Neoantigen-specific tumor-infiltrating lymphocytes in gastrointestinal cancers: a phase 2 trial

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 .

Extended Data Figure 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 [13^th] 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 (e.g. F6, with ~15% CD8 reactivity vs. TMG3) were of inappropriate phenotype (e.g. 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 Figure 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 reports^, 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 Figure 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 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 (gold) or non-responders (blue). 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 (orange for RECIST response, blue 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 Figure 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 orange and non-responder-enriched pathways in blue. 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 orange indicating RECIST response and blue indicating non-response to TIL.

Figure 1.. CONSORT diagram.

Diagram shows patients with metastatic epithelial cancer who underwent surgical resection of metastatic tumors for the purpose of treatment with TIL during the period reported. Laboratory and treatment details provided for patients with metastatic gastrointestinal cancer (including esophageal, gastric, pancreatic, cholangiocarcinoma, and colorectal cancer).

Figure 2.. Clinical activity of treatment.

A. Percent changes in RECIST 1.0-defined target lesion measurements following SEL-TIL administration. B. Percent changes in RECIST 1.0-defined target lesion measurements following selected TIL + pembrolizumab (SEL-TIL + P) administration. In A. and B, baseline imaging was performed a median of 9 days prior to cell infusion (IQR 8–11d), and lesions were first assessed at 4–6 weeks post-TIL administration, then at 8–12 weeks and every 2–3 months thereafter until progression of disease. NED: No evidence of disease. C. Swimmer’s plot showing overall survival following treatment with SEL-TIL or SEL-TIL + P. Surviving patients are indicated by black arrows. Time of partial response (PR) is denoted by diamonds, with open diamonds the unofficial imaging-based PR start and solid diamonds the confirmed PR time point. “×” refers to time of disease progression, and “+” refers to timing of a post-TIL surgical intervention. Data cutoff was May 2024. The four patients with >48 month survival, including two previously reported cases, extend to 126¹⁴, 106¹⁵, 86, and 76 months, respectively.

Figure 3.. Evidence of tumor regression at different metastatic sites.

A. Baseline (left) and post-treatment (right) cross-sectional imaging of a patient with cholangiocarcinoma in the SEL-TIL + P arm. Arrows indicate tumors in bone (top) and paraaortic lymph nodes (bottom). Resolution in both sites at 9 months. The site of the bone metastasis displayed evidence of healing. Additional sites of disease included liver, lung, and adrenal gland (not shown). B. Baseline (left) and post-treatment cross-sectional imaging of a patient with rectal cancer in the SEL-TIL + P arm. Circles highlight tumors in liver. Reduction in both sites at 10 months (right). The patient had additional disease present in lungs and lymph nodes (not shown) and progressed with a new site of disease at 11 months. C. Baseline (left) and post-treatment (right) cross-sectional imaging of a patient with colon cancer in the SEL-TIL + P arm. Arrows indicate tumors in right (top) and left (bottom) lung. As shown, two nodules completely resolved and others were smaller at 7 months. Other sites of disease included liver and a colonic recurrence. Eventual progression of disease at 10 months.

Figure 4.. Characteristics of treatment of SEL-TIL + P arm.

Data (n=34) are divided by clinical response (PR: partial response; NR: no response). Shapes denote diagnosis (diamond: Cohort A/Upper GI, square: Cohort B/HPB, circle: Cohort C/Lower GI). Median values (A-C) are indicated by red bars. Comparisons calculated by Mann-Whitney 2-sided test (A-D); NS denotes p-values > 0.05. A. Numbers of reactive cells infused among all TIL (left), CD4 TIL (center, * p=0.041), and CD8 TIL (right). B. PD-1 expression was assessed on cryopreserved TIL samples. Cells were thawed and rested overnight without cytokines prior to staining with anti-PD-1 clone EH12.2H7 (BioLegend). C. Numbers of CD39-CD69-cells in CD3+, CD4+, and CD8+ TIL (left to right). D. Total selected targets recognized by infusion products in SEL-TIL + P cohort based on experimental testing (* p=0.013), with box plots showing 25^th-75^th percentile, whiskers denoting range, and black bars indicating median. E. Increasing likelihood of partial response (%) with an increasing number of targets (p=0.021, chi-square test for trend).