A Novel Cell-Mediated Immunotherapy for Treatment of Lung and Breast Cancer Title: A novel cell-mediated immunotherapy for treatment of lung and breast cancer Main author: Indu Venugopal Co-Authors: Claire Gormley; Michael J. McGuire; Kathlynn C. Brown Co-Authors' Institutions:  Biological Sciences Division, SRI International, 140 Research Drive, Harrisonburg, VA 22802 USA Abstract: Cell-mediated (CM) cancer immunotherapies are now generating effective treatments for several cancers which were previously thought to be untreatable. Major examples include Dendritic Cell vaccines, PD-1 and CTLA-4 inhibitors, injection of live or attenuated virus into tumors, and adoptive T-cell therapies. The principle of most CM immunotherapies is simple - to activate the body’s adaptive immune system to generate a cytotoxic T lymphocyte response against tumor antigens, resulting in the elimination of the tumor. However, the complexity of the tumor micro-environment and adverse side effects make it extremely difficult to harness the specificity of the immune system for tumor treatment. Also, concerns regarding cost, efficacy and safety have impeded their development into effective clinical therapies. To address these weaknesses, we have developed a novel immunotherapy capable of delivering previously encountered antigenic peptides specifically to cancer cells and facilitating their presentation through the MHC class I pathway. Our therapy utilizes a synthetic nanoparticle delivery system comprising of three components: a neutral stealth liposome, encapsulated synthetic immunogenic HLA class I restricted peptides derived from measles virus (MV), and a tumor-targeting peptide on the external surface of the liposome. The targeting peptide results in accumulation of the liposomes specifically inside cancer cells, and facilitates presentation of the MV-derived immunogenic peptides specifically in HLA class I molecules. We refer to this system as TALL (Targeted Antigen Loaded Liposomes). Therefore, TALL can generate a secondary immune response specifically against the targeted tumor cells in a patient who has been previously vaccinated against or infected by MV. In short, we are attempting to trick the immune system into responding as though the cancer cell is infected with MV without the use of a viral particle. To prove this concept, we synthesized a liposome encapsulating H250, an immunogenic HLA class I restricted peptide identified from measles hemagglutinin protein. These liposomes were targeted specifically to breast and lung cancer cells via our targeting peptide, which was identified using phage-display methodology. Treatment of lung cancer cells with TALL results in functional presentation of H250 in both MHC and HLA class I molecules. Our in vitro and in vivo studies indicate that presentation of H250 is dependent on the cancer targeting peptide; liposomes that lack the targeting peptide did not accumulate in the cancer cells and presentation of H250 was abrogated. We have shown that treatment with TALL substantially reduces growth of LLC1(lung) and 4T1(triple-negative breast) tumors in vaccinated C57BL/6 and Balb/c mice respectively, compared to vehicle treatment. As TALL can provide a potent synthetic antigen specifically to tumor cells, it can turn immune-cold tumors into immune-hot tumors, which is capable of eliciting a strong immune response. We have therefore conducted a pilot study to determine the efficacy of combining TALL with checkpoint inhibitors to reduce the tumor burden in mice bearing orthotopic 4T1 tumors. Checkpoint inhibitors are known to be far less efficacious in immune-cold tumors such as triple negative breast cancer. The results of the study indicated a significant reduction in the tumor burden of mice treated with the combination therapy compared to when either treatment is used alone. The outcome of our TALL treatment is a robust cytotoxic T lymphocyte response directed specifically against the tumor. This approach has advantages over current immunotherapies: 1) It bypasses the need to identify tumor-associated antigens or educate the immune system through a primary immune response; 2) It is anticipated to be effective against tumors with a low mutational load, making it efficacious on early stage as well as metastatic cancer; 3) It does not use live virus, viral subunits or biologically derived material, allowing for complete synthetic manufacturing. It also does not require isolation, culture, or ex vivo manipulation of patient’s cells, which reduces production time and costs.