T cell therapy

Abstract

To provide methods for the identification of patients who may benefit from a modified manufacturing process for CAR T cell product production, and to provide methods for optimization of immunotherapy products.SOLUTION: Disclosed are methods of predicting a likelihood of survival of a patient in need of chimeric antigen receptor (CAR) T cell therapy comprising measuring a level of CD27+CD28+ naive Th cells in an apheresis product from the patient, the patient being determined to have an increased likelihood of survival if the level of CD27+CD28+ naive Th cells is over a cut-off percentage value measured as a percentage of total leukocytes.SELECTED DRAWING: None

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 135,711, filed January 10, 2021, which is incorporated by reference in its entirety.

[0002] The present disclosure relates to diagnostic and prognostic methods, compositions for immunotherapy, methods for improving such compositions, and immunotherapeutic methods using same. [Background technology]

[0003] Human cancers, by their very nature, are composed of normal cells that have undergone genetic or epigenetic transformation to become abnormal cancer cells. In doing so, the cancer cells begin to express proteins and other antigens that differ from those expressed by normal cells. These abnormal tumor antigens can be used by the body's natural immune system to specifically target and kill the cancer cells. However, cancer cells use various mechanisms to prevent immune cells, such as T and B lymphocytes, from successfully targeting the cancer cells.

[0004] Human T cell therapy relies on enriched or engineered human T cells to target and kill cancer cells in patients. To increase the ability of T cells to target and kill specific cancer cells, methods have been developed to engineer T cells to express constructs that direct the T cells to specific target cancer cells. Chimeric antigen receptors (CARs) contain binding domains that can interact with specific tumor antigens. and T cell receptor (TCR), which allows T cells to express specific tumor antigens. It can target and kill cancer cells that are affected.

[0005] There is a need to understand how attributes of CAR-positive T cells, TCR-positive T cells and other cell-based immunotherapies, as well as the patient's immunological status, correlate with clinical outcomes. Summary of the Invention

[0006] In one embodiment, the present disclosure provides that the recovery of apheresis material from cancer patients prior to any cancer therapy may provide an improved source of cells for immunotherapy, such as CAR T cell immunotherapy. In one embodiment, the present disclosure provides that immunotherapy (e.g., CAR T cell immunotherapy) may be administered as part of an early, if not the earliest, line of treatment to maximize the efficacy of the immunotherapy, where there is a negative impact of other therapies on the quality of immune cells from the apheresis product that may be used to generate the immunotherapy (e.g., CAR T cells). In one embodiment, the present disclosure provides for the recovery of apheresis material from cancer patients at the diagnostic stage, where the method improves the quality of the immunotherapy derived from the apheresis material. In one embodiment, the present disclosure provides predictive biomarkers that allow for pre-treatment blood tests to be performed on immunotherapy patients that help stratify patients based on predicted response (e.g., objective response and sustained response) to the immunotherapy (e.g., CAR T cell therapy). In one embodiment, the present method allows for the identification of patients likely to achieve a sustained response to CAR T cell therapy alone. In one embodiment, the present method allows for the identification of patients that may benefit from combination therapy. In one embodiment, the combination therapy is given up front (not after failure of first / second line therapy) to maximize efficacy. In one embodiment, the method allows for the identification of patients who could benefit from modified manufacturing processes for CAR T cell product production, modifications to the process resulting in a T cell product more suitable for immunotherapy. In one embodiment, it allows for the identification of patients who may be better candidates for allogeneic or pre-made CAR T cells.

[0007] In one embodiment, the disclosure provides a method for optimization of an immunotherapy product. In one embodiment, the immunotherapy product comprises CAR T cells. In one embodiment, the levels of pre-treatment biomarkers in the patient's blood (e.g., an apheresis sample) are correlated with features of an immunotherapy product (e.g., a CAR T cell product) prepared from the patient's blood that are related to immunotherapy response. In one embodiment, the levels of pre-treatment biomarkers in the patient's blood are used to inform modifications to the manufacturing process of the immunotherapy cells (e.g., CAR T cells). In one embodiment, the modifications to the manufacturing process result in enrichment of certain T cells in the immunotherapy product, which in turn results in an immunotherapy product with better CAR T cell efficacy. In one embodiment, the immunotherapy product comprises autologous CAR T cells. In one embodiment, the immunotherapy product comprises allogeneic CAR T cells. In one embodiment, the immunotherapy comprises T cell receptor modified T cells. In one embodiment, the immunotherapy comprises tumor infiltrating lymphocytes (TILs). In one embodiment, the immunotherapy product comprises induced pluripotent stem cells (iPSCs). In one embodiment, the immunotherapy is used to treat cancer. In one embodiment, the cancer is a leukemia or lymphoma. In one embodiment, the cancer is a solid tumor.

[0008] In one embodiment, the manufacturing process is adjusted to increase input material. In one embodiment, the manufacturing process is adjusted to a cell selection process to enrich the immunotherapy product in T cells with a particular phenotype. In one embodiment, the manufacturing process is adjusted to deplete the immunotherapy product of bone marrow cells. In one embodiment, the bone marrow cells are intermediate monocytes. In one embodiment, the adjustment to the manufacturing process includes an adjustment to the immune cell growth media composition. In some embodiments, the adjustment to the manufacturing process includes an adjustment to the length of the manufacturing process. In one embodiment, the adjustment to the process helps to overcome negative product factors such as low lymphocyte counts and / or low percentages of specialized cell subsets, such as CD4+CD27+CD28+T cells and CD4+CD127+CD25dim CD27+CD28+CCR7+CD45RA+T cells, and / or low intermediate monocyte CD14+CD16+ cells, or combinations thereof, in the blood or apheresis cell population.

[0009] In one embodiment, the method provides for adjustment of infused T cell dose based on pre-treatment biomarkers to overcome potential mechanisms of treatment resistance. In one embodiment, the pre-treatment biomarkers measured by flow cytometry include levels of pre-manufactured PBMC populations such as CD3+CD4+CD127+CD25dim CCR7+CD45RA+CD27+CD28+(CD27+CD28+ naive Th); CD3-CD19-CD56-CD11c+CD14+CD16+(intermediate monocytes); CD3+CD4+CD127dim CD25+CCR7+CD45RA-CD27-CD28+(CD27-CD28+TEMRA Treg); lymphocyte to leukocyte / ratio (hematology baseline cell count); and / or lymphocyte to monocyte ratio (hematology baseline cell count).

[0010] In one embodiment, the disclosure provides a therapeutic method that integrates other therapies after CAR T cell infusion aimed at overcoming mechanisms of therapeutic resistance associated with negative predictive biomarkers. In one embodiment, the biomarkers include CD3+CD4+CD127+CD25dim CCR7+CD45RA+CD27+CD28+(CD27+CD28+ naive Th);CD3-CD19-CD56-CD11c+CD14+CD16+(intermediate monocytes);CD3+CD4+CD127dim CD25+CCR7+CD45RA-CD27-CD28+(CD27-CD28+TEMRA Treg);Lymphocyte to leukocyte ratio (hematology baseline cell count);Lymphocyte to monocyte ratio (hematology baseline cell count). In one embodiment, the other treatment(s) include monoclonal antibodies with or without immune modulators such as gamma chain receptor cytokines (e.g., IL-15), myeloid cell modulators (e.g., JAK / STAT inhibitors, agents that modulate deleterious myeloid cell subsets such as intermediate monocytes), bispecific engagers, iMiDs (e.g., lenalidomide, pomalidomide). Anti-CD20 antibodies, including CD47 blockade with or without anti-CD20 antibodies.

[0011] In some embodiments, the population of T cells is obtained from apheresis material. In some embodiments, the method further comprises engineering the population of T cells to express a CAR. In some embodiments, the CAR T cells are engineered to express a chimeric antigen receptor that targets a tumor antigen. In some embodiments, the chimeric antigen receptor is a tumor-associated surface antigen, such as 5T4, alpha fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD8, CLL-1, c-Met, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, EGFR variant III (EGFRvII I), ELF2M, endoglin, ephrinB2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2 / neu), fibroblast-associated protein (fap), FLT3, folate-binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipid, gp36, HBV-specific antigen, HCV-specific antigen, HER1-HER2, HER2-HER3 combination, HERV-K, high molecular weight melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigen, human telomerase reverse transcriptase, IGFI receptor, IGF-II, IL-11Rα, IL-13R-a2, influenza virus-specific antigen;CD38, insulin growth factor (IGFl)-1, intestinal carboxylesterase, kappa chain, LAGA-la, lambda chain, Lassa virus specific antigen, lectin-reactive AFP, lineage-specific or tissue-specific antigens such as CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mut Targeting a tumor antigen selected from hsp70-2, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostate specific antigen (PSA), prostate carcinoma tumor antigen-1 (PCTA-1), prostate specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecules, survivin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin and Al domain of tenascin-C (TnC Al), thyroglobulin, tumor stromal antigens, vascular endothelial growth factor receptor-2 (VEGFR2), virus specific surface antigens such as HIV specific antigens (such as HIV gpl20), and any derivative or variant of these surface antigens;

[0012] In some embodiments, the malignancy is one or more of a solid tumor, a sarcoma, a carcinoma, a lymphoma, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), T-cell lymphoma, B-cell acute lymphoblastic leukemia ("BALL"), T-cell ALL, or any combination thereof. Acute lymphocytic leukemia ("TALL"), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Kittbart lymphoma, diffuse large cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell lymphoma or large cell lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, myelodysplasia and myelodysplastic syndromes, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma) myeloma or indolent myeloma), monoclonal gammopathy of undetermined significance (MGUS), plasmacytoma (e.g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloid light chain amyloidosis, POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, PEP syndrome), or a combination thereof.

[0013] In some embodiments, the therapeutically effective dose is between 75 and 200×10 6 In some embodiments, the therapeutically effective dose is between 2×10 6 In some embodiments, the engineered T cells are autologous or allogeneic T cells. In some embodiments, the response is measured within about 1 month, about 3 months, about 6 months, about 9 months, or about 12 months after administration of the engineered T cells.

[0014] Further exemplary embodiments are provided below. 1. A method of optimizing immunotherapy product (e.g., CAR T cell) manufacturing, in which product T cell population compatibility is improved by increasing the levels of naive phenotype CD27+CD28+ Th cells (CCR7+CD45RA+) in the pre-manufacturing PBMC population.

[0015] 2. The method of embodiment 1, wherein this may be accomplished by enriching for naive phenotype CD27+CD28+ Th cells (CCR7+CD45RA+) following subject apheresis, by increasing the amount of apheresis material collected until a threshold of naive phenotype CD27+CD28+ Th cells (CCR7+CD45RA+) is reached and the manufacturing process begins, by selecting the administration dose of CAR T cells not through the total number of CARs per kg, but instead by utilizing the number of naive phenotype CD27+CD28+ Th cells (CCR7+CD45RA+) per kg, and / or by adjusting the T cell product manufacturing conditions (such as, but not limited to, the length of manufacturing and / or the composition of the growth medium) to increase the levels of naive phenotype CD27+CD28+ Th cells (CCR7+CD45RA+).

[0016] 3. Methods to stratify cancer patients as better candidates for allogeneic / off-the-shelf CAR or TCR T cells to overcome the lack of sufficient positive factors such as CD27+CD28+Th cells of naive phenotype (CCR7+CD45RA+) in pre-manufactured PBMC populations obtained from patients.

[0017] 4. To overcome the lack of sufficient positive factors, such as CD27+CD28+Th cells of naive phenotype (CCR7+CD45RA+) in pre-manufacturing PBMC populations obtained from patients, enhance the activity of CAR or TCR T cells or reduce the effect of negative factors to enhance the activity of CAR A method for stratifying patients who may be better candidates for combination therapy that can improve the clinical efficacy of T therapy.

[0018] 5. The method of embodiment 4, wherein the combination therapy can be selected from, but is not limited to, immunotherapy (including checkpoint inhibitors, anti-PD-1, anti-PD-L1, anti-CTLA-4, etc., or any combination thereof), SRC kinase inhibitors (such as dasatinib), T cell bispecific antibodies, anti-CD20 monoclonal antibodies (such as rituximab), anti-4-1BB, anti-CD47, TGF-beta inhibitors or dominant negative TGF-beta, mTOR / AKT agonists, histone deacetylase inhibitors, cyclophosphamide, fluorouracil, gemcitabine, doxorubicin, taxanes and any other form of chemotherapy or radiotherapy, small molecule inhibitors or antibodies targeting enhancement of anti-tumor immunity.

[0019] 6. Patients have a naive phenotype (CCR7+CD45RA+) in the pre-production PBMC population 6. The method of any one of embodiments 3-5, wherein the patient is stratified for manufacturing optimization based on the percentage of CD27+CD28+ Th cells in the primary tumor and / or identified as a patient who would benefit from allogeneic / off-the-shelf CAR or TCR T cell or combination therapy to maximize efficacy of the cell therapy.

[0020] 7. A method for predicting the inflammatory status of a cancer patient and / or the clinical efficacy of the patient's CAR or TCR T cells by quantifying intermediate monocytes and / or total monocytes in the patient's pre-manufacturing PBMC product.

[0021] 8. The method of embodiment 7, wherein the method is used as an indicator of the potential use of anti-inflammatory drugs to neutralize inflammatory signaling in the periphery.

[0022] 9. The method of embodiment 8, wherein the anti-inflammatory agent is selected from, but not limited to, antibodies against the IL-6 pathway (such as tocilizumab and siltuximab), corticosteroids (such as dexamethasone), antibodies inhibiting the TNF pathway (such as etanercept, infliximab), anakinra, and anti-GM-CSF (such as lenzilumab).

[0023] 10. A method for predicting IPI score in cancer patients, wherein the level of intermediate monocytes (% of white blood cells) in a pre-production PBMC population is enriched in patients with higher IPI scores and is a marker for patients with higher IPI scores.

[0024] 11. A method for predicting the clinical efficacy of CAR and TCR T cells, comprising quantifying intermediate monocytes and / or total monocytes in a pre-manufacturing PBMC product, thereby allowing estimation of the patient's tumor burden, which has been shown to be a negative indicator of the clinical efficacy of CAR T cells.

[0025] 12. The method of embodiment 11, wherein the levels of intermediate monocytes and / or total monocytes indicate the use of additional therapeutic agents to help overcome a greater putative tumor burden, such as chemotherapy, radiation antibody and small molecule based therapies, immunotherapies (including but not limited to checkpoint inhibitors, bispecific engagers), and cell therapies (including but not limited to CAR-T, TCR based lymphocytes and tumor infiltrating lymphocytes) where tumor burden has been shown to be a negative prognostic and / or predictive biomarker.

[0026] 13. A method for quantifying biomarkers that allow estimation of the patient's hypoxic state (e.g., intermediate monocytes and / or total monocytes), which have been shown to be negative indicators of the clinical efficacy of CAR or TCR T cells.

[0027] 14. The method of embodiment 13, wherein the level of intermediate monocytes and / or total monocytes is used as an indicator of a supplemental therapeutic agent to overcome the hypoxic tumor microenvironment (TME).

[0028] 15. The method of any one of embodiments 13 and 14, wherein the supplemental therapeutic agent is selected from, but is not limited to, a metabolic regulator that establishes a more normal TME, a VEGF inhibitor (such as bevacizumab), a HIF inhibitor, and an LDH inhibitor.

[0029] 16. A method for predicting response to immunotherapy (e.g., CAR T cell therapy), wherein monocytes, particularly intermediate monocytes, in a pre-manufacturing PBMC population negatively correlate with T cell characteristics in the TME, and CD27+CD28+ naive Th cells and lymphocytes positively correlate with T cell characteristics in the TME that are associated with response.

[0030] 17. T cell features in the TME associated with response include activated CD8+ T cells The method of embodiment 16, including, but not limited to, genes associated with the subset (CD3+CD8+PD-1+Lag3+ / - Tim3- cells) and activated T cell signature (e.g., CXCL10, CXC11, GZMA, GZMB, GZMK and Immunosign21).

[0031] 18. A method for elucidating the overall status of the TME in cancer patients, wherein the levels of peripheral blood biomarkers allow estimation of the tumor immune status into various classes such as immune desert, bone marrow imbalance, immunosuppression, etc. within the TME.

[0032] 19. The method of embodiment 18, wherein these biomarkers are used to select potential combination therapies, which may be selected from, but are not limited to, immunotherapy (including checkpoint inhibitors, anti-PD-1, anti-PD-L1, anti-CTLA-4, etc., or any combination thereof), SRC kinase inhibitors (such as dasatinib), T-cell bispecific antibodies, anti-CD20 monoclonal antibodies (such as rituximab), anti-4-1BB, anti-CD47, TGF-beta inhibitors or dominant negative TGF-beta, mTOR / AKT agonists, histone deacetylase inhibitors, cyclophosphamide, fluorouracil, gemcitabine, doxorubicin, taxanes and any other form of chemotherapy or radiotherapy, small molecule inhibitors or antibodies targeting enhancement of anti-tumor immunity.

[0033] 20. A method to quantify a simple biomarker (CD27+CD28+ naive Th) that allows estimation of the patient's final infusion bag naive status after manufacturing and the T cell-rich tumor immune status, both of which have been shown to be positive indicators of the clinical efficacy of CAR T cells, and low levels of these CD27+CD28+ naive Th cells indicate the potential use of anti-inflammatory drugs, off-the-shelf / allogeneic CAR or TCR T cells, manufacturing optimization, or combination therapies that help modify the tumor microenvironment to improve the efficacy of CAR T cells.

[0034] 21. A method for predicting inflammatory status, wherein intermediate monocytes in a pre-manufacturing PBMC population positively correlate with pre-treatment inflammation (INTL8, ferritin, CRP, amyloid A) / tumor hypoxia (LDH) and negatively correlate with pre-treatment defined T cell-enriched tumor immune landscape (e.g., activated T cell signature, CD3+CD8+PD1+LAG3-TIM3- cells; GZMA, TGIT, LAG3, CXCL10, GZMB, PRF1, STAT1, EOMES, CXCL9, GZMK, CXCL11, HAVCR2, CD3D, IS21).

[0035] 22. A method in which high levels of intermediate monocytes indicate the use of anti-inflammatory drugs (such as corticosteroids or tocilizumab) and / or immunomodulatory drugs (e.g., TME modulating drugs [such as checkpoint inhibitors, drugs that target suppressor myeloid cells and enhance antigen presentation, drugs that stabilize the vasculature, or drugs that normalize tumor metabolism]) to help overcome insufficient TIC.

[0036] 23. The method of embodiment 22, wherein the drug is administered before, during and / or after immunotherapy.

[0037] 24. Patients are screened for CAR or TCR immunization based on the levels of intermediate monocytes in the pre-manufacturing PBMC population. A method for predicting whether a patient is likely to respond to T cell therapy, wherein the level of intermediate monocytes in a pre-production PBMC population is positively associated with pre-treatment tumor burden, which is itself negatively associated with response.

[0038] 25. A method for estimating a patient's tumor burden using the levels of intermediate monocytes and / or total monocytes in a pre-manufacturing PBMC population, which in turn has been shown to be a negative indicator of clinical efficacy of CAR T cells, and the levels of intermediate monocytes have been shown to be a negative prognostic and / or predictive biomarker for tumor burden, chemotherapy, radioactive antibody and small molecule based therapy, immunotherapy, The methods serve as indicators for the use of additional therapeutic agents to help overcome the greater putative tumor burden, such as immunotherapy (including but not limited to checkpoint inhibitors, bispecific engagers), and cell therapy (including but not limited to CAR-T, TCR-based and tumor infiltrating lymphocytes).

[0039] 26. A method for predicting the likelihood of survival of a patient in need of CAR T cell therapy based on the level of CD27+CD28+ naive Th cells (% of white blood cells) in the apheresis product used to prepare a CAR T cell product, wherein the level of CD27+CD28+ naive Th cells (% of white blood cells) in the apheresis product / pre-manufacturing PBMC population is a predictive marker for improved overall survival (OS) and progression free survival (PFS) (optimal cut-off) (i.e., there is a positive association between them, i.e., subjects with pre-treatment CD27+CD28+ naive Th cells above the listed cut-off are more likely to survive than subjects below the selected cut-off).

[0040] 27. A method for predicting PFS in a patient in need of CAR or TCR T cell therapy based on the level of CD27+CD28+ naive Th cells (% of white blood cells) in the apheresis product used to prepare a CAR or TCR T cell product, wherein complete response rate, objective response rate, and CAR or TCR T cell expansion are improved for subjects above a selected cutoff.

[0041] 28. A method of stratification whereby subjects with low levels (e.g., below 0.27%) of CD27+CD28+ naive Th cells would benefit from alternative forms of treatment (combination therapy, allogeneic CAR T cells, etc.) or manufacturing optimization to improve chances of survival.

[0042] 29. The method of embodiment 28, wherein the low level is below the median of evaluable clinical trial subjects, or between 0.1-0.5%, 0.5-1.0%, 1-1.5%, 1.5-2%, 2-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-50%, etc., or below 95-100%.

[0043] 30. A method in which subjects with intermediate monocyte levels (% of white blood cells) in the apheresis product below a cutoff of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%, preferably between 1-5%, and even more preferably below approximately 3%, are predicted to have a greater chance of survival than subjects above the cutoff.

[0044] 31. A method of predicting OS and PFS in a subject in need of CAR or TCR T cell therapy, comprising measuring the level of intermediate monocytes (% of white blood cells) in an apheresis product used to prepare a CAR or TCR T cell product, and determining whether the level is above or below a cutoff, wherein complete response rate and objective response rate, as well as CAR or TCR T proliferation for those subjects below the approximately 3% cutoff, are improved.

[0045] 32. A method of patient stratification, in which subjects with high levels of intermediate monocytes (greater than approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%, preferably between 1-5%, and even more preferably levels greater than approximately 3%) may benefit from another form of treatment (e.g. combination therapy with immunotherapy, allogeneic CAR T cells, etc.) or manufacturing optimization to improve chances of survival.

[0046] 33. To determine the ratio of CD27+CD28p+ naive Th cells (% of white blood cells) in the apheresis product to intermediate monocytes (% of white blood cells) used in the preparation of the CAR or TCR T cell product, and whether the levels are approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 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, and determining whether the CAR T cell proliferation rate is above or below a cutoff of 0.6, 0.7, 0.8, 0.9, 1, 1-5, 5-10, 10-20, preferably 0.05-0.2, 0.2-0.25, 0.25-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-1, 1-5, 5-10, 10-15 etc., 95-100, 100-200, 200-300 etc., more preferably 0.1-1, and even more preferably 0.1705.

[0047] 34. The method of embodiment 33, wherein for selected subjects above a cutoff of 0.1705, there is an improvement in complete response rate, objective response rate, and CAR or TCR T cell expansion.

[0048] 35. A method of patient stratification, in which subjects with low levels of CD27+CD28+ naive Th cells (e.g., levels of approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1-5, 5-10, 10-20, preferably 0.05-0.2, 0.2-0.25, 0.25-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-1, 1-5, 5-10, 10-15, more preferably 0.1-1, even more preferably 0.1705) may benefit / be recommended for another form of treatment (combination therapy, allogeneic CAR T cells, etc.) or manufacturing optimization to improve chances of survival.

[0049] 36. A method of predicting an objective response in a subject in need of CAR T cell therapy comprising measuring CD27+CD28+ naive Th levels and low intermediate monocyte levels, whereby a CD27+CD28+ naive Th level of / greater than 0.08% (levels above the median, or levels above 0.05%, 0.1%, 0.2-1%, 1-5%, 5-10%, 10-15%, 15-20%, etc., 95-100%) and / or a level of intermediate monocytes of / below 3% (levels below the median, or below 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, etc., 95%-100%) indicates an increased likelihood of an objective response.

[0050] 37. The method of embodiment 36, wherein these levels are used to stratify patients who may benefit from pre-made / allogeneic CAR or TCR T cells, immunomodulators, bispecific engagers, combination therapy, etc.

[0051] 38. A method for measuring levels of intermediate monocytes in pre-treatment apheresis PBMCs and CAR or TCR T cell proliferation and using them to actively follow patients after infusion to estimate what the long-term response will be and whether supplemental therapeutics may be useful, comprising measuring high levels of intermediate monocytes in the pre-production PBMC population (wherein high levels are levels above the median of intermediate monocytes in the general population, where the median may be 0-1%, 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, 9-10%, 10-15%, 15-20%, etc., preferably about 1.7-1.8%) and low levels of CAR T cell proliferation (wherein low levels are levels above the median of intermediate monocytes in the general population, preferably about 1.7-1.8%). How levels below the median level of T cell proliferation (median values ​​are 0-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100) correlate with the highest rate of non-responders.

[0052] 39. The method of embodiment 38, wherein the method estimates response based on baseline intermediate monocyte levels and CAR or TCR expansion after infusion.

[0053] 40. Increased CAR T cell peak proliferation (where the expanded level is greater than that of the general CAR 40. The method of embodiment 39, wherein subjects with levels above the median level of CAR T cell proliferation in the RT cell treatment population, where the median is between 0-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, preferably between 40-50), and lower intermediate monocyte levels (wherein low levels are levels below the median intermediate monocytes in the general population, where the median can be 0-1%, 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, 9-10%, 10-15%, 15-20%, etc., preferably about 1.7-1.8%) had increased sustained response rates and decreased relapse or non-responder rates compared to other quadrants.

[0054] 41. Methods used to measure levels of intermediate monocytes in pre-treatment apheresis PBMCs and CAR T cell proliferation and actively follow patients post-infusion to estimate what the continued response is, the likelihood of relapse, and whether supplemental therapeutics may be useful based on the above correlations.

[0055] 42. The subject has a baseline tumor burden above the median level, high intermediate monocytes (above the median, the median can be approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 10-15%, 15-20%, 20-25%, etc., 95-100%, preferably approximately 1.1%) and low CAR-T cell peak proliferation (below the median, the median can be approximately 5-10%, 10-15%, 15-20%, 20-25%, 25-30%). , 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, etc., 95-100%, preferably about 43%.) the likelihood of response is low (1%-10%, 10-20%, 20-30%, 30-40%, 40-50% sustained response rate and 1%-10%, 10-20%, 20-30%, 30-40%, 40-50% objective response rate), the method of embodiment 41.

[0056] 43. The method of embodiment 42, in which the levels of intermediate monocytes in pre-treatment apheresis PBMCs, baseline tumor burden, and CAR or TCR T cell proliferation are measured and actively followed in patients after infusion, and used to estimate, based on the above correlations, what the likelihood of sustained and objective responses is, and whether additional therapeutic agents may be useful.

[0057] 44. CAR or TCR in subjects in need of CAR or TCR T cell therapy 1. A method of predicting likelihood of response to T cell therapy, comprising: measuring the level of CD27+CD28+ naive Th (% of white blood cells) in a pre-production PBMC population; and predicting a high likelihood of response if the level is above an optimal cut-off point (e.g., 0.1036%, 0-0.1%, 0.1%, -0.5%, 0.5%, -1.0%, 1.0-5%, 5-10%, 10-15%, 10-20%, 20-30%, 30-40%, 40-50%) or above the median (e.g., 0.89%, 0-0.1%, 0.1%, -0.5%, 0.5%, -1.0%, 1.0-5%, 5-10%, 10-15%, 10-20%, 20-30%, 30-40%, 40-50%).

[0058] 45. A method for selecting patients for manufacturing optimization, combination therapy, or off-the-shelf / allogeneic CAR or TCR T cell therapy if the level of CD27+CD28+ naive Th (% of white blood cells) in a pre-manufacturing PBMC population is below a selected cutoff point or median range, which is also an indication of a lower likelihood of sustained response.

[0059] 46. ​​A method for predicting likelihood of response to CAR or TCR T cell therapy in a subject in need of CAR or TCR T cell therapy, comprising measuring the level of intermediate monocytes (% of white blood cells) in a pre-production PBMC population and determining whether this level is within an optimal cut point (e.g., 3.02%, 0-0.1%, 0.1%, -0.5%, 0.5%, -1.0%, 1.0%). and predicting a high likelihood of response if the median monocyte (% of white blood cells) in the pre-manufacturing PBMC population level is below -5%, 5-10%, 10-15%, 10-20%, 20-30%, 30-40%, 40-50%) or below the median (e.g., 1.77%, 0-0.1%, 0.1%, -0.5%, 0.5%, -1.0%, 1.0-5%, 5-10%, 10-15%, 10-20%, 20-30%, 30-40%, 40-50%), wherein the median monocyte (% of white blood cells) in the pre-manufacturing PBMC population level is lower in subjects with a continued (sustained) response compared to subjects who experience a relapse or are non-responders.

[0060] 47. A method for measuring levels of intermediate monocytes (% of white blood cells) in a pre-manufacturing PBMC population for use in methods for selecting manufacturing optimization, combination therapy, or off-the-shelf / allogeneic CAR or TCR T cell therapy for subjects above a range with a lower likelihood of sustained response.

[0061] 48. A method for predicting response to CAR or TCR T cell therapy based on the levels of CD27+CD28+ naive Th cells in a pre-manufacturing PBMC population, whereby patients with higher levels of these cells are more than likely not to respond to CAR T therapy and less likely to not require intervention, and patients with lower levels may need to consider further modifications to therapy such as combination therapy, optimized manufacturing approaches, off-the-shelf / allogeneic CAR or TCR T cells, next generation CAR constructs, etc.

[0062] 49. A method of predicting response to CAR or TCR T cell therapy, whereby patients with higher levels of CD27+CD28+ naive Th cells (greater than 0-0.005%, 0.005-0.010%, 0.01%-0.05%, 0.05-0.1%, 0.1-0.5%, 0.5%-1.0%, 1-5%, 5-10%, 10-15%, preferably greater than 0.1%) are predicted to be more responsive to CAR T therapy and less likely to require intervention.

[0063] 50. A method for stratifying patients with lower levels (below 0-0.005%, 0.005-0.010%, 0.01%-0.05%, 0.05-0.1%, 0.1-0.5%, 0.5%-1.0%, 1-5%, 5-10%, 10-15%, preferably below 0.08%) of CD27+CD28+ naive Th cells, where further modifications to the treatment such as combination therapy, optimized manufacturing approaches, off-the-shelf / allogeneic CAR T cells, next generation CAR constructs, etc. are considered.

[0064] 51. A therapeutic approach to preserve CD27+CD28+ naive Th cells in the peripheral / tumor environment and prior to the production of an apheresis PBMC product for CAR T therapy by transferring earlier chemotherapy agents that would otherwise significantly deplete CD27+CD28+ naive Th cells to a later line of treatment.

[0065] 52. A method showing that the levels of CD27+CD28+ naive Th cells in pre-manufacturing apheresis PBMC products, along with the positive impact these cells have on product suitability at the time of apheresis, can be used to freeze apheresis bags prior to initiating any treatment for subjects with cancer to obtain the best transfer cells for CAR or TCR T cell therapy.

[0066] 53. A method for predicting response to CAR or TCR T therapy and the need for further intervention, whereby patients with lower levels of intermediate monocytes of these cells (below 0-1%, 1-5%, 5-10%, 10-15%, 15-20%, preferably below 3%) are more responsive to CAR T therapy and less likely to require further intervention. In one embodiment, patients with higher levels may need to consider further modifications to treatment such as combination therapy, optimized manufacturing approaches, off-the-shelf / allogeneic CAR or TCR T cells, next generation CAR constructs, etc.

[0067] 54. The method of embodiment 53, wherein previous chemotherapy agents that expand these cells are transferred to later lines of treatment to prevent these cells from expanding in the periphery / TME prior to CAR or TCR T therapy, and the apheresis bag should be frozen to obtain the best incoming cells for CAR T cell therapy before any therapy is initiated for the subject with cancer.

[0068] 55. A method for assessing prognosis, further showing that the International Prognostic Index (IPI) score and the level of intermediate monocytes in the apheresis product are positively associated, and that these cells are associated with subjects with a worse prognosis, and that intermediate monocytes are positively associated with baseline tumor burden.

[0069] 56. The method of embodiment 55, wherein the level of intermediate monocytes indicates a less than optimal state for the efficacy of CAR or TCR T cells, and further intervention / optimization may be required to improve the efficacy of CAR T therapy if the level of these cells is above 3% (or above 0-1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%).

[0070] 57. The method of any one of embodiments 55 and 56, wherein the patient is stratified based on the level of intermediate monocytes for manufacturing optimization versus removal of intermediate monocytes and increasing the level of naive product cells, use of next generation CAR constructs, and / or use of combination therapy with immunomodulators or checkpoint blockade, pre-made / allogeneic CARs or TCR T cells, etc.

[0071] 58. A method for predicting OS and PFS for CAR or TCR T cell therapy in a subject in need of CAR or TCR T cell therapy, comprising measuring the level of CD27-CD28+TEMRA Treg cells (% of white blood cells) in an apheresis product and determining the likelihood of survival and PFS based on whether the level is above or below a cutoff, wherein the level of CD27-CD28+TEMRA Treg cells (% of white blood cells) in the apheresis product is positively associated with OS and PFS and is a predictive marker for OS and PFS.

[0072] 59. The method of embodiment 58, wherein for CD27-CD28+TEMRA Tregs, subjects with higher levels of these cells (e.g., above a threshold of 0.17%) have higher complete response rates, objective response rates, and sustained response rates.

[0073] 60. The method of embodiment 59, wherein the cutoff threshold is 0.17%.

[0074] 61. The method of embodiment 59, wherein the cutoff is approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1-5, 5-10, 10-20, preferably between 0.05-0.2, 0.2-0.25, 0.25-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-1, 1-5, 5-10, 10-15, etc., 95-100, 100-200, 200-300, etc., more preferably about 0.1705.

[0075] 62. Patients were stratified according to which low levels of CD27-CD28+TEMRA The method of any one of embodiments 59-61, wherein a subject with Tregs may benefit from another form of therapy (such as combination therapy, allogeneic CAR or TCR T cells), manufacturing optimization, next generation CARs, etc., to improve the chances of survival with CAR therapy.

[0076] 63. A method for predicting CAR or TCR T cell proliferation and therapeutic response, whereby CD27+CD28+ naive Th cells are positively associated with CAR T peak proliferation, This has been shown to positively correlate with response, indicating that these cells have a positive impact on response.

[0077] 64. The method of embodiment 63, wherein low levels of peak proliferation of both CD27+CD28+ naive Th cells and CAR T cells correlate with higher non-responder rates, while increased levels of both result in higher response rates.

[0078] 65. A method of using the level of intermediate monocytes to stratify patients for manufacturing optimization and to reduce this population in the product to enhance final CAR T cells, thereby improving CAR or TCR expansion and response rates, where there is a correlation between the level of intermediate monocytes in the apheresis product versus CAR-T peak and CAR-T peak / baseline tumor burden, and there is a negative correlation between the level of intermediate monocytes and CAR T cell peak expansion (normalized by tumor burden). The level of intermediate monocytes is negatively associated with CAR T peak expansion (CAR / TB) which has been shown to positively correlate with response, indicating that these cells should have a negative impact on response and CAR function after infusion, and high intermediate monocytes are an indication of additional therapeutics, next generation CAR constructs, utilization of off-the-shelf / allogeneic CAR or TCR T cells, or manufacturing optimization to improve efficacy.

[0079] 66. A method of predicting response to CAR or TCR T cell therapy by measuring levels of intermediate monocytes and CAR T peak proliferation levels, whereby high levels (above the median or greater than 0-1%, 1-5%, 5-10%, 10-15%, 15-20%, preferably greater than 3%) of intermediate monocytes and low levels (below the median or less than 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, preferably less than 43%) of CAR-T peak in the apheresis product correlate with a higher non-responder rate, whereas reduced intermediate monocyte levels and increased CAR T peak proliferation result in a higher response rate.

[0080] 67. A method for predicting likelihood of complete response, objective response, and continued response to CAR or TCR T cell therapy in a subject in need of CAR or TCR T cell therapy, comprising measuring a lymphocyte to white blood cell ratio in a baseline hematology cell count and predicting the likelihood of complete response, objective response, and continued response based on the ratio. In one embodiment, when the ratio is above the optimal cutoff (e.g., the optimal cutoff may be about 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, etc.), the likelihood of a complete response, objective response, and sustained response is higher than when the ratio is below the cutoff (e.g., the cutoff may be about 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, etc.).

[0081] 68. A method of patient stratification in which subjects with low levels of lymphocytes relative to white blood cells are treated with alternative forms of therapy (combination therapy, allogeneic CAR T cells, next generation CAR constructs, etc.) to improve chances of survival / response and / or are subjected to optimized manufacturing to improve product suitability.

[0082] 69. A method of patient stratification, whereby a low level of leukocytes to lymphocytes (below the median, or below 1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, etc., preferably below 5.2) indicates a higher likelihood of having a toxic event, and indicates prophylactic administration of anti-inflammatory drugs (e.g., tocilizumab, steroids) to the patient to prevent toxicity.

[0083] 70. A method for predicting response to CAR or TCR T cell therapy by measuring the lymphocyte to white blood cell ratio in baseline hematology cell counts, whereby the ratio is negatively associated with tumor burden and thereby positively associated with response.

[0084] 71. A method of stratifying patients for additional interventions to improve efficacy when pre-manufacturing PBMC lymphocyte-to-leukocyte ratios are low (below the median, or below 1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, etc., preferably below 5.2) and / or patients have high tumor burden.

[0085] 72. Methods for estimating levels of inflammatory cytokines CRP, ferritin, IL 6. CRP, ferritin and IL6 in cancer patients associated with poorer prognosis by measuring lymphocyte to white blood cell ratio in baseline hematology cell counts (lymphocyte to white blood cell ratio in baseline hematology cell counts is negatively associated with cytokines previously shown to be pharmacodynamic markers negatively correlated with response in DLBCL, and optionally, patients are selected for administration of anti-inflammatory drugs before and / or after CAR T cell therapy if low levels of lymphocytes to white blood cells are quantified).

[0086] 73. A method for predicting a patient's myeloid cell levels, wherein the lymphocyte to white blood cell ratio in baseline hematology cell counts is negatively associated with myeloid cells (more specifically, intermediate monocytes, which are negatively associated with response) and positively associated with CD8 and EM / effector T cells.

[0087] 74. The method of embodiment 73, wherein patients with a low lymphocyte to leukocyte ratio (below the median or below 1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, etc., preferably below 5.2) are considered for combination therapy attempting to neutralize activity of the myeloid compartment and / or for optimization of manufacturing processes to deplete those populations in the product.

[0088] 75. A method of stratification in cancer treatment, wherein subjects with low levels of lymphocytes to monocytes are administered another form of therapy in addition to or as an alternative to CAR T cell therapy (e.g., combination therapy, allogeneic CAR T cells, next generation CAR constructs, etc.) to improve chances of survival, and / or the subject is subjected to optimized manufacturing of the CAR T cell product to improve product suitability, wherein the ratio of lymphocytes to monocytes in baseline hematology cell counts is positively associated with OS and PFS and may serve as a predictive biomarker for OS and PFS.

[0089] 76. A method for predicting response in which higher complete response rates, objective response rates, and ongoing response rates are observed in subjects with a lymphocyte-to-monocyte ratio greater than 0.79 or a ratio between 0-0.5, 0.5-1.0, 1.0-1.5, 1.5-2.0, 2-5, 5-10, 10-15, etc.

[0090] 77. A method for predicting response to immunotherapy (e.g., CAR T cells), wherein low levels of monocytes to lymphocytes (below the median, or below 1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, preferably below 8%) indicate a higher likelihood of having a toxic event and indicate prophylactic use of anti-inflammatory drugs (e.g., tocilizumab, steroids) to prevent toxicity.

[0091] 78. A method to quantify the lymphocyte to monocyte ratio in baseline hematology cell counts that allows for estimation of a patient's tumor burden, which has been shown to be a negative indicator of the clinical efficacy of CAR T cells.

[0092] 79. The method of embodiment 78, wherein the lymphocyte to monocyte ratio in the baseline hematology cell count may indicate the use of additional therapeutic agents to help overcome a greater estimated tumor burden, such as chemotherapy, radioactive antibody and small molecule based therapies, immunotherapies (including but not limited to checkpoint inhibitors, bispecific engagers), and cellular therapies (including but not limited to CAR-T, TCR-based lymphocytes and tumor infiltrating lymphocytes) where tumor burden has been shown to be a negative prognostic and / or predictive biomarker.

[0093] 80. By measuring the lymphocyte to monocyte ratio in baseline hematology cell counts, the levels of CRP and IL6 in the serum of cancer patients and / or the efficacy and safety of immunotherapy (e.g., CAR 1. A method for predicting prognosis of a patient with a T cell therapy, wherein CRP and IL6 levels are negatively associated with lymphocyte to monocyte ratio levels in baseline hematology cell counts and positively associated with poor prognosis.

[0094] 81. A method of stratifying patients, in which if low levels of lymphocyte to monocyte counts (or levels below the median, or levels below 0.05%, 0.05-0.1%, 0.1-0.5%, 0.5-1.0%, 1-5%, 5-10%, 10-15%, preferably below 0.78) are quantified, the patient is administered an anti-inflammatory drug.

[0095] 82. A method for predicting the levels of myeloid cells, CD8 and / or EM / effector cells in the final infusion product by measuring the lymphocyte to monocyte ratio in baseline hematology cell counts, which is negatively associated with myeloid cells and positively associated with CD8 and EM / effector T cells, and optionally further using the method to stratify patients for combination therapies attempting to abrogate activity of the myeloid compartment and / or for optimization of pre-manufacturing materials to deplete these populations.

[0096] 83. A method for predicting suitability and response of a T cell product in cancer patients, comprising: a lymphocyte to monocyte ratio in baseline hematology cell counts that correlates negatively with intermediate monocytes and with apheresis populations associated with response including CD27-CD28+TEMRA and Tregs, and CD27+CD28+ naive and Th cells; and a high level of monocytes to lymphocytes (or above the median, or 0-0.5, 0.5-1.0, 1.0 ... wherein a median level (greater than 0.5, 1.5-2.0, 2-5, 5-10, 10-15, preferably greater than 0.8%) indicates incoming apheresis material that tracks positively with product suitability and efficacy, and low levels (below the median or below 0-0.5, 0.5-1.0, 1.0-1.5, 1.5-2.0, 2-5, 5-10, 10-15, preferably below 0.78%) indicates a need for manufacturing optimization, combination therapy, or next generation CAR or TCR therapy.

[0097] One embodiment of the present disclosure relates to a method for predicting the likelihood of survival of a patient in need of chimeric antigen receptor (CAR) T-cell therapy, comprising measuring a level of CD27+CD28+ naive Th cells in an apheresis product from the patient and determining the likelihood of survival of the patient at least in part from the level of CD27+CD28+ naive Th cells in the apheresis product. In such an embodiment, the patient is determined to have an increased likelihood of survival if said level of CD27+CD28+ naive Th cells exceeds a cutoff percentage value measured as a percentage of total white blood cells, and the patient is determined to have a decreased likelihood of survival if the level of CD27+CD28+ naive Th cells is below the cutoff percentage value.

[0098] One embodiment of the present disclosure is a method for treating a patient in need of the above-mentioned chimeric antigen receptor (CAR) T cell therapy. For methods of predicting the chances of survival of a patient, the cut-off percentage value is approximately 0-0.1%, 0.1%-0.5%, 0.5%-1.0%, 1.0-5%, 5-10%, 10-15%, 10-20%, 20-30%, 30-40%, 40-50%, or more preferably approximately 0.27%.

[0099] One embodiment of the present disclosure relates to a method for predicting the likelihood of survival of a patient in need of the above-mentioned chimeric antigen receptor (CAR) T-cell therapy, further comprising measuring a level of intermediate monocytes in an apheresis product from the patient and determining the likelihood of survival of the patient at least in part from the level of intermediate monocytes in the apheresis product. In such a method, the patient is determined to have an increased likelihood of survival if said level of intermediate monocytes is below a cutoff percentage value measured as a percentage of total white blood cells, and the patient is determined to have a decreased likelihood of survival if the level of intermediate monocytes is above the cutoff percentage value.

[0100] One embodiment of the present disclosure relates to a method for predicting the likelihood of survival of a patient in need of said chimeric antigen receptor (CAR) T-cell therapy, wherein the cut-off percentage value is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%, preferably between 1-5%, and even more preferably below approximately 3%.

[0101] One embodiment of the present disclosure relates to a method for predicting the likelihood of survival of a patient in need of the above-mentioned chimeric antigen receptor (CAR) T-cell therapy, further comprising measuring the level of CD27-CD28+TEMRA Treg cells in an apheresis product from the patient, and determining the likelihood of survival of the patient at least in part from the level of CD27-CD28+TEMRA Treg cells in the apheresis product. In such a method, if the level of CD27-CD28+TEMRA Treg cells is above a cutoff percentage value measured as a percentage of total white blood cells, the patient is determined to have an increased likelihood of survival, and if the level of CD27-CD28+TEMRA Treg cells is below the cutoff percentage value, the patient is determined to have a decreased likelihood of survival.

[0102] One embodiment of the present disclosure relates to a method for predicting the likelihood of survival of a patient in need of said chimeric antigen receptor (CAR) T-cell therapy, wherein the cut-off percentage value is approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1-5%, 5-10%, 10-20%, preferably between 0.05-0.2%, 0.2-0.25%, 0.25-0.5%, 0.5-0.6%, 0.6-0.7%, 0.7-0.8%, 0.8-0.9%, 0.9-1%, 1-5%, 5-10%, 10-15%, more preferably approximately 0.1705%.

[0103] One embodiment of the present disclosure relates to a method for predicting the likelihood of survival of a patient in need of the above-mentioned chimeric antigen receptor (CAR) T-cell therapy, further comprising measuring a lymphocyte-to-leukocyte ratio in the patient's baseline hematological count and determining the likelihood of survival of the patient at least in part from the lymphocyte-to-leukocyte ratio. In such a method, if the lymphocyte-to-leukocyte ratio is above a cutoff value, the patient is determined to have an increased likelihood of survival, and if the lymphocyte-to-leukocyte ratio is below the cutoff value, the patient is determined to have a decreased likelihood of survival.

[0104] One embodiment of the present disclosure relates to a method for predicting the likelihood of survival of a patient in need of the above-mentioned chimeric antigen receptor (CAR) T-cell therapy, wherein the cut-off value is 1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, and preferably below 5.2%.

[0105] One embodiment of the present disclosure relates to a method for predicting the likelihood of survival of a patient in need of the above-mentioned chimeric antigen receptor (CAR) T-cell therapy, further comprising measuring a lymphocyte-to-monocyte ratio in the patient's baseline hematological count and determining the likelihood of survival of the patient at least in part from the lymphocyte-to-monocyte ratio. In such a method, if the lymphocyte-to-monocyte ratio is above a cutoff value, the patient is determined to have an increased likelihood of survival, and if the lymphocyte-to-monocyte ratio is below the cutoff value, the patient is determined to have a decreased likelihood of survival.

[0106] One embodiment of the present disclosure relates to a method for predicting the likelihood of survival of a patient in need of the above-mentioned chimeric antigen receptor (CAR) T-cell therapy, wherein the cutoff value is between 0 to 0.5, 0.5 to 1.0, 1.0 to 1.5, 1.5 to 2.0, 2 to 5, 5 to 10, 10 to 15, preferably 0.79.

[0107] One embodiment of the present disclosure relates to a method of manufacturing an immunotherapy product comprising preparing an apheresis product from a blood sample from a subject, measuring the level of CD27+CD28+ naive Th cells in the apheresis product, and increasing the amount of CD27+CD28+ naive Th cells recovered for processing if the level of CD27+CD28+ naive Th cells in the apheresis product is below a cutoff percentage value measured as a percentage of total white blood cells in the apheresis product.

[0108] One embodiment of the present disclosure relates to a method of producing the above immunotherapy product, wherein the cut-off percentage value is approximately 0-0.1%, 0.1%, -0.5%, 0.5%, -1.0%, 1.0-5%, 5-10%, 10-15%, 10-20%, 20-30%, 30-40%, 40-50%, or more preferably approximately 0.27%.

[0109] One embodiment of the present disclosure relates to a method for producing an immunotherapy product as described above, comprising measuring a level of intermediate monocytes in an apheresis product, and if the level of intermediate monocytes in the apheresis product is above a cutoff percentage value, measured as a percentage of total white blood cells in the apheresis product, reducing the level of intermediate monocytes in the apheresis product prior to further processing.

[0110] One embodiment of the present disclosure relates to a method of producing the above immunotherapy product, wherein the cut-off percentage value is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20%, preferably 1-5%, and even more preferably approximately 3%.

[0111] One embodiment of the present disclosure relates to a method for producing an immunotherapy product as described above, further comprising measuring the level of CD27-CD28+TEMRA Treg cells in the apheresis product, and increasing the amount of CD27-CD28+TEMRA Treg cells recovered for processing if the level of CD27-CD28+TEMRA Treg cells in the apheresis product is below a cutoff percentage value measured as a percentage of total white blood cells in the apheresis product.

[0112] One embodiment of the present disclosure relates to a method of producing the above immunotherapy product, wherein the cut-off percentage value is between approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1-5%, 5-10%, 10-20%, preferably 0.05-0.2%, 0.2-0.25%, 0.25-0.5%, 0.5-0.6%, 0.6-0.7%, 0.7-0.8%, 0.8-0.9%, 0.9-1%, 1-5%, 5-10%, 10-15%, more preferably approximately 0.1705%.

[0113] One embodiment of the present disclosure is a method of treating a malignant tumor in a patient, comprising administering to the patient an apheresis vector. determining whether to administer an effective dose of T cells comprising a chimeric receptor, or an effective dose of T cells comprising a chimeric receptor and a combination therapy to the patient based at least in part on the level of CD27+CD28+ naive Th cells in the apheresis product, and administering the effective dose of T cells comprising a chimeric receptor, or an effective dose of T cells comprising a chimeric receptor and a combination therapy based on the determining step. In such a method, if the level of CD27+CD28+ naive Th cells exceeds a cutoff percentage value measured as a percentage of total white blood cells, the patient is administered an effective dose of T cells comprising a chimeric receptor, and if the level of CD27+CD28+ naive Th cells is below the cutoff percentage value, the patient is administered an effective dose of T cells comprising a chimeric receptor and a combination therapy.

[0114] One embodiment of the present disclosure relates to a method of treating a malignant tumor in a patient as described above, wherein the cutoff percentage value is approximately 0-0.1%, 0.1%, -0.5%, 0.5%, -1.0%, 1.0-5%, 5-10%, 10-15%, 10-20%, 20-30%, 30-40%, 40-50%, or more preferably approximately 0.27%.

[0115] One embodiment of the present disclosure relates to a method of treating a malignancy in a patient as described above, further comprising: measuring a level of intermediate monocytes in an apheresis product from the patient; determining whether to administer an effective dose of T cells comprising a chimeric receptor, or an effective dose of T cells comprising a chimeric receptor and a combination therapy to the patient based at least in part on the level of intermediate monocytes in the apheresis product; and administering an effective dose of T cells comprising a chimeric receptor, or an effective dose of T cells and a combination therapy based on the determining step. In such a method, if the level of intermediate monocytes is below a cutoff percentage value measured as a percentage of total white blood cells, the patient is administered an effective dose of T cells comprising a chimeric receptor, and if the level of intermediate monocytes is above the cutoff percentage value, the patient is administered an effective dose of T cells comprising a chimeric receptor and a combination therapy.

[0116] In one embodiment of the present disclosure, relating to the method of treating a malignant tumor in a patient as described above, the cut-off percentage value is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20%, preferably between 1-5%, and even more preferably approximately 3%.

[0117] One embodiment of the present disclosure relates to a method of treating a malignancy in a patient as described above, further comprising: measuring a level of CD27-CD28+TEMRA Treg cells in an apheresis product from the patient; determining whether to administer an effective dose of T cells comprising a chimeric receptor or an effective dose of T cells comprising a chimeric receptor and a combination therapy to the patient based at least in part on the level of CD27-CD28+TEMRA Treg cells in the apheresis product; and administering an effective dose of T cells comprising a chimeric receptor or an effective dose of T cells and a combination therapy based on the determining step. In such a method, if the level of CD27-CD28+TEMRA Treg cells is above a cutoff percentage value measured as a percentage of total white blood cells, the patient is administered an effective dose of T cells comprising a chimeric receptor and a combination therapy, and if the level of CD27-CD28+TEMRA Treg cells is below the cutoff percentage value, the patient is administered an effective dose of T cells comprising a chimeric receptor and a combination therapy.

[0118] One embodiment of the present disclosure relates to a method for treating a malignant tumor in a patient as described above, wherein the cut-off percentage value is approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1-5%, 5-10%, 10-20%, preferably 0.05-0.2%, 0.2-0.25%, 0.25-0.5%, 0.5-0.6%, 0.6-0.7%, 0.7-0.8%, 0.8-0.9%, 0.9-1%, 1-5%, It is preferably 5 to 10%, more preferably 10 to 15%, and even more preferably about 0.1705%.

[0119] One embodiment of the present disclosure relates to a method of treating a malignancy in a patient as described above, further comprising: measuring a lymphocyte-to-leukocyte ratio in the patient's baseline hematological count; determining whether the patient should be administered an effective dose of T cells comprising a chimeric receptor, or an effective dose of T cells comprising a chimeric receptor and a combination therapy based at least in part on the lymphocyte-to-leukocyte ratio; and administering the effective dose of T cells comprising a chimeric receptor, or an effective dose of T cells and a combination therapy based on the determining step. In such a method, if the lymphocyte-to-leukocyte ratio is above a cutoff value, the patient is administered an effective dose of T cells comprising a chimeric receptor, and if the lymphocyte-to-leukocyte ratio is below the cutoff value, the patient is administered an effective dose of T cells comprising a chimeric receptor and a combination therapy.

[0120] One embodiment of the present disclosure relates to a method for treating a malignant tumor in a patient as described above, wherein the cutoff value is 1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, preferably 5.2%.

[0121] One embodiment of the present disclosure relates to a method of treating a malignancy in a patient as described above, further comprising: measuring a lymphocyte-to-monocyte ratio in the patient's baseline hematological count; determining whether the patient should be administered an effective dose of T cells comprising a chimeric receptor, or an effective dose of T cells comprising a chimeric receptor and a combination therapy based at least in part on the lymphocyte-to-monocyte ratio; and administering the effective dose of T cells comprising a chimeric receptor, or an effective dose of T cells and a combination therapy based on the determining step. In such a method, if the lymphocyte-to-monocyte ratio is above a cutoff value, the patient is administered an effective dose of T cells comprising a chimeric receptor, and if the lymphocyte-to-monocyte ratio is below the cutoff value, the patient is administered an effective dose of T cells comprising a chimeric receptor and a combination therapy.

[0122] One embodiment of the present disclosure relates to a method for treating a malignant tumor in a patient, wherein the cutoff value is 0 to 0.5, 0.5 to 1.0, 1.0 to 1.5, 1.5 to 2.0, 2 to 5, 5 to 10, 10 to 15, and preferably 0.79. One embodiment of the present disclosure relates to a method for treating a malignant tumor in said patient, wherein the combination therapy comprises immunotherapy, an SRC kinase inhibitor, a T cell bispecific antibody, an anti-CD20 monoclonal antibody, anti-4-1BB, anti-CD47, a TGF-beta inhibitor or a dominant negative TGF-beta beta, an mTOR / AKT agonist, a histone deacetylase inhibitor, cyclophosphamide, fluorouracil, gemcitabine, doxorubicin, a taxane, chemotherapy or radiation therapy, a small molecule inhibitor, an antibody targeted to enhance anti-tumor immunity, or an anti-inflammatory drug. [Brief description of the drawings]

[0123] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0124] [Figure 1A] Overview of flow cytometry markers utilized in the analysis of apheresis material from clinical studies. [Figure 1B] Overview of NK / monocyte / DC subset flow cytometry markers and gating strategies utilized for analysis of apheresis material from clinical trials. [Figure 1C] Schematic diagram of comparisons and key findings between pre-existing immune system status (as determined from blood), tumor immune status, and axicabtagene ciloleucel product attributes.

[0125] [Diagram 2] Associations between pre-treatment immune populations and key product attributes. Heatmap (y-axis) of selected immune populations from pre-production PBMC populations of clinical trial subjects compared to axicabtagene ciloleucel product attributes (x-axis) by Spearman rank correlation. Red values ​​represent positive correlation and blue values ​​represent negative correlation. % of CD27+CD28+ naive Th cells in pre-production PBMC population correlates with CD27+CD28+ naive product phenotype.

[0126] [Diagram 3] Associations between pre-manufacturing immune populations and immune populations at baseline, inflammatory cytokines, CAR T cell proliferation, and baseline tumor burden. Heatmaps (y-axis) of selected immune populations from pre-manufacturing PBMC populations of clinical trial subjects were compared to immune populations at baseline, inflammatory cytokines, CAR T cell proliferation, and baseline tumor burden (x-axis) by Spearman rank correlation. Red values ​​represent positive correlations and blue values ​​represent negative correlations. % intermediate and total monocytes in pre-manufacturing PBMC populations correlate with pre-treatment circulating inflammatory markers, tumor burden, and hypoxia (LDH), and CD27+CD28+ naive Th, CD27-CD28+ TEMRA Treg, and B cells correlate positively with CAR T cell proliferation.

[0127] [Figure 4] Association between pre-manufacturing immune populations and signatures of the tumor microenvironment. Heatmap (y-axis) of selected immune populations from pre-manufacturing PBMC populations of clinical trial subjects compared to gene expression profiling by nanostring (x-axis) of the tumor microenvironment by Spearman rank correlation. Red values ​​represent positive correlation and blue values ​​represent negative correlation. The % of CD27+CD28+ naive Th and CD27-CD28+ TEMRA Treg cells in the pre-manufacturing PBMC population is associated with a TME enriched in T cell ("hot" TME) and myeloid signatures, whereas the monocyte population in the pre-manufacturing PBMC population is associated with an immune TME devoid of immune cells, i.e., immune desert (intermediate monocyte) or an imbalanced TME with a predominance of myeloid signatures.

[0128] [Diagram 5]Volcano plot of naive Th cells in pre-manufacturing PBMC populations of clinical trial subjects compared to baseline cytokines, baseline laboratory chemistry values, axicabtagene ciloleucel product attributes and baseline pre-treatment TME signature. The x-axis represents Spearman's rank correlation between values ​​and the y-axis represents the significance of the correlation. Naive Th was subsetted pre-manufacturing and positively associated with % naive T cells in product infusion bag, a T cell-rich tumor immune landscape and negatively associated with pre-treatment inflammatory / tumor hypoxia. IS 21, Immunosign 21 gene expression signature.

[0129] [Figure 6] Volcano plot of intermediate monocytes in pre-manufacturing PBMC populations of clinical trial subjects compared to baseline cytokines, baseline laboratory chemistry values, axicabtagene ciloleucel product attributes and pre-treatment TME signatures. The x-axis represents Spearman's rank correlation between values ​​and the y-axis represents the significance of the correlation. Pre-manufacturing intermediate monocytes are positively associated with pre-treatment inflammatory / tumor hypoxia and negatively associated with pre-treatment T cell-rich tumor immune landscape.

[0130] [Figure 7A] Overall survival curves of clinical trial subjects grouped by pre-manufacturing %CD27+CD28+ naive Th cells. Kaplan-Meier overall survival curves with optimal cut-off selection of %CD27+CD28+ naive Th cells in pre-manufacturing PBMC population (with significance determined by Log-Rank test). Rates of complete response, objective response, progressive ongoing response, grade 3+ toxicity and CAR T-cell expansion were determined for subjects with %CD27+CD28+ naive Th cells in the pre-manufacturing PBMC population above or below the optimal cut-off. [Figure 7B]Progression-free survival curves of clinical trial subjects grouped by pre-production %CD27+CD28+ naive Th cells. Kaplan-Meier progression-free survival curves using optimal cut-off selection of %CD27+CD28+ naive Th cells in the pre-production PBMC population (with significance determined by Log-Rank test). Rates of complete response, objective response, ongoing response, grade 3+ toxicity and CAR T-cell expansion were determined for subjects with %CD27+CD28+ naive Th cells in the pre-production PBMC population above or below the optimal cut-off.

[0131] [Figure 8A] Overall survival curves of clinical trial subjects grouped by pre-manufacturing intermediate monocyte %. Kaplan-Meier overall survival curves using optimal cutoff selection for intermediate monocyte % in the pre-manufacturing PBMC population (with significance determined by Log-Rank test). Rates of complete response, objective response, ongoing response, grade 3+ toxicity, and CAR T-cell expansion were determined for subjects with intermediate monocyte % in the pre-manufacturing PBMC population above or below the optimal cutoff. [Figure 8B] Progression-free survival curves of clinical trial subjects grouped by pre-manufacturing % intermediate monocytic cells. Kaplan-Meier progression-free survival curves using optimal cutoff selection for % intermediate monocytic cells in the pre-manufacturing PBMC population (with significance determined by Log-Rank test). Rates of complete response, objective response, ongoing response, grade 3+ toxicity and CAR T-cell expansion were determined for subjects with % intermediate monocytic cells in the pre-manufacturing PBMC population above or below the optimal cutoff.

[0132] [Figure 9A]Overall survival curves of clinical trial subjects grouped by the ratio of CD27+CD28+ naive Th cells to intermediate monocytes pre-manufacturing. Kaplan-Meier overall survival curves with optimal cut-off selection for the ratio of CD27+CD28+ naive Th cells to intermediate monocytes in the pre-manufacturing PBMC population (with significance determined by Log-Rank test). Percentage of complete response, objective response, ongoing response, grade 3+ toxicity, and CAR T-cell expansion were determined for subjects above and below the optimal cut-off. [Figure 9B] Progression-free survival curves of clinical trial subjects grouped by the ratio of CD27+CD28+ naive Th cells to intermediate monocytes pre-manufacturing. Kaplan-Meier progression-free survival curves using optimal cut-off selection for the ratio of CD27+CD28+ naive Th cells to intermediate monocytes in the pre-manufacturing PBMC population (with significance determined by Log-Rank test). Percentage of complete response, objective response, ongoing response, grade 3+ toxicity, and CAR T-cell expansion were determined for subjects above and below the optimal cut-off.

[0133] [Figure 10A] Scatter plot of %CD27+CD28+ naive Th vs. % intermediate monocytes in pre-manufacturing PBMC populations colored by objective response status. Linear combination (black line) between %CD27+CD28+ naive Th and % intermediate monocytes in pre-manufacturing PBMC populations. Blue box encloses the region showing that responders cluster on the curve with high %CD27+CD28+ naive Th cells and low % intermediate monocytes. [Figure 10B] Scatter plot of %CD27+CD28+ naive Th versus % intermediate monocytes in pre-manufacturing PBMC populations colored by continued response status. Linear combination between %CD27+CD28+ naive Th and % intermediate monocytes in pre-manufacturing PBMC populations (black line).

[0134] [Figure 11]Scatter plot of % intermediate monocytes vs. peak CAR T cell proliferation in pre-manufacturing PBMC populations colored by continued response status. The scatter plot is divided into quadrants (Q1-Q4) based on intermediate monocyte status and peak CAR T cell proliferation above or below the respective median of their covariates. Note the high SPD cluster of non-responders with high intermediate monocytes and low CAR-T peaks, and the high SPD cluster of responders with high CAR-T peaks. SPD-two-way product sum.

[0135] [Figure 12A] Sustained response rate from % intermediate monocyte quadrants versus peak CAR-T cell proliferation scatter plot from FIG. 11 . [Figure 12B] Sustained response rate from % intermediate monocyte quadrants versus peak CAR T cell proliferation scatter plot from Figure 11 for subjects with baseline tumor burden above the median level (SPDhi). [Figure 12C] Sustained response rate from % intermediate monocyte quadrant versus peak CAR T cell proliferation scatter plot from Figure 11 for subjects with baseline tumor burden below the median level (SPD lo).

[0136] [Figure 13] Violin plot of CD27+CD28+ naive Th (% of leukocytes) in pre-manufacturing PBMCs grouped by response category.

[0137] [Figure 14] Violin plot of intermediate monocytes (% of leukocytes) in pre-manufacturing PBMCs grouped by response category.

[0138] [Figure 15A] Boxplot of CD27+CD28+ naive Th (% of leukocytes) in pre-production PBMCs grouped by number of prior lines of treatment, showing their enrichment in patients who received 2 or fewer lines of treatment. [Figure 15B]Box plot of CD27+CD28+ naive Th (% of leukocytes) in pre-production PBMCs grouped by IPI score. [Figure 15C] Boxplot of CD27+CD28+ naive Th (% of leukocytes) in pre-manufacturing PBMCs grouped by baseline tumor burden (SPD).

[0139] [Figure 16A] Boxplot of intermediate monocytes. [Figure 16B] Box plot of intermediate monocytes (% of leukocytes) in pre-production PBMCs grouped by IPI score, showing their enrichment in patients with higher IPI scores (% of leukocytes) in pre-production PBMCs grouped by number of prior lines of therapy. [Figure 16C] Box plot of intermediate monocytes (% of leukocytes) in pre-production PBMCs grouped by baseline tumor burden (SPD).

[0140] [Figure 17A] Overall survival curves of clinical trial subjects grouped by pre-manufacturing %CD27-CD28+TEMRA Treg cells. Kaplan-Meier overall survival curves with optimal cut-off selection of %CD27-CD28+TEMRA Treg cells in the pre-manufacturing PBMC population (with significance determined by Log-Rank test). Rates of complete response, objective response, ongoing response, grade 3+ toxicity and CAR T cell expansion were determined for subjects with %CD27-CD28+TEMRA Treg cells in the pre-manufacturing PBMC population above or below the optimal cut-off. [Figure 17B]Progression-free survival curves of clinical trial subjects grouped by pre-production %CD27-CD28+TEMRA Treg cells. Kaplan-Meier progression-free survival curves using optimal cutoff selection of %CD27-CD28+TEMRA Treg cells in the pre-production PBMC population (with significance determined by Log-Rank test). Rates of complete response, objective response, ongoing response, grade 3+ toxicity and CAR T cell expansion were determined for subjects with %CD27-CD28+TEMRA Treg cells in the pre-production PBMC population above or below the optimal cutoff.

[0141] [Figure 18A] Scatter plot of %CD27+CD28+ naive Th versus peak CAR T cell proliferation in pre-manufacturing PBMC populations colored by ongoing response status. Linear association between %CD27+CD28+ naive Th and peak CAR T cell proliferation in pre-manufacturing PBMC populations (black line). [Figure 18B] Scatter plot of %CD27+CD28+ naive Th versus peak CAR T cell proliferation in pre-manufacturing PBMC populations colored by objective response status. Linear association between %CD27+CD28+ naive Th and peak CAR T cell proliferation in pre-manufacturing PBMC populations (black line). Blue boxes indicate areas of high peak CAR T cell proliferation and high response rate with CD27+CD28+ naive Th cells. [Figure 18C] Scatter plot of %CD27+CD28+ naive Th vs. peak CAR T-cell proliferation / baseline tumor burden in pre-manufacturing PBMC populations, colored by continued response status. Linear association between %CD27+CD28+ naive Th and peak CAR T-cell proliferation / baseline tumor burden (as determined by two-way product sum, SPD) in pre-manufacturing PBMC populations (black line); [Figure 18D]Scatter plot of %CD27+CD28+ naive Th vs. peak CAR T cell proliferation / baseline tumor burden in pre-manufacturing PBMC populations colored by objective response status. Linear association between %CD27+CD28+ naive Th and peak CAR T cell proliferation / baseline tumor burden in pre-manufacturing PBMC populations (black line). Blue boxes indicate areas of high response rate with high peak CAR T cell proliferation / baseline tumor burden and CD27+CD28+ naive Th cells.

[0142] [Figure 19A] Scatter plot of % intermediate monocytes vs. peak CAR T cell proliferation in pre-manufacturing PBMC populations colored by continued response status. Linear association between % intermediate monocytes and peak CAR T cell proliferation in pre-manufacturing PBMC populations (black line). Blue boxes indicate a cluster of high continued response rates with high peak CAR-T cell proliferation and low intermediate monocytes. [Figure 19B] Scatter plot of % intermediate monocytes vs. peak CAR T cell proliferation in pre-manufacturing PBMC populations colored by objective response status. Linear association between % intermediate monocytes and peak CAR T cell proliferation in pre-manufacturing PBMC populations (black line). Blue boxes indicate clusters of high response rates with high peak CAR T cell proliferation and low intermediate monocytes: [Figure 19C] Figure 1 shows a scatter plot of % intermediate monocytes in pre-manufacturing PBMC populations versus peak CAR T-cell proliferation / baseline tumor burden, colored by ongoing response status. Linear association between % intermediate monocytes and peak CAR T-cell proliferation / baseline tumor burden in pre-manufacturing PBMC populations (black line). Blue boxes indicate a cluster of high ongoing response rates with high peak CAR-T-cell proliferation / baseline tumor burden and low intermediate monocytes. [Figure 19D]Scatter plot of % intermediate monocytes vs. peak CAR T cell proliferation / baseline tumor burden in pre-manufacturing PBMC populations, colored by objective response status. Linear association between % intermediate monocytes and peak CAR T cell proliferation / baseline tumor burden in pre-manufacturing PBMC populations (black line). Blue boxes indicate clusters of high response rates with high peak CAR T cell proliferation / baseline tumor burden and low intermediate monocytes.

[0143] [Figure 20A] Overall survival curves for clinical trial subjects grouped by % lymphocytes to % white blood cells at baseline. Kaplan-Meier overall survival curves with optimal cut-off selection for % lymphocytes to % white blood cells at baseline (with significance determined by Log-Rank test). Rates of complete response, objective response, ongoing response, grade 3+ toxicity, and CAR T-cell proliferation were determined for subjects above and below the optimal cut-off. [Figure 20B] Overall survival curves for clinical trial subjects grouped by % lymphocytes to % white blood cells at baseline. Kaplan-Meier overall survival curves with optimal cut-off selection for % lymphocytes to % white blood cells at baseline (with significance determined by Log-Rank test). Rates of complete response, objective response, ongoing response, grade 3+ toxicity, and CAR T-cell proliferation were determined for subjects above and below the optimal cut-off.

[0144] [Figure 21] Violin plot of % lymphocytes to % white blood cells at baseline grouped by response category for evaluable clinical trial subjects. % lymphocytes to % white blood cells is positively associated with response.

[0145] [Figure 22]Violin plot of % lymphocytes vs % white blood cells at baseline grouped by worst grade toxicity. Lymphocytes vs. white blood cells in baseline hematology cell counts trend towards a negative association with worst grade toxicity. CRS-cytokine release syndrome. NE-neurological events.

[0146] [Diagram 23] Association between lymphocytes and leukocytes in baseline hematology cell counts and tumor burden by SPD at baseline. The % lymphocytes to leukocytes is negatively associated with tumor burden as shown by the scatter plot (left) and box plots grouped by category of tumor burden SPD at baseline.

[0147] [Figure 24] FIG. 1 shows box plots of the relationship between % lymphocytes to leukocytes in baseline hematology cell counts and number of prior lines of therapy grouped. % lymphocytes to leukocytes is negatively associated with number of prior lines of therapy.

[0148] [Diagram 25] Volcano plot of baseline serum cytokines and their Spearman rank correlation with % lymphocytes to leukocytes in baseline hematology cell counts. % lymphocytes to leukocytes is negatively associated with inflammatory and acute phase cytokines such as CRP, ferritin, and IL6.

[0149] [Figure 26] Volcano plots of pre-production PBMC populations and their Spearman rank correlation with % lymphocytes to leukocytes in baseline hematology cell counts. % lymphocytes to leukocytes in baseline hematology cell counts negatively correlates with myeloid cells and positively correlates with CD8 and EM / effector T cells.

[0150] [Figure 27A]Overall survival curves for clinical trial subjects grouped by lymphocyte to monocyte ratio at baseline. Kaplan-Meier overall survival curves with optimal cut-off selection for lymphocyte to monocyte ratio at baseline (with significance determined by Log-Rank test). Rates of complete response, objective response, ongoing response, grade 3+ toxicity, and CAR T-cell proliferation were determined for subjects above and below the optimal cut-off. [Figure 27B] Progression-free survival curves for clinical trial subjects grouped by baseline monocyte to lymphocyte ratio. Kaplan-Meier progression-free survival curves with optimal cutoff selection for baseline monocyte to lymphocyte ratio (with significance determined by Log-Rank test). Rates of complete response, objective response, ongoing response, grade 3+ toxicity, and CAR T-cell proliferation were determined for subjects above and below the optimal cutoff.

[0151] [Figure 28] Violin plot of monocyte to lymphocyte ratios at baseline grouped by response category for evaluable clinical trial subjects. The monocyte to lymphocyte ratio is positively associated with response.

[0152] [Figure 29] Violin plot of lymphocyte to monocyte ratio at baseline grouped by worst grade toxicity. Lymphocyte to monocyte ratio in baseline hematology cell counts trends towards a negative association with worst grade toxicity. CRS-cytokine release syndrome. NE-neurological event.

[0153] [Diagram 30] Association between lymphocyte to monocyte ratio in baseline hematology cell counts and tumor burden by SPD at baseline. The lymphocyte to monocyte ratio is negatively associated with tumor burden as shown by the scatter plot (left) and box plots grouped by category of tumor burden SPD at baseline.

[0154] [Diagram 31] 1 is a box plot of the relationship between lymphocyte to monocyte ratio in baseline hematology cell counts and grouped number of prior lines of therapy. The lymphocyte to monocyte ratio is negatively associated with the number of prior lines of therapy.

[0155] [Diagram 32] Volcano plots of baseline serum cytokines and their Spearman rank correlation with lymphocyte to monocyte ratio in baseline hematology cell counts. The lymphocyte to monocyte ratio is negatively associated with inflammatory and acute phase cytokines such as CRP and IL6.

[0156] [Diagram 33] Volcano plots of pre-production PBMC populations and their Spearman rank correlation with lymphocyte to monocyte ratios in baseline hematology cell counts. Lymphocyte to monocyte ratios in baseline hematology cell counts are negatively associated with myeloid cells and positively associated with CD8 and EM / effector T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0157] The present disclosure is based in part on the discovery of pre-infusion attributes of apheresis material and engineered CAR T cells (e.g., T cell compatibility), as well as pre-treatment characterization of patient immune factors that may be associated with clinical efficacy and toxicity, including durable responses, grade > 3 cytokine release syndrome, and grade > 3 neurological events. definition

[0158] In order that this disclosure may be more readily understood, certain terms are first defined below. Additional definitions for the following terms, as well as other terms, are found throughout the specification.

[0159] As used in this specification and the accompanying embodiments, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0160] As used herein, unless otherwise stated or clear from the context, the term "or" is understood to be inclusive and includes both "or" and "and."

[0161] The term "and / or" as used herein should be interpreted as a specific disclosure of each of the two specified features or components, whether or not the other is present. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include A and B, A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0162] As used herein, the terms "for example" and "i.e." are used merely as examples and are not intended to be limiting, and should not be construed as referring only to the items explicitly listed herein.

[0163] Terms such as "more than," "at least," and "more than," for example, "at least one," are intended to mean, but are not limited to, at least 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113 , 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or any value greater than the recited value. Any greater number or fraction in between is also included.

[0164] Conversely, the term "less than" includes each value less than the recited value. For example, "100 or fewer nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 55, 56, 57, 58, 59 ... Included are 3, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Any smaller number or fraction in between is also included.

[0165] Terms such as “plurality,” “at least two,” “two or more,” and “at least a second” are intended to mean, but are not limited to, at least, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 110, 111, 11 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 5, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 1 This is understood to include 38, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more, as well as any higher numbers or fractions therebetween.

[0166] Throughout this specification the words "comprising" or "comprises" or variations such as "comprising" are understood to mean the inclusion of the stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Whenever an embodiment is described herein with the language "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also presented.

[0167] As used herein, unless specifically stated or clear from the context, the term "about" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "and" may be used to refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. "Around" may mean within a range of 1x or more standard deviations as practiced in the art. "About" or "approximately" may mean within a range of up to 10% (i.e., ±10%). Thus, "about" may be understood to be 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is presented in this disclosure, unless otherwise indicated, the meaning of "about" or "approximately" should be assumed to be within an acceptable range of error for that particular value or composition.

[0168] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof, as appropriate (such as tenths and hundredths of integers), unless otherwise stated.

[0169] Units, prefixes and symbols used herein are presented using the format accepted by the Systeme International de Unites (SI). Numerical ranges are inclusive of the numbers defining the range.

[0170] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, e.g., Juo, "The Concise Dictionary of Biomedicine and Molecular Biology", 2nd Edition, (2001), CRC Press; "The Dictionary of Cell & Molecular Biology", 5th Edition, (2013), Academic Press; and "The Oxford Dictionary Of Biochemistry And Molecular Biology", Cammack et al., eds., 2nd Edition, (2006), Oxford. The University Press provides one of skill in the art with a general dictionary for many of the terms used in this disclosure.

[0171] "Administering" refers to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example, by injection or infusion. Exemplary routes of administration for the compositions disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example, by injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and substernal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered via a parenteral route, for example, orally. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, intravaginal, rectal, sublingual, or topical. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time. In one embodiment, the CAR T cell therapy is administered via an "infusion product" that includes the CAR T cells.

[0172] The term "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains, or antigen-binding molecules thereof, interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which are embedded in more conserved regions, called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains comprise a binding domain that interacts with an antigen. The constant regions of Abs can mediate the binding of immunoglobulins to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0173] Antibodies include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, Fabs, and the like. Antibodies include, but are not limited to, antibody fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In some embodiments, the antibodies described herein refer to polyclonal antibody populations.

[0174] An "antigen-binding molecule," "antigen-binding portion," or "antibody fragment" refers to any molecule that contains an antigen-binding portion (e.g., CDR) of the antibody from which the molecule is derived. An antigen-binding molecule may contain an antigen complementarity determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules containing a peptide-binding domain) are another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a hyperproliferative disease or a viral or bacterial antigen. In some embodiments, the antigen-binding molecule binds to CD19. In further embodiments, the antigen-binding molecule is an antibody fragment that specifically binds to an antigen and includes one or more of its complementarity determining regions (CDRs). In further embodiments, the antigen binding molecule is a single chain variable fragment (scFv). In some embodiments, the antigen binding molecule comprises or consists of an avimer.

[0175] "Antigen" refers to any molecule that can elicit an immune response or be bound by an antibody or antigen-binding molecule. The immune response can include either antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will readily appreciate that any macromolecule, including virtually any protein or peptide, can function as an antigen. Antigens can be expressed endogenously, i.e., by genomic DNA, or recombinantly. Antigens can be specific to a particular tissue, such as cancer cells, or can be broadly expressed. Additionally, fragments of larger molecules can act as antigens. In some embodiments, the antigen is a tumor antigen.

[0176] The term "neutralizing" refers to an antigen-binding molecule, scFv, antibody, or fragment thereof that binds to a ligand and prevents or reduces the biological action of that ligand. In embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof directly blocks a binding site on the ligand or otherwise alters the ability of the ligand to bind through indirect means (such as structural or energetic changes in the ligand). In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof prevents the protein to which it is bound from performing a biological function.

[0177] The term "autologous" refers to any material derived from the same individual that is later reintroduced. For example, the engineered autologous cell therapy (eACT™) method described herein involves the collection of lymphocytes from a patient, which are then engineered to express, for example, a CAR construct, and then administered to the same patient.

[0178] The term "allogeneic" refers to any material derived from one individual and then introduced into another individual of the same species, e.g., allogeneic T cell transplantation.

[0179] The terms "transduction" and "transduced" refer to the process by which foreign DNA is introduced into a cell via a viral vector (see Jones et al., Genetics: principles and analysis, Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0180] "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth leads to the formation of malignant tumors that may invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" may include tumors. In this application, the term cancer is synonymous with malignant tumor. Examples of cancers that may be treated by the methods disclosed herein include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, myelomas, and other white blood cell malignancies. In some embodiments, the methods disclosed herein are directed to treating a variety of cancers, including, for example, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, cancer of the fallopian tubes, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, [adding other solid tumors] multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, and parathyroid cancer. , adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of cancers. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is NHL. Certain cancers may be responsive to chemotherapy or radiation therapy, or certain cancers may be refractory. Refractory cancer refers to cancer that is not amenable to surgical intervention; refractory cancers either do not respond to chemotherapy or radiation therapy in the first place or the cancer becomes unresponsive over time.

[0181] As used herein, "anti-tumor effect" refers to a biological effect that can be presented as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall survival or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with tumors. Anti-tumor effect can also refer to the prevention of tumor development, e.g., a vaccine.

[0182] As used herein, "cytokine" refers to a non-antibody protein released by one cell in response to contact with a specific antigen, where the cytokine interacts with a second cell to mediate a response in the second cell. As used herein, "cytokine" is meant to refer to a protein released by one cell population that acts on another cell as an intercellular mediator. Cytokines can be expressed endogenously by cells or administered to a subject. Cytokines can be released by immune cells, such as macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, proinflammatory cytokines, effectors, and acute phase proteins. For example, homeostatic cytokines, such as interleukin (IL) 7 and IL-15, can promote survival and proliferation of immune cells, and proinflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of proinflammatory cytokines include IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast proliferation fibroblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble These include soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA). Examples include, but are not limited to:

[0183] A "chemokine" is a type of cytokine that mediates chemotaxis or directional movement of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1 alpha, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC or CCL17).

[0184] As used herein, "chimeric receptor" refers to an engineered surface-expressed molecule capable of recognizing a specific molecule. Chimeric antigen receptors (CARs) contain binding domains capable of interacting with specific tumor antigens, and engineered T-cell receptors are T cell receptors (TCRs) allow T cells to target cancer cells expressing specific tumor antigens. The cells can be targeted and killed. In one embodiment, the T cell therapy is based on T cells engineered to express a chimeric antigen receptor (CAR) or T cell receptor (TCR) that includes (i) an antigen binding molecule, (ii) a costimulatory domain, and (iii) an activation domain. The costimulatory domain may include an extracellular domain, a transmembrane domain, and an intracellular domain, and the extracellular domain includes a hinge domain that may be truncated.

[0185] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent, e.g., an engineered CAR T cell, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from developing a disease or promotes regression of the disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disability or incapacity due to disease affliction. Such terms can be used interchangeably. The ability of a therapeutic agent to promote regression of a disease can be evaluated using a variety of methods known to those of skill in the art, for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0186] As used herein, the term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell-toxic) lymphocyte that represents a major component of the genetic immune system. NK cells reject tumor and virus-infected cells. They act through the process of apoptosis, or programmed cell death. They were called "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without antibody involvement). Their T cell receptors (TCR) differentiate from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TCs, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells or killer T cells), memory T cells ((i) stem memory T cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Rα+, like naive cells, but also express large amounts of CD95, IL-2Rβ, CXCR3 and LFA-1, making them memory cells). They exhibit a number of unique functional attributes. ), (ii) central memory TCM cells express L-selectin and CCR7 and secrete IL-2 but not IFNγ or IL-4, and (iii) effector memory TCM cells, however, do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Treg, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. On the other hand, B cells play a major role in humoral immunity (involving antibodies). B cells produce antibodies and antigens, play the role of antigen-presenting cells (APC), and turn into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, from which the name is derived.

[0187] The term "genetically engineered" or "engineered" refers to a method of modifying the genome of a cell, including, but not limited to, deleting a coding or non-coding region or portion thereof, or inserting a coding region or portion thereof. In some embodiments, the modified cell is a lymphocyte, e.g., a T cell, and can be obtained from either a patient or a donor. The cell can be modified to express an exogenous construct, e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR), which is integrated into the genome of the cell.

[0188] "Immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including Abs, cytokines, and complement) produced either by these cells or the liver, resulting in the selective targeting, binding, damaging, destroying, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0189] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of suffering from or relapsing from a disease by methods that include inducing, enhancing, suppressing, or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy may include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT™), and allogeneic T cell transplantation. However, one of skill in the art will appreciate that the pretreatment methods disclosed herein enhance the efficacy of any transplanted T cell therapy. Examples of T cell therapy are described in U.S. Patent Application Publication Nos. 2014 / 0154228 and 2002 / 0006409, U.S. Patent No. 7,741,465, U.S. Patent No. 6,319,494, U.S. Patent No. 5,728,388, and WO 2008 / 081035. In some embodiments, the immunotherapy comprises CAR T cell therapy, hi some embodiments, the CAR T cell therapy product is administered by infusion.

[0190] T cells for immunotherapy can be derived from any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, splenic tissue, and tumors. Additionally, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood drawn from a subject using a variety of techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. Further methods of isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0191] The term "engineered autologous cell therapy" or "eACT™", also known as adoptive cell transfer, is the process of harvesting a patient's own T cells and subsequently genetically modifying them to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. T cells can be engineered, for example, to express a chimeric antigen receptor (CAR). CAR-positive (+) T cells are engineered to express an extracellular single-chain variable fragment (scFv) specific for a particular tumor antigen linked to an intracellular signaling moiety that includes at least one co-stimulatory domain and at least one activation domain. CAR The scFv can be designed to target, for example, CD19, a transmembrane protein expressed by cells of the B-cell lineage, including all normal B cells and B-cell malignancies such as, but not limited to, diffuse large B-cell lymphoma (DLBCL), not otherwise specified, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma, NHL, CLL, and non-T-cell ALL. Exemplary CAR T cell therapies and constructs are described in U.S. Patent Application Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which references are incorporated by reference in their entireties.

[0192] As used herein, a "patient" includes any human suffering from cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0193] As used herein, the term "in vitro cells" refers to any cells cultured ex vivo. In particular, in vitro cells can include T cells. The term "in vivo" means within a patient.

[0194] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids, with no limit on the maximum number of amino acids that may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, for example, and longer chains, generally referred to in the art as proteins, of which there are many varieties. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0195] As used herein, "stimulation" refers to a primary response induced by the binding of a stimulatory molecule to its cognate ligand, which mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell, e.g., a T cell receptor (TCR) / CD3 complex, that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen-presenting cell (e.g., an APC, a dendritic cell, a B cell, etc.), specifically binds to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, such as, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands include, but are not limited to, anti-CD3 antibodies, peptide-loaded MHC class I molecules, superagonist anti-CD2 antibodies, and superagonist anti-CD28 antibodies.

[0196] As used herein, a "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, results in a T cell response, including but not limited to, proliferation and / or upregulation or downregulation of key molecules.

[0197] As used herein, a "costimulatory ligand" includes a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory molecule on a T cell. Binding of a costimulatory ligand provides a signal that mediates a T cell response, such as, but not limited to, proliferation, activation, and differentiation. A costimulatory ligand induces a signal, for example, by binding of the T cell receptor (TCR) / CD3 complex to a peptide-loaded major histocompatibility complex (MHC) molecule, in addition to the primary signal provided by the stimulatory molecule. Costimulatory ligands can include, but are not limited to, 3 / TR6, 4-1BB ligand, an agonist or antibody that binds to the Toll ligand receptor, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), a ligand that specifically binds to B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or programmed death (PD)L1. In certain embodiments, the costimulatory ligand includes, but is not limited to, an antibody that specifically binds to a costimulatory molecule present on a T cell, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, a ligand that specifically binds to CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT). Some examples include:

[0198] A "costimulatory molecule" refers to a molecule that specifically binds a costimulatory ligand, thereby mediating a costimulatory response by a T cell, including, but not limited to, proliferation, by binding to a cognate binding partner on a T cell. Costimulatory molecules include 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD33, CD45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, and CD3 (A alpha, beta, delta, epsilon, gamma, zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8 alpha, CD8 beta, CD9, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, LIGHT (tumor necrosis factor superfamily member 14, TNFSF14), LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18) , MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule, SLAM (SLAMF1, CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0199] The terms "reduce" and "decrease" are used interchangeably herein and refer to any change less than the original. "Reduce" and "decrease" are relative terms and require a comparison of pre- and post-measurements. "Reduce" and "decrease" include complete depletion. Similarly, the terms "increase, increase" refer to any change greater than the original value. "Increase," "higher," and "lower" are relative terms and require a comparison between pre- and post-measurements and / or between reference standards. In some embodiments, the reference value is obtained from the general population, which may be the general population of patients. In some embodiments, the reference value is a quartile analysis of the general patient population.

[0200] "Treatment" or "treating" of a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, mitigating, ameliorating, inhibiting, slowing or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical manifestations associated with a disease. In some embodiments, "treatment" or "treating" includes partial remission. In other embodiments, "treatment" or "treating" includes complete remission.

[0201] As used herein, the term "polyfunctional T cells" refers to cells that co-secrete at least two proteins from a pre-specified panel per cell in combination with the amount of each protein produced (i.e., a combination of the number of proteins secreted and their intensity). In some embodiments, single cell functional profiles are determined for each evaluable population of engineered T cells. Profiles include effector (granzyme B, IFN-γ, MIP-1α, perforin, TNF-α, TNF-β), stimulatory (GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21), regulatory (IL-4, IL-10, IL-13, IL-22, TGF-beta1, sCD137, sCD40L), chemoattractant (CCL-11, IP-10, MIP In some embodiments, the functional profile of each cell allows for the calculation of other metrics, including breakdown of each sample by cell multifunctionality (i.e., what percentage of cells secrete multiple cytokines compared to non-secreting or monofunctional cells) and breakdown of samples by functional group (i.e., which mono- and polyfunctional groups are secreted by cells in the sample and their frequency).

[0202] As used herein, the term "quartile" or "quadrant" is a statistical term that describes the division of observations into four defined intervals based on the values ​​of the data and how they compare to the entire set of observations.

[0203] As used herein, the term "study day 0" is defined as the day a subject receives their first CAR T cell infusion. The day before study day 0 is study day -1. Both days after enrollment and before study day -1 are consecutive negative integer values.

[0204] As used herein, the term "objective response" refers to complete response (CR), partial response (PR), or no response. The criteria are based on the revised IWG response criteria for malignant lymphoma.

[0205] As used herein, the term "complete response" refers to the complete resolution of the disease such that it is no longer detectable by radiographic and clinical laboratory evaluation. There is no evidence of cancer at a given time point.

[0206] As used herein, the term "partial response" refers to a greater than 30% reduction in tumor without complete resolution. The criteria are based on the revised IWG response criteria for malignant lymphoma, with PR defined as "at least a 50% reduction in the sum of the products of diameters (SPD) of up to six major nodes or nodal masses." These nodes or masses should be selected according to all of the following: they should be clearly measurable in at least two perpendicular dimensions; if possible, they should be from different regions of the body; they should include areas of mediastinal and retroperitoneal disease whenever these sites are involved.

[0207] As used herein, the term "non-responder" refers to a subject who has not experienced a CR or PR following CAR T cell infusion.

[0208] As used herein, the term "durable response" refers to subjects with a sustained response by at least 1 year follow-up after CAR T cell infusion; 6 months f / u is only available for Z1, C3, as f / u is no longer available for this cohort. Nevertheless, the conclusion remains the same.

[0209] As used herein, the term "relapse" refers to a subject who achieves a complete response (CR) or partial response (PR) and then experiences disease progression.

[0210] As used herein, the proliferation and persistence of CAR T cells in peripheral blood can be monitored by qPCR analysis, for example, using CAR-specific primers for the scFv portion of the CAR (e.g., the heavy chain of the CD19 binding domain) and its hinge / CD28 transmembrane domain. Alternatively, it can be measured by enumeration of CAR cells / unit of blood volume.

[0211] As used herein, scheduled blood draws for CAR T cells are prior to CAR T cell infusion, 7 days, 2 weeks (14 days), 4 weeks (28 days), 3 months (90 days), 6 months (1 The time intervals may be 80 days, 12 months (360 days), and 24 months (720 days).

[0212] As used herein, "CAR T cell peak" is defined as the maximum absolute number of CAR+PBMCs / μL in serum achieved after day 0.

[0213] As used herein, "time to peak CAR T cells" is defined as the number of days from day 0 to the day the CAR T cells peak.

[0214] As used herein, the "area under the curve (AUC) of the levels of CAR T cells from day 0 to day 28" is defined as the area under the curve in a plot of the levels of CAR T cells versus scheduled visits from day 0 to day 28. This AUC measures the total levels of CAR T cells over time.

[0215] As used herein, scheduled blood draws for cytokines are prior to or on the day of conditioning chemotherapy (day -5), days 0, 1, 3, 5, 7, every other day during hospitalization if any, week 2 (day 14), and week 4 (day 28).

[0216] As used herein, "baseline" of a cytokine is defined as the last value measured prior to conditioning chemotherapy.

[0217] As used herein, fold change from baseline on day X is defined as:

number

[0218] As used herein, "post-baseline cytokine peak" is defined as the maximum level of cytokine in serum achieved after baseline (day -5) through day 28.

[0219] As used herein, "time to peak cytokine" following CAR T cell infusion is defined as the number of days from day 0 to the day peak cytokine is obtained.

[0220] As used herein, the "area under the curve (AUC) of cytokine levels" from day -5 to day 28 is defined as the area under the curve in a plot of cytokine levels versus scheduled visits from day -5 to day 28. This AUC measures the total level of cytokines over time. Assuming that cytokines and CAR+ T cells are measured at specific discrete time points, the trapezoidal rule can be used to estimate the AUC.

[0221] Various aspects of the disclosure are described in further detail in the following subsections. Pre-treatment attributes

[0222] Pre-treatment attributes of the apheresis and engineered cells (T cell attributes) and patient immune factors measured from patient samples can be used to assess the probability of clinical outcomes, including response and toxicity. Pre-treatment attributes derived from pre-manufactured apheresis material, engineered CAR T cells, and patient immune and clinical factors (including but not limited to cell populations, serum chemokines / cytokines, blood chemistry, etc.) may be used to assess the probability of clinical outcomes, including response and toxicity. This information may also be used to assess the probability of clinical outcomes, including response and toxicity, for both hematological malignancies and solid tumor indications. It may be utilized to optimize the manufacturing process of T cells, allogeneic CAR T cells, iPSCs, and potentially TCRs and TILs.

[0223] In one embodiment, the disclosure provides that the percentage of CD27+CD28+ Th cells of naive phenotype (CCR7+CD45RA+) in a pre-production PBMC population (i.e., a population of PBMCs from which a T cell product is prepared) positively correlates with phenotypic markers of product T cell compatibility, including doubling time and viability, CD4 / CD8 ratio, and percentage of CD8 and CD4 naive T cells. Thus, the disclosure provides a method of manufacturing optimization that improves product T cell population compatibility by increasing the levels of CD27+CD28+ Th cells of naive phenotype (CCR7+CD45RA+) in a pre-production PBMC population. In one embodiment, this may be done by enriching for naive phenotype CD27+CD28+ Th cells (CCR7+CD45RA+) following subject apheresis, by increasing the amount of apheresis material collected until a threshold of naive phenotype CD27+CD28+ Th cells (CCR7+CD45RA+) is reached and the manufacturing process begins, by selecting the administration dose of CAR T cells not through the total number of CARs per kg but instead by utilizing the number of naive phenotype CD27+CD28+ Th cells (CCR7+CD45RA+) per kg, and / or by adjusting the T cell product manufacturing conditions (such as, but not limited to, the length of manufacturing and / or the composition of the growth medium) to increase the levels of naive phenotype CD27+CD28+ Th cells (CCR7+CD45RA+). In one embodiment, the present disclosure also provides a method to stratify patients who may be better candidates for allogeneic / off-the-shelf CAR T cells to overcome the lack of sufficient positive factors such as CD27+CD28+Th cells of naive phenotype (CCR7+CD45RA+) in the pre-manufacturing PBMC population. In one embodiment, the present disclosure provides a method to stratify patients who may be better candidates for combination therapy that can enhance the activity of CAR T cells or reduce the impact of negative factors to improve the clinical efficacy of CAR T therapy.In one embodiment, the combination therapy may be selected from checkpoint inhibitors (including but not limited to anti-PD-1, anti-PD-L1, anti-CTLA-4, etc., or any combination thereof), SRC kinase inhibitors (e.g., dasatinib), anti-CD20 monoclonal antibodies, anti-4-1BB, anti-CD47, lenzilumab, TGF-beta inhibitors or dominant negative TGF-beta, mTOR / AKT agonists, histone deacetylase inhibitors, cyclophosphamide, fluorouracil, gemcitabine, doxorubicin, taxanes. In one embodiment, the present disclosure provides for stratifying patients for manufacturing optimization based on the percentage of naive phenotype CD27+CD28+Th cells (CCR7+CD45RA+) in the pre-manufacturing PBMC population, or identification of subjects who would benefit from allogeneic / off-the-shelf CAR T cells or combination therapy to maximize CAR T cell efficacy.

[0224] In one embodiment, the present disclosure provides that the percentage of intermediate monocytes and total monocytes in a pre-production PBMC population is positively associated with pre-treatment inflammatory markers. Thus, the present disclosure provides a method to quantify simple biomarkers (intermediate monocytes and / or total monocytes) that allow for the estimation of a patient's inflammatory state, which has been shown to be a negative indicator of clinical efficacy of CAR T cells. In one embodiment, the method is used as an indicator of the potential use of anti-inflammatory drugs to abrogate inflammatory signaling in the periphery. In one embodiment, the preferred anti-inflammatory drugs are selected from antibodies against IL-6 (such as tocilizumab), corticosteroids, dexamethasone, siltuximab, etanercept, infliximab, anakinra, and anti-GM-CSF.

[0225] In one embodiment, the present disclosure provides that the level of intermediate monocytes (% of white blood cells) in a pre-manufacturing PBMC population is enriched in patients with higher IPI scores and is a marker for patients with higher IPI scores.

[0226] In one embodiment, the present disclosure provides that the percentage of intermediate monocytes and total monocytes in a pre-manufacturing PBMC population is positively associated with tumor burden (baseline two-way sum of products). Thus, the present disclosure provides a method to quantify biomarkers (e.g., intermediate monocytes and / or total monocytes) that allow for an estimation of a patient's tumor burden, which has been shown to be a negative indicator of clinical efficacy of CAR T cells. In one embodiment, the levels of intermediate monocytes and / or total monocytes may indicate the use of additional therapeutic agents to help overcome a larger estimated tumor burden, such as chemotherapy, radiation antibody and small molecule based therapies, immunotherapies (including but not limited to checkpoint inhibitors, bispecific engagers), and cell therapies (including but not limited to CAR-T, TCR-based lymphocytes and tumor infiltrating lymphocytes) where tumor burden has been shown to be a negative prognostic and / or predictive biomarker.

[0227] In one embodiment, the present disclosure provides that the percentage of intermediate monocytes and total monocytes in a pre-manufacturing PBMC population is positively associated with hypoxia (as indicated by serum LDH concentration). Thus, the present disclosure provides a method to quantify biomarkers (e.g., intermediate monocytes and / or total monocytes) that allow for the estimation of a patient's hypoxic state, which has been shown to be a negative indicator of the clinical efficacy of CAR T cells. In one embodiment, the level of intermediate monocytes and / or total monocytes is used as an indicator of a supplemental therapeutic agent to overcome the hypoxic environment. In one embodiment, the supplemental therapeutic agent is selected from a metabolic regulator, a HIF inhibitor, and an LDH inhibitor that establish a more normal environment.

[0228] In one embodiment, the present disclosure provides that monocytes, particularly intermediate monocytes, in pre-manufacturing PBMC populations are negatively associated with T cell features in the tumor microenvironment (TME), while CD27+CD28+ naive Th cells and lymphocytes are positively associated with T cell features in the TME associated with response. In one embodiment, T cell features in the TME that are associated with response include activated CD8+ T cell subsets (CD3+CD8+PD-1+Lag3+ / -Tim3- cells) and genes associated with activated T cell signatures (e.g., CXCL10, CXC11, GZMA, GZMB, GZMK, and Immunosign21 Galon et al. ASCO, 2020). Thus, the present disclosure provides a method to elucidate the overall state of the tumor microenvironment from peripheral blood biomarkers and allow estimation of the tumor immune status into various classes, such as immune desert, bone marrow imbalance, and immune suppression. These biomarkers may then be useful to select potential combination drugs that can help improve the tumor microenvironment and / or protect the CAR T cells from immune checkpoint or exhaustion, such as immune checkpoint inhibitors and SRC kinase inhibitors (e.g., dasatinib), such as checkpoint inhibitors (including but not limited to anti-PD-1, anti-PD-L1, anti-CTLA-4, etc., or any combination thereof), lenzilumab, TGF-beta inhibitors or dominant negative TGF-beta, histone deacetylase inhibitors, amino acid depletion, cyclophosphamide, fluorouracil, gemcitabine, doxorubicin, or taxanes.

[0229] In one embodiment, the present disclosure provides that the levels of CD27+CD28+ Th cells of naive phenotype in pre-manufacturing PBMC populations positively correlate with the percentage of naive T cells in the product infusion bag and T cell-rich tumor immune context (all markers shown are markers of activated T cells) and negatively correlate with pre-treatment inflammation (INTL8, PRF) / tumor hypoxia (LDH). Thus, the present disclosure provides a method to quantify a simple biomarker (CD27+CD28+ naive Th) that allows for the estimation of the patient's final infusion bag after manufacturing and the T cell-rich tumor immune context, both of which have been shown to be positive indicators of clinical efficacy of CAR T cells. These low levels of CD27+CD28+ naive Th cells may indicate the potential use of anti-inflammatory drugs or combination therapies to help modify the tumor microenvironment to improve CAR T cell efficacy.

[0230] In one embodiment, the disclosure provides that intermediate monocytes in pre-manufacturing PBMC populations are positively associated with pre-treatment inflammation (INTL8, ferritin, CRP, amyloid A) / tumor hypoxia (LDH) and negatively associated with pre-treatment defined T cell-enriched tumor immune landscape (e.g., activated T cell signature, CD3+CD8+PD1+LAG3-TIM3- cells; GZMA, TGIT, LAG3, CXCL10, GZMB, PRF1, STAT1, EOMES, CXCL9, GZMK, CXCL11, HAVCR2, CD3D, IS21). Thus, the disclosure provides methods that demonstrate high levels of intermediate monocytes using anti-inflammatory drugs (such as corticosteroids or tocilizumab) and / or immunomodulatory drugs (e.g., TME modulating drugs [such as checkpoint inhibitors, drugs that target suppressive myeloid cells and enhance antigen presentation, drugs that stabilize the vasculature, or normalize tumor metabolism, etc.]) to help overcome insufficient TIC. In one embodiment, the drug is administered prior to immunotherapy. In one embodiment, the drug is administered before, during, and / or after immunotherapy.

[0231] In one embodiment, the present disclosure provides that the level of intermediate monocytes in a pre-manufacturing PBMC population is positively associated with pre-treatment tumor burden, which itself is negatively associated with response. Thus, the present disclosure provides a method for predicting whether a patient is likely to respond to CAR T cell therapy based on the level of intermediate monocytes in a pre-manufacturing PBMC population. The present disclosure also provides a method for estimating a patient's tumor burden using the level of intermediate monocytes and / or total monocytes in a pre-manufacturing PBMC population, which has been shown to be a negative indicator of clinical efficacy of CAR T cells. In one embodiment, the level of intermediate monocytes serves as an indicator for the use of additional therapeutic agents to help overcome a larger estimated tumor burden, such as chemotherapy, radiation antibody and small molecule based therapies, immunotherapies (including but not limited to checkpoint inhibitors, bispecific engagers), and cell therapies (including but not limited to CAR-T, TCR-based lymphocytes, and tumor infiltrating lymphocytes) where tumor burden has been shown to be a negative prognostic and / or predictive biomarker.

[0232] In one embodiment, the present disclosure provides that the level of CD27+CD28+ naive Th cells (% of white blood cells) in the apheresis product / pre-manufacturing PBMC population was a predictive marker for improved OS and PFS (optimal cut-off). There is a positive association between them, i.e., subjects with pre-treatment CD27+CD28+ naive Th cells above the listed cut-off are more likely to survive than subjects below the selected cut-off. Thus, the present disclosure provides a method for predicting the likelihood of survival of a patient in need of CAR T-cell therapy based on the level of CD27+CD28+ naive Th cells (% of white blood cells) in the apheresis product used to prepare the CAR T-cell product. Also, thus, the present disclosure provides a method for predicting the progression-free survival of a patient in need of CAR T-cell therapy based on the level of CD27+CD28+ naive Th cells (% of white blood cells) in the apheresis product used to prepare the CAR T-cell product. The disclosure also provides that there is an improvement in complete response rate, objective response rate and CAR T cell expansion for subjects above selected cutoffs (see numbers below survival plots). In one embodiment, the disclosure provides a method of stratification where subjects with low levels (e.g., below 0.27%) of CD27+CD28+ naive Th cells may benefit from another form of treatment (combination therapy, allogeneic CAR T cells, etc.) to improve chances of survival. In one embodiment, a low level is below the median or below 0.1-0.5%, 0.5-1.0%, 1-1.5%, 1.5-2%, 2-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-50%, etc., or below 95-100%.

[0233] In one embodiment, the present disclosure provides that intermediate monocyte levels (% of white blood cells) in the apheresis product were predictive markers for OS and PFS (optimal cut-off). Thus, the present disclosure provides that intermediate monocyte levels (% of white blood cells) in the apheresis product were approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%, preferably 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 100%, 15%, 20%, 25%, 3 ... The present disclosure provides a method for predicting OS and PFS in a subject in need of CAR T cell therapy, comprising measuring the level of intermediate monocytes (% of white blood cells) in the apheresis product used to prepare the CAR T cell product and determining whether the level is above or below the cutoff. The present disclosure also provides that there is an improvement in complete response rate and objective response rate, as well as CAR T expansion, in subjects below the approximately 3% cutoff. The present disclosure also provides a method of patient stratification, whereby subjects with high levels of intermediate monocytes (levels above approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20%, preferably between 1-5%, and even more preferably levels above approximately 3%) may benefit from another form of treatment (e.g., combination therapy with immunotherapy, allogeneic CAR T cells, etc.) to improve chances of survival.

[0234] In one embodiment, the present disclosure provides that the ratio of CD27pCD28p naive Th cells (% of white blood cells) to intermediate monocytes (% of white blood cells) in the apheresis product shows a positive association with predictive markers of OS and PFS and serves as a predictive marker. There was a better survival / response / proliferation rate for subjects with levels above a selected cutoff of approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1-5, 5-10, 10-20, preferably between 0.05-0.2, 0.2-0.25, 0.25-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-1, 1-5, 5-10, 10-15, etc., 95-100, 100-200, 200-300, etc., more preferably, even more preferably, 0.1705, compared to those below the cutoff. Thus, the present disclosure provides a method of predicting OS and PFS, response, and CAR T cell proliferation rate in a subject in need of CAR T cell therapy, comprising: The method includes measuring the ratio of CD27pCD28p naive Th cells (% of white blood cells) to intermediate monocytes (% of white blood cells) in an apheresis product used to prepare a T cell product, and determining whether the level is above or below a cutoff of approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1-5, 5-10, 10-20, preferably between 0.05-0.2, 0.2-0.25, 0.25-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-1, 1-5, 5-10, 10-15 etc., 95-100, 100-200, 200-300 etc., more preferably 0.1-1, even more preferably 0.1705. The present disclosure also provides that for subjects above the selected cutoff of 0.1705, there is an improvement in complete response rate, objective response rate and CAR T cell expansion. Thus, the present disclosure provides a method of patient stratification whereby subjects with low levels of CD27+CD28+ naive Th cells (e.g., levels between approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1-5, 5-10, 10-20, preferably 0.05-0.2, 0.2-0.25, 0.25-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-1, 1-5, 5-10, 10-15, more preferably 0.1-1, even more preferably 0.1705) may benefit from another form of treatment (combination therapy, allogeneic CAR T cells, etc.) to improve chances of survival.

[0235] In one embodiment, the disclosure provides that the level of CD27+CD28+ naive Th cells in a pre-production PBMC population is negatively associated with the level of intermediate monocytes. Furthermore, subjects with high CD27+CD28+ naive Th levels and low intermediate monocyte levels had an increased rate of objective responders. Thus, the disclosure provides a method of predicting an objective response in a subject in need of CAR T cell therapy, comprising measuring CD27+CD28+ naive Th levels and low intermediate monocyte levels, thereby predicting an objective response of 0.08% (above the median, or 0.05%, 0.1%, 0.2-1%, 1-5%, 5-10%, 1 CD27+CD28+ naive Th levels above / at levels greater than 0-15%, 15-20%, etc., 95-100%) and / or median monocyte levels below / at 3% (below the median, or below 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, etc., 95%-100%) indicate an increased likelihood of an objective response. In one embodiment, these levels are used to stratify patients who may benefit from off-the-shelf / allogeneic CAR T cells, immunomodulators, bispecific engagers, combination therapies, etc.

[0236] In one embodiment, the disclosure provides that high levels of intermediate monocytosin in pre-manufacturing PBMC populations (wherein high levels are levels above the median intermediate monocytes in the general population, where the median can be 0-1%, 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, 9-10%, 10-15%, 15-20%, etc., preferably about 1.7-1.8%) and low levels of CAR T cell proliferation (wherein low levels are levels below the median level of CAR T cell proliferation in the general population, where the median is 0-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100) correlate with the highest rate of non-responders. Thus, the present disclosure provides a method to predict response based on baseline intermediate monocyte levels and CAR proliferation after infusion. Thus, the present disclosure provides a method to measure the levels of intermediate monocytes in pre-treatment apheresis PBMCs and CAR T cell proliferation, and use them to actively follow patients after infusion to predict what the long-term response will be and whether additional therapeutics may be useful.

[0237] There was an increased sustained response rate and decreased relapse or non-responder rate in subjects with increased CAR-T cell peak proliferation (wherein expanded levels are levels above the median level of CAR T cell expansion in a general CAR T cell therapy population, the median being between 0-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, preferably between 40-50) and decreased intermediate monocyte levels (wherein low levels are levels below the median intermediate monocytes in the general population, the median can be 0-1%, 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, 9-10%, 10-15%, 15-20%, etc., preferably about 1.7-1.8%) compared to other quadrants. Thus, the present disclosure provides methods to measure the levels of intermediate monocytes in pre-treatment apheresis PBMCs and CAR T cell proliferation, and use them to actively follow patients post-infusion to predict what the continued response, likelihood of relapse is, and whether additional therapeutics may be useful based on the above correlations.

[0238] In one embodiment, the subject has a baseline tumor burden above the median level, high intermediate monocytes (above the median, the median can be approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 10-15%, 15-20%, 20-25%, etc., 95-100%, preferably approximately 1.1%) and low CAR-T cell peak proliferation (below the median, the median can be approximately 5-10%, 10-15%, 15-20%, 20-25%, etc., 95-100%, preferably approximately 1.1%). %, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, etc., may be 95-100%, preferably around 43%), the likelihood of response is low (1%-10%, 10-20%, 20-30%, 30-40%, 40-50% sustained response rate and 1%-10%, 10-20%, 20-30%, 30-40%, 40-50% objective response rate). Thus, the present disclosure provides methods to measure the levels of intermediate monocytes in pre-treatment apheresis PBMCs, baseline tumor burden, and CAR T cell proliferation, and actively follow patients post-infusion to use in estimating, based on the above correlations, what the likelihood of sustained and objective response is and whether supplemental therapeutics may be useful.

[0239] In one embodiment, the present disclosure provides that there was a correlation between CD27+CD28+ naive Th (% of white blood cells) in the pre-production PBMC population and the response category. CD27+CD28+ naive Th cell levels are higher in responder patients compared to non-responder patients. Thus, the present disclosure provides a method for the treatment of CAR T cell therapy in subjects in need of CAR T cell therapy. Provided is a method of predicting likelihood of response to T cell therapy, comprising measuring the level of CD27+CD28+ naive Th (% of white blood cells) in a pre-production PBMC population and predicting a high likelihood of response if the level is above an optimal cutoff point (e.g., 0.1036%, 0-0.1%, 0.1%, -0.5%, 0.5%, -1.0%, 1.0-5%, 5-10%, 10-15%, 10-20%, 20-30%, 30-40%, 40-50%) or above the median (e.g., 0.89%, 0-0.1%, 0.1%, -0.5%, 0.5%, -1.0%, 1.0-5%, 5-10%, 10-15%, 10-20%, 20-30%, 30-40%, 40-50%). Thus, the present disclosure provides methods to select patients for manufacturing optimization, combination therapy, or off-the-shelf / allogeneic CAR T cell therapy if the level of CD27+CD28+ naive Th (% of white blood cells) in the pre-manufacturing PBMC population is below a selected cutoff point or median range, which is also an indication of a lower likelihood of sustained response.

[0240] In one embodiment, the present disclosure provides that there was an association between the level of intermediate monocytes (% of white blood cells) in the pre-production PBMC population and response category. Intermediate monocytes were lower in responding patients compared to non-responding patients. Thus, the disclosure provides a method of predicting likelihood of response to CAR T cell therapy in a subject in need of CAR T cell therapy, the method comprising measuring the level of intermediate monocytes (% of white blood cells) in a pre-production PBMC population and predicting a high likelihood of response if the level is below an optimal cut point (e.g., 3.02%, 0-0.1%, 0.1%, -0.5%, 0.5%, -1.0%, 1.0-5%, 5-10%, 10-15%, 10-20%, 20-30%, 30-40%, 40-50%) or below the median (e.g., 1.77%, 0-0.1%, 0.1%, -0.5%, 0.5%, -1.0%, 1.0-5%, 5-10%, 10-15%, 10-20%, 20-30%, 30-40%, 40-50%). Additionally, intermediate monocyte (% of white blood cells) levels in the pre-manufacturing PBMC population are lower in subjects with sustained (sustained) responses compared to subjects who experience relapse or are non-responders. Thus, the present disclosure provides the ability to optimize manufacturing, combination therapy, or off-the-shelf / allogeneic CAR for subjects with intermediate monocyte (% of white blood cells) levels in the pre-manufacturing PBMC population above a range where sustained responses are less likely. Methods are provided that can be used in methods of selecting a T cell therapy.

[0241] In one embodiment, levels of CD27+CD28+ naive Th cells trend negatively with features shown to be associated with worse prognosis (IPI score, tumor burden, prior lines of therapy). Thus, the present disclosure provides a method to predict response to CAR T cell therapy based on the levels of CD27+CD28+ naive Th cells in a pre-manufacturing PBMC population, such that patients with higher levels of these cells are more likely than not to respond to CAR T therapy and less likely than not to require intervention. Those with lower levels may need to consider further modifications to therapy, such as combination therapy, optimized manufacturing approaches, off-the-shelf / allogeneic CAR T cells, next-generation CAR constructs, etc.

[0242] In one embodiment, the present disclosure provides that the level of naive Th cells in the apheresis product was negatively associated with the number of prior lines of therapy. First (Z12) or second (Z7) line DLBCL may have higher levels of naive T cells at leukapheresis. The present disclosure provides that subjects may have higher levels of these cells in the blood with fewer lines of therapy, and that response rates may be improved if CAR T cells are utilized as an earlier line of therapy (1st / 2nd line). Higher IPI score tend to have lower CD27+CD28+ naive Th cells. CD27+CD28+ naive Th cells show a weak negative association with baseline tumor burden. Thus, the present disclosure provides that the levels of CD27+CD28+ naive Th cells in pre-manufacturing apheresis PBMC products trend negatively with features (IPI score, tumor burden, prior lines of therapy) that have been shown to be associated with poorer prognosis ... Methods are provided to predict response to T cell therapy whereby patients with higher levels of these cells (greater than 0-0.005%, 0.005-0.010%, 0.01%-0.05%, 0.05-0.1%, 0.1-0.5%, 0.5%-1.0%, 1-5%, 5-10%, 10-15%, preferably greater than 0.1%) should be more responsive to CAR T therapy and less likely to require intervention. The disclosure also provides a method of stratifying patients where patients with lower levels (below 0-0.005%, 0.005-0.010%, 0.01%-0.05%, 0.05-0.1%, 0.1-0.5%, 0.5%-1.0%, 1-5%, 5-10%, 10-15%, preferably below 0.08%) are considered for further modifications to treatment such as combination therapy, optimized manufacturing approaches, off-the-shelf / allogeneic CAR T cells, next generation CAR constructs, etc. Additionally, the disclosure provides a method of treatment where other prior chemotherapeutic agents that significantly deplete these cells are transferred to later lines of treatment to preserve CD27+CD28+ naive Th cells in the peripheral / tumor environment for pre-manufactured apheresis PBMC product and CAR T treatment. Thus, the present disclosure provides a method showing that the levels of CD27+CD28+ naive Th cells in pre-manufactured apheresis PBMC products, along with the positive impact these cells have on product suitability at the time of apheresis, can be used to freeze apheresis bags prior to initiating any treatment for a subject with cancer to obtain the best transfer cells for CAR-T cell therapy.

[0243] In one embodiment, the present disclosure provides that the levels of intermediate monocyte populations in the apheresis product were associated with disease burden and moderately increased with the number of prior lines of therapy. Thus, the present disclosure provides for active tracking of intermediate monocytes, a feature shown to be associated with worse prognosis (tumor burden, prior lines of therapy). Thus, the present disclosure provides a method for predicting response to CAR T therapy and the need for further intervention, whereby patients with lower levels of these cells (below 0-1%, 1-5%, 5-10%, 10-15%, 15-20%, preferably below 3%) are more responsive to CAR T therapy and less likely to require further intervention. In one embodiment, patients with higher levels may need to consider additional modifications to their therapy, such as combination therapy, optimized manufacturing approaches, off-the-shelf / allogeneic CAR T cells, next generation CAR constructs, etc. The present disclosure provides that prior chemotherapeutic agents that increase these cells should be transferred to later lines of therapy to prevent these cells from increasing in the peripheral / tumor environment prior to CAR T therapy. This data, along with the negative impact that these cells have on product suitability during apheresis, indicates that apheresis bags should be frozen before initiating any treatment in subjects with cancer to obtain the best incoming cells for CAR-T cell therapy.

[0244] In one embodiment, the present disclosure provides that the level of intermediate monocytes in the apheresis product is positively associated with the number of prior lines of therapy. Subjects are expected to have lower levels of intermediate monocytes with fewer prior lines of therapy, and due to the negative association of these cells with response, this also indicates that CAR T cell response rates may be even higher if utilized as an earlier line of therapy (1st / 2nd line).

[0245] In one embodiment, the present disclosure provides that the International Prognostic Index (IPI) score was positively associated with the level of intermediate monocytes in the apheresis product, further indicating that these cells are associated with subjects with a worse prognosis. Intermediate monocytes were positively associated with baseline tumor burden. Thus, the present disclosure provides that the level of intermediate monocytes indicates a less than optimal state for CAR T cell efficacy, and that if the level of these cells is above 3% (or above 0-1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%), further intervention / optimization may be required to improve the efficacy of CAR T therapy. The present disclosure also provides that based on the level of intermediate monocytes, patients can be stratified for removal of intermediate monocytes and increasing the level of naive product cells, use of next generation CAR constructs, and / or manufacturing optimization versus use of combination therapy with immunomodulators or checkpoint blockade, off-the-shelf / allogeneic CAR T cells, etc.

[0246] In one embodiment, the present disclosure provides a method for detecting CD27-CD28+TEMRA in an apheresis product. The present disclosure provides that the level of Treg cells (% of white blood cells) is positively associated with OS and PFS and may be a predictive marker for OS and PFS. The present disclosure provides that for CD27-CD28+TEMRA Tregs, subjects with higher levels of these cells (e.g., above a threshold of 0.17) have higher complete response rates, objective response rates, and sustained response rates. Thus, the present disclosure provides a method for the treatment of CAR T cell therapy in subjects in need of CAR T cell therapy. A method for predicting OS and PFS for T cell therapy is provided, comprising measuring the level of CD27-CD28+TEMRA Treg cells (% of white blood cells) in the apheresis product and determining the probability of survival and PFS based on whether the level is above or below a cutoff. In one embodiment, the cutoff is 0.17. In one embodiment, the cutoff is approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1-5, 5-10, 10-20, preferably between 0.05-0.2, 0.2-0.25, 0.25-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-1, 1-5, 5-10, 10-15, etc., 95-100, 100-200, 200-300, etc., more preferably approximately 0.1705. The present disclosure provides methods of stratifying patients who have low levels of CD27-CD28+TEMRA Tregs that may benefit from alternative forms of therapy (combination therapy, allogeneic CAR T cells, etc.), manufacturing optimization, next generation CARs, etc., to improve chances of survival with CAR therapy.

[0247] In one embodiment, the present disclosure provides that there was a correlation between the level of CD27+CD28+ naive Th cells in the apheresis product versus CAR-T peak and CAR-T peak / baseline tumor burden. A positive correlation was observed between CD27+CD28+ naive Th cells and CAR T cell peak proliferation (normalized by tumor burden). Thus, the present disclosure provides a method to predict CAR T cell proliferation, whereby CD27+CD28+ naive Th cells are positively associated with CAR T peak proliferation, which has been shown to positively correlate with response, indicating that these cells have a positive impact on response.

[0248] In one embodiment, the present disclosure provides that low levels of peak proliferation of both CD27+CD28+ naive Th cells and CAR T cells correlate with higher non-responder rates, while increased levels of both result in higher response rates.

[0249] In one embodiment, the present disclosure provides that there was a correlation between the level of intermediate monocytes in the apheresis product vs. CAR-T peak and CAR-T peak / baseline tumor burden. There was a negative correlation between the level of intermediate monocytes and CAR T cell peak proliferation (normalized by tumor burden). Thus, the present disclosure provides that the level of intermediate monocytes is negatively associated with CAR T peak proliferation (CAR / TB), which has been shown to be positively correlated with response, indicating that these cells should have a negative impact on response and CAR function after infusion. The present disclosure provides a method to use the level of intermediate monocytes to stratify patients for manufacturing optimization and reduce this population in the product to enhance final CAR T cells, thereby improving CAR expansion and response rates. Also, as previously mentioned, high intermediate (high int.) Monocytes may be an indicator for the use of additional therapeutic agents or next generation CAR constructs to improve efficacy.

[0250] In one embodiment, the disclosure provides a method of predicting response to CAR T cell therapy by measuring levels of intermediate monocytes and CAR T peak proliferation levels, whereby high levels of intermediate monocytes (above the median or 0-1%, 1-5%, 5-10%, 10-15%, 15-20%, preferably above 3%) and low levels (below the median or 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, preferably below 43%) of CAR-T peak in the apheresis product correlate with a higher non-responder rate, whereas lower intermediate monocyte levels and increased CAR T peak proliferation result in a higher response rate.

[0251] In one embodiment, the present disclosure provides that the ratio of lymphocytes to white blood cells in baseline hematology cell counts is positively associated with OS and PFS and can serve as a predictive marker for OS and PFS (optimal cut-off). The lymphocytes to white blood cells in baseline hematology cell counts were positively associated with complete response, objective response, and sustained response. Thus, the present disclosure provides a method for predicting the likelihood of complete response, objective response, and sustained response to CAR T-cell therapy in a subject in need thereof, comprising measuring the ratio of lymphocytes to white blood cells in baseline hematology cell counts and predicting the likelihood of complete response, objective response, and sustained response based on the ratio. In one embodiment, when the ratio is above the optimal cutoff (e.g., the optimal cutoff may be about 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, etc.), the likelihood of a complete response, objective response, and sustained response is higher than when the ratio is below the cutoff (e.g., the cutoff may be about 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, etc.). In one embodiment, the present disclosure provides a method of patient stratification where subjects with low levels of lymphocytes relative to white blood cells are treated with alternative forms of therapy (combination therapy, allogeneic CAR T cells, next generation CAR constructs, etc.) to improve chances of survival / response and / or are subjected to optimized manufacturing to improve product suitability.

[0252] In one embodiment, the disclosure provides that the lymphocyte to white blood cell ratio in baseline hematology cell counts had a weak negative association with worst grade toxicity. Thus, the disclosure provides a method of patient stratification whereby low levels of lymphocytes to white blood cells (below the median, or below 1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, etc., preferably below 5.2) indicate a higher likelihood of having a toxic event and indicate prophylactic administration of anti-inflammatory drugs (e.g., tocilizumab, steroids) to the patient to prevent toxicity.

[0253] In one embodiment, the disclosure provides a method of predicting response to CAR T cell therapy by measuring lymphocyte to leukocyte ratio in baseline hematology cell counts, whereby the ratio is negatively associated with tumor burden and thereby positively associated with response. Thus, the disclosure provides a method of stratifying patients for additional interventions to improve efficacy if the pre-manufacturing PBMC lymphocyte to leukocyte ratio is low (below the median or below 1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, etc., preferably below 5.2) and / or the patient has a high tumor burden.

[0254] In one embodiment, the present disclosure provides that the lymphocyte to white blood cell ratio in baseline hematology cell counts was negatively associated with the number of prior lines of therapy. Since an increased number of prior chemotherapy drugs will deplete these cells, it is suggested that CAR T therapy should be considered in the first or second line setting for best efficacy, or chemotherapy that depletes these cells should be considered as a potential option after CAR T cell therapy. This data, along with the positive impact that these cells have on the suitability of the product upon apheresis, indicates that apheresis bags can be frozen to obtain the best incoming cells for CAR-T cell therapy prior to initiating any treatment for subjects with cancer.

[0255] These data indicate that utilization of CAR-T cells in earlier lines of treatment may result in improved objective and durable responses due to higher response rates and positive predictors of product suitability with fewer lines of treatment.

[0256] In one embodiment, the present disclosure provides that the lymphocyte to white blood cell ratio in baseline hematology cell counts was negatively associated with CRP, ferritin, and IL6. CRP, ferritin, and IL6 have previously been shown to be pharmacodynamic markers that negatively correlate with response in DLBCL. Thus, the present disclosure provides a method to estimate the levels of these inflammatory cytokines associated with worse prognosis in patients by measuring the lymphocyte to white blood cell ratio in baseline hematology cell counts. Also, if the level of lymphocytes to white blood cells is low, the patient is selected to administer anti-inflammatory drugs before and / or after CAR T cell therapy.

[0257] In one embodiment, the disclosure provides a method for predicting the level of myeloid cells in a patient, where the lymphocyte to white blood cell ratio in a baseline hematology cell count is negatively associated with myeloid cells (more specifically, intermediate monocytes, which are negatively associated with response) and positively associated with CD8 and EM / effector T cells. Additionally, the method provides that patients with low lymphocyte to white blood cell ratios (below the median, or below 1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, etc., preferably below 5.2) are considered for combination therapies that seek to neutralize activity of the myeloid compartment and / or for optimization of manufacturing processes to deplete those populations in products.

[0258] In one embodiment, the present disclosure provides that the ratio of lymphocytes to monocytes in baseline hematology cell counts is positively associated with OS and PFS and can serve as a predictive biomarker for OS and PFS. Thus, the present disclosure provides that subjects with low levels of lymphocytes to monocytes may benefit from CAR T cell therapy (e.g., combination therapy, allogeneic CAR T cell therapy) to improve chances of survival. The disclosure also provides a method of stratification in cancer treatment, where subjects are administered another form of therapy in addition to or as an alternative to the CAR T cell (e.g., T cells, next generation CAR constructs, etc.) and / or the subjects are subjected to optimized manufacturing of the CAR T cell product to improve product suitability. The disclosure also provides a method of predicting response, where higher complete response rates, objective response rates and sustained response rates are observed in subjects with a lymphocyte to monocyte ratio above 0.79. In one embodiment, the ratio is between 0-0.5, 0.5-1.0, 1.0-1.5, 1.5-2.0, 2-5, 5-10, 10-15, etc.

[0259] In one embodiment, the disclosure provides that the lymphocyte to monocyte ratio in baseline hematology cell counts had a weak negative association with worst grade toxicity. Figure 29. Thus, the disclosure provides a method for predicting response to immunotherapy (e.g., CAR T cells), where low levels of monocytes to lymphocytes (below the median, or below 1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, preferably below 8%) indicate a higher likelihood of having a toxic event and indicate prophylactic use of anti-inflammatory drugs (e.g., tocilizumab, steroids) to prevent toxicity.

[0260] In one embodiment, the present disclosure provides that the ratio of lymphocytes to monocytes in baseline hematology cell counts was negatively associated with tumor burden. Figure 30. Thus, similar to previous tumor burden (negatively tracking biomarkers for negative features of response), it refers to the possibility of additional interventions to improve efficacy in cases of low L to L and high TB. Thus, the present disclosure provides a method to quantify the ratio of lymphocytes to monocytes in baseline hematology cell counts that allows for estimation of a patient's tumor burden, which has been shown to be a negative indicator of clinical efficacy of CAR T cells. In one embodiment, the ratio of lymphocytes to monocytes in baseline hematology cell counts may indicate the use of additional therapeutic agents to help overcome a larger estimated tumor burden, such as chemotherapy, radioactive antibody and small molecule based therapies, immunotherapies (including but not limited to checkpoint inhibitors, bispecific engagers), and cell therapies (including but not limited to CAR-T, TCR-based lymphocytes and tumor infiltrating lymphocytes) where tumor burden has been shown to be a negative prognostic and / or predictive biomarker.

[0261] In one embodiment, the disclosure provides that the lymphocyte to monocyte ratio in baseline hematology cell counts was negatively associated with the number of prior lines of therapy. Figure 31. This suggests that the use of CAR-T cells as a first or second line therapy may result in even better response rates. Thus, CAR T therapy should be considered in the first or second line setting to have the best efficacy, since an increase in the number of prior chemotherapy drugs reduces these cells, or chemotherapy that reduces these cells should be considered as a potential option after CAR T cell therapy. This data, along with the positive impact that these cells have on the suitability of the product at the time of apheresis, indicates that apheresis bags can be frozen before starting any treatment for a subject with cancer to obtain the best incoming cells for CAR-T cell therapy. In that way, if the subject receives treatment prior to CAR T therapy, the subject still has a more effective starting material than subjects who received other treatments before collecting the CAR T apheresis material.

[0262] In one embodiment, the disclosure provides that the lymphocyte to monocyte ratio in baseline hematology cell count was negatively associated with CRP and IL6. Figure 32. Thus, the disclosure provides a method of predicting the levels of CRP and IL6 in the serum of a cancer patient and / or immunotherapy (e.g., CAR T cell therapy) prognosis by measuring the lymphocyte to monocyte ratio in baseline hematology cell count, where the levels of CRP and IL6 are negatively associated with the levels of lymphocyte to monocyte ratio in baseline hematology cell count and positively associated with poor prognosis. The disclosure also provides a method of stratifying patients, administering anti-inflammatory drugs to the patient if low levels of lymphocyte to monocyte (or levels below the median, or below 0.05%, 0.05-0.1%, 0.1-0.5%, 0.5-1.0%, 1-5%, 5-10%, 10-15%, preferably below 0.78) are quantified.

[0263] In one embodiment, the disclosure provides that the ratio of lymphocytes to monocytes in baseline hematology cell counts is negatively associated with myeloid cells and positively associated with CD8 and EM / effector T cells. Figure 33. The disclosure provides that myeloid cells, CD8 and EM / effector T cells are negatively correlated with response. Thus, the disclosure provides a method to predict the levels of myeloid cells, CD8 and / or EM / effector cells in the final infusion product by measuring the ratio of lymphocytes to monocytes in baseline hematology cell counts, which is negatively associated with myeloid cells and positively associated with CD8 and EM / effector T cells. This method can further be used to stratify patients for combination therapies that attempt to neutralize the activity of the myeloid compartment and / or for optimization of pre-manufacturing materials to deplete these populations.

[0264] In one embodiment, the present disclosure provides a method for determining whether the lymphocyte to monocyte ratio in baseline hematology cell counts is negatively associated with intermediate monocytes, CD27-CD28+TEMRA and Tregs, and CD27+ We provide that CD28+ naive and Th cells showed a weak correlation with apheresis populations associated with response. Thus, high levels of lymphocytes to monocytes in pre-production PBMC populations (above the median or above between 0-0.5, 0.5-1.0, 1.0-1.5, 1.5-2.0, 2-5, 5-10, 10-15, preferably above 0.8%) may indicate inflow apheresis material to actively track product suitability and response. Low levels (below the median or below between 0-0.5, 0.5-1.0, 1.0-1.5, 1.5-2.0, 2-5, 5-10, 10-15, preferably below 0.78%) may indicate the need for production optimization, combination therapy, or next generation CAR therapy. T cell compatibility

[0265] In some embodiments, endogenous cytocompatibility is assessed based on the ability of CAR T cells to expand during non-specific stimulation in vitro (e.g., shorter doubling time), the differentiation state of the CAR T cells (favorable juvenile phenotype), the level of specialized CAR T cell subsets in the CAR T cell population (e.g., the number of CD8 and naive-like CD8 cells (e.g., CD8+CCR7+CD45RA+ T cells) in the infusion product), and in vivo CAR T cell proliferation rates.

[0266] In one embodiment, T cell compatibility is the ability of the cells to proliferate rapidly. In the context of engineered T cells, in one embodiment, T cell compatibility is a measure of how quickly an engineered T cell population proliferates prior to treatment. As described herein, T cell compatibility is an attribute of engineered T cells that correlates with clinical outcome. In some embodiments, T cell compatibility is measured by doubling time or proliferation rate. An exemplary derivation of T cell "compatibility" measured as a T cell population doubling time (DT) during the manufacturing process is provided below.

number

[0267] The period can be defined as the total manufacturing time frame minus 3 days (essentially the number of days of produced cells in culture after transduction and before harvesting and cryopreservation). Recombinant IL-2 (following non-specific stimulation with, for example, anti-CD3 antibodies) can be used to drive polyclonal T cell expansion toward achieving the target dose. The shorter the DT, the more compatible the engineered T cells are. The in vitro expansion rate can be calculated using the following formula: Growth rate = ln(2) / doubling time In the above examples, the growth rate is provided in units of "rate / day" or " / day".

[0268] In some embodiments, the in vivo proliferation rate is measured by enumerating CAR cells / unit of blood volume. In some embodiments, the in vivo proliferation rate is measured by the number of CAR gene copies per μg of host DNA. In some embodiments, the in vivo proliferation rate is measured by enumerating CAR cells / unit of blood volume.

[0269] As described herein, during manufacturing, T cells can be first non-specifically stimulated with anti-CD3 antibodies in the presence of IL2 and then expanded in growth medium supplemented with IL2. As described herein, low doubling time is positively associated with objective response compared to non-response. The median DT of responders was 1.6 days, whereas non-responders had a median DT of 2.1 days. Quartile analysis of response by DT showed that all patients (100%) in the lowest DT quartile achieved an objective response, whereas 80% of all non-responders were in the third and fourth quartiles of DT. Thus, the present disclosure provides a method for (a) measuring the doubling time of T cell populations in an infusion product to obtain values ​​and (b) assessing first-line treatment resistance based on the values. and determining in which quartile of the population the patient fails. In some embodiments, the evaluation is performed against a reference standard. In some embodiments, the method further comprises administering an effective dose of CAR T cells to the patient, the effective dose being determined using the value / values ​​above. In some embodiments, a higher doubling time is associated with first-line therapy resistance. In some embodiments, a product doubling time >1.6 days is associated with non-response. In some embodiments, in patients with high tumor burden, patients with objective or durable response have a doubling time of less than 2 days. In some embodiments, a doubling time >2 days is associated with relapse or non-response. In some embodiments, CD28+CD27+T in the apheresis starting material is associated with a doubling time of less than 2 days. N The higher the number of cells, the better (shorter) the doubling time of the injected product.

[0270] As described herein, higher peak proliferation of CAR T cells in peripheral blood, which generally occurs within 2 weeks after CAR T cell infusion, is associated with both objective and sustained responses, defined as sustained response with a minimum follow-up of 1 year. Peak number of CAR T cells in blood correlated with response. Cumulative CAR T cell levels over the first 28 days, as measured by area under the curve (AUC) in blood, were also associated with better objective and sustained responses to treatment. Thus, the present disclosure provides a method for measuring the number of CAR T cells in peripheral blood to obtain (a) values ​​of CAR T cells in peripheral blood. (b) assessing the therapeutic response based on the value. In another aspect, the disclosure provides a method for determining whether a patient will respond to CAR T cell therapy, comprising: (a) measuring peak CAR T cell levels in the blood after CAR T administration to obtain a value; (b) normalizing the value to pre-treatment tumor burden; and (c) determining whether the patient will achieve a sustained response based on the normalized value. In some embodiments, the value is positively associated with sustained response and separates a subset of patients with a higher (about 60%) probability of achieving a sustained response from a lower (about 10%) probability of achieving a sustained response. In some embodiments, the CAR T cell level is determined by the number of CAR T cells per unit blood volume. The in vivo proliferation rate is calculated by counting the number of T cells. In one embodiment, a higher peak proliferation of CAR T cells in peripheral blood means a peak proliferation value that falls within a higher quartile. In some embodiments, the in vivo proliferation rate is measured by enumerating CAR cells / unit of blood volume. In some embodiments, the in vivo proliferation rate is measured by CAR gene copy number per μg of host DNA. In some embodiments, the evaluation or determination comprises determining in which quartile of the population the patient fails. In some embodiments, the evaluation is performed against a reference standard.

[0271] As described herein, higher peak CAR-T cell expansion is associated with severe neurotoxicity, but not CRS. Thus, in one embodiment, the disclosure provides a method of predicting severe neurotoxicity, comprising: (a) measuring peak CAR T cell expansion following CAR T cell therapy to obtain a value; and (b) predicting neurotoxicity based on the value. In one embodiment, the method further comprises administering an agent that prevents or reduces neurotoxicity in combination with CAR T cell therapy.

[0272] As described herein, a higher expansion rate of CAR T cells during manufacturing is associated with greater in vivo CAR T cell expansion and higher probability of long-term remission (long-term remission / sustained response means in response for more than a year). As described herein, product doubling time is negatively associated with CAR T cell expansion in vivo after infusion. As described herein, product doubling time is negatively associated with peak CAR T cells normalized to tumor burden. As described herein, product doubling time is negatively associated with CAR-T cell AUC. In some embodiments, in vivo expansion rate is measured by enumerating CAR cells / unit of blood volume. In some embodiments, in vivo expansion rate is measured by enumerating CAR cells / unit of blood volume. is measured by the number of CAR gene copies per μg of host DNA. Thus, in some embodiments, the disclosure provides a method of determining whether a patient will respond to CAR T cell therapy, comprising: (a) measuring the proliferation rate of CAR T cells during manufacturing or the peak CAR T cell level in the blood after CAR T administration to obtain a value; and (b) determining whether the patient will achieve a durable response based on the value.

[0273] As described herein, among patients with high tumor burden, a greater proportion of patients who achieve an objective or sustained response have a shorter product doubling time (<2 days) compared to patients who relapsed or did not respond. Thus, in some embodiments, the present disclosure provides a method for determining whether a patient will respond to CAR T cell therapy, comprising: (a) measuring peak CAR T cell levels in the blood after CAR T administration to obtain a value; (b) normalizing the value to pre-treatment tumor burden; and (c) determining whether the patient will achieve a sustained response based on the normalized value. In some embodiments, the value is positively associated with sustained response, separating the subset of patients with a higher (about 60%) probability of achieving a sustained response from the subset of patients with a lower (about 10%) probability of achieving a sustained response. In some embodiments, the CAR T cell level is determined by the number of CAR T cells per unit blood volume. It is calculated by counting the number of T cells. In some embodiments, the evaluation comprises determining in which quartile of the population the patient fails. In some embodiments, the evaluation is performed against a reference standard.

[0274] As described herein, doubling time is positively correlated with the frequency of T cell differentiation subsets in the final infusion bag. Doubling time is correlated with the frequency of effector memory T (T EM ) cells and naive-like T (T N ) cells. As described herein, T cell compatibility of endogenous products, as measured by product doubling time, is positively associated with less differentiated products, affecting the ability of CAR T cells to expand in vivo to a sufficient effector to target ratio to support tumor eradication. Thus, in one embodiment, the present disclosure provides a method of improving a patient's response to CAR T cell therapy with an infusion product, comprising manipulating a cell population to decrease the doubling time of the infusion product and / or administering to the patient an infusion product having a lower doubling time compared to a baseline value.

[0275] As described herein, the inherent ability of pretreatment to measure T cell proliferation as measured by product doubling time is a key attribute of product T cell compatibility. Compared to other product characteristics, DT was most strongly associated with the frequency of T cell differentiation subsets in the final infusion bag. Specifically, DT was positively associated with the frequency of effector memory T (TEM) cells and negatively associated with the frequency of naive-like T (TN) cells. As described herein, baseline tumor burden is positively associated with the differentiation phenotype in the final infusion product. As described herein, product composition and clinical outcomes are related to the patient's pretreatment immune status. Thus, in one embodiment, the disclosure provides a method of reducing tumor burden following treatment with CAR T cell therapy, comprising administering an infusion product with an increased frequency of naive-like T (TN) cells in the infusion product compared to a baseline value. In another embodiment, the disclosure provides a method of predicting or estimating the differentiation phenotype of the final infusion product, comprising measuring the patient's baseline tumor burden to obtain a value and estimating or predicting the differentiation phenotype based on the value. In one embodiment, the scale further comprises adjusting the effective dose of CAR T cells in the final product based on the value. T cell phenotype

[0276] As described herein, the T cell phenotype in the manufacturing starting material (apheresis) can be related to T cell compatibility (DT). The total percentage of Tn-like cells and Tcm cells (CCR7+ cells) is inversely proportional to DT. The percentage of Tem (CCR7-CD45RA-) cells is directly related to DT. Thus, in some embodiments, the pre-treatment attributes are the percentage of Tn-like cells and T In some embodiments, the percentage of Tn-like cells and Tcm cells is determined by the percentage of CCR7+ cells. In some embodiments, the percentage of CCR7+ cells is measured by flow cytometry.

[0277] In some embodiments, the pre-treatment attribute is % of Tem (CCR7-CD45RA-) cells. In some embodiments, the % of Tem cells is determined by the percentage of CCR7-CD45RA- cells. In some embodiments, the percentage of CCR7-CD45RA- cells is measured by flow cytometry.

[0278] As described herein, manufacturing doubling time and product T cell compatibility are directly related to the differentiation state of a patient's T cells prior to enrollment in CAR T cell therapy. Accordingly, the present disclosure provides a method of predicting the T cell compatibility of a manufactured product, comprising determining the differentiation state of a patient's T cells prior to CAR T cell therapy (e.g., in an apheresis product) and predicting the T cell compatibility during manufacturing based on the differentiation state.

[0279] As described herein, the greater the proportion of effector memory T cells in the apheresis product within total CD3+ T cells or CD4 and CD8 subsets, the longer the product doubling time. As described herein, the T cell phenotype in the starting material is more immature, but the product T cell fitness is better. As described herein, the greater the proportion of effector memory T cells that express important costimulatory molecules. N CD27+CD28+T, representing an immunologically competent subset of cells N The cells correlate positively with product doubling time. As described herein, there is a direct correlation across all major phenotypic groups, including the proportion of T cell subsets defined by differentiation markers of CD3, CD4, and CD8 subpopulations in the apheresis product to the phenotype of the final product. As described herein, CD25 T cells, which likely represent regulatory T cells in the apheresis material, hi The percentage of T cells with CD4 expression negatively correlates with the CD8 T cell output in the product. As described herein, tumor burden following CAR T cell therapy is positively associated with the differentiation phenotype of the end product.

[0280] As described herein, the number of infused CD8+ T cells normalized to tumor burden correlates with sustained response and expansion of CAR T cells against tumor burden. More specifically, quartile analysis of number of infused CD8 T cells / pretreatment tumor burden showed a sustained response rate of 16% in the lowest quartile compared to 58% in the highest quartile.

[0281] As described herein, the number of specialized T cells infused, primarily CD8+ T N The cell population positively impacts durable clinical efficacy with CAR T cell therapy. As described herein, a higher number of product CD8+ T cells are required to achieve complete tumor resolution and establish sustained responses in patients with higher tumor burden. As described herein, in patients with high tumor burden, sustained responses are associated with significantly higher numbers of infused CD8 T cells compared to patients who respond and then relapse. As described herein, the number of infused TN cells normalized to tumor burden is positively associated with sustained responses. As described herein, the CD4:CD8 ratio is positively associated with sustained responses. As described herein, the total number of CD8 T cells in the product normalized to pretreatment tumor burden is positively associated with sustained responses. Among the CD8 T cells, T N The number of cells is most significantly associated with durable response. The present disclosure provides several additional associations that can be used for one or more methods of improving CAR T cell infusion products, determining effective doses, and / or predicting durable response based on one or more of these associations. See Table 1.

[0282] [Table 1] a Analytes in LOG2 transformation are shown.

[0283] Thus, the present disclosure provides methods of improving sustained clinical efficacy (e.g., sustained response) of CAR T cell therapy in a patient, comprising preparing and / or administering to the patient an effective dose of CAR T cell therapy, where the effective dose is determined based on the number of specialized T cells and / or the CD4CD8 ratio in the infusion product. In some embodiments, the specialized T cells are CD8+ T cells, preferably T N It is a cell.

[0284] In another embodiment, the disclosure provides a method of determining how a patient will respond to a treatment, comprising: (a) characterizing the number of specialized T cells in an infusion product to obtain one or more values; and (b) determining how the patient will respond based on the one or more values. In another embodiment, the disclosure provides a method of treating a malignancy in a patient, comprising measuring a T cell phenotype in a population of T cells obtained from the patient (e.g., apheresis material). In some embodiments, the method further comprises determining whether the patient will respond to chimeric antigen receptor treatment based on the percentage of a particular T cell type measured. In some embodiments, the T cell phenotype is measured before engineering the cells to express a chimeric antigen receptor (CAR) (e.g., apheresis material). In some embodiments, the T cell phenotype is measured after engineering the cells to express a chimeric antigen receptor (CAR) (e.g., engineered T cells containing a CAR). Tumor burden

[0285] Tumor-related parameters (e.g., tumor burden, serum LDH as a marker of hypoxia / cell death, inflammatory markers associated with tumor burden and myeloid cell activity) may be associated with clinical outcome. In one aspect, the disclosure provides a method of treating a malignant tumor in a patient, comprising measuring the patient's tumor burden prior to administration of CAR T cell therapy. In some embodiments, the method further comprises determining whether the patient will respond to CAR T cell therapy based on the level of tumor burden compared to a reference level. In some embodiments, the reference level is about 1,000 mm 2 , about 2,000 mm2 , about 3,000 mm 2 , about 4,000 mm 2 is less than.

[0286] As described herein, higher tumor burden is associated with a higher probability of recurrence within 1 year of treatment in subjects who achieve OR, as well as a higher probability of grade 3+ neurotoxicity. In an embodiment, the tumor burden is a pre-treatment tumor burden of about 4,000 mm 2 Super, about 5,000mm 2 Super, about 6,000mm 2 Super, about 7,000mm 2 Over or about 8,000 mm 2 If >100%, it may be used to assess the probability of recurrence in responding patients.

[0287] As described herein, low tumor burden prior to CAR T cell therapy is a positive predictor of durable response. As described herein, in the highest tumor burden quartile, patients who achieved a durable response had more than 3-fold higher peak CAR T cell expansion compared to patients who relapsed or had no response. As described herein, there is a lower rate of durable response at comparable peak CAR-T cell levels in patients with higher tumor burden compared to patients with lower tumor burden. As described herein, durable responders had a higher peak CAR T cell / tumor burden ratio compared to non-responders or responders who subsequently relapsed within one year after treatment. As described herein, complete responders had a higher peak CAR T cell / tumor burden ratio compared to partial responders or non-responders. Thus, the present disclosure also provides a method of determining whether a patient will become a non-responder, have a durable response, or relapse within one year after administration of CAR T cell therapy, comprising measuring the peak CAR T cell / tumor burden ratio and making a determination based on those levels. As described herein, objective response rates and durable responses correlate with increasing peak CAR T cell levels. As described herein, patients in the lowest quartile of peak CAR T cell / tumor burden ratio have a lower durable response (12%) than the upper quartile (>50%). As described herein, durable responses in refractory large cell lymphoma treated with anti-CD19 CAR T cell therapy containing a CD28 costimulatory domain benefit from early CAR T cell expansion commensurate with tumor burden.

[0288] As described herein, tumor burden was positively associated with severe neurotoxicity, with rates increasing from quartile 1 to quartile 3, but they decreased in the highest quartile, generally reflecting the association between CAR T cell expansion and tumor burden in the overall population.

[0289] As described herein, peak CAR T cell levels normalized to either pre-treatment tumor burden or body weight are strongly associated with efficacy, with the latter being associated with NE of grade 3 or higher. Thus, the present disclosure also provides a method of determining whether a patient exhibits a durable response after administration of a CAR T cell therapy, comprising measuring peak CAR T cell levels normalized to either pre-treatment tumor burden or body weight, and making a determination based on those levels. The present disclosure also provides a method of determining whether a patient exhibits NE of grade 3 or higher after administration of a CAR T cell therapy, comprising measuring peak CAR T cell levels normalized to pre-treatment tumor body weight, and making a determination based on those levels. Response and efficacy measures

[0290] In some embodiments, the methods described herein may provide a clinical benefit to a subject. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of patients achieve a clinical benefit. In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 0%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of patients and any unlisted percentages therebetween achieve a clinical benefit. In some embodiments, the response rate is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 9.5%, 10.5%, 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%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, %, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 25 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any other unrecited percentage and any range between 1% and 100%. In some embodiments, the response rate is between 0%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100%. In some embodiments, the response rate is between 0%-1.%, 1%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 8%-9%, 9%-10%, 10%-15%, 15%-20%, 20-25%, 25%-30%, 35-40%, etc., and between 95%-100%.

[0291] In some embodiments, the quartiles for the peak CAR T cell ranges are those shown in the figures and tables, as well as 0-15, 15-35, etc., 40-100, 0-40 40-50 40-60 40-70, 40-80, 40-90, 40-100, 40-110, 40-120, 40-130, 40-140, 40-150, 40-300, 40-1000, 80-160, 50-100, 50-110, 50-120, 50-130, 50-140, 50-150, 50-160, 50-170, 50-180, 50-190, 50-200, 60-100, 60-110, 60-120 , 60~130, 60~140, 60~150, 60~160, 60~170, 60~180, 60~190, 60~200, 70~100, 70~110, 70~120, 70~130, 70~140, 70~150, 70~160, 70~170, 70~180, 70~190, 70~200, 80~100, 80~110, 80~120, 80~130, 80~140, 80~150, 80~1 60, 80~170, 80~180, 80~190, 80~200, 90~100, 90~110, 90~120, 90~130, 90~140, 90~150, 90~160, 90~170, 90~180, 90~190, 90~200, 100~110, 100~120, 100~130, 100~140, 100~150, 100~160, 100~170, 100~180, 100~190 , 100-200, etc., 50-70, 60-80, 70-90, 80-100, 90-110, 100-120, 110-130, 120-150, 130-160, 140-170, 150-180, 160-190, 170-200, 180-210, 190-210, 200-220, 210-230, 220-240, or 230-250, etc., and any unrecited range therebetween.In some embodiments, the quartiles of the ranges for CCL2 and CXCL10 are those in the figures and tables, as well as 0-100, 100-200, 200-300, 400-500 500-600, 600-700, etc., or 0-50, 50-100, 100, 150, 200, 300, 400, 500, 549, 549-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-2100, 1900-2200, 2200-2300, 2300-2400, 2400-2500, 2500-2600, 2600-2700, 2700-2800, 2800-3000, 2900-3100, 3100-3200, 3200-3300, 3300-3400, 3400-3500, 3500-3600, 3600-3700, 3700-3800, 3800-4000, 3900 400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000, 2000-2200, 2200-2300, 2300-2400, 2400-2600, 2600-2800, 2800-3000, etc., or any other unrecited range therebetween. In some embodiments, the quartiles of tumor burden are those in the figures and tables, as well as 0-500, 500-1000, 1000-1500, etc., 1000-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, etc., 8000-10000, 10000-20000, etc., and therebetween. In some embodiments, the quartiles of the ferritin ranges are those shown in the figures and tables, as well as 0-50, 50-100, 100, 150, 200, 300, 400, 500, 549, 549-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000, 2000-2100, 2100-2200, 2200-2300, 2300-2400, 2400-2500, 2500-3000, 2600-3100, 2700-3200, 2700-3300, 2800-3400, 2800-3500, 2900-3600, 3000-3700, 3100-3800, 3200-3900, 3300-4000, 3400-4100, 3500-4200, 3600-4300, 3700-4400, 3800-4500, 3900-5000, 3900-5100 In some embodiments, the IFNγ, infused naive-like T cells, infused CD8 T cells, infused CD4 ... The quartiles of T-cell ranges are those in the figures and tables: <0.1, 0.1-0.2, 0.2-0.3, 0.3-0.4, 0.4-0.5, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.91-1.0, 1.0-1.1, etc., 99.9-100, 1-5, 5-10, 10-15, 15-20, 25-30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-175, or 175-200, 10-30, 30-50, 50-70, 70-90, etc., 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 40-41, 41-42, 42-43, 43-44, 44-45, 45-46, 46-47, 47-48, 48-49, 49-50, 50-51, 51-52, 52-53, 53-54, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, etc., and any other unrecited ranges therebetween. In some embodiments, quartiles for peak CAR T cells / tumor burden, peak CAR T cells / body weight, infused naive-like T cells / tumor burden, infused CD8 T cells / tumor burden, infused CD4 T cells / tumor burden, and infused CD3 T cells / tumor burden are shown in the figures and tables, as well as ranges from 0.001 to 0.005, 0.005~0.010, 0.010~0.020, 0.020~0.030, 0.030~0.040, 0.040~0.050, 0.05~0.06, 0.06~0.07, 0.07~0.08, 0.08~0.09, 0.09~0.10, 0.1~0.11, 0 .11~0.12, 0.12~0.13, 0.13~0.14, 0.14~0.15, 0.15~0.16, 0.16~0.17, 0.17~0.18, 0.18~0.19, 0.19~0.20, 0.5~2.5, 0.05~0.1, 0.1~0.2, 0.2~0.3, 0.3 ~0.4, 0.4-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-1.0, 1-2, 2-3, 3-4, 4-5, etc., and any unlisted ranges therebetween, and the median is 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0. 47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98、0.9. In some embodiments, the quartiles of the ranges for LDH and infused CD3 T cells are those shown in the figures and tables, as well as 0-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500, etc., and up to 1000, 150-250, 250-350, 350-450, 450-550, etc., 1-500, 1-1000, 25-100, 25-200, 25-300, 350-450, 450-550, etc. ~300, 25~400, 25~500, 25~1000, 100~150, 100~200, 100~300, 100~400, 100~500, 100~1000, etc., 100~5000, 100~4900, 100~4800, 100~4700, 100~4600, 100~4500, 100~4400, 100~4300, 100~4200, 100~4100, 100~4 000, 100~3900, 100~3800, 100~3700, 100~3600, 100~3500, 100~3400, 100~3300, 100~3200, 100~3100, 100~3000, 100~2900, 100~2800, 100~2700, 100~2600, 100~2500, 100~2400, 100~2300, 100~2200, 100~21 00, 100~2000, 100~1900, 100~1800, 100~1700, 100~1600, 100~1500, 100~1400, 100~1300, 100~1200, 100~1100, 100~1000, 100~900, 100~800, 100~700, 100~600, 100~500, 100~400, 100~300, 100~200, 500~10,000, 500~7500, 500~5000, 500~4900, 500~4800, 500~4700, 500~4600, 500~4500, 500~4400, 500~4300, 500~4200, 500~4100, 500~4000, 500~3900, 500~3800, 500~3700, 500~3600, 500~3500, 500~3400, 500~3300, 500~3200, 500~3100, 500~3000, 500~2900, 500~2800, 5 00-2700, 500-2600, 500-2500, 500-2400, 500-2300, 500-2200, 500-2100, 500-2000, 500-1900, 500-1800, 500-1700, 500-1600, 500-1500, 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-900, 500-800, 500-700, or 500-600, etc., and any other unrecited ranges therebetween. In some embodiments, the quartiles of the IL-6 ranges are those in the figures and tables, as well as 0-1, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, etc., 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, etc., 6-1, 6-2, 6-3, 6-4, 6-6, 6-6, 6-7, etc., 6.7-10, 6.7-20, 6.7-30, 6.7-80, 6.7-90, 6.7-100, 6.7-110, 6.77-120, 6.7-130, etc., and any other unrecited ranges therebetween. In some embodiments, the quartiles of ranges for infused CD3 T cells are those in the figures and tables, as well as 0-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, etc., 100-240, 100-150, 100-260, etc., 300-400, 300-500, 300-600, 300-700, 300-800, etc., and any other unrecited ranges therebetween. In some embodiments, the quartiles of doubling times are those in the figures and tables, as well as <2, <2.1, <2.2, <2.3, <2.4, <2.5, etc., greater than 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 to less than 2, etc., and any other ranges therebetween. The IFNγ quartiles for the co-culture range are 200-300, 300-400, 400-500, 500-600, etc., 300-500, 300-1000, 300-1500, 300-2000, 300-2500, 300-3000, 300-3500, 300-3600, etc., 2000-3000, 3000-4000, 4000-5000, 4000-6000, etc., 6000-7000, 6000-8000, 6000-9000, etc., 8000-15000, 8000-16000, 8000-17000, 8000-18000, etc., and and any other unrecited range therebetween. In some embodiments, any of these ranges can be modified by the term about or approximately.

[0292] The clinical benefit may be an objective response or a sustained clinical response, defined as a sustained response at a median follow-up of one year. In some embodiments, the response, blood levels of CAR T cells, or immune-related factors are determined by follow-up at about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration of the engineered CAR T cells. In some embodiments, the response, blood levels of CAR T cells, or immune-related factors are determined by follow-up at about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks after administration of the engineered CAR T cells. In some embodiments, the response, the level of CAR T cells in the blood and / or immune-related factors is determined by follow-up at about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months after administration of the engineered CAR T cells. In some embodiments, the response, the level of CAR T cells in the blood and / or immune-related factors is determined by follow-up at about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, or about 5 years after administration of the engineered CAR T cells. Response and efficacy measures

[0293] In some embodiments, the methods described herein may provide a clinical benefit to a subject. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of patients achieve a clinical benefit. In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 0%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of patients and any unlisted percentages therebetween achieve a clinical benefit. In some embodiments, the response rate is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 9.5%, 10.5%, 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%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 109 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any other unrecited percentage and any range between 1% and 100%. In some embodiments, the response rate is between 0%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100%. In some embodiments, the response rate is between 0%-1.%, 1%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 8%-9%, 9%-10%, 10%-15%, 15%-20%, 20-25%, 25%-30%, 35-40%, etc., and between 95%-100%.

[0294] In some embodiments, the quartiles for the peak CAR T cell ranges are those in the figures and tables, as well as 0-15, 15-35, etc., 40-100, 0-40 40-50 40-60 40-70, 40-80, 40-90, 40-100, 40-110, 40-120 , 40~130,40~140,40~150,40~300,40~1000,80~160,50~100,50~110,50~120,50~130,50~140,50~150,50~160,50~170,50~180,50~190,50~200,60~100,60~110,60~120,60~130,60~140,60~150,60~160, 60~170, 60~180, 60~190, 60~200, 70~100, 70~110, 70~120, 70~130, 70~140, 70~150, 70~160, 70~170, 70~180, 70~190, 70~200, 80~100, 80~110, 80~120, 80~130, 80~140, 80~150, 80~160, 80~170, 80~180, 80 ~190, 80~200, 90~100, 90~110, 90~120, 90~130, 90~140, 90~150, 90~160, 90~170, 90~180, 90~190, 90~ 200, 100~110, 100~120, 100~130, 100~140, 100~150, 100~160, 100~170, 100~180, 100~190, 100~200, etc. 50-70, 60-80, 70-90, 80-100, 90-110, 100-120, 110-130, 120-150, 130-160, 140-170, 150-180, 160-190, 170-200, 180-210, 190-210, 200-220, 210-230, 220-240, or 230-250, etc., and any unrecited range therebetween.In some embodiments, the quartiles of the ranges for CCL2 and CXCL10 are those in the figures and tables, as well as 0-100, 100-200, 200-300, 400-500 500-600, 600-700, etc., or 0-50, 50-100, 100, 150, 200, 300, 400, 500, 549, 549-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-2100, 1900-2200, 2200-2300, 2300-2400, 2400-2500, 2500-2600, 2600-2700, 2700-2800, 2800-3000, 2900-3100, 3100-3200, 3200-3300, 3300-3400, 3400-3500, 3500-3600, 3600-3700, 3700-3800, 3800-4000, 3900 400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000, 2000-2200, 2200-2300, 2300-2400, 2400-2600, 2600-2800, 2800-3000, etc., or any other unrecited range therebetween. In some embodiments, the tumor burden quartiles are those in the figures and tables, as well as 0-500, 500-1000, 1000-1500, etc., 1000-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, etc., 8000-10000, 10000-20000, etc., and any other unrecited ranges therebetween. In some embodiments, the quartiles of the ferritin ranges are those in the figures and tables, as well as 0-50, 50-100, 100, 150, 200, 300, 400, 500, 549, 549-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000, 2000-2100, 2100-2200, 2200-2300, 2300-2400, 2400-2500, 2500-2600, 2600-2700, 2700-2800, 2800-2900, 2900-3000, 3000-3100, 3100-3200, 3200-3300, 3300-3400, 3490-3500, 3500-3600, 3600-3700, 3700-3800, 3800-3900, 3900-4000, 4000-4100, 4100-4200, 00-1200, etc., 100,000-200,000, 200,000-500,000, 500,000, or 400,000-500,000, etc., 1000000-1500000, 1500000-1600000, etc., 2000000-10000000, 2000000-15000000, etc., and any other unrecited ranges therebetween.In some embodiments, the quartiles of ranges for IFNγ, infused naive-like T cells, infused CD8 T cells, infused CD4 T cells are those in the figures and tables, <0.1, 0.1-0.2, 0.2-0.3, 0.3-0.4, 0.4-0.5, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.91-1.0, 1.0-1.1, etc., 99.9-100, 1-5, 5-10, 10-15, 15-20, 25-30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, etc. 00, 100-125, 125-150, 150-175, or 175-200, 10-30, 30-50, 50-70, 70-90, etc., 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 40-41, 41-42, 42-43, 43-44, 44-45, 45-46, 46-47, 47-48, 48-49, 49-50, 50-5. In some embodiments, the peak CAR T cells / tumor burden, peak CAR T cells / body weight, infused naive-like T cells / tumor burden, infused CD8 T cells / tumor burden, infused CD4 T cells / tumor burden, and infused CD3 T cells / tumor burden are expressed as 1, 51-52, 52-53, 53-54, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, etc., units, and any other unrecited ranges therebetween. Quartiles for T cell / tumor burden are those in the figures and tables, as well as 0.001–0.005, 0.005–0.010, 0.010–0.020, 0.020–0.030, 0.030–0.040, 0.040–0.050, 0.05–0.06, 0.06–0.07, 0.07–0.08, 0.08–0.09, 0.09–0.10, 0.1–0.11, 0.11–0.12, 0.12–0.13, 0.13–0.14, and 0.14–0.15. , 0.15-0.16, 0.16-0.17, 0.17-0.18, 0.18-0.19, 0.19-0.20, 0.5-2.5, 0.05-0.1, 0.1-0.2, 0.2-0.3, 0.3-0.4, 0.4-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-1.0, 1-2, 2-3, 3-4, 4-5, etc., and any unlisted ranges therebetween, with the median being 0.001, 0.0015, 0 .002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.09 5, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.In some embodiments, the quartiles of the ranges for LDH and infused CD3 T cells are those shown in the figures and tables, as well as 0-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500, etc., and up to 1000, 150-250, 250-350, 350-450, 450-550, etc., 1-500, 1-1000, 25-100, 25-200, 25-300, 350-450, 450-550, etc. 300, 25~400, 25~500, 25~1000, 100~150, 100~200, 100~300, 100~400, 100~500, 100~1000, etc., 100~5000, 10 0~4900, 100~4800, 100~4700, 100~4600, 100~4500, 100~4400, 100~4300, 100~4200, 100~4100, 100~4000 , 100~3900, 100~3800, 100~3700, 100~3600, 100~3500, 100~3400, 100~3300, 100~3200, 100~3100, 100~3000, 100~2900, 100~2800, 100~2700, 100~2600, 100~2500, 100~2400, 100~2300, 100~2200, 100~2100, 10 0~2000, 100~1900, 100~1800, 100~1700, 100~1600, 100~1500, 100~1400, 100~1300, 100~1200, 100~1100, 100~1000, 100~900, 100~800, 100~700, 100~600, 100~500, 100~400, 100~300, 100~200, 500~10,000, 5. 00~7500, 500~5000, 500~4900, 500~4800, 500~4700, 500~4600, 500~4500, 500~4400, 500~4300, 500~4200, 500~4100, 500~4000, 500~3900, 500~3800, 500~3700, 500~3600, 500~3500, 500~3400, 500~3300, 500~3200, 500~3100, 500~3000, 500~2900, 500~2800, 500 ~2700, 500-2600, 500-2500, 500-2400, 500-2300, 500-2200, 500-2100, 500-2000, 500-1900, 500-1800, 500-1700, 500-1600, 500-1500, 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-900, 500-800, 500-700, or 500-600, etc., and any other unrecited ranges therebetween. In some embodiments, the quartiles of the IL-6 ranges are those in the figures and tables, as well as 0-1, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, etc., 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, etc., 6-1, 6-2, 6-3, 6-4, 6-6, 6-6, 6-7, etc., 6.7-10, 6.7-20, 6.7-30, 6.7-80, 6.7-90, 6.7-100, 6.7-110, 6.77-120, 6.7-130, etc., and any other unrecited ranges therebetween. In some embodiments, the quartiles of ranges for infused CD3 T cells are those in the figures and tables, as well as 0-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, etc., 100-240, 100-150, 100-260, etc., 300-400, 300-500, 300-600, 300-700, 300-800, etc., and any other unrecited ranges therebetween. In some embodiments, the quartiles of doubling times are those in the figures and tables, as well as <2, <2, <2.1, <2.2, <2.3, <2.4, <2.5, etc., greater than 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 to less than 2, etc., and any other ranges therebetween. The IFNγ quartiles for the co-culture range are 200-300, 300-400, 400-500, 500-600, etc., 300-500, 300-1000, 300-1500, 300-2000, 300-2500, 300-3000, 300-3500, 300-3600, etc., 2000-3000, 3000-4000, 4000-5000, 4000-6000, etc., 6000-7000, 6000-8000, 6000-9000, etc., 8000-15000, 8000-16000, 8000-17000, 8000-18000, etc., and any other unlisted ranges in between. In some embodiments, any of these ranges can be modified by the terms about or approximately.

[0295] The clinical benefit may be an objective response or a sustained clinical response, defined as a sustained response at a median follow-up of one year. In some embodiments, the response, blood levels of CAR T cells, or immune-related factors are determined by follow-up at about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration of the engineered CAR T cells. In some embodiments, the response, blood levels of CAR T cells, or immune-related factors are determined by follow-up at about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks after administration of the engineered CAR T cells. In some embodiments, the response, the level of CAR T cells in the blood and / or immune-related factors is determined by follow-up at about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months after administration of the engineered CAR T cells. In some embodiments, the response, the level of CAR T cells in the blood and / or immune-related factors is determined by follow-up at about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, or about 5 years after administration of the engineered CAR T cells. Chimeric Antigen Receptor

[0296] Chimeric antigen receptors (CARs) are genetically engineered receptors. These engineered receptors can be inserted into and expressed by immune cells, including T cells and other lymphocytes, according to techniques known in the art. In CARs, a single receptor can be programmed to recognize and, when bound to a specific antigen, activate the immune cell to attack and destroy the cells bearing that antigen. If these antigens are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells. Chimeric antigen receptors may incorporate costimulatory (signaling) domains to enhance their efficacy. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (supra), Song et al. al.,Blood 119:696-706(2012);Kalos et al.,Sci.Transl.Med.3:95(2011),Porter et al.,N.Engl.J.Med.365:725-33(2011),and Gross et al. al., Annu.Rev.Pharmacol.Toxicol.56:59-83 (2016).

[0297] In some embodiments, the costimulatory domain comprising a truncated hinge domain ("THD") further comprises part or all of a member of the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof.

[0298] In some embodiments, the THD is derived from a human complete hinge domain ("CHD"). In other embodiments, the THD is derived from a rodent, mouse, or primate (e.g., non-human primate) CHD of a costimulatory protein. In some embodiments, the THD is derived from a chimeric CHD of a costimulatory protein.

[0299] The costimulatory domain for the CAR of the present disclosure may further comprise a transmembrane domain and / or an intracellular signaling domain. The transmembrane domain may be fused to the extracellular domain of the CAR. The costimulatory domain may be fused to the intracellular domain of the CAR as well. In some embodiments, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein and minimize interaction with other members of the receptor complex. The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Particularly useful transmembrane regions in the present disclosure include 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, ligand that specifically binds to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, N It may be derived from (i.e., may include) Kp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or combination thereof.

[0300] Optionally, a short linker may form a link between any or some of the extracellular, transmembrane, and intracellular domains of the CAR. In some embodiments, the linker may be derived from glycine-glycine-glycine-glycine-glycine-serine (SEQ ID NO:2) (G4S)n or repeats of GSTSGSGKPGSGEGSTKG (SEQ ID NO:1). In some embodiments, the linker comprises 3-20 amino acids and an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to GSTSGSGKPGSGEGSTKG (SEQ ID NO:1).

[0301] The linkers described herein can also be used as peptide tags. The linker peptide sequence can be of any suitable length for connecting one or more proteins of interest, and is preferably designed to be sufficiently flexible to allow for proper folding and / or function and / or activity of one or both of the peptides it connects. Thus, the linker peptide can have a length of 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, or 20 or less amino acids. In some embodiments, the linker peptide comprises a length of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids. In some embodiments, the linker comprises at least 7 and no more than 20 amino acids, at least 7 and no more than 19 amino acids, at least 7 and no more than 18 amino acids, at least 7 and no more than 17 amino acids, at least 7 and no more than 16 amino acids, at least 7 and no more than 15 amino acids, at least 7 and no more than 14 amino acids, at least 7 and no more than 13 amino acids, at least 7 and no more than 12 amino acids, or at least 7 and no more than 11 amino acids. In certain embodiments, the linker comprises 15-17 amino acids, and in certain embodiments, 16 amino acids. In some embodiments, the linker comprises 10-20 amino acids. In some embodiments, the linker comprises 14-19 amino acids. In some embodiments, the linker comprises 15-17 amino acids. In some embodiments, the linker comprises 15-16 amino acids. In some embodiments, the linker comprises 16 amino acids. In some embodiments, the linker comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0302] In some embodiments, a spacer domain is used. In some embodiments, the spacer domain is derived from CD4, CD8a, CD8b, CD28, CD28T, 4-1BB, or other molecules described herein. In some embodiments, the spacer domain may include a chemically derived dimerizer to control expression upon addition of a small molecule. In some embodiments, no spacer is used.

[0303] The intracellular (signaling) domain of the engineered T cells of the present disclosure can then provide a signal to the activation domain that activates at least one of the normal effector functions of an immune cell. The effector function of a T cell can be, for example, a cytolytic activity or a helper activity including secretion of cytokines.

[0304] In certain embodiments, suitable intracellular signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptor, immunoglobulin proteins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, ligand that specifically binds to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Lyl08), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp 30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or combination thereof. antigen binding molecule

[0305] A suitable CAR can bind to an antigen (such as a cell surface antigen) by incorporating an antigen-binding molecule that interacts with its target antigen. In some embodiments, the antigen-binding molecule is an antibody fragment thereof, such as one or more single-chain antibody fragments ("scFv"). An scFv is a single-chain antibody fragment that has the variable regions of the heavy and light chains of an antibody linked together. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45:131-136. An scFv retains the ability of the parent antibody to specifically interact with the target antigen. An scFv is useful in chimeric antigen receptors, since it can be engineered to be expressed as part of a single chain with other CAR components. Id. See also Krause et al., J. Exp. Med., Volume 10, 1997, pp. 1111-1115. 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161:2791-2797. It will be understood that an antigen-binding molecule is typically contained within the extracellular portion of the CAR such that it can recognize and bind to an antigen of interest. Bispecific and multispecific CARs are contemplated within the scope of the present disclosure and have specificity for two or more targets of interest.

[0306] In some embodiments, the polynucleotide encodes a CAR comprising a (truncated) hinge domain and an antigen binding molecule that specifically binds to a target antigen. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the antigen is a tumor-associated surface antigen, such as 5T4, alpha fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD8, CLL-1, c-Met, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, EGFR variant III (EGFRvIII), ELF2M, endoglin, ephrin B2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2 / neu), fibroblast-associated protein (fap), FLT3, folate-binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipid, gp36, HBV-specific antigen, HCV-specific antigen, HER1-HER2, HER2-HER3 combination, HERV-K, high molecular weight melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigen, human telomerase reverse transcriptase, IGFI receptor, IGF-II, IL-11Rα, IL-13R-a2, influenza virus-specific antigen;CD38, insulin growth factor (IGFl)-1, intestinal carboxylesterase, kappa chain, LAGA-la, lambda chain, Lassa virus specific antigen, lectin-reactive AFP, lineage-specific or tissue-specific antigens such as CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mut Targeting a tumor antigen selected from hsp70-2, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostate specific antigen (PSA), prostate carcinoma tumor antigen-1 (PCTA-1), prostate specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecules, survivin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin and Al domain of tenascin-C (TnC Al), thyroglobulin, tumor stromal antigens, vascular endothelial growth factor receptor-2 (VEGFR2), virus specific surface antigens such as HIV specific antigens (such as HIV gpl20), and any derivative or variant of these surface antigens; Engineered T Cells and Uses

[0307] The cells of the present disclosure can be obtained via T cells obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, tumors, or differentiated in vitro. Additionally, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood drawn from a subject using various techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. In some embodiments, cells collected by apheresis are washed to remove the plasma fraction and placed into an appropriate buffer or medium for subsequent processing. In some embodiments, the cells are washed with PBS. As will be appreciated, the washing step may be performed, for example, using a semi-automated flow-through centrifuge, such as the Cobe™ 2991 cell processor, Baxter CytoMate™, etc. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers or other saline solutions with or without buffers. In some embodiments, undesirable components of the apheresis sample are removed. Further methods of isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0308] In some embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, for example using centrifugation through a PERCOLL™ gradient. In some embodiments, specific subpopulations of T cells, such as CD4+, CD8+, CD28+, CD45RA+, and CD45RO+ T cells, are further isolated by positive or negative selection techniques known in the art. For example, enrichment of T cell populations by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. In some embodiments, cell sorting and / or selection may be performed by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In some embodiments, flow cytometry and cell sorting are performed to isolate the cell population of interest for use in the present disclosure.

[0309] In some embodiments, PBMCs are used directly for genetic modification of immune cells (such as CARs) using the methods described herein. In some embodiments, after isolating PBMCs, T lymphocytes are further isolated and both cytotoxic and helper T lymphocytes are sorted into subpopulations of naive, memory, and effector T cells before or after genetic modification and / or expansion.

[0310] In some embodiments, CD8+ cells are further sorted into naive cells, central memory cells, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, expression of phenotypic markers of central memory T cells includes expression of CCR7, CD3, CD28, CD45RO, CD62L, and CD127, and is negative for granzyme B. In some embodiments, central memory T cells are CD8+, CD45RO+, and CD62L+ T cells. In some embodiments, effector T cells are negative for CCR7, CD28, CD62L, and CD127, and positive for granzyme B and perforin. In some embodiments, CD4+ T cells are further classified into subpopulations. For example, CD4+ T helper cells can be sorted into naive cells, central memory cells, and effector cells by identifying cell populations with cell surface antigens.

[0311] In some embodiments, immune cells, e.g., T cells, are genetically modified after isolation using known methods, or immune cells are activated and expanded (or differentiated in the case of precursor cells) in vitro before being genetically modified. In another embodiment, immune cells, e.g., T cells, are genetically modified with a chimeric antigen receptor (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) as described herein, and then activated and / or expanded in vitro. Methods for activating and expanding T cells are known in the art and described, for example, in U.S. Pat. Nos. 6,905,874, 6,867,041, and 6,797,514, and WO 2012 / 079000, the entire contents of which are incorporated herein by reference. In general, such methods include contacting PBMCs or isolated T cells with stimulatory and costimulatory agents, e.g., anti-CD3 and anti-CD28 antibodies, typically bound to beads or other surfaces, in a culture medium containing appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies bound to the same bead function as "surrogate" antigen-presenting cells (APCs). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulator system for physiological activation of human T cells. In other embodiments, T cells are activated and stimulated to grow with feeder cells and appropriate antibodies and cytokines, for example, using methods described in U.S. Pat. Nos. 6,040,177 and 5,827,642 and WO 2012 / 129514, the contents of which are incorporated herein by reference in their entirety.

[0312] In some embodiments, the T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient suffering from cancer or a tumor. In some embodiments, the donor subject is a human patient not suffering from cancer or a tumor.

[0313] In some embodiments, the composition comprising the engineered T cells comprises a pharma- ceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and / or adjuvant. In some embodiments, the composition comprises an excipient.

[0314] In some embodiments, the composition is selected for delivery via the digestive tract, such as parenterally, by inhalation, or orally. Preparation of such pharmaceutically acceptable compositions is within the capabilities of one of ordinary skill in the art. In some embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8. In some embodiments, when parenteral administration is contemplated, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the composition described herein, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. In some embodiments, the parenteral injection vehicle is sterile distilled water in which the composition described herein, with or without at least one additional therapeutic agent, is formulated as a properly preserved sterile isotonic solution. In some embodiments, the preparation involves formulating the desired molecule with beads or liposomes, which are polymeric compounds (such as polylactic acid or polyglycolic acid) that allow for controlled or sustained release of the product, which are then delivered by depot injection. In some embodiments, an implantable drug delivery device is used to introduce the desired molecule.

[0315] In some embodiments, the method of treating cancer in a subject in need of treatment comprises T cell therapy. In some embodiments, the T cell therapy disclosed herein is an engineered autologous cell therapy (eACT™). According to this embodiment, the method may include harvesting blood cells from the patient. The isolated blood cells (e.g., T cells) can then be engineered to express a CAR disclosed herein. In certain embodiments, the CAR T cells are administered to the patient. In some embodiments, the CAR T cells treat the tumor or cancer in the patient. In some embodiments, the CAR T cells reduce the size of the tumor or cancer.

[0316] In some embodiments, donor T cells for use in T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for use in T cell therapy are obtained from a subject that is not a patient. In certain embodiments, the T cells are tumor infiltrating lymphocytes (TILs), engineered autologous T cells (eACT™), allogeneic T cells, xenogeneic T cells, or any combination thereof.

[0317] In some embodiments, the engineered T cells are administered in a therapeutically effective amount. For example, a therapeutically effective amount of engineered T cells is at least about 10 4 Cells, at least about 10 5 Cells, at least about 10 6 Cells, at least about 10 7 Cells, at least about 10 8 Cells, at least about 10 9 , or at least about 10 10 In another embodiment, the therapeutically effective amount of T cells can be about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, or about 10 8 In some embodiments, the therapeutically effective amount of T cells is about 2×10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7cells / kg, or 9 x 10 7 cells / kg.

[0318] In some embodiments, a therapeutically effective amount of engineered viable T cells is about 1×10 per kg of body weight. 6 ~about 2×10 6 The maximum dose is approximately 1 x 10 8 These are engineered live T cells. Treatment method

[0319] The methods disclosed herein may be used to treat cancer in a subject, to reduce the size of a tumor, to kill tumor cells, to prevent tumor cell proliferation, to prevent tumor growth, to eliminate a tumor from a patient, to prevent tumor recurrence, to prevent tumor metastasis, to induce remission in a patient, or any combination thereof. In some embodiments, the methods induce a complete response. In other embodiments, the methods induce a partial response.

[0320] Cancers that may be treated include tumors that are not vascularized, tumors that are not yet substantially vascularized, or tumors that are vascularized. Cancers may also include solid or non-solid tumors. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is a cancer of the white blood cells. In other embodiments, the cancer is a cancer of the plasma cells. In some embodiments, the cancer is a leukemia, lymphoma, or myeloma. In some embodiments, the cancer is a cancer of the acute lymphoblastic leukemia (ALL) (including non-T cell ALL), acute lymphocytic leukemia (ALL) and hemophagocytic lymphohistiocytosis (HLH), B-cell prolymphocytic leukemia, B-cell acute lymphocytic leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic or acute leukemia (ALL), chronic myelogenous leukemia (CLM), chronic or acute leukemia (CLH), chronic myelogenous leukemia (CLH ... Blastomatous disease, chronic or acute leukemia, diffuse large B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), hairy cell leukemia, hemophagocytic syndrome (macrophage activation syndrome (MAS), Hodgkin's disease, large cell granuloma, leukocyte adhesion deficiency, malignant lymphoproliferative state, MALT lymphoma, mantle cell lymphoma, marginal lymphoma, monoclonal gammopathy of undetermined significance (MGUS), Myeloid disorders including, but not limited to, multiple myeloma, myelodysplasia and myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytoma (e.g., dysplasmocytosis, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple myeloma) myeloma), POEMS syndrome (Crow-Fukase syndrome, Takatsuki's disease, PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoblastic leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma, Waldenstrom's macroglobulinemia, or a combination thereof.

[0321] In some embodiments, the cancer is a myeloma. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is a leukemia. In some embodiments, the cancer is acute myeloid leukemia.

[0322] In some embodiments, the cancer is non-Hodgkin's lymphoma. In some embodiments, the cancer is relapsed / refractory NHL. In some embodiments, the cancer is mantle cell lymphoma.

[0323] In some embodiments, the method further comprises administering a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent selected is a lymphodepleting (preconditioning) chemotherapeutic agent. Beneficial preconditioning treatment regimens, along with correlating beneficial biomarkers, are described in U.S. Provisional Patent Applications 62 / 262,143 and 62 / 167,750, and U.S. Patent Nos. 9,855,298 and 10,322,146. No. 5,393,996, which are incorporated herein by reference in their entireties. These include, for example, a method of pretreating a patient in need of T cell therapy, comprising administering a specified beneficial dose of cyclophosphamide (200 mg / m 2 / day~2000mg / m 2 / day) and the specified dose of fludarabine (20 mg / m 2 / day~900mg / m 2 / day) to the patient. One such dosing regimen comprises administering to the patient about 500 mg / m2 of engineered T cells prior to administering to the patient a therapeutically effective amount of engineered T cells. 2 / day cyclophosphamide, and approximately 60 mg / m 2 Another embodiment includes treating a patient with serum cyclophosphamide and fludarabine on days -4, -3, and -2 prior to T cell administration, at 500 mg / m per day during that period. 2 Cyclophosphamide at a dose of 30 mg / m2 per day 2Another embodiment includes cyclophosphamide on day -2 and fludarabine on days -4, -3, and -2 prior to administration of the T cells at a dose of 900 mg / m per day during that period. 2 Cyclophosphamide dose of 25 mg / m2 and 25 mg / m2 2 In another embodiment, conditioning includes cyclophosphamide and fludarabine on days -5, -4, and -3 prior to administration of the T cells, at 500 mg / m per day during that period. 2 Cyclophosphamide at a dose of 30 mg / m2 per day 2 Includes body surface area doses of fludarabine.

[0324] In some embodiments, the antigen binding molecule, the transduced (or otherwise engineered) cell (such as a CAR), and the chemotherapeutic agent are each administered in an amount effective to treat the disease or condition of the subject.

[0325] In some embodiments, compositions comprising the CAR-expressing immune effector cells disclosed herein can be administered in conjunction with a variety of chemotherapeutic agents. Exemplary chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, metholedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards, such as cyclosporine, cyclosporine, cyclopentasiloxane ... , Chlorambucil, Chlornaphazine, Chlorophosphamide, Estramustine, Ifosfamide, Mechlorethamine, Mechlorethamine Oxide Hydrochloride, Melphalan, Novenbitine, Phenesterine, Prednimustine, Trofosfamide, Uracil Mustard; Nitrosoureas, e.g., Carmustine, Chlorozotocin, Fotemustine, Lomustine, Nimustine, Ranimustine; Antibiotics, e.g., Aclacinomycin, Actinomycin, Ausramycin, Azaserine, Bleomycin, Cactinomycin cin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detrevicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, pofilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubeni mex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmoful, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU;Androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenergics, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine, bestravcil; bisantrene; edatrexate; ;defofamine;demecolcine;diaziquone;elformitin;elliptinium acetate;ethoglucide;gallium nitrate;hydroxyurea;lentinan;lonidamine;mitoguazone;mitoxantrone;mopidamol;nitracrine;pentostatin;phenamet;pirarubicin;podophyllic acid;2-ethylhydrazide;procarbazine;polysaccharide K (PSK);razoxane;sizofiran;spirogermanium;tenuazonic acid;triazicon;2,2',2''-trichlorotriethylamine;urethane;vindesine;dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacytosine;arabinosides ("Ara-C");cyclophosphamide;thiotepa;taxoids, e.g., paclitaxel (TAXOL(TM), Bristol-Myers Squibb) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylomitin (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukin diftitox); esperamycin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.In some embodiments, compositions comprising the CAR-expressing immune effector cells disclosed herein may be administered in conjunction with anti-hormonal agents that act to regulate or inhibit hormone action on tumors, e.g., anti-estrogens such as tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens, e.g., flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Combinations of chemotherapy agents are also administered as appropriate, including, but not limited to, CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®) and prednisone, R-CHOP (CHOP plus rituximab) and G-CHOP (CHOP plus obinutuzumab).

[0326] In some embodiments, the chemotherapeutic agent is administered simultaneously with or within one week after administration of the engineered cells. In other embodiments, the chemotherapeutic agent is administered 1-4 weeks, or 1 week-1 month, 1 week-2 months, 1 week-3 months, 1 week-6 months, 1 week-9 months, or 1 week-12 months after administration of the engineered cells or nucleic acid. In some embodiments, the chemotherapeutic agent is administered at least one month prior to administration of the cells or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

[0327] A variety of additional therapeutic agents can be used in conjunction with the compositions or agents / treatments described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), pidilizumab (CureTech), and atezolizumab (Roche).

[0328] Additional therapeutic agents suitable for use in combination with the compositions and methods disclosed herein include, in addition to antithymocyte globulin, lenzilumab, and mavrilimumab, ibrutinib (IMBRUVICA®), ofatumumab (ARZERRA®), rituximab (RITUXAN®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), trastuzumab elastase (TRAS®), trastuzumab tetanib ... Mutansine (KADCYLA®), imatinib (GLEEVEC®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, , gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib mTOR inhibitors such as toceranib, vandetanib, entrectinib, carbozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, everolimus and temsirolimus; hedgehog inhibitors such as sonidegib and vismodegib; CDK inhibitors such as the CDK inhibitor (palbociclib); and inhibitors of GM-CSF, CSF1, GM-CSFR or CSF1R, but are not limited to these.

[0329] In some embodiments, the treatment further comprises a bridging therapy, the bridging therapy being a therapy between the conditioning treatment and the composition disclosed herein, in some embodiments, the bridging therapy comprises CHOP, G-CHOP, R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone), corticosteroids, bendamustine, platinum compounds, anthracyclines, and / or phosphoinositide 3-kinase (PI3K) inhibitors. In some embodiments, the PI3K inhibitor is selected from duvelisib, idelalisib, venetoclax, pitilisib (GDC-0941), copanlisib, PX-866, buparlisib (BKM120), piralalisib (XL-147), GNE-317, alpelisib (BYL719), INK1117, GSK2636771, AZD8186, SAR260301, and taselisib (GDC-0032). In some embodiments, the AKT inhibitor is perifosine, MK-2206. In one embodiment, the mTOR inhibitor is selected from everolimus, sirolimus, temsirolimus, ridaforolimus. In some embodiments, the dual PI3K / mTOR inhibitor is selected from BEZ235, XL765, and GDC-0980. In some embodiments, the PI3K inhibitor is selected from duvelisib, idelalisib, venetoclax, pitilisib (GDC-0941), copanlisib, PX-866, buparlisib (BKM120), piralalisib (XL-147), GNE-317, alpelisib (BYL719), INK1117, GSK2636771, AZD8186, SAR260301, and taselisib (GDC-0032).

[0330] In some embodiments, the bridging therapy is selected from the group consisting of acalabrutinib, brentuximab vedotin, copanlisib hydrochloride, nelarabine, belinostat, bendamustine hydrochloride, carmustine, bleomycin sulfate, bortezomib, zanubrutinib, carmustine, chlorambucil, copanlisib hydrochloride, denilukin diptitox, dexamethasone, doxorubicin hydrochloride, duvelisib, pratrexate, obinutuzumab, ibritumomab tiuxetan, ibrutinib, idelalisib, recombinant interferon alpha-2b, romidepsin, lenalidomide, meclomid, These include, but are not limited to, retamine hydrochloride, methotrexate, mogamulizumab-kpc, plerixafor, nelarabine, obinutuzumab, denyl iptitox, pembrotuzumab, plerixafor, polatuzumab vedotin-piiq, mogamulizumab-kpc, prednisone, rituximab, hyaluronidase, romidepsin, bortezomib, venetoclax, vinblastine sulfate, vorinostat, zanubrutinib, CHOP, COPP, CVP, EPOCH, R-EPOCH, HYPER-CVAD, ICE, R-ICE, R-CHOP, R-CVP, and combinations thereof.

[0331] In some embodiments, the composition comprising the engineered CAR T cells is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolic acid. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylate. Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporoxifene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), and minocycline.

[0332] In some embodiments, the compositions described herein are administered with a cytokine. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (luteinizing hormone), and the like. hormone, LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; murelan inhibitor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF), e.g., NGF-beta; platelet growth factor; transforming growth factor (TGF), e.g., TGF-α and TGF-β; insulin-like growth factor-I and growth factor-II; erythropoietin (EPO, Epogen®, Procrit®); bone morphogenetic factor; interleukin-1 (IL-1); Examples of cytokines include interferons, such as interferon-alpha, beta, and gamma; colony stimulating factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage CSF (GM-CSF); and granulocyte CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, tumor necrosis factors, such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines. monitoring

[0333] In some embodiments, administration of the chimeric receptor T cell immunotherapy occurs in a certified health care facility.

[0334] In some embodiments, the methods disclosed herein include monitoring the patient daily for at least 7 days after infusion at a certified medical facility for signs and symptoms of CRS and neurotoxicity, as well as other adverse reactions to the CAR T cell therapy. In some embodiments, the symptoms of neurotoxicity are selected from encephalopathy, headache, tremors, dizziness, aphasia, delirium, insomnia, and anxiety. In some embodiments, the symptoms of adverse reactions are selected from the group consisting of fever, hypotension, tachycardia, hypoxia, and chills, including cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, cardiac failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), seizures, encephalopathy, headache, tremors, dizziness, aphasia, delirium, insomnia anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia. In some embodiments, patients are instructed to remain near a qualified medical facility for at least four weeks following the infusion. Clinical outcomes

[0335] In some embodiments, the clinical outcome is a complete response. In some embodiments, the clinical outcome is a durable response. In some embodiments, the clinical outcome is a complete response. In some embodiments, the clinical outcome is non-response. In some embodiments, the clinical outcome is a partial response. In some embodiments, the clinical outcome is an objective response. In some embodiments, the clinical outcome is survival. In some embodiments, the clinical outcome is recurrence.

[0336] In some embodiments, objective response (OR) is determined according to the revised IWG Response Criteria for Lymphoma (Cheson, 2007) and determined by the IWG Response Criteria for Lymphoma (Cheson et al. Journal of Clinical Oncology 32, no. 27 (September 2014) 3059-3067). Duration of response is evaluated. Progression-free survival (PFS) is evaluated by investigator assessment according to the Lugano Response Classification Criteria. Preventing or Managing Serious Adverse Reactions

[0337] In some embodiments, the disclosure provides a method of preventing the onset or reducing the severity of an adverse reaction based on the level of one or more attributes. In this regard, the disclosed method may include administration of a "prophylactically effective amount" of tocilizumab, corticosteroid therapy, or an anticonvulsant for toxicity prevention. In some embodiments, the method includes administering an inhibitor of GM-CSF, CSF1, GM-CSFR, or CSF1R, lenzilumab, mavrilimumab, a cytokine, and / or an anti-inflammatory agent. The pharmacological and / or physiological effect may be prophylactic, i.e., the effect completely or partially prevents a disease or its symptoms. A "prophylactically effective amount" may refer to an amount effective to achieve a desired prophylactic result (e.g., prevention of the onset of an adverse reaction) at the dosage and duration required.

[0338] In some embodiments, the method includes managing an adverse reaction, hi some embodiments, the adverse reaction is selected from the group consisting of cytokine release syndrome (CRS), neurotoxicity, hypersensitivity reactions, serious infections, cytopenias, and hypogammaglobulinemia.

[0339] In some embodiments, the signs and symptoms of the adverse reaction are selected from the group consisting of fever, hypotension, tachycardia, hypoxia, and chills, and include cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, cardiac failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia. Cytokine release syndrome (CRS)

[0340] In some embodiments, the methods include preventing or reducing the severity of CRS in chimeric receptor therapy, hi some embodiments, the engineered CAR T cells are inactivated after administration to the patient.

[0341] In some embodiments, the method includes identifying CRS based on clinical symptoms. In some embodiments, the method includes evaluating and treating fever, hypoxia, and other causes of hypotension. Patients experiencing grade 2 or higher CRS (e.g., hypotension unresponsive to fluids or hypoxia requiring supplemental oxygenation) should be monitored with continuous cardiac telemetry and pulse oximetry. In some embodiments, for patients experiencing severe CRS, consider performing an echocardiogram to assess cardiac function. In cases of severe or life-threatening CRS, intensive care supportive therapy may be considered.

[0342] In some embodiments, the method includes monitoring the patient daily for at least 7 days after the infusion at a certified medical facility for signs and symptoms of CRS. In some embodiments, the method includes monitoring the patient for signs or symptoms of CRS for 4 weeks after the infusion. In some embodiments, the method includes advising the patient to seek immediate medical attention whenever signs or symptoms of CRS occur. In some embodiments, the method includes initiating treatment with supportive care, tocilizumab, or tocilizumab and corticosteroids as indicated at the first sign of CRS. Neurotoxicity (NT)

[0343] In some embodiments, the method includes monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method includes excluding other causes of neurological symptoms. Patients experiencing grade 2 or higher neurotoxicity should be monitored with continuous cardiac telemetry and pulse oximetry. In cases of severe or life-threatening neurotoxicity, intensive supportive care is provided. In some embodiments, symptoms of neurotoxicity are selected from encephalopathy, headache, tremors, dizziness, aphasia, delirium, insomnia, and anxiety.

[0344] In some embodiments, the method comprises monitoring the patient daily for at least seven days after the infusion at a qualified medical facility for signs and symptoms of neurotoxicity, hi some embodiments, the method comprises monitoring the patient for signs or symptoms of neurotoxicity for four weeks after the infusion. Secondary malignancies

[0345] In some embodiments, patients treated with CAR T cells (e.g., directed against CD19) or other genetically modified autologous T cell immunotherapy may develop a secondary malignancy. In certain embodiments, patients treated with CAR T cells (e.g., directed against CD19) or other genetically modified allogeneic T cell immunotherapy may develop a secondary malignancy. In some embodiments, the method includes lifelong monitoring for secondary malignancies.

[0346] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present disclosure. In the event that any definitions or terms provided in a reference incorporated by reference differ from the terms and explanations provided herein, the terms and definitions shall control.

[0347] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references cited throughout this application are hereby expressly incorporated by reference.

[0348] The disclosure provided by this application can be used in a variety of ways in addition to or in combination with the methods described above. Below is a summary of exemplary methods that can be derived from the disclosure provided by this application. Methods and compositions for generating and optimizing products for increased clinical efficacy and / or reduced toxicity

[0349] Please refer to the preceding paragraphs

[0111] to

[0157] and the examples. Methods for increasing efficacy and / or reducing toxicity of T cell therapy

[0350] In one embodiment, the disclosure provides a method of increasing efficacy and / or reducing toxicity of T cell immunotherapy (e.g., CAR T cell immunotherapy), comprising reducing the subject's tumor burden prior to CAR-T cell immunotherapy. In one embodiment, reducing the subject's tumor burden comprises administration of a bridging therapy. In one embodiment, the bridging therapy comprises a therapy between conditioning and administration of the T cells. In one embodiment, the bridging therapy comprises CHOP, R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone), G-CHOP (obinutuzumab, cyclophosphamide, doxorubicin, vincristine, and prednisolone), corticosteroids, bendamustine, platinum compounds, anthracyclines, venetoclax, zanubrutinib, and / or phosphoinositide 3-kinase (PI3K) inhibitors, and inhibitors of the PI3K / Akt / mTOR pathway. In one embodiment, the PI3K inhibitor is selected from duvelisib, idelalisib, venetoclax, pitilisib (GDC-0941), copanlisib, PX-866, buparlisib (BKM120), piralalisib (XL-147), GNE-317, alpelisib (BYL719), INK1117, GSK2636771, AZD8186, SAR260301, and taselisib (GDC-0032).In one embodiment, the bridging therapy is selected from the group consisting of acalabrutinib, brentuximab vedotin, copanlisib hydrochloride, nelarabine, belinostat, bendamustine hydrochloride, carmustine, bleomycin sulfate, bortezomib, zanubrutinib, carmustine, chlorambucil, copanlisib hydrochloride, denilukin diptitox, dexamethasone, doxorubicin hydrochloride, duvelisib, pratrexate, obinutuzumab, ibritumomab tiuxetan, ibrutinib, idelalisib, recombinant interferon alfa-2b, romidepsin, lenalidomide, mechlorethene, These include cefotaxime, methotrexate, mogamulizumab-kpc, plerixafor, nelarabine, obinutuzumab, denyl iptitox, pembrotuzumab, plerixafor, polatuzumab vedotin-piiq, mogamulizumab-kpc, prednisone, rituximab, hyaluronidase, romidepsin, bortezomib, venetoclax, vinblastine sulfate, vorinostat, zanubrutinib, CHOP, COPP, CVP, EPOCH, R-EPOCH, HYPER-CVAD, ICE, R-ICE, R-CHOP, R-CVP, and combinations thereof.

[0351] In one embodiment, the disclosure provides a method of increasing efficacy and / or reducing toxicity of T cell immunotherapy (e.g., CAR T cell immunotherapy), comprising reducing a systemic inflammatory condition in a subject prior to the T cell immunotherapy. In one embodiment, the therapy is CAR T cell therapy. In one embodiment, the method comprises administering an anti-inflammatory therapy to the subject prior to the CAR-T cell immunotherapy. Examples of anti-inflammatory therapies are provided elsewhere in this disclosure.

[0352] In one embodiment, the present disclosure provides a method of increasing efficacy and / or reducing toxicity of T cell immunotherapy (e.g., CAR T cell immunotherapy), comprising reducing myeloid cell activity in a subject prior to CAR-T cell immunotherapy. Methods are provided for increasing efficacy and / or reducing toxicity of T cell immunotherapy (e.g., CAR T cell immunotherapy), comprising reducing MCP-1 and / or IL-6 activity prior to or early thereafter administration of RT cells. In one embodiment, reducing myeloid cell activity, MCP-1 and / or IL-6 activity comprises administering to the subject a monoclonal antibody against MCP-1, IL-6, IL-1, CSF1R, GM-CSF and / or a small molecule. Examples of such agents are described elsewhere in this disclosure. In one embodiment, the small molecule is a JAK / STAT inhibitor. In one embodiment, the JAK / STAT inhibitor is selected from tofacitinib, ruxolitinib, filgotinib, baricitinib, peficitinib, oclacitinib, upazitinib, solcitinib, decernotinib, SHR0302, AC 430, PF-06263276, BMS-986165, lestaurtinib, PF-06651600, PF-04965841, abrocitinib, statix, peptidomimetics, and combinations thereof.

[0353] In one embodiment, the disclosure provides a method of increasing efficacy and / or reducing toxicity of T cell immunotherapy (e.g., CAR T cell immunotherapy), comprising reducing activity of activated T cells in the subject / product prior to CAR-T cell immunotherapy. In one embodiment, this can be accomplished by separating / removing differentiated cells (effector memory and / or effector cells), enriching the product for young T cells (CCR7+), removing or reducing the percentage and number of differentiated T cells in the T cell product infusion bag by separation techniques; and / or treating the product T cells during or after the manufacturing process with pharmacological agents or biological response modifiers (e.g., JAK / STAT inhibitors) that will reduce excess T cell activity.

[0354] In one embodiment, the present disclosure provides methods for increasing efficacy and / or decreasing toxicity of T cell immunotherapy (e.g., CAR T cell immunotherapy) including increasing the dosage of T cell immunotherapy in a manner commensurate with tumor burden and / or re-administering patients with high tumor burden. Methods for measuring and classifying tumor burden are described elsewhere in this disclosure.

[0355] In one embodiment, the disclosure provides a method of increasing efficacy and / or decreasing toxicity of T cell immunotherapy (e.g., CAR T cell immunotherapy), comprising: (a) identifying a subject positive for a marker(s) of toxicity in response to T cell immunotherapy; and (b) decreasing IL-15 elevation after pre-treatment and pre-T cell immunotherapy in the subject. In one embodiment, the marker of toxicity in response to T cell immunotherapy is high tumor burden. In one embodiment, the marker of toxicity in response to T cell immunotherapy is elevated pre-treatment levels of inflammatory markers. In one embodiment, the inflammatory markers are selected from IL6, CRP, and ferritin. In one embodiment, a decrease in IL-15 elevation after pre-treatment and pre-T cell immunotherapy is achieved by selection of a pre-treatment protocol. In one embodiment, the pre-treatment protocol comprises cyclophosphamide, fludarabine, bendamustine, anti-human thymocyte globulin, carmustine, radiation, etoposide, cytarabine, melphalan, rituximab, or a combination thereof. Methods for manipulating the composition of specific T cell subsets in a T cell product to improve the treatment of subjects with the T cell product

[0356] In one embodiment, the disclosure provides a method of treating malignancies combining any of the above methods of predicting response and / or toxicity, a method of manipulating the composition of a T cell product, and administration of a T cell therapy (e.g., a T cell infusion product).

[0357] In one embodiment, the present disclosure provides an improvement to an infusion product comprising engineered lymphocytes, and optionally a method of treating cancer in a subject with an infusion product comprising engineered lymphocytes, the method comprising:

[0358] Measuring the level of one or more attributes in a population of lymphocytes from the apheresis product; and / or

[0359] Measuring the level of one or more attributes in a population of engineered lymphocytes (e.g., CAR T cells) during manufacture of the final infusion product and / or in the final infusion product; and / or

[0360] Manipulating the composition of T cell infusion products to improve efficacy and reduce treatment-related toxicity; and / or

[0361] determining or predicting a patient's response to treatment with the engineered lymphocytes based on the measured level of one or more attributes compared to a reference level; optionally,

[0362] administering to the subject a therapeutically effective dose of the engineered lymphocytes, the therapeutically effective dose being determined based on the level of one or more attributes of the population of engineered lymphocytes in the infusion product and / or the T cells in the apheresis product.

[0363] In one embodiment, the engineered lymphocytes target a tumor antigen. In one embodiment, the tumor antigen is a tumor-associated surface antigen, such as 5T4, alpha fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD8, CLL-1, c-Met, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, EGFR variant III (EGFRvIII), ELF2M, ... endoglin, ephrinB2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2 / neu), fibroblast-associated protein (fap), FLT3, folate-binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipid, gp36, HBV-specific antigen, HCV-specific antigen, HER1-HER2, HER2-HER3 combination, HERV-K, high molecular weight melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigen, human telomerase reverse transcriptase, IGFI receptor, IGF-II, IL-11Rα, IL-13R-a2, influenza virus-specific antigen;CD38, insulin growth factor (IGFl)-1, intestinal carboxylesterase, kappa chain, LAGA-la, lambda chain, Lassa virus specific antigen, lectin-reactive AFP, lineage-specific or tissue-specific antigens such as CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mut Selected from hsp70-2, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostate specific antigen (PSA), prostate carcinoma tumor antigen-1 (PCTA-1), prostate specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecules, survivin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin and Al domain of tenascin-C (TnC Al), thyroglobulin, tumor stromal antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus specific surface antigens such as HIV specific antigens (such as HIV gpl20), and any derivative or variant of these surface antigens. In one embodiment, the target antigen is CD19;

[0364] In one embodiment, the cancer is a solid tumor, a sarcoma, a carcinoma, a lymphoma, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), T-cell lymphoma, B-cell acute lymphocytic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Kitt-Barr lymphoma, diffuse large cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell lymphoma or large cell lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, bone marrow necrosis factor lymphoma, osteosarcoma, osteoporosis ... Myelodysplasia and myelodysplastic syndromes, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), monoclonal gammopathy of undetermined significance (MGUS), plasmacytomas (e.g., plasma cell dyscrasia, isolated myeloma, isolated plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloid light chain amyloidosis, POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, PEP syndrome), or a combination thereof. In one embodiment, the cancer is diffuse large B-cell lymphoma (DLBCL) not otherwise specified (relapsed or refractory), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, follicular lymphoma, or DLBCL arising from mantle cell lymphoma.

[0365] In one embodiment, a therapeutically effective amount or effective dose of engineered lymphocytes (e.g., CAR T cells) is at least about 10 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 cells, at least about 10 8 cells, at least about 109 cells, or at least about 10 10 In one embodiment, the therapeutically effective amount or effective dose of engineered lymphocytes (e.g., CAR T cells) can be about 10 4 cells, approximately 10 5 cells, approximately 10 6 cells, approximately 10 7 cells or about 10 8 In one embodiment, a therapeutically effective amount or effective dose of engineered lymphocytes (e.g., CAR T cells) is about 2×10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 cells / kg, or approximately 9 x 10 7 In one embodiment, the therapeutically effective amount or effective dose of engineered lymphocytes (e.g., CAR T cells) can be about 1×10 cells / kg of body weight. 6 ~Approx. 2×10 6 may be about 1×10 live engineered lymphocytes (e.g., CAR T cells), with the maximum dose being about 1×10 8 In one embodiment, the therapeutically effective dose may be 75-200×10 6 These are engineered lymphocytes. EXAMPLES

[0366] A clinical trial was conducted in which patients with relapsed / refractory NHL were treated with axicabtagene ciloleucel, a CD19-directed genetically modified autologous T cell immunotherapy that involves the patient's own T cells being harvested and genetically modified ex vivo by retroviral transduction to express a chimeric antigen receptor (CAR) containing an anti-CD19 single-chain variable fragment (scFv) linked to the costimulatory domains of CD28 and CD3 zeta.

[0367] Patients had diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, or transformed follicular lymphoma with refractory disease despite receiving recommended prior therapy. The patient received a conditioning regimen of low-dose cyclophosphamide and fludarabine followed by 2 × 10 6 They received a target dose of anti-CD19 CAR T cells. (Neelapu, SS et al. 2017, N Engl J Med 2017;377(26):2531-44).

[0368] In the following examples, biomarker data from clinical trial patients were analyzed according to an extended statistical analysis plan for correlation of responses and parameters differentially associated with treatment efficacy and toxicity, and with product suitability. Several correlations were identified. Available samples from patients in the clinical study (NCT02348216) were analyzed. Safety and efficacy results were previously reported. (Neelapu, SS et al. 2017, N Engl J Med 2017;377(26):2531-44;Locke FL et al.2019;Lancet Oncol.2019 Jan;20(1):31-42.doi:10.1016 / S1470-2045(18)30864-7.Epub 2018 Dec 2). A sustained response refers to a patient who had a continuing response at least 1 year after axicabtagene ciloleucel infusion. A relapse refers to a patient who achieved a CR or PR and then experienced disease progression. Patients who achieved stable or progressive disease as their best response are included in the non-responder category.

[0369] Meanwhile, traditional prognostic factors for LBCL were not associated with outcome in pivotal clinical studies (Neelapu et al. NEJM. 2017), although other attributes such as chimeric antigen receptor (CAR) T-cell compatibility and composition (CCR7+CD45RA+ T cells), reduced preTx tumor burden, and immune tumor microenvironment (TME) with the presence of activated CD8+PD-1+LAG-3+ / -TIM-3- T cells were associated with efficacy (Locke et al., Blood Advances, 2020 https: / / doi.org / 10.1182 / bloodadvances.2020002394 and Galon et al., ASCO, 2020 https: / / ascopubs.org / doi / abs / 10.1200 / JCO.2020.38.15_suppl.3022). A systemic investigation of factors influencing CAR-T cell compatibility and TIC in LBCL revealed associations between pretreatment immune cell characteristics in the blood on the one hand and key features of axicabtagene ciloleucel and TIC, respectively, that influence the clinical response to CAR T cell intervention.

[0370] The existing characteristics of the immune system were systematically analyzed by multiparametric flow cytometry of apheresized peripheral blood mononuclear cells (PBMCs) that served as starting material for the axicabtagene ciloleucel manufacturing process in clinical studies (N=101). Apheresized PBMCs were maintained in liquid nitrogen before thawing for antibody staining. The panels utilized in this analysis characterized T cells (CD27, CD28, CCR7, and CD45RA), myeloid cell subsets, NK, NKT, and memory compartments of B cells (Figures 1A and 1B). As previously described (Rossi et al,Cancer Res July 1 2018 (78)(13 Supplement)LB-016;DOI:10.1158 / 1538-7445.AM2018-LB-016,Galon et al,Journal of Clinical Oncology 2020 (38)(15_suppl),3022-3022 DOI:10.1200 / JCO.2020.38.15_suppl.3022 Journal of Clinical Oncology 38,no.15_suppl(May 20, 2020)3022-3022, analysis of pre-treatment TICs was performed by multiplex immunohistochemistry (N=18) and gene expression analysis (N=30). Axicabtagene ciloleucel characteristics related to T cell compatibility were analyzed by measuring doubling time and viability during production (N=145), as well as final product T cell phenotype by flow cytometry (including percentage and total number of infused CCR7+CD45RA+ T cells). These Correlation analysis between covariates and parameters from routine hematology, as well as features from routine hematology, was performed by Spearman rank correlation and Wilcoxon tests. The effect of different variables on survival was evaluated by the Kaplan-Meier method with optimal cut-point selection.

[0371] Two important pre-existing features of the immune system were identified, consisting of the percentage of naive phenotype CD27+CD28+Th cells (CCR7+CD45RA+) and intermediate monocytes (CD14+CD16+), both measurable in pre-production PBMC populations, and positively and negatively associated, respectively, with determinants of axicabtagene ciloleucel clinical efficacy. More specifically, the frequency of naive phenotype CD27+CD28+Th cells in pre-production PBMC populations was positively associated with pre-treatment T cell signatures in the TME, as was the percentage of product CCR7+CD45RA+T cells. Furthermore, this metric was positively associated with the rate of ongoing response, progression-free survival, and overall response following axicabtagene ciloleucel. Conversely, the percentage of intermediate monocytes (CD14+CD16+) in the pre-manufacturing PBMC population correlated directly with negative predictive markers such as pre-treatment serum levels of LDH, IL-6 and CRP, and inversely with survival and T cell signatures in the TME.

[0372] Pre-production PBMCs, most notably CD27 + CD28 + The pre-existing state of the immune system, examined by investigation of the frequency of naive Th cells and the frequency of intermediate monocytes, respectively positively and negatively influenced important tumor microenvironment and product determinants supporting the clinical efficacy of axicabtagene ciloleucel. Overall, these results provide an important link between the pre-existing state of the immune system and the clinical efficacy of autologous CAR T cell therapy in LBCL, providing insight into how this treatment modality may overcome tumors associated with poor prognostic markers or provide therapeutic optimization to improve its performance. Figure 1C. Example 1

[0373] The percentage of naive phenotype CD27+CD28+ Th cells (CCR7+CD45RA+) in pre-production PBMC populations correlated positively with phenotypic markers of product T cell compatibility, including doubling time and viability, CD4 / CD8 ratio, and percentage of CD8 and CD4 naive T cells (Figure 2). Final product cells were characterized by the same parameters. The percentage of intermediate and total monocytes in the pre-manufacturing PBMC populations positively correlated with pre-treatment inflammatory markers, tumor burden (baseline sum of two-way products (SPD)) and hypoxia (as indicated by serum LDH concentrations). Figure 3. Relative proportions of T cell subsets vs myeloid cell subsets in the pre-manufacturing PBMC populations that are differentially associated with pre-treatment tumor immune landscape. Figure 4. Monocytes, particularly intermediate monocytes, are negatively associated with T cell signatures of the TME, whereas CD27+CD28+ naive Th cells and lymphocytes are positively associated with T cell signatures of the TME that are associated with response. Pre-manufacturing naive Th subsets correlated positively with the percentage of naive T cells in the product infusion bag, the T cell-enriched tumor immune landscape (all shown). Figure 5. Pre-manufacturing intermediate monocytes positively correlated with pre-treatment inflammation (INTL8, PRF) / tumor hypoxia (LDH) and negatively correlated with pre-treatment defined as T cell-rich tumor immune landscape (all markers shown are markers of activated T cells). Figure 6. Pre-manufacturing intermediate monocytes had a negative association with lymphocytes and lymphocyte to monocyte ratio (later in this literature shown to positively correlate with response / survival). Also, a positive association was observed with pre-treatment tumor burden which itself is negatively associated with response. Example 2

[0374] CD27+CD28+ naive Th cells (% of leukocytes) in the apheresis product were predictive markers for improved OS (Figure 7A) and PFS (Figure 7B) (optimal cutoffs). There was a positive association between them, i.e. subjects with pretreatment CD27+CD28+ naive Th cells above the listed cutoff were more likely to survive than those below the selected cutoff. The level of intermediate monocytes (% of white blood cells) in the apheresis product was also a predictive marker for OS (Figure 8A) and PFS (optimal cutoff) (Figure 8B). The current data suggest that subjects with intermediate monocyte levels below the listed cutoff were more likely to survive than those above the cutoff. The ratio of CD27pCD28p naive Th cells (% of white blood cells) to intermediate monocytes (% of white blood cells) in the apheresis product showed a positive association with and serves as a predictive marker for OS (Figure 9A) and PFS (optimal cutoff) (9B). There was a better survival / response / proliferation rate for subjects with levels above the selected cutoff compared to those below it. We also investigated the relationship between CD27+CD28+ naive Th cells (% of white blood cells) versus intermediate monocytes (% of white blood cells, CD14+CD16+) in the apheresis product in non-responders, ongoing responders, and relapsed patients. Figure 10A and Figure 10B. CD27+CD28+ naive Th cells are negatively associated with intermediate monocytes. Furthermore, subjects with high CD27+CD28+ naive Th levels and low intermediate monocyte levels have an increased proportion of objective responders (top left part of Figure 10B). The frequency of intermediate monocytes may have a greater negative impact on efficacy in subjects with tumors with a large sum of two-way products (SPD). In Figure 12B.Q2, we observed that high intermediate monocytes and low CAR T cell proliferation correlated with the highest rate of non-responders. Figure 11. Subjects with increased CAR-T cell peak proliferation and lower intermediate monocyte levels (Q4) had increased ongoing response rates and decreased relapse or non-responder rates compared to other quadrants. Figure 11 and Figure 12A. These quadrants of CAR-T cell peak proliferation and intermediate monocytes can be viewed in the context of high (Figure 12B) or low (Figure 12C) tumor burden. When viewed in the further context of high baseline tumor burden, the above trend of decreased intermediate monocytes and increased CAR T cell proliferation corresponding to increased ongoing response rates is amplified, although subjects with high intermediate monocytes and low CAR T cell peak proliferation have an even lower ongoing response rate.Figure 12B. The trend remains in the setting of low tumor burden, but the sustained response rate is higher due to the need to overcome a smaller tumor burden. Figure 12B and C. Example 3

[0375] There was an association between CD27+CD28+ naive Th (% of white blood cells) and response category. Figure 13. CD27+CD28+ naive Th cell levels are higher in responding patients compared to non-responding patients. There was also an association between intermediate monocytes (% of white blood cells) and response category. Figure 14. Intermediate monocytes are lower in responding patients compared to non-responding patients. Furthermore, levels are lower in subjects with ongoing (sustained) response compared to subjects who experience relapse or are non-responders. Example 4

[0376] The naive Th cell population in the apheresis product was negatively associated with the number of prior lines of therapy. First (Z12) or second (Z7) line DLBCL may have higher levels of naive T cells at leukapheresis. Figure 15A, Figure 15B, Figure 15C. The data in Figure 15A can show that subjects have higher levels of these cells in the blood with fewer lines of therapy, indicating that response rates may be improved if CAR T cells are utilized as an earlier line of therapy (1st / 2nd line). Higher IPI scores tend to have lower CD27+CD28+ naive Th cells. CD27+CD28+ naive Th cells show a weak negative association with baseline tumor burden. Figure 15B. Example 5

[0377] The intermediate monocyte population in the apheresis product was associated with disease burden (FIG. 16C) and moderately increased with the number of prior lines of therapy. Intermediate monocytes are positively associated with the number of prior lines of therapy. Subjects with fewer prior lines of therapy would be expected to have lower levels of intermediate monocytes. Due to the negative association of these cells with response, this also indicates that CAR T cell response rates may be even higher if utilized as an earlier line of treatment (1st / 2nd line). Figure 16A. International Prognostic Index (IPI) score and intermediate monocytes were positively associated, further indicating that these cells are associated with subjects with a worse prognosis. Figure 16B. Intermediate monocytes were positively associated with baseline tumor burden. Figure 16C. Example 6

[0378] The level of CD27-CD28+TEMRA Treg cells (% of white blood cells) in the apheresis product was positively associated with and may be a predictive marker for OS (Figure 17A) and PFS (Figure 17B) (optimal cutoff). Using this cutoff for CD27-CD28+TEMRA Tregs, subjects with higher levels of these cells have higher complete response, objective response, and ongoing response rates. Example 7

[0379] There was an association between CD27+CD28+ naive Th cells in the apheresis product vs. CAR-T peak (Figures 18A and 18B) and CAR-T peak / baseline tumor burden (Figures 18C and 18D). A positive association was observed between CD27+CD28+ naive Th cells and CAR T cell peak proliferation (normalized by tumor burden, see also Figures 18C-D). Low levels of both correlate with higher non-responders, while increased levels of both result in higher response rates. An association was observed between intermediate monocytes vs. CAR-T peak and CAR-T peak / baseline tumor burden. There was a positive association between intermediate monocytes and CAR T cell peak proliferation (normalized by tumor burden, see also Figures 19C-D). Low levels of both correlate with higher non-responders, while increased levels of both result in higher response rates. Figure 19. Example 8

[0380] Apheresis composition and baseline hematology cell counts. [Table 2-1] [Table 2-2] [Table 3] Example 9

[0381] Lymphocytes vs. leukocytes in baseline hematology cell counts were positively associated with OS (Figure 20A) and PFS (Figure 20B) and may serve as a predictive marker for these (optimal cutoff). Lymphocytes vs. leukocytes in baseline hematology cell counts were positively associated with complete response, objective response, and ongoing response. Figure 21. Example 10

[0382] Lymphocytes vs. leukocytes in baseline hematology cell counts were weakly negatively associated with worst grade toxicity. Figure 22. Lymphocytes vs. leukocytes in baseline hematology cell counts were negatively associated with tumor burden. Figure 23. Lymphocytes vs. leukocytes in baseline hematology cell counts were negatively associated with number of prior lines of therapy. Figure 24. These data indicate that utilization of CAR-T cells in earlier lines of therapy may result in improved objective and durable responses due to positive predictors of higher response and product suitability with fewer lines of therapy. Lymphocytes vs. leukocytes in baseline hematology cell counts were positively associated with CD8 and effector cells in product. Lymphocytes vs. leukocytes in baseline hematology cell counts were negatively associated with CRP, ferritin, and IL6. CRP, ferritin, and IL6 have previously been shown to be pharmacodynamic markers that negatively correlate with response in DLBCL. Figure 25. Lymphocytes vs. leukocytes in baseline hematology cell counts were negatively associated with myeloid cells (more specifically, intermediate monocytes, which are negatively associated with response) and positively associated with CD8 and EM / effector T cells. Figure 26. Lymphocytes vs. leukocytes in baseline hematology cell counts were negatively associated with intermediate monocytes and showed weak correlations with apheresis populations associated with response, including CD27-CD28+TEMRA and Tregs, as well as CD27+CD28+naive and Th cells. B cell levels are most likely not the population driving lymphocyte levels, as they showed weak or no associations. High B cell levels positively correlate with response. Lymphocytes vs. leukocytes in baseline hematology had limited or no association with CAR T peak cell proliferation and naive product T cells. Due to the limited associations between these features, we may be able to use them in combination to better stratify patients. Example 11

[0383] Lymphocytes vs. monocytes in baseline hematology cell counts were positively associated with OS (Figure 27A) and PFS (Figure 27B) and may serve as a predictive biomarker for these (optimal cutoff) (positive association). Lymphocytes vs. monocytes in baseline hemato...

Claims

1. 1. A method for predicting the likelihood of survival of a patient in need of chimeric antigen receptor (CAR) T cell therapy, comprising: measuring the level of CD27+CD28+ naive Th cells in an apheresis product from said patient; determining said likelihood of survival of said patient at least in part from said level of CD27+CD28+ naive Th cells in said apheresis product; The patient is determined to have an increased chance of survival if the level of CD27+CD28+ naive Th cells exceeds a cutoff percentage value measured as a percentage of total white blood cells, and the patient is determined to have a decreased chance of survival if the level of CD27+CD28+ naive Th cells is below the cutoff percentage value.

2. 2. The method of claim 1, wherein the cut-off percentage value is approximately 0-0.1%, 0.1%-0.5%, 0.5%-1.0%, 1.0-5%, 5-10%, 10-15%, 10-20%, 20-30%, 30-40%, 40-50%, or more preferably approximately 0.27%.

3. measuring the level of intermediate monocytes in the apheresis product from the patient; determining the likelihood of survival of the patient at least in part from the level of intermediate monocytes in the apheresis product; 2. The method of claim 1, wherein the patient is determined to have an increased chance of survival if the level of intermediate monocytes is below a cutoff percentage value measured as a percentage of total white blood cells, and the patient is determined to have a decreased chance of survival if the level of intermediate monocytes is above the cutoff percentage value.

4. 4. The method of claim 3, wherein said cut-off percentage value is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%, preferably between 1 and 5%, and even more preferably below approximately 3%.

5. measuring the level of CD27-CD28+ TEMRA Treg cells in the apheresis product from the patient; determining said likelihood of survival of said patient at least in part from said level of CD27-CD28+ TEMRA Treg cells in said apheresis product; 2. The method of claim 1, wherein if the level of CD27-CD28+ TEMRA Treg cells is above a cutoff percentage value measured as a percentage of total white blood cells, the patient is determined to have an increased chance of survival, and if the level of CD27-CD28+ TEMRA Treg cells is below the cutoff percentage value, the patient is determined to have a decreased chance of survival.

6. 6. The method of claim 5, wherein said cut-off percentage value is between approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1-5%, 5-10%, 10-20%, preferably 0.05-0.2%, 0.2-0.25%, 0.25-0.5%, 0.5-0.6%, 0.6-0.7%, 0.7-0.8%, 0.8-0.9%, 0.9-1%, 1-5%, 5-10%, 10-15%, more preferably approximately 0.1705%.

7. measuring the lymphocyte to leukocyte ratio in the patient's baseline hematology count; determining said likelihood of survival of said patient at least in part from said lymphocyte to white blood cell ratio; 2. The method of claim 1, wherein if the lymphocyte to white blood cell ratio is above a cutoff value, the patient is determined to have an increased chance of survival, and if the lymphocyte to white blood cell ratio is below the cutoff value, the patient is determined to have a decreased chance of survival.

8. 8. The method of claim 7, wherein the cut-off value is 1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, preferably below 5.2%.

9. measuring the lymphocyte to monocyte ratio in the patient's baseline hematology count; determining said likelihood of survival of said patient at least in part from said lymphocyte to monocyte ratio; 2. The method of claim 1, wherein if the lymphocyte to monocyte ratio is above a cutoff value, the patient is determined to have an increased chance of survival, and if the lymphocyte to monocyte ratio is below the cutoff value, the patient is determined to have a decreased chance of survival.

10. 10. The method of claim 9, wherein the cut-off value is between 0 and 0.5, 0.5 and 1.0, 1.0 and 1.5, 1.5 and 2.0, 2 and 5, 5 and 10, 10 and 15, preferably 0.

79.

11. 1. A method for producing an immunotherapy product, comprising: Preparing an apheresis product from a blood sample from a subject; Measuring the level of CD27+CD28+ naive Th cells in the apheresis product; and and increasing the amount of CD27+CD28+ naive Th cells collected for processing if the level of CD27+CD28+ naive Th cells in the apheresis product is below a cutoff percentage value measured as a percentage of total white blood cells in the apheresis product.

12. 12. The method of claim 11, wherein the cut-off percentage value is approximately 0-0.1%, 0.1%-0.5%, 0.5%-1.0%, 1.0-5%, 5-10%, 10-15%, 10-20%, 20-30%, 30-40%, 40-50%, or more preferably approximately 0.27%.

13. measuring the level of intermediate monocytes in the apheresis product; 12. The method of claim 11, further comprising: if the level of intermediate monocytes in the apheresis product is above a cutoff percentage value, measured as a percentage of total white blood cells in the apheresis product, reducing the level of intermediate monocytes in the apheresis product prior to further processing.

14. 14. The method of claim 13, wherein the cut-off percentage value is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%, preferably between 1 and 5%, even more preferably approximately 3%.

15. measuring the level of CD27-CD28+ TEMRA Treg cells in the apheresis product; and If the level of CD27-CD28+ TEMRA Treg cells in the apheresis product is below a cutoff percentage value measured as a percentage of total white blood cells in the apheresis product, then CD27-CD28+ TEMRA Treg cells are collected for processing. The method of claim 11 , further comprising increasing the amount of MRA Treg cells.

16. 16. The method of claim 15, wherein said cut-off percentage value is between approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1-5%, 5-10%, 10-20%, preferably 0.05-0.2%, 0.2-0.25%, 0.25-0.5%, 0.5-0.6%, 0.6-0.7%, 0.7-0.8%, 0.8-0.9%, 0.9-1%, 1-5%, 5-10%, 10-15%, more preferably about 0.1705%.

17. 1. A method for treating a malignant tumor in a patient, comprising: measuring the level of CD27+CD28+ naive Th cells in an apheresis product from said patient; determining, at least in part from the level of CD27+CD28+ naive Th cells in the apheresis product, whether to administer to the patient an effective dose of T cells comprising a chimeric receptor, or an effective dose of T cells comprising a chimeric receptor and a combination therapy; administering, based on said determining step, the effective dose of T cells comprising a chimeric receptor, or the effective dose of T cells and the combination therapy; The patient is administered the effective dose of T cells comprising a chimeric receptor if the level of CD27+CD28+ naive Th cells exceeds a cutoff percentage value measured as a percentage of total white blood cells, and the patient is administered the effective dose of T cells comprising a chimeric receptor and the combination therapy if the level of CD27+CD28+ naive Th cells is below the cutoff percentage value.

18. 18. The method of claim 17, wherein the cut-off percentage value is approximately 0-0.1%, 0.1%-0.5%, 0.5%-1.0%, 1.0-5%, 5-10%, 10-15%, 10-20%, 20-30%, 30-40%, 40-50%, or more preferably approximately 0.27%.

19. measuring the level of intermediate monocytes in the apheresis product from the patient; determining, at least in part from the level of intermediate monocytes in the apheresis product, whether to administer to the patient an effective dose of T cells comprising a chimeric receptor, or an effective dose of T cells comprising a chimeric receptor and a combination therapy; administering the effective dose of T cells comprising a chimeric receptor, or the effective dose of T cells and the combination therapy based on the determining step; 18. The method of claim 17, wherein the patient is administered the effective dose of T cells comprising a chimeric receptor if the level of intermediate monocytes is below a cutoff percentage value measured as a percentage of total white blood cells, and the patient is administered the effective dose of T cells comprising a chimeric receptor and the combination therapy if the level of intermediate monocytes is above the cutoff percentage value.

20. 20. The method of claim 19, wherein the cut-off percentage value is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%, preferably between 1 and 5%, even more preferably approximately 3%.

21. measuring the level of CD27-CD28+ TEMRA Treg cells in the apheresis product from the patient; determining, at least in part from the level of CD27-CD28+ TEMRA Treg cells in the apheresis product, whether to administer to the patient an effective dose of T cells comprising a chimeric receptor, or an effective dose of T cells comprising a chimeric receptor and a combination therapy; administering the effective dose of T cells comprising a chimeric receptor, or the effective dose of T cells and the combination therapy based on the determining step; 18. The method of claim 17, wherein the patient is administered the effective dose of T cells comprising a chimeric receptor if the level of CD27-CD28+ TEMRA Treg cells is above a cutoff percentage value measured as a percentage of total white blood cells, and the patient is administered the effective dose of T cells comprising a chimeric receptor and the combination therapy if the level of CD27-CD28+ TEMRA Treg cells is below the cutoff percentage value.

22. 22. The method of claim 21, wherein said cut-off percentage value is between approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1-5%, 5-10%, 10-20%, preferably 0.05-0.2%, 0.2-0.25%, 0.25-0.5%, 0.5-0.6%, 0.6-0.7%, 0.7-0.8%, 0.8-0.9%, 0.9-1%, 1-5%, 5-10%, 10-15%, more preferably about 0.1705%.

23. measuring the lymphocyte to leukocyte ratio in the patient's baseline hematology count; determining, at least in part from the lymphocyte to white blood cell ratio, whether to administer to the patient an effective dose of T cells comprising a chimeric receptor, or an effective dose of T cells comprising a chimeric receptor and a combination therapy; administering the effective dose of T cells comprising a chimeric receptor, or the effective dose of T cells and the combination therapy based on the determining step; 18. The method of claim 17, wherein the patient is administered the effective dose of T cells comprising a chimeric receptor if the lymphocyte to white blood cell ratio is above a cutoff value, and the patient is administered the effective dose of T cells comprising a chimeric receptor and the combination therapy if the lymphocyte to white blood cell ratio is below the cutoff value.

24. 24. The method according to claim 23, wherein the cut-off value is 1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, preferably 5.2%.

25. measuring the lymphocyte to monocyte ratio in the patient's baseline hematology count; determining, at least in part from the lymphocyte to monocyte ratio, whether to administer to the patient an effective dose of T cells comprising a chimeric receptor, or an effective dose of T cells comprising a chimeric receptor and a combination therapy; administering the effective dose of T cells comprising a chimeric receptor, or the effective dose of T cells and the combination therapy based on the determining step; 18. The method of claim 17, wherein the patient is administered the effective dose of T cells comprising a chimeric receptor if the lymphocyte to monocyte ratio is above a cutoff value, and the patient is administered the effective dose of T cells comprising a chimeric receptor and the combination therapy if the lymphocyte to monocyte ratio is below the cutoff value.

26. 26. The method of claim 25, wherein the cut-off value is between 0 and 0.5, 0.5 and 1.0, 1.0 and 1.5, 1.5 and 2.0, 2 and 5, 5 and 10, 10 and 15, preferably 0.

79.

27. The combination therapy may be any combination of immunotherapy, SRC kinase inhibitors, T cell bispecific antibodies, anti-CD20 monoclonal antibodies, anti-4-1BB, anti-CD47, TGF-beta inhibitors or dominant negative TGF-beta, mTOR / AKT agonists, histone deacetylase inhibitors, cyclophosphamide, fluorouracil, gemcitabine, doxorubicin, tachyarrhythmidine, cyclophosphamide ...

27. The method of any one of claims 17 to 26, comprising administering to the patient a therapeutically effective amount of a medicament for the treatment of a tumor, chemotherapy or radiation therapy, a small molecule inhibitor, an antibody targeted to enhance anti-tumor immunity, or an anti-inflammatory drug.

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