An Fc-engineered CD40 agonist, injected directly into tumors, was safe in a first-in-human trial and pushed two metastatic cancers into complete remission — including a melanoma patient still disease-free at 28 months. The drug worked by building tertiary lymphoid structures, organized immune hubs, inside tumors. Explicyte's multiplex immunofluorescence made those structures visible in patient biopsies, confirming that mature TLS appeared in responders' injected and even non-injected lesions, but were absent in a non-responder.
The first-in-human phase 1 trial (NCT04059588) was led by Juan Osorio, David Knorr, and Jeffrey Ravetch’s group across Memorial Sloan Kettering Cancer Center and Rockefeller University, building on more than a decade of antibody-engineering work on CD40 agonism. Explicyte Immuno-oncology (Bordeaux) ran the multiplex immunofluorescence on patient FFPE biopsies — the readout that revealed tertiary lymphoid structures in responders — on an automated Ventana Discovery XT workflow with Opal/TSA multiplexing and a TLS-focused panel (CD8, CD4, CD20, CD21, CD23). It’s exactly the kind of spatial, multiplex IHF readout Explicyte’s translational platform is built to deliver, from TLS induction and maturation through dendritic-cell activation and chemokine programs.
2141-V11 is a human IgG1 anti-CD40 agonist carrying five point mutations that increase binding to the inhibitory receptor FcγRIIB, boosting CD40 cross-linking and agonistic signaling while limiting systemic toxicity. In this phase 1 trial the team delivered it intratumorally across four dose levels (0.7–10 mg) to 12 patients with metastatic cancer rather than systemically.
The drug was well tolerated with no dose-limiting toxicities; adverse events were mostly low-grade (fever, chills, injection-site reactions). Six of 12 patients showed tumor reduction, including two complete responses — an 89-year-old melanoma patient (remission lasting ~28 months) and a 67-year-old breast cancer patient whose liver and nodal metastases cleared and whose tumor markers normalized. Regression appeared in both injected and distant non-injected lesions, an abscopal effect.
Explicyte’s multiplex immunofluorescence on FFPE biopsies (Ventana Discovery XT, Opal/TSA, PhenoImager HT, inForm analysis) mapped mature tertiary lymphoid structures in both complete responders — organized aggregates of CD11c+ dendritic cells, CD4+/CD8+ T cells, B cells, and CD21+ follicular dendritic cells. Crucially, TLS also formed in the melanoma patient’s non-injected lesions, while a non-responder’s tumor lacked them entirely — turning an abstract mechanism into a measurable tissue readout that tracked with response.
In CD40/FcγR humanized mice bearing bilateral E0771 breast tumors, intratumoral 2141-V11 induced de novo TLS in most injected tumors (5/6) but none in non-injected tumors or controls (0/6), drove rejection of both injected and distant tumors, and produced durable immune memory — with single-cell RNA-seq showing expansion of mature CCR7+ dendritic cells in TLS-bearing tumors.
The trial reframes TLS from a favorable bystander biomarker into something a therapy can deliberately build — and shows a local injection can generate body-wide, durable immunity.
For drug developers, the work validates Fc engineering as a route to CD40 agonists that are both safer and more active, and it positions multiplex IHF as a practical, tissue-level readout for TLS-directed therapies. The findings support advancing 2141-V11 into phase 2 and offer a template for measuring TLS induction, maturation, and spatial immune remodeling in future immunotherapy programs.
