Extended Data Fig. 1: TIL selection process.

a. Schematic of tumor resection, TIL growth, and TIL screening pipeline. Tumors were surgically removed and dissected into small fragments, which were grown in IL-2 for TIL fragment culture expansion. Additional tumor fragments were sequenced by whole exome and RNA-seq. Based on tumor mutation calling, candidate neoepitopes were generated in vitro (25-amino acid peptides or minigene constructs with mutation-encoded amino acid at the center [13th] position). Candidate neoepitopes are expressed by autologous dendritic cells in pools (peptide pools [PP] or tandem minigenes [TMG]). TIL fragment cultures are then co-cultured with these candidate neoepitope-expressing dendritic cells or PDTO if available and TIL demonstrating specific TCR-mediated activation (IFNγ release or induction of cell surface 4-1BB (CD137) or OX40 (CD134) following co-culture were selected for potential treatment. b. Example of TIL screening for tumor 4493. From tumor 4493, 12/24 TIL cultures expanded to numbers sufficient for testing. Based on the corresponding tumor sequencing, 48 candidate neoepitopes were screened in 3 PPs and 3 TMGs. Reactivity was observed against TMG3 (CD8 + TIL exhibiting 4-1BB) and PPs 1 and 2 (CD4 + TIL showing 4-1BB/OX40 induction). TIL fragments selected for treatment are indicated with arrows. Cultures not selected for treatment that appear reactive (for example F6, with ~15% CD8 reactivity vs. TMG3) were of inappropriate phenotype (for example F6 was <20% CD8 or <3% reactive in total). PDTO was not available for this patient. c. Example of TIL infusion product retrospective testing for tumor 4493. Cryopreserved TIL were separated into CD8+ and CD4+ fractions and co-cultured with autologous dendritic cells expressing multiple concentrations of neoantigenic peptides within their ‘selected’ target TMGs and PPs. The peak activation value (4-1BB for CD8, 4-1BB and/or OX40 for CD4) subtracting out vehicle control (DMSO) was considered the specific reactivity value against a neoantigen. Left, TMG3 reactivity was mediated by CD8 + TIL reactive to mutant DOP1A. Center, PP2 reactivity was mediated by CD4 + TIL reactive to mutant ZFP36L1. Right, PP1 reactivity was mediated by CD4 + TIL reactive to mutant PANK4. d. Reactivity calculations for example infusion product 4493 from C. Peak CD8 reactivity value against mutant DOP1A and CD4 reactivity against mutant ZFP36L1 and PANK4 was used to calculate numbers of reactive CD8 (left), CD4 (center), and all TIL (right). e. Overall clinical schema illustrating timing of cyclophosphamide (Cy), fludarabine (F), TIL, interleukin-2 (IL-2), and pembrolizumab (P) when added. f. Partial response of pancreatic ductal adenocarcinoma liver metastases following treatment with SEL-TIL + P. Magnetic resonance imaging (MRI) of the pre-treatment (left) and post-treatment (right) liver. Post-treatment images were obtained 5 months after 4493 TIL infusion.