#METABOLOMICS WORKBENCH imssr_20250530_135845 DATATRACK_ID:5976 STUDY_ID:ST003963 ANALYSIS_ID:AN006511 PROJECT_ID:PR002483 VERSION 1 CREATED_ON June 11, 2025, 10:24 am #PROJECT PR:PROJECT_TITLE Metabolomics of CAR-T cells and their products in leukemia patients PR:PROJECT_TYPE Untargeted metabolomics PR:PROJECT_SUMMARY Although most patients with relapsed/refractory B-cell acute lymphoblastic PR:PROJECT_SUMMARY leukemia (B-ALL) receiving CD19-targeted chimeric antigen receptor (CAR) T cell PR:PROJECT_SUMMARY therapy achieve remission, loss of CAR T cell functionality and subsequent PR:PROJECT_SUMMARY relapse remains an unmet therapeutic need. We applied an integrative approach to PR:PROJECT_SUMMARY study the immunometabolism of pre- and post-infusion CD19-CAR T cells of PR:PROJECT_SUMMARY patients with relapsed/refractory B-ALL. Pre-infusion CAR T cells of long-term PR:PROJECT_SUMMARY responders (LTR) had increased oxidative phosphorylation, fatty acid oxidation, PR:PROJECT_SUMMARY and pentose phosphate pathway activities, higher mitochondrial mass, tighter PR:PROJECT_SUMMARY cristae, and lower mTOR expression compared to products of short-term PR:PROJECT_SUMMARY responders. Post-infusion CAR T cells in bone marrow (BM) of LTR had high PR:PROJECT_SUMMARY immunometabolic plasticity and mTOR-pS6 expression supported by the BM PR:PROJECT_SUMMARY microenvironment. Transient inhibition of mTOR during manufacture induced PR:PROJECT_SUMMARY metabolic reprogramming and enhanced anti-tumor activity of CAR T cells. Our PR:PROJECT_SUMMARY findings provide insight into immunometabolic determinants of long-term response PR:PROJECT_SUMMARY and suggest a therapeutic strategy to improve long-term remission. PR:INSTITUTE Translational Genomics Research Institute PR:LAST_NAME Pirrotte PR:FIRST_NAME Patrick PR:ADDRESS 445 N 5TH STREET, Phoenix, Arizona PR:EMAIL ims@tgen.org PR:PHONE 6023438454 PR:FUNDING_SOURCE NIH K12 grant no. 5K12CA001727–29, The Hyundai Hope on Wheels Young PR:FUNDING_SOURCE Investigator Award, NCI grant no.P30CA033572 PR:PROJECT_COMMENTS Study 1 of 4 PR:CONTRIBUTORS Lior Goldberg, Eric R. Haas, Jiaqi Wu, Bryan Garcia, Ryan Urak, Vibhuti Vyas, PR:CONTRIBUTORS Ruby Espinosa, Tamara Munoz, Shirley Bierkatz, Jamie R. Wagner, Jinny Paul, Mary PR:CONTRIBUTORS C. Clark, Dat Ngo, Ibrahim Aldoss, Stephen J. Forman & Xiuli Wang #STUDY ST:STUDY_TITLE Immunometabolic determinants of long-term response in preinfusion CAR-T cells of ST:STUDY_TITLE the leukemia patients receiving CD19 CAR T cell therapy ST:STUDY_TYPE Untargeted metabolomics ST:STUDY_SUMMARY To interrogate the immunometabolic properties of pre-infusion CAR-T cell ST:STUDY_SUMMARY products of both long-term and short-term responders, pre-infusion CAR-T cells ST:STUDY_SUMMARY were subjected untargeted metabolomics. We saw TCA metabolites, nucleotide ST:STUDY_SUMMARY biosynthesis intermediates, sphingolipid metabolism, and intermediates in ST:STUDY_SUMMARY central energy metabolism were significantly higher in long-term responders and ST:STUDY_SUMMARY nucleotide catabolic and tRNA metabolism intermediates were significantly ST:STUDY_SUMMARY decreased in long-term responders compared to short-term responders suggesting ST:STUDY_SUMMARY higher oxidative phosphorylation and fatty acid oxidation and pentose phosphate ST:STUDY_SUMMARY pathway contributing to higher antileukemic activity of CAR-T cells in long-term ST:STUDY_SUMMARY responders. ST:INSTITUTE Translational Genomics Research Institute ST:LAST_NAME Pirrotte ST:FIRST_NAME Patrick ST:ADDRESS 445 N 5TH STREET, Phoenix, Arizona ST:EMAIL ims@tgen.org ST:PHONE 16023438454 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 33-52 SU:GENDER Male and female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS CARTG10xUPN505 CARTG10xUPN505_HILICneg Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG10xUPN505xHILICneg_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG10xUPN505 CARTG10xUPN505_HILICpos Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG10xUPN505xHILICpos_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG11xUPN397 CARTG11xUPN397_HILICneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG11xUPN397xHILICneg_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG11xUPN397 CARTG11xUPN397_HILICpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG11xUPN397xHILICpos_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG12xUPN544 CARTG12xUPN544_HILICneg Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG12xUPN544xHILICneg_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG12xUPN544 CARTG12xUPN544_HILICpos Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG12xUPN544xHILICpos_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG13xUPN518 CARTG13xUPN518_HILICneg Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG13xUPN518xHILICneg_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG13xUPN518 CARTG13xUPN518_HILICpos Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG13xUPN518xHILICpos_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG14xUPN543 CARTG14xUPN543_HILICneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG14xUPN543xHILICneg_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG14xUPN543 CARTG14xUPN543_HILICpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG14xUPN543xHILICpos_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG1xUPN188 CARTG1xUPN188_HILICneg Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG1xUPN188xHILICneg_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG1xUPN188 CARTG1xUPN188_HILICpos Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG1xUPN188xHILICpos_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG2xUPN293 CARTG2xUPN293_HILICneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG2xUPN293xHILICneg_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG2xUPN293 CARTG2xUPN293_HILICpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG2xUPN293xHILICpos_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG3xUPN379 CARTG3xUPN379_HILICneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG3xUPN379xHILICneg_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG3xUPN379 CARTG3xUPN379_HILICpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG3xUPN379xHILICpos_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG4xUPN209 CARTG4xUPN209_HILICneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG4xUPN209xHILICneg_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG4xUPN209 CARTG4xUPN209_HILICpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG4xUPN209xHILICpos_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG5xUPN431 CARTG5xUPN431_HILICneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG5xUPN431xHILICneg_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG5xUPN431 CARTG5xUPN431_HILICpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG5xUPN431xHILICpos_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG6xUPN549 CARTG6xUPN549_HILICneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG6xUPN549xHILICneg_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG6xUPN549 CARTG6xUPN549_HILICpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG6xUPN549xHILICpos_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG7xUPN466 CARTG7xUPN466_HILICneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG7xUPN466xHILICneg_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG7xUPN466 CARTG7xUPN466_HILICpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG7xUPN466xHILICpos_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG8xUPN495 CARTG8xUPN495_HILICneg Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG8xUPN495xHILICneg_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG8xUPN495 CARTG8xUPN495_HILICpos Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG8xUPN495xHILICpos_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG9xUPN552 CARTG9xUPN552_HILICneg Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG9xUPN552xHILICneg_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG9xUPN552 CARTG9xUPN552_HILICpos Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01A_R_CARTG9xUPN552xHILICpos_28DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG10xUPN505 CARTG10xUPN505_RPneg Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG10xUPN505xRPneg_30DEC3_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG10xUPN505 CARTG10xUPN505_RPpos Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG10xUPN505xRPpos_29DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG11xUPN397 CARTG11xUPN397_RPneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG11xUPN397xRPneg_30DEC3_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01_20240103210342.raw SUBJECT_SAMPLE_FACTORS CARTG11xUPN397 CARTG11xUPN397_RPpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG11xUPN397xRPpos_29DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG12xUPN544 CARTG12xUPN544_RPneg Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG12xUPN544xRPneg_30DEC3_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG12xUPN544 CARTG12xUPN544_RPpos Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG12xUPN544xRPpos_29DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG13xUPN518 CARTG13xUPN518_RPneg Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG13xUPN518xRPneg_30DEC3_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG13xUPN518 CARTG13xUPN518_RPpos Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG13xUPN518xRPpos_29DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG14xUPN543 CARTG14xUPN543_RPneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG14xUPN543xRPneg_30DEC3_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG14xUPN543 CARTG14xUPN543_RPpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG14xUPN543xRPpos_29DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG1xUPN188 CARTG1xUPN188_RPneg Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG1xUPN188xRPneg_30DEC3_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG1xUPN188 CARTG1xUPN188_RPpos Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG1xUPN188xRPpos_29DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG2xUPN293 CARTG2xUPN293_RPneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG2xUPN293xRPneg_30DEC3_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01_20240103204528.raw SUBJECT_SAMPLE_FACTORS CARTG2xUPN293 CARTG2xUPN293_RPpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG2xUPN293xRPpos_29DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG3xUPN379 CARTG3xUPN379_RPneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG3xUPN379xRPneg_30DEC3_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG3xUPN379 CARTG3xUPN379_RPpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG3xUPN379xRPpos_29DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG4xUPN209 CARTG4xUPN209_RPneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG4xUPN209xRPneg_30DEC3_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG4xUPN209 CARTG4xUPN209_RPpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG4xUPN209xRPpos_29DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG5xUPN431 CARTG5xUPN431_RPneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG5xUPN431xRPneg_30DEC3_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG5xUPN431 CARTG5xUPN431_RPpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG5xUPN431xRPpos_29DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG6xUPN549 CARTG6xUPN549_RPneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG6xUPN549xRPneg_30DEC3_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG6xUPN549 CARTG6xUPN549_RPpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG6xUPN549xRPpos_29DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG7xUPN466 CARTG7xUPN466_RPneg Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG7xUPN466xRPneg_30DEC3_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG7xUPN466 CARTG7xUPN466_RPpos Sample source:CAR-T cells | Outcome:Short term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG7xUPN466xRPpos_29DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG8xUPN495 CARTG8xUPN495_RPneg Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG8xUPN495xRPneg_30DEC3_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG8xUPN495 CARTG8xUPN495_RPpos Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG8xUPN495xRPpos_29DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG9xUPN552 CARTG9xUPN552_RPneg Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG9xUPN552xRPneg_30DEC3_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw SUBJECT_SAMPLE_FACTORS CARTG9xUPN552 CARTG9xUPN552_RPpos Sample source:CAR-T cells | Outcome:Long term responders RAW_FILE_NAME(File name)=160_0002_01C_R_CARTG9xUPN552xRPpos_29DEC23_CELL_HUM_LUM_0000_04AC_15MIN_KP_KP_01.raw #COLLECTION CO:COLLECTION_SUMMARY De-identified clinical samples were collected from a subset of adult patients CO:COLLECTION_SUMMARY with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) who CO:COLLECTION_SUMMARY received investigational CD19-specific CAR T cells in a phase 1/2 clinical trial CO:COLLECTION_SUMMARY (NCT02146924) at City of Hope. For CAR T cells, PBMC were depleted of CD14+ CO:COLLECTION_SUMMARY monocytes and CD25+ Tregs, followed by positive selection of CD62L+ T cells. CO:COLLECTION_SUMMARY Tcm-derived CAR T cells were purified, transduced, formulated, and released. The CO:COLLECTION_SUMMARY CAR T cells were used for metabolomics profiling. CO:SAMPLE_TYPE CAR-T cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Preinfusion CAR-T cells from leukemia patient exhibiting long-term and shor-term TR:TREATMENT_SUMMARY treatment response were used for metabolomics profiling. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For CAR-T cells, metabolites were extracted from 3x10^6 cells using of 0.4 mL of SP:SAMPLEPREP_SUMMARY solvent mixture (water: methanol: acetonitrile, 1:2.25:0.75, v/v/v) and 3 SP:SAMPLEPREP_SUMMARY freeze-thaw (30 seconds each) cycles. The resultant metabolite extracts from CAR SP:SAMPLEPREP_SUMMARY T cells were recovered by centrifuging at 13,226 xg at 4°C for 15 min. For SP:SAMPLEPREP_SUMMARY HILIC chromatography, the extract was directly used LC-MS analysis. For reversed SP:SAMPLEPREP_SUMMARY phase chromatography, the extract was dried under vacuum centrifugation and the SP:SAMPLEPREP_SUMMARY dried extract was reconstituted in 20% Acetonitrile, 80% water and used used for SP:SAMPLEPREP_SUMMARY LC-MS analysis. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD The metabolites were extracted using 1 part of water and 3 part of solvent SP:EXTRACTION_METHOD mixture, acetonitrile: methanol (3:1) was added for cell lysis and protein SP:EXTRACTION_METHOD removal SP:EXTRACT_ENRICHMENT 1x SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION For reversed phase chormatography, the metabolites were reconstituted in SP:SAMPLE_RESUSPENSION water-acetonitrile (80:20, v/v) and for HILIC chromatography, clarified neat SP:SAMPLE_RESUSPENSION extract was sued SP:SAMPLE_DERIVATIZATION none SP:SAMPLE_SPIKING The samples were spiked with isotopically labelled internal standards #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY HILIC chromatography CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um) CH:SOLVENT_A 95% water/5% acetonitrile; 10mM ammonium acetate; 0.1% acetic acid CH:SOLVENT_B 95% acetonitrilie/5% water; 10mM ammonium acetate; 0.1% acetic acid CH:FLOW_GRADIENT 99% B for 1 min, 99–85% B for 3 min, 85–75% B for 3 min, 75–30% B CH:FLOW_GRADIENT for 3 min, 30% B for 1 min, 30-99%B for 1 min and 99%B for 4 min CH:FLOW_RATE 0.35 ml/min CH:COLUMN_TEMPERATURE 45 CH:INJECTION_TEMPERATURE 4 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Fusion Lumos Tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Xcalibur Compound discoverer 3.2 MS:CAPILLARY_TEMPERATURE 325 MS:ION_SOURCE_TEMPERATURE 275 MS:ION_SPRAY_VOLTAGE 3500 MS:MS_RESULTS_FILE ST003963_AN006511_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END