Background CD40 is a pivotal target in cancer immunotherapy, central to dendritic cell activation and subsequent T cell priming against tumor antigens. MP0317, a bispecific DARPin (designed ankyrin repeat protein), selectively activates CD40 within the tumor microenvironment (TME) by engaging fibroblast activation protein (FAP) on cancer-associated fibroblasts, thereby aiming to mitigate toxicity seen with systemic CD40 agonists. The present biomarker analyses aimed to decipher the mechanism of TME-localized CD40 pathway activation in patients treated with MP0317.

Methods Biomarker analyses were performed on data from a completed Phase 1 dose-escalation trial (NCT05098405) of MP0317 monotherapy (0.03–10 mg/kg) in 46 adults with advanced solid tumors, selected on literature-based predicted FAP expression. Paired pre- and post-treatment tumor biopsies underwent RNA sequencing and multiplex immunofluorescence (mIF) analysis, while peripheral blood biomarkers were analyzed using immunoassays and flow cytometry. Correlation analyses were conducted between peripheral and intra-tumor clinical biomarkers.

Results In tumor biopsies, MP0317 colocalization with FAP and CD40 was confirmed by mIF. The presence of MP0317, particularly at doses ≥1.5 mg/kg, was associated with a significant increase in dendritic cell abundance in the TME. Transcriptomic analysis using bulk RNA sequencing data revealed that MP0317 treatment induces substantial remodeling of the TME, exemplified by an increase in antigen-presenting cells (dendritic and B cells), plasma cells, and T follicular helper (Tfh) cells, alongside activated IFNγ signaling. In keeping with these findings, gene set enrichment scores for mature dendritic cells increased post-treatment. Elevated serum levels of chemoattractant markers CXCL10 and CXCL9 post-MP0317 treatment were consistently observed, with a statistically significant correlation for CXCL10 between serum levels and tumor mRNA expression. Blood immunophenotyping showed that the transient B cell reduction observed in the periphery one-week post-dosing was accompanied by significant upregulation of CD69 and CD54 surface markers. These activation markers were sustained during MP0317 treatment, indicative of B cell activation after tissue migration. In blood, T, NK, and monocyte cell numbers remained within normal ranges, and no rise in proinflammatory cytokines was detected, supporting previously reported favorable MP0317 safety data.

Conclusions The biomarker analyses of this Phase 1 study of MP0317 revealed localized CD40 activation within the TME post treatment with doses ≥1.5 mg/kg and demonstrated MP0317`s pharmacodynamic effects, characterized by an increase in antigen-presenting cells, IFNγ signature, plasma and Tfh cells within the TME, alongside correlations between intra-tumoral and peripheral clinical biomarkers. These findings support further clinical investigation of MP0317, in combination with complementary anticancer therapies.

Trial Registration NCT05098405.