludarabine is in accordance with the administration of T cell therapy. In other embodiments, the administration of cyclophosphamide begins at least 7 days, at least 6 days, at least 5 days, at least 4 days, at least 3 days, at least 2 days, or at least 1 day before the administration of cyclophosphamide. Administration of Famid and Fludarabine should be at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days prior to administration of T-cell therapy. Or at least 14 days prior. In one embodiment, administration of cyclophosphamide and fludarabine begins 7 days prior to administration of T cell therapy. In another embodiment, administration of cyclophosphamide and fludarabine begins 5 days prior to administration of T cell therapy.

[0080] In one particular embodiment, administration of cyclophosphamide begins about 7 days prior to administration of T cell therapy and administration of fludarabine begins about 5 days prior to administration of T cell therapy. In another embodiment, administration of cyclophosphamide begins about 5 days prior to administration of T cell therapy and administration of fludarabine begins about 5 days prior to administration of T cell therapy. This begins approximately 5 days before cell therapy begins.

[0081] The timing of administration of each component can be adjusted to maximize efficacy. Generally, cyclophosphamide and fludarabine can be administered daily. In some embodiments, cyclophosphamide and fludarabine can be administered for about 2 days, about 3 days, about 4 days, It is administered daily for about 5 days, about 6 days, or about 7 days. In another embodiment, cyclophosphamide is administered daily for two days and fludarabine is administered daily for five days. In another embodiment, cyclophosphamide and fludarabine are both administered daily for about three days.

[0082] As described herein, the day that T cell therapy is administered to a patient is referred to as day 0. In some embodiments, cyclophosphamide is administered to a patient on days 0 to 7 and 6 (i.e., days -7 and -6). In other embodiments, cyclophosphamide is administered on days -5, -4, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -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, -49, -40, -41, -42, -43, -44, -45, -46, -47, -48, -49, -49, -44, -45, -46, -47, -48, -49, -49, -50, -51, -52, -53, -54, -55, -56, -57, -58, -59, -60, -61, -62, -63, -64, -65, -66, -66, -67, -68, -69, -69, -70, -71, -72, -73, -74, -75, -76, -77, -78, -79, -86, -87, -88, -89, -90, -91, -92, -93, -94, -95, -96, -97, -98, -9 and on day -3. In some embodiments, fludarabine is administered to the patient on day -5, day -4, day -3, day -2, and day -1. In other embodiments, fludarabine is administered to the patient on day -5, day -4, and day -3.

[0083] Cyclophosphamide and fludarabine can be administered on the same day or on different days.When cyclophosphamide and fludarabine are administered on the same day, cyclophosphamide can be administered before or after fludarabine.In one embodiment, cyclophosphamide is administered to patients on days -7 and -6, and fludarabine is administered to patients on days -5, -4, -3, -2, and -1.In another embodiment, cyclophosphamide is administered to patients on days -5, -4, and -3, and fludarabine is administered to patients on days -5, -4, and -3.

[0084] In certain embodiments, cyclophosphamide and fludarabine can be administered simultaneously or sequentially. In one embodiment, cyclophosphamide is administered to the patient before fludarabine. In another embodiment, cyclophosphamide is administered to the patient after fludarabine.

[0085] Cyclophosphamide and fludarabine may be administered by any route, including intravenously (IV). In some embodiments, cyclophosphamide is administered by IV over about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 90 minutes, or about 120 minutes. In some embodiments, fludarabine is administered by IV over about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 90 minutes, or about 120 minutes.

[0086] In certain embodiments, the T cell therapy is administered after administration of cyclophosphamide and fludarabine. In some embodiments, T cell therapy comprises adoptive cell therapy. In certain embodiments, adoptive cell therapy includes tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered cell therapy, and the like. Autologous cell therapy (eACT), and allogeneic T cell transplantation. In one particular embodiment, eACT is In another embodiment, the method comprises administering engineered antigen-specific chimeric antigen receptor (CAR)-positive T cells. eACT involves the administration of engineered antigen-specific T cell receptor (TCR) positive (+) T cells. In some embodiments, the engineered T cells treat a tumor in a patient.

[0087] In one particular embodiment, the present invention provides a patient with a dose of about 500 mg / m 2 / day dose of cyclophosphamide and and approximately 60 mg / m 2 1. A method for treating a patient in need of T cell therapy, comprising administering fludarabine at a dose of 100 mg / day to a patient in need of T cell therapy. In another aspect, the present invention provides a method of conditioning a patient with cyclophosphamide administered on days -5, -4, and -3, and fludarabine administered on days -5, -4, and -3. 2 / day dose of cyclophosphamide and approximately 60 mg / m 2 / day and administering a dose of fludarabine to a patient in need of T-cell therapy. In another aspect, the present invention provides a method of administering cyclophosphamide to a patient on days -7 and -6, and fludarabine on days -5, -4, -3, -2, and -1. 2 / day dose of cyclophosphamide and approximately 30 mg / m 2 / day dose of Fulda Methods for conditioning a patient in need of T cell therapy, including administering rabies In another embodiment, the present invention provides a method of treating a patient with cyclophosphamide at about 300 mg / m 2 / day dose of cyclophosphamide and approximately 60 mg / m 2 Fludarabine was administered at a dose of 100 mg / day. and a method for conditioning a patient in need of T cell therapy, the method comprising: In this case, cyclophosphamide is administered on days −7 and −6, and fludarabine is administered on days −5, −4, −3, −2, and −1.

[0088] Various other interventions can be included in the methods described herein.For example, it is well known that cyclophosphamide and fludarabine can cause adverse events in patients after administration.It is within the scope of the present invention that compositions can also be administered to patients to reduce some of these adverse events.In some embodiments, this method further comprises administering saline to patients.Saline can be used to reduce the risk of cyclophosphamide and / or fludarabine administration. either before or after cyclophosphamide and / or fludarabine In certain embodiments, saline may be administered to the patient both before and after administration of cyclophosphamide and / or fludarabine. In one particular embodiment, the saline solution is administered on each infusion day in combination with cyclophosphamide and / or Before administration of fludarabine and after administration of cyclophosphamide and / or fludarabine is administered to the patient.

[0089] The saline solution may be administered to the patient by any route, including, for example, intravenously or orally. In some embodiments, the method includes administering about 0.1 L, about 0.2 L, about 0.3 L, about 0.4 L, about 0.5 L, about 0.6 L, about 0.7 L, about 0.8 L, about 0.9 L, about 1 L, about 1.1 L, about 1.2 L, about 1.3 L, about 1.4 L, about 1.5 L, about 1.6 L, about 1.7 L, about 1.8 L, about 1.9 L, or about 2.0 L of saline. The NaCl in the saline solution may be at a final concentration of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9 L, about 1 L, about 1.1 L, about 1.2 L, about 1.3 L, about 1.4 L, about 1.5 L, about 1.6 L, about 1.7 L, about 1.8 L, about 1.9 L, or about 2.0 L. The composition can be dissolved to about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0%. In one embodiment, the method includes administering 1.0 L of 0.9% NaCl saline to the patient. In a particular embodiment, the method includes administering 1.0 L of 0.9% NaCl saline to the patient prior to administration of cyclophosphamide and / or fludarabine on each infusion day. and 0.9% NaCl solution after administration of cyclophosphamide and / or fludarabine. The method includes administering 1.0 L of saline.

[0090] Additionally, adjuvants and excipients may also be administered to the patient. For example, mesna (2-sulfonyl 2-methylpropional). sodium phenylethanesulfonate) occurs after treatment with cyclophosphamide It is an adjunct agent that acts as an antidote to prevent possible hemorrhagic cystitis and hematuria. Cyclophosphamide can be converted in vivo to uremic metabolites such as acrolein. Mesna helps detoxify these metabolites by reacting its sulfhydryl group with vinyl groups. Mesna also increases the urinary excretion of cysteine. In certain embodiments, the method further comprises administering mesna to the patient. Mesna can be administered before administration of cyclophosphamide and / or fludarabine, after administration of cyclophosphamide and / or fludarabine, or before administration of cyclophosphamide and / or fludarabine. In one embodiment, mesna can be administered both intravenously and orally (by mouth). For example, oral mesna is given with oral cyclophosphamide. It can be done.

[0091] Additionally, exogenous cytokines may also be administered to the patient in the methods described herein. As noted above, reducing the number of endogenous lymphocytes may promote the proliferation, activity, and proliferation of adoptively transferred T cells. It is hypothesized that this increases the bioavailability of endogenous molecules, such as cytokines, which can be favorably activated and transported. Thus, various cytokines can be administered to the patient. In one embodiment, the method further comprises administering one or more doses of IL-2, IL-15, IL-7, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof. In a specific embodiment, the method further comprises administering one or more doses of IL-2, IL-15, IL-7, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof. The dose of IL-2 is at least about 10,000 IU / kg, at least about 10,000 IU / kg, and at least about 10,000 IU / kg. at least about 50,000 IU / kg, at least about 100,000 IU / kg, at least about 200,000 IU / kg, at least It can be at least about 400,000 IU / kg, at least about 600,000 IU / kg, at least about 700,000 IU / kg, at least about 800,000 IU / kg, or at least about 1,000,000 IU / kg.

[0092] Cyclophosphamide and fludarabine Cyclophosphamide (ENDOXAN®, CYTOXAN®, PROCYTOX®, NEOSAR®, REVIMMUNE®, CYCLOBLASTIN®) is a potent immunosuppressant. Cyclophosphamide is an alkylating agent that is a nitrogen mustard derivative with potent anti-cancer activity. It acts as an antineoplastic agent and is used to treat various types of cancer, including lymphoma, multiple myeloma, leukemia, mycosis fungoides, neuroblastoma, ovarian cancer, eye cancer, and breast cancer, as well as autoimmune disorders.

[0093] After administration to patients, cyclophosphamide is converted in the liver to acrolein and phosphoramide. Together, these metabolites cross-link DNA by adding an alkyl group to the nitrogen atom at the 7-position of the imidazole ring of the guanine base in both resting and dividing cells. As a result, DNA replication is inhibited, leading to cell death.

[0094] In the present invention, the dose of cyclophosphamide may be adjusted depending on the desired effect, for example, to regulate endogenous lymphocyte depletion and / or to control the severity of adverse events. For example, the dose of cyclophosphamide can be about 300 mg / m 2 / day, approximately 900 mg / m 2 In some embodiments, the dose of cyclophosphamide can be less than about 350 mg / m 2 / day~about 2000mg / m 2 / day, at least about 400 mg / m 2 / day~about 2000mg / m 2 / day, approximately 450m g / m 2 / day~about 2000mg / m 2 / day, about 500mg / m 2 / day~about 2000mg / m 2 / day, about 550mg / m 2 / day~about 2000mg / m 2 / day, or approximately 600 mg / m 2 / day~about 2000mg / m 2 In another embodiment, the dose of cyclophosphamide is about 350 mg / m 2 / day~about 1500mg / m 2 / day, about 350mg / m 2 / day~about 1000mg / m 2 / day, about 400mg / m 2 / day~about 900mg / m 2 / day, about 450mg / m 2 / day~about 800mg / m 2 / day, approximately 450mg / m 2 / day~about 700mg / m 2 / day, about 500mg / m 2 / day~about 600mg / m 2 / day, or approximately 300 mg / m 2 / day~about 500mg / m 2 In certain embodiments, the dose of cyclophosphamide is about 350 mg / m 2 / day, about 400mg / m 2 / day, about 450mg / m 2 / day, about 500mg / m 2 / day, about 550mg / m 2 / day, about 600mg / m 2 / day, approximately 650mg / m 2 / day, about 700mg / m 2 / day, about 800mg / m 2 / day, approximately 900mg / m 2 / day, or approximately 1000 mg / m 2 In one particular embodiment, the dose of cyclophosphamide is about 200 mg / m 2 In one particular embodiment, cyclophosphamide is administered at a dose of 100 mg / day. The dose of amide is approximately 300 mg / m 2In another embodiment, the dose of cyclophosphamide is Approximately 500mg / m 2 In other embodiments, the dose of cyclophosphamide is about 200 mg / m 2 / day~about 2000mg / m 2 / day, about 300mg / m 2 / day~about 2000mg / m 2 / day, about 400mg / m 2 / day~about 2000mg / m 2 / day, about 500mg / m 2 / day~about 2000mg / m 2 / day, about 600mg / m 2 / day~about 2000mg / m 2 / day, about 700mg / m 2 / day~about 2000mg / m 2 / day, about 800mg / m 2 / day~about 2000mg / m 2 / day, approximately 900mg / m 2 / day~about 2000mg / m 2 / day, about 1000mg / m 2 / day~about 2000mg / m 2 / day, approximately 1100mg / m 2 / day~about 2000mg / m 2 / day, about 1200mg / m 2 / day~about 2000mg / m 2 / day, approximately 1300mg / m 2 / day~about 2000mg / m 2 / day, approximately 1400mg / m 2 / day~about 2000mg / m 2 / day, about 1500mg / m 2 / day~about 2000mg / m 2 / day, approximately 1600mg / m 2 / day~about 2000mg / m 2 / day, approximately 1700mg / m 2 / day~about 2000mg / m 2 / day, approximately 1800mg / m 2 / day~about 2000mg / m 2 / day, approximately 1900mg / m 2 / day~about 2000mg / m 2 / day, about 200mg / m 2 / day ~ approx. 1900mg / m 2 / day, about 400mg / m 2 / day~about 1800mg / m 2 / day, about 500mg / m 2 / day~about 1700mg / m 2 / day, about 600mg / m 2 / day~about 1600mg / m 2 / day, about 700mg / m 2 / day~about 1500mg / m 2 / day, about 800mg / m 2 / day ~about 1400mg / m 2 / day, approximately 900mg / m 2 / day ~ approx. 1300mg / m 2 / day, about 1000mg / m 2 / day ~ approx. 1200mg / m 2 / day , about 1100mg / m 2 / day ~ approx. 1200mg / m 2 / day, or approximately 1110 mg / m 2 / day~about 1150mg / m 2 / day.

[0095] Fludarabine phosphate (FLUDARA®) is a phosphate-rich drug whose sugar moiety is ribose or deoxyribose. Fludarabine is a synthetic purine nucleoside that differs from physiological nucleosides by containing arabinose instead of ribose. It acts as a purine antagonist antimetabolite and is used to treat various types of hematological malignancies, including various lymphomas and leukemias.

[0096] After administration to patients, fludarabine is rapidly dephosphorylated to 2-fluoro-ara-A, which is then phosphorylated intracellularly by deoxycytidine kinase to the active triphosphate, 2-fluoro-ara-ATP. This metabolite is then converted to the ribonuclease α, which is then activated by DNA polymerase alpha, ribonuclease 2, and ribonucleotide 1 (RI). Fludarabine interferes with DNA replication by inhibiting DNA synthesis, possibly through inhibition of thrombin reductase and DNA primase. As a result, administration of fludarabine inhibits the growth of dividing cells. This leads to increased cell death in the

[0097] In the present invention, the dose of fludarabine can be adjusted depending on the desired effect. For example, the dose of fludarabine is 30 mg / m 2 / day, higher than 900 mg / m 2 / days In some embodiments, the dose of fludarabine is about 35 mg / m 2 / day~about 900mg / m 2 / day, Approximately 40mg / m 2 / day~about 900mg / m 2 / day, about 45mg / m 2 / day~about 900mg / m 2 / day, about 50mg / m 2 / day~about 900mg / m 2 / day, about 55mg / m 2 / day~about 900mg / m 2 / day, or approximately 60 mg / m 2 / day~about 900mg / m 2 / day In another embodiment, the dose of fludarabine is about 35 mg / m 2 / day~about 900mg / m 2 / day, about 35mg / m 2 / day~about 800mg / m 2 / day, about 35mg / m 2 / day~about 700mg / m 2 / day, about 35mg / m 2 / day~about 600mg / m 2 / day, about 35mg / m 2 / day~about 500mg / m 2 / day, about 35mg / m 2 / day ~ approx. 400mg / m 2 / day, about 35mg / m 2 / day ~ approx. 300mg / m 2 / day, about 35mg / m 2 / day~about 200mg / m 2 / day, about 35mg / m 2 / day~about 100mg / m 2 / day, about 40mg / m 2 / day ~about 90mg / m 2 / day, about 45mg / m 2 / day~about 80mg / m 2 / day, about 45mg / m 2 / day~about 70mg / m 2 / day, or approximately 50 mg / m 2 / day~about 60mg / m 2 In certain embodiments, the dose of fludarabine is about 35 mg / m 2 / day, about 40mg / m 2 / day, about 45mg / m 2 / day, about 50mg / m 2 / day, about 55mg / m 2 / day, about 60mg / m 2 / day, about 65mg / m 2 / day, about 70mg / m 2 / day, about 75mg / m 2 / day, about 80mg / m 2 / day, about 85mg / m 2 / day, about 90mg / m 2 / day, approximately 95mg / m 2 / day, about 100mg / m 2 / day, about 200mg / m 2 / day, or approximately 300 mg / m 2 / day. Other states In this setting, the fludarabine dose is approximately 110 mg / m 2 / day, 120mg / m 2 / day, 130mg / m 2 / day, 140mg / m 2 / day, 150mg / m 2 / day, 160mg / m 2 / day, 170mg / m 2 / day, 180mg / m 2 / day, or 190 mg / m 2 / day In some embodiments, the dose of fludarabine is about 210 mg / m 2 / day, 220mg / m 2 / day, 230mg / m 2 / day, 240mg / m 2 / day, 250mg / m 2 / day, 260mg / m 2 / day, 270mg / m2 / day, 280mg / m 2 / day, or 290 mg / m 2 In one particular embodiment, the dose of fludarabine is about 20 mg / m 2 / day. In one embodiment, the dose of fludarabine is about 30 mg / m 2 In another embodiment, the dose of fludarabine is about 60 mg / m 2 In another embodiment, the dose of fludarabine is about 25 mg / m 2 / day.

[0098] The dose of cyclophosphamide and fludarabine can be increased or decreased together or independently.For example, the dose of cyclophosphamide can be increased while the dose of fludarabine is decreased, or the dose of cyclophosphamide can be decreased while the dose of fludarabine is increased.Alternatively, the doses of both cyclophosphamide and fludarabine can be increased or decreased together.

[0099] In some embodiments, the dose of cyclophosphamide is 100 mg / m 2 / day (or 110 mg / m 2 / day , 120 mg / m 2 / day, 130mg / m 2 / day or 140 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0100] In some embodiments, the dose of cyclophosphamide is 150 mg / m 2 / day (or 160 mg / m 2 / day , 170 mg / m 2 / day, 180mg / m 2 / day or 190 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0101] In some embodiments, the dose of cyclophosphamide is about 200 mg / m 2 / day (or 210 mg / m 2 / day, 220mg / m 2 / day, 230mg / m 2 / day or 240 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0102] In some embodiments, the dose of cyclophosphamide is 250 mg / m 2 / day (or 260 mg / m 2 / day , 270 mg / m 2 / day, 280mg / m 2 / day or 290 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0103] In some embodiments, the dose of cyclophosphamide is 300 mg / m 2 / day (or 310 mg / m 2 / day , 320 mg / m 2 / day, 330mg / m 2 / day or 340 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0104] In some embodiments, the dose of cyclophosphamide is 350 mg / m 2 / day (or 360 mg / m 2 / day , 370 mg / m 2 / day, 380mg / m 2 / day or 390 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0105] In some embodiments, the dose of cyclophosphamide is 400 mg / m 2 / day (or 410 mg / m 2 / day , 420 mg / m 2 / day, 430mg / m 2 / day or 440 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0106] In some embodiments, the dose of cyclophosphamide is 450 mg / m 2 / day (or 460 mg / m 2 / day , 470 mg / m 2 / day, 480mg / m 2 / day or 490 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0107] In some embodiments, the dose of cyclophosphamide is 500 mg / m 2 / day (or 510 mg / m 2 / day , 520 mg / m 2 / day, 530mg / m 2 / day or 540 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0108] In some embodiments, the dose of cyclophosphamide is 550 mg / m 2 / day (or 560 mg / m 2 / day , 570 mg / m 2 / day, 580mg / m 2 / day or 590 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0109] In some embodiments, the dose of cyclophosphamide is 600 mg / m 2 / day (or 610 mg / m 2 / day , 620 mg / m 2 / day, 630mg / m 2 / day or 640 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0110] In some embodiments, the dose of cyclophosphamide is 650 mg / m 2 / day (or 660 mg / m 2 / day , 670 mg / m 2 / day, 680mg / m 2 / day, or 690 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0111] In some embodiments, the dose of cyclophosphamide is 700 mg / m 2 / day (or 710 mg / m 2 / day , 720 mg / m 2 / day, 730mg / m 2 / day, or 740 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0112] In some embodiments, the dose of cyclophosphamide is 750 mg / m 2 / day (or 760 mg / m 2 / day , 770 mg / m 2 / day, 780mg / m 2 / day, or 790 mg / m 2 / day) and the fludarabine dose was 5 mg / m2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0113] In some embodiments, the dose of cyclophosphamide is 800 mg / m 2 / day (or 810 mg / m 2 / day , 820 mg / m 2 / day, 830mg / m 2 / day or 840 mg / m 2 / day) and the dose of fludarabine was 5m g / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0114] In some embodiments, the dose of cyclophosphamide is 850 mg / m 2 / day (or 860 mg / m 2 / day , 870 mg / m 2 / day, 880mg / m 2 / day, or 890 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0115] In some embodiments, the dose of cyclophosphamide is 900 mg / m 2 / day (or 910 mg / m 2 / day , 920 mg / m 2 / day, 930mg / m 2 / day or 940 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m2 / day, or 75 mg / m 2 / day.

[0116] In some embodiments, the dose of cyclophosphamide is 950 mg / m 2 / day (or 960 mg / m 2 / day , 970 mg / m 2 / day, 980mg / m 2 / day or 990 mg / m 2 / day) and the fludarabine dose was 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day , 40 mg / m 2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0117] In some embodiments, the dose of cyclophosphamide is 1000 mg / m 2 / day (or 1010 mg / m 2 / day, 1020mg / m 2 / day, 1030mg / m 2 / day, or 1040 mg / m 2 / day) and the dose of fludarabine is 5 mg / m 2 / day, 10mg / m 2 / day, 15mg / m 2 / day, 20mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, 35mg / m 2 / day, 40mg / m2 / day, 45mg / m 2 / day, 50mg / m 2 / day, 55mg / m 2 / day, 60mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, or 75 mg / m 2 / day.

[0118] In other embodiments, the dose of cyclophosphamide is 100 mg / m 2 / day~650mg / m 2 / day and the fludarabine dose is 10 mg / m 2 / day~50mg / m 2 In other embodiments, the dose of cyclophosphamide is 150 mg / m 2 / day~600mg / m 2 / day and the fludarabine dose is 20 mg / m 2 / day~50mg / m 2 In other embodiments, the dose of cyclophosphamide is 200 mg / m 2 / day~550mg / m 2 / day and the fludarabine dose is 20 mg / m 2 / day~40mg / m 2 In other embodiments, the dose of cyclophosphamide is 250 mg / m 2 / day~550mg / m 2 / day and the fludarabine dose is 15 mg / m 2 / day~45mg / m 2 / day.

[0119] In certain embodiments, the dose of cyclophosphamide is 1000 mg / m 2 / day and the fludarabine dose is 60 mg / m 2 / day, 65mg / m 2 / day, 70mg / m 2 / day, 75mg / m 2 / day, 80mg / m 2 / day, 85mg / m 2 / day, 90mg / m 2 / day, 95mg / m 2 / day, 100mg / m 2 / day, 105mg / m 2 / day, 110mg / m 2 / day, 115mg / m 2 / day, 120mg / m 2 / day, 125mg / m 2 / day, 130mg / m 2 / day, 135mg / m 2 / day, 140mg / m 2 / day, 145mg / m 2 / day, 150mg / m 2 / day, 155mg / m 2 / day, 160mg / m 2 / day, 165mg / m 2 / day, 170mg / m 2 / day, 175mg / m 2 / day, 180mg / m 2 / day, 185mg / m 2 / day, 190mg / m 2 / day, 195mg / m 2 / day, 200mg / m 2 / day, 205mg / m 2 / day, 210mg / m 2 / day, 215mg / m 2 / day, 220mg / m 2 / day, 225mg / m 2 / day, 230mg / m 2 / day, 235mg / m 2 / day, 240mg / m 2 / day, 245mg / m 2 / day, or 250 mg / m 2 / day.

[0120] In some embodiments, the dose of cyclophosphamide is 200 mg / m 2 / day and fludarabine The dose is 20 mg / m 2 In some embodiments, the dose of cyclophosphamide is 200 mg / m 2 / day and the fludarabine dose is 30 mg / m 2 / day. In some embodiments, cyclophosphamide The dose of Famid is 300 mg / m2 / day and the fludarabine dose is 30 mg / m 2 / day. Other states In such cases, the dose of cyclophosphamide is 300 mg / m 2 / day and the fludarabine dose is 60 mg / m 2 In other embodiments, the dose of cyclophosphamide is 500 mg / m 2 / day and the fludarabine dose is 30 mg / m 2 In yet other embodiments, the dose of cyclophosphamide is 500 mg / m 2 / day and the fludarabine dose is 60 mg / m 2 / day. In some embodiments, cyclophosphamide The dose of Famid is approximately 1110 mg / m 2 / day and the fludarabine dose is 25 mg / m 2 In some embodiments, the dose of cyclophosphamide is about 2000 mg / m 2 / day and the fludarabine dose is 25 mg / m 2 In some embodiments, the dose of cyclophosphamide is 30 mg / kg / day and the dose of fludarabine is 25 mg / m 2 / day.

[0121] T cell therapy The present invention provides a method for enhancing the efficacy of T cell therapy by conditioning a patient by administering cyclophosphamide and fludarabine to the patient. Conditioning regimens generally favor homeostatic proliferation, activation, and trafficking of T cells. A variety of different T cell therapies can benefit from the conditioning methods described herein, as these serve to modify the immune environment through the induction of molecules that can Those skilled in the art will also appreciate that this conditioning regimen is applicable to any method of treating a patient that involves administering one or more T cells to the patient.

[0122] For example, but not by way of limitation, the conditioning regimens described herein include tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), allogeneic T cell transplantation, non-T cell transplantation, and The efficacy of T cell therapy can be enhanced by adoptive T cell therapy selected from the group consisting of: TIL immunotherapy broadly includes any method of selecting, in vitro enriching, and administering to a patient autologous or allogeneic T cells that are capable of binding to tumor cells. TIL cells can be either autologous or allogeneic. Autologous cell therapy involves the sole administration of T cells capable of targeting tumor cells from the patient. Adoptive T cell therapy involves isolating T cells from a patient, enriching the T cells in vitro, and administering the T cells back into the same patient. Allogeneic T cell transplantation involves the use of ex vivo expanded natural T cells or The method may include the transplantation of genetically engineered T cells. Autologous cell therapy is adoptive T cell therapy in which a patient's own lymphocytes are isolated, genetically modified to express tumor-targeting molecules, expanded in vitro, and administered back to the patient. Non-T cell transplantation can be autologous or allogeneic therapy using non-T cells, such as, but not limited to, natural killer (NK) cells. This may include therapy.

[0123] In one particular embodiment, the T cell therapy of the present invention is engineered autologous cell therapy (eACT™). According to this embodiment, the method can include collecting blood cells from the patient prior to administration of cyclophosphamide and fludarabine. The isolated blood cells (e.g., T cells) can then be engineered to express chimeric antigen receptor ("engineered CAR T cells") or T cell receptor ("engineered TCR T cells"). In certain embodiments, the engineered CAR T cells or engineered TCR T cells are administered to a patient after administration of cyclophosphamide and fludarabine. In some embodiments, the engineered T cells treat a tumor in a patient.

[0124] In one embodiment, T cells can be engineered to express a chimeric antigen receptor. The chimeric antigen receptor can comprise a binding molecule for a tumor antigen. The binding molecule can be an antibody or an antigen-binding molecule thereof. For example, the antigen-binding molecule can be an scFv, Fab, Fab', Fv, F(ab')2, and dAb, as well as any fragment or combination thereof. You can choose.

[0125] The chimeric antigen receptor can further comprise a hinge region derived from an IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 alpha. In one particular embodiment, the hinge region is derived from the hinge region of IgG4.

[0126] The chimeric antigen receptor can also include a transmembrane domain. The transmembrane domain can be that of any transmembrane molecule that is a co-receptor on immune cells or that of a member of the immunoglobulin superfamily. In certain embodiments, the transmembrane domain is derived from the transmembrane domain of CD28, CD8 alpha, CD4, or CD19. In one particular embodiment, the transmembrane domain comprises a domain derived from the CD28 transmembrane domain.

[0127] The chimeric antigen receptor may further comprise one or more costimulatory signaling domains. For example, costimulatory signaling regions include CD28, OX-40, 41BB, CD27, inducible T cell costimulatory molecule (ICOS), CD3 gamma, CD3 delta, CD3 epsilon, CD247, and Ig alpha (CD79a). , or the signaling region of an Fc gamma receptor. In one particular embodiment, the costimulatory signaling region is the CD28 signaling region.

[0128] In one embodiment, the chimeric antigen receptor further comprises a CD3 zeta signaling domain. .

[0129] Chimeric antigen receptors can be engineered to target specific tumor antigens. In some embodiments, tumor antigens include CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha Fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2 / Neu, folate-binding protein, HIV-1 envelope Glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-llR alpha, kappa chain, Ram In one particular embodiment, the tumor antigen is selected from the group consisting of CD19, EGFRvIII, CSPG4, ERBB2, EGFRvIII, VEGFR2, a HER2-HER3 combination, a HER1-HER2 combination, and any combination thereof.

[0130] In another embodiment, T cell therapy involves administering to a patient engineered T cells expressing a T cell receptor ("engineered TCR T cells"). The T cell receptor (TCR) can include a binding molecule for a tumor antigen. In some embodiments, the tumor antigen is CD19, CD20, ROR1, or CD22. carcinoembryonic antigen, alpha fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate specific antigen, melanoma associated antigen, mutant p53, mutant ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R alpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 combination, HER1-HER2 combination, and any combination thereof.

[0131] In one embodiment, the TCR comprises a binding molecule to a viral oncogene. The viral oncogene is selected from human papillomavirus (HPV), Epstein-Barr virus (EBV), and human T-lymphotropic virus (HTLV).

[0132] In yet another embodiment, the TCR comprises a binding molecule to a testicular, placental, or fetal tumor antigen. In one particular embodiment, the testicular, placental, or fetal tumor antigen is NY-ESO-1, synovial sarcoma X-ray protein 1 (SYN)-1. and any combination thereof. are selected.

[0133] In another embodiment, the TCR comprises a binding molecule for a lineage-specific antigen. The lineage-specific antigen is selected from the group consisting of T-cell recognized melanoma antigen 1 (MART-1), gp100, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), and any combination thereof.

[0134] In one embodiment, the T cell therapy expresses a chimeric antigen receptor that binds to CD19, or engineered CAR T cells further comprising a CD28 costimulatory domain and a CD3-zeta signaling region In certain embodiments, the T cell therapy comprises administering to the patient KTE-C19.

[0135] T cell therapy encompassed by the present invention involves the transfer of T cells into a patient. The T cells are administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells, e.g., engineered CAR+ T cells or engineered The activated TCR+ T cells were at least approximately 10 4 pieces, at least about 10 5 pieces, at least about 10 6 Fewer pieces Both are about 10 7 pieces, at least about 10 8 pieces, at least about 10 9 pieces, or at least about 10 10 Individual In another embodiment, a therapeutically effective amount of T cells, e.g., engineered CAR+ T cells or engineered The number of activated TCR+ T cells is approximately 10 4 pieces, about 10 5 pieces, about 10 6 pieces, about 10 7 pieces, or about 10 8 It is an individual. In one embodiment, a therapeutically effective amount of T cells, e.g., engineered CAR+ T cells or engineered TCR+ T cells, is about 1 x 10 5 pieces / kg, approximately 2×10 5 pieces / kg, approximately 3×10 5 pieces / kg, approximately 4×10 5 pieces / kg, approximately 5×10 5 pieces / kg, approximately 6×10 5 pieces / kg, approximately 7×10 5 pieces / kg, approximately 8×10 5 pieces / kg, approximately 9×10 5 pieces / kg, approximately 1×10 6 pieces / kg, approximately 2×10 6 pieces / kg, approximately 3×10 6 pieces / kg, approximately 4×10 6 pieces / kg, approximately 5×10 6pieces / kg, approximately 6×10 6 pieces / kg, approximately 7×10 6 pieces / kg, approximately 8×10 6 pieces / kg, approximately 9×10 6 pieces / kg, approximately 1×10 7 pieces / kg, approximately 2×10 7 pieces / kg, approximately 3×10 7 pieces / kg, approximately 4×10 7 pieces / kg, approximately 5×10 7 pieces / kg, approximately 6×10 7 pieces / kg, approximately 7×10 7 pieces / kg, approximately 8×10 7 pieces / kg, or approximately 9 x 10 7 In a specific embodiment, T cells, e.g., engineered CAR+ T cells or engineered TCR+ T cells, are administered in a dose of 100 mg / kg. The therapeutically effective dose of cells is approximately 2 × 10 6 Pieces / kg.

[0136] In other embodiments, a therapeutically effective amount of T cells, e.g., engineered CAR+ T cells or engineered TCR+ T cells, , about 1.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 2.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 3.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 4.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 5.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 6.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 7.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 8.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 9.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approx. 0.5×10 6 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 2×10 6 / kg~about 9×10 7 / kg, about 3×10 6 / kg~about 9×10 7 / kg, about 4×10 6 / kg~about 9×10 7 / kg, about 5×10 6 / kg~about 9×10 7 / kg, about 6×10 6 / kg~about 9×10 7 / kg, about 7×10 6 / kg~about 9×10 7 / kg, about 8×10 6 / kg~about 9×10 7 / kg, about 9×10 6 / kg~about 9×10 7 / kg, about 1×10 7 / kg~about 9×10 7 / kg, about 2×10 7 / kg~about 9×10 7 / kg, about 3×10 7 / kg~about 9×10 7 / kg, about 4×10 7 / kg~about 9×10 7 / kg, about 5×10 7 / kg~about 9×10 7 / kg, about 6×10 7 / kg~about 9×10 7 / kg, about 7×10 7 / kg~about 9×10 7 / kg, about 8×10 7 / kg~about 9×10 7 / kg, about 2×10 6 / kg~about 8×10 7 / kg, about 2×10 6 / kg~about 7×10 7 / kg, about 2×10 6 / kg~about 6×10 7 / kg, about 2×10 6 / kg~about 5×10 7 / kg, about 2×10 6 / kg~about 4×10 7 / kg, about 2×10 6 / kg~about 3×10 7 / kg, about 2×106 pieces / kg ~ approx. 2×10 7 pieces / kg, approximately 2×10 6 pieces / kg ~ approx. 1×10 7 pieces / kg, approximately 2×10 6 pieces / kg ~ approx. 9×10 6 pieces / kg, approximately 2×10 6 pieces / kg ~ approx. 8×10 6 pieces / kg, approximately 2×10 6 pieces / kg ~ approx. 7×10 6 pieces / kg, approximately 2×10 6 pieces / kg ~ approx. 6×10 6 pieces / kg, approximately 2×10 6 pieces / kg ~ approx. 5×10 6 pieces / kg, approximately 2×10 6 pieces / kg ~ approx. 4×10 6 pieces / kg, approximately 2×10 6 pieces / kg ~ approx. 3×10 6 pieces / kg, approximately 3×10 6 pieces / kg ~ approx. 8×10 7 pieces / kg, approximately 4×10 6 pieces / kg ~ approx. 7×10 7 pieces / kg, approximately 5×10 6 pieces / kg ~ approx. 6×10 7 pieces / kg, approximately 6×10 6 pieces / kg ~ approx. 5×10 7 pieces / kg, approximately 7×10 6 pieces / kg ~ approx. 4×10 7 pieces / kg, approximately 8×10 6 pieces / kg ~ approx. 3×10 7 pieces / kg, or approximately 9 x 10 6 pieces / kg ~ approx. 2×10 7 In one embodiment, a therapeutically effective amount of engineered CAR T cells is about 0.8 x 10 T cells / kg. 6 pieces / kg ~ approx. 1.2×10 6 In one particular embodiment, a therapeutically effective amount of engineered CAR T The cells were 2.0 x 10 5 In one particular embodiment, the therapeutically effective amount of engineered CAR T cells is 1.0 x10 6 Pieces / kg.

[0137] Cytokine levels The present invention provides a method for treating T-cell proliferation, comprising administering to a patient cyclophosphamide and fludarabine. A method of conditioning a patient in need of T cell therapy is described. Administration of cyclophosphamide and fludarabine increases the levels of endogenous cytokines and modifies the immune environment in a way that favors homeostatic proliferation, activation, and trafficking of T cells. After the transplanted T cells are administered to the patient, they stimulate increased levels of endogenous cytokines. exposed to in.

[0138] Various cytokines can be concentrated in the serum of patients after administration of cyclophosphamide and fludarabine. In some embodiments, patients after administration of cyclophosphamide and fludarabine and / or T cell therapy have increased serum concentrations of interleukin (IL) 15, IL-7, IL-10, IL-5, IL-8, IL-1, IL-1b, IL-2, IL-3, IL-4, IL-6, IL-9, IL-11, IL-12, IL-12p40, IL-12p70, IL-13, IL-14, IL-16, IL-17, IL-17a, IL-20, IL-21, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), MCP-4, gamma-inducible protein 10 (IP-10), placenta growth factor receptor 2 (PLR2), leukocyte chemoattractant protein 1 (MCP-2), leukocyte chemoattractant protein 1 (MCP-3), leukocyte chemoattractant protein 1 (MCP-4), placenta growth factor receptor 2 (PGR2), leukocyte chemoattractant protein 1 (MCP-4), placenta growth factor receptor 2 (PGR2), leukocyte chemoattractant protein 1 (MCP-5), placenta growth factor receptor 2 (PGR2), placenta growth factor receptor 3 (PGR2), placenta growth factor receptor 4 (PGR2), placenta growth factor receptor 5 (PGR2), placenta growth factor receptor 6 (PGR2), placenta growth factor receptor 7 (PGR2), placenta growth factor receptor 8 (PGR2), placenta growth factor receptor 9 (P factor (PLGF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), C reaction CRP, vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, macrophage inflammatory protein MIP-1β, MIP-1b, leukemia inhibitory factor (LIF), oncostatin M (OSM), interleukin-1β (IL-1β), Interferon (IFN) alpha, IFN-beta, IFN-gamma, tumor necrosis factor (TNF) alpha, TNF-beta, CD154, lymphotoxin (LT) beta, 4-1BB ligand (4-1BBL), proliferation-inducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-related ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L, TNF- and ApoL-related leukocyte proliferation TNF-related apoptosis-inducing ligand 1 (TALL-1), TNF-related apoptosis-inducing ligand (TRAIL), chemokine (CC mochi) CCL1, macrophage inflammatory protein 1 alpha (MIP-1a or CCL3), CCL5, monocyte-specific chemokine 3 (MCP3 or CCL7), monocyte chemoattractant protein 2 (MCP-2 or CCL8), CCL13, thymus and activation-regulated chemokine (TARC or CCL17), CCL22, FGF2, eotaxin, MDC, granzin A, granzin B, perforin, SAA, MCP-4, and In some embodiments, after administration of cyclophosphamide and fludarabine, the patient exhibits increased serum levels of IL-15 and / or IP-10. In some embodiments, after administration of cyclophosphamide and fludarabine, the patient exhibits increased serum levels of IL-15 and / or IP-10. and after administration of fludarabine, patients show decreased serum levels of perforin.

[0139] In some embodiments, the present invention provides a method for the treatment of homeostatic cytokines in patients in need of T cell therapy. In certain embodiments, the homeostatic cytokines include interleukin 7 (IL-7), interleukin 15 (IL-15), interleukin 10 (IL-10), interleukin 20 (IL-20), interleukin 30 (IL-30), interleukin 40 (IL-40), interleukin 50 (IL-50), interleukin 60 (IL-50), interleukin 70 (IL-50), interleukin 80 (IL-50), interleukin 90 (IL-50), interleukin 100 (IL-50), interleukin 110 (IL-50), interleukin 120 (IL-50), interleukin 130 (IL-50), interleukin 140 (IL-50), interleukin 150 (IL-50), interleukin 160 (IL-50), interleukin 170 (IL-50), interleukin 180 (IL-50), interleukin 190 (IL-50), interleukin 200 (IL-50), interleukin 210 (IL-50), interleukin 220 (IL-50), interleukin 230 (IL-50), interleukin 240 (IL-50), interleukin 250 (IL-50), interleukin 260 (IL-50), interleukin 270 (IL-50), interleukin 280 (IL-50), interleukin 290 (IL-50), interleukin 310 (IL-50), interleukin 320 (IL-50), interleukin 330 (IL-50), interleukin 340 (IL-50), interleukin 350 (IL-50), interleukin 360 ( Interleukin 5 (IL-5), gamma-inducible protein 10 (IP-10), interleukin 8 (IL-8), Monocyte chemotactic protein 1 (MCP-1), placental growth factor (PLGF), C-reactive protein (CRP), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), or any combination thereof.

[0140] In one embodiment, the serum level of IL-7 in the patient is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, or at least 90-fold after administration of cyclophosphamide and fludarabine compared to the serum level of IL-7 before administration. In a specific embodiment, the level of IL-7 is increased by at least about 2-fold compared to the serum level of IL-7 before administration of cyclophosphamide and fludarabine. In another embodiment, In one embodiment, the level of IL-7 is increased by administering exogenous IL-7 to the patient. In one particular embodiment, the level of IL-7 is increased by administering cyclophosphamide, fludarabine, and exogenous IL-7 to the patient.

[0141] In one embodiment, the serum level of IL-15 in a patient is determined by administering cyclophosphamide and fludara. In certain embodiments, the serum level of IL-15 increases by at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, or at least 90-fold after administration compared to the serum level of IL-15 before administration. compared with serum levels of IL-15 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-15 is increased by at least about 10-fold. and increased by at least approximately 20-fold compared to serum levels of IL-15 before administration of fludarabine. In another embodiment, the level of IL-15 is measured after administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-15 is increased by at least about 30-fold compared to the serum level of IL-15 prior to administration. In another embodiment, the level of IL-15 is increased by administering exogenous IL-15 to the patient. In one particular embodiment, In this study, the levels of IL-15 are increased by administering cyclophosphamide, fludarabine, and exogenous IL-15 to patients.

[0142] In one embodiment, the serum level of IL-10 in a patient is determined by administering cyclophosphamide and fludara. At least two-fold and at least three-fold increase in serum IL-10 levels after administration compared to pre-administration , at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, In certain embodiments, the level of IL-10 is increased by at least 9-fold, at least 10-fold, or at least 20-fold compared to the serum level of IL-10 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-10 is increased by at least about 2-fold compared to the serum level of IL-10 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-10 is increased by at least about 3-fold compared to the serum level of IL-10 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-10 is increased by at least about 5-fold compared to the serum level of IL-10 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-10 is increased by at least about 20 ... In one particular embodiment, the level of IL-10 is increased by administering to the patient cyclophosphamide, fludarabine, and exogenous IL-10. and increases.

[0143] In one embodiment, the serum level of IL-5 in the patient is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold At least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 30 times, In certain embodiments, the level of IL-5 is increased by at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold. In another embodiment, the level of IL-5 is increased by at least about 5-fold compared to the serum level of IL-5 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-5 is increased by at least about 10-fold compared to the serum level of IL-5 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-5 is increased by at least about 30-fold compared to the serum level of IL-5 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-5 is increased by at least about 10 ... exogenous IL-5 to the patient. In one particular embodiment, the level of IL-5 is increased by administering to the patient cyclophosphamide, fludarabine, and exogenous IL-5.

[0144] In one embodiment, the serum level of IP-10 in a patient is determined after treatment with cyclophosphamide and fludara. At least two-fold and at least three-fold increase in serum IP-10 levels after administration compared to pre-administration levels , at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, or at least 30-fold In certain embodiments, IP-10 levels are increased by 2-fold following treatment with cyclophosphamide and fludara. In another embodiment, the level of IP-10 is increased by at least about two-fold compared to the serum level of IP-10 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IP-10 is increased by at least about three-fold compared to the serum level of IP-10 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IP-10 is increased by at least about three-fold compared to the serum level of IP-10 before administration of cyclophosphamide and fludarabine. In another embodiment, IP-10 levels are increased by approximately four-fold in both cyclophosphamide and fluoxetine. The serum level of IP-10 is increased by at least about 7-fold compared to the serum level before administration of fludarabine. In another embodiment, the level of IP-10 is increased by administering exogenous IP-10 to the patient. In a particular embodiment, the level of IP-10 is increased by administering exogenous IP-10 to the patient. It is increased by administering exogenous IP-10.

[0145] In one embodiment, the serum level of IL-8 in the patient is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold after administration of cyclophosphamide and fludarabine compared to the serum level of IL-8 before administration. In certain embodiments, the level of IL-8 is increased by at least about two-fold compared to the serum level of IL-8 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-8 is increased by at least about 5-fold compared to the serum level of IL-8 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-8 is increased by at least about 10-fold compared to the serum level of IL-8 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-8 is increased by at least about 20-fold compared to the serum level of IL-8 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-8 is increased by at least about 40-fold compared to the serum level of IL-8 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-8 is increased by at least about 60-fold compared to the serum level of IL-8 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of IL-8 is increased by administering exogenous IL-8 to the patient. In one particular embodiment, the level of IL-8 is increased by administering cyclophosphamide, fludarabine, and exogenous IL-8 to the patient.

[0146] In one embodiment, the serum level of MCP-1 in a patient is determined by administering cyclophosphamide and fludara. Compared to serum levels of MCP-1 before administration of the bottle, the serum levels after administration are at least 1.5-fold and at least 2-fold higher. times, at least three times, at least four times, at least five times, at least six times, at least seven times, In certain embodiments, the level of MCP-1 is increased by at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold. In another embodiment, the level of MCP-1 is increased by at least about two-fold compared to the serum level of MCP-1 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of MCP-1 is increased by at least about three-fold compared to the serum level of MCP-1 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of MCP-1 is increased by at least about three-fold compared to the serum level of MCP-1 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of MCP-1 is increased by at least about 7-fold compared to the serum level of MCP-1 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of MCP-1 is increased by administering exogenous MCP-1 to the patient. In one particular embodiment, the level of MCP-1 is increased by administering exogenous MCP-1 to the patient after administration of cyclophosphamide, fludarabine, and It is increased by administering exogenous MCP-1.

[0147] In one embodiment, the serum level of PLGF in the patient is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, or less after administration of cyclophosphamide and fludarabine compared to the serum level of PLGF before administration. In certain embodiments, the level of PLGF is increased by at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold. In another embodiment, the level of PLGF is increased by at least about 1.5-fold compared to the serum level of PLGF before administration of cyclophosphamide and fludarabine. The serum level of PLGF increases by at least about two-fold compared to the level before administration of fludarabine. In another embodiment, the level of PLGF is increased by at least about 3-fold compared to the serum level of PLGF before administration of cyclophosphamide and fludarabine. is increased by administering exogenous PLGF to the patient. In one particular embodiment, the level of PLGF is increased by administering cyclophosphamide, fludarabine, and exogenous PLGF to the patient.

[0148] In one embodiment, the serum level of CRP in a patient is determined by administering cyclophosphamide and fludarabine. Compared with serum CRP levels before administration of the drug, the serum CRP level after administration is at least 1.5-fold, at least 2-fold, or In certain embodiments, the level of CRP is increased by at least 3-fold, at least 4-fold, at least 5-fold, at least about 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold compared to the serum level of CRP before administration of cyclophosphamide and fludarabine. In another embodiment, the level of CRP is increased by at least about 1.5-fold compared to the serum level of CRP before administration of cyclophosphamide and fludarabine. In another embodiment, the level of CRP is increased by at least about 2-fold compared to the serum level of CRP before administration of cyclophosphamide and fludarabine. In another embodiment, the CRP is increased by at least about 5-fold compared to the serum level of CRP before administration. The levels of CRP were compared with serum levels before administration of cyclophosphamide and fludarabine. In another embodiment, the level of CRP is increased by at least about 10-fold compared to the serum level of CRP before administration of cyclophosphamide and fludarabine. In another embodiment, the level of CRP is measured after administration of cyclophosphamide and fludarabine. The serum level of CRP is increased by at least about 25-fold compared to the previous serum level of CRP. In another embodiment, the level of CRP is increased by administering exogenous CRP to the patient. In one particular embodiment, the level of CRP is increased by administering cyclophosphamide, fludarabine, and exogenous CRP to the patient. increases by

[0149] In one embodiment, the serum levels of sICAM-1 in a patient are determined by administering cyclophosphamide and fludarabine. Compared to the serum level of sICAM-1 before administration of rabin, the level of sICAM-1 increases by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold after administration. compared with serum levels of sICAM-1 before administration of cyclophosphamide and fludarabine In another embodiment, the level of sICAM-1 is increased by at least about 1.5-fold compared to the serum level of sICAM-1 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of sICAM-1 is increased by at least about 2-fold compared to the serum level of sICAM-1 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of sICAM-1 is increased by at least about three-fold compared to the serum level of sICAM-1 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of sICAM-1 is increased by at least about four-fold compared to the serum level of sICAM-1 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of sICAM-1 is increased by administering exogenous sICAM-1 to the patient. In one particular embodiment, the level of sICAM-1 is increased by administering cyclophosphamide, fludarabine, and exogenous sICAM-1 to the patient. It increases by

[0150] In one embodiment, the serum level of sVCAM-1 in a patient is determined by administering cyclophosphamide and fludarabine. at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, or at least 5-fold higher serum levels of sVCAM-1 after administration of rabin compared to serum levels before administration of rabin In certain embodiments, the level of sVCAM-1 is increased by 4.5-fold, or at least 5-fold. Compared with serum levels of sVCAM-1 before administration of rofosfamide and fludarabine, In another embodiment, the level of sVCAM-1 is increased by at least about 2-fold compared to the serum level of sVCAM-1 before administration of cyclophosphamide and fludarabine. In another embodiment, the level of sVCAM-1 is increased by at least about 2-fold compared to the serum level of sVCAM-1 before administration of cyclophosphamide and fludarabine. The serum level of sVCAM-1 is increased by at least about three-fold compared to the previous serum level. In another embodiment, the level of sVCAM-1 is increased by administering exogenous sVCAM-1 to the patient. In , levels of sVCAM-1 are increased by administering cyclophosphamide, fludarabine, and exogenous sVCAM-1 to patients.

[0151] In some embodiments, one or more of the following administration of cyclophosphamide and fludarabine: Levels of several cytokines may predict how patients will respond to T-cell therapy. For example, increases in certain cytokines after administration of cyclophosphamide and fludarabine indicate that patients are more likely to respond to T cell therapy. In another example, a decrease or no change in the levels of certain cytokines after administration of cyclophosphamide and fludarabine may indicate that a patient is likely to respond to T cell therapy. An increase in one or more cytokines and a decrease in one or more different cytokines after administration of cyclophosphamide and fludarabine can indicate that the patient is more or less likely to respond to T cell therapy. Thus, a patient's cytokine profile may influence their response to T cell therapy. Responsiveness can be demonstrated.

[0152] In some embodiments, the levels of IL-15 after administration of cyclophosphamide and fludarabine are More than about a 3-fold, more than about a 4-fold, more than about a 5-fold, more than about a 10-fold, more than about a 15-fold, or more than about a 20-fold increase in In another embodiment, the cyclophosphamide-containing antibody is a cyclophosphamide-containing antibody that is more likely to respond to T cell therapy. A greater than approximately 2-fold, greater than approximately 3-fold, greater than approximately 4-fold, greater than approximately 5-fold, or greater than approximately 6-fold increase in IP-10 levels after administration of falciparum and fludarabine indicates that the patient is more likely to respond to T-cell therapy. In yet another embodiment, a decrease in MIP-1b levels following administration of cyclophosphamide and fludarabine indicates that the patient is less likely to respond to T cell therapy.

[0153] In some embodiments, the serum levels of any one or more cytokines are determined by cyclophosphite. One or more days before administration of sulfamide and fludarabine, and cyclophosphamide The measurement is performed on one or more days selected from the day of administration of cyclophosphamide and fludarabine to 21 days after administration of cyclophosphamide and fludarabine.

[0154] One aspect of the present invention is to improve the availability of homeostatic cytokines in patients in need of T cell therapy. Another aspect of the present invention includes a method of improving the efficacy of T cell therapy comprising administering to a patient a treatment that increases the levels of one or more homeostatic, pro-inflammatory cytokines or chemokines selected from IL-15, IL-7, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, and sVCAM-1. Those skilled in the art will recognize the benefits of T cell therapy, including, but not limited to, the use of T cell therapy described herein. Use of cyclophosphamide and fludarabine, and administration of one or more exogenous steroids to patients administration of cytokines, inducing expression of one or more endogenous cytokines or administration of one or more compositions for preventing the onset of inflammatory bowel disease, administration of one or more transgenic cells capable of expressing one or more recombinant cytokines, and administering the composition to a patient. It will be appreciated that homeostatic cytokine levels can be increased by a number of different methods, including any other method that has the effect of increasing homeostatic cytokine levels.

[0155] In some embodiments, the present invention includes a method of conditioning a patient in need of T cell therapy, comprising administering to the patient cyclophosphamide and fludarabine and one or more doses of an isolated or recombinant cytokine. The isolated or recombinant cytokine can be any cytokine. In one embodiment, the cytokine is a homeostatic cytokine. In another embodiment, the cytokine is a pro-inflammatory cytokine. In yet another embodiment, the cytokine is a chemokine. In one particular embodiment, a patient in need of T cell therapy is The method of conditioning a patient in need thereof comprises administering to the patient cyclophosphamide and fludarabine and one or more doses of an isolated or recombinant cytokine. wherein the cytokines include IL-2, IL-15, IL-7, IL-10, IL-5, IP-10, IL-8, MCP The cytokines are selected from IL-15, IL-7, IP-10, MCP-1, CRP, PLGF, sICAM-1, sVCAM-1, and any combination thereof, such as IL-15, IL-7, IP-10, MCP-1, CRP, and PLGF. One or more doses of isolated or recombinant cytokines can be administered before T cell therapy, or after T cell therapy, or any combination thereof.

[0156] In one aspect, the method of conditioning a patient in need of T cell therapy comprises administering to the patient a stimulatory agent. administering clofosfamide and fludarabine and one or more doses of IL-2 In some embodiments, the dose of IL-2 is at least about 10,000 IU / kg, at least about 50,000 IU / kg, at least about 100,000 IU / kg, at least about 200,000 IU / kg, at least about 400,000 IU / kg, at least about 600,000 IU / kg, at least about 700,000 IU / kg, at least about 800,000 IU / kg, or at least about 1,000,000 IU / kg. In one embodiment, the dose of IL-2 is at least about 700,000 IU / kg. In one particular embodiment, the dose of IL-2 is about 720,000 IU / kg. In some embodiments, IL-2 is administered every 8 hours up to 15 times, or until toxicity prevents additional administration. is administered to the patient.

[0157] Cancer treatment The methods of the present invention can be used to treat cancer in a subject, reduce tumor size, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, remove tumors from patients, prevent tumor recurrence, prevent tumor metastasis, induce remission in patients, or any combination thereof. In certain embodiments, the methods induce complete responses. In other embodiments, the methods induce partial responses.

[0158] Cancers that can be treated include tumors that are not vascularized, not yet substantially vascularized, or vascularized. Cancers can also include solid or non-solid tumors. In certain embodiments, the cancer is bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, T-cell rich cancer, Primary mediastinal large B-cell lymphoma (TCRBCL), primary mediastinal large B-cell lymphoma (PMBCL), non-Hodgkin's lymphoma tumors, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancers The tumor may be selected from a tumor derived from a cancer, and a combination of said cancers.

[0159] In one embodiment, this method can be used to treat tumors, where the tumor is lymphoma or leukemia. Lymphoma and leukemia are blood cancers that specifically affect lymphocytes. All white blood cells in the blood originate from a single type of pluripotent hematopoietic stem cell found in the bone marrow. This stem cell produces both myeloid and lymphoid progenitor cells, which then give rise to the various types of white blood cells found in the body. White blood cells that develop from myeloid progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells, and plasma cells. White blood cells that develop from lymphoid progenitor cells include megakaryocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, and macrophages. Lymphoma and leukemia can affect one or more of these cell types in patients.

[0160] Lymphomas are generally divided into at least two subgroups: Hodgkin's lymphoma and non-Hodgkin's lymphoma. Non-Hodgkin's lymphoma (NHL) is a type of lymphoma caused by B lymphocytes, T lymphocytes, and It is a heterogeneous group of cancers that originate in lymphocytes or natural killer cells. Cell lymphoma accounts for 80-85% of reported cases. Approximately 69 new cases of NHL were reported in 2013. It was estimated that there would be 740 cases and over 19,000 disease-related deaths. Lymphoma is the most prevalent hematologic malignancy and the seventh leading site of new cancers among men and women, accounting for 4% of all new cancer cases and 3% of cancer-related deaths.

[0161] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, accounting for approximately 30% of NHL cases. Approximately 22,000 new cases of DLBCL are diagnosed each year in the United States. It is classified as an aggressive lymphoma in which the majority of patients are cured with conventional chemotherapy (NCCN Guidelines NHL 2014).

[0162] First-line therapy for DLBCL typically results in an objective response rate of approximately 80% and a complete response rate of approximately 50%. % (Coiffier 2002), anthracyclines using rituximab such as R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) Approximately one-third of patients have refractory disease to initial therapy or relapse after R-CHOP (Sehn 2005). For patients who relapse after a response to first-line therapy, Approximately 40-60% of patients can achieve a secondary response with additional chemotherapy. The standard of care for second-line therapy for patients eligible for autologous stem cell transplantation (ASCT) includes rituximab and combination chemotherapy, such as R-ICE (rituximab, ifosfamide, carboplatin, and etoposide) and R-DHAP (rituximab, dexamethasone, cytarabine, and cisplatin), which have an objective response rate of approximately 63% and a complete response rate of approximately 26%, respectively (Gisselbrecht et al., 2013). Patients who respond to second-line therapy and are deemed well-suited for transplant undergo consolidation with high-dose chemotherapy and ASCT, which cures approximately half of transplanted patients (Gisselbrecht 2010). Patients who fail ASCT have a very poor prognosis and require curative options. No limbs.

[0163] Primary mediastinal large B-cell lymphoma (PMBCL) is a distinct clinical and pathological entity compared with DLBCL. PMBCL is thought to arise from thymic (medullary) B cells and accounts for approximately 3% of patients diagnosed with DLBCL. PMBCL occurs in relatively young adults, typically in their 40s. Gene expression profiling has been shown to be associated with PMBCL. It has been suggested that the deregulated pathways in PMBCL overlap with those in Hodgkin lymphoma. Initial therapy for this disease generally involves an anthracycline-containing regimen in combination with rituximab, such as infusional dose-adjusted etoposide, doxorubicin, and cyclophosphamide (DA-EPOCH-R) in combination with vincristine, prednisone, and rituximab, with or without regional radiation therapy.

[0164] Follicular lymphoma (FL), a B-cell lymphoma, is the most common indolent (indolent) form of NHL, accounting for approximately 20% to 30% of all NHL. Some patients with FL are more likely to develop Histological transformation to DLBCL (TFL) is aggressive and associated with poor outcomes. Histologic transformation occurs at a rate of approximately 3% per year for 15 years, and the risk of transformation increases in subsequent years. The biological mechanism of histological transformation is unknown. Initial treatment of TFL is Although influenced by prior treatment for follicular lymphoma, it generally involves anthracycline-containing regimens in combination with rituximab to eliminate the aggressive component of the disease.

[0165] Treatment options for relapsed / refractory PMBCL and TFL are similar to those in DLBCL. Given the low prevalence of these diseases, there have been no large prospective randomized trials in these patient populations. Patients with chemotherapy-refractory disease may be more likely to have refractory DLBCL than those with chemotherapy-refractory disease. have a similar or worse prognosis to patients with

[0166] In summary, subjects with refractory aggressive NHL (e.g., DLBCL, PMBCL, and TFL) represent a large unmet medical need, and further research with novel treatments in this population is warranted.

[0167] Thus, in some embodiments, the methods can be used to treat lymphoma or leukemia, where the lymphoma or leukemia is a B-cell malignancy. In certain embodiments, the lymphoma or leukemia is selected from the group consisting of B-cell chronic lymphocytic leukemia / small cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., Waldenstrom's macroglobulinemia), splenic marginal zone lymphoma, hairy cell leukemia, plasma cell neoplasms (e.g., plasma cell myeloma (i.e., multiple myeloma), or plasmacytoma), extranodal marginal zone B-cell lymphoma (e.g., MALT lymphoma), nodal marginal zone B-cell lymphoma, follicular lymphoma (FL), ... Transformed follicular lymphoma (TFL), primary cutaneous follicle center lymphoma, mantle cell lymphoma , diffuse large B-cell lymphoma (DLBCL), Epstein-Barr virus positive DLBCL, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma (PMBCL), intravascular large B-cell lymphoma Cellular lymphoma, ALK+ large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma Large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, B-cell lymphoma Kitt lymphoma / leukemia, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, Aggressive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides / Sézary disease syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, peripheral T-cell lymphoma, hematologic Tumor immunoblastic T-cell lymphoma, anaplastic large cell lymphoma, B-cell lymphoblastic leukemia / lymphoma B-cell lymphoblastic leukemia / lymphoma with recurrent genetic abnormalities, T-cell lymphoblastic leukemia In some embodiments, the cancer is selected from hematologic / lymphoma and Hodgkin's lymphoma. , is refractory to one or more prior treatments, and / or the cancer is one or more recurrence after previous treatment with

[0168] In certain embodiments, the cancer is selected from follicular lymphoma, transformed follicular lymphoma, diffuse large B-cell lymphoma, and primary mediastinal (thymic) large B-cell lymphoma. In one particular embodiment, the cancer is diffuse large B-cell lymphoma.

[0169] In some embodiments, the cancer is treated with chemotherapy, radiation therapy, immunotherapy (T cell therapy and / or or antibody or antibody-drug conjugate), autologous stem cell transplant, or any combination thereof, or the cancer is subsequently refractory to one or more of In one particular embodiment, the cancer is refractory diffuse large B-cell lymphoma. It is a pancreatic cancer.

[0170] In one particular aspect, the present invention provides a method for treating a patient with a CAR cell-associated leukemia virus (LEV) virus, comprising administering to the patient a therapeutically effective amount of engineered CAR cells. Any of the doses described herein (e.g., about 200 mg / m 2 / day, about 300mg / m 2 / day, about 400mg / m 2 / day, about 500mg / m 2 / day, about 600mg / m 2 / day, about 700mg / m 2 / day, about 800mg / m 2 / day, or approximately 900 mg / m 2 / day) cyclophosphamide and any of the doses described herein (e.g., about 20 mg / m 2 / day, about 25mg / m2 / day, about 30mg / m 2 / day, about 35mg / m 2 / day, about 40mg / m 2 / day, about 45mg / m 2 / day, about 50mg / m 2 / day, about 55mg / m 2 / day, about 60mg / m 2 100 mg / day of fludarabine daily for 3 days. and a method of treating a patient with a CAR-associated inflammatory cytokine, wherein the engineered CAR cells express a chimeric antigen receptor. However, the chimeric antigen receptor binds to CD19 and further contains a CD28 costimulatory domain and a CD3-zeta signaling region.

[0171] In another aspect, the present invention includes a method of treating a patient with lymphoma, comprising the steps of: (i) administering to a patient any dose described herein (e.g., about 200 mg / m 2 / day, about 300mg / m 2 / day, about 400mg / m 2 / day, about 500mg / m 2 / day, about 600mg / m 2 / day, about 700mg / m 2 / day, about 800mg / m 2 / day, or approximately 900 mg / m 2 / day) cyclophosphamide and any of the doses described herein (e.g., about 20 mg / m 2 / day, about 25mg / m 2 / day, about 30mg / m 2 / day, about 35mg / m 2 / day, about 40mg / m 2 / day, about 45mg / m 2 / day, about 50mg / m 2 / day, about 55mg / m 2 / day, about 60mg / m 2 / day) fludarabine; and (ii) administering to the patient a therapeutically effective amount of engineered CAR cells, wherein the engineered CAR cells express a chimeric antigen receptor, wherein the chimeric antigen receptor binds to CD19 and further comprises a CD28 costimulatory domain and a CD3-zeta signaling region.

[0172] In yet another aspect, the invention provides a method for treating a cancer, comprising administering to a patient a therapeutically effective amount of engineered CAR cells. and methods of treating patients with lymphoma, wherein the patient is administered any of the doses described herein (e.g., about 200 mg / m 2 / day, about 300mg / m 2 / day, about 400mg / m 2 / day, about 500mg / m 2 / day, about 600mg / m 2 / day, about 700mg / m 2 / day, about 800mg / m 2 / day, or approximately 900 mg / m 2 / day) cyclophosphamide and any of the doses described herein (e.g., about 20 mg / m 2 / day, about 25mg / m 2 / day, about 30mg / m 2 / day, Approximately 35mg / m 2 / day, about 40mg / m 2 / day, about 45mg / m 2 / day, about 50mg / m 2 / day, about 55mg / m 2 / day, about 60mg / m 2 The cells were conditioned with fludarabine (100 mg / day), and the engineered CAR cells express a chimeric antigen receptor that binds to CD19 and further contains a CD28 costimulatory domain and a CD3-zeta signaling region.

[0173] kit Cyclophosphamide and cyclophosphamide for use in preconditioning for T cell therapy Also included within the scope of the present invention are kits, e.g., pharmaceutical kits, containing fludarabine and riboflavin. The kits typically include a label and instructions indicating the intended use of the contents of the kit. The term "label" includes any writing or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0174] In some embodiments, the present invention provides a method for conditioning a patient in need of T cell therapy. (i) cyclophosphamide, (ii) fludarabine, and (iii) engineered CAR cells. Patients in need of cell therapy may be administered any of the doses described herein (e.g., 200 mg / m 2 / day~2000mg / m 2 / day) cyclophosphamide and any dose described herein (e.g., 20 mg / m 2 / day~900mg / m 2 kit containing instructions for administering fludarabine (100 mg / day) daily for 3 days provide.

[0175] In another aspect, the present invention provides a kit for conditioning a patient in need of T cell therapy. (i) cyclophosphamide, (ii) fludarabine, and (iii) engineered CAR cell therapy. Patients in need thereof may be administered any of the doses described herein (e.g., 200 mg / m 2 / day~2000mg / m 2 / day) for 2 days from day -7 to day -6 and any dose described herein (e.g., 20 mg / m 2 / day~900mg / m 2 The kit includes instructions for administering fludarabine at a dose of 0.1 mg / day daily for 5 days from day -5 to day -1.

[0176] In another aspect, the present invention provides a kit for conditioning a patient in need of T cell therapy. (i) cyclophosphamide, (ii) fludarabine, and (iii) engineered TCR cell therapy. Patients in need thereof may be administered any of the doses described herein (e.g., 200 mg / m 2 / day~2000mg / m 2 / day) cyclophosphamide and any dose described herein (e.g., 20 mg / m 2 / day~900mg / m 2 The kit includes instructions for administering fludarabine at a dose of 100 mg / day daily for 3 days. do.

[0177] In another aspect, the present invention provides a kit for conditioning a patient in need of T cell therapy. (i) cyclophosphamide, (ii) fludarabine, and (iii) engineered TCR cell therapy. Patients in need thereof may be administered any of the doses described herein (e.g., 200 mg / m 2 / day~2000mg / m 2 / day) for 2 days from day -7 to day -6 and any dose described herein (e.g., 20 mg / m 2 / day~900mg / m 2 The kit includes instructions for administering fludarabine at a dose of 0.1 mg / day daily for 5 days from day -5 to day -1.

[0178] In some embodiments, the present invention provides a method for conditioning a patient in need of T cell therapy. (i) cyclophosphamide, (ii) fludarabine, and (iii) engineered CAR cells. Patients requiring cell therapy should be given 300 mg / m before the therapy. 2 / day dose of cyclophosphamide and and 30 mg / m 2 The kit contains instructions for administering fludarabine at a dose of 1 / day for three days. Provide.

[0179] In another aspect, the present invention provides a kit for conditioning a patient in need of T cell therapy. (i) cyclophosphamide, (ii) fludarabine, and (iii) engineered CAR cell therapy. 300 mg / m before therapy in patients requiring 2 Cyclophosphamide at a dose of 100 mg / day was administered from day -7 to 2 days through day -6 and 30 mg / m 2 The kit includes instructions for administering fludarabine at a dose of 1 / day daily for 5 days from day -5 to day -1.

[0180] In another aspect, the present invention provides a kit for conditioning a patient in need of T cell therapy. (i) cyclophosphamide, (ii) fludarabine, and (iii) engineered TCR cell therapy. 500 mg / m before therapy in patients requiring 2 / day dose of cyclophosphamide and 30 mg / m 2 The kit includes instructions for administering fludarabine at a dose of 100 mg / day daily for three days.

[0181] In another aspect, the present invention provides a kit for conditioning a patient in need of T cell therapy. (i) cyclophosphamide, (ii) fludarabine, and (iii) engineered TCR cell therapy. 500 mg / m before therapy in patients requiring 2 Cyclophosphamide at a dose of 100 mg / day was administered from day -7 to 2 days through day -6 and 30 mg / m 2 The kit includes instructions for administering fludarabine at a dose of 1 / day daily for 5 days from day -5 to day -1.

[0182] In some embodiments, the present invention provides a method for conditioning a patient in need of T cell therapy. (i) cyclophosphamide, (ii) fludarabine, and (iii) engineered CAR cells. Patients requiring cell therapy should be given 300 mg / m before the therapy. 2 / day dose of cyclophosphamide and and 60 mg / m 2 The kit contains instructions for administering fludarabine at a dose of 1 / day for three days. Provide.

[0183] In another aspect, the present invention provides a kit for conditioning a patient in need of T cell therapy. (i) cyclophosphamide, (ii) fludarabine, and (iii) engineered CAR cell therapy. 300 mg / m before therapy in patients requiring 2 Cyclophosphamide at a dose of 100 mg / day was administered from day -7 to 60 mg / m for 2 days through day -6 2 The kit includes instructions for administering fludarabine at a dose of 1 / day daily for 5 days from day -5 to day -1.

[0184] In another aspect, the present invention provides a kit for conditioning a patient in need of T cell therapy. (i) cyclophosphamide, (ii) fludarabine, and (iii) engineered TCR cell therapy. 500 mg / m before therapy in patients requiring 2 / day dose of cyclophosphamide and 60 mg / m 2 The kit includes instructions for administering fludarabine at a dose of 100 mg / day daily for three days.

[0185] In another aspect, the present invention provides a kit for conditioning a patient in need of T cell therapy. (i) cyclophosphamide, (ii) fludarabine, and (iii) engineered TCR cell therapy. 500 mg / m before therapy in patients requiring 2 Cyclophosphamide at a dose of 100 mg / day was administered from day -7 to 60 mg / m for 2 days through day -6 2The kit includes instructions for administering fludarabine at a dose of 1 / day daily for 5 days from day -5 to day -1.

[0186] In certain embodiments, the kit comprises saline and cyclophosphamide and / or or administering saline to the patient either before or after administration of fludarabine or both before and after administration of cyclophosphamide and / or fludarabine. In some embodiments, the kit further comprises instructions for administering mesna and cyclophosphamide and / or fludarabine prior to administration of cyclophosphamide and / or fludarabine. After administration of darabine, or before administration of cyclophosphamide and / or fludarabine The patient's prescription further includes instructions for administering mesna to the patient both before and after administration.

[0187] Biomarker-based diagnostic methods The present invention also includes a method for identifying a subject suitable for T cell therapy. 200 mg / m 2 ~2000mg / m 2 , e.g., 200 mg / m 2 , 300 mg / m 2 , 400 mg / m 2 , 500 mg / m 2 , 600 mg / m 2 , 700 mg / m 2 , 800 mg / m 2 , 900 mg / m 2 , 1000 mg / m 2 , or 1110 mg / m 2 A dose of cyclophosphamide amide, and 20 mg / m 2 ~900mg / m 2 , e.g., 20 mg / m 2 , 25 mg / m 2 , 30 mg / m 2 , 35 mg / m 2 , 40 mg / m 2 , 45 mg / m 2, or 50 mg / m 2 and administering a dose of fludarabine to a patient suitable for T cell therapy. wherein the patient has increased serum levels of IL-15, IP-10, and / or In another aspect, the present invention provides a method for treating a patient with T cell therapy after the patient exhibits decreased serum levels of IL-7 and / or perforin. 2 ~2000mg / m 2 , e.g., 200 mg / m 2 , 300 mg / m 2 , 400 mg / m 2 , 500 mg / m 2 , 600 mg / m 2 , 700 mg / m 2 , 800 mg / m 2 , 900 mg / m 2 , 1000 mg / m 2 , or 1110 mg / m 2 cyclophosphamide at a dose of 20 mg / m 2 ~900mg / m 2 , e.g., 20 mg / m 2 , 25 mg / m 2 , 30 mg / m 2 , 35 mg / m 2 , 40 mg / m 2 , 45 mg / m 2 , or 50 mg / m 2 and (ii) administering T cell therapy after the patient exhibits increased serum levels of IL-15, IP-10, and / or IL-7 and / or decreased serum levels of perforin. In other aspects, the invention includes a method for treating cancer in a patient suitable for T cell therapy, comprising (i) preconditioning the patient by administering fludarabine at a dose of 200 mg / m 2 ~2000mg / m 2 , e.g., 200 mg / m 2 , 300 mg / m 2 , 400 mg / m 2 , 500 mg / m 2, 600 mg / m 2 , 700 mg / m 2 , 800 mg / m 2 , 900 mg / m 2 , 1000 mg / m 2 , or 1110 mg / m 2 cyclophosphamide at a dose of 20 mg / m 2 ~900mg / m 2 , e.g., 20 mg / m 2 , 25 mg / m 2 , 30 mg / m 2 , 35 mg / m 2 , 40 mg / m 2 , 45 mg / m 2 , or 50 mg / m 2 (ii) if the patient does not exhibit sufficient serum levels of IL-15, IP-10, and / or IL-7 after administration in (i), administering an additional amount of cyclophosphamide and / or or administering fludarabine or administering IL-15, IP-10, and / or IL-7. (iii) Patients had increased serum levels of IL-15, IP-10, and and / or IL-7 in a patient suitable for T cell therapy, including administering T cell therapy after the patient has expressed IL-7. In certain embodiments, the present invention is directed to a method for treating cancer in a patient having elevated serum levels of a compound selected from the group consisting of MCP-1, CRP, PLGF, IP-10, and any combination thereof. If the patient exhibits at least one additional cytokine of the T cell type, the patient is administered T cell therapy.

[0188] The present invention provides a 200 mg / m 2 ~2000mg / m 2 , e.g., 200 mg / m 2 , 300 mg / m 2 , 500 mg / m 2 , 400 mg / m 2 , 600 mg / m 2 , 700 mg / m2 , 800 mg / m 2 , 900 mg / m 2 , 1000 mg / m 2 , or 1110 mg / m 2 Dosage of cyclophosphamide, and 20 mg / m 2 ~900mg / m 2 , e.g., 20 mg / m 2 , 25 mg / m 2 , 30 mg / m 2 , 35 mg / m 2 , 40 mg / m 2 , 45 mg / m 2 , or 50 mg / m 2 The present invention further provides a method for identifying a patient suitable for T cell therapy, comprising administering a dose of fludarabine to the patient, wherein the patient is In another embodiment, the patient is treated with T cell therapy after exhibiting increased serum levels of IL-15, IP-10, and / or IL-7 and / or decreased serum levels of perforin. (i) 200 mg / m 2 ~2000mg / m 2 , e.g., 200 mg / m 2 , 300 mg / m 2 , 400 mg / m 2 , 500 mg / m 2 , 600 mg / m 2 , 700 mg / m 2 , 800 mg / m 2 , 900 mg / m 2 , 1000 mg / m 2 , or 1110 mg / m 2 cyclophosphamide at a dose of 20 mg / m 2 ~900mg / m 2 , e.g., 20 mg / m 2 , 25 mg / m 2 , 30 mg / m 2 , 35 mg / m 2 , 40 mg / m 2 , 45 mg / m 2 , or 50 mg / m 2and (ii) administering T cell therapy after the patient exhibits increased serum levels of IL-15, IP-10, and / or IL-7 and / or decreased serum levels of perforin. In another aspect, the present invention provides a method for identifying a patient suitable for T cell therapy, comprising (i) administering fludarabine at a dose of 200 mg / m 2 ~2000mg / m 2 , e.g., 200 mg / m 2 , 300 mg / m 2 , 400 mg / m 2 , 500 mg / m 2 , 600 mg / m 2 , 700 mg / m 2 , 800 mg / m 2 , 900 mg / m 2 , 1000 mg / m 2 , or 1110 mg / m 2 cyclophosphamide at a dose of 20 mg / m 2 ~900mg / m 2 , e.g., 20 mg / m 2 , 25 mg / m 2 , 30 mg / m 2 , 35 mg / m 2 , 40 mg / m 2 , 45 mg / m 2 ,Ma or 50 mg / m 2 (ii) if the patient does not exhibit sufficient serum levels of IL-15, IP-10, and / or IL-7, administering an additional amount of cyclophosphamide or fludarabine or administering an effective amount of IL-15, IP-10, and / or IL-7. and (iii) administering T cell therapy after the patient exhibits increased serum levels of MCP-1, CRP, PLGF, IP-10, and / or IL-7. and at least one additional compound selected from the group consisting of any combination of compounds with increased serum levels. If the patient exhibits a cytokine response, T cell therapy is administered to the patient.

[0189] The methods of the invention further comprise measuring serum levels of IL-15, IP10, perforin, and / or IL-7. In one embodiment, the serum level of IL-7 in the patient is at least 2-fold higher after administration of cyclophosphamide and fludarabine compared to the serum level of IL-7 before administration. fold, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times In another embodiment, the serum level of IL-15 in the patient is increased by at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, or at least 90 fold. Compared with the serum levels of IL-15 before administration of cyclophosphamide and fludarabine, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, or at least 90-fold increase after administration do.

[0190] In other embodiments, 200 mg / m 2 ~2000mg / m 2 , e.g., 200 mg / m 2 , 300 mg / m 2 , 400 mg / m 2 , 500 mg / m 2 , 600 mg / m 2 , 700 mg / m 2 , 800 mg / m 2 , 900 mg / m 2 , 1000 mg / m 2 , or 1110 mg / m 2 of doses Cyclophosphamide and 20 mg / m 2 ~900mg / m 2, e.g., 20 mg / m 2 , 25 mg / m 2 , 30 mg / m 2 , 35 mg / m 2 , 40 mg / m 2 , 45 mg / m 2 , or 50 mg / m 2 the serum level of MCP-1 in the patient increases after administration of a dose of fludarabine that is at least 1.5-fold, at least 2-fold, at least 3-fold, or less than the serum level of MCP-1 before administration of cyclophosphamide and fludarabine. In some embodiments, the serum level of PLGF in the patient is increased by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold after administration of cyclophosphamide and fludarabine compared to the serum level of PLGF before administration. In certain embodiments, the serum level of CRP in the patient is increased by at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold compared to the serum level of CRP before administration of cyclophosphamide and fludarabine. In yet other embodiments, the serum level of IP-10 in the patient increases by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least about 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold after administration, compared to the serum level of IP-10 before administration of cyclophosphamide and fludarabine. At least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times , at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, At least a 20-fold or at least a 30-fold increase.

[0191] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references cited throughout this application are expressly incorporated herein by reference. [Example]

[0192] Example 1 A Phase 1 / 2 single-arm, open-label study was designed to determine the safety and feasibility of anti-CD19 CAR+ T cells administered to subjects with B-cell malignancies.

[0193] Subjects who signed informed consent and met the study entry criteria were enrolled in the study and underwent leukapheresis to obtain PBMCs for anti-CD19 CAR+ T cell production. Subjects were treated with conditioning chemotherapy before admission for a single infusion of anti-CD19 CAR+ T cells on day 0. Some subjects were then treated with interleukin-2 (Group 1 only) 3 hours after the anti-CD19 CAR+ T-cell infusion. If there was a partial response (PR) or complete response (CR) after the first infusion, followed by disease progression, retreatment with a second dose of anti-CD19 CAR+ T cells was permitted.

[0194] Three groups of subjects were enrolled: Group 1 received 3 x 10 anti-CD19 CAR+ T cells; 6 ~30×10 6 Eight subjects received anti-CD19 CAR+ T cells at doses ranging from 60 to 120 mg / kg (2220 to 4440 mg / m), including one subject who was re-treated. 2 ) high-dose cyclophosphamide for 2 days followed by 25 mg / m2 These subjects received 720,000 IU / kg of fludarabine for 5 days to stimulate the proliferation of anti-CD19 CAR+ T cells after administration. High-dose interleukin-2 (IL-2) was also administered (every 8 hours for up to 15 doses or until toxicity prevented additional doses).

[0195] Group 2 received high-dose cyclophosphamide and fludarabine, with various doses of Administration of anti-CD19 CAR+ T cells (anti-CD19 CAR+ T cells 1 × 10 6 ~5×10 6 (pieces / kg) after interleukin The study included 15 subjects who did not receive IVF-2, including 2 subjects from Group 1 who were re-treated.

[0196] Group 3 was a reduced conditioning regimen of 300 mg / m 2 Cyclophosphamide and and 30 mg / m 2 These included 11 subjects who received 1 × 10 fludarabine (all administered concomitantly for 3 days without IL-2). 6 anti-CD19 CAR+ T cells, 4 days later People are 2 x 10 6 received an infusion of anti-CD19 CAR+ T cells.

[0197] demographics Subject demographics and disease characteristics are provided in Table 1. Thirty-two subjects were enrolled; 19 subjects (59%) had DLBCL or PMBCL, 7 subjects (22%) had CLL, and 6 subjects (19%) had other indolent NHL, including indolent follicular lymphoma and splenic marginal zone lymphoma. The subjects had refractory disease (84%) and had received a median of three lines of prior therapy. All subjects with aggressive NHL had received prior anti-CD20 therapy, platinum-based chemotherapy, and 95% had previously received anthracycline-based chemotherapy;

[0198] Pharmacokinetics qPCR analysis was used to assess the number of anti-CD19 CAR+ T cells in the peripheral blood at various time points after the first dose on day 0, and was verified by a standard curve generated by flow cytometry using an antibody reagent specific for the scFv present in the anti-CD19 CAR construct (Kochenderfer et al., "B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells," Blood 119:2709-20 (2012)).

[0199] In group 1, anti-CD19 CAR+ T cells 3 × 10 6 ~30×10 6 In the first six subjects, circulating anti-CD19 CAR+ T cells reached a maximum of 0.02–1% of total PBMCs within 2 weeks of infusion. The highest numbers of anti-CD19 CAR+ T cells (28 × 10 anti-CD19 CAR+ T cells, respectively) were detected at higher levels, then rapidly disappeared and were undetectable after 50 days. 6 cells / kg and anti-CD19 CAR+ T cells 30 × 10 6 Subjects 7 and 8 who received 100 mg / kg of IgG reached >10% of their total PBMCs. They had a higher peak percentage of anti-CD19 CAR+ T cells and a longer persistence of anti-CD19 CAR+ T cells in the blood (>130 and 180 days, respectively).

[0200] Table 1. Demographics of Clinical Trial Subjects TIFF2025148404000001.tif146166

[0201] In Group 2, in the absence of interleukin-2 treatment, anti-CD19 CAR+ T cells were The cells showed similar proliferation in peripheral blood within 1 week, followed by disappearance and complete disappearance from the circulation within several weeks (Table 2).

[0202] Overall, there was a significant correlation between the dose of anti-CD19 CAR+ T cells and their proliferation and persistence in peripheral blood. Similarly, so far, there has been no clear relationship between anti-CD19 CAR+ T cells, respectively. The dose of anti-CD19 CAR+ T cells, the proliferation or persistence of anti-CD19 CAR+ T cells in the blood, and the effects of this treatment There was no apparent relationship between clinical response or toxicity.

[0203] Table 2. Expansion and persistence of anti-CD19 CAR+ T cells in peripheral blood of subjects in Group 2 TIFF2025148404000002.tif31155

[0204] In groups 1 and 2, there was no secondary proliferation after the primary expansion of anti-CD19 CAR+ T cells 7–14 days after infusion. There is no evidence of oncogenic transformation due to the treatment. Results for Group 3 are still available at the time of data cutoff. It wasn't available.

[0205] efficacy Clinicians evaluated 32 subjects for safety and 29 subjects for efficacy. The overall response rate for the 29 subjects evaluable for efficacy was 76%. Eleven of the 29 subjects (38%) achieved a CR, and 11 of 29 subjects (38%) achieved a PR (Figures 2A and 2B; Table 3).

[0206] Sixteen of 29 evaluable subjects (55%) remained in response from their initial treatment, with 12 subjects (including those who were retreated) having a response duration of more than 1 year (Table 3). All patients were re-treated after progression, and all are currently in response (17.4 to over 52.2 months).

[0207] As shown in Table 3, 17 of 19 subjects with refractory aggressive DLBCL / PMBCL were evaluable for disease response (1 subject was unevaluable; 1 subject had not yet been evaluated). Of these 17 subjects, 11 (65%) had a response, and 6 / 17 (35%) subjects had a CR. The median duration of response was 7.3 months.

[0208] Six of seven evaluable subjects with CLL (86%) had a response, with four of seven subjects (57%) achieving a CR (Table 3). The median duration of response was 22.2 months, with responses ongoing for more than 27 months. Four of seven subjects (57%) are still responding, including three subjects in the previous study (Table 3).

[0209] Table 3. Objective response rate and duration of response by tumor type TIFF2025148404000003.tif88159 "+" indicates that the response is still ongoing.

[0210] Five of five evaluable subjects with indolent NHL (100%) responded, with subject 1 Five of the patients (20%) achieved a CR. The median duration of response was 18.8 months (Table 3). Five patients (5 of 5; 100%) continued to respond, with two patients experiencing responses longer than 45 months (Table 4).

[0211] safety Adverse events Thirty-two subjects were treated with anti-CD19 CAR+ T cells, with no adverse events reported for the last subject treated. Not yet reported. The overall safety summary includes all 32 treated subjects. The group summary is provided twice for subjects 1010003 and 1010004 (once when these subjects were treated in group 1 and once when these subjects were treated in group 2) (anti-CD19 CAR+ T cells were used). Includes safety data for the treatment of rheumatoid arthritis (rheumatoid arthritis, rheumatoid arthritis, and rheumatoid arthritis).

[0212] Summary of adverse events A summary of adverse events is provided in Table 4. Overall, 31 subjects (97%) experienced any adverse event. 0 subjects (0%) experienced grade 3 as the worst grade, and 29 subjects (91%) experienced grade 4 as the worst grade. Twenty subjects (63%) experienced adverse events related to anti-CD19 CAR+ T cells; six subjects (19%) experienced a worst-case grade of 3, eight subjects (25%) experienced a worst-case grade of 4, and no subjects experienced a grade 5 event. Sixteen subjects (50%) experienced serious adverse events; three subjects (9%) experienced a worst-case grade of 3, and nine subjects (28%) experienced a worst-case grade of 4. Most patients experienced grade 4, and two patients (6%) experienced grade 5.

[0213] (Table 4) Summary of adverse events TIFF2025148404000004.tif90169

[0214] Dose-limiting toxicity The incidence of DLTs within groups 1, 2, and 3 was 38%, 40%, and 0%, respectively. Except for subject 1010002, DLTs were primarily neurotoxicity, with two cases of elevated creatinine and one event each of hypoxia and hypotension. Table 6 provides a list of DLTs. In group 3, no DLTs were reported. In group 3, 2 × 10 6 Conditioning regimen using anti-CD19 CAR+ T cells at 100 cells / kg was tested.

[0215] Table 5. Dose-limiting toxicities TIFF2025148404000005.tif110166

[0216] Cytokine release syndrome Cytokine release is induced by activated T cells engaging CD19 targets. Using a search strategy, adverse events that occurred after treatment and could be attributed to CRS included fever, febrile neutrophils, Adverse events potentially attributable to cytokine release included cytopenia, hypotension, acute vascular leak syndrome, elevated creatinine, renal failure, hypoxia, and pleural effusion. Twenty-eight subjects (88%) reported adverse events, including ≥ grade 3 events in 24 subjects (75%) and serious events in 6 subjects (19%). IL-2 (used in Arm 1) and conditioning chemotherapy were associated with a significant adverse event rate. Adverse events from combination therapy, such as steroid therapy (leading to febrile neutropenia), potentially confound this analysis.

[0217] Clinical signs of CRS typically occurred during the first week after anti-CD19 CAR+ T-cell infusion and were significantly higher in patients with CR. This was uncommon in elephants. Only 1 of 11 subjects in Group 3 experienced Grade 3 hypotension. and 4 experienced grade 3 fever. Events of acute vascular leak syndrome, oliguria, elevated creatinine, and renal failure were reported only by subjects in groups 1 and 2.

[0218] Neurological adverse events Neurological adverse events, primarily aphasia / language disorders, confusion, motor neuropathy, and somnolence, were observed in all three groups. Thirteen subjects (41%) had severe, ≥ Grade 3 neurotoxicity. Eleven subjects (34%) experienced serious events.

[0219] The subject who died from neurotoxicity had a CNS cerebrovascular ischemic event in the setting of influenza A virus infection, which the investigator considered unrelated to the anti-CD19 CAR+ T cells. did.

[0220] Five subjects (16%) with neurotoxic events required airway protection for the neurological adverse event. All of these subjects were in Groups 1 and 2. All of the subjects in Group 3 were intubated and required mechanical ventilation. There were no subjects.

[0221] Neurological adverse events occurred between days 2 and 17 after anti-CD19 CAR+ T-cell infusion, with a median of 6 days. However, one subject experienced an episode of rheumatoid arthritis at day 110 after anti-CD19 CAR+ T cell infusion. Given the time of onset, symptoms, and the context of the brain MRI findings, the investigator considered this event to be related to fludarabine and not attributable to the anti-CD19 CAR+ T cells. The median time for neurological adverse events to resolve to grade 1 or less was 14 days after infusion. It was.

[0222] death Two subjects died within 30 days of chemotherapy and anti-CD19 CAR+ T cell infusion. Subject 11 died 18 days after the investigational treatment from a cerebral infarction accompanied by viral pneumonia, influenza A infection, E. coli infection, respiratory distress, and hypoxia. The patient had PMBCL with extensive fibrous mediastinal lymphoma involvement and died 16 days after treatment on the study. The cause of death was not determined at the time of the autopsy, and the autopsy report stated that the cause of death was likely due to mediastinal involvement of PMBCL. The investigators concluded that the cause was cardiac arrhythmia. None of the events were related to the anti-CD19 CAR+ T cells. And then I didn't see it.

[0223] Example 2 Cyclophosphamide 300 mg / m in selected patients 2 / day and fludarabine 30 mg / m 2 / day included Conditioning chemotherapy was administered for 3 days from day -5 to day -3. On day 0, the first subset of patients (patients 22-28) (Table 6) received a 10-day supply of new chemotherapy. A second subset of patients (patients 29–32) received fresh anti-CD19 CAR+ T cells and were treated for 6 days. received cryopreserved anti-CD19 CAR+ T cells.

[0224] Table 6. Status and outcome data for patients 22–28. TIFF2025148404000006.tif61157DLBCL=Diffuse large B-cell lymphoma; FL=Follicular lymphoma; PR=Partial response; CR=Complete response; PD=Progressive disease

[0225] Patient sera were tested by Luminex using the Millipore HCD8MAG15K17PMX kit (T1, T2, immunomodulatory cytokines, chemokines, immune effectors). Before and after treatment, interleukin 15 (IL-15), monocyte chemoattractant protein 1 (MCP-1) , gamma-inducible protein 10 (IP-10), placental growth factor (PLGF), soluble intercellular adhesion molecule 1 (sICAM-1), C-reactive protein (CRP), vascular endothelial growth factor D (VEGF-D), and macrophage inflammatory protein 1β (MIP-1β) levels were measured.

[0226] Among patients 22-28, patients 22-25 and 27 showed at least a partial response, and patients 26 and 28 showed progressive disease after treatment. For patients 22-26, the levels of IL-15, MCP-1, and PLGF in the patient's serum showed at least some increase (Figures 4A, 4B, and 4D), while In patients with IL-10, sICAM-1, CRP, VEGF-D, and MIP-1β levels increased in some patients and remained stable or decreased in others (Figures 4C and 4E–4H). Only IL-15 was measured in patients 27 and 28 (Figure 4A).

[0227] Some differences in marker levels were observed between responding patients, who showed either a partial or complete response, and non-responding patients with progressive disease. IL-15 levels were significantly higher than baseline. The levels of IL-15 increased by an average of about 35-fold in responder patients compared to non-responder patients, ranging from about 10-fold to about 55-fold, while non-responder patients each had a less than about 10-fold increase in IL-15 levels (Figure 5A). MCP-1 levels in responders increased by an average of about 5-fold, ranging from about 2-fold to about 7-fold, while non-responders (patient 26) had a less than 4-fold increase in MCP-1 levels (Figure 5B). levels increased by an average of about 3.5-fold, ranging from about 2-fold to about 7-fold, while non-responders There was essentially no change in serum IP-10 levels (Figure 5C). PLGF levels in responders increased by an average of about 30-fold, ranging from a slight increase of less than about 2-fold to a greater than about 100-fold increase, while non-responders had only a slight increase in serum PLGF levels (Figure 5D). sICAM-1 levels in responders increased by an average of about 3-fold, ranging from essentially unchanged to a 4.5-fold increase. In contrast, serum sICAM-1 levels were essentially unchanged in non-responders (Figure 5E). CRP levels in responders increased by an average of approximately 10-fold, ranging from essentially unchanged to an approximately 25-fold increase, whereas serum CRP levels in non-responders were essentially unchanged (Figure 5F). VEGF-D levels in responders increased by an average of approximately 3-fold, ranging from essentially unchanged to an approximately 6-fold increase, whereas serum CRP levels in non-responders were essentially unchanged (Figure 5F). Non-responders had essentially unchanged serum VEGF-D levels (Figure 5G). MIP-1β levels in responders increased by an average of approximately 1.5-fold, ranging from essentially unchanged to an approximately 3-fold increase. , whereas serum MIP-1β levels were reduced by only approximately 50% in non-responders ( Fig. 5H ).

[0228] Patients 30-33 received cryopreserved cells produced over a 6-day period and were randomly assigned to receive cryopreserved cells on selected days ranging from day -6 to day 18. In addition, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFNγ or IFNG), interleukin 10 (IL-10), IL-15, interleukin 2 (IL-2), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 8 (IL-8), IP-10, MCP-1, MIP-1β, serum granzyme A (GRNZA), serum granzyme B (GRNZB), PLGF, CRP, monocyte chemotactic factor (MCF), and monocyte chemotactic factor (MCF). We measured the levels of various cytokines, chemokines, effectors, inflammatory markers, and adhesion molecules, including mitochondrial protein 4 (MCP-4), interleukin 16 (IL-16), thymus and activation-regulated chemokine (TARC), eotaxin-3, sICAM-1, soluble vascular adhesion molecule 1 (sVCAM-1), and serum amyloid A (SAA) (Figures 6A-6V).

[0229] Example 3 To ameliorate the degree and duration of lymphodepletion observed in Group 3 of Example 1, the dose of conditioning chemotherapy in Cohort A1 was increased to cyclophosphamide 500 mg / m 2 and fludarabine 30 mg / m 2 and both were administered at a target dose of 2 × 10 anti-CD19 CAR+ T cells. 6 The dose of cyclophosphamide used in this regimen (Cohort A1) was approximately 38% lower than that used in the cyclophosphamide 30 mg / kg conditioning regimen of Group 2 from Example 1 (incidence of dose-limiting toxicity (DLT) 29%), and used the same lower fludarabine dose as Group 3 in Example 1.

[0230] Evaluation of higher doses of conditioning chemotherapy and / or different doses of anti-CD19 CAR+ T cells will proceed based on evaluation of the incidence of DLTs and profit / loss. The CAR vector construct is identical to the construct described in Example 1. This example demonstrates the rapid, closed, bead-free process. A clinical trial designed to test the safety and efficacy of anti-CD19 CAR+ T cells generated by The characteristics of the T cell product are preserved when the process is completed.

[0231] Experimental design A Phase 1 / 2 Multicenter Study Evaluating the Safety and Efficacy of KTE-C19 in Subjects with Refractory NHL An open-label study will be conducted. The study will be divided into two distinct phases, called Phase 1 and Phase 2. do.

[0232] During Phase 1, approximately 6-24 subjects with DLBCL, PMBCL, or TFL will be enrolled to evaluate the safety of the KTE-C19 regimen. The sponsor's internal Safety Review Team (SRT) will review the safety data. The study will review the data and make recommendations regarding further Phase 1 studies and progression to Phase 2, as shown in Figure 3. cormorant.

[0233] During Phase 2, subjects will be enrolled in two separate cohorts, designated Cohort 1 and Cohort 2. Cohort 1 will enroll adult subjects with refractory DLBCL, and Cohort 2 will enroll adult subjects with refractory PMBCL and TFL. TFL is defined as subjects who have previously received chemotherapy for follicular lymphoma.

[0234] Regardless of the phase of the study, each subject will follow the same study treatment schedule and procedural requirements. Each subject will undergo the following study periods: Screening / Leukapheresis Period; Conditioning Chemotherapy Period; Investigational Product (IP) Treatment Period; Post-Treatment Evaluation Period; and Long-Term Follow-Up Period. Go through and move forward.

[0235] Test duration For individual subjects, the length of participation will be a maximum of 28 days for the screening period, 5-7 days for the conditioning chemotherapy treatment period, and the KTE-C19 treatment period (which includes a 7-day inpatient recovery period). (including gestational age), post-treatment evaluation period, and long-term follow-up period (up to 15 years of survivorship surveillance). nothing.

[0236] Subjects will be followed for 3 months after treatment for any adverse events. After 3 months, subjects will be followed at intervals outlined in the Schedule of Assessments (SOA) for targeted adverse events / serious adverse events (e.g., hematological, neurological, secondary malignancies, infections, or autoimmune disorders) and for the presence of replication-competent retrovirus (RCR) in the subject's blood. The need for long-term follow-up will depend on the treatment. This is based on the potential persistence of the gene transfer vector in the implanted subject.

[0237] Study completion is defined as the time when the last subject completes the long-term follow-up visit, is considered lost to follow-up, withdraws consent, or dies. All subjects in Phase 2 Cohort 1 and the overall study population complete the 6-month disease response assessment, are lost to follow-up, withdraw from the study, or die, whichever occurs first. The primary analysis will be performed at the time of death.

[0238] Eligibility Inclusion criteria for subjects included: a) Histologically confirmed aggressive B, as defined by WHO 2008, including the following types: Cellular NHL: DLBCL non-specific type, T cell / histiocyte-rich B large cell lymphoma, DLBCL associated with chronic inflammation, Senile Epstein-Barr virus (EBV)+ DLBCL; primary mediastinal (thymic) B-large cell lymphoma; or transformation of follicular lymphoma to DLBCL; b) Stable disease (duration of stable disease) as the best response to the most recent chemotherapy-containing regimen must be ≤12 months since previous autologous SCT) or progressive disease; and ≤12 months since previous autologous SCT chemotherapy-refractory disease, defined as one or more of disease progression or recurrence; c) Subjects must have received sufficient prior therapy, including, at a minimum, an anti-CD20 monoclonal antibody and an anthracycline-containing chemotherapy regimen, unless the tumor is determined by the investigator to be CD20 negative; d) subjects with transformed FL must have received prior chemotherapy for follicular lymphoma and subsequently have chemotherapy-resistant disease after transformation to DLBCL; e) At least one measurable lesion according to the revised IWG response criteria for malignant lymphoma; previously irradiated lesions are measurable only if progression is documented after completion of radiation therapy. Consider it possible; f) brain MRI showing no evidence of central nervous system lymphoma; g) At the time leukapheresis is planned for the subject, at least 2 weeks must have elapsed since either previous radiation therapy or systemic therapy; h) Toxicity from previous treatment must be stable or resolved to ≤ Grade 1 (excluding clinically insignificant toxicities such as alopecia); i) Subjects must be 18 years of age or older; j) Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; k) Subjects must have the following laboratory values: (i) ANC ≥ 1000 / uL; (ii) platelet count ≥ 50,000 / uL; (iii) serum creatinine ≤ 1.5 mg / dL, serum ALT / AST ≤ 2.5 ULN, and Gilbert's Adequate renal, hepatic, and cardiac function, defined as total bilirubin ≤ 1.5 mg / dl, excluding subjects with syndrome; and iv) cardiac ejection fraction ≥ 50% as determined by echo and no evidence of pericardial effusion; and l) Women of childbearing potential must have a negative serum or urine pregnancy test.

[0239] Exclusion criteria for subjects included: a) History of malignancy other than nonmelanoma skin cancer or carcinoma in situ (e.g., cervical, bladder, breast) or follicular lymphoma, unless disease-free for at least 3 years; b) a history of Richter transformation of CLL; c) autologous stem cell transplantation within 6 weeks of informed consent; d) history of allogeneic stem cell transplant; e) Previous CD19 targeting, excluding subjects who received KTE-C19 in this study and are eligible for retreatment therapy; f) previous chimeric antigen receptor therapy or other genetically modified T cell therapy; g) History of severe immediate hypersensitivity reactions caused by aminoglycosides; h) Clinically significant active infection (e.g., uncomplicated UTI, bacterial pharyngitis are acceptable) or were currently receiving IV antibiotics or had received IV antibiotics within 7 days prior to enrollment may be prescribed (prophylactic antibiotics, antivirals, and antifungals are permitted); i) A history of infection with HIV, hepatitis B virus (HBsAg positive), or hepatitis C virus (anti-HCV positive) I know; j) Subjects with detectable cerebrospinal fluid malignant cells or brain metastases, or a history of cerebrospinal fluid malignant cells or brain metastases; k) history of seizure disorder, cerebrovascular ischemia / hemorrhage, dementia, cerebellar disease, or any autoimmune disease with CNS involvement; l) Subjects with lymphoma spread to the atria or ventricles; m) Urgent treatment required due to mass effects such as intestinal obstruction or vascular compression; n) Primary immunodeficiency disorders; o) Any medical condition that may interfere with determining the safety or efficacy of the study treatment; p) Current or anticipated systemic corticosteroid therapy; Standard doses of topical and inhaled corticosteroids and physiological substitutes are acceptable for elephants; corticosteroid doses of prednisone 5 mg / day or more or other corticosteroids equivalent doses of are not tolerated; q) History of severe immediate hypersensitivity reaction to any of the drugs used in this study; r) live vaccines ≤6 weeks before the start of the conditioning regimen; s) Due to the potential adverse effects of preparative chemotherapy on the fetus or infant, women of childbearing potential who are pregnant or breastfeeding; women who have been sterilized or who have been postmenopausal for at least 2 years are not considered to be of childbearing potential; t) Subjects of both sexes who do not wish to practice birth control from the time of consent until 6 months after completion of KTE-C19; and u) In the investigator's judgment, the subject is unlikely to complete all protocol-required study visits or procedures, including follow-up visits, or to comply with the study's participation requirements.

[0240] Additionally, biomarker analysis was performed on blood and tumor samples to identify potential risk factors for KTE-C19. Evaluating predictive and pharmacodynamic markers. Prognostic markers in aggressive NHL. may also be assessed. Baseline leukapheresis samples and final KTE-C19 Samples will be deposited and analyzed by immunophenotyping and / or gene expression profiling. The remaining sample may be stored for future exploratory analysis of DNA, RNA, or protein markers. Archived tumor tissue will be collected for central path review. Additional analyses include CD19 expression, gene expression profiling, and These may include analysis of DNA markers, RNA markers, or protein The remaining tumor sample may be stored for future exploratory analysis of markers.

[0241] Treatment Protocol schedule Approximately 5 x 10 mononuclear cells were obtained for the production of leukapheresis (KTE-C19). 9 ~10×10 9 Targeting the individual Obtain white blood cells from each subject by apheresis (with a goal of 12-15 liters). The leukapheresis product is processed to enrich for the T cell-containing PBMC fraction. The T cells are then stimulated to proliferate and transduced with a retroviral vector to deliver the CAR gene. The T cells are then expanded and cryopreserved to generate the investigational product. After completion of the conditioning chemotherapy regimen, subjects will receive their respective KTE-C19 infusions.

[0242] Test Procedures Subjects received a non-myeloablative conditioning regimen consisting of cyclophosphamide and fludarabine to induce lymphocyte depletion, providing an optimal environment for the growth of KTE-C19. Subjects were administered 100 mg of 10 ... Conditioning chemotherapy with cyclophosphamide and fludarabine will be initiated over 5 days. The 5-day conditioning chemotherapy regimen will be administered in an outpatient setting. The 7-day conditioning chemotherapy regimen may be administered as an outpatient or inpatient regimen at the investigator's discretion. It can be administered as an in-patient regimen.

[0243] Phase 1: In cohorts A1 and A2, subjects received the following 5-day conditioning chemotherapy regimen: Receive: 1 L of 0.9% NaCl saline given before cyclophosphamide on the day of infusion followed by 500 mg / m on days -5, -4, and -3. 2 Cyclophosph amide IV over 60 minutes; followed by 30 mg / m on days -5, -4, and -3 2 of fludarabine IV over 30 minutes; followed by an additional 1 L of 0.9% NaCl saline upon completion of the fludarabine infusion (Figure 3). In certain cases, institutional guidelines may require the use of mesna (2-mercaptoethanesulfonic acid). sodium phosphate) can be added.

[0244] In Cohort A3, subjects receive the following 5-day chemotherapy regimen: IV hydration with 1 L of 0.9% NaCl saline given before cyclophosphamide on the day of infusion; followed by 300 mg / m on days -5, -4, and -3. 2 of cyclophosphamide IV over 60 minutes; followed by 30 mg / m on days -5, -4, and -3 2 of fludarabine IV over 30 minutes; followed by an additional 1 L of 0.9% NaCl saline upon completion of the fludarabine infusion. In certain cases, mesna may be added per institutional guidelines.

[0245] For subjects enrolled in cohorts A1, A2, or A3, days -2 and -1 are considered to be day 0. This is a rest day before KTE-C19 injection.

[0246] In cohorts B1 and B2, subjects will receive the following 7-day chemotherapy regimen: IV hydration with a recommended 2.6 ml / kg / hr (maximum 200 ml / hr) of 0.9% NaCl saline administered as a continuous infusion starting 11 hours before the cyclophosphamide infusion, with hydration continuing until 24 hours after the final cyclophosphamide infusion; 30 mg / kg (1110 mg / m) on days -7 and -6. 2 ) cyclophosphamide IV given as a 120-minute infusion; followed by 25 mg / m on days -5, -4, -3, -2, and -1. 2 of fludarabine is given IV and infused over 30 minutes In certain cases, mesna may be added per institutional guidelines.

[0247] For subjects enrolled in cohorts B1 or B2, there will be no rest day between the last day of chemotherapy (day -1) and the day 0 KTE-C19 infusion.

[0248] For KTE-C19, subjects in cohorts A1, A3, or B1 will receive a target dose of 2 x 10 anti-CD19 CAR+ T cells. 6 cells / kg (±20%; anti-CD19 CAR+ T cells 1.6×10 6 cells / kg~anti-CD19 CAR+ T cells 2.4×10 6 Patients received KTE-C19 treatment, consisting of a single infusion of CAR-transduced autologous T cells administered intravenously at 100x the dose (100x the dose / kg). A minimum dose of 1 x 10 anti-CD19 CAR+ T cells was used. 6 may be administered at a dose of 100 / kg. For subjects eligible for the study, a maximum flat dose of 2 x 10 anti-CD19 CAR+ T cells was administered. 8 Individuals are administered.

[0249] Subjects in cohorts A2 or B2 will receive a target dose of 1 x 10 anti-CD19 CAR+ T cells. 6 pieces / kg(±20 %;anti-CD19 CAR+ T cells 0.8×10 6cells / kg~anti-CD19 CAR+ T cells 1.2×10 6 Patients will receive KTE-C19 treatment, consisting of a single infusion of CAR-transduced autologous T cells administered intravenously at a minimum dose of 0.5 x 10 anti-CD19 CAR+ T cells (cells / kg). 6 For subjects weighing more than 100 kg, a maximum flat dose of 1 x 10 anti-CD19 CAR+ T cells may be administered. 8 Individuals are administered.

[0250] Phase 2: The KTE-C19 regimen determined to be safe by the SRT in Phase 1 will be advanced into the Phase 2 portion of the trial.

[0251] Retreatment Subjects who achieve PR or CR may be eligible for a second course of conditioning chemotherapy and KTE-C19 if their disease subsequently progresses (and the relapse is not known to be in CD19- malignant cells). To qualify for a second course of treatment, subjects will be re-evaluated and Patients should continue to meet the original study eligibility criteria, with the exception of any exclusion criteria related to their previous CAR therapy. and should not have received subsequent chemotherapy for the treatment of lymphoma. In addition, any toxicity related to fludarabine or cyclophosphamide, except for alopecia, should be stable or resolved to less than Grade 1 before retreatment. Maximum of 1 per subject A retreatment course of 100 mg / kg / day may occur. Subjects enrolled in Phase 2 will receive the same KTE-C19 regimen. Subjects enrolled in Phase 1 will receive the KTE-C19 regimen selected for Phase 2. If a Phase 2 regimen has not yet been selected, subjects will receive the final KTE-C19 regimen determined to be safe by SRT.

[0252] Subjects who experienced DLT in Phase 1 or comparable toxicity in Phase 2 were excluded. A subject is ineligible for retreatment. Furthermore, if the subject is known to have neutralizing antibodies, the subject is ineligible for retreatment. However, if non-neutralizing HAMA or HABA antibodies develop, the subject may be retreated if the subject meets the eligibility criteria.

[0253] Post-treatment assessment After completing the KTE-C19 infusion and being discharged (typically on day 8), all subjects will participate in a post-treatment evaluation period. Starting from day 0 (infusion of KTE-C19), subjects will be followed up at week 2, week 4 (±3 days) The patients returned to the clinic at 2 months (±1 week), and 3 months (±1 week). t test); PET-CT for disease determination; physical examination and vital signs; laboratory tests including chemistry panel, CBC with differential, β-HCG pregnancy test (serum or urine) for all women of childbearing potential, anti-KTE-C19 antibody, lymphocyte subsets, cytokine levels, anti-CD19 CAR+ T cell, and replication-competent retrovirus (RCR) analysis; reporting of adverse / serious adverse events; and reporting of concomitant medications. description; and including the collection of fresh tumor specimens for subjects who signed an optional portion of the agreement. can be done.

[0254] Circulating abundance, proliferation, persistence, and immunophenotype of transduced anti-CD19 CAR+ T cells The phenotype is monitored primarily by PCR analysis, supplemented by flow cytometry. Serum cytokine levels are also assessed from blood. The following cytokines may be included in the panel: pro-inflammatory and immunomodulatory cytokines IL-6, TNFα, IL-8, IL-1, IL-2, GM-CSF, IL-15, IL-17α, IFNγ, IL-12p40 / p70; immune effector molecules granzymes A, B, perforin, sFasL; acute phase response correlates CRP, SAA, and chemokines MIP-1α, MIP-3α, IP-10, eotaxin, MCP-4. KTE-C19 delivers retroviral vectors The presence of replication-competent retrovirus (RCR) in the blood of treated patients is also monitored, as it contains transduced T cells.

[0255] If a subject is eligible for retreatment with KTE-C19, the final scan before retreatment will be the eligibility criteria for retreatment. This is considered a baseline for assessing subsequent responses.

[0256] If at any time during the post-treatment evaluation period the subject fails to respond to treatment (i.e., CR or PR) or progresses after response, the subject proceeds directly to the 3-month visit and to the long-term follow-up visit. Patients will be followed for disease outcomes during the observation period.

[0257] All subjects will be followed for survival and disease status during the long-term follow-up period, if applicable. Subjects will begin the long-term follow-up period after completing the 3-month visit of the post-treatment evaluation period (whether they responded to treatment or proceeded directly to the 3-month visit due to disease progression). Starting from day 0 (KTE-C19 infusion), subjects will return to the clinic every 3 months (± 2 weeks) until month 18; every 6 months (± 1 month) from months 24 to 60; and beginning at year 6, i.e., month 72 (± 3 months), subjects will return to the clinic annually for up to 15 years. At these visits: Complete the following procedures: physical exam; PET-CT scan; disease determination; laboratory tests including CBC with differential, anti-KTE-C19 antibody, lymphocyte subset, anti-CD19 CAR+ T cell, and RCR analysis; neurological, hematological Report of target adverse events / serious adverse events until disease progression (24 months or until disease progression, whichever occurs first), including steroids, infections, autoimmune disorders, and secondary malignancies; description of target concomitant medications, including gamma globulins, immunosuppressants, anti-infectives, vaccines, and any therapy for treatment of progressive disease (for 2 years after disease progression).

[0258] Evaluations include baseline PET-CT scans of the neck, chest, abdomen, and pelvis, along with appropriate imaging of all other areas of disease. Subjects undergo their first planned PET-CT tumor assessment after KTE-C19 injection 4 weeks after KTE-C19 injection, and at regular intervals as described above.

[0259] Bone marrow aspirate and biopsy will be performed in subjects who have achieved a CR. Subjects must have bone marrow involvement of lymphoma prior to treatment according to the revised IWG response criteria for malignant lymphoma. Bone marrow aspirate and biopsy should be performed only when the patient is first treated or if new abnormalities in the peripheral blood count or blood smear raise clinical suspicion of lymphoma spread to the bone marrow after treatment. To assign a CR to treatment, the bone marrow aspirate and biopsy must show no morphologic evidence of disease or, if morphology is equivocal, immunohistochemistry must be negative.

[0260] Test evaluation items major The primary endpoint for Phase 1 was the incidence of adverse events defined as dose-limiting toxicities (DLTs). The primary endpoint for Phase 2 was the incidence of adverse events as assessed by the investigator. The revised IWG treatment response criteria for advanced lymphoma are classified as complete or partial response. The objective response rate (ORR) is defined as the incidence of either All subjects who do not meet the criteria for objective response will be considered non-responders.

[0261] secondary The objective response rate among subjects in Phase 1 will be summarized. The objective response rate among subjects in Phase 2 will be assessed by the IRRC. The objective response rate is defined as the occurrence of either a complete or partial response according to the revised IWG Response Criteria for Malignant Lymphoma as assessed by the IRRC. Duration of response (DOR) for subjects experiencing an objective response is defined as the rate at which the objective response is achieved. All subjects who do not meet the criteria for objective response by the cutoff date of the analysis data are considered non-responders. The revised IWG response criteria for malignant lymphoma were used to evaluate the response rate from the date of first objective response. Patients who do not meet the criteria for progression or death by the analysis data cutoff date will be censored at the date of last evaluable disease and their response will be recorded as ongoing.

[0262] Dose-limiting toxicity (DLT) Dose-limiting toxicities were defined as the following KTE-C19-related events occurring within the first 30 days of KTE-C19 infusion: a) Grade 4 neutropenia lasting longer than 21 days from the date of cell transplantation; b) Grade 4 thrombocytopenia persisting for more than 35 days from the date of cell transplantation; c) Any condition requiring intubation, including Grade 4 confusion requiring intubation to secure an airway Any KTE-C19-related adverse event will be considered a DLT; d) All other grade 3 toxicities lasting longer than 3 days and the following conditions not considered DLTs: All grade 4 toxicities except: (i) aphasia / language disorder or confusion / cognitive impairment that was grade 1 or less within 2 weeks and recovered to baseline within 4 weeks; (ii) fever grade 3; (iii) lymphopenia, decreased hemoglobin, neutropenia, and thrombocytopenia met the above definition of DLT. (iv) myelosuppression, defined as neutropenia and thrombocytopenia (including bleeding in the setting of a platelet count less than 50 × 109 / L and documented bacterial infection in the setting of neutropenia), unless otherwise noted; (iv) immediate hypersensitivity reactions occurring within 2 hours of cell infusion (related to cell infusion) and reversible to Grade 2 or less within 24 hours of cell administration with standard therapy; and (v) Grade 3 or or hypogammaglobulinemia 4.

[0263] CRS will be graded according to the revised grading system (Lee 2014). Adverse events attributable to CRS will be mapped to an overall CRS grading decision for DLT determination.

[0264] During Phase 1, the safety of the KTE-C19 regimen will be evaluated in patients with DLBCL, PMBCL, or TFL. Approximately 6-24 subjects will be enrolled. Subjects in each cohort will be evaluated for DLT within the first 30 days following completion of the subject's respective KTE-C19 infusion. If the subject DLT rate is ≤1 in 6 subjects, cohort B1 may be investigated or the study may proceed to phase 2 of the clinical trial. The decision will be based on overall benefit / risk and available biomarker data.

[0265] However, if two of the six enrolled subjects experience a protocol-defined DLT during Phase 1, SRT will be administered to two additional sets of three subjects at the same dose administered to the first six subjects. In this scenario, the first 9 If ≤2 of 12 subjects or ≤3 of 12 subjects experience DLT, then enter additional cohorts, or Advance to Phase 2 of the trial.

[0266] If the DLT rate for subjects is >2 / 6, >3 / 9, or >4 / 12, additional subjects will be considered. Other KTE-C19 methods can be investigated in elephants 6–12 (Figure 3). The same DLT rules as above apply.

[0267] Example 4 The desired cell dose was achieved by transducing autologous lymphocytes with a g-murine retrovirus carrying the anti-CD19 CAR construct gene to generate a T cell product, which was subsequently expanded. At harvest or after co-culture with CD19+ cells, flow cytometry and multiplex sampling of co-culture supernatants were performed. The anti-CD19 CAR+ T cell product was characterized by cytokine analysis. CAR+ T cells for product characterization were co-cultured with K562-CD19 cells or K562-NGFR control cells at an effector-to-target ratio of 1:1. The standard incubation time was 18 hours. Patients with relapsed / refractory B-cell malignancies were conditioned with cyclophosphamide and fludarabine and then received anti-CD19 CAR+ T cells.

[0268] Cytokine and chemokine levels were measured using the EMDmillipore Luminex® xMAP® multiplex assay. Data acquisition and analysis were performed using a Luminex 200™ instrument and xPONENT® 3.1 data analysis software. For IL-7: Human IL-7 was measured using the Quantikine HS ELISA Kit (HS750) according to the manufacturer's guidelines. Samples were used neat. Circulating CAR T cell numbers were measured by quantitative PCR analysis. 300 mg / m on days -5 and -4. 2 Cyclophosphamide and 30 mg / m on days -5, -4, and -3 2 Patients received a preconditioning regimen consisting of fludarabine. Before administration of cyclophosphamide and fludarabine on days -12 to -5 ("Pre"), CAR+ T Patient serum was collected immediately prior to administration of cells ("post") and on selected days up to day 18 after administration of CAR+ T cells. As shown in Figure 6, serum concentrations of GF-CSF, IL-2, MCP-1, IL-6, IL-10, MCP-4, CRP, IFN-gamma, granzyme A, IL-15, IL-5, granzyme B, IL-8, IP-10, MIP-1b, PLGF, IL-16, TARC, eotaxin-3, sICAM-1, sVCAM-1, and SAA were measured before and after conditioning and on selected days after administration of CAR+ T cells. Concentrations of certain cytokines were measured at 300 mg / m 2 Cyclophosphamide and 30 mg / m 2 Fuldarabhi It was found that the serum of patients after conditioning with acetaminophen increased (Fig. 7A-7I). In particular, the concentrations of IL-15, IL-7, PLGF, CRP, and MCP-1 significantly increased after conditioning with cyclophosphamide and fludarabine (Figures 7A–7D, 7G, 18A, and 18C–18E). Increases were also observed in the concentrations of IL-5, IL-10, IP-10, and s-ICAM1. (Figures 7E-7F, 7H-7I, and 18B). Conversely, perforin was inhibited by cyclophosphamide and Preconditioning with fludarabine was found to decrease serum levels (Figure 18F). As shown in Figure 18G, serum concentrations of various other analytes were observed to increase or decrease after preconditioning. Additional patients were treated, and the results are shown in Figures 11-17. In addition, increased serum levels of IL-15 (Figure 19A) and IP-10 (Figure 19B) and decreased serum levels of perforin (Figure 19C) after preconditioning were found to significantly correlate with positive objective responses in patients treated with CAR T cells.

[0269] Peripheral blood lymphocytes (PBLs) and serum after CAR+ T cell infusion were analyzed by flow cytometry. The site of anti-CD19 CAR+ T cells before infusion was assessed by ELISA and multiplex cytokine analysis. Kine production was compared with the K562-NGFR negative control (Figure 8). T1, T2, and immune homeostatic cytokines GM-CSF, IL-2, IFN-gamma, IL-5, IL-4, and IL-13, as well as pro-inflammatory cytokines and chemokines TNF-alpha, IL-6, granzyme B, MIP-1b(beta), MIP-1a(alpha). Concentrations of CD19, sCD137, and sCD137 were higher in anti-CD19 CAR+ T cell samples than in negative controls (Figures 8A-8L). In addition, engagement of target antigens by product T cells prior to infusion regulates their activity. Upregulation of receptors such as CD107a(alpha), 401BB, and PD-1, which can resulting in fusion (Figures 9A-9C).

[0270] A BD FACSCanto II utilizing FlowJo software was used for data acquisition and analysis. Multicolor flow cytometry was performed using the same CAR+ T cell product as the previous study. The shorter manufacturing process resulted in a CAR+ T cell product with a higher representation of CD4+, naive, and central memory T cells (Figure 10). After infusion, CAR+ T cells primarily differentiate into differentiated T cells and some central memory or niche T cells. The diversified subset composition including immune T cells is shown (Figure 10).

[0271] Anti-CD19 CD28 Zeta CAR+ T cells are clinically effective and have been shown to be effective in treating both lymphoma and leukemia. Induce long-lasting responses in patients with cyclophosphamide and fludara. Sustained clinical responses can occur without long-lasting CAR+ T cells in the circulation and restore normal B cells. Bottle conditioning favors homeostatic proliferation, activation, and trafficking of T cells. The CAR+ T cell treatment modifies the immune environment by inducing molecules that can Within a short time, this leads to a rapid increase in circulating cytokines and chemokines and subsequent recovery.

[0272] Example 5 300 mg / m 2 or more cyclophosphamide and 30 mg / m 2 or more doses of fludarabine Studies are conducted to assess the safety and efficacy of treating subjects with a non-myeloablative conditioning regimen. Lymphocyte depletion is further induced and maximized by in vivo expansion of KTE-C19. Doses of these conditioning chemotherapeutic agents are used to create a suitable environment.

[0273] Enrolled subjects will undergo leukopheresis to obtain PBMCs for the production of anti-CD19 CAR+ T cells. Subjects then received 500 mg / m administered on days -5 to -3. 2 / day cyclophosphamide and 60 mg / m 2 Subjects will then receive conditioning chemotherapy containing fludarabine / day. Subjects will then receive anti-CD19 CAR+ T cells / kg IV on day 0. As a starting dose, subjects will receive anti-CD19 CAR+ T cells 2 × 10 6 A starting dose of 100 mg / kg (±20%) may be administered, and the starting dose can then be increased or decreased depending on the subject's response.

[0274] After conditioning chemotherapy and administration of anti-CD19 CAR+ T cells, adverse effects, serum serum Subjects are monitored for cytokine levels, T cell counts, and disease response. IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, IL-21, MCP-1, IP-10, PLGF, sICAM-1, CRP, VEGF, VEGF-C, VEGF-D, sVCAM-1, MIP-1β, FGF2, IL-1b, Otaxin, GM-CSF, IFN gamma, IL-12p40, MDC, IL-12p70, IL-13, IL-17A, MIP-1a, TNFa, TNFb, granzyme A, granzyme B, perforin, SAA, MCP-4, and TARC Serum levels of various cytokines, chemokines, effectors, inflammatory markers, and adhesion molecules, including cyclophosphamide, fludarabine, and anti-CD19 CAR+ T cells, will be measured before and after conditioning to determine the effect of conditioning chemotherapy. Cells are collected before and after each administration and all levels are compared to the levels before conditioning chemotherapy. Disease response is compared to each patient's cytokine profile after conditioning to determine disease response and one or more sites after conditioning. To confirm any correlation between the levels of ATP and ATP.

[0275] The occurrence of adverse effects is closely monitored to determine the maximum tolerated dose of cyclophosphamide and fludarabine. Adverse effects may be medically controlled as necessary. The dose of one or both of cyclophosphamide and fludarabine may be increased or decreased to improve clinical efficacy and limit adverse effects. Any subject who shows disease progression following an initial partial response may receive a second treatment with the same or different levels of cyclophosphamide and / or fludarabine. Throughout this application, various publications are identified by author name and date in parentheses. or patent number or patent publication number. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into this application to more fully describe the state of the art as known to those skilled in the art as of the date of the invention described and claimed herein. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present invention. All of the various aspects, embodiments, and options described herein can be combined in any and all variations.

[0276] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present invention.

[0277] Having generally described the invention, a further understanding can be obtained by reference to the examples provided herein, which are for illustrative purposes only and are not intended to be limiting.

Claims

1. 200 mg / m 2 / day ~ 2000mg / m 2 / day dose of cyclophosphamide and 20 mg / m 2 / day ~ 900mg / m 2 and administering fludarabine at a dose of 100 mg / day to a patient in need of T cell therapy. How to condition.

2. The method of claim 1, wherein administration of cyclophosphamide and fludarabine (i) reduces endogenous lymphocytes, (ii) increases serum levels of homeostatic cytokines, (iii) enhances effector function of T cells administered after conditioning, (iv) enhances activation and / or availability of antigen-presenting cells, or (v) any combination thereof.

3. 200 mg / m 2 / day ~ 2000mg / m 2 / day dose of cyclophosphamide and 20 mg / m 2 / day ~ 900mg / m 2 1. A method for treating a patient in need of T cell therapy, comprising administering fludarabine at a dose of 100 mg / day to a patient in need of T cell therapy. A method for reducing endogenous lymphocytes in the

4. 4. The method of claim 3, wherein the endogenous lymphocytes comprise regulatory T cells, B cells, natural killer cells, CD4+ T cells, CD8+ T cells, or any combination thereof.

5. 200 mg / m 2 / day ~ 2000mg / m 2 / day dose of cyclophosphamide and 20 mg / m 2 / day ~ 900mg / m 2 1. A method for treating a patient in need of T cell therapy, comprising administering fludarabine at a dose of 100 mg / day to a patient in need of T cell therapy. A method for increasing serum levels of homeostatic cytokines in mice.

6. Homeostatic cytokines include interleukin 7 (IL-7), interleukin 15 (IL-15), interleukin 10 (IL-10), interleukin 5 (IL-5), gamma-inducible protein 10 (IP-10), Interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1), placental growth factor (PLGF), C-reactive protein (CRP), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1) or any combination thereof.

7. The serum level of IL-7 in the patient is at least two-fold, at least three-fold, or at least four-fold higher after administration of cyclophosphamide and fludarabine compared to the serum level of IL-7 before administration. , at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times 7. The method of claim 6, wherein the increase in the expression level is at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, or at least 90-fold.

8. Serum levels of IL-15 in patients before administration of cyclophosphamide and fludarabine 8. The method of claim 6 or 7, wherein the serum level of IL-15 is increased by at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, or at least 90-fold after administration compared to the serum level of IL-15 in the control group.

9. Serum levels of IL-10 in patients before administration of cyclophosphamide and fludarabine at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, after administration compared to the serum level of IL-10 in The method of any one of claims 6 to 8, wherein the increase is at least 10-fold, or at least 20-fold.

10. The serum level of IL-5 in the patient is at least two-fold, at least three-fold, or at least four-fold higher after administration of cyclophosphamide and fludarabine compared to the serum level of IL-5 before administration. , at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, 10. The method of any one of claims 6 to 9, wherein the increase in the expression level of the IgG antibody is at least 10-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold.

11. Serum levels of IP-10 in patients before administration of cyclophosphamide and fludarabine at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, The method of any one of claims 6 to 10, wherein the increase is at least 10-fold, at least 15-fold, at least 20-fold, or at least 30-fold.

12. 12. The method of any one of claims 6-11, wherein the serum level of IL-8 in the patient is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold after administration of cyclophosphamide and fludarabine compared to the serum level of IL-8 before administration.

13. Serum levels of MCP-1 in patients before administration of cyclophosphamide and fludarabine at least 1.5-fold, at least 2-fold, or at least 1.5-fold or at least 2-fold higher serum MCP-1 levels after administration compared to serum MCP-1 levels in the control group also increases by 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold The method of any one of claims 6 to 12.

14. The serum level of PLGF in the patient is at least 1.5-fold, at least 2-fold, or at least 3-fold higher after administration of cyclophosphamide and fludarabine compared to the serum level of PLGF before administration. fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, or at least 100 fold increase.

15. The serum level of CRP in patients is at least 1.5-fold, at least 2-fold, or at least 3-fold higher after administration of cyclophosphamide and fludarabine compared to the serum level of CRP before administration. , at least 4 times, at least 5 times, at least about 9 times, at least 10 times, at least 15 times , at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold increase.

16. Serum levels of sICAM-1 in patients receiving cyclophosphamide and fludarabine At least 1.5-fold, at least 2-fold, or less sICAM-1 serum levels after administration compared to pre-administration at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 16. The method of any one of claims 6 to 15, wherein the antibody titer is increased by at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, or at least 30 fold.

17. Serum levels of sVCAM-1 in patients receiving cyclophosphamide and fludarabine At least 1.5-fold, at least 2-fold, or less sVCAM-1 serum levels after administration compared to pre-administration at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, or 17. The method of any one of claims 6 to 16, wherein the expression level of the IL-1 receptor is increased by at least 5-fold.

18. 200 mg / m 2 / day ~ 2000mg / m 2 / day dose of cyclophosphamide and 20 mg / m 2 / day~900 mg / m 2 1. A method for treating a patient in need of T cell therapy, comprising administering fludarabine at a dose of 100 mg / day to a patient in need of T cell therapy. A method for enhancing the effector function of administered T cells in a mouse model.

19. 200 mg / m 2 / day ~ 2000mg / m 2 / day dose of cyclophosphamide and 20 mg / m 2 / day ~ 900mg / m 2 1. A method for treating a patient in need of T cell therapy, comprising administering fludarabine at a dose of 100 mg / day to a patient in need of T cell therapy. A method for enhancing the activation and / or availability of antigen-presenting cells in a subject.

20. The dose of cyclophosphamide was 300 mg / m 2 / day, and higher than 2000 mg / m 2 / day lower, please 20. The method of any one of claims 1 to 19.

21. The fludarabine dose was 30 mg / m 2 / day, higher than 900 mg / m 2 21. The method of any one of claims 1 to 20, wherein the dose is less than 100 mg / day.

22. The dose of cyclophosphamide is approximately 350 mg / m 2 / day ~ approx. 2000mg / m 2 / day, at least about 400 mg / m 2 / day ~ approx. 2000mg / m 2 / day, approximately 450mg / m 2 / day ~ approx. 2000mg / m 2 / day, about 500mg / m 2 / day ~ approx. 2000mg / m 2 / day, about 550mg / m 2 / day ~ approx. 2000mg / m 2 / day, or approximately 600 mg / m 2 / day ~ approx. 2000mg / m 2 22. The method of any one of claims 1 to 21, wherein the daily dose is 100 mg / day.

23. The dose of cyclophosphamide is approximately 350 mg / m 2 / day ~ approx. 1500mg / m 2 / day, about 350mg / m 2 / day ~ approx. 1000mg / m 2 / day, about 400mg / m 2 / day ~ approx. 900mg / m 2 / day, approximately 450mg / m 2 / day ~ approx. 800mg / m 2 / day, approximately 450mg / m 2 / day ~ approx. 700mg / m 2 / day, about 500mg / m 2 / day ~ approx. 600mg / m 2 / day, or approximately 300 mg / m 2 / day ~ approx. 500mg / m 2 22. The method of any one of claims 1 to 21, wherein the daily dose is 100 mg / day.

24. The dose of cyclophosphamide is approximately 350 mg / m 2 / day, about 400mg / m 2 / day, about 450mg / m 2 / day, about 500mg / m 2 / day, about 550mg / m 2 / day, about 600mg / m 2 / day, approximately 650mg / m 2 / day, about 700mg / m 2 / day, about 800mg / m 2 / day, approximately 900mg / m 2 / day, or approximately 1000 mg / m 2 24. The method of claim 23, wherein the daily dose is 100 mg / day.

25. The dose of fludarabine is approximately 35 mg / m 2 / day ~ approx. 900mg / m 2 / day, about 40mg / m 2 / day ~ approx. 900mg / m 2 / day, about 45mg / m 2 / day ~ approx. 900mg / m 2 / day, about 50mg / m 2 / day ~ approx. 900mg / m 2 / day, about 55mg / m 2 / day ~ approx. 900mg / m 2 / day, or approximately 60 mg / m 2 / day ~ approx. 900mg / m 2 / day How to do it.

26. The dose of fludarabine is approximately 35 mg / m 2 / day ~ approx. 900mg / m 2 / day, about 35mg / m 2 / day ~ approx. 800mg / m 2 / day, about 35mg / m 2 / day ~ approx. 700mg / m 2 / day, about 35mg / m 2 / day ~ approx. 600mg / m 2 / day, about 35mg / m 2 / day ~ approx. 500mg / m 2 / day, about 35mg / m 2 / day ~ approx. 400mg / m 2 / day, about 35mg / m 2 / day ~ approx. 300mg / m 2 / day, about 35mg / m 2 / day ~Approx. 200mg / m 2 / day, about 35mg / m 2 / day ~ approx. 100mg / m 2 / day, about 40mg / m 2 / day ~ approx. 90mg / m 2 / day, about 45mg / m 2 / day ~ approx. 80mg / m 2 / day, about 45mg / m 2 / day ~ approx. 70mg / m 2 / day, or approximately 50 mg / m 2 / day ~ approx. 60mg / m 2 25. The method of any one of claims 1 to 24, wherein the daily dose is 100 mg / day.

27. The dose of fludarabine is approximately 35 mg / m 2 / day, about 40mg / m 2 / day, about 45mg / m 2 / day, about 50mg / m 2 / day, about 55mg / m 2 / day, about 60mg / m 2 / day, about 65mg / m 2 / day, about 70mg / m 2 / day, about 75mg / m 2 / day, about 80mg / m 2 / day, about 85mg / m 2 / day, approximately 90mg / m 2 / day, approximately 95mg / m 2 / day, about 100mg / m 2 / day, about 200mg / m 2 / day, or approximately 300 mg / m 2 27. The method of claim 26, wherein the daily dose is 100 mg / day.

28. Cyclophosphamide doses of approximately 500 mg / m 2 / day and fludarabine at a dose of 60 mg / m 2 / day The method of any one of claims 1 to 27, wherein

29. The cyclophosphamide dose and the fludarabine dose should be administered for at least 1 day. at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or 29. The method of any one of claims 1 to 28, wherein the compound is administered daily for at least 7 days.

30. The dose of cyclophosphamide and the dose of fludarabine are administered daily for about 3 days. The method of any one of claims 1 to 28.

31. 29. The method of any one of claims 1 to 28, wherein the dose of cyclophosphamide is administered before, after, or simultaneously with the dose of fludarabine.

32. 32. The method of claim 31, wherein the dose of cyclophosphamide is administered before the dose of fludarabine.

33. 33. The method of any one of claims 1 to 32, further comprising administering one or more doses of IL-2.

34. 34. The method of claim 33, wherein each dose of IL-2 is at least about 10,000 IU / kg, at least about 50,000 IU / kg, at least about 100,000 IU / kg, at least about 200,000 IU / kg, at least about 400,000 IU / kg, at least about 600,000 IU / kg, at least about 700,000 IU / kg, at least about 800,000 IU / kg, or at least about 1,000,000 IU / kg.

35. 10. The method of claim 1, further comprising administering one or more doses of IL-15, IL-7, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof. The method of any one of claims 1 to 34.

36. T-cell therapy is selected from tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T-cell transplantation, and is administered with cyclophosphamide and fludarabine.

36. The method of any one of claims 1 to 35, further comprising administering said T cell therapy to a patient after administering said T cell therapy.

37. 37. The method of any one of claims 1 to 36, further comprising collecting blood cells from the patient prior to administration of cyclophosphamide and fludarabine.

38. 38. The method of claim 37, further comprising engineering blood cells to express a chimeric antigen receptor ("engineered CAR T cells") or a T cell receptor ("engineered TCR T cells").

39. After receiving cyclophosphamide and fludarabine, patients received either engineered CAR T cells or 36. The method of any one of claims 1 to 35, further comprising administering engineered TCR T cell therapy.

40. 40. The method of claim 36 or 39, wherein the T cell therapy treats a tumor in the patient.

41. Administration of cyclophosphamide and / or fludarabine is required prior to administration of T-cell therapy (Day 0). At least 7 days, at least 6 days, at least 5 days, at least 4 days, at least 3 days, at least 41. The method of any one of claims 1 to 40, wherein the method begins at least two days, or at least one day, before the administration of the compound.

42. 42. The method of claim 41, wherein administration of cyclophosphamide begins about 7 days prior to administration of T cell therapy and administration of fludarabine begins about 5 days prior to day 0.

43. 43. The method of any one of claims 1-42, wherein cyclophosphamide is administered to the patient for about 2 days, starting about 7 days and 6 days before day 0.

44. Fludarabine was administered to patients for approximately 5 days on days 5, 4, 3, 2, and 1 prior to day 0. The method of any one of claims 1 to 43,

45. 42. The method of claim 41, wherein administration of cyclophosphamide and fludarabine begins about 5 days before day 0.

46. Cyclophosphamide is administered to the patient for approximately 3 days on days 5, 4, and 3 prior to Day 0; 46. ​​The method of any one of claims 1 to 41 and 45.

47. 47. The method of any one of claims 1-41 and 45-46, wherein fludarabine is administered to the patient for about 3 days on days 5, 4, and 3 prior to day 0.

48. Cyclophosphamide and fludarabine were administered at a dose of 300 mg / m without or with cyclophosphamide and fludarabine. 2 / day cyclophosphamide and 30 mg / m 2 / day's Fulda 48. The method of any one of claims 39-47, wherein the method induces improved anti-tumor efficacy of the T cell therapy compared to the anti-tumor efficacy of the T cell therapy following administration of rabin.

49. After receiving cyclophosphamide and fludarabine and / or T-cell therapy, patients had increased serum concentrations of IL-15, IL-7, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, and CRP. , sICAM-1, sVCAM-1, IL-1, IL-2, IL-3, IL-4, IL-6, IL-9, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-20 , granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), vascular endothelial growth factor D (VEGF-D), macrophage inflammatory protein 1β (MIP-1β), leukemia inhibition factor (LIF), oncostatin M (OSM), interferon (IFN) alpha, IFN-beta, IFN-gamma, tumor necrosis factor (TNF) alpha, TNF-beta, CD154, lymphotoxin (LT) beta, 4-1BB ligand (4-1BBL), proliferation-inducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-related ligand (GITRL), tumor necrosis factor-stimulating factor (TNF-stimulating factor S), 48. The method of any one of claims 39-47, wherein the cytokine or pro-inflammatory factor is selected from the group consisting of Parr family member 14 (TNFSF14), OX40L, TNF- and ApoL-related leukocyte-expressed ligand 1 (TALL-1), TNF-related apoptosis-inducing ligand (TRAIL), chemokine (C-C motif) ligand (CCL) 1, macrophage inflammatory protein 1 alpha (MIP-1a or CCL3), CCL5, monocyte-specific chemokine 3 (MCP3 or CCL7), monocyte chemoattractant protein 2 (MCP-2 or CCL8), CCL13, thymus- and activation-regulated chemokine (TARC or CCL17), CCL22, and any combination thereof.

50. 50. The method of any one of claims 1-42 and 45-49, wherein cyclophosphamide and fludarabine are administered simultaneously or sequentially.

51. 51. The method of claim 50, wherein cyclophosphamide is administered to the patient before or after fludarabine.

52. 52. The method of any one of claims 39 to 51, wherein the engineered CAR T cell expresses a chimeric antigen receptor. How to post.

53. 53. The method of claim 52, wherein the chimeric antigen receptor comprises a binding molecule for a tumor antigen.

54. 54. The method of claim 53, wherein the binding molecule is an antibody or an antigen-binding molecule thereof.

55. The binding molecule is an antibody selected from the group consisting of scFv, Fab, Fab', Fv, F(ab')2, and dAb.

55. The method of claim 54, wherein the antibody is a prototypic binding molecule.

56. The method of any one of claims 52 to 55, wherein the chimeric antigen receptor comprises a hinge region.

57. The hinge region is IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 57. The method of claim 56, wherein the

58. 58. The method of claim 57, wherein the hinge region is that of IgG4.

59. The method of any one of claims 52 to 58, wherein the chimeric antigen receptor comprises a transmembrane domain.

60. 60. The method of claim 59, wherein the transmembrane domain is the transmembrane domain of CD28, CD8 alpha, CD4, or CD19.

61. 61. The method of claim 60, wherein the transmembrane domain is the transmembrane domain of CD28.

62. The method of any one of claims 52-61, wherein the chimeric antigen receptor further comprises a costimulatory signaling region.

63. The costimulatory signaling domain is CD28, OX-40, 41BB, CD27, inducible T cell costimulatory molecule (ICOS), CD3 gamma, CD3 delta, CD3 epsilon, CD247, Ig alpha (CD79a), or Fc gamma 63. The method of claim 62, wherein the signal transduction region is a receptor.

64. 64. The method of claim 63, wherein the costimulatory signaling region is the signaling region of CD28.

65. The method of any one of claims 52 to 64, wherein the chimeric antigen receptor further comprises a CD3 zeta signaling domain.

66. The tumor antigen is CD19 CD20, ROR1, CD22, carcinoembryonic antigen, alpha fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2 / Neu, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R alpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 combination, HER1-HER2 combination, and any combination thereof.

66. The method of any one of claims 52 to 65, wherein the compound is selected from the group consisting of:

67. The method of any one of claims 38-66, wherein the engineered CAR T cells reduce tumor size. Law.

68. The method of any one of claims 38 to 51, wherein the engineered TCR T cells express a T cell receptor.

69. The method of claim 68, wherein the T cell receptor comprises a binding molecule for a tumor antigen.

70. The tumor antigen is CD19 CD20, ROR1, CD22, carcinoembryonic antigen, alpha fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2 / Neu, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R alpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 combination, HER1-HER2 combination, and any combination thereof.

70. The method of claim 69, wherein the compound is selected from the group consisting of:

71. The method of claim 68, wherein the T cell receptor comprises a binding molecule for a viral oncogene. Law.

72. 72. The method of claim 71, wherein the viral oncogene is selected from human papillomavirus (HPV), Epstein-Barr virus (EBV), and human T-lymphotropic virus (HTLV).

73. The T cell receptor comprises a binding molecule for a testicular, placental, or fetal tumor antigen. Item 69. The method described in item 68.

74. 74. The method of claim 73, wherein the testicular, placental, or fetal cancer antigen is selected from NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), and melanoma antigen (MAGE).

75. 69. The method of claim 68, wherein the T cell receptor comprises a binding molecule for a lineage-specific antigen.

76. The lineage-specific antigens are melanoma antigen 1 (MART-1), gp100, and prostate-specific antigen, which are recognized by T cells. selected from prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and prostate stem cell antigen (PSCA), 76. The method of claim 75.

77. Any of claims 38-51 and 68-76, wherein the engineered TCR T cells reduce tumor size. The method described in claim 1.

78. A therapeutically effective amount of engineered CAR T cells is at least about 10 4 pieces, at least about 10 5 At least about 10 6 pieces, at least about 10 7 pieces, at least about 10 8 pieces, at least about 10 9 pieces, or at least about 10 10 The method of any one of claims 38 to 67, wherein

79. A therapeutically effective amount of engineered CAR T cells is approximately 10 4 pieces, about 10 5 pieces, about 10 6 pieces, about 10 7 pieces, about 10 8 pieces, about 10 9 pieces, or about 10 10 The method of any one of claims 38 to 67 and 78, wherein

80. A therapeutically effective dose of engineered CAR T cells was approximately 2 x 10 6 pieces / kg, approximately 3×10 6 pieces / kg, approximately 4×10 6 pieces / kg, approx. 5 x10 6 pieces / kg, approximately 6×10 6 pieces / kg, approximately 7×10 6 pieces / kg, approximately 8×10 6 pieces / kg, approximately 9×10 6 pieces / kg, approximately 1×10 7 pieces / kg, approximately 2×10 7 pieces / kg, approximately 3×10 7 pieces / kg, approximately 4×10 7 pieces / kg, approximately 5×10 7 pieces / kg, approximately 6×10 7 pieces / kg, approximately 7×10 7 pieces / kg, approximately 8×10 7 pieces / kg, or approximately 9 x 10 7 Claims 38 to 67 and 78: The method according to any one of the preceding claims.

81. The tumor is bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, Myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancer, and and a tumor derived from said combination of cancers.

82. 71. The method of claim 66 or 70, wherein the tumor antigen is CD19.

83. 82. The method of claim 81, wherein the tumor is a lymphoma or leukemia.

84. Lymphoma or leukemia is classified as B-cell chronic lymphocytic leukemia / small cell lymphoma, B-cell prolymphocyte lymphoma, or lymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., Waldenstrom's macroglobulinemia) leukemia), splenic marginal zone lymphoma, hairy cell leukemia, plasma cell neoplasms (e.g., plasma cell myeloma (i.e., multiple myeloma), or plasmacytoma), extranodal marginal zone B-cell lymphoma (e.g., MALT lymphoma), nodal marginal zone B-cell lymphoma, follicular lymphoma, transformed Follicular lymphoma, primary cutaneous follicle center lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Epstein-Barr virus positive DLBCL, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, HHV8-associated multicentric Large B-cell lymphoma, Burkitt lymphoma / leukemia, and T lymphoma arising in Castleman disease cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK cell leukemia, Adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides / Sézary syndrome, primary cutaneous anaplastic large Cell lymphoma, lymphomatoid papulosis, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, B-lymphoblastic leukemia / lymphoma, B-lymphoblastic leukemia / lymphoma with recurrent genetic abnormalities Lymphoblastic leukemia / lymphoma, T-cell lymphoblastic leukemia / lymphoma, and Hodgkin's lymphoma 84. The method of claim 83, wherein the cancer is selected from the group consisting of: pancreatic cancer, bronchial asthma, and pulmonary fibrosis.

85. Prior to administering a therapeutically effective amount of the engineered CAR T cells to the patient, the patient is administered a therapeutically effective amount of about 500 mg / m 2 / day cyclophosphamide and approximately 60 mg / m 2 administering fludarabine / day daily for 3 days and a method of treating a patient with a tumor, wherein the engineered CAR T cells express a chimeric antigen receptor. wherein the chimeric antigen receptor binds to CD19 and further comprises a CD28 costimulatory domain and a CD3-zeta signaling region.

86. 1. A method of treating a patient with lymphoma, comprising: (i) about 500 mg / m 2 / day cyclophosphamide and approximately 60 mg / m 2 / day fludarabine, and (ii) administering to the patient a therapeutically effective amount of engineered CAR T cells, The engineered CAR T cells express a chimeric antigen receptor, which is a receptor for CD19. and further comprising a CD28 costimulatory domain and a CD3-zeta signaling region.

87. administering to the patient a therapeutically effective amount of engineered CAR T cells.

1. A method of treating a patient with about 500 mg / m 2 / day cyclophosphamide and approximately 60 mg / m 2 and the engineered CAR T cells express a chimeric antigen receptor that binds to CD19 and further comprises a CD28 costimulatory domain and a CD3-zeta signaling region.

88. The method of any one of claims 1 to 87, further comprising administering saline to the patient. Law.

89. 89. The method of claim 88, wherein saline is administered before administration of cyclophosphamide, after administration of cyclophosphamide, or both before and after administration of cyclophosphamide.

90. 90. The method of claim 88 or 89, wherein saline is administered before administration of fludarabine, after administration of fludarabine, or both before and after administration of fludarabine.

91. 91. The method of any one of claims 1-90, further comprising administering to the patient mesna (sodium 2-mercaptoethanesulfonate).

92. Mesna is administered before cyclophosphamide and fludarabine, and after cyclophosphamide and fludarabine.

92. The method of claim 91, wherein the compound is administered after administration of cyclophosphamide and fludarabine, or both before and after administration of cyclophosphamide and fludarabine.

93. (i) cyclophosphamide, (ii) fludarabine, and (iii) engineered CAR T-cell therapy Patients receiving 200 mg / m 2 / day ~ 2000mg / m 2 / day dose of cyclophosphamide and and 20 mg / m 2 / day ~ 900mg / m 2 1. A kit comprising: a 100mg / day dose of fludarabine; and instructions for administering the 100mg / day dose of fludarabine daily for three days.

94. Cyclophosphamide doses of approximately 500 mg / m 2 / day, and the fludarabine dose is approximately 60 mg / m 2 / 94. The kit of claim 93, wherein the day is

95. 95. The kit of claim 93 or 94, further comprising NaCl.

96. 96. The kit of any one of claims 93-95, further comprising mesna.

97. Cyclophosphamide dose 200 mg / m 2 93. The method of any one of claims 1-19, 29-47, 49-84, 91, and 92, wherein the daily dose is 100 mg / day.

98. Claims 1 to 19, 29 to 47, 49 to 84, 91, 92, and claims ...

98. The method of any one of claims 97 to 98.

99. Prior to administering a therapeutically effective amount of the engineered CAR T cells to the patient, the patient is administered a therapeutically effective amount of about 200 mg / m 2 / day cyclophosphamide and approximately 20 mg / m 2 administering fludarabine / day daily for 3 days and a method of treating a patient with a tumor, wherein the engineered CAR T cells express a chimeric antigen receptor. wherein the chimeric antigen receptor binds to CD19 and further comprises a CD28 costimulatory domain and a CD3-zeta signaling region.

100. The dose of cyclophosphamide was 1000 mg / m 2 / day ~ 2000mg / m 2 / day, claims 1 to 23 and 25 The method of any one of claims 1 to 27, 29 to 47, 49 to 84, and 88 to 92.

101. Cyclophosphamide dose of approximately 1110 mg / m 2 102. The method of any one of claims 1-23, 25-27, 29-47, 49-84, 88-92, and 100, wherein the dose is 100 / day.

102. Claims 1-23, 25-27, 29-47, 49, wherein the dose of cyclophosphamide is about 30 mg / kg / day The method of any one of claims 84, 88-92, and 100.

103. Fludarabine doses of approximately 25 mg / m 2 / day, claims 1 to 23, 29 to 47, 49 to 84, 88 to 92, and the method of any one of 100 to 102.

104. Fludarabine doses of approximately 30 mg / m 2 / day, claims 1 to 23, 29 to 47, 49 to 84, 88 to 92, and the method of any one of 100 to 102.

105. Fludarabine doses of approximately 60 mg / m 2 / day, claims 1 to 23, 29 to 47, 49 to 84, 88 to 92, and the method of any one of 100 to 102.

106. After administration of cyclophosphamide and fludarabine, patients developed increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or or any combination thereof, or reduced serum levels of perforin and / or MIP-1b.

107. 1110 mg / m 2 / day ~ 2000mg / m 2 / day dose of cyclophosphamide and 25 mg / m 2 and administering to the patient in need of T cell therapy a dose of fludarabine per day.

10. A method of treating a patient with cyclophosphamide and fludarabine, wherein the patient has increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof, or decreased serum levels of perfomance, after administration of cyclophosphamide and fludarabine. phospho- and / or MIP-1b.

108. 1110 mg / m 2 / day dose of cyclophosphamide and 25 mg / m 2 / day dose of fludarabine to said patient in need of T cell therapy. 。

109. 200 mg / m 2 / day dose of cyclophosphamide and 20 mg / m 2 / day dose of fludarabine and a method for conditioning a patient in need of T cell therapy, the method comprising administering to said patient a compound ... 。

110. 2000 mg / m 2 / day dose of cyclophosphamide and 25 mg / m 2 / day dose of fludarabine to said patient in need of T cell therapy. wherein, after administration of cyclophosphamide and fludarabine, the patient exhibits increased serum levels of IL-7, IL-15, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, or any combination thereof, or decreased serum levels of perforin and / or MIP-1b.

111. Patients were given cyclophosphamide at a dose of 30 mg / kg / day and 25 mg / m 2 2. A method of conditioning a patient in need of T cell therapy comprising administering fludarabine at a dose of 100 mg / day to said patient.

112. A therapeutically effective amount of engineered CAR T cells is approximately 1.0 x 10 cells 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 2.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 3.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 4.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 5.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 6.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 7.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 8.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 9.0×10 5 pieces / kg ~ approx. 2×10 8 pieces / kg, approx. 0.5×10 6 pieces / kg ~ approx. 2×10 8 pieces / kg, approximately 2×10 6 pieces / kg ~ approx. 9×10 7 pieces / kg, approximately 3×10 6 pieces / kg ~ approx. 9×10 7 pieces / kg, approximately 4×10 6 pieces / kg ~ approx. 9×10 7 pieces / kg, approximately 5×10 6 pieces / kg ~ approx. 9×10 7 pieces / kg, approximately 6×10 6 pieces / kg ~ approx. 9×10 7 pieces / kg, approximately 7×10 6 pieces / kg ~ approx. 9×10 7 pieces / kg, approximately 8×10 6 pieces / kg ~ approx. 9×10 7 pieces / kg, approximately 9×10 6 pieces / kg ~ approx. 9×10 7 pieces / kg, approximately 1×10 7 pieces / kg ~ approx. 9×10 7 pieces / kg, approximately 2×10 7 pieces / kg ~ approx. 9×10 7 pieces / kg, approximately 3×10 7 pieces / kg ~ approx. 9×10 7 / kg, about 4×10 7 pieces / kg~about 9×10 7 / kg, about 5×10 7 pieces / kg~about 9×10 7 / kg, about 6×10 7 pieces / kg~about 9×10 7 / kg, about 7×10 7 pieces / kg~about 9×10 7 / kg, about 8×10 7 pieces / kg~about 9×10 7 / kg, about 2×10 6 pieces / kg~about 8×10 7 / kg, about 2×10 6 / kg~about 7×10 7 / kg, about 2×10 6 pieces / kg~about 6×10 7 / kg, about 2×10 6 / kg~about 5×10 7 / kg, about 2×10 6 / kg~about 4×10 7 / kg, about 2×10 6 / kg~about 3×10 7 / kg, about 2×10 6 / kg~about 2×10 7 / kg, about 2×10 6 / kg~about 1×10 7 / kg, about 2×10 6 pieces / kg~about 9×10 6 / kg, about 2×10 6 pieces / kg~about 8×10 6 / kg, about 2×10 6 / kg~about 7×10 6 / kg, about 2×10 6 pieces / kg~about 6×10 6 / kg, about 2×10 6 / kg~about 5×10 6 / kg, about 2×10 6 / kg~about 4×10 6 / kg, about 2×10 6 / kg~about 3×10 6 / kg, about 3×10 6 pieces / kg~about 8×10 7 / kg, about 4×10 6 pieces / kg~about 7×10 7 pieces / kg, approximately 5×10 6 pieces / kg ~ approx. 6×10 7 pieces / kg, approximately 6×10 6 pieces / kg ~ approx. 5×10 7 pieces / kg, approximately 7×10 6 pieces / kg ~ approx. 4×10 7 pieces / kg, approximately 8×10 6 pieces / kg ~ approx. 3×10 7 pieces / kg, or approximately 9 x 10 6 pieces / kg ~ approx. 2×10 7 Claims 38 to 67: The method according to any one of the preceding claims.

113. A therapeutically effective dose of engineered CAR T cells is approximately 0.8 x 10 cells 6 cells / kg ~ T cells approximately 1.2×10 6 The method of any one of claims 38 to 67, wherein the amount is 100 mg / kg.

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