Checkpoint inhibitor (CPI) immunotherapy has achieved remarkable clinical success, yet its efficacy in 'immunologically cold' tumours has been modest. Interleukin (IL)-12 is a powerful cytokine that activates the innate and adaptive arms of the immune system, yet its administration has been associated with immune-related adverse events. Here, we show that the intravenous administration of a collagen-binding domain fused to IL-12 (CBD-IL-12) in mice bearing aggressive murine tumours accumulates in the tumour stroma, owing to exposed collagen in the disordered tumour vasculature. In comparison with the administration of unmodified IL-12, CBD-IL-12 induced sustained intratumoral levels of interferon-, markedly reduced its systemic levels as well as organ damage, and led to superior anticancer efficacy, eliciting complete regression of CPI-unresponsive breast tumours. Furthermore, CBD-IL-12 potently synergized with CPI to eradicate large established melanoma, induced antigen-specific immunological memory, and controlled tumour growth in a genetically engineered mouse model of melanoma. CBD-IL-12 may potentiate CPI immunotherapy for immunologically cold tumours.

Immune checkpoint blockade exhibits considerable antitumor activity, but previous studies have reported instances of severe treatment-related adverse events. We sought to explore local immune checkpoint blockade, with an antibody (Ab) form that would be retained intra- or peritumorally, limiting systemic exposure. To accomplish this, we conjugated the checkpoint blockade Abs to an extracellular matrix (ECM)-super-affinity peptide derived from placenta growth factor-2 (PlGF-2). We show enhanced tissue retention and lower Ab concentrations in blood plasma after PlGF-2 conjugation, reducing systemic side effects such as the risk of autoimmune diabetes. Peritumoral injections of PlGF-2-anti-CTLA4 (cytotoxic T lymphocyte antigen 4) and PlGF-2-anti-PD-L1 (programmed death ligand 1) Abs delayed tumor growth and prolonged survival compared to the unmodified Abs in genetically engineered murine tumor models of melanoma and breast cancer. The PlGF-2-Abs increased tumor-infiltrating activated CD8 and CD4 T cells, resulting in a delay of distant tumor growth as well. This simple and translatable approach of engineered ECM-binding Abs may present a viable and safer approach in checkpoint blockade.

Cancer immunotherapy with immune checkpoint inhibitors (CPI) and interleukin (IL)-2 has demonstrated clinical efficacy but is frequently accompanied with severe adverse events caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-cytotoxic T-lymphocyte antigen 4 antibody (αCTLA4) + anti-programmed death-ligand 1 antibody (αPD-L1) and the cytokine IL-2 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokine) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain, harnessing the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. We show that intravenously (i.v.) administered CBD protein accumulated mainly in tumors. CBD conjugation or fusion decreases the systemic toxicity of both αCTLA4+αPD-L1 combination therapy and IL-2, for example eliminating hepatotoxicity with the CPI molecules and ameliorating pulmonary edema with IL-2. Both CBD-CPI and CBD-IL-2 suppressed tumor growth compared to their unmodified forms in multiple murine cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8+ T cells. In an orthotopic breast tumor model, combination treatment with CPI and IL-2 eradicated tumors in 9 of 13 animals with the CBD-modified drugs, whereas it did so in only 1 of 13 animals with the unmodified drugs. Thus, the A3 domain of VWF can be used to improve safety and efficacy of systemically-administered tumor drugs with high translational promise.

Although a clinical breakthrough for cancer treatment, it remains that a minority of patients respond to checkpoint inhibitor (CPI) immunotherapy. The composition of tumor-infiltrating immune cells has been identified as a key factor influencing CPI therapy success. Thus, enhancing tumor immune cell infiltration is a critical challenge. A lack of the chemokine CCL4 within the tumor microenvironment leads to the absence of CD103+ dendritic cells (DCs), a crucial cell population influencing CPI responsiveness. Here, we use a tumor stroma–targeting approach to deliver CCL4; by generating a fusion protein of CCL4 and the collagen-binding domain (CBD) of von Willebrand factor, we show that CBD fusion enhances CCL4 tumor localization. Intravenous CBD-CCL4 administration recruits CD103+ DCs and CD8+ T cells and improves the antitumor effect of CPI immunotherapy in multiple tumor models, including poor responders to CPI. Thus, CBD-CCL4 holds clinical translational potential by enhancing efficacy of CPI immunotherapy.

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