Methods of conditioning patients for t cell therapy

Abstract

This invention provides a method for increasing the efficacy of T-cell therapy in patients who require it. The invention includes a method for conditioning a patient prior to T-cell therapy, wherein the conditioning comprises administering a composition of cyclophosphamide and fludarabine.

Description

Technical Field

[0001] This invention relates to a method for pre-conditioning patients requiring cancer treatment (e.g., T-cell therapy). More specifically, this invention relates to a method for improving the efficacy of T-cell therapy (including engineered CAR T-cell therapy) by first administering a conditioning chemotherapy regimen to patients requiring T-cell therapy, the regimen comprising cyclophosphamide and fludarabine.

[0002] [[Government Rights Statement]]

[0003] This invention was made during the execution of a Cooperative Research and Development Agreement with the National Cancer Institute (NCI) and an agency of the Department of Health and Human Services. The U.S. government owns certain rights to this invention. Prior Technology

[0004] Human cancers are essentially composed of normal cells that have undergone genetic or non-hereditary transformations into abnormal cancer cells. In this state, cancer cells begin to exhibit proteins and other antigens that differ from those of normal cells. The body's innate immune system can use these abnormal tumor antigens to specifically target and kill cancer cells. However, cancer cells use various mechanisms to prevent immune cells (such as T lymphocytes and B lymphocytes) from successfully targeting them.

[0005] Human T-cell therapy relies on concentrated or modified T cells to target and kill cancer cells in patients. Various technologies have been developed to concentrate natural T cells or genetically modified T cells that target tumor antigens to specifically target known cancer antigens. These therapies have demonstrated moderate (though promising) effectiveness in tumor size and patient survival. However, it has proven difficult to predict whether a given T-cell therapy will be effective in every patient.

[0006] Cyclophosphamide can be administered alone or in combination with other drugs, including carmustine (BCNU) and etoposide (VP-16). For monotherapy, cyclophosphamide can be administered intravenously (IV) at a dose of 40 to 50 mg / kg (1.5 to 1.8 g / m2) for 10 to 20 mg / kg / day for 2 to 5 days.

[0007] Recent studies have shown that preconditioning patients with one or more immunosuppressive chemotherapy drugs before T-cell infusion can increase the effectiveness of transplanted T-cells (Rosenberg et al., Clin. Cancer. Res. (2011)). However, current methods rely on high doses of toxic and nonspecific drugs, which can cause pain and sometimes fatal adverse events. Therefore, it remains necessary to identify effective preconditioning regimens for modified T-cell therapy. Summary of the Invention

[0008] This disclosure provides a method for conditioning a patient requiring T-cell therapy, comprising administering to the patient cyclophosphamide at a dose between 200 mg / m² / and 2000 mg / m² / day and fludarabine at a dose between 20 mg / m² / and 900 mg / m² / day.

[0009] This disclosure also provides a method for reducing endogenous lymphocytes in a patient requiring T-cell therapy, comprising administering to the patient cyclophosphamide at a dose between 200 mg / m² / and 2000 mg / m² / day and fludarabine at a dose between 20 mg / m² / and 900 mg / m² / day.

[0010] This disclosure also provides a method for increasing serum levels of homeostatic cytokines in patients requiring T-cell therapy, the method comprising administering to the patient cyclophosphamide at a dose between 200 mg / m² / and 2000 mg / m² / day and fludarabine at a dose between 20 mg / m² / and 900 mg / m² / day.

[0011] In some embodiments, the in vivo 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.

[0012] This disclosure also provides a method for enhancing administered T-cell effector function in patients requiring T-cell therapy, the method comprising administering to the patient cyclophosphamide at a dose between 200 mg / m² / and 2000 mg / m² / day and fludarabine at a dose between 20 mg / m² / and 900 mg / m² / day.

[0013] This disclosure also provides a method for activating and / or enhancing the effectiveness of antigen-presenting cells in patients requiring T-cell therapy, the method comprising administering to the patient cyclophosphamide at a dose between 200 mg / m² / and 2000 mg / m² / day and fludarabine at a dose between 20 mg / m² / and 900 mg / m² / day.

[0014] In some 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.

[0015] This disclosure also provides a method for treating a patient with lymphoma, comprising administering approximately 500 mg / m² / day of cyclophosphamide and approximately 60 mg / m² / day of fludarabine to the patient for three days prior to administering a therapeutically effective amount of engineered CAR T cells, wherein the engineered CAR T cells exhibit a chimeric antigen receptor that binds to CD19 and additionally includes a CD28 co-stimulatory domain and a CD3ζ signaling region.

[0016] This disclosure also provides a method for treating a patient with lymphoma, comprising (i) administering to the patient approximately 200 mg / m² / day of cyclophosphamide and approximately 20 mg / m² / day of fludarabine, and (ii) administering to the patient a therapeutically effective amount of engineered CAR T cells, wherein the engineered CAR T cells exhibit a chimeric antigen receptor that binds to CD19 and additionally includes a CD28 co-stimulatory domain and a CD3ζ signaling region.

[0017] This disclosure also provides a method for treating a patient with lymphoma, comprising (i) administering to the patient approximately 300 mg / m² / day of cyclophosphamide and approximately 30 mg / m² / day of fludarabine, and (ii) administering to the patient a therapeutically effective amount of engineered CAR T cells, wherein the engineered CAR T cells exhibit a chimeric antigen receptor that binds to CD19 and additionally includes a CD28 co-stimulatory domain and a CD3ζ signaling region.

[0018] This disclosure also provides a method for treating a patient with lymphoma, comprising (i) administering to the patient approximately 300 mg / m² / day of cyclophosphamide and approximately 60 mg / m² / day of fludarabine, and (ii) administering to the patient a therapeutically effective amount of engineered CAR T cells, wherein the engineered CAR T cells exhibit a chimeric antigen receptor that binds to CD19 and additionally includes a CD28 co-stimulatory domain and a CD3ζ signaling region.

[0019] This disclosure also provides a method for treating a patient with lymphoma, comprising (i) administering to the patient approximately 500 mg / m² / day of cyclophosphamide and approximately 60 mg / m² / day of fludarabine, and (ii) administering to the patient a therapeutically effective amount of engineered CAR T cells, wherein the engineered CAR T cells exhibit a chimeric antigen receptor that binds to CD19 and additionally includes a CD28 co-stimulatory domain and a CD3ζ signaling region.

[0020] This disclosure also provides a method for treating a patient with lymphoma, comprising administering to the patient a therapeutically effective amount of engineered CAR T cells, wherein the patient has been conditioned with approximately 500 mg / m² / day of cyclophosphamide and approximately 60 mg / m² / day of fludarabine, and wherein the engineered CAR T cells express a chimeric antigen receptor that binds to CD19 and additionally includes a CD28 co-stimulatory domain and a CD3ζ signaling region.

[0021] This disclosure also provides a kit comprising (i) cyclophosphamide, (ii) fludarabine, and (iii) instructions for administering cyclophosphamide at a dose between 200 mg / m² / day and 2000 mg / m² / day and fludarabine at a dose between 20 mg / m² / day and 900 mg / m² / day for three days prior to treatment in patients requiring engineered CAR T-cell therapy. Simple Explanation of the Diagram

[0022] Figure 1 shows a schematic diagram of a CAR-engineered T cell, illustrating an exemplary CAR-engineered T cell and its construction. In this exemplary engineered CAR T cell, the target-binding domain includes an antibody-derived scFv domain, the co-stimulatory domain is derived from CD28, and the essential activation domain is derived from CD3ζ (zeta). A viral vector can carry the CAR vector construct and then integrate it into the T cell genome. This CAR construct can then be expressed as a transmembrane protein by the T cell.

[0023] Figure 2A shows the optimal response to anti-CD19 CAR+ T cells in B-cell malignancies, and Figure 2B shows the patient response to anti-CD19 CAR+ T cells in B-cell malignancies, illustrating the disease response in patients after treatment with anti-CD19 CAR+ T cells. For optimal responses in patients with B-cell malignancies, see Figure 2A, expressed as a percentage change in disease symptoms. Dashed lines indicate complete response (CR). Shaded bars indicate partial response. White bars indicate stable disease (SD). Black bars indicate progressive disease (PD). Figure 2B shows the patient's disease response several months after CAR+ T cell infusion. Solid black bars indicate partial response (PR), and gray bars indicate complete response (CR). Breaks in bars marked "PD" indicate patients experiencing progressive disease. Inverted triangles indicate T cell infusion time. Solid circles indicate B cell recovery time. White circles indicate the time it takes for CAR+ T cells to be cleared from the patient's blood. Horizontal arrows indicate that the patient's response is ongoing.

[0024] Figure 3 provides a sample illustration from a Phase 1 clinical trial, which is intended to determine the safety, efficacy, and dose-limiting toxicities of patients treated with 500 mg / m² / day of cyclophosphamide, 30 mg / m² / day of fludarabine, and 2 × 10⁶ anti-CD19 CAR+ T cells / kg.

[0025] Figures 4A to 4H show the serum levels of selected cytokines before and after conditioning with cyclophosphamide at 300 mg / m² / day and fludarabine at 30 mg / m² / day. The following serum levels are shown before and after administration of cyclophosphamide at 300 mg / m² / day and fludarabine at 30 mg / m² / day: interleukin-15 (IL-15; Figure 4A), monocyte chemotactic protein 1 (MCP-1; Figure 4B), γ-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). Pre-treatment serum was collected between -12 and -5 days prior to T-cell therapy, and post-treatment serum was collected on day 0 (Figures 4A to 4H).

[0026] Figures 5A to 5H show the serum levels of selected cytokines before and after conditioning, illustrating the fold changes in serum levels of the selected cytokines analytes in patients who responded to or did not respond to subsequent T-cell therapy after conditioning with cyclophosphamide at 300 mg / m² / day and fludarabine at 30 mg / m² / day. The figures show the 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) in responders and non-responders. Horizontal lines indicate mean values ​​(Figures 5A to 5H). Figure 5A shows individual patient changes in IL-15 and labels each patient's disease responsiveness next to each data point as partial response (PR), complete response (CR), stable disease (SD), or progressive disease (PD).

[0027] Figures 6A to 6V show the serum concentrations of selected cytokines in patients who received cyclophosphamide at 300 mg / m² / day and fludarabine at 30 mg / m² / day before receiving T-cell therapy on day 0, at different time points from day -10 to day 18. The serum concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF; Fig. 6A), IL-2 (Fig. 6B), MCP-1 (Fig. 6C), IL-6 (Fig. 6D), IL-10 (Fig. 6E), MCP-4 (Fig. 6F), CRP (Fig. 6G), interferon-γ (IFNγ; Fig. 6H), granzyme A (Fig. 6I), IL-15 (Fig. 6J), IL-5 (Fig. 6K), granzyme B (Fig. 6L), IL-8 (Fig. 6M), IP-10 (Fig. 6N), MIP-1b (Fig. 6O), PLGF (Fig. 6P), IL-16 (Fig. 6Q), thymus and activation-regulated chemokine (TARC; Fig. 6R), eosin-3 (Fig. 6S), sICAM-1 (Fig. 6T), soluble vascular adhesion molecule 1 (sVCAM-1; Fig. 6U), and (SAA; Fig. 6V) are shown.

[0028] Figures 7A to 7I show the serum levels of selected cytokines before and after administration of cyclophosphamide (300 mg / m² / day) and fludarabine (30 mg / m² / day). The serum concentrations of the selected cytokines analytes are shown before and after administration of cyclophosphamide (300 mg / m² / day) and fludarabine (30 mg / m² / day). Post-administration serum was collected prior to the intended 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 one patient. Horizontal bars show the mean (Figures 7A to 7I). The p-values ​​of the Wilcoxon matched-pairs signed rank test were applied to the analytes before and after conditioning, and the corresponding p-values ​​are shown (Figures 7A to 7I). Some IL-7 values ​​were greater than the upper limit of quantification (ULOQ; Figure 7B).

[0029] Figures 8A to 8L show the in vitro production of cytokines from anti-CD19 CAR+ T cells stimulated by K562 cells. The figures illustrate the in vitro production of various cytokines from CD19 CAR+ T cells compared to the negative control group (K562-NGFR) stimulated by K562 cells, as analyzed by the analyte (K562-CD19). The 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 α (TNF α; Figure 8G), IL-6 (Figure 8H), granzyme B (Figure 8I), MIP-1 β (Figure 8J), MIP-1 α (Figure 8K), and soluble CD137 (Figure 8L) used in the control and anti-CD19 CAR+ T cells are shown. This was used to label T1, T2, and in vivo constant cytokines (Figs. 8A to 8F), as well as pro-inflammatory cytokines and chemokines (Figs. 8G to 8L). Pre-infusion data were collected by co-culturing product T cells with K562-CD19 or control group K562-NGFR cells and measuring the concentrations of the listed analytes in the culture medium (Figs. 8A to 8L).

[0030] Figures 9A to 9C show the percentage of anti-CD19 CAR+ T cells (K562-CD19) exhibiting various cytokines after binding to the target antigen, induced by anti-CD19 CAR+ T cells, compared to the negative control group (K562-NGFR). The percentage of cells exhibiting CD107 α (Figure 9A), 4-1BB (Figure 9B), and programmed cell death 1 (PD-1; Figure 9C) is also shown. Data were collected before infusion by co-culturing product T cells with K562-CD19 or control group K562-NGFR cells and measuring the concentration of selected activation markers in the culture medium (Figures 9A to 9C). The p-values ​​shown indicate the results of pairwise T assays comparing K562-CD19 test cells with K562-NGFR negative control group cells (Figures 9A to 9C).

[0031] Figure 10 illustrates the subset composition of product T cells, showing various characteristics of product T cells and peripheral blood lymphocytes (PBL) from the perspective of manufacturing time (days). This data includes the percentage of anti-CD19 CAR+ T cells detected in the product-paired PBL; the CD8 / CD4 ratio in the product-paired PBL; the relative prevalence of naïve central memory T cells (Tcm), effector memory T cells (Tem), and effector T cells (Teff) in the anti-CD19 CAR+ CD8+ T cell population; and the relative prevalence of naïve central memory T cells (Tcm), effector memory T cells (Tem), and effector T cells (Teff) in the anti-CD19 CAR+ CD4+ T cell population (Figure 10). Phenotypic analysis of product T cells before infusion and PBL during peak proliferation in the blood on anti-CD19 CAR+ T cells is also presented (Figure 10). The p-value represents the rank of the association between manufacturing time and T cell subset composition.

[0032] Figures 11A through 11G show an overview of the performance of cytokines, chemokines, and other markers observed after conditioning NHL patients according to the present invention. The performance of IL-15 (Figure 11A), CRP (C-reactive protein; Figure 11B), IL-16 (Figure 11C), IL-7 (Figure 11D), PLGF (placental growth factor; Figure 11E), perforin (Figure 11F), and MCP-1 (monocyte chemoattractant protein-1; Figure 11G) is shown.

[0033] Figure 12 illustrates the quantification of changes observed in cytokines, chemokines, and other markers after conditioning with cyclophosphamide and fludarabine according to the invention.

[0034] Figures 13A and 13B show the fold changes in circulating IL-15 and perforin following modulatory chemotherapy in relation to objective response. p-values ​​were not adjusted for fold change. Analysis was performed on the markers measured prior to CAR T-cell infusion.

[0035] Figure 14 illustrates the biomarker analysis of cytokines, chemokines, and effector molecules. Markers were ranked within each biomarker category using the Wilcoxon sign-rank test, ordered from lowest to highest p-value. Markers with modified p-values ​​<0.05 are presented in most patients. Of the 41 markers measured, only 7 showed changes in most patients and were associated with p-values ​​<0.05. Analysis was performed on the measured markers prior to CAR T-cell infusion.

[0036] Figures 15A to 15H illustrate the sequential induction and clearance of homeostatic, inflammatory, and regulatory cytokines, chemokines, and immune effector molecules in the immune system. Representative markers are shown. Of the 41 markers measured, 22 showed at least a 2-fold increase from baseline in at least 50% of patients after CAR T-cell therapy. These 22 markers are 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. For homeostatic cytokines and chemokines, a peak was observed on days 3 to 4.

[0037] Figures 16A to 16H illustrate the sequential induction and clearance of immunomodulatory, inflammatory, and regulatory cytokines, chemokines, and immune effector molecules in vivo. Representative markers are shown. Of the 41 markers measured, 22 showed at least a 2-fold increase from baseline in at least 50% of patients after CAR T-cell therapy. These 22 markers are 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. For immunomodulatory cytokines and chemokines, peaks were observed from day 5 to day 7. "ULOQ": Upper limit of quantification.

[0038] Figure 17 shows the changes in treatment-related biomarkers and clinical response induced by anti-CD19 CAR T cells according to the present invention. The maximum fold change in marker levels from baseline (pre-conditioned) after CAR T cell therapy is shown. Each line represents an individual. The maximum fold change in all 41 assessed markers was compared between the responder and non-responder groups using the Wilcoxon rank-sum test. p-values ​​are not fold-adjusted and only biomarkers with p < 0.10 are shown: p-values ​​< 0.05 for IL-7 and sICAM-1. This association also applies to changes in the absolute levels of IL-7 (p = 0.0165), IL-15 (p = 0.0314), and IL-15 (p = 0.041).

[0039] Figures 18A to 18G show the changes in the levels of cytokines, chemokines, pro-inflammatory markers, and perforin* induced by low-dose conditioning, as well as the changes in these levels in the analytes before and after conditioning with cyclophosphamide and fludarabine. Figures 18A to 18F show the preceding and following levels of the following: IL-15 (Figure 18A), IP-10 (Figure 18B), CRP (Figure 18C), IL-7 (Figure 18D), MCP-1 (Figure 18E), and perforin (Figure 18F). Figure 18G summarizes the changes in serum levels of each analyte and the corresponding p-values.

[0040] Figures 19A to 19D show the opsonization-related regulation of IL-15, IP-10, and perforin, illustrating the association between changes in the amounts of the analytes after opsonization and the objective response to CAR T-cell therapy for IL-15 (Figure 19A), IP-10 (Figure 19B), and perforin (Figure 19C). Figure 19D summarizes the statistical significance of the data presented in each of Figures 19A to 19C. Implementation

[0041] This invention relates to a method for conditioning patients requiring T-cell therapy (e.g., engineered CAR T-cell therapy, such as autologous cell therapy (eACTTM)), the method comprising administering cyclophosphamide and fludarabine prior to T-cell therapy. Preconditioning the patient with these doses of cyclophosphamide and fludarabine prior to T-cell therapy reduces the number of endogenous lymphocytes and increases the serum levels of homeostatic cytokines and / or pro-immune factors present in the patient, thereby improving the efficacy of T-cell therapy. Once administered to the patient, this preconditioning creates a more suitable microenvironment for the proliferation of transplanted T cells. Preconditioning at the doses described herein unexpectedly reduces the number of endogenous lymphocytes while minimizing the toxicities associated with cyclophosphamide and fludarabine treatment. This invention relates to reducing the dose of cyclophosphamide and fludarabine used for preconditioning prior to T-cell therapy. Administering specific doses of cyclophosphamide and fludarabine induces optimal cytokine efficacy against transplanted T cells, while providing lower overall toxicity for patients receiving T-cell therapy.

[0042] [, definition , ]

[0043] To facilitate a better understanding of this invention, certain terms are first defined. Unless otherwise explicitly provided herein, each of the following terms as used in this application should have the following meanings. Additional definitions are explicitly stated in this application.

[0044] The term "and / or" as used herein is understood to mean the specific disclosure of each of two specifically designated features or components, with or without the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following states: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0045] Please understand that, where a state is described using the term "comprising", other similar states described using the terms "composed of" and / or "substantially composed of" are also provided.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. 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 Biolchemistry and Molecular Biology, Revised, 2000, Oxford University Press provide a general dictionary for those skilled in the art of using many of the terms used in this invention.

[0047] Units, prefixes, and symbols are represented in their form as accepted by the Système International de Unites (SI). Numerical ranges include the numbers that define the range. The headings provided herein are not intended to limit the various aspects of the invention as may be learned by referring to the entire specification. Therefore, the terms defined below are defined more fully by referring to the entire specification.

[0048] The term "activation" refers to the state of immune cells (such as T cells) that have been adequately stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. Furthermore, the term "activated T cell" also refers to T cells that have undergone cell division.

[0049] "Administration" means the physical introduction of a pharmaceutical preparation into an individual using any of the methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, or other non-enteric routes of administration (e.g., by injection or infusion). The phrase "non-enteric administration" as used herein means a mode of administration other than enteral and local administration (typically by injection), including but not limited to: intravenous, intramuscular, intra-arterial, intramenstrual, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, and in vivo electroporation. In some embodiments, the formulation is administered via an enteral route (e.g., orally). Other enteral routes include local, epidermal, or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual, or local administration. Alternatively, it can be given once, multiple times, and / or over a long period of time.

[0050] As used herein, an "adverse event" (AE) is any unfavorable and generally unplanned or adverse sign (including abnormal test results), symptom, medical occurrence, or illness associated with the use of this medical treatment. An adverse event is defined as a worsening of an existing medical condition. Worsening indicates an increase in the severity, frequency, and / or duration of an existing medical condition, or is associated with a more severe outcome.

[0051] The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to antigens. Typically, an antibody may comprise at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or its antigen-binding portion. Each H chain contains a heavy chain variable region (abbreviated 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 VL) and a light chain constant region. The light chain constant region contains a constant domain CL. The VH and VL regions can be further divided into highly variable regions (called complementarity-determining regions (CDRs)) and reserved regions (called framework regions (FRs)). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain variable region and light chain variable region contain binding domains that interact with the antigen. The constant region of the Ab can mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (such as effector cells) and the first component (C1q) of the typical complement system.

[0052] Immunoglobulins can be obtained from well-known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subtypes are also well-known in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to an Ab class or subclass (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. The term "antibody" includes, for example, natural and non-natural Abs; monoclonal and polyclonal Abs; chimeric and anthropomorphic Abs; human and non-human Abs; fully synthetic Abs; and single-chain Abs. Non-human Abs can be anthropomorphized using recombinant methods to reduce their immunogenicity in humans. Unless otherwise specified, the term "antibody" also includes antigen-binding fragments or portions of any of the above-described immunoglobulins, including monovalent and bivalent fragments or portions, and single-chain Abs.

[0053] "Antigen-binding molecule" or "antibody fragment" refers to any part of an antibody that is less than the whole. Antigen-binding molecules may include antigenic complementarity-determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules.

[0054] "Antigen" refers to any molecule that can induce an immune response or be bound by antibodies. An immune response may include antibody production or activation of cells with specific immune function, or both. Those skilled in the art will readily understand that any macromolecule (including virtually all proteins or peptides) can act as an antigen. Antigens can be expressed endogenously (i.e., expressed by genomic DNA) or recombinantly. Antigens may be specific to certain tissues (e.g., cancer cells) or can be expressed broadly. Furthermore, fragments of larger molecules can act as antigens. In one embodiment, the antigen is a tumor antigen.

[0055] The term "autologous" refers to any substance derived from an individual that is subsequently introduced into the same individual. For example, the engineered autologous cell therapy (eACTTM) described herein involves collecting lymphocytes from a patient, then engineering those lymphocytes to represent, for example, a CAR construct, and then administering them back to the same patient.

[0056] The term "allogeneic" refers to any substance derived from one individual that is later introduced into another individual of the same species, such as allogeneic T-cell transplantation.

[0057] "Cancer" refers to a variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" can include tumors. Examples of cancers that can be treated using the methods of this invention include, but are not limited to, cancers of the immune system, including lymphoma, leukemia, and other malignant white blood cell tumors. In some embodiments, the method of the present invention can be used to reduce the size of tumors derived from, for example, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, 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 (SMZL), esophageal cancer, small bowel cancer, endocrine system cancers, thyroid cancer, parathyroid cancer. Prostate 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) cancers, primary CNS lymphoma, tumor angiogenesis, spinal cord axonoma, brainstem glioma, pituitary adenoma, Karposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer (including asbestos-induced cancer), other B-cell malignancies, and combinations of the above cancers. Certain cancers may respond to chemotherapy or radiation therapy, or the cancer may be refractory to treatment. Refractory cancers are those that cannot be improved by surgery, those that do not respond to chemotherapy or radiation therapy initially, or those that become unresponsive over time.

[0058] The term "anti-tumor effect" as used here refers to biological effects that can exist in the following forms: reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, reduction in the number of metastases, increase in overall survival or survival without progressive disease, increase in life expectancy, or improvement in various physical symptoms associated with tumors. Anti-tumor effects can also refer to the prevention of tumor development (e.g., vaccines).

[0059] The term “progressive disease-free survival” (which may be abbreviated as PFS) as used here refers to the time from the date of treatment to the date of progressive disease (according to the revised IWG Response Criteria for Malignant Lymphoma) or the date of death from any cause.

[0060] Assessing "progressive disease" by measuring malignant lesions on radiographs or by other methods should not be reported as an adverse event. Death due to progressive disease without signs or symptoms should be reported as primary neoplastic disease (e.g., DLBCL).

[0061] The term “time to treatment response” (DOR) used here refers to the time from an individual’s first objective response to the date of diagnosis of progressive disease (according to the revised IWG Response Criteria for Malignant Lymphoma) or death.

[0062] The term "overall survival" (OS) used here refers to the time from the date of treatment to the date of death.

[0063] As used herein, "cytokines" refers to non-antibody proteins released by a cell in response to a contact-specific antigen, wherein the cytokine interacts with a second cell to mediate the response in that second cell. Cytokines can be expressed endogenously within cells and can be administered to an individual. Cytokines can be released by immune cells (including macrophages, B cells, T cells, and cytotoxic cells) to extend the immune response. 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) promote immune cell survival and proliferation, and pro-inflammatory cytokines 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)γ. 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)-α, TNF-β, 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 placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute-phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0064] Chemokines are a type of cytokine that mediate cellular chemotaxis or directional movement. Examples of chemokines include, but are not limited to: IL-8, IL-16, eosin, eosin 3, macrophage-derived chemokines (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1α (MIP-1α, MIP-1a), MIP-1β (MIP-1b), γ-inducible protein 10 (IP-10), and thymus and activation-regulating chemokines (TARC or CCL17).

[0065] Other examples of analytes and cytokines in this invention include, but are not limited to: chemokine (CC motif) ligands (CCL)1, CCL5, monocyte-specific chemokine 3 (MCP-3 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), and leukemia inhibitory factor (L-CSF). IF), oncogene M (OSM), CD154, lymphotoxin (LT)β, 4-1BB ligand (4-1BBL), proliferation-inducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-associated ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L, TNF- and ApoL-associated leukocyte-associated ligand 1 (TALL-1), or TNF-associated apoptosis-inducing ligand (TRAIL).

[0066] The terms "serum volume" and "serum concentration" are used interchangeably herein and refer to the amount of the analyte in an individual's serum. The serum volume of a given analyte can be measured using any method known in the art. For example, the serum volume of cytokines can be measured using an enzyme immunosorbent assay (ELISA). In one particular embodiment, the serum volume of cytokines can be measured using the EMDmillipore LUMINEX® xMAP® multiplex assay.

[0067] The term "dosing interval" as used herein refers to the time elapsed between multiple doses of the compound described herein administered to an individual. Therefore, the dosing interval can be indicated as a range.

[0068] The dosage described here may be expressed as a "weight-based dose" or a "body surface area (BSA)-based dose." A weight-based dose is the dose administered to a patient calculated based on the patient's weight, for example, mg / kg. A BSA-based dose is the dose administered to a patient calculated based on the patient's body surface area, for example, mg / m². For human administration, these two dosage measurement forms can be converted by multiplying the weight-based dose by 37 or dividing the BSA-based dose by 37. For example, a dose of 60 mg / kg administered to a human individual is equivalent to a dose of the same drug administered to the same individual at 2220 mg / m².

[0069] The term "dosage frequency" as used herein refers to the frequency at which the dose of the compound disclosed herein is administered within a given time period. Dosage frequency can be indicated as the number of doses per given time period. For example, cyclophosphamide can be administered as follows: once daily for 5 consecutive days, once daily for 4 consecutive days, once daily for 3 consecutive days, once daily for 2 consecutive days, or once daily. In some embodiments, cyclophosphamide is administered as once daily for 3 consecutive days or once daily for 2 consecutive days. Fludarabine can be administered as follows: once daily for 8 consecutive days, once daily for 7 consecutive days, once daily for 6 consecutive days, once daily for 5 consecutive days, once daily for 4 consecutive days, once daily for 3 consecutive days, once daily for 2 consecutive days, or once daily. In other embodiments, fludarabine is administered as once daily for 5 consecutive days or once daily for 3 consecutive days.

[0070] The "therapeutic effective dose," "therapeutic amount," "effective dose," or "therapeutic effective dose" of a drug or therapeutic agent is any amount that, when used alone or in combination with another therapeutic agent, protects an individual from the onset of disease or promotes disease remission (evidence being a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods), or prevents infirmity or disability caused by disease suffering. The ability of a therapeutic agent to promote disease remission can be assessed by using various methods familiar to a skilled physician, such as human individuals during clinical trials, animal model systems that predict efficacy in humans, or by analyzing the activity of the drug in in vitro assays.

[0071] The term "lymphocyte" as used here includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic lymphocyte that represents a major component of the innate immune system. NK cells repel tumor cells and virus-infected cells. NK cells function through apoptosis, or programmed cell death. NK cells are called "natural killers" because they can kill cells without activation. T cells play a major role in cell-mediated immunity (not involving antibodies). The T cell receptor (TCR) of T cells distinguishes them from other lymphocyte types. The thymus, a specialized organ of the immune system, is a key factor in T cell maturation. T cells are classified into six types: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T-killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), and memory T cells (i) stem memory TCM cells (similar to naïve cells) are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but also exhibit high levels of CD95, IL-2Rβ, CXCR3, and LFA-1, and show many characteristic functional properties of memory cells); (ii) central memory TCM cells exhibit L-selectin and CCR7, secrete IL-2, but do not produce IFNγ or IL-4; and (iii) effector memory TCM cells do not exhibit L-selectin or CCR7, but produce IFNγ-like substances. Effector cytokines of γ and IL-4, regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKTs), and γδ T cells. On the other hand, B cells play a major role in humoral immunity (involving antibodies). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and transform into memory B cells after activation by antigen-antigen interactions. In mammals, immature B cells are formed in the bone marrow, hence their name.

[0072] The terms "genetically engineered" or "engineered" refer to methods of modifying the genome of cells, including but not limited to deleting coding or non-coding regions or portions thereof, or inserting coding regions or portions thereof. In some embodiments, the modified cells are lymphocytes (e.g., T cells), which may be obtained from a patient or donor. Cells may be modified to express exogenous constructs, such as chimeric antigen receptors (CARs) or T-cell receptors (TCRs), which are inserted into the cell's genome.

[0073] "Immune response" refers to the action of cells of the immune system (such as T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, basophils, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or any of them in the liver, which selectively target, bind to, destroy, eliminate, and / or eliminate invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or in the context of autoimmunity or pathological inflammation, or normal human cells or tissues.

[0074] The term "immunotherapy" refers to the treatment of an individual suffering from a disease or at risk of gaining or experiencing a relapse of a disease by means of methods that involve inducing, enhancing, suppressing, or modifying an immune response. Examples of immunotherapy include, but are not limited to, T-cell therapy. T-cell therapy may include adoptive T-cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T-cell transplantation. However, those skilled in the art will understand that the conditioning methods described herein will enhance the effectiveness of any T-cell transplantation therapy. For a description of examples of T-cell therapy, see: US Patent Publication Nos. 2014 / 0154228 and 2002 / 0006409, US Patent No. 5,728,388, and International Publication No. WO 2008 / 081035.

[0075] T cells for immunotherapy can be derived from any source well-known in the field. For example, T cells can be differentiated in vitro from a population of hematopoietic stem cells, or T cells can be obtained from an individual. T cells can be obtained from peripheral blood mononuclear globulins, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In addition, T cells can be derived from one or more T cell lines available in the field. T cells can also be obtained from units of blood collected from an individual using certain techniques well-known to those in the art (e.g., FICOLL™ separation and / or blood cell separation methods). For disclosure of other methods for isolating T cells for T cell therapy, please see US Patent Publication No. 2013 / 0287748, the entire contents of which are incorporated herein by reference.

[0076] The term "engineered autologous cell therapy" (abbreviated as "eACTTM," also known as adoptive cell transfer) is a method of collecting a patient's T cells and then genetically modifying them to identify and target one or more antigens expressed on the surface of one or more specific tumor cells or malignant tumor cells. T cells can be engineered to express, for example, chimeric antigen receptors (CARs) or T-cell receptors (TCRs). CAR-positive (+) T cells are engineered to express extracellular single-stranded variant fragments (scFvs) that are specific to a particular tumor antigen linked to an intracellular signaling portion containing a costimulatory domain and an activation domain. The costimulatory domain may be derived from, for example, CD28, and the activation domain may be derived from, for example, CD3ζ (Figure 1). In some embodiments, the CAR is designed to have two, three, four, or more costimulatory domains. CAR scFvs can be designed to target, for example, CD19, a transmembrane protein expressed by cells in the B-cell lineage, which includes all normal B cells and B-cell malignancies (including, but not limited to, NHL, CLL, and non-T-cell ALL). For a description of examples of CAR+T cell therapies and constructs, please see US Patent Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, the entire contents of which are incorporated herein by reference.

[0077] The term "patient" as used here includes people with cancer (such as lymphoma or leukemia). The term "individual" is used interchangeably with "patient" here.

[0078] The terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to compounds composed of amino acid residues covalently linked by peptide bonds. Proteins or peptides must contain at least two amino acids, and there is no limit to the maximum number of amino acids, which may contain the sequence of a protein or peptide. Polypeptides include any peptide or protein that contains two or more amino acids linked together by peptide bonds. The terms used herein refer to both short chains (often also called peptides, oligopeptides, and oligomers) and long chains (often also called proteins). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0079] The term "stimulus" as used here refers to the primary response induced by the binding of a stimulating molecule to its ligand, where this binding mediates a message-transmitting event. A "stimulating molecule" is a molecule on T cells (e.g., the T cell receptor (TCR) / CD3 complex) that specifically binds to a homologous stimulating ligand present on antigen-presenting cells. A "stimulating ligand" is a ligand that, when present on antigen-presenting cells (e.g., APCs, dendritic cells, B cells, etc.), specifically binds to a stimulating molecule on T cells, thereby mediating a primary response by the T cell, which includes, but is not limited to, activation, initiation of an immune response, and proliferation. Stimulating ligands include, but are not limited to, MHC Class I molecules with peptides, anti-CD3 antibodies, super-stimulating anti-CD28 antibodies, and super-stimulating anti-CD2 antibodies.

[0080] The term "co-stimulatory message" as used here refers to a message that combines with an initial message (e.g., TCR / CD3 binding) to lead to a T cell response, such as, but not limited to, the proliferation and / or upregulation or downregulation of key molecules.

[0081] The term "co-stimulatory ligand" as used here includes molecules on antigen-presenting cells that specifically bind to homologous co-stimulatory molecules on T cells. The binding of a co-stimulatory ligand provides a message that mediates T cell responses (including but not limited to proliferation, activation, and differentiation). Co-stimulatory ligands induce a message in addition to the initial message provided by a stimulatory molecule, such as the message provided by the binding of the T cell receptor (TCR) / CD3 complex to a peptide-containing major histocompatibility complex (MHC) molecule. Costimulatory ligands may include, but are not limited to: CD7, B7-1 (CD80), B7-2 (CD86), programmed cell death (PD) L1, PD-L2, 4-1BB ligand, OX40 ligand, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30 ligand, CD40, CD70, CD83, human leukocyte antigen G (HLA-G), MHC class I chain-associated protein A (MICA), MHC class I chain-associated protein B (MICB), herpesvirus entry vector (HVEM), lymphotoxin β receptor, 3 / TR6, immunoglobulin transcript (ILT) 3, ILT4, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited 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 ligands that specifically bind to CD83.

[0082] "Costimulatory molecules" are binding companions on T cells that specifically bind to costimulatory ligands, thereby enabling T cells to mediate costimulatory responses (e.g., but not limited to 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 I molecules, B- and T-lymphocyte attenuation factors (BTLA), and Toll ligand receptors.

[0083] The terms "conditioning" and "preconditioning" are used interchangeably here to indicate preparing a patient for T-cell therapy in a suitable state. Conditioning as used herein includes, but is not limited to: reducing the number of endogenous lymphocytes after conditioning, removing cytokine buildup, increasing serum levels of one or more homeostatic cytokines or pro-inflammatory factors, enhancing the effector function of administered T cells, enhancing antigen-presenting cell activation and / or effectiveness before T-cell therapy, or any combination thereof. In one embodiment, "conditioning" includes increasing the serum levels of one or more cytokines, such as 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 chemoattractant 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. In another embodiment, "conditioning" includes increasing the serum levels of IL-7, IL-15, IP-10, MCP-1, PLGF, CRP, or any combination thereof.

[0084] The terms "reducing" and "decreasing" are used interchangeably here to indicate any change less than before. "Reducing" and "decreasing" are relative terms used to make comparisons between before and after measurement. "Reducing" and "decreasing" include complete depletion.

[0085] "Treatment" or "treating" an individual refers to any type of intervention or procedure or administration of an active agent to that individual with the aim of reversing, alleviating, improving, suppressing, mitigating, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or conditions, or biochemical indicators associated with the disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission.

[0086] The use of "one of several" (e.g., or) means one, two, or any combination of the options. The indefinite article "a" or "an" as used herein means "one or more" of any detailed or enumerated elements.

[0087] The terms "about" or "comprising essentially of" refer to a value or composition within an acceptable margin of error as determined by someone of ordinary skill in the art, where the value or composition depends in part on how it is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "comprising essentially of" may mean a standard deviation of less than or greater than 1 according to practice in the art. Alternatively, "about" or "comprising essentially of" may mean a range of up to 10% (i.e., ±10%). For example, about 3 mg may include any number between 2.7 mg and 3.3 mg (10%). Additionally, particularly in relation to biological systems or procedures, the term may mean a value of up to one order of magnitude or up to five times. When a specific value or composition is provided in the claims and patent claims, unless otherwise indicated, "about" or "comprising essentially of" shall be assumed to mean within an acceptable margin of error for that specific value or composition.

[0088] Any concentration range, percentage range, proportion range or integer range mentioned herein shall be understood to include any integer within the range and, where appropriate, its fractional (e.g., one-tenth and one-hundredth of an integer) value, unless otherwise indicated.

[0089] Various aspects of the invention are described in more detail in the following sections.

[0090] [, Methods of Invention , ]

[0091] This invention relates to a method for conditioning a patient requiring T-cell therapy, comprising administering cyclophosphamide and fludarabine to the patient. This invention demonstrates that conditioning a patient with cyclophosphamide at a dose between about 200 mg / m² / day and about 2000 mg / m² / day, and fludarabine at a dose between about 20 mg / m² / day and about 900 mg / m² / day, enhances the effectiveness of subsequent T-cell therapy administered to the patient, while reducing the incidence and / or severity of adverse events associated with higher doses of cyclophosphamide and / or fludarabine.

[0092] This invention identifies the reduction of endogenous lymphocytes by administering cyclophosphamide and fludarabine prior to T-cell therapy. The reduced endogenous lymphocytes may 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 can suppress the antitumor effect of secondary metastatic T cells. Endogenous lymphocytes compete with secondary metastatic T cells for antigens and supporting cytokines. Pretreatment with cyclophosphamide and fludarabine removes this competition, resulting in an increase in endogenous cytokine levels. Once secondary metastatic T cells are administered to the patient, these T cells are exposed to increased levels of endogenous homeostatic cytokines or pro-inflammatory factors. Furthermore, cyclophosphamide and fludarabine treatment induces tumor cell death, leading to an increase in tumor antigens in the patient's serum. This enhances the antigen-presenting cell activation and / or efficacy in the patient prior to T-cell therapy. Without being bound by any theoretical framework, the immune environment is improved by using cyclophosphamide and fludarabine to modulate the immune system by introducing molecules that promote the homeostatic proliferation, activation, and transport of T cells.

[0093] Previous studies used high doses of cyclophosphamide and fludarabine to reduce endogenous lymphocyte counts. However, these harsh treatment regimens were associated with serious and potentially fatal adverse events. Surprisingly, we found that our approach increased the effectiveness of secondary metastatic T cells while reducing the incidence and severity of adverse events.

[0094] In some embodiments, administration of cyclophosphamide and fludarabine reduces endogenous lymphocytes. In some embodiments, administration of cyclophosphamide and fludarabine increases the effectiveness of homeostatic cytokines in vivo. In some embodiments, administration of cyclophosphamide and fludarabine enhances the effector function of T cells administered after this conditioning. In some embodiments, administration of cyclophosphamide and fludarabine enhances antigen-presenting cell activation and / or effectiveness.

[0095] In one embodiment, the present invention includes a method for conditioning a patient requiring T-cell therapy, the method comprising administering to the patient cyclophosphamide at a dose between about 200 mg / m² / day and about 2000 mg / m² / day, and fludarabine at a dose between about 20 mg / m² / day and about 900 mg / m² / day. In another embodiment, the present invention includes a method for conditioning a patient requiring T-cell therapy, the method comprising administering to the patient cyclophosphamide at a dose between about 200 mg / m² / day and about 2000 mg / m² / day (e.g., 200 mg / m² / day, 300 mg / m² / day, or 500 mg / m² / day), and fludarabine at a dose between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 500 mg / m² / day). Fludarabine (mg / m2 / day or 60 mg / m2 / day) was administered to patients who, after administration of the cyclophosphamide and fludarabine, showed 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. In one embodiment, the present invention includes a method for conditioning a patient requiring T-cell therapy, the method comprising administering to the patient cyclophosphamide at a dose between about 1110 mg / m² / day and about 2000 mg / m² / day, and fludarabine at a dose between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day). In another embodiment, the present invention includes a method for conditioning a patient requiring T-cell therapy, the method comprising administering to the patient cyclophosphamide at a dose between about 1110 mg / m² / day and about 2000 mg / m² / day and fludarabine at a dose between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day), wherein the patient, after administration of the cyclophosphamide and fludarabine, 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.In one embodiment, the present invention includes a method for conditioning a patient requiring T-cell therapy, the method comprising administering to the patient a dose of cyclophosphamide equal to or higher than about 30 mg / kg / day and lower than 60 mg / kg / day, and a dose of fludarabine between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day).

[0096] In another embodiment, the present invention includes a method for reducing or depleting endogenous lymphocytes in a patient requiring T-cell therapy, the method comprising administering to the patient a dose of cyclophosphamide between about 200 mg / m² / day and about 2000 mg / m² / day, and a dose of fludarabine between about 20 mg / m² / day and about 900 mg / m² / day. In another embodiment, the present invention includes a method for reducing or depleting endogenous lymphocytes in a patient requiring T-cell therapy, the method comprising administering to the patient a dose of cyclophosphamide between about 200 mg / m² / day and about 2000 mg / m² / day (e.g., 200 mg / m² / day, 300 mg / m² / day, or 500 mg / m² / day), and a dose of cyclophosphamide between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day). Fludarabine (30 mg / m² / day, or 60 mg / m² / day) was administered to a patient who, after administration of the cyclophosphamide and fludarabine, showed an increase in 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 a decrease in serum levels of perforin and / or MIP-1b. In one embodiment, the present invention includes a method for reducing or depleting endogenous lymphocytes in a patient requiring T-cell therapy, the method comprising administering to the patient a dose of cyclophosphamide between about 1110 mg / m² / day and about 2000 mg / m² / day, and a dose of cyclophosphamide between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day). Fludarabine ( / m2 / day), wherein the patient showed 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 the cyclophosphamide and fludarabine.In one embodiment, the present invention includes a method for reducing or depleting endogenous lymphocytes in a patient requiring T-cell therapy, the method comprising administering to the patient a dose equal to or higher than about 30 mg / kg / day and lower than 60 mg / kg / day of cyclophosphamide and a dose of fludarabine between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day), wherein the patient, after administration of the cyclophosphamide and fludarabine, 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. .

[0097] In other embodiments, the present invention includes a method for increasing the effectiveness of in vivo constant cytokines in a patient requiring T-cell therapy, the method comprising administering to the patient cyclophosphamide at a dose between about 200 mg / m² / day and about 2000 mg / m² / day (e.g., 200 mg / m² / day, 300 mg / m² / day, or 500 mg / m² / day) and fludarabine at a dose between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day). In another embodiment, the present invention includes a method for increasing the effectiveness of in vivo constant cytokines in a patient requiring T-cell therapy, the method comprising administering to the patient a dose of cyclophosphamide at a dose between about 200 mg / m² / day and about 2000 mg / m² / day (e.g., 200 mg / m² / day, 300 mg / m² / day, or 500 mg / m² / day) and a dose of cyclophosphamide at a dose between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, or 2000 mg / m² / day). Fludarabine (m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day) was administered to patients who, after administration of the cyclophosphamide and fludarabine, showed 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. In one embodiment, the present invention includes a method for increasing the efficacy of in vivo constant cytokines in a patient requiring T-cell therapy, the method comprising administering to the patient a dose of cyclophosphamide between about 1110 mg / m² / day and about 2000 mg / m² / day and a dose of fludarabine between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day), wherein the patient, after administration of the cyclophosphamide and fludarabine, 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.In one embodiment, the present invention includes a method for increasing the efficacy of in vivo constant cytokines in a patient requiring T-cell therapy, the method comprising administering to the patient a dose equal to or higher than about 30 mg / kg / day and lower than 60 mg / kg / day of cyclophosphamide and a dose of fludarabine between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day), wherein the patient, after administration of the cyclophosphamide and fludarabine, 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.

[0098] In one particular embodiment, the present invention includes a method for enhancing the effector function of administered T cells in a patient requiring T-cell therapy, the method comprising administering to the patient cyclophosphamide at a dose between about 200 mg / m² / day and about 2000 mg / m² / day (e.g., 200 mg / m² / day, 300 mg / m² / day, or 500 mg / m² / day) and fludarabine at a dose between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day). In another embodiment, the present invention includes a method for enhancing the effector function of administered T cells in a patient requiring T-cell therapy, the method comprising administering to the patient cyclophosphamide at a dose between about 200 mg / m² / day and about 2000 mg / m² / day (e.g., 200 mg / m² / day, 300 mg / m² / day, or 500 mg / m² / day) and cyclophosphamide at a dose between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, or 2000 mg / m² / day). Fludarabine administered at 2 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day, wherein the patient showed 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 the cyclophosphamide and fludarabine. In one embodiment, the present invention includes a method for enhancing the effector function of administered T cells in a patient requiring T-cell therapy, the method comprising administering to the patient a dose of cyclophosphamide between about 1110 mg / m² / day and about 2000 mg / m² / day, and a dose of cyclophosphamide between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day). Fludarabine (g / m2 / day) was administered to patients who, after administration of the cyclophosphamide and fludarabine, showed 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.In one embodiment, the present invention includes a method for enhancing the effector function of administered T cells in a patient requiring T-cell therapy, the method comprising administering to the patient a dose equal to or higher than about 30 mg / kg / day and lower than 60 mg / kg / day of cyclophosphamide and a dose of fludarabine between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day), wherein the patient, after administration of the cyclophosphamide and fludarabine, 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. .

[0099] In some embodiments, the present invention includes a method for activating and / or enhancing the effectiveness of antigen-presenting cells in a patient requiring T-cell therapy, the method comprising administering to the patient a dose of cyclophosphamide at a level between about 200 mg / m² / day and about 2000 mg / m² / day (e.g., 200 mg / m² / day, 300 mg / m² / day, or 500 mg / m² / day) and a dose of fludarabine at a level between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day). In another embodiment, the present invention includes a method for activating and / or enhancing the effectiveness of antigen-presenting cells in a patient requiring T-cell therapy, the method comprising administering to the patient a dose of cyclophosphamide at a level between about 200 mg / m² / day and about 2000 mg / m² / day (e.g., 200 mg / m² / day, 300 mg / m² / day, or 500 mg / m² / day) and a dose of cyclophosphamide at a level between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day). Fludarabine (30 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day) was administered to patients who, after administration of the cyclophosphamide and fludarabine, showed 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. In one embodiment, the present invention includes a method for activating and / or enhancing the effectiveness of antigen-presenting cells in a patient requiring T-cell therapy, the method comprising administering to the patient a dose of cyclophosphamide between about 1110 mg / m² / day and about 2000 mg / m² / day, and a dose of cyclophosphamide between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day). Fludarabine (mg / m2 / day) was administered to patients who, after administration of the cyclophosphamide and fludarabine, showed 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.In one embodiment, the present invention includes a method for activating and / or enhancing the effectiveness of antigen-presenting cells in a patient requiring T-cell therapy, the method comprising administering to the patient a dose equal to or higher than about 30 mg / kg / day and lower than 60 mg / kg / day of cyclophosphamide and a dose of fludarabine between about 20 mg / m² / day and about 900 mg / m² / day (e.g., 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, or 60 mg / m² / day), wherein the patient, after administration of the cyclophosphamide and fludarabine, 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. .

[0100] The method of this invention includes administering cyclophosphamide and fludarabine prior to T-cell therapy. The timing of administration of each component can be adjusted to maximize the effect. As described herein, the day of administration of T-cell therapy is designated as day 0. Cyclophosphamide and fludarabine can be administered at any time prior to T-cell therapy. In some embodiments, cyclophosphamide and fludarabine are administered 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 prior to T-cell therapy. In other embodiments, cyclophosphamide and fludarabine are administered 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 T-cell therapy. In one embodiment, cyclophosphamide and fludarabine are administered 7 days prior to T-cell therapy. In another implementation, cyclophosphamide and fludarabine are administered 5 days prior to the administration of T-cell therapy.

[0101] In one particular embodiment, cyclophosphamide is administered approximately 7 days prior to T-cell therapy, and fludarabine is administered approximately 5 days prior to T-cell therapy. In another embodiment, cyclophosphamide is administered approximately 5 days prior to T-cell therapy, and fludarabine is administered approximately 5 days prior to T-cell therapy.

[0102] The timing of administration of each component can be adjusted to maximize the effect. Typically, cyclophosphamide and fludarabine can be administered daily. In some embodiments, cyclophosphamide and fludarabine are administered daily for approximately 2, 3, 4, 5, 6, or 7 days. In one specific embodiment, cyclophosphamide is administered daily for 2 days, and fludarabine is administered daily for 5 days. In another embodiment, cyclophosphamide and fludarabine are administered daily for approximately 3 days.

[0103] As described herein, the date on which the patient receives T-cell therapy is designated as day 0. In some embodiments, cyclophosphamide is administered to the patient on days 7 and 6 prior to day 0 (i.e., day -7 and day -6). In other embodiments, cyclophosphamide is administered to the patient on days -5, -4, and -3. In some embodiments, fludarabine is administered to the patient on days -5, -4, -3, -2, and -1. In other embodiments, fludarabine is administered to the patient on days -5, -4, and -3.

[0104] Cyclophosphamide and fludarabine can be administered on the same day or different days. If cyclophosphamide and fludarabine are administered on the same day, cyclophosphamide can be administered before or after fludarabine. In one embodiment, cyclophosphamide is administered to the patient on days -7 and -6, and fludarabine is administered to the patient on days -5, -4, -3, -2, and -1. In another embodiment, cyclophosphamide is administered to the patient on days -5, -4, and -3, and fludarabine is administered to the patient on days -5, -4, and -3.

[0105] In some embodiments, cyclophosphamide and fludarabine may 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.

[0106] Cyclophosphamide and fludarabine can be administered via any route, including intravenous (IV) injection. In some embodiments, cyclophosphamide is administered IV at the following times: approximately 30 minutes, approximately 35 minutes, approximately 40 minutes, approximately 45 minutes, approximately 50 minutes, approximately 55 minutes, approximately 60 minutes, approximately 90 minutes, and approximately 120 minutes. In some embodiments, fludarabine is administered IV at the following times: approximately 10 minutes, approximately 15 minutes, approximately 20 minutes, approximately 25 minutes, approximately 30 minutes, approximately 35 minutes, approximately 40 minutes, approximately 45 minutes, approximately 50 minutes, approximately 55 minutes, approximately 60 minutes, approximately 90 minutes, and approximately 120 minutes.

[0107] In some embodiments, T-cell therapy is administered to the patient after administration of cyclophosphamide and fludarabine. In some embodiments, the T-cell therapy comprises adoptive cell therapy. In some embodiments, the adoptive cell therapy is selected from tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T-cell transplantation. In one particular embodiment, eACT comprises administration of engineered antigen-specific chimeric antigen receptor (CAR) positive (+) T cells. In another embodiment, eACT comprises administration of engineered antigen-specific T-cell receptor (TCR) positive (+) T cells. In some embodiments, engineered T cells treat tumors in the patient.

[0108] In one particular embodiment, the present invention includes a method for conditioning a patient requiring T-cell therapy, the method comprising administering to the patient a dose of about 500 mg / m² / day of cyclophosphamide and a dose of about 60 mg / m² / day of fludarabine, wherein cyclophosphamide is administered on days -5, -4, and -3, and fludarabine is administered on days -5, -4, and -3. In another embodiment, the present invention includes a method for conditioning a patient requiring T-cell therapy, the method comprising administering to the patient a dose of about 500 mg / m² / day of cyclophosphamide and a dose of about 60 mg / m² / day of fludarabine, wherein cyclophosphamide is administered on days -7 and -6, and fludarabine is administered on days -5, -4, -3, -2, and -1. In another embodiment, the present invention includes a method for conditioning a patient requiring T-cell therapy, the method comprising administering to the patient a dose of about 500 mg / m² / day of cyclophosphamide and a dose of about 30 mg / m² / day of fludarabine, wherein cyclophosphamide is administered on days -7 and -6, and fludarabine is administered on days -5, -4, -3, -2, and -1. In another embodiment, the present invention includes a method for conditioning a patient requiring T-cell therapy, the method comprising administering to the patient a dose of about 300 mg / m² / day of cyclophosphamide and a dose of about 60 mg / m² / day of fludarabine, wherein cyclophosphamide is administered on days -7 and -6, and fludarabine is administered on days -5, -4, -3, -2, and -1.

[0109] Various other interventions may be included in the methods described herein. For example, it is well known that cyclophosphamide and fludarabine may cause adverse events in patients after administration. The scope of the invention includes the possibility of administering the composition to patients to reduce some of these adverse events. In some embodiments, the method further includes administering saline solution to the patient. Saline solution may be administered to the patient before or after administration of cyclophosphamide and / or fludarabine, or both before and after administration of cyclophosphamide and / or fludarabine. In some embodiments, saline solution may be administered concurrently with administration of cyclophosphamide and / or fludarabine. In one particular embodiment, saline solution is administered to the patient on each infusion day before and after administration of cyclophosphamide and / or fludarabine.

[0110] The saline solution can be administered to the patient via any route, including intravenous or oral administration. 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 solution. The NaCl in the saline solution can be dissolved to 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%, 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 comprises administering 1.0 L of 0.9% NaCl saline solution to the patient. In a particular embodiment, the method comprises administering 1.0 L of 0.9% NaCl saline solution to the patient on each infusion day before and after administration of cyclophosphamide and / or fludarabine.

[0111] Additionally, adjuvants and excipients may be administered to the patient. For example, mesna (sodium 2-mercaptoethanesulfonate) is an adjuvant with antidote properties to inhibit hemorrhagic cystitis and hematuria that may occur after treatment with cyclophosphamide. Cyclophosphamide is converted into uremic metabolites (e.g., acrolein) in the body. These metabolites are detoxified by the reaction of the sulfhydryl group and vinyl group of mesna. Mesna also increases cysteine ​​levels in urine. In some embodiments, the method further includes administering mesna to the patient. Mesna may be administered before, after, or before and after administration of cyclophosphamide and / or fludarabine. In one embodiment, mesna is administered intravenously or orally (orally). For example, oral mesna may be administered together with oral cyclophosphamide.

[0112] Furthermore, exogenous cytokines may also be administered to the patient in the methods described herein. As mentioned above, it is assumed that reducing the number of endogenous lymphocytes increases the bioavailability of endogenous molecules (e.g., cytokines), which may benefit the proliferation, activation, and transport of secondary metastatic T cells. Therefore, various cytokines may 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 particular embodiment, the method comprises administering one or more doses of IL-2. The IL-2 dose may be 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.

[0113] [, Cyclophosphamide and fludarabine , ]

[0114] Cyclophosphamide (ENDOXAN®, CYTOXAN®, PROCYTOX®, NEOSAR®, REVIMMUNE®, CYCLOBLASTIN®) is a nitrogen mustard derivative alkylating agent with potent immunosuppressive activity. Cyclophosphamide has antitumor effects and is used to treat various cancers, including lymphoma, multiple myeloma, leukemia, mycosis fungoides, neuroblastoma, ovarian cancer, eye cancer, breast cancer, and autoimmune diseases.

[0115] Once administered to a patient, cyclophosphamide is converted into acrolein and phosphamide in the liver. Adding an alkyl group to the guanine base of DNA at the 7th nitrogen atom position of the imidazole causes these metabolites to cross-link with DNA in both quiescent and dividing cells. Therefore, DNA replication is inhibited, leading to cell death.

[0116] In this invention, the cyclophosphamide dosage can be adjusted according to the desired effect, for example, to regulate the reduction of endogenous lymphocytes and / or control the severity of adverse events. For example, the cyclophosphamide dosage can be higher than about 300 mg / m² / day and lower than about 900 mg / m² / day. In some embodiments, the cyclophosphamide dosage is about 350 mg / m² / day to about 2000 mg / m² / day, at least about 400 mg / m² / day to about 2000 mg / m² / day, about 450 mg / m² / day to about 2000 mg / m² / day, about 500 mg / m² / day to about 2000 mg / m² / day, about 550 mg / m² / day to about 2000 mg / m² / day, or about 600 mg / m² / day to about 2000 mg / m² / day. In another embodiment, the cyclophosphamide dosage is about 350 mg / m² / day to about 1500 mg / m² / day, about 350 mg / m² / day to about 1000 mg / m² / day, about 400 mg / m² / day to about 900 mg / m² / day, about 450 mg / m² / day to about 800 mg / m² / day, about 450 mg / m² / day to about 700 mg / m² / day, about 500 mg / m² / day to about 600 mg / m² / day, or about 300 mg / m² / day to about 500 mg / m² / day. In some embodiments, the cyclophosphamide dose is about 350 mg / m² / day, about 400 mg / m² / day, about 450 mg / m² / day, about 500 mg / m² / day, about 550 mg / m² / day, about 600 mg / m² / day, about 650 mg / m² / day, about 700 mg / m² / day, about 800 mg / m² / day, about 900 mg / m² / day, or about 1000 mg / m² / day. In one particular embodiment, the cyclophosphamide dose is about 200 mg / m² / day. In one particular embodiment, the cyclophosphamide dose is about 300 mg / m² / day. In another embodiment, the cyclophosphamide dose is about 500 mg / m² / day.In other embodiments, the dosage of cyclophosphamide is about 200 mg / m² / day to about 2000 mg / m² / day, about 300 mg / m² / day to about 2000 mg / m² / day, about 400 mg / m² / day to about 2000 mg / m² / day, about 500 mg / m² / day to about 2000 mg / m² / day, about 600 mg / m² / day to about 2000 mg / m² / day, about 700 mg / m² / day to about 2000 mg / m² / day, about 800 mg / m² / day to about 2000 mg / m² / day. 0 mg / m² / day, approximately 900 mg / m² / day to approximately 2000 mg / m² / day, approximately 1000 mg / m² / day to approximately 2000 mg / m² / day, approximately 1100 mg / m² / day to approximately 2000 mg / m² / day, approximately 1200 mg / m² / day to approximately 2000 mg / m² / day, approximately 1300 mg / m² / day to approximately 2000 mg / m² / day, approximately 1400 mg / m² / day to approximately 2000 mg / m² / day, approximately 1500 mg / m² / day to approximately 2000 mg / m² / day g / m² / day, approximately 1600 mg / m² / day to approximately 2000 mg / m² / day, approximately 1700 mg / m² / day to approximately 2000 mg / m² / day, approximately 1800 mg / m² / day to approximately 2000 mg / m² / day, approximately 1900 mg / m² / day to approximately 2000 mg / m² / day, approximately 200 mg / m² / day to approximately 1900 mg / m² / day, approximately 400 mg / m² / day to approximately 1800 mg / m² / day, approximately 500 mg / m² / day to approximately 1700 mg / m² / day 2 mg / m² / day, approximately 600 mg / m² / day to approximately 1600 mg / m² / day, approximately 700 mg / m² / day to approximately 1500 mg / m² / day, approximately 800 mg / m² / day to approximately 1400 mg / m² / day, approximately 900 mg / m² / day to approximately 1300 mg / m² / day, approximately 1000 mg / m² / day to approximately 1200 mg / m² / day, approximately 1100 mg / m² / day to approximately 1200 mg / m² / day, or approximately 1110 mg / m² / day to approximately 1150 mg / m² / day.

[0117] Fludarabine hydrochloride (FLUDARA®) is a synthetic purine nucleoside that differs from physiological nucleosides in that its sugar portion is arabinose instead of ribose or deoxyribose. Fludarabine has purine antagonist and antimetabolite effects and is used to treat various hematologic malignancies, including various lymphomas and leukemias.

[0118] Once administered to a patient, fludarabine is rapidly dephosphorylated to 2-fluoro-ara-A, and then phosphorylated intracellularly by deoxycytidine kinase to the active triphosphate 2-fluoro-ara-ATP. This metabolite then interferes with DNA replication (e.g., by inhibiting DNA polymerase α, ribonucleotide reductase, and DNA priming enzymes), thereby inhibiting DNA synthesis. Therefore, administration of fludarabine leads to increased cell death in dividing cells.

[0119] In this invention, the fludarabine dosage can be adjusted according to the desired effect. For example, the fludarabine dosage can be higher than 30 mg / m² / day and lower than 900 mg / m² / day. In some embodiments, the fludarabine dosage can be about 35 mg / m² / day to about 900 mg / m² / day, about 40 mg / m² / day to about 900 mg / m² / day, about 45 mg / m² / day to about 900 mg / m² / day, about 50 mg / m² / day to about 900 mg / m² / day, about 55 mg / m² / day to about 900 mg / m² / day, or about 60 mg / m² / day to about 900 mg / m² / day. In other embodiments, the fludarabine dosage is approximately 35 mg / m² / day to approximately 900 mg / m² / day, approximately 35 mg / m² / day to approximately 800 mg / m² / day, approximately 35 mg / m² / day to approximately 700 mg / m² / day, approximately 35 mg / m² / day to approximately 600 mg / m² / day, approximately 35 mg / m² / day to approximately 500 mg / m² / day, approximately 35 mg / m² / day to approximately 400 mg / m² / day, approximately 35 mg / m² / day to approximately 300 mg / m² / day, approximately 35 mg / m² / day to approximately 200 mg / m² / day, approximately 35 mg / m² / day to approximately 100 mg / m² / day, approximately 40 mg / m² / day to approximately 90 mg / m² / day, approximately 45 mg / m² / day to approximately 80 mg / m² / day, approximately 45 ... The dosage is approximately 35 mg / m² / day to about 40 mg / m² / day, or approximately 50 mg / m² / day to about 60 mg / m² / day. In some embodiments, the fludarabine dosage is approximately 35 mg / m² / day, approximately 40 mg / m² / day, approximately 45 mg / m² / day, approximately 50 mg / m² / day, approximately 55 mg / m² / day, approximately 60 mg / m² / day, approximately 65 mg / m² / day, approximately 70 mg / m² / day, approximately 75 mg / m² / day, approximately 80 mg / m² / day, approximately 85 mg / m² / day, approximately 90 mg / m² / day, approximately 95 mg / m² / day, approximately 100 mg / m² / day, approximately 200 mg / m² / day, or approximately 300 mg / m² / day. In other embodiments, the fludarabine dose is approximately 110 mg / m² / day, 120 mg / m² / day, 130 mg / m² / day, 140 mg / m² / day, 150 mg / m² / day, 160 mg / m² / day, 170 mg / m² / day, 180 mg / m² / day, or 190 mg / m² / day. In some embodiments, the fludarabine dose is approximately 210 mg / m² / day, 220 mg / m² / day, 230 mg / m² / day, 240 mg / m² / day, 250 mg / m² / day, 260 mg / m² / day, 270 mg / m² / day, 280 mg / m² / day, or 290 mg / m² / day. In one particular embodiment, the fludarabine dose is approximately 20 mg / m² / day.In one specific embodiment, the fludarabine dose is approximately 30 mg / m² / day. In another embodiment, the fludarabine dose is approximately 60 mg / m² / day. In yet another embodiment, the fludarabine dose is approximately 25 mg / m² / day.

[0120] The dosage of cyclophosphamide can be increased or decreased together with or independently of fludarabine. For example, the dosage of cyclophosphamide can be increased while the dosage of fludarabine is decreased, or the dosage of cyclophosphamide can be decreased while the dosage of fludarabine is increased. Alternatively, the dosage of cyclophosphamide and fludarabine can be increased or decreased together.

[0121] In some embodiments, the cyclophosphamide dose is 100 mg / m² / day (or about 110 mg / m² / day, 120 mg / m² / day, 130 mg / m² / day, or 140 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0122] In some embodiments, the cyclophosphamide dose is 150 mg / m² / day (or 160 mg / m² / day, 170 mg / m² / day, 180 mg / m² / day, or 190 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0123] In some embodiments, the cyclophosphamide dose is about 200 mg / m² / day (or 210 mg / m² / day, 220 mg / m² / day, 230 mg / m² / day, or 240 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0124] In some embodiments, the cyclophosphamide dose is 250 mg / m² / day (or 260 mg / m² / day, 270 mg / m² / day, 280 mg / m² / day, or 290 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0125] In some embodiments, the cyclophosphamide dose is 300 mg / m² / day (or 310 mg / m² / day, 320 mg / m² / day, 330 mg / m² / day, or 340 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0126] In some embodiments, the cyclophosphamide dose is 350 mg / m² / day (or 360 mg / m² / day, 370 mg / m² / day, 380 mg / m² / day, or 390 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0127] In some embodiments, the cyclophosphamide dose is 400 mg / m² / day (or 410 mg / m² / day, 420 mg / m² / day, 430 mg / m² / day, or 440 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0128] In some embodiments, the dosage of cyclophosphamide is 450 mg / m² / day (or 460 mg / m² / day, 470 mg / m² / day, 480 mg / m² / day, or 490 mg / m² / day) and the dosage of fludarabine is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0129] In some embodiments, the dosage of cyclophosphamide is 500 mg / m² / day (or 510 mg / m² / day, 520 mg / m² / day, 530 mg / m² / day, or 540 mg / m² / day) and the dosage of fludarabine is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0130] In some embodiments, the dosage of cyclophosphamide is 550 mg / m² / day (or 560 mg / m² / day, 570 mg / m² / day, 580 mg / m² / day, or 590 mg / m² / day) and the dosage of fludarabine is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0131] In some embodiments, the cyclophosphamide dose is 600 mg / m² / day (or 610 mg / m² / day, 620 mg / m² / day, 630 mg / m² / day, or 640 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0132] In some embodiments, the cyclophosphamide dose is 650 mg / m² / day (or 660 mg / m² / day, 670 mg / m² / day, 680 mg / m² / day, or 690 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0133] In some embodiments, the cyclophosphamide dose is 700 mg / m² / day (or 710 mg / m² / day, 720 mg / m² / day, 730 mg / m² / day, or 740 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0134] In some embodiments, the cyclophosphamide dose is 750 mg / m² / day (or 760 mg / m² / day, 770 mg / m² / day, 780 mg / m² / day, or 790 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0135] In some embodiments, the cyclophosphamide dose is 800 mg / m² / day (or 810 mg / m² / day, 820 mg / m² / day, 830 mg / m² / day, or 840 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0136] In some embodiments, the cyclophosphamide dose is 850 mg / m² / day (or 860 mg / m² / day, 870 mg / m² / day, 880 mg / m² / day, or 890 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0137] In some embodiments, the cyclophosphamide dose is 900 mg / m² / day (or 910 mg / m² / day, 920 mg / m² / day, 930 mg / m² / day, or 940 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0138] In some embodiments, the cyclophosphamide dose is 950 mg / m² / day (or 960 mg / m² / day, 970 mg / m² / day, 980 mg / m² / day, or 990 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0139] In some embodiments, the cyclophosphamide dose is 1000 mg / m² / day (or 1010 mg / m² / day, 1020 mg / m² / day, 1030 mg / m² / day, or 1040 mg / m² / day) and the fludarabine dose is 5 mg / m² / day, 10 mg / m² / day, 15 mg / m² / day, 20 mg / m² / day, 25 mg / m² / day, 30 mg / m² / day, 35 mg / m² / day, 40 mg / m² / day, 45 mg / m² / day, 50 mg / m² / day, 55 mg / m² / day, 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, or 75 mg / m² / day.

[0140] In other embodiments, the cyclophosphamide dose is between 100 mg / m² / day and 650 mg / m² / day, and the fludarabine dose is between 10 mg / m² / day and 50 mg / m² / day. In other embodiments, the cyclophosphamide dose is between 150 mg / m² / day and 600 mg / m² / day, and the fludarabine dose is between 20 mg / m² / day and 50 mg / m² / day. In other embodiments, the cyclophosphamide dose is between 200 mg / m² / day and 550 mg / m² / day, and the fludarabine dose is between 20 mg / m² / day and 40 mg / m² / day. In other embodiments, the cyclophosphamide dose is between 250 mg / m² / day and 550 mg / m² / day, and the fludarabine dose is between 15 mg / m² / day and 45 mg / m² / day.

[0141] In some embodiments, the cyclophosphamide dose is 1000 mg / m² / day, and the fludarabine dose is 60 mg / m² / day, 65 mg / m² / day, 70 mg / m² / day, 75 mg / m² / day, 80 mg / m² / day, 85 mg / m² / day, 90 mg / m² / day, 95 mg / m² / day, 100 mg / m² / day, 105 mg / m² / day, 110 mg / m² / day, 115 mg / m² / day, 120 mg / m² / day, 125 mg / m² / day, 130 mg / m² / day, 135 mg / m² / day, 140 mg / m² / day, 145 mg / m² / day, 150 mg / m² / day, 155 mg / m² / day, or 160 mg / m² / day. / day, 165mg / m2 / day, 170mg / m2 / day, 175mg / m2 / day, 180mg / m2 / day, 185mg / m2 / day, 190mg / m2 / day, 195mg / m2 / day, 200mg / m2 / day, 205mg / m2 / day, 210mg / m2 / day, 215mg / m2 / day, 220mg / m2 / day, 225mg / m2 / day, 230mg / m2 / day, 235mg / m2 / day, 240mg / m2 / day, 245mg / m2 / day, or 250mg / m2 / day.

[0142] In some embodiments, the dosage of cyclophosphamide is 200 mg / m² / day and the dosage of fludarabine is 20 mg / m² / day. In some embodiments, the dosage of cyclophosphamide is 200 mg / m² / day and the dosage of fludarabine is 30 mg / m² / day. In some embodiments, the dosage of cyclophosphamide is 300 mg / m² / day and the dosage of fludarabine is 30 mg / m² / day. In other embodiments, the dosage of cyclophosphamide is 300 mg / m² / day and the dosage of fludarabine is 60 mg / m² / day. In other embodiments, the dosage of cyclophosphamide is 500 mg / m² / day and the dosage of fludarabine is 30 mg / m² / day. In yet another embodiment, the dosage of cyclophosphamide is 500 mg / m² / day and the dosage of fludarabine is 60 mg / m² / day. In some embodiments, the dosage of cyclophosphamide is approximately 1110 mg / m² / day and the dosage of fludarabine is 25 mg / m² / day. In some embodiments, the dosage of cyclophosphamide is approximately 2000 mg / m² / day and the dosage of fludarabine is 25 mg / m² / day. In some embodiments, the dosage of cyclophosphamide is 30 mg / m² / day and the dosage of fludarabine is 25 mg / m² / day.

[0143] [, T-cell therapy , ]

[0144] This invention provides a method for conditioning a patient by administering cyclophosphamide and fludarabine to enhance the effectiveness of T-cell therapy. Generally, various T-cell therapies can benefit from the conditioning methods described herein because conditioning protocols can modify the immune environment by introducing molecules that promote the homeostatic proliferation, activation, and transport of T cells. Those skilled in the art will understand that this conditioning protocol can be applied to any treatment of a patient involving the administration of one or more T cells.

[0145] For example, and without limitation, the conditioning regimens described herein can enhance the effectiveness of T-cell therapy, which can be adoptive T-cell therapy selected from the group consisting of: tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), allogeneic T-cell transplantation, non-T-cell transplantation, and any combination thereof. Adoptive T-cell therapy generally includes any method of selecting, in vitro concentrated, and administering autologous or allogeneic T cells that can recognize and bind to tumor cells to a patient. TIL immunotherapy is an adoptive T-cell therapy in which lymphocytes capable of infiltrating tumor tissue are isolated, in vitro concentrated, and administered to a patient. TIL cells can be autologous or allogeneic. Autologous cell therapy is an adoptive T-cell therapy that involves singling tumor-targeting T cells from a patient, concentrating the T cells in vitro, and administering the T cells back to the same patient. Allogeneic T-cell transplantation can include natural T cells or genetically engineered T cells that have proliferated in vitro (ex vivo). As detailed above, engineered autologous cell therapy is an adoptive T-cell therapy in which lymphocytes from a patient are isolated, genetically modified to express tumor-targeting molecules, proliferated in vitro, and reintroduced into the same patient. Non-T-cell transplantation can include autologous or allogeneic therapies utilizing non-T cells (such as, but not limited to, natural killer (NK) cells).

[0146] In one specific embodiment, the T-cell therapy of the present invention is engineered autologous cell therapy (eACTTM). According to this embodiment, the method may include collecting blood cells from the patient prior to administration of cyclophosphamide and fludarabine. The isolated blood cells (e.g., T cells) are then engineered to express a chimeric antigen receptor (“engineered CAR T cells”) or a T-cell receptor (“engineered TCR T cells”). In one specific embodiment, engineered CAR T cells or engineered TCR T cells are administered to the patient after administration of cyclophosphamide and fludarabine. In some embodiments, engineered T cells treat tumors in the patient.

[0147] In one embodiment, T cells can be engineered to express a chimeric antigen receptor. This chimeric antigen receptor may contain a binding molecule to a tumor antigen. The binding molecule may be an antibody or an antigen-binding molecule. For example, the antigen-binding molecule may be selected from scFv, Fab, Fab', Fv, F(ab')2, and dAb, and any fragment or combination thereof.

[0148] The chimeric antigen receptor may additionally include a hinge region. This hinge region may be derived from the following hinge regions: IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8α. In one particular embodiment, the hinge region is derived from the hinge region of IgG4.

[0149] Chimeric antigen receptors may also include a transmembrane domain. The transmembrane domain may be a transmembrane domain of any transmembrane molecule (a coreceptor on an immune cell) or a transmembrane domain of a member of the immunoglobulin superfamily. In some embodiments, the transmembrane domain is derived from the following transmembrane domains: CD28, CD8α, CD4, or CD19. In a particular embodiment, the transmembrane domain includes a domain derived from the CD28 transmembrane domain.

[0150] Chimeric antigen receptors may additionally include one or more co-stimulatory signaling regions. For example, the co-stimulatory signaling region may be the signaling region of CD28, OX-40, 41BB, CD27, inducible T-cell co-stimulatory factor (ICOS), CD3γ, CD3δ, CD3ε, CD247, Igα (CD79a), or Fcγ receptors. In one particular embodiment, the co-stimulatory signaling region is the CD28 signaling region.

[0151] In one embodiment, the chimeric antigen receptor further includes a CD3ζ message transduction domain.

[0152] Chimeric antigen receptors can be engineered to target specific tumor antigens. In some embodiments, the tumor antigen is selected from 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 mantle glycoprotein gp120, HIV-1 mantle glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R α, κ chain, λ chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 combination, HER1-HER2 combination, and any combination thereof. In one particular embodiment, the tumor antigen is CD19.

[0153] In another embodiment, the T-cell therapy includes administering engineered T cells ("engineered TCR T cells") that express a T-cell receptor to the patient. The T-cell receptor (TCR) may include a binding molecule to a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of: CD19, CD20, ROR1, CD22, carcinoembryonic antigen, α-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 mantle glycoprotein gp120, HIV-1 mantle glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R α, κ chain, λ chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 combination, HER1-HER2 combination, and any combination thereof.

[0154] In one embodiment, the TCR comprises a binding molecule to a viral oncogene. In a particular embodiment, the viral oncogene is selected from human papillomavirus (HPV), epidemiologic virus (EBV), and human T-lymphotropic virus (HTLV).

[0155] 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 selected from the group consisting of: NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), and any combination thereof.

[0156] In another embodiment, the TCR comprises a binding molecule to a lineage-specific antigen. In one particular embodiment, the lineage-specific antigen is selected from the group consisting of: melanoma antigen 1 (MART-1), gp100, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), and prostate stem cell antigen (PSCA), and any combination thereof, which are recognized by T cells.

[0157] In one embodiment, the T-cell therapy includes administering engineered CAR T cells to a patient. These engineered CAR T cells exhibit a chimeric antigen receptor that binds to CD19 and additionally includes a CD28 co-stimulatory domain and a CD3ζ signaling region. In a particular embodiment, the T-cell therapy includes administering KTE-C19 to a patient.

[0158] The T-cell therapy included in this invention involves transferring T cells to a patient. The T cells can be administered at a therapeutically effective amount. For example, a therapeutically effective amount of T cells (e.g., engineered CAR+ T cells or engineered TCR+ T cells) may be at least about 10⁴ cells, at least about 10⁵ cells, at least about 10⁶ cells, at least about 10⁷ cells, at least about 10⁸ cells, at least about 10⁹ cells, or at least about 10¹⁰ cells. In another embodiment, a therapeutically effective amount of T cells (e.g., engineered CAR+ T cells or engineered TCR+ T cells) is about 10⁴ cells, about 10⁵ cells, about 10⁶ cells, about 10⁷ cells, or about 10⁸ cells. In one particular embodiment, the therapeutically effective amount of T cells (e.g., engineered CAR+ T cells or engineered TCR+ T cells) is approximately 1 × 10⁵ cells / kg, approximately 2 × 10⁵ cells / kg, approximately 3 × 10⁵ cells / kg, approximately 4 × 10⁵ cells / kg, approximately 5 × 10⁵ cells / kg, approximately 6 × 10⁵ cells / kg, approximately 7 × 10⁵ cells / kg, approximately 8 × 10⁵ cells / kg, approximately 9 × 10⁵ cells / kg, approximately 1 × 10⁶ cells / kg, approximately 2 × 10⁶ cells / kg, and approximately 3 × 10⁶ cells / kg. / kg, approximately 4 × 10⁶ cells / kg, approximately 5 × 10⁶ cells / kg, approximately 6 × 10⁶ cells / kg, approximately 7 × 10⁶ cells / kg, approximately 8 × 10⁶ cells / kg, approximately 9 × 10⁶ cells / kg, approximately 1 × 10⁷ cells / kg, approximately 2 × 10⁷ cells / kg, approximately 3 × 10⁷ cells / kg, approximately 4 × 10⁷ cells / kg, approximately 5 × 10⁷ cells / kg, approximately 6 × 10⁷ cells / kg, approximately 7 × 10⁷ cells / kg, approximately 8 × 10⁷ cells / kg, or approximately 9 × 10⁷ cells / kg. In one particular embodiment, the therapeutically effective amount of T cells (e.g., engineered CAR+ T cells or engineered TCR+ T cells) is approximately 2 × 10⁶ cells / kg.

[0159] In other embodiments, the therapeutically effective amount of T cells (e.g., engineered CAR+ T cells or engineered TCR+ T cells) is from about 1.0 × 10⁵ cells / kg to about 2 × 10⁸ cells / kg, from about 2.0 × 10⁵ cells / kg to about 2 × 10⁸ cells / kg, from about 3.0 × 10⁵ cells / kg to about 2 × 10⁸ cells / kg, from about 4.0 × 10⁵ cells / kg to about 2 × 10⁸ cells / kg, from about 5.0 × 10⁵ cells / kg to about 2 × 10⁸ cells / kg, from about 6.0 × 10⁵ cells / kg to about 2 × 10⁸ cells / kg, and from about 7.0 × 10⁵ cells / kg to about 2 × 10⁸ cells / kg. g to about 2×10⁸ cells / kg, from about 8.0×10⁵ cells / kg to about 2×10⁸ cells / kg, from about 9.0×10⁵ cells / kg to about 2×10⁸ cells / kg, from about 0.5×10⁶ cells / kg to about 2×10⁸ cells / kg, from about 2×10⁶ cells / kg to about 9×10⁷ cells / kg, from about 3×10⁶ cells / kg to about 9×10⁷ cells / kg, from about 4×10⁶ cells / kg to about 9×10⁷ cells / kg, from about 5×10⁶ cells / kg to about 9×10⁷ cells / kg, from about 6×10⁶ cells / kg to about 9×10⁷ cells / kg / kg, from about 7×10⁶ cells / kg to about 9×10⁷ cells / kg, from about 8×10⁶ cells / kg to about 9×10⁷ cells / kg, from about 9×10⁶ cells / kg to about 9×10⁷ cells / kg, from about 1×10⁷ cells / kg to about 9×10⁷ cells / kg, from about 2×10⁷ cells / kg to about 9×10⁷ cells / kg, from about 3×10⁷ cells / kg to about 9×10⁷ cells / kg, from about 4×10⁷ cells / kg to about 9×10⁷ cells / kg, from about 5×10⁷ cells / kg to about 9×10⁷ cells / kg, from about 6× 107 cells / kg to about 9×107 cells / kg, from about 7×107 cells / kg to about 9×107 cells / kg, from about 8×107 cells / kg to about 9×107 cells / kg, from about 2×106 cells / kg to about 8×107 cells / kg, from about 2×106 cells / kg to about 7×107 cells / kg, from about 2×106 cells / kg to about 6×107 cells / kg, from about 2×106 cells / kg to about 5×107 cells / kg, from about 2×106 cells / kg to about 4×107 cells / kg, from about 2×106 cells / kg to about 9 ... g to about 3×10⁷ cells / kg, from about 2×10⁶ cells / kg to about 2×10⁷ cells / kg, from about 2×10⁶ cells / kg to about 1×10⁷ cells / kg, from about 2×10⁶ cells / kg to about 9×10⁶ cells / kg, from about 2×10⁶ cells / kg to about 8×10⁶ cells / kg, from about 2×10⁶ cells / kg to about 7×10⁶ cells / kg, from about 2×10⁶ cells / kg to about 6×10⁶ cells / kg, from about 2×10⁶ cells / kg to about 5×10⁶ cells / kg, from about 2×10⁶ cells / kg to about 4×10⁶ cells / kg The therapeutically effective amount of engineered CAR T cells is from about 2 × 10⁶ cells / kg to about 3 × 10⁶ cells / kg, from about 3 × 10⁶ cells / kg to about 8 × 10⁷ cells / kg, from about 4 × 10⁶ cells / kg to about 7 × 10⁷ cells / kg, from about 5 × 10⁶ cells / kg to about 6 × 10⁷ cells / kg, from about 6 × 10⁶ cells / kg to about 5 × 10⁷ cells / kg, from about 7 × 10⁶ cells / kg to about 4 × 10⁷ cells / kg, from about 8 × 10⁶ cells / kg to about 3 × 10⁷ cells / kg, or from about 9 × 10⁶ cells / kg to about 2 × 10⁷ cells / kg. In one embodiment, the therapeutically effective amount of engineered CAR T cells is from about 0.8 × 10⁶ cells / kg to about 1.2 × 10⁶ T cells / kg. In a particular embodiment, the therapeutically effective amount of engineered CAR T cells is...The T cell count is 2.0 × 10⁵ cells / kg. In one particular embodiment, the therapeutically effective amount of engineered CAR T cells is 1.0 × 10⁶ cells / kg.

[0160] [, Cytokine levels , ]

[0161] This invention describes a method for conditioning a patient requiring T-cell therapy, comprising administering cyclophosphamide and fludarabine to the patient. Administering cyclophosphamide and fludarabine prior to T-cell therapy increases the levels of endogenous cytokines, thereby improving the immune environment to facilitate the homeostatic proliferation, activation, and transport of T cells. Once the patient is administered secondary-transferred T cells, these T cells are exposed to the increased levels of endogenous cytokines.

[0162] Administration of cyclophosphamide and fludarabine can concentrate various cytokines in the patient's serum. In some embodiments, the patient exhibits increased serum concentrations of cytokines or pro-inflammatory factors selected from: 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-macrophage colony-stimulating factor (G-CSF), monocyte chemotactic protein 1 (MCP-1), MCP-4, γ-inducible protein 10 (IP-10), placental growth factor (PLGF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), C-reactive protein (CRP), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, macrophage inflammatory protein 1β (MIP-1) β, MIP-1b), leukemia inhibitory factor (LIF), oncogene M (OSM), interferon (IFN) α, IFN-β, IFN-γ, tumor necrosis factor (TNF) α, TNF-β, CD154, lymphotoxin (LT) β, 4-1BB ligand (4-1BBL), proliferation-inducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-associated ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L, TNF- and ApoL-associated leukocyte phenotype ligand 1 (TALL-1), TNF-associated apoptosis-inducing ligand (TRAIL), chemokine (CC motif) ligand (CCL) 1, macrophage inflammatory protein 1 α (MIP-1a or CCL3), CCL5, monocyte-specific chemokine 3 (MCP-3 or CCL7), monocyte chemoattractant protein 2 (MCP-2 or CCL8), CCL13, thymus and activated regulatory chemokine (TARC or CCL17), CCL22, FGF2, eosin, MDC, granzine A, granzine B, perforin, SAA, MCP-4, and any combination thereof. In some embodiments, the patient showed increased serum levels of IL-15 and / or IP-10 after administration of cyclophosphamide and fludarabine. In some embodiments, the patient showed decreased serum levels of perforin after administration of cyclophosphamide and fludarabine.

[0163] In some embodiments, the present invention includes a method for increasing the effectiveness of in vivo constant cytokines in patients requiring T-cell therapy. In some embodiments, the in vivo constant cytokines are 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 chemoattractant 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.

[0164] In one embodiment, the serum IL-7 level in the patient increases by at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 times compared to the pre-administration level after administration of cyclophosphamide and fludarabine. In a particular embodiment, the IL-7 level increases by at least about 2 times compared to the serum IL-7 level before administration of cyclophosphamide and fludarabine. In another embodiment, the patient is given exogenous IL-7 to increase the IL-7 level. In a particular embodiment, the patient is given cyclophosphamide, fludarabine, and exogenous IL-7 to increase the IL-7 level.

[0165] In one embodiment, the serum IL-15 level in the patient increases by at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, or at least 90 times compared to the serum IL-15 level before administration of cyclophosphamide and fludarabine. In a particular embodiment, the IL-15 level increases by at least about 10 times compared to the serum IL-15 level before administration of cyclophosphamide and fludarabine. In another embodiment, the IL-15 level increases by at least about 20 times compared to the serum IL-15 level before administration of cyclophosphamide and fludarabine. In yet another embodiment, the IL-15 level increases by at least about 30 times compared to the serum IL-15 level before administration of cyclophosphamide and fludarabine. In another embodiment, the patient is given exogenous IL-15 to increase the IL-15 level. In a particular embodiment, the patient is given cyclophosphamide, fludarabine, and exogenous IL-15 to increase the IL-15 level.

[0166] In one embodiment, the serum IL-10 level in the patient increases by at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 20 times compared to the serum IL-10 level before administration of cyclophosphamide and fludarabine. In a particular embodiment, the IL-10 level increases by at least about 2 times compared to the serum IL-10 level before administration of cyclophosphamide and fludarabine. In another embodiment, the IL-10 level increases by at least about 3 times compared to the serum IL-10 level before administration of cyclophosphamide and fludarabine. In another embodiment, the IL-10 level increases by at least about 5 times compared to the serum IL-10 level before administration of cyclophosphamide and fludarabine. In yet another embodiment, the IL-10 level increases by at least about 20 times compared to the serum IL-10 level before administration of cyclophosphamide and fludarabine. In another embodiment, the patient is given exogenous IL-10 to increase the IL-10 level. In a particular embodiment, the patient is given cyclophosphamide, fludarabine, and exogenous IL-10 to increase the IL-10 level.

[0167] In one embodiment, the serum IL-5 level in the patient increases by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times compared to the pre-administration IL-5 level after administration of cyclophosphamide and fludarabine. In a particular embodiment, the IL-5 level increases by at least about 5 times compared to the pre-administration IL-5 serum level. In another embodiment, the IL-5 level increases by at least about 10 times compared to the pre-administration IL-5 serum level. In yet another embodiment, the IL-5 level increases by at least about 30 times compared to the pre-administration IL-5 serum level. In another embodiment, the IL-5 level is at least about 100 times higher than the serum IL-5 level before administration of cyclophosphamide and fludarabine. In another embodiment, the patient is given exogenous IL-5 to increase the IL-5 level. In a particular embodiment, the patient is given cyclophosphamide, fludarabine, and exogenous IL-5 to increase the IL-5 level.

[0168] In one embodiment, the serum IP-10 level in the patient increases by at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 30 times compared to the pre-administration level of cyclophosphamide and fludarabine. In a particular embodiment, the IP-10 level increases by at least about 2 times compared to the pre-administration level of cyclophosphamide and fludarabine. In another embodiment, the IP-10 level increases by at least about 3 times compared to the pre-administration level of cyclophosphamide and fludarabine. In another embodiment, the IP-10 level increases by at least about 4 times compared to the pre-administration level of cyclophosphamide and fludarabine. In yet another embodiment, the IP-10 level increases by at least about 7 times compared to the pre-administration level of cyclophosphamide and fludarabine. In another embodiment, the patient is given exogenous IP-10 to increase the amount of IP-10. In a particular embodiment, the patient is given cyclophosphamide, fludarabine, and exogenous IP-10 to increase the amount of IP-10.

[0169] In one embodiment, the serum IL-8 level in the patient increases by at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 times compared to the serum IL-8 level before administration of cyclophosphamide and fludarabine. In a particular embodiment, the IL-8 level increases by at least about 2 times compared to the serum IL-8 level before administration of cyclophosphamide and fludarabine. In another embodiment, the IL-8 level increases by at least about 5 times compared to the serum IL-8 level before administration of cyclophosphamide and fludarabine. In yet another embodiment, the IL-8 level increases by at least about 10 times compared to the serum IL-8 level before administration of cyclophosphamide and fludarabine. In another embodiment, the IL-8 level is at least about 20 times higher than the serum IL-8 level before administration of cyclophosphamide and fludarabine. In another embodiment, the IL-8 level is at least about 40 times higher than the serum IL-8 level before administration of cyclophosphamide and fludarabine. In another embodiment, the IL-8 level is at least about 60 times higher than the serum IL-8 level before administration of cyclophosphamide and fludarabine. In another embodiment, the patient is given exogenous IL-8 to increase the IL-8 level. In a particular embodiment, the patient is given cyclophosphamide, fludarabine, and exogenous IL-8 to increase the IL-8 level.

[0170] In one embodiment, the serum MCP-1 level in the patient increases by at least 1.5 times, 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, or at least 20 times compared to the pre-administration MCP-1 level after administration of cyclophosphamide and fludarabine. In a particular embodiment, the MCP-1 level increases by at least about 2 times compared to the pre-administration MCP-1 level. In another embodiment, the MCP-1 level increases by at least about 3 times compared to the pre-administration MCP-1 level. In another embodiment, the MCP-1 level increases by at least about 5 times compared to the pre-administration MCP-1 level. In yet another embodiment, the MCP-1 level increases by at least about 7 times compared to the pre-administration MCP-1 level. In another embodiment, the patient is given exogenous MCP-1 to increase the amount of MCP-1. In a particular embodiment, the patient is given cyclophosphamide, fludarabine, and exogenous MCP-1 to increase the amount of MCP-1.

[0171] In one embodiment, the serum PLGF level in the patient increases by at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times after administration of cyclophosphamide and fludarabine compared to the serum PLGF level before administration. In a particular embodiment, the PLGF level increases by at least about 1.5 times compared to the serum PLGF level before administration of cyclophosphamide and fludarabine. In another embodiment, the PLGF level increases by at least about 2 times compared to the serum PLGF level before administration of cyclophosphamide and fludarabine. In yet another embodiment, the PLGF level increases by at least about 3 times compared to the serum PLGF level before administration of cyclophosphamide and fludarabine. In another embodiment, the patient is given exogenous PLGF to increase the PLGF level. In a particular embodiment, the patient is given cyclophosphamide, fludarabine, and exogenous PLGF to increase the PLGF level.

[0172] In one embodiment, the serum CRP level in the patient increases by at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least about 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times after administration of cyclophosphamide and fludarabine compared to the pre-administration CRP level. In a particular embodiment, the CRP level increases by at least about 1.5 times compared to the pre-administration CRP level. In another embodiment, the CRP level increases by at least about 2 times compared to the pre-administration CRP level. In yet another embodiment, the CRP level increases by at least about 5 times compared to the pre-administration CRP level. In another embodiment, the CRP level increases at least about 9 times compared to the serum CRP level before administration of cyclophosphamide and fludarabine. In another embodiment, the CRP level increases at least about 10 times compared to the serum CRP level before administration of cyclophosphamide and fludarabine. In another embodiment, the CRP level increases at least about 25 times compared to the serum CRP level before administration of cyclophosphamide and fludarabine. In another embodiment, exogenous CRP is administered to the patient to increase the CRP level. In a particular embodiment, cyclophosphamide, fludarabine, and exogenous CRP are administered to the patient to increase the CRP level.

[0173] In one embodiment, the serum sICAM-1 level in the patient increases by at least 1.5 times, 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 20 times, at least 25 times, or at least 30 times after administration of cyclophosphamide and fludarabine compared to the level before administration. In a particular embodiment, the sICAM-1 level increases by at least about 1.5 times compared to the serum sICAM-1 level before administration of cyclophosphamide and fludarabine. In another embodiment, the sICAM-1 level increases by at least about 2 times compared to the serum sICAM-1 level before administration of cyclophosphamide and fludarabine. In yet another embodiment, the sICAM-1 level increases by at least about 3 times compared to the serum sICAM-1 level before administration of cyclophosphamide and fludarabine. In another embodiment, the sICAM-1 level is at least about four times higher than the serum sICAM-1 level before administration of cyclophosphamide and fludarabine. In another embodiment, the patient is given exogenous sICAM-1 to increase the sICAM-1 level. In a particular embodiment, the patient is given cyclophosphamide, fludarabine, and exogenous sICAM-1 to increase the sICAM-1 level.

[0174] In one embodiment, the serum sVCAM-1 level in the patient increases by at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, or at least 5 times after administration of cyclophosphamide and fludarabine compared to the level before administration. In a particular embodiment, the sVCAM-1 level increases by at least about 1.5 times compared to the serum sVCAM-1 level before administration of cyclophosphamide and fludarabine. In another embodiment, the sVCAM-1 level increases by at least about 2 times compared to the serum sVCAM-1 level before administration of cyclophosphamide and fludarabine. In another embodiment, the sVCAM-1 level increases by at least about 3 times compared to the serum sVCAM-1 level before administration of cyclophosphamide and fludarabine. In another embodiment, exogenous sVCAM-1 is administered to the patient to increase the sVCAM-1 level. In one particular implementation, the patient was administered cyclophosphamide, fludarabine, and exogenous sVCAM-1 to increase the amount of sVCAM-1.

[0175] In some implementations, the levels of one or more cytokines following administration of cyclophosphamide and fludarabine can be used to predict a patient's response to T-cell therapy. For example, an increase in a specific cytokine after administration of cyclophosphamide and fludarabine may indicate a greater likelihood of a response to T-cell therapy. In another instance, a decrease or no change in the level of a specific cytokine after administration of cyclophosphamide and fludarabine may indicate a less likely response to T-cell therapy. It is also possible that an increase in one or more cytokines and a decrease in one or more different cytokines after administration of cyclophosphamide and fludarabine may indicate a greater or less likely response to T-cell therapy. In this manner, a patient's cytokine profile can indicate responsiveness to T-cell therapy.

[0176] In some embodiments, an increase in IL-15 levels greater than about 3 times, 4 times, 5 times, 10 times, 15 times, or 20 times after administration of cyclophosphamide and fludarabine indicates that the patient is more likely to respond to T-cell therapy. In other embodiments, an increase in IP-10 levels greater than about 2 times, 3 times, 4 times, 5 times, or 6 times after administration of cyclophosphamide 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 after administration of cyclophosphamide and fludarabine indicates that the patient is less likely to respond to T-cell therapy.

[0177] In some embodiments, serum levels of any one or more cytokines are measured on one or more days prior to administration of cyclophosphamide and fludarabine and on one or more days during the period from administration of cyclophosphamide and fludarabine to 21 days after administration of cyclophosphamide and fludarabine.

[0178] One embodiment of the present invention includes a method for increasing the effectiveness of in vivo constant cytokines in a patient requiring T-cell therapy. Another embodiment of the present invention includes a method for improving the efficacy of T-cell therapy, comprising administering to a patient a treatment that increases the levels of one or more of the following in vivo constant cytokines, pro-inflammatory cytokines, or chemokines: 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 that many different methods can be used to increase the levels of in vivo constant cytokines, including but not limited to: the use of cyclophosphamide and fludarabine as described herein; administering to a patient one or more exogenous cytokines; administering one or more components that induce the expression of one or more endogenous cytokines or prevent their degradation; administering one or more transgenic cells that express one or more recombinant cytokines; and any other method that has the effect of increasing the levels of endogenous cytokines in a patient.

[0179] In some embodiments, the present invention includes a method of conditioning a patient requiring T-cell therapy, the method comprising administering cyclophosphamide and fludarabine to the patient and one or more doses of isolated or recombinant cytokines. The isolated or recombinant cytokines may 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 a particular embodiment, the method of conditioning a patient requiring T-cell therapy comprises administering cyclophosphamide and fludarabine to the patient and one or more doses of isolated or recombinant cytokines, wherein the cytokines are selected from IL-2, IL-15, IL-7, IL-10, IL-5, IP-10, IL-8, MCP-1, PLGF, CRP, sICAM-1, sVCAM-1, and any combination thereof (e.g., IL-15, IL-7, IP-10, MCP-1, CRP, and PLGF). One or more doses of isolated or recombinant cytokines may be administered before, after, or in any combination thereof, T-cell therapy.

[0180] In one embodiment, the method of conditioning a patient requiring T-cell therapy comprises administering cyclophosphamide and fludarabine, along with one or more doses of IL-2. In some embodiments, the IL-2 dose 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 IL-2 dose is at least about 700,000 IU / kg. In a particular embodiment, the IL-2 dose is about 720,000 IU / kg. In some implementations, IL-2 is administered to the patient every 8 hours until 15 doses are administered or until toxicity prevents additional administration.

[0181] [, Cancer treatment , ]

[0182] The method of this invention can be used to treat cancer in an individual, reduce tumor size, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, eliminate tumors from a patient, prevent tumor recurrence, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In some embodiments, the method induces a complete response. In other embodiments, the method induces a partial response.

[0183] Treatable cancers include non-vascularized, non-solidly vascularized, or vascularized tumors. The cancer may also include solid or non-solid tumors. In some embodiments, the cancer may be selected from the following derived tumors: bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, 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, T-cell rich B-cell lymphoma (TCRBCL), primary mediastinal large B-cell lymphoma (PMBCL), non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, and endocrine cancer. Systemic cancers, 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) cancers, primary CNS lymphoma, tumor angiogenesis, spinal cord axonoma, brainstem glioma, pituitary adenoma, Karposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including asbestos-induced cancers), and combinations of the above cancers.

[0184] In one implementation, the method can be used to treat a tumor, wherein the tumor is lymphoma or leukemia. Lymphoma and leukemia are blood cancers that specifically attack lymphocytes. All white blood cells in the blood originate from a type of pluripotent hematopoietic stem cell found in the bone marrow. These stem cells produce bone marrow precursor cells and lymphocyte precursor cells, which then derive from various white blood cells found in the body. White blood cells originating from bone marrow precursor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells, and plasma cells. White blood cells originating from lymphocyte precursor cells include megakaryocytes, basophils, neutrophils, eosinophils, monocytes, and macrophages. Lymphoma and leukemia can attack one or more of these cell types in a patient.

[0185] Lymphomas are typically divided into at least two subgroups: Hodgkin's lymphoma and non-Hodgkin's lymphoma. Non-Hodgkin's lymphoma (NHL) is a heterogeneous group of cancers originating from B lymphocytes, T lymphocytes, or natural killer cells. In the United States, B-cell lymphoma accounts for 80 to 85% of reported cases. In 2013, an estimated 69,740 new cases of NHL and over 19,000 deaths were associated with the disease. Non-Hodgkin's lymphoma is the most prevalent hematologic malignancy, ranking seventh among new cancer cases in both men and women, accounting for 4% of all new cancer cases and 3% of cancer-related deaths.

[0186] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, accounting for approximately 30% of NHL cases. In the United States, approximately 22,000 new DLBCL cases are diagnosed each year. These new DLBCL cases are classified as aggressive lymphomas, and the majority of patients are cured with conventional chemotherapy (NCCN guidelines NHL 2014).

[0187] First-line therapy for DLBCL typically includes regimens containing anthracyclines such as rituximab, like R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). This regimen has an objective response rate of approximately 80% and a complete response rate of approximately 50% (Coiffier 2002). About one-third of patients who undergo R-CHOP experience initial therapy refractory disease or relapse (Sehn 2005). In patients who relapse after responding to first-line therapy, approximately 40 to 60% can achieve a second response with additional chemotherapy. Standards of care for second-line therapy in eligible patients undergoing autologous stem cell transplantation (ASCT) include rituximab and combination therapies such as R-ICE (rituximab, ifosamide, carboplatin, and etoposide) and R-DHAP (rituximab, dexamethasone, cytarabine, and cisplatin), each with an objective response rate of approximately 63% and a complete response rate of approximately 26% (Gisselbrecht 2010). Patients who respond to second-line therapy and are deemed adequately suited for transplantation receive adjuvant therapy using high-dose chemotherapy and ASCT, which is curative in approximately half of transplant recipients (Gisselbrecht 2010). Patients who fail ASCT have a very poor prognosis and no cure.

[0188] Primary mediastinal large B-cell lymphoma (PMBCL) has different clinical presentations, pathological features, and molecular characteristics from DLBCL. PMBCL is believed to originate from thymic (bone marrow) B cells and accounts for approximately 3% of patients diagnosed with DLBCL. PMBCL typically occurs in young adults over 40 years of age, with a slightly higher incidence in women. Genetic profiling suggests deregulated pathways in PMBCL that overlap with Hodgkin's lymphoma. Initial treatment for PMBCL typically includes anthracycline-containing antibiotic regimens using rituximab, such as dose-adjusted etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone, and rituximab, and radiation therapy with or without access to the relevant area.

[0189] Follicular lymphoma (FL), a type of B-cell lymphoma, is the most common indolent (slow-growing) form of non-Hodgkin's lymphoma (NHL), accounting for approximately 20% to 30% of all NHL cases. Some FL patients undergo tissue transformation (TFL) into the more aggressive and associated with poorer outcomes, DLBCL. Despite a continued decline in the risk of TFL transformation over several years, TFL transformation to DLBCL occurs at an annualized rate of approximately 3% over 15 years. The biological mechanisms of TFL transformation remain unknown. Initial treatment for TFL, while influenced by prior follicular lymphoma treatment, typically includes regimens using anthracycline-containing antibiotics such as rituximab to eliminate the aggressive component of the disease.

[0190] Treatment options for relapsed / refractory PMBCL and TFL are similar to those for DLBCL. Due to the low prevalence of these diseases, large-scale prospective randomized studies have not been conducted on these patient populations. Patients with chemotherapy-resistant disease have similar or worse prognoses to those with refractory DLBCL.

[0191] In general, individuals with refractory aggressive NHL (such as DLBCL, PMBCL, and TFL) have greater unmet medical needs and are more likely to be approved for further research using novel treatments in these populations.

[0192] Therefore, in some embodiments, this method can be used to treat lymphoma or leukemia, wherein the lymphoma or leukemia is a B-cell malignancy. In some embodiments, the lymphoma or leukemia is selected from: B-cell chronic lymphocytic leukemia / small cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., Waldenström macroglobulinemia). Macroglobulinemia), splenic marginal zone lymphoma, hairy cell leukemia, plasmacytoma (e.g., plasmacytic myeloma (i.e., multiple myeloma) or plasmacytoma), extranodal marginal zone B-cell lymphoma (e.g., MALT lymphoma), intranodal marginal zone B-cell lymphoma, follicular lymphoma (FL), transformed follicular lymphoma (TFL), primary cutaneous follicular central lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Estane-Barr virus-positive DLBCL, lymphomatoid granuloma, primary mediastinal (thymic) large B-cell lymphoma (PMBCL), intravascular large B-cell lymphoma, AL K+ large B-cell lymphoma, plasmablastic lymphoma, primary exudative lymphoma, large B-cell lymphoma originating from HHV8-associated multicentric Castleman's disease, Burkitt's 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, intestinal lesion-associated T-cell lymphoma, hepatocellular T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides / Sezary syndrome, primary cutaneous undifferentiated large cell lymphoma. Lymphomatoid papules, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, B-lymphoblastic leukemia / lymphoma, B-lymphoblastic leukemia / lymphoma with recurrent gene abnormalities, T-lymphoblastic leukemia / lymphoma, and Hodgkin's lymphoma. In some embodiments, the cancer is refractory to one or more prior treatments and / or the cancer has relapsed after one or more prior treatments.

[0193] In some 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.

[0194] In some embodiments, the cancer is refractory to one or more of the following or has relapsed after treatment with one or more of the following: chemotherapy, radiotherapy, immunotherapy (including T-cell therapy and / or treatment using antibodies and / or antibody-drug conjugates), autologous stem cell transplantation, and any combination thereof. In one particular embodiment, the cancer is refractory diffuse large B-cell lymphoma.

[0195] In one particular embodiment, the present invention includes a method for treating a patient with lymphoma, the method comprising administering to the patient daily any dose of cyclophosphamide (e.g., about 200 mg / m² / day, about 300 mg / m² / day, about 400 mg / m² / day, about 500 mg / m² / day, about 600 mg / m² / day, about 700 mg / m² / day, about 800 mg / m² / day, or about 900 mg / m² / day) and any dose of cyclophosphamide (e.g., about 20 mg / m² / day) as described herein, prior to administering the patient a therapeutically effective amount of engineered CAR cells. Fludarabine was administered for 3 days at doses of approximately 25 mg / m² / day, approximately 30 mg / m² / day, approximately 35 mg / m² / day, approximately 40 mg / m² / day, approximately 45 mg / m² / day, approximately 50 mg / m² / day, approximately 55 mg / m² / day, and approximately 60 mg / m² / day. The engineered CAR cells exhibited a chimeric antigen receptor that binds to CD19 and additionally includes the CD28 co-stimulatory domain and the CD3ζ signaling region.

[0196] In another embodiment, the present invention includes a method of treating a patient with lymphoma, the method comprising (i) administering to the patient any dose of cyclophosphamide as described herein (e.g., about 200 mg / m² / day, about 300 mg / m² / day, about 400 mg / m² / day, about 500 mg / m² / day, about 600 mg / m² / day, about 700 mg / m² / day, about 800 mg / m² / day, or about 900 mg / m² / day) and any dose of cyclophosphamide as described herein (e.g., about 20 mg / m² / day). (i) Fludarabine at doses of approximately 25 mg / m² / day, approximately 30 mg / m² / day, approximately 35 mg / m² / day, approximately 40 mg / m² / day, approximately 45 mg / m² / day, approximately 50 mg / m² / day, approximately 55 mg / m² / day, and approximately 60 mg / m² / day, and (ii) the patient was administered a therapeutically effective amount of engineered CAR cells, wherein the engineered CAR cells exhibit a chimeric antigen receptor that binds to CD19 and additionally includes a CD28 co-stimulatory domain and a CD3ζ signaling region.

[0197] In yet another embodiment, the invention includes a method of treating a patient with lymphoma, the method comprising administering to the patient a therapeutically effective amount of engineered CAR cells, wherein any dose of cyclophosphamide described herein (e.g., about 200 mg / m² / day, about 300 mg / m² / day, about 400 mg / m² / day, about 500 mg / m² / day, about 600 mg / m² / day, about 700 mg / m² / day, about 800 mg / m² / day, or about 900 mg / m² / day) has been administered. The patient was treated with any dose of fludarabine described herein (e.g., about 20 mg / m² / day, about 25 mg / m² / day, about 30 mg / m² / day, about 35 mg / m² / day, about 40 mg / m² / day, about 45 mg / m² / day, about 50 mg / m² / day, about 55 mg / m² / day, about 60 mg / m² / day), wherein the engineered CAR cells expressed a chimeric antigen receptor that binds to CD19 and additionally includes the CD28 co-stimulatory domain and the CD3ζ signaling region.

[0198] [, Set , ]

[0199] The scope of this invention also includes kits (e.g., pharmaceutical kits) containing pre-conditioned cyclophosphamide and fludarabine for T-cell therapy. Kits typically include a label indicating the intended use and instructions for use of the kit contents. The term "label" includes any written or recorded material on or supplied with the kit, or accompanying the kit.

[0200] In some embodiments, the present invention provides a kit for conditioning a patient requiring T-cell therapy, the kit comprising: (i) cyclophosphamide, (ii) fludarabine, and (iii) instructions for administering cyclophosphamide (at any dose described herein, e.g., between 200 mg / m² / day and 2000 mg / m² / day) and fludarabine (at any dose described herein, e.g., between 20 mg / m² / day and 900 mg / m² / day) daily for three days prior to engineered CAR cell therapy.

[0201] In other embodiments, the present invention provides a kit for conditioning a patient requiring T-cell therapy, the kit comprising: (i) cyclophosphamide, (ii) fludarabine, and (iii) instructions for administering cyclophosphamide to the patient requiring the therapy at any dose described herein (e.g., between 200 mg / m² / and 2000 mg / m² / day) daily for two days from day -7 to day -6 prior to engineered CAR cell therapy, and for administering fludarabine to the patient at any dose described herein (e.g., between 20 mg / m² / and 900 mg / m² / day) daily for five days from day -5 to day -1.

[0202] In other embodiments, the present invention provides a kit for conditioning a patient requiring T-cell therapy, the kit comprising: (i) cyclophosphamide, (ii) fludarabine, and (iii) instructions for administering cyclophosphamide (at any dose described herein, e.g., between 200 mg / m² / day and 2000 mg / m² / day) and fludarabine (at any dose described herein, e.g., between 20 mg / m² / day and 900 mg / m² / day) daily for three days prior to engineered TCR cell therapy.

[0203] In other embodiments, the present invention provides a kit for conditioning a patient requiring T-cell therapy, the kit comprising: (i) cyclophosphamide, (ii) fludarabine, and (iii) instructions for administering cyclophosphamide to the patient requiring the therapy at any dose described herein (e.g., between 200 mg / m² / and 2000 mg / m² / day) for two days daily from day -7 to day -6 prior to engineered TCR cell therapy, and for administering fludarabine to the patient at any dose described herein (e.g., between 20 mg / m² / and 900 mg / m² / day) daily for five days from day -5 to day -1.

[0204] In some embodiments, the present invention provides a kit for conditioning patients requiring T-cell therapy, the kit comprising: (i) cyclophosphamide, (ii) fludarabine, and (iii) instructions for administering cyclophosphamide at a dose of 300 mg / m² / day and fludarabine at a dose of 30 mg / m² / day to patients requiring the therapy for three days prior to engineered CAR cell therapy.

[0205] In other embodiments, the present invention provides a kit for conditioning a patient requiring T-cell therapy, the kit comprising: (i) cyclophosphamide, (ii) fludarabine, and (iii) instructions for administering cyclophosphamide at a dose of 300 mg / m² / day to the patient requiring the therapy for two days from day -7 to day -6 prior to engineered CAR cell therapy, and for administering fludarabine at a dose of 30 mg / m² / day to the patient for five days from day -5 to day -1.

[0206] In other embodiments, the present invention provides a kit for conditioning patients requiring T-cell therapy, the kit comprising: (i) cyclophosphamide, (ii) fludarabine, and (iii) instructions for administering cyclophosphamide at a dose of 500 mg / m² / day and fludarabine at a dose of 30 mg / m² / day to patients requiring the therapy daily for three days prior to engineered TCR cell therapy.

[0207] In other embodiments, the present invention provides a kit for conditioning a patient requiring T-cell therapy, the kit comprising: (i) cyclophosphamide, (ii) fludarabine, and (iii) instructions for administering cyclophosphamide at a dose of 500 mg / m² / day to the patient requiring the therapy for two days from day -7 to day -6 prior to engineered TCR cell therapy, and for administering fludarabine at a dose of 30 mg / m² / day to the patient for five days from day -5 to day -1.

[0208] In some embodiments, the present invention provides a kit for conditioning patients requiring T-cell therapy, the kit comprising: (i) cyclophosphamide, (ii) fludarabine, and (iii) instructions for administering cyclophosphamide at a dose of 300 mg / m² / day and fludarabine at a dose of 60 mg / m² / day to patients requiring the therapy for three days prior to engineered CAR cell therapy.

[0209] In other embodiments, the present invention provides a kit for conditioning a patient requiring T-cell therapy, the kit comprising: (i) cyclophosphamide, (ii) fludarabine, and (iii) instructions for administering cyclophosphamide at a dose of 300 mg / m² / day to the patient requiring the therapy for two days from day -7 to day -6 prior to engineered CAR cell therapy, and for administering fludarabine at a dose of 60 mg / m² / day to the patient for five days from day -5 to day -1.

[0210] In other embodiments, the present invention provides a kit for conditioning patients requiring T-cell therapy, the kit comprising: (i) cyclophosphamide, (ii) fludarabine, and (iii) instructions for administering cyclophosphamide at a dose of 500 mg / m² / day and fludarabine at a dose of 60 mg / m² / day to patients requiring the therapy daily for three days prior to engineered TCR cell therapy.

[0211] In other embodiments, the present invention provides a kit for conditioning a patient requiring T-cell therapy, the kit comprising: (i) cyclophosphamide, (ii) fludarabine, and (iii) instructions for administering cyclophosphamide at a dose of 500 mg / m² / day to the patient requiring the therapy for two days from day -7 to day -6 prior to engineered TCR cell therapy, and for administering fludarabine at a dose of 60 mg / m² / day to the patient for five days from day -5 to day -1.

[0212] In some embodiments, the kit further includes normal saline and instructions for administering the normal saline to the patient before or after administration of cyclophosphamide and / or fludarabine, or before and after administration of cyclophosphamide and / or fludarabine. In some embodiments, the kit further includes mesna and instructions for administering mesna to the patient before, after, or before and after administration of cyclophosphamide and / or fludarabine.

[0213] [, Diagnosis using biomarkers , ]

[0214] The present invention also includes a method for identifying individuals suitable for T-cell therapy. In one embodiment, the present invention includes a method for treating cancer in a patient suitable for T-cell therapy, the method comprising administering to the patient a dose between 200 mg / m² and 2000 mg / m² (e.g., 200 mg / m², 300 mg / m², 400 mg / m², 500 mg / m², 600 mg / m², 700 mg / m², 800 mg / m², 900 mg / m², 1000 mg / m², or 1110 mg / m²). The patient was pre-conditioned with cyclophosphamide (m2) and fludarabine at doses between 20 mg / m2 and 900 mg / m2 (e.g., 20 mg / m2, 25 mg / m2, 30 mg / m2, 35 mg / m2, 40 mg / m2, 45 mg / m2, or 50 mg / m2), and then treated with T-cell therapy after the patient showed increased serum levels of IL-15, IP-10, and / or IL-7, and / or decreased serum levels of perforin. In another embodiment, the present invention includes a method for treating cancer in a patient suitable for T-cell therapy, the method comprising (i) administering to the patient a dose between 200 mg / m² and 2000 mg / m² (e.g., 200 mg / m², 300 mg / m², 400 mg / m², 500 mg / m², 600 mg / m², 700 mg / m², 800 mg / m², 900 mg / m², 1000 mg / m², or 1110 mg / m²). (ii) Pre-condition the patient with cyclophosphamide at a dose between 20 mg / m2 and 900 mg / m2 (e.g., 20 mg / m2, 25 mg / m2, 30 mg / m2, 35 mg / m2, 40 mg / m2, 45 mg / m2, or 50 mg / m2), and (ii) administer T-cell therapy after the patient shows an increase in serum levels of IL-15, IP-10, and / or IL-7, and / or a decrease in serum levels of perforin.In other embodiments, the present invention relates to a method for treating cancer in a patient suitable for T-cell therapy, the method comprising (i) administering to the patient a dose of cyclophosphamide between 200 mg / m² and 2000 mg / m² (e.g., 200 mg / m², 300 mg / m², 400 mg / m², 500 mg / m², 600 mg / m², 700 mg / m², 800 mg / m², 900 mg / m², 1000 mg / m², or 1110 mg / m²) and a dose of cyclophosphamide between 20 mg / m² and 900 mg / m² (e.g., 20 mg / m², ... (ii) Pre-conditioning the patient with fludarabine at 25 mg / m², 30 mg / m², 35 mg / m², 40 mg / m², 45 mg / m², or 50 mg / m²; (ii) administering an additional dose of cyclophosphamide and / or fludarabine or IL-15, IP-10, and / or IL-7 after the administration in (i) when the patient does not show adequate serum levels of IL-15, IP-10, and / or IL-7; and (iii) administering T-cell therapy after the administration in (ii) when the patient shows increased serum levels of IL-15, IP-10, and / or IL-7. In some embodiments, T-cell therapy is administered to the patient when the patient shows increased serum levels of at least one additional cytokine selected from the group consisting of MCP-1, CRP, PLGF, IP-10, and any combination thereof.

[0215] The present invention further provides a method for identifying patients suitable for T-cell therapy, the method comprising administering to the patient a dose between 200 mg / m² and 2000 mg / m² (e.g., 200 mg / m², 300 mg / m², 500 mg / m², 400 mg / m², 600 mg / m², 700 mg / m², 800 mg / m², 900 mg / m², 1000 mg / m², or 1110 mg / m²). 2) Cyclophosphamide and fludarabine at doses between 20 mg / m2 and 900 mg / m2 (e.g., 20 mg / m2, 25 mg / m2, 30 mg / m2, 35 mg / m2, 40 mg / m2, 45 mg / m2, or 50 mg / m2), wherein the patient shows increased serum levels of IL-15, IP-10, and / or IL-7, and / or decreased serum levels of perforin, before being treated with T-cell therapy. In other embodiments, the method of the present invention is to identify patients suitable for T-cell therapy, the method comprising (i) administering to the patient a dose between 200 mg / m² and 2000 mg / m² (e.g., 200 mg / m², 300 mg / m², 400 mg / m², 500 mg / m², 600 mg / m², 700 mg / m², 800 mg / m², 900 mg / m², 1000 mg / m², or 1110 mg / m²). (i) Cyclophosphamide and fludarabine at doses between 20 mg / m2 and 900 mg / m2 (e.g., 20 mg / m2, 25 mg / m2, 30 mg / m2, 35 mg / m2, 40 mg / m2, 45 mg / m2, or 50 mg / m2), and (ii) T-cell therapy after the patient shows increased serum levels of IL-15, IP-10, and / or IL-7, and / or decreased serum levels of perforin.In other embodiments, the present invention relates to a method for identifying a patient suitable for T-cell therapy, the method comprising (i) administering to the patient a dose of cyclophosphamide between 200 mg / m² and 2000 mg / m² (e.g., 200 mg / m², 300 mg / m², 400 mg / m², 500 mg / m², 600 mg / m², 700 mg / m², 800 mg / m², 900 mg / m², 1000 mg / m², or 1110 mg / m²) and a dose of cyclophosphamide between 20 mg / m² and 900 mg / m². (e.g., 20 mg / m², 25 mg / m², 30 mg / m², 35 mg / m², 40 mg / m², 45 mg / m², or 50 mg / m²) of fludarabine, (ii) administering an additional dose of cyclophosphamide or fludarabine or an effective dose of IL-15, IP-10, and / or IL-7 when the patient does not show adequate serum levels of IL-15, IP-10, and / or IL-7, and (iii) administering T-cell therapy after the patient shows increased serum levels of IL-15, IP-10, and / or IL-7. In some embodiments, T-cell therapy is administered to the patient when the patient shows increased serum levels of at least one additional cytokine selected from the group consisting of MCP-1, CRP, PLGF, IP-10, and any combination thereof.

[0216] The method of the present invention further includes measuring serum levels of IL-15, IP-10, perforin, and / or IL-7. In one embodiment, the serum IL-7 level in the patient increases by at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, or at least 90 times after administration of cyclophosphamide and fludarabine compared to the serum IL-7 level before administration. In another embodiment, the serum IL-15 level in the patient increases by at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, or at least 90 times after administration of cyclophosphamide and fludarabine compared to before administration.

[0217] In other embodiments, the serum MCP-1 level in the patient increased by at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, or at least 7 times compared to the pre-administration level after administration of cyclophosphamide at a dose between 200 mg / m² and 2000 mg / m² (e.g., 200 mg / m², 300 mg / m², 400 mg / m², 500 mg / m², 600 mg / m², 700 mg / m², 800 mg / m², 900 mg / m², 1000 mg / m², or 1110 mg / m²) and fludarabine at a dose between 20 mg / m² and 900 mg / m² (e.g., 20 mg / m², 25 mg / m², 30 mg / m², 35 mg / m², 40 mg / m², 45 mg / m², or 50 mg / m²). At least 8 times, at least 9 times, at least 10 times, at least 15 times, or at least 20 times. In some embodiments, the serum PLGF level in the patient increases by at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times after administration of cyclophosphamide and fludarabine compared to the serum PLGF level before administration. In some embodiments, the serum CRP level in the patient increases by at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least about 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times after administration of cyclophosphamide and fludarabine compared to the serum IP-10 level before administration. In other embodiments, the serum IP-10 level in the patient increases by 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 20 times, or at least 30 times after administration of cyclophosphamide and fludarabine compared to the serum IP-10 level before administration.

[0218] The following embodiments are provided to further illustrate the present invention and should not be construed as limiting the invention further. All references cited in this application are expressly incorporated herein by reference.

[0219] [Example]

[0220] Example 1

[0221] Design a phase 1 / 2, single-arm, open-label clinical trial to determine the safety and feasibility of administering anti-CD19 CAR+ T cells to individuals with B-cell malignancies.

[0222] Individuals who signed informed consent and met the study eligibility criteria participated in the study and underwent leukocyte ablation to obtain PBMCs for the production of anti-CD19 CAR+ T cells. Individuals treated with conditioning chemotherapy prior to admission were prepared for a single infusion of anti-CD19 CAR+ T cells on day 0. Then, 3 hours after the anti-CD19 CAR+ T cell infusion, some individuals (Group 1 only) were treated with interleukin-2. If a partial response (PR) or complete response (CR) occurred after the first infusion followed by progressive disease, a second dose of anti-CD19 CAR+ T cells was administered.

[0223] Three groups of individuals participated. Group 1 consisted of 8 individuals (one of whom had undergone retreatment), who were administered anti-CD19 CAR+ T cells ranging from 3 × 10⁶ to 30 × 10⁶ cells / kg. The conditioning regimen following anti-CD19 CAR+ T cell administration consisted of a high dose of cyclophosphamide for two days at 60 to 120 mg / kg (2220 to 4440 mg / m²), followed by fludarabine for five days at 25 mg / m². Following anti-CD19 CAR+ T cell administration, these individuals also received a high dose of interleukin-2 (IL-2) at 720,000 IU / kg (every 8 hours until 15 doses or until toxicity prevents further administration) to stimulate its proliferation.

[0224] Group 2 consisted of 15 individuals (2 of whom were from Group 1 and had undergone retreatment), who received varying doses of anti-CD19 CAR+ T cells, ranging from 1×10⁶ to 5×10⁶ anti-CD19 CAR+ T cells / kg, followed by high doses of cyclophosphamide and fludarabine, but without interleukin-2.

[0225] Group 3 consisted of 11 individuals who received a reduction conditioning regimen of 300 mg / m² cyclophosphamide and 30 mg / m² fludarabine, administered for 3 comorbidity days without IL-2. The first 7 and last 4 of these individuals received anti-CD19 CAR+ T cell infusions of 1 × 10⁶ and 2 × 10⁶ anti-CD19 CAR+ T cells, respectively.

[0226] Demographics

[0227] Individual demographic characteristics and disease traits are shown in Table 1. Thirty-two individuals participated, of whom 19 (59%) had DLBCL or PMBCL, 7 (22%) had CLL, and 6 (19%) had other indolent NHLs (including indolent follicular lymphoma and splenic marginal zone lymphoma). The majority of individuals had refractory disease (84%) and a median of three prior first-line treatments. All individuals with aggressive NHL had previously received anti-CD20 therapy, platinum-based chemotherapy, and 95% had previously received anthracycline-based chemotherapy.

[0228] Pharmacokinetics

[0229] The number of anti-CD19 CAR+ T cells in peripheral blood at various time points after the initial administration on day 0 was assessed using qPCR analysis, and confirmed by a standard curve generated by flow cytometry using an antibody reagent specific to 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 cimeric-antigen-receptor-transduced T cells," Blood 119:2709-20 (2012)).

[0230] In Group 1, infusions ranged from 3 × 10⁶ to 30 × 10⁶ anti-CD19 CAR+ T cells / kg. In the first six individuals, circulating anti-CD19 CAR+ T cells were detected at high levels within 2 weeks post-infusion, reaching up to 0.02% to 1% of total PBMCs, before rapidly declining and becoming undetectable after 50 days. Individuals 7 and 8, who received the highest doses of anti-CD19 CAR+ T cells (28 × 10⁶ and 30 × 10⁶ anti-CD19 CAR+ T cells / kg, respectively), exhibited a higher peak percentage of anti-CD19 CAR+ T cells reaching >10% of total PBMCs and longer-term persistence of anti-CD19 CAR+ T cells in the blood (>130 and 180 days, respectively).

[0231]

[0232] In group 2 (without interleukin-2 treatment), anti-CD19 CAR+ T cells showed similar proliferation in peripheral blood within 2 weeks, followed by decline and complete disappearance from circulation within a few weeks (Table 2).

[0233] In summary, there is no clear relationship between the dose of anti-CD19 CAR+ T cells and their proliferation and persistence in peripheral blood. Similarly, to date, there is no clear relationship between the dose of anti-CD19 CAR+ T cells, their proliferation or persistence in the blood, and clinical response or toxicity associated with this treatment.

[0234]

[0235] In groups 1 and 2, anti-CD19 CAR+ T cells did not undergo secondary proliferation following the first phase of proliferation 7–14 days post-infusion. To date, there is no evidence that oncogenic transformation in subjects is attributable to genomic insertion of CAR-expressing retroviruses. Results for group 3 were not available at the data cutoff.

[0236] efficacy

[0237] Clinicians assessed safety in 32 individuals and efficacy in 29 individuals. The overall response rate for the 29 individuals for whom efficacy was evaluable was 76%. Of the 29 individuals, 11 (38%) achieved complete response (CR) and 11 out of 29 individuals (38%) achieved partial response (PR) (Figures 2A and 2B; Table 3).

[0238] Of the 29 evaluable individuals, 16 (55%) maintained a response since their first treatment, and 12 individuals (including those who had been retreated) had a response that lasted for more than one year (Table 3). Three responsive individuals were retreated after progression, and all of them maintained a response (17.4 months to more than 52.2 months).

[0239] As indicated in Table 3, 17 out of 19 individuals with refractory aggressive DLBCL / PMBCL had evaluable disease responses (1 individual was not evaluable; 1 individual had not yet been evaluated). Of these 17 individuals, 11 (65%) had a response, and 6 out of 17 (35%) achieved complete remission (CR). The median duration of response was 7.3 months.

[0240] Of the 7 evaluable individuals with CLL, 6 (86%) responded, and 4 out of 7 (57%) achieved complete remission (CR) (Table 3). The median duration of response was 22.2 months, and 4 out of 7 (57%) remained responsive, including 3 who maintained a response for more than 27 months (Table 3).

[0241]

[0242] Of the 5 evaluable individuals with indolent NHL, 5 (100%) responded, and 1 / 5 (20%) achieved complete remission (CR). The median duration of response was 18.8 months (Table 3). Five individuals (5 / 5; 100%) maintained a response, with 2 of them responding for more than 45 months (Table 4).

[0243] Security

[0244] Adverse events

[0245] Thirty-two individuals were treated with anti-CD19 CAR+ T cells, and no adverse events were reported by the time the last individual was treated. The overall safety summary includes all 32 treated individuals. The summary for the cohort includes safety data from two separate events for individuals 1010003 and 1010004: one when these individuals were treated in group 1 and the second when these individuals were treated in group 2 (re-treated with anti-CD19 CAR+ T cells).

[0246] Summary of adverse events

[0247] Please refer to Table 4 for a summary of adverse events. Overall, 31 individuals (97%) experienced any adverse event, 0 individuals (0%) experienced the worst grade 3, 29 individuals (91%) experienced the worst grade 4, and 2 individuals (6%) experienced fatal adverse events. 20 individuals (63%) experienced anti-CD19 CAR+ T cell-related adverse events; 6 individuals (19%) experienced the worst grade 3, 8 individuals (25%) experienced the worst grade 4, and no individuals experienced a grade 5 event. 16 individuals (50%) experienced serious adverse events; 3 individuals (9%) experienced the worst grade 3, 9 individuals (28%) experienced the worst grade 4, and 2 individuals (6%) experienced the worst grade 5.

[0248]

[0249] Dose-limiting toxicity

[0250] The incidence of DLT in groups 1, 2, and 3 was 38%, 40%, and 0%, respectively. Apart from individual 1010002, DLT was primarily neurotoxicity, with two cases of hypercreatinine and one case of hypoxia and hypotension. Table 6 provides a list of DLTs. No DLT was reported in group 3. The conditioning regimen in group 3 was studied using 2 × 10⁶ anti-CD19 CAR+ T cells / kg.

[0251]

[0252] Cytokine release syndrome

[0253] Upon binding to the CD19 target, activated T cells induce cytokine release. Using a broad search strategy, sudden adverse events attributable to CRS during treatment included fever, febrile neutropenia, hypotension, acute vasoperfusion syndrome, hypercreatinine, renal failure, hypoxia, and pleural effusion. 28 individuals (88%) reported adverse events attributable to cytokine release, of which 24 individuals (75%) reported one. Grade 3 events, and 6 individuals (19%) experienced serious events. Adverse events caused by co-treatment (e.g., IL-2 (used in Group 1) and modulating chemotherapy (causing febrile hyponeutropenia)) may confound this analysis.

[0254] Clinical manifestations of CRS typically appear within the first week after anti-CD19 CAR+ T-cell infusion, but are less common in Group 3 individuals. Only one of the eleven individuals in Group 3 experienced Grade 3 hypotension, and four experienced Grade 3 fever. Acute vascular leakage syndrome, oliguria, hypercreatinine, and renal failure events were reported only in Groups 1 and 2.

[0255] Neurological adverse events

[0256] Neurological adverse events were observed in all three groups, primarily aphasia / speech disorders, confusion, motor neuropathy, and somnolence. Thirteen individuals (41%) experienced severe adverse events. Grade 3 neurotoxicity, and 11 individuals (34%) experienced serious events.

[0257] Individuals who died from neurotoxicity had CNS cerebral ischemia events in the context of viral influenza A infection. Researchers believe this event was unrelated to anti-CD19 CAR+ T cells.

[0258] Regarding neurotoxic adverse events, five individuals (16%) with neurotoxic events required mechanical ventilation to protect their airways; all of these individuals were in groups 1 and 2. No individuals in group 3 were intubated.

[0259] Neurological adverse events had a median onset time of 6 days between days 2 and 17 following anti-CD19 CAR+ T-cell infusion (except for one individual who developed grade 4 myelitis), and onset on day 110 following infusion. Considering the onset time, presentation, and brain MRI findings, the researchers concluded that the events were related to fludarabine rather than attributed to the anti-CD19 CAR+ T-cells. The median time for neurological adverse events to resolve to grade 1 or better was 14 days post-infusion.

[0260] die

[0261] Two individuals died within 30 days of receiving chemotherapy and anti-CD19 CAR+ T-cell infusion. Individual 2 died 18 days after treatment in the study due to cerebral infarction complicated by viral pneumonia, influenza A infection, E. coli infection, dyspnea, and hypoxia. Individual 11, suffering from PMBCL complicated by extensive fibrotic mediastinal lymphoma, died 16 days after treatment in the study. The cause of death was not determined at the autopsy, and the autopsy report concluded that the cause of death was likely arrhythmia and PMBCL invasion of the mediastinum. The researchers concluded that none of the events were related to anti-CD19 CAR+ T-cell infusion.

[0262] Example 2

[0263] Selected patients were given a conditioning therapy consisting of cyclophosphamide at 300 mg / m² / day and fludarabine at 30 mg / m² / day. This conditioning therapy was administered for three days, from day -5 to day -3. On day 0, the first subgroup of patients (patients 22–28) (Table 6) received freshly produced anti-CD19 CAR+ T cells on day 10, and the second subgroup of patients (patients 29–32) received cryopreserved anti-CD19 CAR+ T cells on day 6.

[0264]

[0265] Patient serum was tested using Luminex with a Millipore HCD8MAG15K17PMX kit (T1, T2, immunomodulatory cytokines, chemokines, and immune effectors). The following levels were measured before and after treatment: interleukin-15 (IL-15), monocyte chemoattractant protein-1 (MCP-1), γ-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).

[0266] Of patients 22–28, patients 22–25 and 27 showed at least a partial response, and patients 26 and 28 showed progressive disease after treatment. In patients 22–26, IL-15, MCP-1, and PLGF levels showed at least some increase in serum (Figs. 4A, 4B, and 4D), while IP-10, sICAM-1, CRP, VEGF-D, and MIP-1β levels increased in some patients and remained stable or decreased in the rest (Figs. 4C and 4E–4H). Only IL-15 was measured in patients 27 and 28 (Fig. 4A).

[0267] Some differences in marker levels were observed between responding patients (partial or complete response) and non-responding patients (with progressive disease). Relative to baseline, IL-15 levels increased by an average of approximately 35-fold in responding patients (ranging from approximately 10-fold to approximately 55-fold), while in non-responding patients, IL-15 levels increased by less than approximately 10-fold (Figure 5A). MCP-1 levels increased by an average of approximately 5-fold in responding patients (ranging from approximately 2-fold to approximately 7-fold), while in non-responding patients (patient 26), MCP-1 levels increased by less than 4-fold (Figure 5B). IP-10 levels increased by an average of approximately 3.5-fold in responding patients (ranging from approximately 2-fold to approximately 7-fold), while serum IP-10 levels remained substantially unchanged in non-responding patients (Figure 5C). PLGF levels increased by an average of approximately 30-fold in responding patients (ranging from a slight increase to approximately 2-fold or less to an increase of more than 100-fold), while serum PLGF levels in non-responding patients showed only a slight increase (Figure 5D). sICAM-1 levels increased by an average of approximately 3-fold in responding patients (ranging from virtually no change to an increase of approximately 4.5-fold), while serum sICAM-1 levels in non-responding patients showed virtually no change (Figure 5E). CRP levels increased by an average of approximately 10-fold in responding patients (ranging from virtually no change to an increase of approximately 25-fold), while serum CRP levels in non-responding patients showed virtually no change (Figure 5F). VEGF-D levels increased by an average of approximately 3-fold in responding patients (ranging from virtually no change to an increase of approximately 6-fold), while serum VEGF-D levels in non-responding patients showed virtually no change (Figure 5G). Serum levels of MIP-1 β increased by an average of about 1.5 times in responding patients (ranging from virtually no change to an increase of about 3 times), while serum levels of MIP-1 β decreased by about 50% in non-responding patients (Figure 5H).

[0268] Patients aged 30 to 33 were administered cryopreserved cells prepared 6 days prior. The following quantities were measured on selected days 6 to 18: various inflammatory cytokines, chemokines, effectors, inflammatory markers, and adhesion molecules, including granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ 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, and MIP-1. β, serum granzyme A (GRNZA), serum granzyme B (GRNZB), PLGF, CRP, mononuclear globulin-4 (MCP-4), interleukin-16 (IL-16), thymus and activated regulatory chemokine (TARC), eotaxin-3, sICAM-1, soluble vascular adhesion molecule-1 (sVCAM-1), and serum amyloid A (SAA) (Figures 6A to 6V).

[0269] Example 3

[0270] To improve the depth and duration of lymphocyte depletion observed in Group 3 of Example 1, the conditioning chemotherapy dose in Group A1 was increased to 500 mg / m² of cyclophosphamide and 30 mg / m² of fludarabine, along with a target dose of 2 × 10⁶ anti-CD19 CAR+ T cells / kg administered over 3 days. The cyclophosphamide dose used in this regimen (Group A1) was approximately 38% lower than the 30 mg / kg cyclophosphamide conditioning regimen in Group 2 of Example 1 (dose-limiting toxicity rate of 29%), while the fludarabine dose was as low as in Group 3 of Example 1.

[0271] Higher modulated chemotherapy doses and / or varying anti-CD19 CAR+ T cell doses were evaluated based on DLT incidence and benefit-risk assessment. The CAR vector construct was identical to that described in Example 1. This example describes an embodiment designed to evaluate the safety and efficacy of anti-CD19 CAR+ T cells generated using a rapid, closed, and bead-less procedure. Closing the procedure preserves the characteristics of the T cell product.

[0272] [Research Design]

[0273] We are conducting a phase 1 / 2 multicenter, open-label clinical trial to evaluate the safety and efficacy of KTE-C19 in individuals with refractory NHL. This study will be divided into two phases, designated Phase 1 and Phase 2.

[0274] During Phase 1, approximately 6 to 24 individuals with DLBCL, PMBCL, or TFL participated to evaluate the safety of the KTE-C19 protocol. The study organizer's internal Safety Review Team (SRT) reviewed the safety data and made recommendations for further research and progress in Phase 2, as shown in Figure 3.

[0275] During Phase 2, individuals participated in two distinct groups designated Group 1 and Group 2. Group 1 included adult individuals with refractory DLBCL, and Group 2 included adult individuals with refractory PMBCL and TFL. TFL was defined as individuals who had previously received chemotherapy for follicular lymphoma.

[0276] Regardless of the study phase, all experiences followed the same study course and procedures. Each experience underwent the following study periods: screening / leukocyte separation; conditioning chemotherapy; treatment with the investigational product (IP); post-treatment evaluation; and long-term follow-up.

[0277] During the research period

[0278] For each individual, the participation period includes a 28-day screening period, a 5-7 day conditioning chemotherapy treatment period, a KTE-C19 treatment period (including a 7-day in-hospital recovery period), a post-treatment assessment period, and a long-term follow-up period (survival monitoring for up to 15 years).

[0279] All adverse events will be tracked for 3 months post-treatment. After 3 months, the individual will be monitored periodically for target / serious adverse events (e.g., hematologic disorders, neurological disorders, secondary malignancies, infections, or autoimmune diseases) and replicating retroviruses (RCRs) in the individual's blood, as outlined in the Society of Assessment (SOA). Extended follow-up may be requested based on the potential persistence of the gene transfer vector in the treated individual.

[0280] Study completion was defined as the last individual completing a follow-up visit during the long-term follow-up period, being deemed lost to follow-up, withdrawing consent, or dying. The primary analysis will be conducted when all individuals in Group 1 of Phase 2 and the entire study population have completed the 6-month disease response assessment, lost to follow-up, withdrawn from the study, or died (whichever occurs first).

[0281] Individual Qualification

[0282] Inclusion criteria for individuals include:

[0283] a) Histological examination confirms aggressive B-cell NHL, including the following types as defined by WHO 2008: DLBCL (unless otherwise specified), large B-cell lymphoma rich in T cells / histocytes, chronic inflammation-associated DLBCL, elderly patient with Estane-Barr virus (EBV) + DLBCL, primary mediastinal (thymic) large B-cell lymphoma, or follicular lymphoma transformed into DLBCL;

[0284] b) Chemotherapy-resistant disease is defined as one or more of the following: stable disease that responds best to regimens containing the latest chemotherapy (during the stable disease period must be...) (12 months) or progressive disease; and disease progression or recurrence of previous autologous SCT. 12 months;

[0285] c) The individual must have previously received appropriate treatment (at least including anti-CD20 monoclonal antibodies, unless the researcher determines the tumor is CD20 negative) and chemotherapy regimens containing anthracyclines;

[0286] d) Individuals with transformed FL who previously underwent chemotherapy for follicular lymphoma and subsequently developed chemotherapy-resistant disease after transformation into DLBCL;

[0287] e) At least one measurable lesion (according to the modified IWG Response Criteria for Malignant Lymphoma); a previously irradiated lesion is considered measurable only if its progression has been documented after completion of radiotherapy.

[0288] f) Brain MRI showed no evidence of central nervous system lymphoma;

[0289] g) When performing individualized white blood cell separation, at least two weeks must have elapsed since any previous radiation therapy or systemic treatment;

[0290] h) Toxicity caused by previous treatment must be stable or return to normal. Category 1 (excluding clinically insignificant toxicities (e.g., hair loss));

[0291] i) Individuals must be at least 18 years old;

[0292] j) An East Coast Cancer Clinical Research Partnership (ECOG) daily physical fitness assessment score of 0 or 1;

[0293] k) Individuals must have the following laboratory values: i) ANC 1000 / μL; ii) Platelet count 50,000 cells / μL; iii) adequate kidney, liver, and heart function, defined as serum creatinine. 1.5 mg / dL, serum ALT / AST 2.5 ULN, with total bilirubin 1.5 mg / dl, except for individuals with Gilbert's syndrome; and iv) left ventricular ejection rate 50% and no evidence of pericardial effusion (measured using ECHO assay); and

[0294] l) Women of childbearing age must have a negative serum or urine pregnancy test.

[0295] Exclusion criteria for individuals include:

[0296] a) Unless there is a history of skin cancer or carcinoma in situ (e.g., cervical cancer, bladder cancer, breast cancer) or malignancy other than follicular lymphoma, or unless the disease has been absent for at least 3 years;

[0297] b) History of Richter's conversion in CLL;

[0298] c) Autologous stem cell transplantation within 6 weeks of signing the informed consent form;

[0299] d) History of allogeneic stem cell transplantation;

[0300] e) Previous CD19 targeted therapy (except for individuals who received KTE-C19 in this study and are eligible for retreatment);

[0301] f) Previous chimeric antigen receptor therapy or other genetically modified T-cell therapy;

[0302] g) A history of severe immediate hypersensitivity reactions attributed to aminoglycosides;

[0303] h) Clinically significant active infection (e.g., uncomplicated UTI, bacterial pharyngitis allowed) or currently receiving IV antibiotics or having received IV antibiotics within 7 days prior to participation (prophylactic antibiotics, antiviral drugs, and antifungal drugs allowed);

[0304] i) A known history of infection with HIV, hepatitis B virus (HBsAg positive), or hepatitis C virus (anti-HCV positive);

[0305] j) Individuals with detectable malignant tumor cells in the cerebrospinal fluid or brain metastases, or individuals with a history of malignant tumor cells in the cerebrospinal fluid or brain metastases;

[0306] k) History of epilepsy, cerebral ischemia / hemorrhagic disease, dementia, cerebellar disease, or any autoimmune disease complicated by CNS involvement;

[0307] l) Individuals with atrial or ventricular lymphoma involvement;

[0308] m) Emergency treatment is required if the tumor mass effect is present (e.g., bowel obstruction or vascular compression);

[0309] n) Primary immunodeficiency;

[0310] o) Any medical condition that is likely to hinder the assessment of the safety or efficacy of the research treatment;

[0311] Dosage and physiological replacement doses of topical and inhaled corticosteroids for individuals with adrenal insufficiency; doses of prednisone or other corticosteroids at or above 5 mg / day are not permitted;

[0312] q) A history of severe immediate allergic reaction to any of the medications used in this study;

[0313] r) Before the treatment plan begins 6-week live virus vaccine;

[0314] s) Women of childbearing age who are pregnant or breastfeeding are not considered fertile due to the potential dangers of this preparatory chemotherapy to the fetus or infant; women who have undergone sterilization or have been menopausal for at least 2 years are not considered fertile.

[0315] t) Individuals who do not wish to undergo sterilization from the date of signing the consent form until six months after the completion of KTE-C19; and

[0316] u) Researchers determine that an individual is unlikely to complete all required research follow-ups or procedures, including follow-up visits or compliance with research participation requirements.

[0317] In addition, biomarker analysis will be performed on blood and tumor samples to evaluate predictive and efficacy indicators for KTE-C19. Prognostic indicators in aggressive NHL can also be assessed. Baseline leukocytes will be isolated and accumulated with final KTE-C19 samples, and analysis can be performed using immunophenotyping and / or gene expression profiling. Remaining samples can be stored for future exploratory analyses of DNA, RNA, or protein markers. Archived tumor tissue will be used as the basis for central pathway review. Additional analyses may include CD19 expression, gene expression profiling, and DNA variation analysis. Remaining tumor samples can be stored for future exploratory analyses of DNA, RNA, or protein markers.

[0318] [Treatment Procedure]

[0319] Scheduling

[0320] Leukocytes were obtained from individuals using leukocyte ablation (12 to 15 liters of blood ablated, targeting approximately 5 x 10⁹ to 10 x 10⁹ monocytes for KTE-C19 production). The ablated leukocyte products from each individual were processed to concentrate a PBMC population containing T cells. The T cells were then stimulated to proliferate and transduced via a retroviral vector to introduce the CAR gene. The T cells were then proliferated and cryopreserved to produce the investigational drug. After completing the individual's conditioning chemotherapy regimen, each individual received a KTE-C19 infusion.

[0321] Study course of treatment

[0322] Individuals will receive a non-myelodestructive conditioning regimen (consisting of cyclophosphamide and fludarabine) to induce lymphocyte depletion and create a favorable environment for KTE-C19 proliferation. The conditioning chemotherapy regimen of cyclophosphamide and fludarabine will be initiated on day -5 (or day -7 for group B) to day -1. This 5-day conditioning chemotherapy regimen is administered on an outpatient basis. The 7-day conditioning chemotherapy regimen may be administered on an outpatient or inpatient basis, at the researcher's discretion.

[0323] Issue 1:

[0324] In groups A1 and A2, individuals will receive the following 5-day conditioning chemotherapy regimen: 1 L of 0.9% NaCl saline will be administered intravenously before cyclophosphamide administration on the infusion day; followed by 500 mg / m² cyclophosphamide intravenously over 60 minutes on days -5, -4, and -3; followed by 30 mg / m² fludarabine intravenously over 30 minutes on days -5, -4, and -3; and finally, 1 L of 0.9% NaCl saline will be administered after the fludarabine infusion is completed (Figure 3). In some cases, mesna (sodium 2-mercaptoethanesulfonate) may be added according to hospital guidelines.

[0325] In group A3, individuals will receive the following 5-day chemotherapy regimen: 1 L of 0.9% NaCl saline will be administered intravenously before cyclophosphamide administration on the infusion day; followed by 300 mg / m² cyclophosphamide intravenously over 60 minutes on days -5, -4, and -3; followed by 30 mg / m² fludarabine intravenously over 30 minutes on days -5, -4, and -3; and finally, 1 L of 0.9% NaCl saline will be administered after the fludarabine infusion is completed. In some cases, mesna may be added according to hospital guidelines.

[0326] For individuals participating in groups A1, A2, or A3, the -2 and -1 days prior to the KTE-C19 infusion (day 0) are rest days.

[0327] In groups B1 and B2, individuals will receive the following 7-day chemotherapy regimen: intravenous infusion of 0.9% NaCl saline, recommended at 2.6 ml / kg / hr (maximum 200 ml / hr), administered continuously from 11 hours before the last cyclophosphamide infusion until 24 hours after the last cyclophosphamide infusion; intravenous infusion of 30 mg / kg (1110 mg / m2) cyclophosphamide over 120 minutes on days -7 and -6; followed by intravenous infusion of 25 mg / m2 fludarabine over 30 minutes on days -5, -4, -3, -2, and -1. In some cases, mesna may be added according to hospital guidelines.

[0328] For individuals participating in group B1 or B2, there is no rest day between the last day of chemotherapy (day -1) and the KTE-C19 infusion (day 0).

[0329] For KTE-C19, individuals in groups A1, A3, or B1 will receive KTE-C19 treatment consisting of: a single intravenous infusion of CAR-transferred autologous T cells at a target dose of 2 × 10⁶ anti-CD19 CAR+ T cells / kg (±20%; from 1.6 × 10⁶ anti-CD19 CAR+ T cells / kg to 2.4 × 10⁶ anti-CD19 CAR+ T cells / kg). A minimum dose of 1 × 10⁶ anti-CD19 CAR+ T cells / kg may be administered. For individuals weighing over 100 kg, a maximum flat dose of 2 × 10⁸ anti-CD19 CAR+ T cells / kg should be administered.

[0330] Individuals in groups A2 or B2 will receive KTE-C19 treatment consisting of: a single intravenous infusion of CAR-transferred autologous T cells, with a target dose of 1 × 10⁶ anti-CD19 CAR+ T cells / kg (±20%; from 0.8 × 10⁶ anti-CD19 CAR+ T cells / kg to 1.2 × 10⁶ anti-CD19 CAR+ T cells / kg). A minimum dose of 0.5 × 10⁶ anti-CD19 CAR+ T cells / kg may be administered. For individuals weighing over 100 kg, a maximum flat dose of 1 × 10⁸ anti-CD19 CAR+ T cells / kg should be administered.

[0331] Issue 2:

[0332] The KTE-C19 protocol, which was determined to be safe by SRT in Phase 1, will be transferred to Phase 2 of this study.

[0333] Retreatment

[0334] Individuals who achieve PR or CR may receive a second course of conditioning chemotherapy and KTE-C19 therapy if they subsequently develop disease (and the recurrence has not yet been confirmed as C19 cancer cells). To be eligible for a second course, individuals should be re-evaluated and continue to meet the original study eligibility criteria (except for exclusion criteria related to previous CAR therapy), and should not have received subsequent chemotherapy for lymphoma. Additionally, any toxicities related to fludarabine or cyclophosphamide should be stable and return to grade 1 or below (except for hair loss) before retreatment. An individual may receive a maximum of one retreatment course. Individuals participating in Phase 2 will receive the same KTE-C19 regimen. Individuals participating in Phase 1 will receive the KTE-C19 regimen selected for Phase 2. If the Phase 2 regimen has not yet been selected, the individual will receive the last KTE-C19 regimen determined to be safe by SRT.

[0335] Individuals experiencing DLT in stage 1 or comparable toxicity in stage 2 are ineligible for retreatment. Additionally, individuals with known neutralizing antibodies are ineligible for retreatment. However, individuals who develop non-neutralizing HAMA or HABA antibodies may be retreated if they meet the eligibility criteria.

[0336] Post-treatment assessment

[0337] Following completion of KTE-C19 infusion and discharge (typically on day 8), all individuals will be followed up during post-treatment evaluation. Starting from day 0 (KTE-C19 infusion), individuals will return for follow-up visits at week 2, week 4 (±3 days), month 2 (±1 week), and month 3 (±1 week). Evaluations may include the Mini-Mental State Examination (MMSE); PET-CT for disease assessment; physical examination and vital signs; laboratory medicine, including Chemistry Panel, CBC with differential, β-HCG pregnancy test (serum or urine) for all women of childbearing age, anti-KTE-C19 antibody, lymphocyte subsets, cytokine levels, anti-CD19 CAR+ T cells, and replication-promoting retrovirus (RCR) analysis; adverse event / serious adverse event reporting; preparation of concomitant medication documentation; and collection of fresh tumor samples from individuals who have signed consent forms (optional).

[0338] First, PCR analysis (in conjunction with flow cytometry) was used to monitor the presence, proliferation, persistence, and immunophenotype of transduced anti-CD19 CAR+ T cells in the blood. Serum levels of cytokines in the blood were also assessed. The following cytokines may be included in the sample: pro-inflammatory and immunomodulatory cytokines IL-6, TNF-α, IL-8, IL-1, IL-2, GM-CSF, IL-15, IL-17a, IFN-γ, and IL-12p40 / p70; immune effector molecules granzymes A and B, perforin, and sFasL; acute-phase response CRP and SAA, and chemokines MIP-1α, MIP-3α, IP-10, eosin, and MCP-4. When KTE-C19 contains T cells transduced with a retroviral vector, the presence of replicating retroviruses (RCRs) in the blood of the treated patients was also monitored.

[0339] If an individual is eligible for KTE-C19 retreatment, the last scan prior to retreatment will be used as the baseline for assessing the response to retreatment.

[0340] At any point during the post-treatment assessment period, if an individual does not improve after treatment (i.e., CR or PR) or progresses after a response, the individual proceeds directly to the 3-month follow-up and is tracked for disease outcomes during the long-term follow-up period.

[0341] Survival and disease status of all individuals will be tracked during the long-term follow-up period, if appropriate. Long-term follow-up will begin after the 3-month follow-up visit following the completion of the post-treatment assessment period (regardless of whether they have improved after treatment or proceed directly to the 3-month follow-up due to progressive disease). From day 0 (KTE-C19 infusion) to month 18, individuals will return every 3 months (±2 weeks); from month 24 to month 60, every 6 months (±1 month); and from year 6 (month 72) (±3 months) to year 15, individuals will return annually. During this follow-up visit, the following procedures will be completed: physical examination; PET-CT scan; disease assessment; laboratory medicine, including CBC with differential, anti-KTE-C19 antibody, lymphocyte subsets, anti-CD19 CAR+T cells, and RCR analysis; reporting of target adverse events / serious adverse events (within 24 months or up to the progression of the disease, whichever comes first), including neurological disorders, hematologic disorders, infections, autoimmune diseases, and secondary malignancies; and documentation of target concomitant medications (within 2 years of the progression of the disease), including gamma globulin, immunosuppressants, anti-infective agents, vaccinations, and any therapies used to treat the progression of the disease.

[0342] The evaluation will include PET-CT scans of the neck, chest, abdomen, and pelvis, along with appropriate imaging of all other areas affected by the disease. Individuals will receive their first planned PET-CT tumor evaluation 4 weeks after KTE-C19 infusion and as described above, following regular KTE-C19 infusions.

[0343] Bone marrow biopsy should be performed on individuals being evaluated for complete remission (CR). According to the revised IWG Response Criteria for Malignant Lymphoma, bone marrow biopsy should only be performed if the individual had pre-existing lymphoma invading the bone marrow prior to treatment, or if new abnormalities in peripheral blood cell counts or blood smears lead to a clinical diagnosis of lymphoma invading the bone marrow. The bone marrow biopsy must show no morphological evidence of disease, or, if morphologically indeterminate, must be immunohistochemically negative, in order to designate CR for treatment.

[0344] [End point of the study]

[0345] Primary endpoint

[0346] The primary endpoint for Phase 1 was the occurrence of adverse events (defined as dose-limiting toxicities (DLT)). The primary endpoint for Phase 2 was the objective response rate (ORR), defined as the rate of complete or partial response as determined by the investigator according to the modified IWG Response Criteria for Malignant Lymphoma. All individuals who did not meet the objective response criteria by the analysis cutoff date were considered non-responders.

[0347] Secondary endpoint

[0348] Objective response rates were summarized for Phase 1 individuals. Objective response rates for Phase 2 individuals were determined by the IRRC and defined as the incidence of a complete or partial response as determined by the IRRC according to the modified IWG Response Criteria for Malignant Lymphoma. All individuals who did not meet the objective response criteria before the analysis cutoff date were considered non-responders. The time to treatment response (DOR) for individuals experiencing an objective response was defined as the date of that individual's first objective response or death (regardless of cause), which was subsequently identified as a progressive disease according to the modified IWG Response Criteria for Malignant Lymphoma. Individuals who did not meet the progression criteria or died before the data analysis cutoff date were reviewed and their responses were expressed as progression on their last evaluable disease assessment date.

[0349] Dose-limiting toxicities (DLT)

[0350] Dose-limiting toxicity is defined as the following KTE-C19-related events that occur within 30 days of KTE-C19 infusion:

[0351] a) Grade 4 neutropenia lasting more than 21 days from the date of cell transfer.

[0352] b) Grade 4 thrombocytopenia lasting more than 35 days from the date of cell transfer.

[0353] c) Any KTE-C19-related adverse event requiring intubation (including a Grade 4 disorder requiring intubation to protect the airway) is considered DLT.

[0354] d) All other Grade 3 toxicities lasting more than 3 days and all Grade 4 toxicities, except for the following conditions not considered DLT: i) Aphasia / speech disorder or confusion / cognitive impairment that resolves to Grade 1 or less within 2 weeks and to baseline within 4 weeks; ii) Grade 3 fever; iii) Myelosuppression (including bleeding with a platelet count of less than 50 × 10⁹ / L and confirmed bacterial infection with a neutropenia condition), defined as lymphopenia, hemoglobin deficiency, neutropenia, or thrombocytopenia, unless neutropenia and thrombocytopenia meet the above DLT definitions; iv) Immediate hypersensitivity reactions (related to cell infusion) occurring within 2 hours of cell infusion that can be reversed to Grade 2 or less within 24 hours of cell infusion with standard therapy; and v) Grade 3 or 4 hypogammaglobulinemia.

[0355] The CRS was classified according to the revised classification system (Lee 2014). Adverse events attributable to the CRS were mapped to the overall CRS classification assessment to determine DLT.

[0356] During Phase 1, approximately 6 to 24 individuals with DLBCL, PMBCL, or TFL participated to evaluate the safety of the KTE-C19 regimen. Individuals in each group were assessed for DLT within 30 days of completing their respective KTE-C19 infusions. If the incidence of DLT in 6 individuals was... If the result is 1, then the study can proceed to Phase B1 or Phase 2 of the trial. This decision is based on the overall benefit / risk ratio and available biomarker data.

[0357] However, if two out of six individuals develop DLT as defined by the procedure during Phase 1, the SRT may recommend adding two groups of three individuals each (up to a total of 12 individuals), administering the same dose as the initial six individuals. In this case, if nine individuals... 2 or if there are 12 individuals If three individuals exhibit DLT, the study progresses to another group or enters phase 2 of the study.

[0358] If the incidence of DLT in an individual is >2 / 6, >3 / 9, or >4 / 12, other KTE-C19 protocols can be explored in an additional 6 to 12 individuals (Figure 3). The same DLT rules apply as described above.

[0359] Example 4

[0360] Autologous lymphocytes were transduced via g-mouse retrovirus carrying the anti-CD19 CAR construct gene, and then proliferated to produce T cell products at the desired cell dose. The anti-CD19 CAR+ T cell products were evaluated by flow cytometry and multiplex cytokine analysis of the supernatant of the co-culture medium at collection or after co-culturing with CD19+ cells. Product characteristics of the CAR+ T cell co-culture medium were analyzed using K562-CD19 cells or K562-NGFR control group cells at a 1:1 efficacy-to-target cell ratio. The standard culture time was 18 hours. Patients with relapsed / refractory B-cell malignancies were treated with cyclophosphamide and fludarabine, followed by administration of anti-CD19 CAR+ T cells.

[0361] 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, the Human IL-7 Quantikine HS ELISA Kit (HS750) was used in purified samples according to manufacturer guidelines. The frequency of circulating CAR T cells was measured using quantitative PCR. Patients were administered a preconditioning regimen consisting of 300 mg / m² cyclophosphamide on days -5 and -4 and 30 mg / m² fludarabine on days -5, -4, and -3. Serum was collected from patients on the following dates: before administration of cyclophosphamide and fludarabine between days -12 and -5 (“before”), before administration of CAR+ T cells between day 0 (“after”), and selected days between CAR+ T cell administration and day 18. Serum concentrations of GM-CSF, IL-2, MCP-1, IL-6, IL-10, MCP-4, CRP, IFNγ, granzyme A, IL-15, IL-5, granzyme B, IL-8, IP-10, MIP-1b, PLGF, IL-16, TARC, eosinophil 3, sICAM-1, sVCAM-1, and SAA were measured before and after conditioning and on selected days after CAR+T cell administration, as shown in Figure 6. It was found that after conditioning with 300 mg / m² cyclophosphamide and 30 mg / m² fludarabine, the concentrations of certain cytokines in the patient's serum increased (Figures 7A to 7I and Figures 18A to 18E). In particular, after conditioning with cyclophosphamide and fludarabine, the concentrations of IL-15, IL-7, PLGF, CRP, and MCP-1 significantly increased (Figures 7A to 7D, 7G, 18A, and 18C to 18E). Increases in the concentrations of IL-5, IL-10, IP-10, and sICAM-1 were also observed (Figures 7E to 7F, 7H to 7I, and 18B). Conversely, perforin was found to decrease after conditioning with cyclophosphamide and fludarabine (Figure 18F). Increases or decreases in serum concentrations of various other analytes were observed after preconditioning, as shown in Figure 18G. Treatment of other patients and their results are shown in Figures 11 to 17. Furthermore, it was found that, after preconditioning, increased serum levels of IL-15 (Fig. 19A) and IP-10 (Fig. 19B) and decreased serum levels of perforin (Fig. 19C) were significantly associated with positive objective response in patients treated with CAR T-cell therapy.

[0362] Peripheral blood lymphocytes (PBLs) and serum levels after CAR+ T cell infusion were assessed using flow cytometry and multiplex cytokine analysis, respectively. Cytokine production in pre-infusion anti-CD19 CAR+ T cells was compared with that in the K562-NGFR negative control group (Figure 8). Anti-CD19 CAR+ T cell samples showed higher concentrations of homeostatic cytokines GM-CSF, IL-2, IFNγ, IL-5, IL-4, and IL-13, as well as pro-inflammatory cytokines and chemokines TNF-α, IL-6, granzyme B, MIP-1b(β), MIP-1a(α), and SCD137 at T1, T2, and immunologically stable levels compared to the negative control group (Figures 8A to 8L). Furthermore, binding of the target antigen to pre-infusion T cell products led to upregulation of modulotropic receptors (e.g., CD107a(α), 401BB, and PD-1) (Figures 9A to 9C).

[0363] Multicolor flow cytometry was performed on BD FACSCanto II (using FlowJo software for data acquisition and analysis). A shorter manufacturing process produced CAR+ T cell products with high expression of CD4+, naive, and central memory T cells (Figure 10). After infusion, the CAR+ T cells showed a diverse subset composition, mainly consisting of differentiated T cells and some central memory or naive T cells (Figure 10).

[0364] Anti-CD19 CD28ζ CAR+ T cells are clinically effective and induce sustained responses in lymphomas and leukemias. Sustained clinical responses can occur even without durable CAR+ T cells in circulation, allowing normal B cells to recover. Osteotherapy with cyclophosphamide and fludarabine modulates the immune environment by introducing molecules that promote the homeostatic proliferation, activation, and transport of T cells. CAR+ T cell therapy results in a rapid increase in circulating cytokines and chemokines within 3 weeks post-treatment, followed by a decline.

[0365] Example 5

[0366] The study aimed to evaluate the safety and efficacy of a non-myeloablative conditioning regimen in treating individuals, consisting of cyclophosphamide at a dose greater than or equal to 300 mg / m² and fludarabine at a dose greater than or equal to 30 mg / m². These conditioning agents were used to further induce lymphocyte depletion and create a more favorable environment for KTE-C19 proliferation in vivo.

[0367] Participants underwent leukocyte ablation to obtain PBMCs for the production of anti-CD19 CAR+ T cells. Individuals then received conditioning chemotherapy consisting of cyclophosphamide at 500 mg / m² / day and fludarabine at 60 mg / m² / day on days -5 to -3. On day 0, individuals received an intravenous dose of anti-CD19 CAR+ T cells / kg. Individuals received a starting dose of 2 x 10⁶ anti-CD19 CAR+ T cells / kg (±20%), which could then be increased or decreased based on individual responsiveness.

[0368] After conditioning chemotherapy and administration of anti-CD19 CAR+ T cells, individual side effects, serum cytokine levels, T cell counts, and disease response were monitored. The following serum levels were measured before and after conditioning to determine the efficacy of the conditioning chemotherapy: various inflammatory cytokines, chemokines, effectors, inflammatory markers, and adhesion molecules, including but not limited to 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, eosinophils, GM-CSF, IFNγ, IL-12p40, MDC, IL-12p70, IL-13, IL-17A, MIP-1a, TNFα, TNFb, granzyme A, granzyme B, perforin, SAA, MCP-4, and TARC. Serum was collected before or after administration of cyclophosphamide, fludarabine, and anti-CD19 CAR+ T cells, and all serum levels were compared with serum levels prior to conditioning chemotherapy. Disease responsiveness was compared with conditioning cytokine profiles for each patient to identify any association between disease responsiveness and conditioning levels of one or more cytokines.

[0369] Close monitoring of side effect incidence is necessary to determine the maximum tolerated dose of cyclophosphamide and fludarabine. Side effects may be medically controlled as needed. The dose of one or both of cyclophosphamide and fludarabine may be increased or decreased to improve clinical efficacy and limit side effects. Any individual exhibiting an initial partial response followed by progressive disease may receive a second treatment at the same or different doses of cyclophosphamide and / or fludarabine. In this patent application, various publications are cited in parentheses with the author's name and date, or patent number, or patent publication number. Full reference to these publications can be found at the end of the specification preceding the claims of the patent application. The disclosures of these publications are incorporated herein by reference in their entirety to more fully illustrate the state of the art known to those skilled in the art up to the date of invention described and claimed herein. However, the references cited herein should not be construed as an admission that such references are prior art to the present invention. The various forms, embodiments, and options described herein may be incorporated into any and all variations.

[0370] All publications, patents, or patent applications mentioned in this patent specification are incorporated herein by reference in such a manner as to be specifically and individually indicated to be incorporated herein by reference. However, the references cited herein should not be construed as an admission that such references are prior art to this invention.

[0371] The invention has now been summarized in general terms, and a further understanding can be obtained by referring to the embodiments provided herein. These embodiments are for illustrative purposes only and are not intended to be limiting.

Claims

1. The use of a chimeric antigen receptor (CAR)-T cell expressing an anti-tumor antigen CAR for manufacturing an agent for treating multiple myeloma cancer in patients who have been exposed to three daily doses of cyclophosphamide at 300 mg / m² / day and three daily doses of fludarabine at 30 mg / m² / day prior to CAR-T cell administration, wherein the tumor antigen is expressed in multiple myeloma cells, and wherein the serum IL-15 level of the patient was measured to be at least 5-fold increased on the day of CAR-T cell administration, but prior to CAR-T cell administration, compared to the serum level prior to cyclophosphamide and fludarabine administration on day 0.

2. As claimed in claim 1, wherein the therapeutically effective amount of the CAR-T cells administered is approximately 10⁴ cells, approximately 10⁵ cells, approximately 10⁶ cells, approximately 10⁷ cells, approximately 10⁸ cells, approximately 10⁹ cells, or approximately 10¹⁰ cells.

3. As claimed in claim 2, wherein the therapeutically effective dose of the anti-CAR-T cell is about 1 x 10⁶ or about 2 x 10⁶ cells / kg.

4. As requested in any of items 1 to 3, wherein cyclophosphamide and fludarabine are administered on days -5, -4, and -3 prior to CAR-T cell administration.

5. A method for predicting whether a patient will respond to CAR-T cell therapy for multiple myeloma cancer, wherein the CAR-T antigen is expressed in multiple myeloma cells and is included prior to CAR-T cell administration, after the patient has been exposed to three daily doses of cyclophosphamide at 300 mg / m² / day and three daily doses of fludarabine at 30 mg / m² / day, and the serum level of IL-15 in a serum sample is measured in vitro, wherein an increase in the serum level of IL-15 prior to administration of the CAR-T cell therapy but on the day of administration of the CAR-T cell therapy is an indication that the individual will respond to the CAR-T cell therapy, and wherein the increase in serum level is at least 5-fold greater than that of IL-15.

6. The method of claim 5, wherein the CAR-T cells are administered at a dose of about 100 x 10⁶ to about 300 x 10⁶ cells.

7. The method of any one of claims 5 to 6, wherein cyclophosphamide and fludarabine are administered on days -5, -4, and -3 prior to CAR-T cell administration.

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