Abstract B112: Targeting immunotherapy to the tumor microenvironment using anti-PDL1 VHH
Authors: Dougan Michael , Ingram Jessica , Ploegh Hidde , Dougan Stephanie K. 
Published: November 2016

Abstract
Cytokines and other immune modulating agents act locally, and delivery of particular immune based therapies to the right cell population in the tumor has been challenging. Many agents have systemic toxicities. Many others fail to arrive in the tumor, and delivering multiple agents to tumor infiltrating lymphocytes is likewise problematic. Pancreatic cancer is the best example of a stroma-rich tumor, where drug delivery often fails. Immunotherapy has shown impressive clinical benefit for many malignancies, particularly for metastatic melanoma; however, pancreatic cancer is among the few cancer types in which anti-CTLA-4 and PD-1 blockade have largely failed. PDAC is fairly average in terms of mutational burden, PDL1 expression, and T cell infiltration, thus the failure to respond to immune checkpoint blockade cannot be explained by these factors. PDAC is uniquely distinguished by a dense stromal reaction, resulting in a nutrient-poor immunosuppressive environment. We hypothesize that targeted delivery of a combination of immunomodulatory agents will change the pancreatic tumor microenvironment to relieve immunosuppression will allow for more effective anti-tumor immune responses.
Alpacas and other camelid species generate single domain antibodies, the variable portions of which can be expressed recombinantly and retain the binding properties of full-size antibodies at 1/10th the size. We have generated high-affinity VHHs specific for mouse and human PD-L1. We show by immuno-positron emission tomography (PET) that small VHHs penetrate deeply into orthotopic pancreatic tumors, and can effectively deliver payloads to the tumor interior. Pancreatic tumor cells, whether growing in culture or growing in an orthotopic tumor, express high levels of PD-L1 at baseline. PD-L1 is largely absent from normal tissues; thus we can use PDL1-VHH as a tumor-specific targeting agent. PDL1-VHH was conjugated to the cytokines IL-2 or IFNγ, and we looked for an effect of these reagents in both melanoma and an orthotopic model of pancreatic cancer. Both reagents reduce the growth of B16 tumors in conjunction with an antibody against the melanoma protein TRP1 (TA99). For pancreatic cancer, aPDL1-IL2 treatment showed a 50% reduction in overall tumor burden, an increase in the intratumoral ratio of CD8+ T cells to CD4+Tregs, and increased T cell trafficking to the tumor microenvironment. aPDL1-IFNγ caused a profound reduction in size of both Panc02 and KPC orthotopic tumors, largely by reprogramming intratumoral macrophages.
Here we exploit the near-ubiquitous expression of PD-L1 by tumors to focus immune therapies on the tumor microenvironment, and propose that this may be a general technique for enhancing responses to immune therapy, while greatly reducing the risk of systemic toxicities.
Citation Format: Michael Dougan, Jessica Ingram, Hidde Ploegh, Stephanie K. Dougan. Targeting immunotherapy to the tumor microenvironment using anti-PDL1 VHH [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B112.