Abstract
Adoptive transfer of unselected autologous tumor-infiltrating lymphocytes (TILs) has mediated meaningful clinical responses in patients with metastatic melanoma but not in cancers of gastrointestinal epithelial origin. In an evolving single-arm phase 2 trial design, TILs were derived from and administered to 91 patients with treatment-refractory mismatch repair proficient metastatic gastrointestinal cancers in a schema with lymphodepleting chemotherapy and high-dose interleukin-2 (three cohorts of an ongoing trial). The primary endpoint of this study was the objective response rate as measured using Response Evaluation Criteria in Solid Tumors 1.0; safety was a descriptive secondary endpoint. In the pilot phase, no clinical responses were observed in 18 patients to bulk, unselected TILs; however, when TILs were screened and selected for neoantigen recognition (SEL-TIL), three responses were seen in 39 patients (7.7% (95% confidence interval (CI): 2.7–20.3)). Based on the high levels of programmed cell death protein 1 in the infused TILs, pembrolizumab was added to the regimen (SEL-TIL + P), and eight objective responses were seen in 34 patients (23.5% (95% CI: 12.4–40.0)). All patients experienced transient severe hematologic toxicities from chemotherapy. Seven (10%) patients required critical care support. Exploratory analyses for laboratory and clinical correlates of response were performed for the SEL-TIL and SEL-TIL + P treatment arms. Response was associated with recognition of an increased number of targeted neoantigens and an increased number of administered CD4+ neoantigen-reactive TILs. The current strategy (SEL-TIL + P) exceeded the parameters of the trial design for patients with colorectal cancer, and an expansion phase is accruing. These results could potentially provide a cell-based treatment in a population not traditionally expected to respond to immunotherapy. ClinicalTrials.gov identifier: NCT01174121.
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Data availability
Next-generation sequencing data for all samples in this study will be deposited in raw fastq format to the database of Genotypes and Phenotypes under study accession number phs001003. Please contact the corresponding authors for additional data. Upon reasonable request, the corresponding authors will respond within 2 weeks.
Change history
15 April 2025
A Correction to this paper has been published: https://doi.org/10.1038/s41591-025-03708-5
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Acknowledgements
This study would not have been possible without the collective efforts of Surgery Branch staff and Clinical Center nursing support, including X. Zhao, F. Cobarde, N. Torres, K. Ezhakunnel, D. Komjathy, E. Abecassis, N. Mesa-Diaz, Z. Zheng, A. Berman, K. Borkowski, M. Dawson, R. Salau, M. Rilko, D. Warga, S. Ramirez, S. Chen, B. Zhu, J. Fisher, M. Chaikin, A.-R. Torres, N. Sellers, T Benzine, S. Chatmon, D. White and additional Surgery Branch alumni. This work was primarily supported by funding from the Intramural Research Program, National Institutes of Health, National Cancer Institute, Center for Cancer Research with additional support through a collaborative research and development agreement with Iovance Biotherapeutics.
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S.L.G., M.R.P., P.F.R., J.C.Y. and S.A.R. conceived of and designed the original and revised clinical study. F.J.L., B.G., M.R.P., T.E.S., M.M.L., S.R., J.J.G., T.D.P., P.F.R., J.C.Y. and S.A.R. developed the methodology. F.J.L., S.L.G., B.G., M.R.P., N.M.R., H.K.H., T.E.S., M.M.L., A.B., A.J.D., V.D., I.S.G., A.M.G., A.A.H., K.J.H., L.M.K., L.L., J.G.R.-W., A.B., S.R., C.D.S., C.D.H., J.M.H., J.J.G., S.S., T.D.P., L.S.M., S.K., P.F.R., N.D.K., M.L.M.K., J.C.Y. and S.A.R. acquired the data (accrued and managed patients, tumor resections, manufactured products, etc.). F.J.L., S.L.G., B.G., M.R.P., N.M.R., J.J.G., S.S., P.F.R., J.C.Y. and S.A.R. analyzed and interpreted the data (statistical analysis, biostatistics and computational analysis). F.J.L., S.L.G., B.G., M.R.P., I.S.G., P.F.R, J.C.Y. and S.A.R. wrote, reviewed and revised the paper. S.L.G., A.J.D., A.M.G., A.A.H., K.J.H., L.M.K., C.D.S., L.S.M., N.D.K., M.M.K., J.C.Y. and S.A.R. were responsible for the clinical care of patients.
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Extended data
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.
Extended Data Fig. 2 Regression of target tumors in patients receiving selected TIL.
a. Waterfall plot of maximal change from baseline of target tumors per RECIST 1.0 post-TIL infusion for SEL (left, n = 39) and SEL + P (right, n = 34) arms. Bars are colored according to primary tumor histology (Lower GI in blue, upper GI in red, HPB in green). Bars labeled with numbers indicate duration of confirmed partial responses, asterisks indicate clinical non-responders with >30% reduction, hexagons indicate the patients with further imaging in panels b-d, and the caret indicates a non-evaluable patient whose disease progressed prior to first follow-up visit. Underlined values represent previously published case reports14,15 b. Regression of diffuse hepatic metastases in a patient with pancreatic ductal adenocarcinoma. Stable hemangioma noted (Hemang). Baseline (left) and six-week follow-up (right) shown. c. Regression of multiple pulmonary nodules and resolution of pleural effusion in a patient with pancreatic ductal adenocarcinoma. Baseline (left) and six-week follow-up (right) shown. d. Regression of pulmonary tumors in a patient with colon cancer. Baseline (left) and 10-month evaluation (right) shown. Patient is a non-responder for development of a new brain metastasis at 6 months (not shown).
Extended Data Fig. 3 Transcriptomic analysis of TIL harvest tumors from SEL-TIL + P arm.
a. Volcano plot of DEGs between TIL harvest lesions of responders (n = 8) and non-responders (n = 25). Dotted lines indicate adjusted p-values < 0.05 (Benjamini-Hochberg corrected to adjust for multiple comparisons) and absolute log2FC > 2. Highlighted genes include selected immune-related genes and those from IPA-indicated pathways (Extended Data Fig. 4a). b. Normalized enrichment scores (NES) of significantly enriched hallmark gene sets (nominal p-value < 0.05, GSEA) in TIL harvest lesions from responders (black) or non-responders (white). Gene sets with false discovery rates < 0.3 are included to correct for multiple comparisons. c. Clustering of bulk tumor RNA-seq data by patient (SEL-TIL + P) according to top and bottom 100 response-associated DEGs. Z-scaled gene expression is indicated red to blue, and clinical response to TIL is shown below cluster plots (black for RECIST response, white for non-response). Responder-enriched clusters 1 and 2 show enhanced expression of response-associated genes, while non-responder-enriched clusters 3 and 4 show heightened expression of non-response-associated genes.
Extended Data Fig. 4 Ingenuity Pathway Analysis (IPA) of response-associated DEGs (SEL-TIL + P) and clustering of SEL-TIL tumor RNA.
a. Top 10 response-associated and non-response associated pathways of DEGs within TIL harvest lesions of SEL-TIL + P arm according to IPA. Pathways with highest significant z-scores are shown and ranked by -log10(p-value), with responder-enriched pathways in black and non-responder-enriched pathways in white. IPA-generated p-values are Benjamini-Hochberg corrected to adjust for multiple comparisons b. TIL harvest tumor RNA samples from SEL-TIL group (n = 36 samples) were clustered according to top and bottom 100 response-associated DEGs of SEL-TIL + P group. Patient response to TIL is shown below, with black indicating RECIST response and white indicating non-response to TIL.
Supplementary information
Supplementary Table 3
Response-associated tumor DEGs (SEL-TIL + P).
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Lowery, F.J., Goff, S.L., Gasmi, B. et al. Neoantigen-specific tumor-infiltrating lymphocytes in gastrointestinal cancers: a phase 2 trial. Nat Med 31, 1994–2003 (2025). https://doi.org/10.1038/s41591-025-03627-5
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DOI: https://doi.org/10.1038/s41591-025-03627-5
