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1 Supplementary appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Chandran SS, Somerville RPT, Yang JC, et al. Treatment of metastatic uveal melanoma with adoptive transfer of tumour-infiltrating lymphocytes: a single-centre, two-stage, single-arm, phase 2 study. Lancet Oncol 2017; published online April 7.
2 Supplementary Figure Appendix p.1 NCT Adoptive transfer trial design Non-myeloablative lymphocyte depleting preparative regimen: Cyclophosphamide (60 mg/kg/day intravenous X 2 days) Fludarabine (25 mg/m 2 /day intravenous X 5 days) Intravenous infusion of TIL Intravenous high-dose IL-2 NCT Adoptive transfer trial design. Beginning on day -7, patients received a nonmyeloablative lymphodepleting conditioning chemotherapy regimen consisting of intravenous cyclophosphamide (Cy, 60 mg/kg) daily for 2 days followed by fludarabine (25 mg/m 2 ) daily for 5 days. One day after completion of their preparative regimen, patients received expanded autologous TIL intravenously (day 0), followed by high-dose IL-2 (720,000 IU/kg) every 8 hours to tolerance. 1
3 Supplementary Figure. CONSORT diagram of enrollment Appendix p.2 Eligible and Consented for Metastasectomy (n=27) Underwent Successful Metastasectomy (n=27) Excluded Due to Insufficient TIL expansion (n=1) No TIL identified in tumor after prior Yttrium bead therapy Successful TIL Expansion (n=26; 96%) Ineligible for TIL Therapy (n=2) New clinically significant cardiac metastasis (n=1) Patient refused therapy (n=1) TIL Reactivity Insufficient (n=3) Treated with TIL Therapy (n=21) 2
4 Supplementary Table. Additional Patient Related Characteristics Appendix p.3 Pt No. Age (yrs) Sex Primary Ocular Tumor Time between primary and met disease (yrs) Time between diagnosis of met disease and TIL therapy (yrs) BAP-1 Status AJCC M stage for Uveal Melanoma Maximum individual tumor diameter (cm) LDH (U/L)* Alk Phos (U/L)* 1 55 F Uveal Wild type M1c F Uveal Wild type M1a M Uveal Mutated M1c M Uveal Wild type M1c F Uveal Wild type M1c F Uveal Wild type M1b M Uveal Mutated M1c M Uveal Wild type M1b M Uveal Mutated M1c F Uveal Mutated M1c F Uveal Mutated M1a M Uveal Wild type M1b F Uveal Wild type M1b F Uveal Wild type M1b M Uveal Wild type M1c M Uveal Wild type M1b M Uveal Wild type M1b M Uveal Wild type M1b M Uveal Mutated M1a M Uveal Mutated M1b M Uveal Mutated M1b *Bolded indicate elevated values Normal ranges: Lactate Dehydrogenase (LDH) U/L Alkaline Phosphatase (Alk Phos) U/L 3
5 Supplementary Figure Appendix p.4 A. B. Administered TIL have predominantly an effector memory (T EM ) phenotype. (A) Phenotype of infused CD8+ and (B) CD4+ T cells in the administered TIL products of metastatic uveal melanoma patients (n=21). Infused TIL underwent flow cytometric analysis to determine the expression of indicated differentiation states: Naïve (CD45RO-CD62L+), central memory (T CM :CD45RO+CD62L+), effector memory (T EM :CD45RO+CD62L-), effector memory RA (T EMRA :CD45RA+CD45RO-CD62L-). Shown are the percentage of live CD3+ gated CD4+ or CD8+ cells staining with the respective markers compared to isotype control. Each dot represents an individual TIL infusion product. The bar represents the mean. 4
6 Supplementary Figure Appendix p.5 In vitro flow cytometric assessment of tumor reactive T cell frequency. The frequency of tumor-reactive T cells within the TIL infusion product was determined by the sum of the flow cytometric measurements of the T cell activation markers, OX40 (CD134) on CD4+ T cells and 4-1BB (CD137) on CD8+ T cells following overnight co-culture with cryopreserved autologous tumor digests minus the background frequency seen against autologous APCs (negative control). Shown are representative flow cytometry plots used to calculate the tumor-reactive T cell frequency in the TIL of Patients #3 and #10. 5
7 Supplementary Figure Appendix p IFN- (pg/ml) R 2 = 0.91 P < % tumor reactive T cells (OX40+CD4+CD3+) + (4-1BB+CD8+CD3+) Correlation between flow cytometric and ELISA based assessment of tumor reactivity in administered TIL. The frequency of tumor reactive T cells in the TIL infusion products (n=18) was determined by flow cytometric measurement of tumor induced T cell activation markers. The % tumor reactive T cells was calculated from the sum of the frequency of OX40+CD4+CD3+ cells and 4-1BB+CD8+CD3+ cells in response to autologous tumor stimulation (minus background reactivity against autologous APCs). The supernatant from these respective co-cultures was assessed by ELISA to determine the tumor induced IFN-g cytokine production. Correlation analysis was used to quantify the relationship between the flow cytometric and ELISA parameters and is presented as R 2 values with their significance (P) level. Each dot represents an individual analyzed TIL infusion product. Each data point represents the mean of triplicate technical measurements from ELISA and flow cytometric assays and are representative of two independent tests with similar results. 6
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