Abstract An attractive strategy for cancer treatment is to strengthen the ability of the patient’s immune system to detect and clear cancerous cells. However, antibody-based immune checkpoint inhibitors have limited effectiveness against some types of tumors such as diffuse large B cell lymphoma and follicular lymphoma, while solid tumors such as sarcomas are often more broadly resistant to both immune checkpoint inhibitors and cell-based immunotherapy. Therefore, further immunotherapy options are necessary for these indications. The objective of our research is to develop a drug conjugate that stimulates the immune response to cancer by targeting BTN3A1. This protein is required for activation of gamma delta T cells and functions as an immune checkpoint in alpha beta T cells. Gamma delta T cells exhibit different patterns of tumor infiltration relative to alpha beta T cells targeted by current immunotherapy approaches, and some recent studies have shown that when gamma delta cells can infiltrate tumors they can even have a stronger impact on overall survival relative to tumor infiltrating alpha beta cells. Therefore, gamma delta T cell activation holds great promise for cancer immunotherapy in cases where alpha beta T cells are ineffective. In contrast to T cells that express the alpha beta T cell receptor and respond to peptide antigens, T cells that express the Vgamma9Vdelta2 T cell receptor respond to small phosphorous-containing compounds known as phosphoantigens (pAgs) and are MHC independent, which can be a benefit to immunotherapy. However, no known direct activators of gamma delta T cells are available for human use. The transmembrane pAg receptor BTN3A1 is critical for TCR mediated Vgamma9Vdelta2 T cell activation. Due to MHC independence and non-traditional antigen response, BTN3A1 ligands offer a potential alternative to boost anti-cancer immunity when immune checkpoint therapy fails. Here, we propose to test the central hypothesis that novel synthetic pHLIP-pAg conjugates can engage BTN3A1 in tumors to trigger an anti- cancer immune response. To develop tumor specific distribution of the pAgs, we will take advantage of the emerging pHLIP (pH (low) insertion peptide) which inserts into tumor cell membranes in the acidic tumor microenvironment. We will synthesize pHLIP-pAg conjugates optimized for in vivo application and characterize their activation of gamma delta T cells. We will examine the activity of these new conjugates in cellular and animal models of lymphoma, assessing both the activity and potential toxicity of these novel agents. Additional studies will evaluate the conjugates against more immunologically cold sarcomas. The ultimate goal is to identify a pHLIP-pAg that will be used for treatment of cancers that are resistant to immune checkpoint inhibitors. These findings will come at a time when the biological understanding of anti-cancer immunity is far from complete, and thus these studies have the potential for dramatic ...