8 | See next page for caption. NATuRE MEDICINE | www.nature.com/naturemedicine Articles Nature MediciNeExtended Data Fig. 8 | (A) Schematic outlining approach for matched PBMC and FFPE tumor tissue TCR analyses. (B) Post-IVS ELISpot (mean SFU per 106 PBMCs +/- SD for duplicate wells) from baseline (W0; orange) and on-treatment (W16; blue) timepoints corresponding to available on-treatment tumor biopsy samples (W16) for patients G1 and G3. TCR clonotypes (paired α-β chains) present at baseline and expanded on-treatment (expanded; orange) or absent at baseline and present on-treatment (de novo primed; blue) and the corresponding single-cell mRNA signatures for CD4 or CD8 expression are shown. Patient G1: CD8 expanded (n=1), CD8 de novo (n=10), CD8 preexisting (n=60, CD4 expanded (n=0), CD4 de novo (n=1), CD4 preexisting (n=3). Patient G3: CD8 expanded (n=2, CD8 de novo (n=13), CD8 preexisting (n=5), CD4 expanded (n=2), CD4 de novo (n=9), CD4 preexisting (n=27), undetermined expanded (n=0), undetermined de novo (n=4), undetermined preexisting (n=5). (C) Violin plots showing analyses of CD3, CD4, and CD8 mRNA expression in cells from baseline (orange) and on-treatment (blue) PBMC samples for patients G1 and G3. (D) UMAP plots showing individual cells expressing TCR clonotypes present in on-treatment samples (closed circles, n=12 for G1, n=27 for G3) and expressing 0, 1, 2, 3, or 4 cytokine transcripts (see also Supplementary T able 3). (E) UMAP plots showing individual cells expressing ≤1 (blue) and ≥2 cytokines (red) and TCR clonotypes of interest (n=12 for G1, n=27 for G3) comparing baseline and on-treatment samples (see also Supplementary T able 3). NATuRE MEDICINE | www.nature.com/naturemedicine Articles Nature MediciNeExtended Data T able 1 | Listing of patient baseline characteristics, Disposition, and Efficacy by DL (as of 31 January 2022) GEA: Gastroesophageal adenocarcinoma; NSCLC: Non-Small Cell Lung Cancer; CRC: Colorectal Cancer; 1Dose Level 1=GRT-C901/GRT-R902 30μg+ Nivolumab; Dose Level 2= GRT-C901/GRT-R902 100μg+ Nivolumab; Dose Level 3= GRT-C901/GRT-R902 100μg+ Nivolumab+ SC Ipilimumab; Dose Level 4= GRT-C901/GRT-R902 300μg+ Nivolumab+ SC Ipilimumab 2Weeks from the prime ChAd68 dosing 3Best of response as per RECIST v 1.1; CR=confirmed response; PR=partial response; SD=stable disease (≥ 16 weeks on treatment); PD=progressive disease; NE=no evidence of disease 4Time from the first dose with GRT-C901 (or GRT-R902 if patient does not receive GRT-C901) to the earliest date of PD or death by any cause 5Time to death (died) or last contact/ assessment (alive) from the 1st study treatment 6This is a confirmed complete response defined as disappearance of target liver lesions and no visible disease on imaging; biopsy at original primary tumor site showed microscopic invasive adenocarcinoma at 4 months; response duration of 6 months followed by worsening of primary esophageal lesion with dysphagia *Concurrent chemotherapy with study treatment: Patient G1 received 3 doses of 5-FU over the first 8 weeks. Patient G8 received 5-FU/bevacizumab (had been receiving for 21 months prior to study treatment). Patient G9 received FOLFIRI/ cetuximab (receiving for 11 months prior to study treatment). Patient G10 received FOLFIRI (receiving for 11 months prior to study treatment). Patient G12 received Paclitaxel/ramucirumab started after 12 weeks of study treatment. NATuRE MEDICINE | www.nature.com/naturemedicine γ α ≥ μ μ μ μ μ ˚ ˚ ˚ ˚