Targeting 4-1BB costimulation to the tumor stroma with bispecific aptamer conjugates for tumor immunotherapy

of a dissertation at the University of Miami. Dissertation supervised by Professor Eli Gilboa. No. of pages in text. (82) Cancer is a multifaceted disease causing millions of deaths each year. Recently, the role of the immune system has been shown to play a major role in development, progression, and tumor elimination. With the recent FDA approval of Ipilumimab, an anti-CTLA-4 antibody, and the strong results of phase 3 clinical trials of an anti-PD-1 antibody, more effort is being put in to tumor immunotherapy. Despite the anti-tumor effects of these immunomodulatory antibodies, there still remain dose limiting toxicities associated with non-specific immune activation. Arguably, targeting these immunomodulatory effects specifically to the tumor could reduce the dose limiting toxicities. Additionally, antibodies are very expensive to generate and can have high batch to batch variability. In contrast, aptamers are easy and cheap to manufacture while reducing the possibility of an antitherapeutic immune response. Our lab has recently published a study showing that targeting immune stimulation to tumor cells using an agonistic 4-1BB aptamer annealed to a Prostate Specific Membrane Antigen (PSMA) specific aptamer would efficiently and effectively reduce tumor burden in animals bearing PSMA expressing tumor cells. However, the caveat with this approach was that these tumor cells were engineered to express a mutant form of PSMA containing a cytoplasmic deletion so the receptor could not internalize. The reason this was done was because the PSMA specific aptamer would bind to PSMA receptor and be internalized, thus masking the therapeutic target, the 4-1BB aptamer. Despite showing tumor reduction and little to no therapeutic toxicity, this approach is not clinically feasible since tumor cells cannot be easily engineered in vivo. This thesis work describes a way to overcome this limitation of internalized receptors by targeting the therapeutic cargo, the 4-1BB specific aptamer, to products not expressed on the tumor cell surface, but to products secreted into the tumor microenvironment. Such products, such as VEGF and Osteopontin, are preferentially secreted into the tumor microenvironment and are indicative of highly aggressive, highly metastatic tumors. Thus, targeting 4-1BB costimulation to areas of high VEGF or Osteopontin expression would potentially activate the tumor specific T cells within the tumor before reaching the cognate MHC restricted antigen expressed on the tumor cell. Additionally, targeting costimulation to the tumor microenvironment represents a much broader approach to tumor therapy since many tumors secrete products like VEGF as compared to tumor specific receptors that exhibit a much more restricted expression pattern (HER2 or PSMA). As such, agonistic 4-1BB specific aptamer were annealed to aptamers specific to murine VEGF or Osteopontin in the attempt to induce anti-tumor immunity in murine tumor models. These bispecific aptamers retain binding and functional activity when annealed together. Importantly, these bispecific aptamers were potent at inducing antitumor immunity and shown to be a feasible option when used in increasingly relevant in vivo tumor models. These bispecific aptamers were also shown to be as potent as nontargeted antibodies at reducing tumor volume and were less toxic than antibody treated mice. In addition to tumor stroma targeted costimulation, aptamer selection was done on murine CD27. Much like 4-1BB, CD27 is a costimulatory molecule found on T cells. SELEX was done using CELL-SELEX. High-throughput sequencing was used after nine rounds of selection in order to identify sequences that were enriched, candidates were chosen and tested for function in in vitro costimulation assays. Overall, this work demonstrates a novel approach to cancer immunotherapy.