Serum metabolomics in relation to chimeric antigen receptor (CAR) T cell therapy

Abstract

Methods and compositions are provided for using metabolomics to determine and improve the likelihood of response to cell therapy and / or to determine and reduce the likelihood of toxicity. [Solution] The embodiments of the present disclosure include methods for measuring certain metabolites in an individual's biological sample to determine whether the individual has a responsive or non-responsive phenotype to cell therapy or to the toxicity of cell therapy. The embodiments also include compositions for increasing the response to cell therapy or reducing the toxicity associated with cell therapy. The embodiments also show that plasma metabolomics correlates with efficacy and toxicity associated with CAR-T therapy for cancer. The present disclosure relates to compositions and methods for predicting a subject's response to CAR T cell therapy by analyzing the subject's plasma metabolomics. The present disclosure further provides therapeutic compositions and methods for treating a subject with cancer to improve the efficacy of CAR T cell therapy.

Description

[Technical field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under P30 CA016672 awarded by the NIH-NCI. The government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 185,412, filed May 7, 2021, and also claims priority to U.S. Provisional Patent Application Serial No. 63 / 257,621, filed October 20, 2021, both of which are incorporated by reference in their entireties herein.

[0003] Embodiments of the present disclosure encompass at least the fields of cell biology, molecular biology, immunology, and medicine. [Background technology]

[0004] Recently, considerable progress has been made in cancer treatment with effective and less toxic targeted strategies. Immunotherapy and chimeric antigen receptor (CAR) T cells are increasingly being evaluated in a variety of tumors in relapsed / refractory and frontline disease settings, primarily in hematological malignancies. Despite impressive therapeutic outcomes in some patients, there is still a large heterogeneity in clinical responses to CAR T cells. Metabolites have emerged as one of the key host factors that could potentially be modulated to enhance responses to immunotherapy as well as reduce the toxicity associated with immunotherapy. Several recent human studies evaluating immunotherapy strategies, such as immune checkpoint inhibitor treatment, have shown significantly better responses and survival in patients with a more diverse gut microbiota. Currently, it is unclear whether plasma metabolomics modulates antitumor responses and toxicity with respect to CAR T cells. There is a paucity of studies linking the role of blood metabolites to the outcome of CAR-T therapy. Summary of the Invention

[0005] The present disclosure relates to systems, compositions and methods that include metabolomics associated with various immunotherapies, such as adoptive cell therapy that includes immune cells expressing chimeric antigen receptors (CARs) or T cell receptors. Certain embodiments include methods for treating individuals and methods for reducing risk for individuals undergoing immunotherapy (such as CAR-related therapy). Certain embodiments include reducing the likelihood of toxicity of cell therapy. In some embodiments, the method includes measuring one or more compositions in an individual, including before undergoing therapy, during the period of receiving therapy, and / or after receiving therapy. The composition may include a biomarker, metabolite, or the like, and in some cases, one or more metabolites serve as biomarkers associated with efficacy and / or toxicity of therapy for an individual. In a specific embodiment, one or more metabolites serve as predictive biomarkers for outcomes associated with CAR-related therapy. In some embodiments, the metabolites include microbial-associated metabolites including indole and / or related indole derivatives, and trimethylamine oxide (TMAO), or non-microbial metabolites including 1-methylnicotinamide, nicotinamide, polyamines, asymmetric dimethylarginine, symmetric dimethylarginine, taurine, 6-phosphogluconic acid, triacylglycerol, N-alpha-L-acetyl L-asparagine, deoxycarnitine, or combinations thereof. Indole derivatives may include tryptophan, serotonin, indole-3-acetaldehyde, indoleacrylic acid, indoxyl sulfate, or combinations thereof. Polyamines may include diacetylspermine, N8-acetylspermidine, putrescine, spermidine, spermine, acetylputresceine, acetylspermidine, acetylspermine, N3AP, cadaverine, acetylcadaverine, diacetylcadaverine, or combinations thereof.

[0006] Certain embodiments of the present disclosure relate to methods for measuring compositions (such as, for example, one or more metabolites) in any biological sample from an individual. In some embodiments, the sample comprises a blood sample. The blood sample may comprise any of the components of blood, including whole blood, serum, and / or plasma. In certain embodiments, measuring one or more metabolites provides a measurement indicating whether an individual will have toxicity in response to a CAR-associated therapy. In certain embodiments, measuring one or more metabolites provides a measurement indicating whether an individual will have an effective therapeutic response to a CAR-associated therapy. In some embodiments, measuring the composition identifies an individual as having a responder phenotype or as having a non-responder phenotype. Also included are embodiments in which the measurement indicates that a treatment should be adjusted for the individual, including adjusting the level of one or more metabolites in the individual, or increasing the level of one or more metabolites in the individual. In some cases for the same individual, the measurement reveals that one or more metabolites should have increased levels for the individual, and one or more other metabolites should have decreased levels for the individual.

[0007] Certain embodiments relate to methods that include administering a predetermined amount of cell therapy to an individual, including any individual encompassed herein, which amount may include a therapeutically effective amount of cell therapy. In some embodiments, the therapeutically effective amount is determined based on measuring the level of one or more metabolites from a sample from the individual. In some embodiments, the therapeutically effective amount is dependent on the individual having a responder phenotype or a non-responder phenotype. In certain embodiments, the responder phenotype comprises a predetermined concentration of at least one metabolite in a biological sample, the concentration being statistically equal to the concentration of the metabolite in a biological sample, including any type of blood sample, of a variety of individuals known to respond to a predetermined amount of cell therapy. In certain embodiments, the non-responder phenotype comprises a predetermined concentration of at least one metabolite in a biological sample, the concentration being statistically higher than the concentration of the metabolite in a biological sample, including any type of blood sample, of a variety of individuals known to respond to a predetermined amount of cell therapy. In certain embodiments, the non-responder phenotype comprises a concentration of at least one metabolite in a biological sample, said concentration being statistically lower than the concentration of said metabolite in biological samples, including any type of blood sample, of various individuals known to respond to a given amount of cell therapy. In some embodiments, such as when an individual has a non-responder phenotype, the individual does not receive therapy, or the therapy is adjusted prior to delivery and / or supplemented; such supplementation may include delivery of one or more compositions capable of increasing or decreasing at least one of the metabolites in the individual.

[0008] In some embodiments, a blood sample is taken from an individual before the cell therapy is administered to the individual, during the time the cell therapy is administered to the individual, and / or after the cell therapy is administered to the individual. At least one metabolite may be measured in the blood sample. In some embodiments, at least one metabolite is measured in a second blood sample. The second blood sample may include a blood sample taken from an individual who has been administered a therapy, including a cell therapy. The individual may have a responder phenotype when the concentration of the metabolite(s) in the blood sample is equal to the concentration of the metabolite(s) in the second blood sample. The individual may have a non-responder phenotype when the concentration of the metabolite(s) in the blood sample is different from the concentration of the metabolite(s) in the second blood sample.

[0009] Certain embodiments relate to methods for reducing the potential toxicity of cell therapy in an individual. The cell therapy may be any cell therapy, including any CAR T cell therapy, as encompassed herein. The toxicity may include cytokine response syndrome, immune effector cell-associated neurotoxicity, or a combination thereof. In some embodiments, the individual is monitored for toxicity during the period the therapy is being delivered, and / or in some cases, including immediately after the therapy is delivered.

[0010] Certain embodiments include one or more cell therapies. In some embodiments, the one or more cell therapies include T cell therapies. In some embodiments, the one or more cell therapies include cells that include at least one engineered receptor. The engineered receptor may include a chimeric antigen receptor (CAR). In some embodiments, the one or more cell therapies include cells that include an enzyme that can increase or decrease at least one metabolic product, including any metabolic product encompassed herein, and / or a nucleic acid that encodes such an enzyme. In some embodiments, the one or more cell therapies include a nucleic acid (RNA or DNA) that encodes a protein that can act as a transcription factor, and / or a cell that includes such a protein. In some embodiments, the one or more cell therapies include cells that include one or more non-mammalian proteins. The cells may be genetically modified to include the enzymes, proteins, and / or nucleic acids. In some embodiments, the one or more cell therapies include cells that are genetically modified to overexpress an enzyme that can increase or decrease at least one metabolic product, including any metabolic product encompassed herein. The enzyme may be endogenous or exogenous to the cell. The cell therapy may be produced after the individual's ability to respond to a CAR-related therapy has been determined, or the cell therapy may be stored, such as by being cryopreserved, and utilized when the need for the therapy is determined.

[0011] Proteins used in the embodiments encompassed herein, including enzymes and / or transcription factors, include, but are not limited to, ornithine decarboxylase (ODC), lysine decarboxylase, arginase, agmatinase, spermidine synthase, spermine synthase, spermine oxidase, spermidine / spermine-N1-acetyltransferase (SSAT1), S-adenosyl-methionine decarboxylase (AdoMetDC), deoxyhypusine synthase (DHPS), deoxyhypusine hydroxylase (DOHH), antizyme inhibitor (AZI), eukaryotic translation initiation factor 5A (eIF5A), ATF4, and / or Gcn2.

[0012] The present disclosure includes an embodiment of a method for treating an individual, comprising: (a) measuring the concentration of at least one metabolite in a blood sample (including a serum sample) from the individual to identify the individual as having a responder phenotype or as having a non-responder phenotype; and (b) administering to the individual a therapeutically effective amount of a cell therapy (including at least cells expressing one or more engineered receptors) either alone or in combination with other drugs or metabolites or chemical compounds as a treatment to generate an effective anti-tumor response or limited toxicity associated with the cell therapy, and the therapeutically effective amount is correlated with the individual having a responder phenotype or a non-responder phenotype. In a specific embodiment, the at least one metabolite comprises a metabolite selected from the group consisting of TMAO, indole, indole derivatives, 1-methylnicotinamide, nicotinamide, polyamines, asymmetric dimethylarginine, symmetric dimethylarginine, taurine, and combinations thereof. The indole derivative may include tryptophan, serotonin, indole-3-acetaldehyde, indole-3-lactate, indole-3-acetate, indoleacrylic acid, indoxyl sulfate, or a combination thereof. The polyamine may include diacetylspermine, N8-acetylspermidine, putrescine, spermidine, spermine, acetylputreceine, acetylspermidine, acetylspermine, N3AP, cadaverine, acetylcadaverine, diacetylcadaverine, or a combination thereof. In a specific embodiment, the responder phenotype comprises a predetermined concentration of at least one metabolite in a blood sample, the concentration being statistically equal to the concentration of said metabolite in blood samples of different individuals known to respond to a given amount of cell therapy. In a specific embodiment, the non-responder phenotype comprises a predetermined concentration of at least one metabolite in a blood sample, the concentration being statistically higher than the concentration of said metabolite in blood samples of different individuals known to respond to a given amount of cell therapy.The non-responder phenotype may include a predetermined concentration of at least one metabolite in a blood sample, said concentration being statistically lower than the concentration of said metabolite in blood samples of various individuals known to respond to a predetermined amount of cell therapy. In certain cases, when an individual is identified as having a non-responder phenotype, therapy is not given to the individual. In some cases, when an individual is identified as having a non-responder phenotype, cell therapy is adjusted. In some cases, when an individual is identified as having a non-responder phenotype, cell therapy is supplemented, such as with one or more metabolites or drugs. In a specific embodiment of the method, at least one of the one or more metabolites is determined to be statistically lower than the concentration of said metabolite in blood samples of various individuals known to respond to a predetermined amount of cell therapy. The cell of the cell therapy may be an immune cell, such as a T cell, a NK cell, a NKT cell, a macrophage, or other hematopoietic cell. The cells of the cell therapy may express one or more engineered receptors, such as chimeric antigen receptors, non-native TCRs, or both. The cells may further comprise a protein capable of increasing or decreasing at least one of the metabolites in an individual. The cells may be genetically modified to express said proteins or metabolites. In a specific embodiment, the protein comprises ornithine decarboxylase (ODC), lysine decarboxylase, arginase, agmatinase, spermidine synthase, spermine synthase, spermine oxidase, spermidine / spermine-N1-acetyltransferase (SSAT1), S-adenosyl-methionine decarboxylase (AdoMetDC), deoxyhypusine synthase (DHPS), deoxyhypusine hydroxylase (DOHH), antizyme inhibitor (AZI), eukaryotic translation initiation factor 5A (eIF5A), ATF4, Gcn2, or a combination thereof. The method may further comprise administering a composition capable of increasing or decreasing at least one of the metabolites.When the composition is capable of increasing at least one of the metabolites, the composition may comprise at least one of the metabolites. In certain embodiments, the blood sample is from the individual before the cell therapy is administered. The method includes the method further comprising measuring the concentration of the metabolite(s) in a second blood sample, the second blood sample comprising a blood sample from the individual after the cell therapy is administered to the individual, and the individual has a responder phenotype when the concentration of the metabolite(s) in the blood sample is equal to the concentration of the metabolite(s) in the second blood sample. The method may further comprise measuring the concentration of the metabolite(s) in a second blood sample, the second blood sample comprising a blood sample from the individual after the cell therapy is administered to the individual, and the individual has a non-responder phenotype when the concentration of the metabolite(s) in the blood sample is different from the concentration of the metabolite(s) in the second blood sample.

[0013]

[0013] Embodiments of the present disclosure include a method for reducing the potential for or risk of toxicity (e.g., cytokine release syndrome, immune effector cell associated neurotoxicity, prolonged cytopenia, hemophagocytic lymphohistiocytosis, or a combination thereof) of a cell therapy (including cells expressing one or more engineered receptors such as a chimeric antigen receptor, a TCR, or both), comprising: (a) measuring the concentration of at least one metabolite in a blood sample (including a serum sample, which may be taken prior to therapy) from an individual to identify the individual as having a responder phenotype or as having a non-responder phenotype; and (b) administering a therapeutically effective amount of the cell therapy to the individual, the therapeutically effective amount being dependent upon the individual having a responder or non-responder phenotype. The individual may or may not be monitored for toxicity. The at least one metabolite may include a metabolite selected from the group consisting of indole derivatives, TMAO, tryptophan, serotonin, 1-methylnicotinamide, nicotinamide, polyamines, asymmetric dimethylarginine, symmetric dimethylarginine, and combinations thereof. The indole derivatives include indole-3-acetaldehyde, indole, indoleacrylic acid, indole-3-lactate, indole-3-acetate, indoxyl sulfate, or combinations thereof. The polyamines may include diacetylspermine, N8-acetylspermidine, putrescine, spermidine, spermine, acetylputreceine, acetylspermidine, acetylspermine, N3AP, cadaverine, acetylcadaverine, diacetylcadaverine, or combinations thereof.The responder phenotype may include a predetermined concentration of at least one metabolite in a blood sample, the concentration being statistically equal to the concentration of the metabolite in blood samples of different individuals known to respond to a given amount of cell therapy. The non-responder phenotype may include a predetermined concentration of at least one metabolite in a blood sample, the concentration being statistically higher than the concentration of the metabolite in blood samples of different individuals known to respond to a given amount of cell therapy. In some cases, the non-responder phenotype may include a predetermined concentration of at least one metabolite in a blood sample, the concentration being statistically lower than the concentration of the metabolite in blood samples of different individuals known to respond to a given amount of cell therapy. When an individual is identified as having a non-responder phenotype, no therapy may be given. The cells of the therapy may be any type of cell, including T cells, NK cells, NKT cells, macrophages, or other hematopoietic cells.

[0014] In some embodiments, the cells of the cell therapy further comprise a protein capable of increasing or decreasing at least one of the metabolites in an individual. The cells may be genetically modified to express one or more proteins, including at least ornithine decarboxylase (ODC), lysine decarboxylase, arginase, agmatinase, spermidine synthase, spermine synthase, spermine oxidase, spermidine / spermine-N1-acetyltransferase (SSAT1), S-adenosyl-methionine decarboxylase (AdoMetDC), deoxyhypusine synthase (DHPS), deoxyhypusine hydroxylase (DOHH), antizyme inhibitor (AZI), eukaryotic translation initiation factor 5A (eIF5A), ATF4, Gcn2, or a combination thereof. In some cases, the method further comprises administering a composition capable of increasing or decreasing at least one of the metabolites. When the composition is capable of increasing at least one of the metabolic products, the composition may comprise at least one of the metabolic products.

[0015] In some embodiments, the method further comprises measuring the concentration of the metabolite(s) in a second blood sample, the second blood sample comprising a blood sample from the individual after the individual has been administered the cell therapy, and the individual has a responder phenotype when the concentration of the metabolite(s) in the blood sample is equal to the concentration of the metabolite(s) in the second blood sample. The method may further comprise measuring the concentration of the metabolite(s) in a second blood sample, the second blood sample comprising a blood sample from the individual after the individual has been administered the cell therapy, and the individual has a non-responder phenotype when the concentration of the metabolite(s) in the blood sample differs from the concentration of the metabolite(s) in the second blood sample.

[0016] In some embodiments, there is a therapeutic composition comprising a cell expressing one or more engineered receptors (one or more chimeric antigen receptors and / or one or more non-native TCRs) and one or more metabolites as encompassed herein. The cell of the therapeutic composition may comprise a genetic modification to express at least one protein capable of increasing or decreasing one or more metabolites. The one or more metabolites may be selected from the group consisting of TMAO, indole, indole derivatives (e.g., tryptophan, serotonin, indole-3-acetaldehyde, indoleacrylic acid, indoxyl sulfate, or combinations thereof), 1-methylnicotinamide, nicotinamide, polyamines (e.g., diacetylspermine, N8-acetylspermidine, putrescine, spermidine, spermine, acetylputreceine, acetylspermidine, acetylspermine, N3AP, cadaverine, acetylcadaverine, diacetylcadaverine, or combinations thereof), asymmetric dimethylarginine, symmetric dimethylarginine, taurine, and combinations thereof. The proteins may include ornithine decarboxylase (ODC), lysine decarboxylase, arginase, agmatinase, spermidine synthase, spermine synthase, spermine oxidase, spermidine / spermine-N1-acetyltransferase (SSAT1), S-adenosyl-methionine decarboxylase (AdoMetDC), deoxyhypusine synthase (DHPS), deoxyhypusine hydroxylase (DOHH), antizyme inhibitor (AZI), eukaryotic translation initiation factor 5A (eIF5A), ATF4, Gcn2, or combinations thereof.

[0017] Embodiments of the present disclosure include a method for treating an individual receiving a first cell therapy, comprising: (a) measuring the concentration of one or more metabolites in at least one blood sample from the individual; and (b) administering a therapeutic composition comprising a metabolite, a bacteria, a second cell therapy, or a combination thereof when the concentration of the metabolite(s) is higher or lower than a baseline level. The at least one metabolite may comprise a metabolite selected from the group consisting of TMAO, indole, indole derivatives (e.g., tryptophan, serotonin, indole-3-acetaldehyde, indoleacrylic acid, indoxyl sulfate, or combinations thereof), 1-methylnicotinamide, nicotinamide, polyamines (e.g., diacetylspermine, N8-acetylspermidine, putrescine, spermidine, spermine, acetylputreceine, acetylspermidine, acetylspermine, N3AP, cadaverine, acetylcadaverine, diacetylcadaverine, or combinations thereof), asymmetric dimethylarginine, symmetric dimethylarginine, taurine, and combinations thereof. In some cases, the baseline level comprises a predetermined concentration of the at least one metabolite in a blood sample of an individual known to be responsive to a predetermined amount of cell therapy. The first cell therapy and / or the second cell therapy may comprise cells expressing one or more engineered receptors (one or more chimeric antigen receptors, non-native TCRs, or both). The cells of the therapy may be any type of cell, including, for example, immune cells such as T cells, NK cells, NKT cells, macrophages, or other hematopoietic cells. The second cell therapy may further include a protein capable of increasing or decreasing at least one of the metabolic products in an individual.Any of the cell therapies may be genetically modified to express one or more proteins, including ornithine decarboxylase (ODC), lysine decarboxylase, arginase, agmatinase, spermidine synthase, spermine synthase, spermine oxidase, spermidine / spermine-N1-acetyltransferase (SSAT1), S-adenosyl-methionine decarboxylase (AdoMetDC), deoxyhypusine synthase (DHPS), deoxyhypusine hydroxylase (DOHH), antizyme inhibitor (AZI), eukaryotic translation initiation factor 5A (eIF5A), ATF4, Gcn2, or combinations thereof. The blood sample may include a serum sample. At least one blood sample may be from the individual prior to administration of the first cell therapy. The level of the metabolite may be at least 2-fold, 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold greater than the baseline level, or at least 2-fold, 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold less than the baseline level.

[0018] An embodiment of the present disclosure includes a method of predicting a response to a cell therapy, comprising: (a) measuring the concentration of at least one metabolite in a blood sample from an individual; and (b) predicting a response to the cell therapy when the metabolite is higher or lower than a baseline level. In a specific embodiment, when the response is predicted to be harmful to the individual, the cell therapy is not administered to the individual, is modified before administration to the individual to make it less harmful, and the individual is given a different therapy, or a combination thereof. In a specific embodiment, when the response is predicted to be harmful to the individual, the individual is given a therapeutically effective amount of (1) one or more metabolites, or (2) a bacterial composition that can change the metabolite or its level, or other synthetic derivatives / products thereof, or that can change its level, or (3) genetically modified immune cells that can change the metabolite in the body to cause a response or toxicity to the therapy. In a specific case, when the response is predicted to be not harmful to the individual, a therapeutically effective amount of the cell therapy is administered to the individual. The level of the metabolite may be at least 2-fold, 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold greater than the baseline level, or at least 2-fold, 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold less than the baseline level.

[0019] The features and technical advantages of the present disclosure have been outlined above rather broadly in order that the detailed description that follows may be better understood. Additional features and advantages will be described hereinafter which form the subject of the claims herein. It should be appreciated by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the inventive design. It should also be recognized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as set forth in the appended claims. The novel features believed to be characteristic of the design disclosed herein, both as to its structure and method of operation, together with further objects and advantages, will be better understood from the following description when considered in connection with the accompanying drawings. It is to be expressly understood, however, that each of the drawings is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure. [Brief description of the drawings]

[0020] For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

[0021] [Figure 1] 1 shows serum metabolites associated with cytokine release syndrome (CRS).

[0022] [Diagram 2] 1 shows serum metabolites associated with immune effector cell-associated neurotoxicity syndrome (ICANS).

[0023] [Diagram 3] 1 shows the association between circulating lysophospholipids and circulating polyamines with progression-free survival and overall survival in patients with B-cell lymphoma treated with CAR-T.

[0024] [Figure 4]We provide the development and validation of an example of a six-marker prognostic metabolite panel for progression-free survival and overall survival in patients with B-cell lymphoma treated with CAR-T.

[0025] [Figure 5A] Figure 5A shows that B cell lymphomas show elevated mRNA expression of polyamine metabolic enzymes, and that high spermidine synthase gene expression is prognostic for poor overall survival. Violin diagram illustrating mRNA expression of polyamine metabolic enzymes (PME) in diffuse large B cell lymphoma and normal B lymphocytes in the Basso lymphoma dataset (Basso et al., 2005). Statistical significance was determined by two-tailed Wilcoxon rank sum test. Abbreviations: ODC1-ornithine decarboxylase 1; AMD1-adenosylmethionine decarboxylase 1; SRM-spermidine synthase; SMS-spermine synthase; SAT1-spermidine / spermine N1-acetyltransferase 1. [Figure 5B] Figure 5B shows that B-cell lymphomas show elevated mRNA expression of polyamine metabolic enzymes, and high spermidine synthase gene expression is prognostic for poor overall survival. Dot plots illustrating the hazard ratio (HR) (95%CI) per unit increase in PME mRNA expression and progression-free survival (PFS) in The Cancer Genome Atlas (TCGA)-Diffuse Large B-cell Lymphoma (DLBCL) transcriptome dataset, and overall survival in the Lenz (Lenz et al., 2008) and Shipp (Shipp et al., 2002) B-cell lymphoma transcriptome datasets. [Figure 5C]Figure 5C shows that B-cell lymphomas show elevated mRNA expression of polyamine metabolic enzymes, and that high spermidine synthase gene expression is prognostic for poor overall survival. Kaplan-Meier survival curves for the association between SRM mRNA expression above or below optimal change-point values ​​(Cecile Contal, 1999) and PFS in the TCGA-DLBCL dataset, and overall survival in the Lenz (Lenz et al., 2008) and Shipp (Shipp et al., 2002) B-cell lymphoma datasets, respectively. [Figure 5D] Figure 5D shows that B-cell lymphomas show elevated mRNA expression of polyamine metabolism enzymes, and high spermidine synthase gene expression is prognostic for poor overall survival. Dot plots illustrate Spearman's rho coefficients (95% CI) for associations between mRNA expression of polyamine metabolism enzymes (ODC1, SRM, and SMS) with respect to gene-based signatures of immune cell infiltrates and immune checkpoint blockade-related genes in the TCGA-DLBCL transcriptome dataset and the Ma DLBCL transcriptome dataset (Ma et al., 2019). Gene-based signatures were according to Bindea et al. (Bindea et al., 2013). Abbreviations: ODC1: ornithine decarboxylase 1; AMD1: adenosylmethionine decarboxylase 1; SRM: spermidine synthase; SMS: spermine synthase; SAT1: spermidine / spermine N1-acetyltransferase 1.

[0026] [Figure 6] Progression-free and overall survival curves for responders and non-responders in the discovery and validation cohorts are shown.

[0027] [Figure 7] 16A-C provide the predictive performance of a six-marker metabolite panel for discriminating CAR-T responders from non-responders.

[0028] [Figure 8] Circulating levels of acetylated polyamines in patients with LBCL after CAR-T cell treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] I Definition Example Following long-standing patent law practice, the words "a" and "an" when used in conjunction with the word comprising, herein, including in the claims, refer to "one or more." Some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. In specific embodiments, any embodiment herein may comprise, consist of, or consist essentially of any combination of markers. Any method or composition described herein may be implemented with respect to any other method or composition described herein, and it is contemplated that different embodiments may be combined.

[0030] As used herein, the terms "or" and "and / or" are utilized to describe multiple elements in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0031] Throughout this application, the term "about" is used according to its plain and ordinary meaning in the field of cell and molecular biology to indicate that the standard deviation of error for the device or method being employed to determine the value.

[0032] The term "engineered" or "engineering" as used herein refers to an entity produced by the hand of man (or a process that produces the same), including cells, nucleic acids, polypeptides, vectors, and the like. In at least some cases, an engineered entity is synthetic and is composed of elements that do not occur in nature or are not constructed in the manner utilized in this disclosure. With respect to cells, the cells may be engineered because they have reduced expression of one or more endogenous genes and / or express one or more heterologous genes (such as synthetic antigen receptors), in which case the engineering is done by the hand of man. With respect to antigen receptors, the antigen receptors are considered engineered because they are composed of multiple components that have been genetically recombined and constructed in a way that does not occur in nature, for example, in the form of a fusion protein of the components that do not occur in nature so constructed.

[0033] As used herein, the term "concentration" can be used interchangeably with the term "level" when referring to the amount of a biomarker or metabolite present in an individual, including the amount of a biomarker or metabolite present in a biological sample taken from an individual. For example, the "level" of a measured metabolite may, in some embodiments, be used interchangeably with the "concentration" of the measured metabolite.

[0034] As used herein, when two values ​​are "statistically" equal or different, the values ​​are equal or different as determined by a suitable statistical method for comparing values. For example, the level of a metabolite may be measured one or more times in a first biological sample to obtain a first value set for the metabolite in the first biological sample. The level of the metabolite may be measured one or more times in a second biological sample to obtain a second value set. To determine whether the first value set is statistically equal or different from the second value set, a statistical test, such as a Student's t-test, may be performed on the two sets. Those skilled in the art may determine the resulting value, such as a p-value, required to reach statistical significance to determine whether the two value sets are statistically equal or different. Other statistical tests may be employed to better compare multiple metabolites or to compare two or more value sets. In some embodiments, a p-value of 0.1, 0.05, 0.01, 0.001, 0.0001 or less is determined to be statistically significant between one or more value sets.

[0035] As used herein, the term "therapeutically effective amount" is synonymous with "effective amount," "therapeutically effective dose," and / or "effective amount," and refers to the amount of therapy that elicits the biological or clinical response desired by the skilled artisan in an individual in need thereof. The appropriate effective amount to be administered for a particular application of the disclosed method can be determined by the skilled artisan using the guidance provided herein. For example, the effective amount can be extrapolated from in vitro and in vivo assays or interpretation of clinical data, as described herein. One skilled in the art will recognize that an individual's condition can be monitored throughout the course of treatment, and the effective amount of the compound or composition disclosed herein administered can be adjusted accordingly.

[0036] As used herein, the terms "therapy", "treatment" or "treating" refer to an intervention in an attempt to change the natural course of the individual or cell being treated, and may be performed either for prophylaxis or during the course of the pathology of a disease or condition. Treatment is performed to achieve one or more of a variety of desired results, such as, for example, prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of a disease, prevention of metastasis, slowing the rate of disease progression, amelioration or alleviation of a disease state, remission or improved prognosis.

[0037] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular features, structures, or characteristics described in connection with an embodiment are included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0038] Various aspects of the present disclosure can be shown in range format. It should be understood that the description in range format is merely for convenience and conciseness, and should not be interpreted as an inflexible limitation on the scope of the present disclosure. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as the individual numerical values ​​within that range, as if they were explicitly written out. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numerical values ​​within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. When a range exists, the range may include the end points of the range.

[0039] The term "individual" refers to an individual in need of treatment. The individual may be a mammal, such as a human, dog, cat, horse, pig, or rodent. The individual may be, for example, a patient having or suspected of having a disease or condition, including any cancer, such as a hematological cancer or solid tumor. The hematological cancer may be lymphoma, including B-cell lymphoma. The individual may have or be suspected of having a disease, including cancer. They may be asymptomatic. They may be of any gender. They may be of a certain age, such as at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 years old or older. II. General Embodiments

[0040] Various embodiments disclosed herein show that plasma metabolomics correlates with efficacy and toxicity associated with treating diseases (e.g., cancer, etc.) with various cell therapies, including at least CAR-T therapy. The present disclosure relates to methods and compositions for treating various diseases, including cancer, whatever the type, by modulating certain metabolites to enhance the efficacy of various cell therapies, including CAR-T therapy. The present disclosure also relates to determining metabolite abundance in plasma and serum as a biomarker for predicting efficacy and / or toxicity associated with CAR-T therapy. The cancer being treated by adoptive cell therapy may be of any type and may be primary, recurrent, refractory, metastatic, and others. The cancer may be a solid tumor or a hematological malignancy, including lymphoma or leukemia. Predictive Metabolite Signatures

[0041] The embodiments of the present disclosure include methods for identifying or predicting individuals who will or will not achieve an effective response to adoptive cell therapy, whatever the type of adoptive cell therapy, including CAR-expressing cells (including T cells, NK cells, etc.). In a specific embodiment, the method predicts individuals who are unlikely to achieve a durable response to adoptive cell therapy, whatever the type of adoptive cell therapy. In various embodiments, the method identifies individuals who are reduced in likelihood of achieving an effective response to adoptive cell therapy, e.g., by comparing with individuals who are not so identified (e.g., individuals who lack the same marker(s) as the individual determined to be reduced in likelihood). In a specific embodiment, the method allows prediction of whether an individual will have a poor response to CAR-T cell therapy when one or more metabolites are measured, including by measuring one or more plasma metabolites. In specific cases, the metabolites include one or more polyamines and / or lysophospholipids, and in certain aspects, the presence of elevated levels of certain polyamines and / or lysophospholipids (e.g., such presence compared to the general population) indicates that an individual will have poorer progression-free survival (PFS) and overall survival (OS), e.g., when compared to individuals who did not have elevated levels of the polyamines and / or lysophospholipids.

[0042] In certain embodiments, one or more checkpoint inhibitor therapies may also be utilized in any of the methods herein, and the one or more checkpoint inhibitor therapies may include one or more lymphoid checkpoint inhibitors, including but not limited to, PD-1 inhibitors, PDl-1 inhibitors, TIM3 inhibitors, LAG3 inhibitors, and / or one or more myeloid checkpoint inhibitors, including but not limited to, CD47 inhibitors, SIR alpha inhibitors. Commercially approved drugs include nivolumab, pembrolizumab, ipilimumab, atezolizumab, and the like, which may be utilized.

[0043] In specific cases, a response is effective when one or more symptoms are alleviated and / or when one or more symptoms are delayed. Efficacy may include a reduction in the number of cancer cells (including a reduction to undetectable levels), a reduction in tumor mass, a reduction in tumor size, an improvement in the individual's quality of life, an extension of the individual's life span, a reduction in the risk of metastasis, prevention of metastasis, delaying the onset of metastasis, combinations thereof, and others. In some embodiments, the individual is also subjected to a step of determining the presence of a marker(s) other than the markers encompassed herein, such as, for example, elevated lactate dehydrogenase (LDH), c-reactive protein (CRP), increased tumor interferon signaling, and / or elevated IL-6 at baseline.

[0044] In various embodiments, an individual in whom one or more certain markers are present or absent in a sample from the individual is given an effective amount of one or more treatments (e.g., surgery, chemotherapy, radiation, hormone therapy, drug therapy, antibodies, and the like), the treatment being given to the individual as a result of determining the presence or absence, respectively, of the one or more markers. The method may or may not include a step of determining that the individual has cancer.

[0045] Measurement of metabolites / proteins and interventions are useful to modulate cell therapy (including CAR-T therapy and associated toxicities therein) to enhance efficacy of the therapy. Various embodiments herein include various metabolomics methods, including metabolomics in blood, serum or plasma samples from individuals. Such methods may be used in clinical decision-making in selecting or managing recipients of various cell therapies, including CAR-T therapy. Various embodiments herein also include compositions, such as cell therapy and / or metabolites. Cell therapy may include CAR-T cells in specific cases. Cell therapy may include engineered cells that are capable of expressing one or more enzymes to modulate metabolite levels. In some embodiments, individuals undergoing cell therapy (such as CAR-T therapy) may benefit from the compositions included herein.

[0046] Included herein are methods of measuring one or more metabolites or correlating one or more metabolites with efficacy or toxicity of a cell therapy that an individual needs, where the level of one or more metabolites is determined from a sample from the individual. In a specific embodiment, the method includes a treatment step in which a treatment is determined based on the measuring or based on the correlating, and a treatment step in which the treatment is subsequently administered. In a specific embodiment, the level of one or more metabolites is evaluated in the individual to facilitate clinical treatment decision-making for the individual. In some cases, the treatment is delivered after the evaluation, whereas in other cases, the treatment is modified after the evaluation and before it is administered to the individual. For example, after determining that an individual may or will have reduced efficacy based on the presence of one or more certain metabolites, the cells of the CAR cell therapy may be changed to another type of cell and / or the cells may be genetically or otherwise modified.

[0047] In a specific case, the cell may be modified to express one or more specific proteins (including enzymes) and / or nucleic acids. The proteins and / or nucleic acids may be capable of changing one or more metabolic products, including any metabolic products encompassed herein, inside and / or outside the cell. The proteins may include ornithine decarboxylase (ODC), arginase, lysine decarboxylase, agmatinase, spermidine synthase, spermine synthase, spermine oxidase, spermidine / spermine-N1-acetyltransferase (SSAT1), S-adenosyl-methionine decarboxylase (AdoMetDC), deoxyhypusine synthase (DHPS), deoxyhypusine hydroxylase (DOHH), antizyme inhibitor (AZI), eukaryotic translation initiation factor 5A (eIF5A), ATF4, Gcn2, or combinations thereof. In some embodiments, the cells contain nucleic acids encoding ornithine decarboxylase (ODC), arginase, agmatinase, spermidine synthase, spermine synthase, spermine oxidase, spermidine / spermine-N1-acetyltransferase (SSAT1), S-adenosyl-methionine decarboxylase (AdoMetDC), deoxyhypusine synthase (DHPS), deoxyhypusine hydroxylase (DOHH), antizyme inhibitor (AZI), eukaryotic translation initiation factor 5A (eIF5A), ATF4, Gcn2, or combinations thereof.

[0048] Additionally or alternatively, the CAR may be modified, for example, to utilize a different extracellular domain, transmembrane domain, and / or intracellular domain. In particular cases, the CAR may be modified to utilize a different scFv, a different transmembrane domain, and / or one or more different costimulatory domains. Additionally or alternatively, the therapy includes CAR-associated cells, but one or more other compositions, such as one or more other compositions including one or more metabolites determined to be deficient in levels or suspected to be deficient in levels, are provided in addition to the cells. The methods of the present disclosure include a method of predicting a response to a cell therapy for an individual. An individual may need to predict whether a cell therapy will be effective or toxic, for example, because the individual has cancer and is in need of a CAR-associated therapy in which the CAR is directed to one or more antigens of the individual's cancer. If it is determined that the CAR cell therapy will not be effective and / or will be toxic to the individual based on an analysis of one or more metabolites, including at least their levels, in some cases, the individual is not provided with the CAR cell therapy. In other cases, when it is determined that the CAR cell therapy will not be effective and / or will be toxic to the individual, the individual may be given a different cancer therapy. In some cases, when it is determined that the CAR cell therapy will not be effective and / or will be toxic to the individual, the CAR cell therapy is modified to be more effective and / or less toxic.

[0049] In certain embodiments, an individual is administered one or more metabolites, including any metabolites encompassed herein. Specific embodiments include methods that include a treatment step in which a metabolite therapy is determined based on measuring or correlating one or more metabolites, and a treatment step in which the metabolite therapy is subsequently administered. In some cases, the metabolite therapy is delivered after evaluation, whereas in other cases, the metabolite therapy is modified after evaluation and prior to administration to the individual. The metabolite therapy may include administering the metabolite, for example, orally (including via diet), by injection (including at least intravenously), or by any other suitable method for administering one or more metabolites in any manner. In specific embodiments, one or more metabolites encompassed herein are administered to enhance the efficacy of CAR T cell therapy and / or to reduce the toxicity of CAR T therapy.

[0050] In certain embodiments, the individual is administered one or more bacterial strains. Specific embodiments include methods that include a treatment step in which bacterial therapy is determined based on measuring or correlating one or more metabolic products, and a treatment step in which the bacterial therapy is administered thereafter. In some cases, bacterial therapy is delivered after evaluation, whereas in other cases, bacterial therapy is modified after evaluation and prior to administration to the individual. Bacterial therapy may include administering bacteria, for example, orally (including through the diet), by injection (including at least intravenously), or by any other suitable method for administering bacteria. Bacterial therapy may include any bacterial strain that can increase or decrease metabolic products in an individual.

[0051] There are many ways to administer or manipulate these metabolites in the human body, including, but not limited to: (1) administering the metabolite(s) directly, such as via iv or orally; (2) diet can also manipulate these metabolites; (3) administering bacteria with modified genes that can alter these metabolites; and / or (4) fecal microbial transplantation (FMT) can modify these metabolites.

[0052] In some embodiments, the survival outcome for an individual with cancer is determined based on measuring the level of one or more metabolites in an individual in need of treatment. The survival outcome may be determined based on the level of metabolite(s) in the absence of treatment or as a result of treatment. Once determined, the clinical course of treatment may or may not be altered depending on the outcome. The survival outcome may be determined to be positive such that treatment is subsequently administered, or the survival outcome may be determined to be non-positive such that treatment is not administered or altered.

[0053] In some embodiments, cell therapy is monitored for efficacy by measuring the level of one or more metabolites in an individual receiving the therapy. In a specific case, the level of one or more metabolites may be determined before cell therapy is administered and after cell therapy is administered. When one or more metabolites are found to indicate that cell therapy is losing efficacy, or is losing efficacy, or is at risk of becoming toxic, or is toxic, one or more measures can be taken to address the change. Metabolite monitoring may be, for example, for a certain duration of time with the therapy. In some cases, monitoring includes measuring the level of one or more metabolites to assess the risk for CRS and / or ICANS or the presence of CRS and / or ICANS. III. Measurement of Metabolites

[0054] Certain embodiments of the present disclosure encompass methods for detecting one or more metabolites in a biological sample from an individual, including measuring the level of one or more metabolites in a biological sample from an individual. Any method known in the art may be used to measure metabolites, including, but not limited to, mass spectrometry, nuclear magnetic resonance (NMR), ion mobility spectrometry, electrochemical detection, Raman spectroscopy, immunoassay and / or radiolabeling. Any mass spectrometry method may be used in the embodiments herein, including, but not limited to, time-of-flight, quadrupole, ion trap, Fourier transform ion cyclotron resonance, electron ionization, atmospheric pressure chemical ionization, electrospray ionization, matrix-assisted laser desorption / ionization, or combinations thereof. In some embodiments, metabolites in a sample are separated by any method, including, but not limited to, chromatography (e.g., gas chromatography, liquid chromatography (e.g., high performance liquid chromatography and ultra high performance liquid chromatography), capillary electrophoresis, or combinations thereof. In some embodiments, separation and measurement of metabolites are performed in tandem, for example by GC-MS, LC-MS, LC-MS / MS, and / or HPLC-MS.

[0055] In some embodiments, the metabolites measured in the first biological sample are compared to a normal value for the measured metabolite. In some embodiments, the normal value is determined by measuring the metabolite in a second biological sample from an individual known to have a desired phenotype. As a non-limiting example, a certain metabolite is measured in a second biological sample from an individual known to have a desired phenotype of responding to cell therapy. In some embodiments, such an individual has a responder phenotype. In some embodiments, the metabolite level measured in the individual is a baseline level. The metabolite level measured in the individual is then compared to the metabolite level measured in the first biological sample. If the metabolite levels in the first and second biological samples are equal, including statistically equal, the individual from whom the first biological sample was taken may be said to have a desired phenotype. If the metabolite levels in the first and second biological samples are different, including statistically different, the individual from whom the first biological sample was taken may be said to not have a desired phenotype. In some embodiments, the individual said to not have a desired phenotype is identified as having a non-responder phenotype. In some cases, the normal values ​​for one or more particular metabolites are based on values ​​from the general population, e.g., as may be determined by standard means or known in the art.

[0056] In some embodiments, the metabolites measured in the first biological sample are compared to a normal value for the measured metabolite. In some embodiments, the normal value is determined by measuring a baseline value of the metabolite. The baseline value may be measured at any time before or during a given time point, including a cell therapy (such as CAR-T therapy). For example, a biological sample may be taken from an individual before the individual starts cell therapy to establish a baseline measurement value for one or more metabolites. In some embodiments, after establishing a baseline level of a certain metabolite, the individual is administered at least one therapy, including any of the therapies (or combinations of therapies) encompassed herein. After administering the therapy, at least one additional biological sample is taken from the individual. Certain metabolites, such as the metabolites measured in the first baseline sample, may be measured in the additional biological sample(s). The levels of each metabolite in each sample may then be compared.

[0057] In some embodiments, an individual may be said to have a responder phenotype when one or more metabolites are unchanged (e.g., equal, including statistically equal, or not different, including not statistically different) between the baseline sample and one or more of the additional samples. In some embodiments, an individual may be said to have a non-responder phenotype when the level of one or more metabolites from the baseline sample is different, including statistically different, from the level in at least one of the additional samples.

[0058] Metabolites that are useful for embodiments encompassed herein include TMAO, indoxyl sulfate, acetylcadaverine, 1-methylnicotinamide, diacetylspermine, Ng,Ng-dimethyl-l-arginine, 5,6-dihydrouridine, nicotinamide, 4,7-dioxo-octanoic acid, N8-acetylspermidine, plus phosphatidylcholine (o-40:7) and / or plus phosphatidylcholine (p-40:6), n-acetyl-l-phenylalanine, 2-hydroxyphenylacetic acid; 4-hydroxyphenylacetate, benzyl alcohol, acetylcholine, acetylspermidine ...choline, acetylspermidine, acetylcholine, acetylcholine, acetylspermidine, acetylcholine, acetylcholine, acetylcholine, acetylcholine, acetylcholine, acetylcholine, acetyl oleamide, phosphatidylcholine (37:5), l-valine, phosphatidylcholine (38:5), l-norvaline, oleamide, norleucine, phosphatidylcholine (36:4), lysophosphatidylcholine (20:4), phosphatidylcholine (38:4); phosphatidylglycerol (40:0), sphingomyelin (38:1), adenosine 5'-monophosphate, 2'-deoxyguanosine 5'-monophosphate, cholesterol ester (20:4), taurine, 3-methoxy-l-tyrosine, 2'-deoxyguanosine 5'-diphosphate; adenosine 3',5'-diphosphate;Adenosine 5'-diphosphate, deoxycorticosterone acetate phosphatidylcholine (33:2), glucosylceramide (36:0), 2-hydroxypyridine, beta-nicotinamide adenine dinucleotide phosphate, azelaic acid, plus phosphatidylcholine (p-36:0), plus phosphatidylethanolamine (o-34:3) and / or plus phosphatidylethanolamine (p-34:2), 10-hydroxydecanoate, plas_phosphatidylethanolamine (o-40:6) and / or plas_phosphatidylethanolamine (o-40:6) Sphatidylethanolamine (p-40:5), phosphatidylcholine (p-33:2), 1-(hydroxymethyl)-5,5-dimethyl-2,4-imidazolidinedione_exogenous, phosphatidylethanolamine (44:11), plas_phosphatidylethanolamine (o-36:5) and / or plas_phosphatidylethanolamine (p-36:4), citrulline, plus phosphatidylethanolamine (o-40:6) and / or plus phosphatidylethanolamine (p-40:5), homoserine, thyritol n-oleoylethanolamine, plus phosphatidylcholine (o-30:1) and / or plus phosphatidylcholine (p-30:0), plus phosphatidylethanolamine (o-38:5) and / or plus phosphatidylethanolamine (p-38:4), lysophosphatidylethanolamine (18:2), nepsilon.nepsilon.nepsilon-trimethyllysine, plus phosphatidylethanolamine (o-36:5) and / or plus phosphatidylethanolamine (p-36:4) ), 8-methoxykynurenate, lysophosphatidylinositol (18:1), ln.gamma.-monomethylarginine, plas_phosphatidylethanolamine (o-40:7) and / or plas_phosphatidylethanolamine (p-40:6), lysophosphatidylcholine (17:2), l-asparagine, prostaglandin e2, sphingomyelin (34:0), creatine phosphate, lysophosphatidylcholine (18:2), glucosylceramide (40:1), 12(s)-hete; 15(s)-hete;5(s)-hete, lactosylceramide (18:1 / 16:0), plus phosphatidylethanolamine (o-38:4) and / or plus phosphatidylethanolamine (p-38:3), lactosylceramide (32:1), triacylglycerol (58:4), sphinganine, d-ribose 5-phosphate;quinate, deoxycarnitine, aspartyl-threonine, phosphatidylcholine (28:0), metformin, phosphatidylcholine phosphorus (40:8), acylcarnitine (c18:0), phylloquinone, deoxycarnitine; phosphocholine, indole-3-acetaldehyde, 6-phosphogluconic acid, sphingomyelin (32:0), glycyl-threonine, acylcarnitine (c4:0), hypoxanthine, 1-methylhistidine; 3-methylhistidine; n(pai)-methyl-l-histidine, dl-5-hydroxylysine, indole, l-methionine, ascorbate, proline destaglandin f2a, serotonin, glycerophosphocholine, 1-aminocyclopropane-1-carboxylate, phosphatidylethanolamine (36:1), ceramide (42:0), 3-amino-5-hydroxybenzoic acid, d-tryptophan, l-tryptophan, n-acetyl-dl-serine, o-acetyl-l-serine, phosphatidylinositol (43:2), triacylglycerol Glycerol (47:0), ceramide (42:0) iso, ll-2,6-diaminoheptanedioate, pyridoxal 5'-phosphate, quinoline, indoleacrylic acid, l-anserine, methylpyrazine, o-butanoyl-r-carnitine, 2'-deoxyguanosine 5'-monophosphate; adenosine 5'-monophosphate; n-acetylneuraminate, urate, ceramide (40:0), 3-(4-hydroxyphenyl)propionic acid, glycerophosphocholine;sn-glycero-3-phosphocholine, n-methyl-d-aspartate, citrate, plus lysophosphatidylethanolamine (p-22:0), plus lysophosphatidylcholine (p-16:0), l-tryptophan, lysophosphatidylcholine (22:5), lysophosphatidylcholine (p-18:0 / 0:0) and / or lysophosphatidylcholine (o-18:1), 5'-deoxyadenosine diacetylspermine, triacylglycerol (51:6), (3'-sulfo)galβ-cer (d18:1 / 18:0(2oh)), phosphatidylcholine (35:2), phosphatidylcholine (33:3), phosphatidylcholine (3 4:4), triacylglycerol (51:5), 4-pyridoxate, phosphatidylcholine (32:2), thymidine-5'-diphospho-alpha-d-glucose, nicotinamide mononucleotide, phosphatidylcholine (33:2), Cl(1'-[15:0 / 15:0],3'-[15:0 / 16:1(9z)])[rac], plus phosphatidylethanolamine (o-38:4) and / or plus phosphatidylethanolamine (p-38:3); plus phosphatidylethanolamine (o-40:7) and / or plus phosphatidylethanolamine (p-40:6), free fatty acids (14:1) (myristelaic acid);Free fatty acids (14:1) (myristoleic acid), 3-cis-hydroxy-b,e-carotene-3'-one, phosphatidylcholine (33:1), phosphatidylcholine (31:0), triacylglycerol (51:4), triacylglycerol (56:4), plus phosphatidylethanolamine (o-34:3) and / or plus phosphatidylethanolamine (p-34:2), galactosylceramide (36:1) and / or or glucosylceramide (36:1), cis-quinseoxepane, phosphatidylethanolamine (o-38:5) and / or plus phosphatidylethanolamine (p-38:4), presumed_kdnalpha2-3galbeta1-4glcbeta-cer (d18:1 / 24:0), plus phosphatidylethanolamine (o-36:5) and / or plus phosphatidylethanolamine (p-36:4), 1,2 -Didecanoyl-sn-glycero-3-phosphocholine, lysophosphatidylinositol (18:1), triacylglycerol (16:0_16:1_18:2), triacylglycerol (51:3), triacylglycerol (55:7), (r,r)-tartaric acid; (s,s)-tartaric acid, deoxyuridine, galβ1-3galβ1-3galβ1-3galβ1-3galβ1-3galβ1-4galβ1-4glcβ-cer(42 :2), plus phosphatidylethanolamine (o-38:4) and / or plus phosphatidylethanolamine (p-38:3), lactosylceramide (32:1), ceramide (42:1) iso, sphinganine, d-ribose 5-phosphate; quinate, triacylglycerol (49:3), deoxycarnitine, n-alpha-acetyl-l-asparagine, phosphatidylcholine (40:8), phylloquinone, acetylcholine;These include, but are not limited to, deoxycarnitine, ceramide (43:1), triacylglycerol (49:2), phosphatidylcholine (35:4), d-glucono-1.5-lactone, lysophosphatidylcholine (15:0), triacylglycerol (56:3), lysophosphatidylcholine (o-16:2(9e,10e) / 0:0)[u], ceramide (42:0), sphingomyelin (42:1), ceramide (42:0) iso, ceramide (40:0), n-methyl-d-aspartic acid, l-carnitine, triacylglycerol (61:6), or combinations thereof;

[0059] In some embodiments, TMAO, indoxyl sulfate, acetylcadaverine, 1-methylnicotinamide, diacetylspermine, Ng,Ng-dimethyl-l-arginine, 5,6-dihydrouridine, nicotinamide, 4,7-dioxo-octanoic acid, N8-acetylspermidine, plus phosphatidylcholine (o-40:7) and / or plus phosphatidylcholine (p-40:6), n-acetyl-l-phenylalanine, 2-hydroxyphenylacetic acid; 4-hydroxyphenylacetate, benzyl alcohol, phosphatidylcholine (37:5), l-valine, phosphatidylcholine (38:5), l-norvaline, oleamide, norleucine, phosphatidylcholine ( One or more metabolites selected from the group consisting of phosphatidylcholine (36:4), lysophosphatidylcholine (20:4), phosphatidylcholine (38:4); phosphatidylglycerol (40:0), sphingomyelin (38:1), adenosine 5'-monophosphate, 2'-deoxyguanosine 5'-monophosphate, cholesterol ester (20:4), taurine, 3-methoxy-l-tyrosine, 2'-deoxyguanosine 5'-diphosphate; adenosine 3'.5'-diphosphate; adenosine 5'-diphosphate, deoxycorticosterone acetate, and combinations thereof, are measured in an individual, including in a biological sample of the individual, to determine whether the individual has a desired phenotype. The desired phenotype may be a response to cell therapy (including CAR-T cell therapy), such as a complete response or a durable complete response.

[0060] In some embodiments, phosphatidylcholine (33:2), glucosylceramide (36:0), 2-hydroxypyridine, beta-nicotinamide adenine dinucleotide phosphate, azelaic acid, plus phosphatidylcholine (p-36:0), plus phosphatidylethanolamine (o-34:3) and / or plus phosphatidylethanolamine (p-34:2), 10-hydroxydecanoate, plas_phosphatidylethanolamine (o-40:6) and / or plas_phosphatidylethanolamine (p-40:5), plus phosphatidylcholine (p-33:2), 1-(hydroxymethyl)-5,5-dimethyl-2,4-imidazolidinedione_exogenous, phosphatidylethanolamine (44:11), plas_phosphatidylethanolamine (o-36:5) and / or plas_phosphatidyl n-oleoylethanolamine (p-36:4), citrulline, plus phosphatidylethanolamine (o-40:6) and / or plus phosphatidylethanolamine (p-40:5), homoserine, thyroxine, n-oleoylethanolamine, plus phosphatidylcholine (o-30:1) and / or plus phosphatidylcholine (p-30:0), plus phosphatidylethanolamine (o-38:5) and / or plus phosphatidylethanolamine (p-38:4), lysophosphatidylethanolamine (18:2), nepsilon.nepsilon.nepsilon-trimethyllysine, plus phosphatidylethanolamine (o-36:5) and / or plus phosphatidylethanolamine (p-36:4), 8-methoxykynurenate, lysophosphatidylinositol (18:1), ln.gamma.-monomethylarginine, plas_phosphatidylethanolamine (o-40:7) and / or plas_phosphatidylethanolamine (p-40:6), lysophosphatidylcholine (17:2), l-asparagine, prostaglandin e2, sphingomyelin (34:0), creatine phosphate, lysophosphatidylcholine (18:2), glucosylceramide (40:1), 12(s)-hete; 15(s)-hete; 5(s)-hete, Lactosylceramide (18:1 / 16:0), plus phosphatidylethanolamine (o-38:4) and / or plus phosphatidylethanolamine (p-38:3), lactosylceramide (32:1), triacylglycerol (58:4), sphinganine, d-ribose 5-phosphate;quinate, deoxycarnitine, aspartyl-threonine, phosphatidylcholine (28:0), metformin, phosphatidylcholine (40:8), acylcarnitine (C18:0), phylloquinone, deoxycarnitine; phosphocholine, indole-3-acetaldehyde, 6-phosphogluconic acid, sphingomyelin (32:0), glycyl-threonine, acylcarnitine (C4:0), hypoxanthine, 1-methylhistidine; 3-methylhistidine; n(pai)-methyl-l-histidine, dl-5-hydroxylysine, indole, l-methionine, ascorbate, prostaglandin f2a, serotonin; serotonin, Glycerophosphocholine, 1-aminocyclopropane-1-carboxylate, phosphatidylethanolamine (36:1); phosphatidylethanolamine (36:1), ceramide (42:0), 3-amino-5-hydroxybenzoic acid, d-tryptophan; l-tryptophan, n-acetyl-dl-serine; o-acetyl-l-serine, phosphatidylinositol (43:2), triacylglycerol (47:0), ceramide (42:0) iso, ll-2.6-Diaminoheptanedioate, pyridoxal 5'-phosphate, quinoline, indoleacrylic acid, l-anserine, methylpyrazine, o-butanoyl-r-carnitine, 2'-deoxyguanosine 5'-monophosphate; adenosine 5'-monophosphate; n-acetylneuraminate, urate, ceramide (40:0), 3-(4-hydroxyphenyl)propionic acid, glycerophosphocholine; sn-glycero-3-phosphocholine, n-methyl-d-aspartate, citrate, plus lysophosphatidylethanolamine One or more metabolites selected from the group consisting of tryptophan (p-22:0), plus lysophosphatidylcholine (p-16:0), l-tryptophan, lysophosphatidylcholine (22:5), lysophosphatidylcholine (p-18:0 / 0:0) and / or lysophosphatidylcholine (o-18:1), 5'-deoxyadenosine, and combinations thereof, are measured in the individual, including in a biological sample of the individual, to determine whether the individual has a desired phenotype. The desired phenotype may be developing or non-developing CRS after cell therapy is administered, including any cell therapy encompassed herein.

[0061] In some embodiments, diacetylspermine, triacylglycerol (51:6), (3'-sulfo)galβ-cer(d18:1 / 18:0(2OH)), phosphatidylcholine (35:2), phosphatidylcholine (33:3), phosphatidylcholine (34:4), triacylglycerol (51:5), 4-pyridoxate, phosphatidylcholine (32:2), thymidine-5'-diphospho-alpha-d-glucose, nicotinamide mononucleotide, phosphatidylcholine (33:2), cl( 1'-[15:0 / 15:0],3'-[15:0 / 16:1(9z)])[rac], plus phosphatidylethanolamine (o-38:4) and / or plus phosphatidylethanolamine (p-38:3); plus phosphatidylethanolamine (o-40:7) and / or plus phosphatidylethanolamine (p-40:6), free fatty acids (14:1) (myristolidic acid); free fatty acids (14:1) (myristoleic acid), 3-cis-hydroxy-b,e-carotene-3'-one, phosphatidylethanolamine (p-40:6), cis-Quinceoxepane, plus phosphatidylcholine (33:1), phosphatidylcholine (31:0), triacylglycerol (51:4), triacylglycerol (56:4), plus phosphatidylethanolamine (o-34:3) and / or plus phosphatidylethanolamine (p-34:2), galactosylceramide (36:1) and / or glucosylceramide (36:1), cis-Quinceoxepane, plus phosphatidylethanolamine (o-38:5) and / or plus phosphatidylethanolamine (p-38 :4), virtual_kdnalpha2-3galbeta1-4glcbeta-cer(d18:1 / 24:0), plus phosphatidylethanolamine(o-36:5) and / or plus phosphatidylethanolamine(p-36:4), 1,2-didecanoyl-sn-glycero-3-phosphocholine, lysophosphatidylinositol(18:1), triacylglycerol(16:0_16:1_18:2), triacylglycerol(51:3), triacylglycerol(55:7), (r,r)-tartaric acid;(S,S)-Tartaric acid, deoxyuridine, galβ1-3galβ1-3galβ1-3galβ1-3galβ1-3galβ1-4galβ1-4glcβ-cer (42:2), plus phosphatidylethanolamine (o-38:4) and / or plus phosphatidylethanolamine (p-38:3), lactosylceramide (32:1), ceramide (42:1) iso, sphinganine, d-ribose 5-phosphate;quinate, triacylglycerol (49:3), deoxycarnitine, n-alpha-acetyl-l-asparagine, phosphatidylcholine (40:8), phylloquinone, acetylcholine; deoxycarnitine, ceramide (43:1), triacylglycerol One or more metabolites selected from the group consisting of lysophosphatidylcholine (49:2), phosphatidylcholine (35:4), d-glucono-1.5-lactone, lysophosphatidylcholine (15:0), triacylglycerol (56:3), lysophosphatidylcholine (o-16:2(9E,10E) / 0:0)[u], ceramide (42:0), sphingomyelin (42:1), ceramide (42:0)iso, ceramide (40:0), n-methyl-d-aspartic acid, l-carnitine, triacylglycerol (61:6), and combinations thereof are measured in the individual, including in a biological sample of the individual, to determine whether the individual has a desired phenotype. The desired phenotype may be developing or non-developing ICANS after cell therapy is administered, including any cell therapy encompassed herein.;

[0062] The biological sample may include any biological sample from an individual suitable for measuring metabolites. The biological sample may include a blood sample (including whole blood, serum and / or plasma), a urine sample, a saliva sample, a biopsy, a tissue sample, a tumor sample, a cerebrospinal fluid sample, or a combination thereof. IV. Sample preparation

[0063] In certain aspects, the method requires obtaining a sample from the subject. The method may include obtaining a blood sample. In some embodiments, the methods provided herein include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excision biopsy, punch biopsy, scraping biopsy or skin biopsy. In other embodiments, the sample may be obtained from any of the tissues provided herein, including but not limited to non-cancerous or cancerous tissues, i.e., non-cancerous or cancerous tissues from serum, gallbladder, mucosa, skin, heart, lung, breast, pancreas, blood, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus or thyroid tissue. In the alternative, the sample may be obtained from any source or other, including but not limited to blood, sweat, hair follicle, buccal tissue, tears, menses, stool or saliva. In certain aspects of the current method, any medical professional, such as a doctor, nurse or medical technician, may obtain the biological sample for testing. Furthermore, such biological samples may be obtained without the assistance of a medical professional.

[0064] A sample may include, but is not limited to, tissue, cells, or biological material from or derived from a subject's cells. A biological sample may be a heterogeneous or homogeneous population of cells or tissue. A biological sample may be obtained using any method known in the art that can provide a sample suitable for the analytical methods described herein. A sample may be obtained by non-invasive methods, including, but not limited to, skin or cervical scraping, buccal swabbing, saliva collection, urine collection, stool collection, menstrual, tear or semen collection.

[0065] The sample may be obtained by methods known in the art. In certain embodiments, the sample is obtained by biopsy. In other embodiments, the sample is obtained by swabbing, endoscopy, scraping, venisection, or any other method known in the art. In some cases, the sample may be obtained using the components of the kit of the method, or may be stored, or may be transported. In some cases, multiple samples, such as multiple esophageal samples, may be obtained for diagnosis by the methods described herein. In other cases, multiple samples, such as one or more samples from one tissue type (e.g., esophagus) and one or more samples from another specimen (e.g., serum), may be obtained for diagnosis by the methods described herein. In some cases, multiple samples, such as one or more samples from one tissue type (e.g., esophagus) and one or more samples from another specimen (e.g., serum), may be obtained at the same time or at different times. The various samples may be obtained and stored at different times and / or analyzed by different methods. For example, a sample may be obtained and analyzed by routine staining methods, or any other method of cytological analysis.

[0066] In some embodiments, the biological sample may be obtained by a physician, nurse, or other medical professional, such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or pulmonologist. The medical professional may prescribe the appropriate test or assay to perform on the sample. In certain aspects, the molecular profiling entity may be consulted as to which assay or test is most appropriately prescribe. In further aspects of the current method, the patient or subject may obtain the biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a stool sample, a buccal sample, or a saliva sample.

[0067] In other cases, samples are obtained by invasive procedures including, but not limited to, biopsy, needle aspiration, endoscopy or venisection. Methods of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy or large core biopsy. In some embodiments, multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.

[0068] The general method for obtaining biological samples is also known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001 (which is incorporated herein by reference in its entirety) describe the general method for biopsy and cytological methods. In one embodiment, the sample is a fine needle aspirate of esophageal tumor or neoplasm or suspected esophageal tumor or neoplasm. In some cases, the fine needle aspirate sample collection procedure may be guided by the use of ultrasound, X-ray, or other imaging devices.

[0069] In some embodiments of the method, the molecular profiling entity may obtain the biological sample directly from the subject, from a medical professional, from a third party, or from a kit provided by the molecular profiling entity or a third party. In some cases, the biological sample may be obtained by the molecular profiling entity after the subject, a medical professional, or a third party obtains the biological sample and sends it to the molecular profiling entity. In some cases, the molecular profiling entity may provide suitable containers and excipients for storage and transport of the biological sample to the molecular profiling entity.

[0070] In some embodiments of the methods described herein, a medical professional need not be involved in the initial diagnosis or sample acquisition. Instead, an individual may obtain a sample through the use of an over-the-counter (OTC) kit. An OTC kit may contain a means for obtaining the sample as described herein, a means for storing the sample for testing, and instructions for the appropriate use of the kit. In some cases, a molecular profiling service fee is included in the purchase price of the kit. In other cases, a molecular profiling service fee is charged separately. A sample suitable for use by a molecular profiling business may be any material that contains tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Various methods are provided for determining the suitability and / or appropriateness of a sample.

[0071] In some embodiments, the subject may be referred to a specialist, such as an oncologist, surgeon, or endocrinologist. The specialist may also obtain a biological sample for testing, or may refer the individual to a testing center or laboratory for sending a biological sample. In some cases, a medical professional may refer the subject to a testing center or laboratory for sending a biological sample. In other cases, the subject may provide the sample. In some cases, a molecular profiling entity may obtain the sample. V. Cell therapy

[0072] Certain embodiments include one or more cell therapies. In some embodiments, the cell therapy includes cells that include at least one engineered receptor. The engineered receptor may include a chimeric antigen receptor (CAR), including one directed to any cancer antigen, and in some cases, the CAR is an anti-CD19 CAR. The engineered receptor may include a protein, including an enzyme and / or a transcription factor, that can increase or decrease one or more metabolites inside and / or outside the cell. The cell therapy may be useful in treating one or more cancers, including hematological cancers, such as leukemia or lymphoma, including at least B-cell lymphoma. In some embodiments, the cell therapy targets cancer cells. In some embodiments, the cell therapy targets CD19, such as by including an anti-CD19 CAR. In some embodiments, the cell therapy includes T cells, including T cells that include one or more CARs.

[0073] In some embodiments, the cell therapy includes cells that contain one or more enzymes that can increase or decrease at least one metabolic product, including any metabolic product encompassed herein. The cells may be genetically engineered or modified to increase or decrease expression of the enzyme(s). The enzymes may be capable of increasing or decreasing metabolic products in an individual, including any individual encompassed herein, such as individuals with responder or non-responder phenotypes. In some embodiments, the enzymes are endogenous to the cells. In some embodiments, the enzymes are exogenous to the cells. In some embodiments, the cells that contain the enzymes also include an engineered receptor. In some embodiments, the cells that contain the enzymes do not include an engineered receptor. The cells may be engineered by the human hand to express one or more CARs and one or more proteins that can decrease or increase the level of one or more metabolic products in the recipient individual.

[0074] Certain embodiments relate to cells comprising a polypeptide or nucleic acid of the disclosure. In some embodiments, the cell is an immune cell. The immune cell may be any type of immune cell, including any cell expressing CD3, such as a T helper cell, an invariant natural killer T (iNKT) cell, a cytotoxic T cell, a regulatory T cell (Treg), a gamma delta T cell, a natural killer (NK) T cell, a neutrophil, or a macrophage. In some embodiments, the cell comprises a T cell, such as a CD4+ T cell or a CD8+ T cell.

[0075] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, and CRL9096), human embryonic kidney (HEK) 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0076] In some cases, the cell is not an immortalized cell line, but instead is a cell (e.g., a primary cell) obtained from an individual.For example, in some cases, the cell is an immune cell obtained from an individual.As an example, the cell is a T lymphocyte obtained from an individual.As another example, the cell is a cytotoxic cell obtained from an individual.As another example, the cell is a stem cell (e.g., a peripheral blood stem cell) or a progenitor cell obtained from an individual. VI. Chimeric Antigen Receptors (CARs)

[0077] Embodiments of the present disclosure include cell therapies in which cells express one or more CARs. In some embodiments, the CAR comprises a) one or more intracellular signaling domains, b) a transmembrane domain, and c) an extracellular domain that comprises one or more antigen binding regions.

[0078] In some embodiments, engineered antigen receptors include CARs for activation, or various CARs, including stimulatory CARs, costimulatory CARs (see International Publication WO2014 / 055668), and / or inhibitory CARs (iCARs, see Fedorov et al. (2013)). CARs generally include an extracellular antigen (or ligand) binding domain that is linked to one or more intracellular signaling components, in some aspects, via a linker and / or transmembrane domain(s). Such molecules typically mimic or approximate the signaling through a natural antigen receptor, the signaling through such receptor in combination with a costimulatory receptor, and / or the signaling through a costimulatory receptor alone.

[0079] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding antigen-specific CAR polypeptides, including in some cases humanized CARs (hCARs) that include an intracellular signaling domain, a transmembrane domain, and an extracellular domain that includes one or more signaling motifs, to reduce immunogenicity. In certain embodiments, the binding region can include a complementarity determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0080] It is intended that the human CAR nucleic acid may be a human gene used to enhance cellular immunotherapy for human patients. In a specific embodiment, the disclosure includes a cDNA or coding region of a full-length CAR, and also includes a vector encoding the CAR. In some cases, the antigen-binding region or domain is a V of a single chain variable fragment (scFv) derived from a particular human monoclonal antibody, such as the human monoclonal antibody described in U.S. Pat. No. 7,109,304, which is incorporated herein by reference. H Chain and V LThe fragments may include fragments of the chains. The fragments can also be many different antigen-binding domains of human antigen-specific antibodies. In a more specific embodiment, the fragments are antigen-specific scFvs encoded by sequences optimized for human codon usage for expression in human cells. The arrangement could be in a multimeric form, such as a diabody or multimer. The multimers are likely formed by cross-pairing the variable portions of the light and heavy chains into a diabody. The hinge portion of the construct can have a number of alternatives, from being completely deleted, to maintaining the first cysteine, to making a proline instead of a serine substitution, to being truncated to the first cysteine. The Fc portion can be deleted. Any protein that is stable and / or dimerizes can serve this purpose. Only one of the Fc domains could be used, for example, either the CH2 or CH3 domain from a human immunoglobulin. Hinge, CH2 and CH3 regions of human immunoglobulins that have been modified to improve dimerization could also be used. Only the hinge portion of an immunoglobulin could also be used. A portion of CD8 alpha could also be used.

[0081] In some embodiments, CARs are constructed that have specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive therapy (e.g., a cancer antigen) and / or an antigen intended to induce a weakened response (e.g., an antigen expressed on the surface of a normal or non-disease cell type). Thus, CARs typically include one or more antigen-binding molecules, such as one or more antigen-binding fragments, antigen-binding domains or portions thereof, or one or more antibody variable domains, and / or antibody molecules, in their extracellular portion. In some embodiments, CARs include antigen-binding portion(s) of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0082] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (which may be referred to as the extracellular domain that includes the antigen-binding region) comprises a cancer-associated antigen binding domain or a pathogen-specific antigen binding domain. The cancer-associated antigen may be of any type, so long as it is expressed on the cell surface of the cancer cell. Exemplary embodiments of antigens include CD19, CD70, HLA-G, CD38, CD123, CLL1, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, carcinoembryonic antigen, alpha fetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD3 ... ERV-K, IL-11R alpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A1 0, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, DAM-10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, NA88-A, MC1R, mda-7, gp75, Gp100, PSA, PSM, tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC 1, MUC2, phosphoinositide 3-kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP,Annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signaling factor 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducer and activator of transcription STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-kappa B (NF-B), Notch receptors (e.g., Notch1-4), NY ESO 1, c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and its regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma metastasis breakpoint, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 and ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, Fucosyl GM1, Mesothelial, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, and LRRN1, or a combination thereof.

[0083] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA or cDNA sources, or can be synthesized (e.g., via PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, since introns have been found to stabilize mRNA. It may also be more advantageous to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0084] It is intended that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector.Various methods are known in the art for stably transfecting cells by electroporation using naked DNA.See, for example, U.S. Patent No. 6,410,319.Naked DNA generally refers to the DNA encoding the chimeric receptor contained in a plasmid expression vector in the appropriate orientation for expression.

[0085] Alternatively, viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors or lentiviral vectors) can be used to introduce chimeric constructs into immune cells.Suitable vectors for use according to the method of the present disclosure are non-replicative in immune cells.Many virus-based vectors are known, and the copy number of the virus maintained in cells is sufficiently low to maintain cell viability, such as, for example, the vectors based on HIV, SV40, EBV, HSV or BPV.

[0086] In some aspects, the antigen-specific binding or recognition component is linked to one or more transmembrane domains and intracellular signaling domains.In some embodiments, CAR comprises a transmembrane domain that is fused to the extracellular domain of the CAR.In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used.Optionally, the transmembrane domain is selected or modified by amino acid substitution to avoid such domain binding to the transmembrane domain of the same or different surface membrane proteins, and to minimize interaction with other components of the receptor complex.

[0087] The transmembrane domain, in some embodiments, is derived from either natural or synthetic sources. If the source is natural, the domain is in some aspects derived from any membrane-bound or transmembrane protein. Transmembrane regions include transmembrane regions from the alpha, beta or zeta chains of the T cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D and DAP molecules (i.e. , the transmembrane region includes at least the transmembrane region(s) of the alpha, beta or zeta chain of a cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D and DAP molecules). Alternatively, the transmembrane domain is synthetic in some embodiments. In some aspects, the synthetic transmembrane domain contains primarily hydrophobic residues, such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain.

[0088] Any costimulatory domain, including one or more costimulatory domains, such as at least those from CD28, 4-1BB, OX40, CD27, and others, may be utilized in the CAR. VII. Genetic Engineering

[0089] Certain aspects of the present disclosure relate to methods and compositions for gene editing (also "genetic engineering"), which are useful for generating one or more genetic modifications in cells. As used herein, "genetic modification" describes a genomic region of a cell that is modified from its native (i.e., endogenous) sequence. Various methods and systems for gene editing are known in the art, including, for example, zinc finger nuclease (ZFN)-based gene editing, transcription activator-like effector nuclease (TALEN)-based gene editing, and CRISPR / Cas-based gene editing. In some embodiments, the methods of the present disclosure include CRISPR / Cas-based gene editing, which includes the use of components of the CRISPR system, such as guide RNA (gRNA) and Cas nuclease.

[0090] In general, a "CRISPR system" collectively refers to transcripts and other elements involved in expression of or directing activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active portion tracrRNA), tracr-mate sequences ("direct repeats", which in the context of an endogenous CRISPR system encompass tracrRNA processed portion direct repeats), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), and / or other sequences and transcripts from the CRISPR locus.

[0091] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner, and a Cas protein (e.g., Cas9) that has nuclease functionality (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes.

[0092] In some embodiments, Cas nuclease and gRNA (including a fusion of a crRNA specific for a target sequence and a fixed tracrRNA) are introduced into a cell. Cas nuclease and gRNA can be indirectly introduced into a cell by introduction of one or more nucleic acids (e.g., vectors) encoding Cas nuclease and / or gRNA. Cas nuclease and gRNA can be directly introduced into a cell by introduction of Cas nuclease protein and gRNA molecule. Generally, a target site at the 5' end of the gRNA targets the Cas nuclease to a target site, e.g., a gene, using complementary base pairing. The target site can be selected based on its position, typically immediately 5' of a protospacer adjacent motif (PAM) sequence, such as NGG or NAG. In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, CRISPR system is characterized by the element that promotes the formation of CRISPR complex at the site of target sequence.Typically, "target sequence" generally refers to the sequence that guide sequence is designed to have complementarity with, and the hybridization between target sequence and guide sequence promotes the formation of CRISPR complex.Full complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of CRISPR complex.

[0093] The CRISPR system can induce double-strand breaks (DSBs) at target sites and subsequently disruption, as discussed herein. In other embodiments, Cas9 variants considered to be "nickases" are used to nick single strands at target sites. Paired nickases can be used, for example, to improve specificity, each guided by a pair of different gRNA targeting sequences, such that a 5' overhang is introduced when a nick is introduced simultaneously. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or activator, to affect gene expression.

[0094] The target sequence can comprise any polynucleotide, for example, DNA or RNA polynucleotide. The target sequence can be located in the nucleus or cytoplasm of a cell, for example, in an organelle of a cell. In general, the sequence or template that can be used for recombination into the target locus that comprises the target sequence is referred to as "editing template" or "editing polynucleotide" or "editing sequence". In some embodiments, the exogenous template polynucleotide may be referred to as editing template. In some embodiments, the recombination is homologous recombination.

[0095] Typically, in the context of an endogenous CRISPR system, the formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in the cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the target sequence). Also, the tracr sequence may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence), but may form part of a CRISPR complex, for example, by hybridization with all or a portion of a tracr mate sequence operably linked to the guide sequence along at least a portion of the tracr sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence (such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned) to hybridize and participate in the formation of a CRISPR complex.

[0096] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into a cell, such that the expression of the elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. Also, the components can be delivered to the cell as proteins and / or RNA. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements can be combined in a single vector with one or more additional vectors that provide any components of the CRISPR system that are not included in the first vector. The vector can include one or more insertion sites (also referred to as "cloning sites"), such as restriction endonuclease recognition sequences. In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences in a cell.

[0097] The vector may include a regulatory element operably linked to an enzyme coding sequence encoding a Cas protein (also called a Cas nuclease). Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12a (Cpf1), Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csx1 ... Examples of Cas9 enzymes include sm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, their homologs, or modified versions thereof. These enzymes are known. For example, the amino acid sequence of S. pyogenes Cas9 protein can be found in the SwissProt database under the accession number Q99ZW2.

[0098] The Cas nuclease may be Cas9 (e.g., from S. pyogenes or S. pneumonia). The Cas nuclease may be Cas12a. The Cas nuclease may direct the cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. The vector may encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartic acid to alanine substitution (D10A) in the RuvCI catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves a single strand). In some embodiments, Cas9 nickases can be used in combination with guide sequences, such as two guide sequences, that target the sense and antisense strands of a DNA target, respectively, allowing both strands to be nicked and used to induce NHEJ or HDR.

[0099] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be of or derived from a particular organism, such as a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence. Different species show a particular bias for a particular codon of a particular amino acid. Codon bias (the difference in codon usage between organisms) often correlates with the translation efficiency of messenger RNA (mRNA), which is believed to depend, among other things, on the properties of the codon to be translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of a selected tRNA in a cell is generally a reflection of the codon that is most frequently used in peptide synthesis. Thus, based on codon optimization, genes can be tailored for optimal gene expression in a given organism.

[0100] In general, a guide sequence is any polybase sequence that has sufficient complementarity with a target polybase sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0101] Optimal alignment can be determined by use of any algorithm suitable for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0102] Cas nuclease can be part of a fusion protein that includes one or more heterologous protein domains. Cas nuclease fusion protein can include any additional protein sequence and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to Cas nuclease include, but are not limited to, epitope tags, reporter gene sequences, and protein domains that have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), autofluorescent proteins including HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). Cas nucleases can be fused to gene sequences encoding proteins or fragments of proteins that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tags, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can form part of fusion proteins containing Cas nucleases are described in U.S. Patent Application Publication No. 2011 / 0059502, which is incorporated herein by reference. VIII. Administration of Therapeutic Compositions

[0103] Certain embodiments encompass administration of one or more therapeutic compositions (which may be referred to as "pharmaceutical compositions"), including after measuring one or more metabolites. In some embodiments, the therapeutic composition is administered to the individual before or after the individual analyzes one or more samples for the level of one or more metabolites. Different aspects may include administering an effective amount of the composition to the individual. In some embodiments, at least one cell therapy, such as any cell therapy herein, may be administered to the individual to protect against or treat a condition (e.g., cancer). In some embodiments, one or more metabolites are administered to the individual to protect against or treat a condition or toxicity. Any cell therapy may be administered to the individual in combination, including sequentially or simultaneously. In some cases, CAR therapy is provided following and / or prior to and / or during administration of one or more metabolites, including one or more metabolites whose levels are determined to be deficient based on analysis of the individual's sample. Any therapeutic composition may be administered in combination with one or more additional therapeutic agents (e.g., one or more chemotherapeutic agents, one or more immunotherapeutic agents, one or more biotherapeutic agents, combinations thereof, etc.). Such compositions are generally dissolved or dispersed in a pharma- ceutically acceptable carrier or aqueous medium.

[0104] In some embodiments, the cell therapy is administered to an individual at a dose between 1 million cells / kg and 1 billion cells / kg. In some embodiments, the cell therapy is administered to an individual at the following doses: The cells are administered to an individual at doses of approximately 1 million cells / kg, 2 million cells / kg, 3 million cells / kg, 4 million cells / kg, 5 million cells / kg, 6 million cells / kg, 7 million cells / kg, 8 million cells / kg, 9 million cells / kg, 10 million cells / kg, 11 million cells / kg, 12 million cells / kg, 13 million cells / kg, 14 million cells / kg, 15 million cells / kg, 16 million cells / kg,17 million pieces / kg, 18 million pieces / kg, 19 million pieces / kg, 20 million pieces / kg, 21 million pieces / kg, 22 million pieces / kg, 23 million pieces / kg, 24 million pieces / kg, 25 million pieces / kg, 26 million pieces / kg, 27 million pieces / kg, 28 million pieces / kg, 29 million pieces / kg, 30 million pieces / kg, 31 million pieces / kg, 32 million pieces / kg, 33 million pieces / kg 34 million pieces / kg, 35 million pieces / kg, 36 million pieces / kg, 37 million pieces / kg, 38 million pieces / kg, 39 million pieces / kg, 40 million pieces / kg, 41 million pieces / kg, 42 million pieces / kg, 43 million pieces / kg, 44 million pieces / kg, 45 million pieces / kg, 46 million pieces / kg, 47 million pieces / kg, 48 million pieces / kg, 49 million pieces / kg, 50 million pieces / kg, 51 million pieces / kg, 52 million pieces / kg, 53 million pieces / kg, 54 million pieces / kg, 55 million pieces / kg, 56 million pieces / kg, 57 million pieces / kg, 58 million pieces / kg, 59 million pieces / kg, 60 million pieces / kg, 61 million pieces / kg, 62 million pieces / kg, 63 million pieces / kg, 64 million pieces / kg 65 million pieces / kg, 66 million pieces / kg, 67 million pieces / kg, 68 million pieces / kg, 69 million pieces / kg, 70 million pieces / kg, 71 million pieces / kg, 72 million pieces / kg, 73 million pieces / kg, 74 million pieces / kg, 75 million pieces / kg, 76 million pieces / kg, 77 million pieces / kg, 78 million pieces / kg, 79 million pieces / kg 80 million / kg, 81 million / kg, 82 million / kg, 83 million / kg, 84 million / kg, 85 million / kg, 86 million / kg, 87 million / kg, 88 million / kg, 89 million / kg, 90 million / kg, 91 million / kg, 92 million / kg, 93 million / kg, 94 million / kg 95 million / kg, 96 million / kg, 97 million / kg, 98 million / kg, 99 million / kg, 100 million / kg, 200 million / kg, 300 million / kg, 400 million / kg, 500 million / kg, 600 million / kg, 700 million / kg, 800 million / kg, 900 million / kg, or 1 billion / kg.

[0105] The phrases "pharmacologically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carriers" include any solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonicity agents and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except in the case where a conventional medium or agent is incompatible with the active ingredient, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the composition.

[0106] Active compound can be formulated for parenteral administration, for example, can be formulated for injection via intravenous, intramuscular, subcutaneous or intraperitoneal route.Typically, such compositions can be prepared as either liquid solution or suspension;solid forms suitable for use in preparing solution or suspension by adding liquid before injection can also be prepared;and preparations can also be emulsified.

[0107] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations, for example, containing aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the formulation must be sterile and fluid to the extent that easy syringability exists. It must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0108] The composition can be formulated in neutral or salt form.Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. The salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0109] The pharmaceutical composition may contain a solvent or dispersion medium, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating agent such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugars or sodium chloride. Prolonged absorption of the injectable composition can be brought about by the use in the composition of an absorption delaying agent, for example, aluminum monostearate or gelatin.

[0110] Sterile injectable solution is prepared by incorporating the required amount of active compound into a suitable solvent, together with various other ingredients as listed above as necessary, and then carrying out filtration sterilization or equivalent procedures.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other ingredients as listed above as required.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which obtains a powder of active ingredient and any additional desired ingredient from the solution previously sterile filtered.

[0111] The composition is typically administered by any common route, including, but not limited to, oral or intravenous administration. Alternatively, administration may be orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal. Such compositions will usually be administered as pharma- ceutically acceptable compositions that include physiologically acceptable carriers, buffers, or other excipients.

[0112] The formulated solutions will be administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above. IX. Kit

[0113] Certain aspects of the present disclosure also relate to kits that contain compositions of the present disclosure or compositions for carrying out the methods disclosed herein. In some embodiments, various kits can be used to evaluate one or more metabolites. In specific embodiments, the kits include one or more metabolites or one or more reagents for producing one or more metabolites. The kits may include means for obtaining or analyzing blood, whatever the means may be. The kits may include vectors, of any kind, including viral (retroviral, lentiviral, adenoviral or adeno-associated viral) or non-viral (plasmid, transposon, etc.), such as vectors that encode part or all of the CAR and / or one or more enzymes that increase or decrease the level of metabolite(s). In certain embodiments, the kit includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more labels, probes, synthetic molecules or In one embodiment, the label or probe comprises a number of inhibitors, reagents, eluents, standards, or whatever values ​​or ranges and combinations that may be derived therein, or comprises at least such a number of labels, probes, synthetic molecules or inhibitors, reagents, eluents, standards, or whatever values ​​or ranges and combinations that may be derived therein, or comprises at most such a number of labels, probes, synthetic molecules or inhibitors, reagents, eluents, standards, or whatever values ​​or ranges and combinations that may be derived therein.

[0114] The kits may include various components that may be individually packaged or placed in containers (such as, for example, tubes, bottles, vials, syringes, or other suitable container means). Individual components may also be provided in the kit in concentrated amounts, and in some embodiments, components are provided individually at the same concentration as they would be in solution with the other components. Concentrations of components may be provided as 1x, 2x, 5x, 10x, or 20x or more.

[0115] Included as part of this disclosure are various kits for using the disclosed probes, reagents, synthetic molecules or inhibitors, eluents, standards for prognostic or diagnostic applications. Any such molecule that corresponds to any metabolite identified herein is specifically contemplated.

[0116] An embodiment of the present disclosure includes a kit for analyzing a pathological sample by evaluating a metabolite profile for the sample, comprising: The kit may further include a reagent for labeling metabolites in the sample. The kit may also include a labeling reagent. The labeling reagent may include, for example, an amine-reactive dye. EXAMPLES

[0117] The following examples are included to clarify certain embodiments of the present invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques found by the inventors to function well in the implementation of the disclosed method, and therefore can be considered to constitute preferred modes for its implementation. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the disclosed specific embodiments, and that many changes can still produce the same or similar results without departing from the spirit and scope of the present disclosure. Example 1: Metabolomics predicts durable complete response and toxicity

[0118] Baseline serum and plasma samples were collected from patients with relapsed / refractory large B-cell lymphoma treated with anti-CD19 CAR-T therapy. Metabolomic profiling was performed on baseline samples to identify associations with response, survival outcomes, and toxicity. As shown in Figure 3, certain plasma metabolites, including taurine, 1-methylnicotinamide, diacetylspermine, Ng-Ng-dimethyl-L-arginine, nicotinamide and N8-acetylspermidine, TMAO, indoxyl sulfate, and acetylcadaverine, were associated with poor or no complete response. Polyamines and lysophospholipids were also highly represented among metabolites prognostic for either PFS or OS. Elevated levels of polyamines acetylspermidine (AcSpmd), diacetylspermidine (DiAcSpmd) and diacetylspermine (DAS) were found to be associated with poorer PFS and OS, whereas high levels of various species of lysophospholipids were inversely associated with better prognosis. Plasma samples from n=43 patients were collected at baseline before CAR-T infusion, and untargeted metabolomic analysis was performed using a Xevo GS-X2 quadrupole time-of-flight (TOF) mass spectrometer (MS) under standardized operating procedures. Figure 3 reveals a volcano plot illustrating the area under the receiver operating characteristic curve (AUC) for each annotated metabolite (x-axis) and the -log(two-tailed Wilcoxon rank sum test p-value) (y-axis) for distinguishing patients with durable CR at 6 months follow-up from those without. Increased polyamines were associated with poor response in patients undergoing CAR-T therapy. In particular, increased 1-methylnicotinamide and other correlated polyamines (such as diacetylspermine and N8-acetylspermine) have been associated with poor responses. Additionally, taurine and other nucleotides have been associated with increased responses. Taurine has been shown to correlate with lymphocyte proliferation. High levels of lysophospholipids, especially odd-chain fatty acids, have been associated with good responses.

[0119] As shown in Figure 1, certain metabolites, including indoleacrylic acid and 6-phosphogluconic acid, are predictive of cytokine release syndrome (CRS). Plasma samples from n=43 patients were collected at baseline before CAR-T infusion, and untargeted metabolomic analysis was performed using a Xevo GS-X2 quadrupole time-of-flight (TOF) mass spectrometer (MS) under standardized operating procedures. Figure 1 reveals a volcano plot illustrating the area under the receiver operating characteristic curve (AUC) for each annotated metabolite (x-axis) and the -log(two-tailed Wilcoxon rank sum test p-value) (y-axis) for distinguishing patients with CR from those without at 6 months follow-up.

[0120] As shown in Figure 2, certain metabolites, including deoxycarnitine, triacylglycerol, and N-alpha-L-acetyl-L-asparagine, are predictive of immune effector cell-associated neurotoxicity syndrome (ICANS). Plasma samples from n=43 patients were collected at baseline before CAR-T infusion, and untargeted metabolomic analysis was performed using a Xevo GS-X2 quadrupole time-of-flight (TOF) mass spectrometer (MS) under standardized operating procedures. Figure 2 reveals a volcano plot illustrating the area under the receiver operating characteristic curve (AUC) for each annotated metabolite (x-axis) and the -log(two-tailed Wilcoxon rank sum test p-value) (y-axis) for distinguishing patients with ICANS from those without at 6 months follow-up. Example 2: Panel for predicting response to CAR-T therapy

[0121] Focusing on polyamines and lysophospholipids, a Cox proportional hazards model with LASSO regularization was used to select features for predicting PFS and to develop a six-marker metabolite panel (M6P) for predicting PFS, consisting of AcSpmd, DiAcSpmd and four lysophospholipids (lysophosphatidylcholine (16:0), lysophosphatidylcholine (14:0), plasmanyllysophosphatidycholines (P-18:0 or O-18:1) and plasmanyl-lysophosphatidycholine (P-18:1 or O-18:2)). In a multivariate Cox proportional hazards model, adjusting for other significant (two-sided p<0.05) variables, M6P score gave a hazard ratio (HR) of 3.65 (95%CI: 1.38-9.67) per unit increase. Metabolite abundance was assayed using mass spectrometry. In summary, it is shown in the embodiments herein that plasma metabolomics is a strong predictor of durability of response and survival and toxicity after CAR-T cell therapy. Modulation of metabolite levels in plasma has great potential for influencing efficacy and toxicity after CAR-T cell therapy. Example 3: Blood-based metabolite signature predictive of aggressive phenotype of B-cell lymphoma and poor response to CAR-T therapy

[0122] Identification of metabolic biomarkers predictive of CAR-T response and model development

[0123] A discovery cohort consisting of plasma collected from 43 patients with r / r LBCL treated with anti-CD19 CAR-T therapy was assembled to profile metabolite signatures predictive of response to CAR-T therapy. Patient and tumor characteristics are provided in Table 1. Axicabtagene ciloleucel and tisagenlecleucel were the CAR-T products for 39 and 4 patients, respectively. No statistical differences were noted between the two groups in age, sex, stage, CRS or ICANs status, circulating levels of LDH or CRP (Vercellino et al., 2020) (Table 1). Three patients died early due to toxicity before response could be evaluated. Overall, among the 40 evaluable patients, 15 (37%) of the 40 patients had ongoing complete response (CR) and 24 (63%) had progressive disease (PD) at 6 months follow-up, respectively. One remaining patient continued to have an ongoing partial response (PR) at 6 months of follow-up.

[0124] Multi-assay untargeted metabolomic analysis of these plasma samples yielded 746 uniquely annotated metabolite signatures. Associations were screened for quantified metabolites with progression-free and overall survival using Cox proportional hazards models. Among metabolites prognostic for either PFS or OS, polyamines and lysophospholipids were highly represented. Elevated levels of the polyamines acetylspermidine (AcSpmd), diacetylspermidine (DiAcSpmd) and diacetylspermine (DAS) were found to be associated with poorer progression-free and overall survival, whereas high levels of various species of lysophospholipids were inversely associated with better prognosis.

[0125] Elevated levels of circulating polyamines have been reported in cancer states and have been shown to be associated with disease aggressiveness (Fahrmann, Bantis et al., 2019; Fahrmann et al., 2021; Fahrmann Vykoukal et al., 2019). Lysophospholipids, particularly lysophosphatidylcholine, are bioactive lipids that are captured and metabolized by cancer cells to promote their growth (Fahrmann, Bantis et al., 2019; Kamphorst et al., 2013; Raynor et al., 2015). Reduced levels of lysophospholipids are frequently reported in the plasma of individuals presenting with various malignancies, including pancreatic, lung, ovarian and colorectal cancer (Fahrmann, Bantis et al., 2019; Kuehn et al., 2016; Zhao et al., 2007).

[0126] Focusing on polyamines and lysophospholipids, a Cox proportional hazards model with LASSO regularization was used to select features for predicting progression-free survival and to develop a biomarker panel for predicting progression-free survival. The resulting six-marker panel (6MetP), consisting of AcSpmd, DiAcSpmd and four lysophospholipids, gave a hazard ratio (HR) of 4.53 (95% CI: 2.07-9.92) per unit increase for PFS in a univariate Cox proportional hazards model (Figure 7). Non-proportional hazards model tests gave statistically insignificant P values. 6MetP had an area under the receiver operating characteristic curve of 0.79 (95% CI: 0.65-0.93) with a sensitivity of 40% at 95% specificity for discriminating individuals who were subsequently non-responsive to CAR-T from those who were responsive to the treatment (Figure 7). Next, using the log-rank test statistic from the Cox model, the optimal change-point was calculated to give the maximum difference in 6MetP between individuals in the two previously defined groups (progressive vs. non-progressive disease). Kaplan-Meier survival curves revealed that patients with 6MP scores above the change-point value had statistically significant (log-rank-Mantel-Cox test, two-sided p<0.0001) worse PFS and worse OS (Figure 4A and Figure 4B). Testing a metabolite biomarker panel in an independent study set of individuals undergoing CAR-T therapy

[0127] Testing of individual candidate metabolites as well as the 6MP panel using established coefficients and change-point values ​​developed in the discovery set to predict PFS after anti-CD19 CAR-T treatment was performed in an independent study set of 28 patients with r / rLBCL (Table 1). The median duration of follow-up in the validation cohort was 12 months (range 0.3-24.8 months). In the validation set, 11 of 28 patients (39%) had ongoing CR at 6 months of follow-up, whereas the remaining 17 patients (61%) had PD or died. The median PFS and median OS among non-responders were 2.9 and 8.25 months, respectively (Figure 6). No statistically significant differences were observed between the two groups with respect to age, sex, stage, CRS or ICANs status, circulating levels of LDH or CRP. Patients who were non-responsive to CAR-T treatment tended to have a higher (2+) ECOG status (2-sided Fisher's exact test p: 0.04) (Table 1).

[0128] In the test set, elevated plasma polyamines were associated with poor PFS and OS, whereas elevated lysophospholipids were a good prognostic indicator (Figure 3). Confirmed 6MetP gave an AUC of 0.71 (95% CI: 0.52-0.90) with a sensitivity of 41% at 95% specificity for discriminating patients who subsequently did not respond to treatment from those who had an objective response. A 6MetP score above the change-point value established in the discovery set was found to be a statistically significant (log-rank-Mantel-Cox two-sided p<0.05) prognostic indicator of poorer PFS and OS in the test set (Figures 4A and 4B). Elevated mRNA expression of spermidine synthase and adenosylmethionine decarboxylase 1 is associated with poor overall survival in patients with B-cell lymphoma.

[0129] Analysis revealed that elevated levels of circulating polyamines were prognostic for poor PFS and OS among patients undergoing CAR-T therapy. We assessed whether the circulating polyamine signature might reflect a less aggressive phenotype that is likely to be responsive to CAR-T treatment. To this end, we evaluated the Basso lymphoma gene expression dataset (Basso et al., 2005) and found that mRNA expression of polyamine metabolic enzymes (PMEs) was statistically significantly higher (Wilcoxon rank sum test, two-sided p<0.05) in DLBCL cells compared to healthy B lymphocytes (Figure 5A): indicating that increased polyamine biosynthesis is a metabolic hallmark of DLBCL. Next, the association was evaluated between gene expression of PMEs and overall survival in two independent B cell lymphoma transcriptome datasets (Shipp et al., 2002; Lenz et al., 2008). In both data sets, elevated mRNA expression of spermidine synthase (SRM) and adenosylmethionine decarboxylase 1 (AMD1), the rate-limiting enzymes in polyamine metabolism, was statistically significantly associated with poor overall survival (Figure 5B-C). Thus, both plasma polyamines, as well as PME mRNA expression, are concordantly associated with poor prognosis among individuals with B-cell lymphoma. Circulating polyamine levels after CAR-T cell infusion in r / r LBCL patients

[0130] A linear mixed model with random intercepts and slopes was built to calculate the association between polyamine levels after CAR-T infusion. The reported values ​​(slope and intercept) in the table represent the average of all calculated coefficients for each patient. p-values ​​were calculated from 10,000 bootstraps of delta values ​​between responders and non-responders.

[0131] Intra-patient levels of AcSpmd, DiAcSpmd and DAS were further assessed up to 16 days after CAR-T cell infusion. The results showed that polyamines remained elevated in patients with progressive disease or who died within 6 months after CAR-T cell treatment compared to patients with ongoing complete responses (Figure 8). [Table 1] material and method Human subjects

[0132] Human plasma samples were collected through an international collaboration between MD Anderson Cancer Center (MDACC) (Houston, USA) and the German Cancer Research Center (DKFZ) (Heidelberg, Germany). Clinical data and patient plasma samples were collected under existing Institutional Research Board (IRB) approved protocols at each institution and were managed according to institutional guidelines and the principles of the Declaration of Helsinki. Response status was determined by the Lugano classification (2014). CRS and ICANS were prospectively graded and addressed according to the CAR-T cell therapy-associated toxicity guidelines. For the MDACC cohort analysis, EDTA plasma was obtained from 43 r / r LBCL patients on the day of CAR-T infusion (day 0). For validation purposes, plasma samples from an additional 20 patients on day 0 of CAR-T therapy from the German (DKFZ) r / r LBCL cohort were analyzed. Metabolomic analysis Sample extraction Primary Metabolites and Biogenic Amines

[0133] Plasma and serum metabolites were extracted from pre-aliquoted biological samples (15 μL) in 96-well microplates (Eppendorf) with 45 μL of LCMS grade methanol (ThermoFisher). Plates were heat sealed, vortexed at 750 rpm for 5 min, and centrifuged at 2000×g for 10 min at room temperature. Supernatants (30 μL) were carefully transferred to the 96-well plates, leaving behind precipitated proteins. Supernatants were further diluted with 60 μL of 100 mM ammonium formate, pH 3 (Fisher Scientific). For hydrophilic interaction liquid chromatography (HILIC) cation analysis, 15 μL of the mixture of supernatant and ammonium formate was diluted with 195 μL of 1:3:8:144 water (GenPure Ultrapure Water System, Thermofisher): LCMS grade methanol (ThermoFisher): 100 mM ammonium formate (pH 3) (Fisher Scientific): LCMS grade acetonitrile (ThermoFisher), whereas for HILIC anion analysis, 15 μL of the mixture of supernatant and ammonium formate was diluted with 90 μL of LCMS grade acetonitrile (ThermoFisher). For C18 analysis, 15 μL of the mixture of supernatant and ammonium formate was diluted with 90 μL of water (GenPure Ultrapure Water System, Thermofisher) for cation and anion mode, respectively. Each sample solution was transferred to a 384-well microplate (Eppendorf) for LCMS analysis. complex lipid

[0134] Pre-aliquoted serum or plasma samples (10 μL) were extracted with 30 μL of LCMS grade 2-propanol (ThermoFisher) in a 96-well microplate (Eppendorf). The plate was heat sealed, vortexed at 750 rpm for 5 min, and centrifuged at 2000×g for 10 min at room temperature. The supernatant (10 μL) was carefully transferred to a 96-well plate, leaving behind precipitated proteins. The supernatant was further diluted with 90 μL of 1:3:2 100 mM ammonium formate (pH 3) (Fischer Scientific): LCMS grade acetonitrile (ThermoFisher): LCMS grade 2-propanol (ThermoFisher) and transferred to a 384-well microplate (Eppendorf) for lipid analysis using LCMS. Untargeted analysis of primary metabolites and biogenic amines

[0135] Untargeted metabolomics analysis was performed on a Waters Acquity™ UPLC system with a 2D column regeneration configuration (I-class and H-class) coupled to a Xevo G2-XS quadrupole time-of-flight (qTOF) mass spectrometer. Chromatographic separation was performed at 45° C. using a HILIC column (Acquity™ UPLC BEH amide, 100 Å, 1.7 μm, 2.1×100 mm, Waters Corporation, Milford, USA) and a C18 column (Acquity™ UPLC HSS T3, 100 Å, 1.8 μm, 2.1×100 mm, Water Corporation, Milford, USA).

[0136] The quaternary solvent system mobile phase was (A) 0.1% formic acid / water, (B) 0.1% formic acid / acetonitrile, and (D) 100 mM ammonium formate, pH 3. Samples were separated using the following gradient profile: for HILIC separation, a starting gradient of 95% B / 5% D was changed linearly to 70% A / 25% B / 5% D over 5 min at a flow rate of 0.4 mL / min and to 100% A over 1 min, followed by another 1 min isocratic gradient at 100% A at a flow rate of 0.4 mL / min to initiate the starting gradient for the next C18 run. For C18 separation, the chromatographic gradient was as follows: starting conditions, 100% A, linear change to 5% A / 95% B over 5 min at a flow rate of 0.4 mL / min, back to 95% B / 5% D over 1 min, followed by a 1 min isocratic gradient at 95% B / 5% D at 0.4 mL / min for the next HILIC run.

[0137] A dual pump was used for column regeneration and equilibration. The solvent mobile phase was (A1) 100 mM ammonium formate (pH 3), (A2) 0.1% formic acid / 2-propanol, and (B1) 0.1% formic acid / acetonitrile. The HILIC column was stripped with 90% A2 at a flow rate of 0.25 mL / min for 5 min, followed by 2 min equilibration with 100% B1 at a flow rate of 0.3 mL / min. The reversed-phase C18 column was regenerated with 95% A1 / 5% B1 at a flow rate of 0.4 mL / min for 2 min, followed by column equilibration with 5% A1 / 95% B1 for 5 min. Untargeted analysis of complex lipids

[0138] For lipidomics assays, untargeted metabolomics analysis was performed on a Waters Acquity™ UPLC system coupled to a Xevo G2-XS quadrupole time-of-flight (qTOF) mass spectrometer. Chromatographic separation was performed at 55° C. using a C18 column (Acquity™ UPLC HSS T3, 100 Å, 1.8 μm, 2.1×100 mm, Water Corporation, Milford, USA). The mobile phases were (A) water, (B) acetonitrile, (C) 2-propanol, and (D) 500 mM ammonium formate (pH 3). The initial elution gradient was 20% A / 30% B / 49% C / 1% D, which changed linearly to 4% A / 14% B / 81% C / 1% D over 4.5 min, followed by an isocratic elution at 4% A / 14% B / 81% C / 1% D for 2.1 min and column equilibration at the initial conditions for 1.4 min. Mass spectrometry data acquisition

[0139] Mass spectrometry data were acquired in the range of 50-800 Da for primary metabolites and 100-2000 Da for complex lipids using "sensitivity" mode in positive and negative electrospray ionization mode. For electrospray acquisition, the capillary voltage was set at 1.5 kV (positive), 3.0 kV (negative), sample cone voltage was set at 30 V, source temperature was set at 120 °C, cone gas flow rate was set at 50 L / h, desolvation gas flow rate was set at 800 L / h, and scan time was 0.5 s in continuous mode. Leucine enkephalin; 556.2771 Da (positive) and 554.2615 Da (negative) were used for lockspray correction, and scans were performed in 0.5 s. Injection volumes for each sample were 3 μL for complex lipids and 6 μL for primary metabolites. Acquisition was performed with instrument auto-gain control to optimize instrument sensitivity over sample acquisition time. Data Processing

[0140] LC-MS and LC-MSe data were processed using Progenesis QI (Nonlinear, Waters). Peak selection and retention time alignment of LC-MS and MSe data were performed using Progenesis QI software (Nonlinear, Waters). Data processing and peak annotation were performed using an in-house automated pipeline as previously described. Annotation was determined by accurate mass and retention time matching using customized libraries created from authentic standards and by matching experimental tandem mass spectrometry data against theoretical fragmentation from NIST MSMS, LipidBlast, or HMDB v3. For complex lipids, retention time patterns characteristic of lipid subclasses were also considered. To correct for injection order drift, each feature was normalized using data from replicate injections of quality control samples collected every 10 injections throughout the run sequence. Measurement data were smoothed by Locally Weighted Scatterplot Smoothing (LOESS) signal correction (QC-RLSC) as previously described. Values ​​are reported as ratios to the median of historical quality control reference samples processed per analytical batch for a given analyte. statistical analysis

[0141] The Cox proportional hazards model with LASSO regularization using the glmnet package in R statistical software was used to select metabolite features for predicting PFS and to develop a biomarker panel for predicting PFS. The coefficients of selected features were derived in the test set and applied to the validation set. To test the proportionality of the hazard assumption of Cox regression, we used the method of Patricia et al.

[0142] A method based on the log-rank statistic, as described by Contal and O'Quigley, was used to determine the optimal change-point value for the model to distinguish patients who had progressive disease from those who had a complete response after CAR-T treatment. Kaplan-Meier survival analysis was performed using R version 1.1.442. The log-rank (Mantel-Cox) test was used to assess for statistical differences between survival curves.

[0143] The area under the receiver operating characteristic curve (AUC) was obtained using R (R version 3.6.0). The 95% confidence intervals presented for the individual performance of each biomarker were based on a bootstrap procedure in which we performed 1000 resamplings with replacement. For comparison of two classes, statistical significance was determined using the Wilcoxon rank sum test. Statistical significance was determined at a p-value of less than 0.05 for all analyses unless otherwise stated. Figures were generated with Graph Pad Prism Version 8.0 (GraphPad Software, Inc., San Diego, CA, USA). References

[0144] The following references, and those cited elsewhere in this specification, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Basso K, Margolin AA, Stolovitzky G, et al. Reverse engineering of regulatory networks in human B cells. Nat Genet 2005;37:382-90. Fahrmann JF, Bantis LE, Capello M, et al. A Plasma-Derived Protein-Metabolite Multiplexed Panel for Early-Stage Pancreatic Cancer. J Natl Cancer Inst 2019;111:372-379. Fahrmann JF, Irajizad E, Kobayashi M, et al. A MYC-Driven Plasma Polyamine Signature for Early Detection of Ovarian Cancer. Cancers (Basel) 2021;13. Fahrmann JF, Vykoukal J, Fleury A, et al. Association between plasma diacetylspermine and tumor spermine synthase with outcome in triple negative breast cancer. J Natl Cancer Inst 2019. Kamphorst JJ, Cross JR, Fan J, et al. Hypoxic and Ras-transformed cells support growth by scavenging unsaturated fatty acids from lysophospholipids. Proc Natl Acad Sci U S A 2013;110:8882-7. Kuhn T, Floegel A, Sookthai D, et al. Higher plasma levels of lysophosphatidylcholine 18:0 are related to a lower risk of common cancers in a prospective metabolomics study. BMC Med 2016;14:13. Lenz G, Wright G, Dave SS, et al. Stromal gene signatures in large-B-cell lymphomas. N Engl J Med 2008;359:2313-23. Raynor A, Jantscheff P, Ross T, et al. Saturated and mono-unsaturated lysophosphatidylcholine metabolism in tumour cells: a potential therapeutic target for preventing metastases. Lipids Health Dis 2015;14:69. Shipp MA, Ross KN, Tamayo P, et al. Diffuse large B-cell lymphoma outcome prediction by gene-expression profiling and supervised machine learning. Nat Med 2002;8:68-74. Vercellino L, Di Blasi R, Kanoun S, et al. Predictive factors of early progression after CAR T-cell therapy in relapsed / refractory diffuse large B-cell lymphoma. Blood Adv 2020;4:5607-5615. Zhao Z, Xiao Y, Elson P, et al. Plasma lysophosphatidylcholine levels: potential biomarkers for colorectal cancer. J Clin Oncol 2007;25:2696-701. Patricia M. Grambsch TMT: Proportional hazards tests and diagnostics based on weighted residuals. Biometrika 81:12, 1994 Cecile Contal JOQ: An application of changepoint methods in studying the effect of age on survival in breast cancer. Computational statistics & data analysis 30:253-270, 1999 Vykoukal J, Fahrmann JF, Gregg JR, et al: Caveolin-1-mediated sphingolipid oncometabolism underlies a metabolic vulnerability of prostate cancer. Nat Commun 11:4279, 2020 Friedman J, Hastie T, Tibshirani R: Regularization Paths for Generalized Linear Models via Coordinate Descent. J Stat Softw 33:1-22, 2010

[0145] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the design as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described herein. As will be readily understood by those skilled in the art from the present disclosure, any currently existing or later developed process, machine, manufacture, composition of matter, means, methods or steps that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized in accordance with the present disclosure. Thus, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods or steps.

[0146] It is contemplated that any method or composition described herein may be implemented with respect to any other method or composition described herein, and that different embodiments may be combined. It is contemplated that the claims as originally filed shall cover any claim that is multiple dependent on any filed claim or combination of filed claims.

Claims

1. 1. An adjunctive method for treating an individual, comprising the steps of: (a) measuring the concentration of at least one metabolite in a blood sample from the individual to identify the individual as having a responder phenotype or as having a non-responder phenotype; and (b) preparing a therapeutically effective amount of cells for cell therapy, the cells being administered to the individual, the therapeutically effective amount being correlated with the individual having a responder or non-responder phenotype, the administering being performed either alone or in combination with one or more metabolites, drugs, chemical compounds, biologics and / or bacteria to modulate response and / or reduce toxicity, wherein at least one metabolite comprises a metabolite selected from the group consisting of TMAO, indole, indole derivatives, 1-methylnicotinamide, nicotinamide, polyamines, asymmetric dimethylarginine, symmetric dimethylarginine, taurine, lysophospholipids, and combinations thereof. Methods including (excluding medical procedures by physicians).

2. 2. The method of claim 1, wherein the responder phenotype comprises a predetermined concentration of at least one metabolite in the blood sample, the concentration being statistically equivalent to the concentration of the metabolite in blood samples of different individuals known to be responsive to a predetermined amount of the cellular therapy.

3. 3. The method of claim 1 or 2, wherein the non-responder phenotype comprises a predetermined concentration of at least one metabolite in the blood sample, the concentration being statistically higher than the concentration of the metabolite in blood samples of a different individual known to respond to a predetermined amount of the cellular therapy.

4. The method of any one of claims 1 to 3, wherein when the individual is identified as having a non-responder phenotype, the therapy is not administered to the individual.

5. The method of claim 4, wherein the cellular therapy is supplemented with one or more metabolic products.

6. 6. The method of claim 5, wherein at least one of the one or more metabolites is determined to be statistically lower than the concentration of the metabolite in blood samples of various individuals known to be responsive to a given amount of the cellular therapy.

7. 10. The method of claim 1, wherein the cells further comprise a protein capable of increasing or decreasing at least one metabolic product in the individual, and the cells are genetically modified to express the protein.

8. 8. The method of claim 7, wherein the protein comprises ornithine decarboxylase (ODC), lysine decarboxylase, arginase, agmatinase, spermidine synthase, spermine synthase, spermine oxidase, spermidine / spermine-N1-acetyltransferase (SSAT1), S-adenosyl-methionine decarboxylase (AdoMetDC), deoxyhypusine synthase (DHPS), deoxyhypusine hydroxylase (DOHH), antizyme inhibitor (AZI), eukaryotic translation initiation factor 5A (eIF5A), ATF4, Gcn2, or a mutant form of each of the proteins, or a combination thereof.

9. A composition for use in the method according to any one of claims 1 to 8, which is capable of increasing or decreasing at least one of said metabolites.

10. The composition of claim 9, wherein the composition comprises at least one of the metabolic products when the composition is capable of increasing at least one of the metabolic products.

11. 1. An adjunct method for reducing the potential toxicity or risk of toxicity of cell therapy, comprising the steps of: (a) measuring the concentration of at least one metabolite in a blood sample from the individual to identify the individual as having a responder phenotype or as having a non-responder phenotype; and (b) preparing a therapeutically effective amount of cells for cell therapy, the cells being administered to the individual, the therapeutically effective amount being dependent on the individual having a responder or non-responder phenotype, and the at least one metabolite comprising a metabolite selected from the group consisting of indole derivatives, tryptophan, serotonin, 1-methylnicotinamide, nicotinamide, polyamines, asymmetric dimethylarginine, symmetric dimethylarginine, taurine, lysophospholipids, and combinations thereof. Methods including (excluding medical procedures by physicians).

12. The method of claim 11 , wherein the blood sample comprises a serum sample.

13. Cells expressing the engineered receptor, and One or more metabolites A therapeutic composition comprising:

14. 14. The therapeutic composition of claim 13, wherein the cells comprise a genetic modification to express at least one protein capable of increasing or decreasing one or more metabolic products.

15. 15. The therapeutic composition of claim 13 or 14, wherein the one or more metabolites are selected from the group consisting of TMAO, indole, indole derivatives, 1-methylnicotinamide, nicotinamide, polyamines, asymmetric dimethylarginine, symmetric dimethylarginine, taurine, lysophospholipids, and combinations thereof.

16. 1. A therapeutic composition comprising a metabolite, a bacterium, a second cell therapy, or a combination thereof, for use in a method for treating an individual receiving a first cell therapy comprising the steps of: (a) measuring the concentration of one or more metabolites in at least one blood sample from said individual; and (b) administering a therapeutic composition comprising a metabolite, a bacteria, a second cellular therapy, or a combination thereof when the concentration of the metabolite(s) is higher or lower than a baseline level, wherein the at least one metabolite comprises a metabolite selected from the group consisting of TMAO, indole, an indole derivative, 1-methylnicotinamide, nicotinamide, a polyamine, asymmetric dimethylarginine, symmetric dimethylarginine, taurine, a lysophospholipid, and combinations thereof. The method includes:

17. 17. The composition method of claim 16, wherein the baseline level comprises a predetermined concentration of at least one metabolite in said blood sample of an individual known to be responsive to a predetermined amount of said cellular therapy.

18. 18. The composition of claim 16 or 17, wherein the first cell therapy and / or the second cell therapy comprises cells expressing one or more engineered receptors.

19. A method for predicting a response to a cell therapy (excluding diagnostic methods by a physician), comprising the steps of: (a) measuring the concentration of at least one metabolite in a blood sample from the individual; and (b) predicting a response to said cell therapy when said metabolites are higher or lower than baseline levels, wherein at least one metabolite comprises a metabolite selected from the group consisting of TMAO, indole, indole derivatives, 1-methylnicotinamide, nicotinamide, polyamines, asymmetric dimethylarginine, symmetric dimethylarginine, taurine, lysophospholipids, and combinations thereof. The method includes:

20. 20. The method of claim 19, wherein when the response is predicted to be harmful to the individual, the cellular therapy is not administered to the individual, is modified prior to administration to the individual to make it less harmful, the individual is given a different therapy, or a combination thereof.

21. 21. The method of claim 19 or 20, wherein when the response is predicted to be deleterious to the individual, the individual is provided with a therapeutically effective amount of (1) one or more metabolic products, or (2) a bacterial composition capable of altering the metabolic products or their levels, or other synthetic derivatives / products thereof or their levels, or (3) genetically modified immune cells that can alter the metabolic products in the body to cause a response or toxicity to the therapy.

22. 20. The method of claim 19, wherein a therapeutically effective amount of the cell therapy is administered to the individual when the response is predicted to be non-detrimental to the individual.

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