Following are public and technical abstracts for the Anti-PD-1 Antibody project funded by the Department of Defense Kidney Cancer Research Program (KCRP) for 2017.
Principal Investigator: Wilson Meng
Institution: Duquesne University
Funding Mechanism: Concept Award
Award Amount: $100,791
Public Abstract
Kidney cancer can be treated by a new class of drugs called immune checkpoint inhibitors, one type of which are called anti-PD1 antibodies. Although many patients with renal cell carcinoma (RCC) have benefited from anti-PD1 antibodies, the majority of the individuals treated with checkpoint inhibitors do not. One reason for the lack of response to anti-PD1 and other inhibitors is the accumulation of adenosine (ADO) in RCC tumors. Released by cells in tumors, ADO impairs the ability of cancer-destroying immune cells to be amplified by anti-PD1 antibodies. Therefore, it is possible that depleting ADO in RCC tumors can increase the sensitivity of the cancer towards anti-PD1 antibodies.
The purpose of this research project is to test whether injecting adenosine deaminase (ADA), the enzyme by which ADO is metabolized, can enhance the therapeutic efficacy of an anti-PD1 antibody in a mouse model of RCC. Direct injection of the two into tumors represents a form of vaccination, generating immunity locally and throughout the body. The key is to keep both ADA and anti-PD1 antibody in the same place at the same time in order to generate synergism.
We have developed an injectable gel called “pG_EAK,” designed to concentrate both anti-PD1 antibody and ADA in tumors. The gel can render concentrations of the agents that cannot be achieved by injecting simple solutions of the two proteins. This technology is referred to as “multiplexing” (mux), and the formulation of the gel containing the two agents is termed mux-alpha-PD1ADA. The goal is to understand the mechanisms and efficacy of mux-alpha-PD1ADA in mice carrying kidney cancer. The hypothesis is that simultaneous and proximal delivery of anti-PD1 and ADA (mux-alpha-PD1ADA) into kidney tumors will induce a body-wide immune response that can reduce the spreading and relapse of the cancer. The hypothesis will be tested in mice inoculated with the classical mouse model of RCC called RENCA. Two tasks will be carried out, the first testing the specific nature of the immune responses generated by mux-alpha-PD1ADA and the second documenting the efficacy of the formulation in inhibiting tumor growth and metastasis, and protection from a second cancer challenge.
The research is novel because the intervention is aimed at neutralizing a metabolite that has not been targeted in kidney cancer. It is distinct from other approaches because it restricts the neutralization inside the tumors in raising the sensitivity of the cancer towards checkpoint inhibitors. The short-term impact is that the experiments will provide evidence for advancing the idea to clinical trials. It may lead to improved formulations of mux-alpha-PD1ADA that are effective in humans. In the long run, the study has the potential to improve the quality of life in patients with refractory RCC by using ADA as an adjuvant.
The research will benefit the American public because new treatments are needed for kidney cancer. More than 14,000 Americans die every year from kidney and renal pelvic cancer. New cases have risen, from 10.4 (per 100,000 persons) in 1992 to 15.3 in 2015. The investigation can benefit current service members of the armed forces who are at high risk of developing kidney cancer. The localized approach provides a treatment alternative for elderly veterans because it is expected to have fewer side effects. Veterans who have exposed to battlefield solvent and chemical agents linked to kidney cancer could also benefit from the results of the research.
Technical Abstract
Despite the immunogenic nature of kidney cancer, the response rates of metastatic renal cell carcinoma (RCC) to immune checkpoint inhibitors, including anti-PD1 antibodies (alpha-PD1), have been modest. Tumor resistance to alpha-PD1 is due in part to the accumulation of adenosine (ADO) generated in the tumor microenvironment (TME). ADO impairs the activation and proliferation of effector T cells, while upregulating PD1 expression on regulatory T cells (Tregs), which generate extracellular ADO via ectonucleotidases. This is a vicious cycle because ADO also stabilizes and expands Tregs, the frequency of which is inversely related to the overall survival of patients with RCC. Thus, suppressing ADO in the TME is expected to enhance the efficacy of alpha-PD1.
The purpose of this concept proposal is to evaluate the impacts of intratumoral (i.t.) injection of alpha-PD1 and adenosine deaminase (ADA), the enzyme by which ADO is metabolized. The advantage of ADA over ADO receptor antagonists is that the enzyme interferes upstream of the pathway, thereby preventing the accumulation of ADO in engaging its diverse leukocyte targets. Studies have shown that i.t. injections of checkpoint inhibitor antibodies can activate T cell responses systemically, thereby turning primary tumors into vaccination sites. Because ADO acts through paracrine actions, it is critical that alpha-PD1 and ADA are concentrated in the TME at the same time for extended durations. Because of the positive interstitial pressure in solid tumors, simply injecting saline solutions of the agents via i.t. will not result in their colocalization.
We have developed an injectable bioaffinity hydrogel in which IgG and Fc fusion proteins can be retained in tumors. The biomaterials strategy entails intermixing the self-assembling peptide AEAEAKAEAEAEAKAK (single amino acid code; hereafter “EAK”) with a second peptide containing a truncated Streptococci protein G (pG) genetically fused with EAK. The bifunctional peptide, called “pG_EAK”, co-assembles with EAK to form coacervates with multivalent Fc-binding sites. When formulated with anti-PD1 IgG and his-tagged ADA (via anti-His IgG), the gel formed by pG_EAK and EAK will enable sustained local concentrations of the agents that cannot be achieved by injecting i.t. or i.v. saline solutions of the two. The “multiplexing” (mux) system, which renders a multivalent (congregating many copies of each in tumors) and multifunctional (targeting PD1 and ADO simultaneously) immune depot, is referred to hereafter as mux-alpha-PD1ADA.
The objective of this project is to delineate the immune mechanisms and therapeutic efficacy of mux-alpha-PD1ADA in a mouse RCC tumor model. The central hypothesis is that simultaneous and proximal delivery of anti-PD1 and ADA (mux-alpha-PD1ADA) into kidney tumors will induce systemic antigen-specific cytotoxic T cell responses and reduce metastasis in an immunocompetent mouse model of RCC. The scientific premise is that sustained suppression of ADO concentrations in the TME will reverse RCC resistance to alpha-PD1. The hypothesis will be tested in mice inoculated with the syngeneic RENCA renal adenocarcinoma cells. In Specific Aim 1, we will determine the impacts of mux-alpha-PD1ADA on antitumor T cell responses. The sub-hypothesis is that mux-alpha-PD1ADA injected into the RCC tumors will amplify antitumor cytotoxic T cell responses superior to those of i.t. or intravenous injection of alpha-PD1 alone in saline. A statistics-based Design of Experiments will be used to identify superior combinations of alpha-PD1 and ADA. In Specific Aim 2, the efficacy of mux-alpha-PD1ADA in inhibiting tumor growth and metastasis will be evaluated. The sub-hypothesis is that mux-alpha-PD1ADA injected into RCC tumors will confer protection from metastasis and a second tumor challenge.
The innovativeness of the research centers on confining ADO removal in the TME, with the catalytic action tied to the local extracellular concentrations of the purine nucleoside. The strategy is enabled by leveraging a novel biomaterials tool in coinciding spatiotemporally ADO degradation with PD1 blockade. The enabling hydrogel is an injectable “multiplexing” platform on which copies of ADA and alpha-PD1 can be concentrated in tumors within which anti-RCC T cells are being shaped by presentations of tumor-associated antigens by infiltrated monocyte-derived cells.
The short-term impact is that the experiments will generate data to reveal the extent to which accelerating ADO catabolism can enhance alpha-PD1 therapies in mouse RCC in vivo. Specifically, from the experiments correlations between ADO concentrations in tumors and anti-RCC T cell responses generated by a given dose of alpha-PD1 will be determined. In the long term, the study has the potential to improve the quality of life in patients with refractory RCC by using ADA as an adjuvant. The localized approach is conducive for clinical development because it reduces off-target toxicities.