#METABOLOMICS WORKBENCH halqudairy_20251101_233651 DATATRACK_ID:6616 STUDY_ID:ST004386 ANALYSIS_ID:AN007329 PROJECT_ID:PR002779 VERSION 1 CREATED_ON November 25, 2025, 7:42 am #PROJECT PR:PROJECT_TITLE PTPRD-based CAR T-cells demonstrate efficacy against IL-1RAP-positive AML PR:PROJECT_TYPE Research Study PR:PROJECT_SUMMARY Acute myeloid leukemia (AML) remains a challenging disease to treat, primarily PR:PROJECT_SUMMARY due to the persistence of leukemic stem cells (LSCs). Interleukin-1 receptor PR:PROJECT_SUMMARY accessory protein (IL-1RAP) is overexpressed on the surface of LSCs across all PR:PROJECT_SUMMARY AML subtypes, making it a compelling and promising marker compared to other PR:PROJECT_SUMMARY common AML targets, as it is not expressed by normal hematopoietic stem cells. PR:PROJECT_SUMMARY IL-1RAP interacts with various protein partners, but among them, only PTPRD PR:PROJECT_SUMMARY (Protein Tyrosine Phosphatase Receptor Type D) has been shown to bind in Trans PR:PROJECT_SUMMARY to IL-1RAP. We designed a CAR with the extracellular domain of PTPRD as the PR:PROJECT_SUMMARY recognition domain. PTPRD CAR was cloned into lentiviral vector backbone PR:PROJECT_SUMMARY containing the 4-1BB co-stimulatory domain. The efficacy of PTPRD CAR-T cell to PR:PROJECT_SUMMARY eliminate specifically IL-1RAP+ cells was demonstrated by high intracellular PR:PROJECT_SUMMARY TNFα expression and elevated IFNγ release, leading to significant cytotoxic PR:PROJECT_SUMMARY effects compared to untransduced T cells in vitro. In a xenograft mouse model, PR:PROJECT_SUMMARY PTPRD CAR-T cells exhibited potent anti-tumor activity, resulting in complete PR:PROJECT_SUMMARY elimination of AML cells and prolonged survival of the mice. In conclusion, our PR:PROJECT_SUMMARY preclinical findings suggest that PTPRD CAR-T cell therapy holds significant PR:PROJECT_SUMMARY promise as a novel and effective strategy for AML treatment. PR:INSTITUTE King Faisal Specialist Hospital and Research Centre (KFSHRC) PR:DEPARTMENT Research and Innovation Centre PR:LABORATORY Research Laboratories PR:LAST_NAME Warda PR:FIRST_NAME Walid PR:ADDRESS Makkah Al Mukarramah Rd, Al Mathar Ash Shamali, Riyadh 12713 PR:EMAIL wwarda@kfshrc.edu.sa;walidwarda1990@gmail.com PR:PHONE 966500858514 PR:FUNDING_SOURCE KFSH&RC Research and Innovation Fund PR:CONTRIBUTORS Walid Warda, Syed Osman Ahmed, Hanan Alqudairy, Ayadole Alaiya, Anas Abdel PR:CONTRIBUTORS Rahman, Reem Almalki, Monther Alhamdoosh, Edward Hitti, Abdullah Aldhfyan, Ahmed PR:CONTRIBUTORS Kotb, Nour Almozain, Falah Almohanna, Farhatullah Syed, Mahmoud Aljurf, Hazzaa PR:CONTRIBUTORS Alzahrani and Jackie Y. Ying #STUDY ST:STUDY_TITLE Metabolomic analysis of CAR T-cells and untransduced T-cells from healthy donor ST:STUDY_TYPE Untargeted Metabolomics ST:STUDY_SUMMARY Untransduced T cells and PTPRD-based CAR T-cells were generated from peripheral ST:STUDY_SUMMARY blood of a healthy donor. Following collection, cells were washed with cold PBS ST:STUDY_SUMMARY and rapidly quenched in liquid nitrogen. Intracellular metabolites were ST:STUDY_SUMMARY extracted and analyzed using untargeted LC-MS. Metabolic features were annotated ST:STUDY_SUMMARY using the Human Metabolome Database (HMDB), and exogenous compounds (for ST:STUDY_SUMMARY example, drugs and food additives) were removed from the dataset. Comparative ST:STUDY_SUMMARY analysis showed that PTPRD CAR T-cells exhibited coordinated enrichment of ST:STUDY_SUMMARY multiple metabolic pathways, including sphingolipid, purine, pyrimidine, ST:STUDY_SUMMARY glycerophospholipid, propanoate, tryptophan, and porphyrin metabolism, relative ST:STUDY_SUMMARY to untransduced T-cells. ST:INSTITUTE King Faisal Specialist Hospital and Research Centre (KFSHRC) ST:DEPARTMENT Research and Innovation Centre ST:LABORATORY Research Laboratories ST:LAST_NAME AlQudairy ST:FIRST_NAME Hanan ST:ADDRESS Al-Takhasussi ST:EMAIL alqudairyhanan@gmail.com ST:PHONE 966500858514 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 1 ST:NUM_MALES 1 ST:STUDY_COMMENTS This is an exploratory study with limited statistical power. #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male #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 HD1 IC_CART_cells_1 Treatment:CAR | Sample source:Blood RAW_FILE_NAME(Raw Data)=IC_CART_cells_1 SUBJECT_SAMPLE_FACTORS HD1 IC_CART_cells_2 Treatment:CAR | Sample source:Blood RAW_FILE_NAME(Raw Data)=IC_CART_cells_2 SUBJECT_SAMPLE_FACTORS HD1 IC_CART_cells_3 Treatment:CAR | Sample source:Blood RAW_FILE_NAME(Raw Data)=IC_CART_cells_3 SUBJECT_SAMPLE_FACTORS HD1 IC_Untransduced_Tcells_1 Treatment:Untreated | Sample source:Blood RAW_FILE_NAME(Raw Data)=IC_Untransduced_Tcells_1 SUBJECT_SAMPLE_FACTORS HD1 IC_Untransduced_Tcells_2 Treatment:Untreated | Sample source:Blood RAW_FILE_NAME(Raw Data)=IC_Untransduced_Tcells_2 SUBJECT_SAMPLE_FACTORS HD1 IC_Untransduced_Tcells_3 Treatment:Untreated | Sample source:Blood RAW_FILE_NAME(Raw Data)=IC_Untransduced_Tcells_3 #COLLECTION CO:COLLECTION_SUMMARY Blood samples from a healthy donor were collected and obtained after written CO:COLLECTION_SUMMARY informed consent (RAC#2240004, KFSH&RC, Riyadh). On day 0, Peripheral blood CO:COLLECTION_SUMMARY mononuclear cells (PBMCs) were separated by density gradient centrifugation CO:COLLECTION_SUMMARY using Ficoll-Paque PLUS (GE Healthcare, reference 17-1440-02), and centrifuged CO:COLLECTION_SUMMARY at 2000×g for 20 minutes at room temperature without brake. Next, CD4+ and CD8+ CO:COLLECTION_SUMMARY cells were magnetically isolated with CD4 Microbeads (Miltenyi™, reference: CO:COLLECTION_SUMMARY 130-045-101) and CD8 Microbeads (Miltenyi™, reference: 130-045-201). Selected CO:COLLECTION_SUMMARY T cells were activated using T cell TransAct™ (Miltenyi™, reference: CO:COLLECTION_SUMMARY 200-076-204). CO:SAMPLE_TYPE Blood (whole) CO:COLLECTION_LOCATION KFSH&RC Research Labs CO:COLLECTION_FREQUENCY one time CO:COLLECTION_DURATION 9 days CO:STORAGE_CONDITIONS Described in summary #TREATMENT TR:TREATMENT_SUMMARY Twenty-four hours after T cell stimulation, CAR lentiviral supernatants were TR:TREATMENT_SUMMARY added. In parallel, control cells (untransduced T cells or UTD) were produced in TR:TREATMENT_SUMMARY the same manner, but without adding the CAR vector. Subsequently, cells were TR:TREATMENT_SUMMARY expanded for 9 days. During the entire process, T cells were cultivated in TR:TREATMENT_SUMMARY TexMACS medium (Miltenyi™, reference: 170-076-306) supplemented with the TR:TREATMENT_SUMMARY recombinant human cytokines IL-7 (1700 UI/mL, Miltenyi™, reference TR:TREATMENT_SUMMARY 170-076-111) and IL-15 (320 UI/mL, Milteny™, reference 170-076-114), 3% of TR:TREATMENT_SUMMARY human serum (Life science production, reference S-101B-HI, EU,) and 1% TR:TREATMENT_SUMMARY penicillin-streptomycin. Over the entire process, quality control tests enabled TR:TREATMENT_SUMMARY in-process assessment of cell count and viability via trypan blue (Gibco, TR:TREATMENT_SUMMARY reference 15250-061). TR:TREATMENT CAR lentiviral supernatants TR:TREATMENT_COMPOUND PTPRD-based CAR #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted following a previously reported protocol SP:SAMPLEPREP_SUMMARY (doi:10.3390/ijms24044219) to extract intracellular metabolites. Briefly, in SP:SAMPLEPREP_SUMMARY intracellular metabolism, medium was removed, and cells were washed with cold SP:SAMPLEPREP_SUMMARY PBS followed by quenching in liquid nitrogen. The cells were extracted by 80% SP:SAMPLEPREP_SUMMARY (v:v) MeOH:H2O and scraped using a cell scraper. The mixture was vortexed in a SP:SAMPLEPREP_SUMMARY Thermomixer (Eppendorf, Germany) for 1 h at 600 rpm on 4 °C. The mixture was SP:SAMPLEPREP_SUMMARY then spun down for 10 min at 4 °C, 10,000 rpm. The supernatants were SP:SAMPLEPREP_SUMMARY transferred to new Eppendorf tubes. The extracts were evaporated completely in a SP:SAMPLEPREP_SUMMARY Speed-Vac (Christ, Germany) and stored at −80 °C until LC-MS analysis. The SP:SAMPLEPREP_SUMMARY samples were reconstituted in 50% mobile phase A:B (A: 0.1% formic acid in dH2O, SP:SAMPLEPREP_SUMMARY B: 0.1% formic acid in 50% MeOH and ACN) for untargeted metabolomics analyses SP:SAMPLEPREP_SUMMARY using LCMS. SP:SAMPLEPREP_PROTOCOL_FILENAME Sample_preparation.pdf SP:SAMPLEPREP_PROTOCOL_COMMENTS sample prep protocol published in AlMalki, R. H., Al-Nasrallah, H. K., Aldossry, SP:SAMPLEPREP_PROTOCOL_COMMENTS A., Barnawi, R., Al-Khaldi, S., Almozyan, S., ... & Al-Alwan, M. (2024). SP:SAMPLEPREP_PROTOCOL_COMMENTS Comparative Analysis of Breast Cancer Metabolomes Highlights Fascin’s Central SP:SAMPLEPREP_PROTOCOL_COMMENTS Role in Regulating Key Pathways Related to Disease Progression. International SP:SAMPLEPREP_PROTOCOL_COMMENTS Journal of Molecular Sciences, 25(14), 7891. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Metabolites were acquired by a Waters Acquity ultra pressure liquid CH:CHROMATOGRAPHY_SUMMARY chromatography (UPLC) system coupled with a Xevo G2-S QTOF mass spectrometer CH:CHROMATOGRAPHY_SUMMARY equipped with an electrospray ionization source (ESI) on positive and negative CH:CHROMATOGRAPHY_SUMMARY (ESI+, ESI−). The metabolites were chromatographed using a Waters Acquity UPLC CH:CHROMATOGRAPHY_SUMMARY HSS T3 (100×2.1mm 1.8µm) column (Waters Ltd., Elstree, UK). the mobile phase CH:CHROMATOGRAPHY_SUMMARY composed of 0.1% formic acid in dH2O as solvent A and solvent B consists of 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid in 50% ACN: MeOH. The mobile phases A and B were pumped to the CH:CHROMATOGRAPHY_SUMMARY column in a gradient mode (0–16 min 95–5% A, 16–19 min 5% A, 19–22 min CH:CHROMATOGRAPHY_SUMMARY 5–95% A) at 300 μL/min flow rate. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 50% methanol/50% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0–16 min 95–5% A, 16–19 min 5% A, 19–22 min 5–95% A CH:FLOW_RATE 300 μL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo-G2-S MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS MS conditions were as follows: the source temperature was 150 °C, the MS:MS_COMMENTS desolvation temperature was 500 °C for both ESI+ and ESI- analysis, capillary MS:MS_COMMENTS voltages were 3.20 kV (ESI+) or 3 kV (ESI−), cone voltage was 40 V, MS:MS_COMMENTS desolvation gas flow was 800.0 L/h, and cone gas flow was 50 L/h. The collision MS:MS_COMMENTS energy of low and high functions was set off, at 10–50 V, respectively, in MSE MS:MS_COMMENTS mode. The mass spectrometer was calibrated, as recommended by the vendor, with MS:MS_COMMENTS sodium formate in the range of 100–1200 Da in both ionization modes. Data MS:MS_COMMENTS Independent Acquisition (DIA) was collected in continuum mode with a Masslynx™ MS:MS_COMMENTS V4.1 workstation (Waters Inc., Milford, MA, USA).The MS raw data were processed MS:MS_COMMENTS following a standard pipeline starting from alignment based on the m/z value and MS:MS_COMMENTS the ion signals’ retention time, peak picking, and signal filtering based on MS:MS_COMMENTS the peak quality using the Progenesis QI v.3.0 software from Waters (Waters MS:MS_COMMENTS Technologies, Milford, MA, USA). MS:MS_RESULTS_FILE ST004386_AN007329_Results.txt UNITS:Peak Area Has m/z:Yes Has RT:Yes RT units:Minutes #END