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Preprint WatchStrongJuly 27th, 2026

Dual stimulation of CD40 and 41BB pathways during ex-vivo TIL expansion enhances CD8+ T cell expansion

Marques Rossetti, R. A.; S. Beatty, M.; Cianne, J.; R. Ali, J.; Harris, K.; Ramadan, A.; Grant, M.; Martinez Planes, E.; Aurelio, J.; Karapetyan, L.; Creelan, B.; Pilon-Thomas, S.; Hwu, P.; Luca, V. C.; Abate-Daga, D.

Simultaneous agonism of the CD40-CD40L axis on tumor-infiltrating B cells and the 4-1BB/TNFRSF9-41BBL axis on T cells, delivered by a single bispecific CD40L EPC6-41BBL trimeric fusion, augments ex vivo CD8 positive T cell expansion, and the authors position this as justification for a phase 1 trial.

Strong contradiction

3 prior failures

Three or more documented clinical failures match this mechanism, or a Phase 3 efficacy failure is on record.

This preprint proposes taking dual CD40 and 4-1BB agonism into a phase 1 trial, and the Claidex record already holds three terminated programs across exactly those two axes. GEN1042, a CD40 by 4-1BB bispecific antibody in metastatic non-CNS solid tumors, was terminated for efficacy failure (gen1042-cd40-4-1bb-immunoradiotherapy-solid-tumors-phase1-2-efficacy-termination). Acasunlimab, a PD-L1 by 4-1BB bispecific in cutaneous melanoma, was terminated on sponsor strategy (acasunlimab-tnfrsf9-pdl1-4-1bb-bispecific-melanoma-phase2-strategic-termination), and PRS-344/S095012, a further 4-1BB by PD-L1 bispecific in solid tumors, ended the same way (prs-344-s095012-tnfrsf9-4-1bb-pdl1-solid-tumors-phase1-2-sponsor-termination). The distinction that matters is compartment. The recorded failures all delivered agonism systemically to patients, where 4-1BB agonism has a long history of hepatotoxicity and narrow therapeutic windows, whereas this preprint applies the same agonism ex vivo during TIL manufacture, which removes the systemic exposure that drove those programs down. A phase 1 proposal built on this work should state explicitly that the ex vivo compartment is the differentiator, because the in-patient version of this exact combination has already failed on efficacy once.

Abstract excerpt

Background Tumor-infiltrating lymphocyte (TIL) therapy has demonstrated clinical efficacy in malignant melanoma; however, inefficient ex vivo expansion remains a major limitation. We previously showed that stimulation of tumor-infiltrating B cells via CD40-CD40L axis improves TIL expansion, and that direct activation of the 41BB-41BBL pathway on T cells enhances CD8+ T cell outgrowth. We hypothesized that adding simultaneous targeting of both pathways would augment the growth and activity of CD8+ cytotoxic T cells. We conducted a study with the objective of determining the feasibility of dual stimulation with human tumors as justification for a Phase I trial. Methods CD40L variants were generated by yeast display selection and evaluated for B cell binding and activation. The effects of CD40L variants on TIL expansion were evaluated using tumors derived from standard of care resections using fragment method. Based on these findings, a bi-specific molecule was designed and generated fusing a CD40L variant and 41BB to the N- and C-termini of a trimeric leucine zipper. The effects of the bi-specific molecule (termed CD40L EPC6-41BBL) on TIL expansion were evaluated in TIL cultures derived from lung tumor and melanoma fragments. TIL phenotypes were assessed by flow cytometry, including high-dimensional FlowSOM analysis, and tumor reactivity by autologous tumor co-culture assays. Results Each of our engineered CD40L variants bound B cells and induced CD80/CD86 expression at levels comparable to wild-type CD40L. Supplementation of TIL cultures with CD40L variants increased the success rate of TIL expansion compared to control. We then developed a bi-specific CD40L EPC6-41BBL molecule capable of binding to both B and T cells. Addition of CD40L EPC6-41BBL significantly increased total TIL yield and improved expansion success rates in both lung tumor and melanoma cultures. In particular, CD40L EPC6-41BBL promoted preferential expansion of CD8+ T cells. High-dimensional analysis revealed enrichment of CD8+ T cell clusters expressing CD39, CD69, TIM3, and CD56 in cultures supplemented with CD40L EPC6-41BBL. Furthermore, treated cultures displayed increased frequencies of CD27+ CD4+ T cells. Functional assessment suggested a trend toward enhanced tumor reactivity in melanoma-derived TIL products expanded with CD40L EPC6-41BBL. Conclusions Simultaneous stimulation of CD40 and 41BB pathways using a novel bi-specific molecule resulted in qualitative and quantitative enhancement of TIL products. These findings support dual targeting of tumor-infiltrating B cells and T cells as a promising strategy to optimize TIL manufacturing for adoptive cell therapy in Phase I trials.

Matching Claidex post-mortems

3 of 3 indexed

This is an automated contradiction flag, not an editorial judgment on the preprint's quality. Flags identify where the preclinical literature and the clinical failure record diverge.