Abstract B003: Enhancing anti-tumoral reactivity with iHPK1 in liver metastasis to revitalize TIL product for therapy

Abstract Background: Tumor-infiltrating lymphocytes (TILs) have demonstrated efficacy in certain cancers. TILs with anti-tumoral activity can be identified even in tumors traditionally resistant to immunotherapy, such as colon cancer or uveal melanoma. However, manufacturing often leads to loss in tumor reactivity. Enhancing T-cell function through pathway-targeting drugs may improve TIL survival and reactivity. We screened candidate drugs using peripheral blood mononuclear cells (PBMCs) from healthy donors to identify effective compounds, then compared the selected drug to standard TIL expansion for impact on TIL characteristics and anti-tumoral reactivity. Methods: 10 Drugs were used for screening. PBMCs sensitized to viral antigens were exposed to increasing drug concentrations. TNFα and IFNγ were measured by flow cytometry (FC) after re-stimulation, selecting the drug with highest reactivity for tumor testing. Liver metastases (LM) from microsatellite stable colorectal cancer and uveal melanoma were cultured and treated with IL-2 alone (Standard, S) or IL-2 + the selected drug (Modified, M; single dose, day 1). After 3 weeks, TILs from S and M were analyzed for phenotypic markers (CCR7, CD45RA) and immune checkpoints (ICPs) (PD1, TIGIT, LAG3, CTLA4, TIM3). TILs were then challenged with autologous tumor tissue, and reactivity (TNFα, IFNγ) was measured by FC. scRNA-seq was used to validate the findings. TILs, when feasible, underwent REP with reactivity reassessed. Results: Of the 10 drugs, iHPK1 increased antigen-specific reactivity dose-dependently (1.45-fold at 500 nM, p < 0.01) and was selected. Up to 183 cultures from 19 LM were initiated. No significant differences were found between S and M cultures in TIL count (54M vs 40M, p=0.5), viability (92% vs 91%, p=0.6), T-cell subsets (CD3⁺: 91% vs 92%, p=0.6; CD4⁺: 75% vs.74%, p=0.8; CD8⁺: 16% vs 21%, p=0.6), or proliferation (33% vs 32%, p=0.7). Upon autologous tumor stimulation, anti-tumoral reactivity was observed in 9.6% of S vs 34% of M cultures (p = 0.01). Patient-level reactivity rates were 15% in S vs 34% in M (p = 0.03). Median %CD4⁺TNFα⁺ was significantly higher in M vs S (0.25 [0.02–3.98] vs 0.04 [0–1.28], p < 0.05). %CD8⁺IFNγ⁺ showed a non-significant increase (0.18 vs 0.02, p = 0.06). ICPs and phenotype markers were unchanged. Anti-tumor TILs found in M (but not S) were sorted from one patient, and B-TCR chains sequenced. Reactive clones were traced to the initial LM via scRNA-seq and showed high exhaustion marker expression (TOX, ENTPD1, TNFRSF9, CXCL13), confirming iHPK1 preserves TIL functionality otherwise lost during expansion. Six paired S and M TILs underwent REP. Anti-tumoral reactivity was retained in 16% vs 50% of S and M cultures. No post-REP differences in ICPs or phenotype were noted. Conclusion: Exhaustion-driven TIL loss during expansion can be mitigated by functional enhancement. Adding iHPK1 at expansion start improved TIL reactivity in LM and may sustain it post-REP, supporting iHPK1 as a modulator to optimize TIL therapy for immunotherapy resistant tumors. Citation Format: Luis P. Del Carpio Huerta, Ainhoa Muñoz, Marcel Costa, Carles Codony, Lidia Franco, Juan Marin, Laura Llado, Emilio Ramos, David G. Garcia, Ferran Losa, Ramon Salazar, Josep M. Piulats. Enhancing anti-tumoral reactivity with iHPK1 in liver metastasis to revitalize TIL product for therapy [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B003.