The concept of selectively targeting extracellular phosphatidylserine (PS) exposure in the tumor microenviron- ment has been demonstrated extensively in the literature for a myriad of solid tumors. Annexin A5 (AnxA5) is a direct, high-affinity PS-binding molecule. Its localization to cells presenting the PS molecule on the extracellular surface of the plasma membrane has been well documented. In fact, AnxA5 has been successfully used as an imaging agent in vivo, detecting cell death. We have access to an engineered variant of AnxA5 lacking cellular internalization. We intend to test this non-internalizing variant with two conjugates that help convert the immunosuppresive environment of a tumor into an immunostimulated one. Our long-term goal is to produce a therapeutic agent that triggers an immunostimulatory response against a tumor and to anchor that agent to the PS exposed on the surface of tumor cells and the tumor vasculature. The objective here in the pursuit of this goal is to establish proof-of-principle that our modified AnxA5 variants can localie immunostimulatory signals in vivo using standard tumor models and allow us to identify the best candidate for clinical development. Our central hypothesis is that our modified AnxA5 variants will be therapeutically effective in directing the patient's immune system to the neoplasia. The rationale for the proposed research is that a chimeric antibody that indirectly binds PS has been tested in clinical trials and has shown sufficient promise, however, its targeting of an intermediary molecule (apolipoprotein H), whose in vivo interaction with PS is poorly understood, has fallen short of expectations. Our approach is to create a fully human direct PS binding molecule armed with immunostimulatory activity. Funding is requested for the following specific aims: 1) Conjugate the AnxA5 variant to candidate effector molecules and characterize the resulting fusion proteins for potency, degradation, aggregation, product identity, [[toxicity and pharmcokinetics]]. 2) Test the AnxA5 fusion constructs in a syngeneic breast cancer model using an isogenic cultured breast cancer cell line (4T1) introduced ortho- topically into the mammary fat pads of mice. 3) Test the AnxA5 fusion constructs in a genetic breast cancer and lung metastasis model initiated by an MMTV-promoter driven expression of polyoma middle T antigen. In both tumor models, mice will be treated intravenously with candidate AnxA5 molecules when the tumor burden reaches its pre-determined half-maximal value. Pharmacological distribution of the targeting agents is deter- mined at time of injection and at time of sacrifice. Tumor growth and response to treatment will be monitored longitudinally while tumor histology and immune cell composition will be determined upon sacrifice. The propo- sed research is significant because extracellular surface PS is inherent to a tumor and its vasculature, even for inoperable tumors, and its expression cannot be mutated nor effected by acquired drug resistance. The proposed research is innovative because it utilizes a modified high affinity direct PS-binding protein, human Annexin A5, to deliver immunostimulators that help the patient's own immune system attack the cancer.
The research proposal is relevant to the National Cancer Institute because the development of more effective cancer therapeutics that can target a broad array of tumors, particularly ones that are inoperable or recurrent, is an unmet medical need. The proposed research is relevant to the mission of the NCI because targeting an inherent structural component of the plasma membrane, phosphatidyl serine, a phospholipid exposed on the surface of tumors and tumor vasculature, provides us the ability to attack a broad array of tumors and avoid the complications of drug resistance and target mutations. The goal here is to use a direct phosphatidyl serine binding protein, Annexin A5, modified to trigger immunostimulatory activity in the tumor microenvironment and assist the immune system in the destruction of the cancer.
