#METABOLOMICS WORKBENCH juliehaines_20230221_121940 DATATRACK_ID:3758 STUDY_ID:ST002490 ANALYSIS_ID:AN004064 PROJECT_ID:PR001607 VERSION 1 CREATED_ON February 21, 2023, 12:40 pm #PROJECT PR:PROJECT_TITLE Simultaneous targeting of PD-1 and IL2Rβγ with radiation therapy to inhibit PR:PROJECT_TITLE pancreatic cancer growth and metastasis PR:PROJECT_SUMMARY In pancreatic ductal adenocarcinoma (PDAC) patients, we show that response to PR:PROJECT_SUMMARY radiation therapy (RT) is characterized by increased IL2Rβ and IL2Rγ and PR:PROJECT_SUMMARY decreased ILR2α expression. The bispecific aPD1-IL2v is a PD-1-targeted IL-2 PR:PROJECT_SUMMARY variant (IL2v) immunocytokine with engineered IL-2 cis-targeted to PD-1 and PR:PROJECT_SUMMARY abolished IL2Rα binding, which enhances tumor-antigen specific T cell PR:PROJECT_SUMMARY activation while reducing regulatory T cell (Treg) suppression. Using aPD1-IL2v PR:PROJECT_SUMMARY in orthotopic PDAC KPC-driven tumor models, we show marked improvement in local PR:PROJECT_SUMMARY and metastatic survival along with profound increase in tumor-infiltrating PR:PROJECT_SUMMARY polyfunctional CD8+ T cell subsets with a transcriptionally and metabolically PR:PROJECT_SUMMARY active phenotype, and preferential activation of antigen-specific CD8+ T cells. PR:PROJECT_SUMMARY In combination with single dose RT, aPD1-IL2v treatment results in a robust, PR:PROJECT_SUMMARY durable expansion of polyfunctional CD8+ T cells, T cell stemness, PR:PROJECT_SUMMARY tumor-specific memory immune response, natural killer (NK) cell activation, and PR:PROJECT_SUMMARY decreased Tregs. These data show that aPD1-IL2v leads to profound local and PR:PROJECT_SUMMARY distant response in PDAC. PR:INSTITUTE University of Colorado Denver PR:LABORATORY Lab of Angelo D'Alessandro in collaboration with lab of Sana Karam PR:LAST_NAME Haines PR:FIRST_NAME Julie PR:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA PR:EMAIL julie.haines@cuanschutz.edu PR:PHONE 3037243339 #STUDY ST:STUDY_TITLE Simultaneous targeting of PD-1 and IL2Rβγ with radiation therapy to inhibit ST:STUDY_TITLE pancreatic cancer growth and metastasis - human plasma metabolomics ST:STUDY_SUMMARY Patient plasma samples were collected as part of a Phase I radiation ST:STUDY_SUMMARY dose-escalation clinical trial (NCT02873598) before, during (6 hours post), and ST:STUDY_SUMMARY post (6 weeks) SBRT. Samples were collected and analyzed per COMIRB19–0328 for ST:STUDY_SUMMARY branched chain amino acids and kynurenine. ST:INSTITUTE University of Colorado Denver ST:LAST_NAME Haines ST:FIRST_NAME Julie ST:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA ST:EMAIL julie.haines@cuanschutz.edu ST:PHONE 3037243339 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - 1 Patient ID:C04 | Timepoint:Baseline RAW_FILE_NAME=BW49-01_r18 SUBJECT_SAMPLE_FACTORS - 4 Patient ID:C05 | Timepoint:Baseline RAW_FILE_NAME=BW49-02_r11 SUBJECT_SAMPLE_FACTORS - 7 Patient ID:C06 | Timepoint:Baseline RAW_FILE_NAME=BW49-03_r5 SUBJECT_SAMPLE_FACTORS - 10 Patient ID:C08 | Timepoint:Baseline RAW_FILE_NAME=BW49-04_r33 SUBJECT_SAMPLE_FACTORS - 13 Patient ID:C09 | Timepoint:Baseline RAW_FILE_NAME=BW49-05_r9 SUBJECT_SAMPLE_FACTORS - 16 Patient ID:C10 | Timepoint:Baseline RAW_FILE_NAME=BW49-06_r22 SUBJECT_SAMPLE_FACTORS - 19 Patient ID:C11 | Timepoint:Baseline RAW_FILE_NAME=BW49-07_r31 SUBJECT_SAMPLE_FACTORS - 22 Patient ID:C13 | Timepoint:Baseline RAW_FILE_NAME=BW49-08_r13 SUBJECT_SAMPLE_FACTORS - 25 Patient ID:C14 | Timepoint:Baseline RAW_FILE_NAME=BW49-09_r35 SUBJECT_SAMPLE_FACTORS - 28 Patient ID:C15 | Timepoint:Baseline RAW_FILE_NAME=BW49-10_r3 SUBJECT_SAMPLE_FACTORS - 31 Patient ID:C16 | Timepoint:Baseline RAW_FILE_NAME=BW49-11_r1 SUBJECT_SAMPLE_FACTORS - 34 Patient ID:C17 | Timepoint:Baseline RAW_FILE_NAME=BW49-12_r23 SUBJECT_SAMPLE_FACTORS - 2 Patient ID:C04 | Timepoint:During RAW_FILE_NAME=BW49-13_r14 SUBJECT_SAMPLE_FACTORS - 5 Patient ID:C05 | Timepoint:During RAW_FILE_NAME=BW49-14_r28 SUBJECT_SAMPLE_FACTORS - 8 Patient ID:C06 | Timepoint:During RAW_FILE_NAME=BW49-15_r21 SUBJECT_SAMPLE_FACTORS - 11 Patient ID:C08 | Timepoint:During RAW_FILE_NAME=BW49-16_r7 SUBJECT_SAMPLE_FACTORS - 14 Patient ID:C09 | Timepoint:During RAW_FILE_NAME=BW49-17_r12 SUBJECT_SAMPLE_FACTORS - 17 Patient ID:C10 | Timepoint:During RAW_FILE_NAME=BW49-18_r30 SUBJECT_SAMPLE_FACTORS - 20 Patient ID:C11 | Timepoint:During RAW_FILE_NAME=BW49-19_r10 SUBJECT_SAMPLE_FACTORS - 23 Patient ID:C13 | Timepoint:During RAW_FILE_NAME=BW49-20_r4 SUBJECT_SAMPLE_FACTORS - 26 Patient ID:C14 | Timepoint:During RAW_FILE_NAME=BW49-21_r32 SUBJECT_SAMPLE_FACTORS - 29 Patient ID:C15 | Timepoint:During RAW_FILE_NAME=BW49-22_r26 SUBJECT_SAMPLE_FACTORS - 32 Patient ID:C16 | Timepoint:During RAW_FILE_NAME=BW49-23_r29 SUBJECT_SAMPLE_FACTORS - 35 Patient ID:C17 | Timepoint:During RAW_FILE_NAME=BW49-24_r15 SUBJECT_SAMPLE_FACTORS - 3 Patient ID:C04 | Timepoint:Post RAW_FILE_NAME=BW49-25_r6 SUBJECT_SAMPLE_FACTORS - 6 Patient ID:C05 | Timepoint:Post RAW_FILE_NAME=BW49-26_r36 SUBJECT_SAMPLE_FACTORS - 9 Patient ID:C06 | Timepoint:Post RAW_FILE_NAME=BW49-27_r20 SUBJECT_SAMPLE_FACTORS - 12 Patient ID:C08 | Timepoint:Post RAW_FILE_NAME=BW49-28_r2 SUBJECT_SAMPLE_FACTORS - 15 Patient ID:C09 | Timepoint:Post RAW_FILE_NAME=BW49-29_r19 SUBJECT_SAMPLE_FACTORS - 18 Patient ID:C10 | Timepoint:Post RAW_FILE_NAME=BW49-30_r8 SUBJECT_SAMPLE_FACTORS - 21 Patient ID:C11 | Timepoint:Post RAW_FILE_NAME=BW49-31_r24 SUBJECT_SAMPLE_FACTORS - 24 Patient ID:C13 | Timepoint:Post RAW_FILE_NAME=BW49-32_r27 SUBJECT_SAMPLE_FACTORS - 27 Patient ID:C14 | Timepoint:Post RAW_FILE_NAME=BW49-33_r25 SUBJECT_SAMPLE_FACTORS - 30 Patient ID:C15 | Timepoint:Post RAW_FILE_NAME=BW49-34_r24 SUBJECT_SAMPLE_FACTORS - 33 Patient ID:C16 | Timepoint:Post RAW_FILE_NAME=BW49-35_r17 #COLLECTION CO:COLLECTION_SUMMARY Patient plasma samples were collected as part of a Phase I radiation CO:COLLECTION_SUMMARY dose-escalation clinical trial (NCT02873598) before, during (6 hours post), and CO:COLLECTION_SUMMARY post (6 weeks) SBRT. Samples were collected and analyzed per COMIRB19–0328. CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY All patients received neoadjuvant chemotherapy prior to SBRT. SBRT doses ranged TR:TREATMENT_SUMMARY from 9Gy × 3 fractions to 11Gy ×3 fractions. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Plasma aliquots were thawed on ice then a 20 uL aliquot treated with 480 uL of SP:SAMPLEPREP_SUMMARY cold 5:3:2 MeOH:acetonitrile:water. Samples were vortexed 30 min at 4 degrees C SP:SAMPLEPREP_SUMMARY then supernatants clarified by centrifugation (10 min, 10,000 g, 4 degrees C) SP:SAMPLEPREP_SUMMARY and transferred to autosampler vials. SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Positive C18 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 2.1 x 150 mm, 1.7 um CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min CH:FLOW_GRADIENT 95-5% B, 3-5 min hold at 5% B. CH:FLOW_RATE 450 uL/min CH:COLUMN_TEMPERATURE 45 CH:SAMPLE_INJECTION 10 uL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, MS:MS_COMMENTS microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, MS:MS_COMMENTS capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas MS:MS_COMMENTS 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were MS:MS_COMMENTS annotated and integrated using Maven in conjunction with the KEGG database. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples 1 4 7 10 13 16 19 22 25 28 31 34 2 5 8 11 14 17 20 23 26 29 32 35 3 6 9 12 15 18 21 24 27 30 33 Factors Patient ID:C04 | Timepoint:Baseline Patient ID:C05 | Timepoint:Baseline Patient ID:C06 | Timepoint:Baseline Patient ID:C08 | Timepoint:Baseline Patient ID:C09 | Timepoint:Baseline Patient ID:C10 | Timepoint:Baseline Patient ID:C11 | Timepoint:Baseline Patient ID:C13 | Timepoint:Baseline Patient ID:C14 | Timepoint:Baseline Patient ID:C15 | Timepoint:Baseline Patient ID:C16 | Timepoint:Baseline Patient ID:C17 | Timepoint:Baseline Patient ID:C04 | Timepoint:During Patient ID:C05 | Timepoint:During Patient ID:C06 | Timepoint:During Patient ID:C08 | Timepoint:During Patient ID:C09 | Timepoint:During Patient ID:C10 | Timepoint:During Patient ID:C11 | Timepoint:During Patient ID:C13 | Timepoint:During Patient ID:C14 | Timepoint:During Patient ID:C15 | Timepoint:During Patient ID:C16 | Timepoint:During Patient ID:C17 | Timepoint:During Patient ID:C04 | Timepoint:Post Patient ID:C05 | Timepoint:Post Patient ID:C06 | Timepoint:Post Patient ID:C08 | Timepoint:Post Patient ID:C09 | Timepoint:Post Patient ID:C10 | Timepoint:Post Patient ID:C11 | Timepoint:Post Patient ID:C13 | Timepoint:Post Patient ID:C14 | Timepoint:Post Patient ID:C15 | Timepoint:Post Patient ID:C16 | Timepoint:Post L-leucine/isoleucine 27215180 28805310 27636740 34797230 28219400 43379600 42851890 36578500 34454600 19722300 19903230 21614110 39810820 34707530 23546040 33257360 25885160 21676690 49355150 53771920 51423520 34932040 18866020 34107500 30690000 46465420 40980990 35473880 49648100 42516120 21356560 57351630 41288030 25407170 70818020 L-valine 60218590 51431160 65579130 57297020 39206380 68792990 45958280 71277590 65534560 35423210 63660700 41626540 63525310 72474180 63148900 59207280 39259580 58136700 63855760 83846550 67293330 55375090 60518910 54950160 58044050 74934730 83326400 77739060 59787490 86874860 54153390 90059580 73366410 53977200 119463100 kynurenine 72157.52 142227.6 189133.3 186570.9 130459.6 198990.8 128218.6 160219.2 102085.6 122405.3 108144.5 313367.5 82897.82 190227.2 125268.2 194202.3 110696.3 113611 258809.7 130823.2 105283.6 170689.9 76949.13 249766.5 116543.8 149513.6 154770.3 237840.7 213216.8 186766.6 177919.4 169804.9 204573.2 128821.3 216468.3 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z medRt L-leucine/isoleucine C00123 132.102 0.6674764 L-valine C00183 118.0865 0.6581454 kynurenine C00328 209.0919 0.6664889 METABOLITES_END #END