Background Vedotin antibody-drug conjugates (ADCs), which incorporate the microtubule-disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable peptide linker, elicit antitumor activity through multiple potential mechanisms of action including MMAE-mediated cytotoxicity and immunogenic cell death (ICD). Mounting preclinical and clinical evidence suggests the combination of a vedotin ADC plus anti-PD1 immunotherapy elicits clinically meaningful responses. Using a preclinical mouse model of anti-PD1 resistance, we build on our understanding of MMAE-induced antitumor immunity and provide rationale for the combination of vedotin ADCs with immune checkpoint therapy in tumors with established anti-PD1 resistance.

Methods An immunocompetent, anti-PD1 resistant murine tumor model (CT26.PD1R) was developed by subjecting CT26 tumors to successive rounds of escalating anti-PD1 treatment in vivo. We assessed the underlying drivers of anti-PD1 resistance by subjecting wild type and CT26.PD1R cultured cell lines to RNA-sequencing analysis. The antitumor activity of a tumor-targeted vedotin ADC – both as a monotherapy and in combination with an anti-PD1 agent – was then evaluated. To define the immune system changes associated with vedotin ADC mediated anti-tumor immunity, tumors and draining lymph nodes were subjected to high-parameter spectral flow cytometry analysis.

Results RNA sequencing analysis on CT26.PD1R cells in culture revealed pronounced suppression of gene networks involved in interferon signaling and antigen presentation machinery. Treatment of CT26.PD1R tumor-bearing mice with a vedotin ADC in combination with an anti-PD1 agent elicited robust anti-tumor activity, while neither the vedotin ADC nor anti-PD1 agent drove any appreciable anti-tumor activity alone. Flow cytometry analysis revealed vedotin ADC treatment elicited activated myeloid clusters within the tumor microenvironment and accumulation of activated DCs in the tumor-draining lymph node (dLN), consistent with MMAE-mediated ICD. Notably, this was accompanied by enhanced CD8+ T cell progenitor priming in the dLN and preservation of an effector-like phenotype on CD8+ tumor-infiltrating lymphocytes (TILs). The combination with anti-PD1 amplified the vedotin-induced changes observed in the dLN and on CD8+ TILs and, crucially, resulted in the restoration of anti-tumor immunity.

Conclusions Here, we highlight preclinical data suggesting that a tumor-targeted vedotin ADC can improve the immune system’s ability to reinvigorate antitumor immunity and sensitize tumors to anti-PD1 therapy in a CT26 model of anti-PD1 resistance. These data are consistent with a model in which MMAE-mediated ICD enhances tumor cell immunogenicity to enable synergy with anti-PD1 therapy, even in the context of an anti-PD1 resistant setting. This nonclinical data supports the evaluation of vedotin ADCs in combination with immunotherapies in the anti-PD1 resistant setting.

Ethics Approval All procedures performed on animals were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and Use Committee or through an ethical review process.