PROJECT SUMMARY The research objective is to engineer a nanoparticle platform to bind cell receptors and inhibit cell signaling more effectively than an antibody. To date, antibodies are universally employed as antagonists due to their high binding affinity for their target cell receptor. However, their large size may be less effective in blocking multiple cell surface receptors that organize as homodimers or colocalize within lipid rafts. We propose that peptide- conjugated liposomes (PCLs) - at an optimal peptide density - may be more effective than FDA-approved antibodies due to their ability to bind and inhibit receptor homodimers via optimal interpeptide spacings and receptor monomers due to cooperative binding. This proposal will evaluate the role of liposome peptide density and cell receptor organization on PCL binding and inhibition in vitro and pharmacokinetics and pharmacodynamics in vivo. In contrast to other liposomal delivery systems that encapsulate and release drugs, the biological activity of PCLs is due to the peptide density and diffusivity of the lipid bilayer. We have previously demonstrated that an optimized PCL bound and inhibited the CXCR4 homodimer, reducing triple negative breast cancer (TNBC) primary tumor growth and metastasis. In this proposal, we will apply PCLs to TNBC immunotherapy. Immune checkpoint inhibitor (ICI) therapy is predicated on strong binding between antibodies and their target receptor, inducing anti-tumor activity. Atezolizumab is FDA approved for use in TNBC to activate the anti-tumor response but only extends progression free survival from 5.5 months with chemotherapy to 7.2 months with chemotherapy and ICI therapy. Further research is needed to improve anti-tumor immune activity in TNBC. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), is present primarily as a homodimer on cell surfaces whereas programmed cell death ligand 1 (PD-L1) and programmed cell death 1 (PD-1) are monomeric, which suggests that different peptide spacings may be necessary to achieve maximal binding and inhibition. Thus, we will synthesize and characterize a series of PCLs that target PD-1 (L-PD1), PD-L1 (L-PDL1), and CTLA-4 (L-CTLA4) with increasing peptide density (9k/µm2, 24k/µm2, 39k/µm2, 53k/µm2, and 74k/µm2). TNBC and activated T cells will be measured for PD-1, PD-L1 or CTLA-4 expression, PCL-cell binding, and inhibition. We will compare PCL biodistribution in an immune competent TNBC tumor mice model and mice depleted of lymphocytes, neutrophils, or macrophages to assess how immune cells affect PCL tumor accumulation. PCL anti-tumor activity will be measured by cytokine expression (aim 1) and changes in tumor immune cell infiltration (aim 2) relative to the FDA-approved, ICI therapy (anti-PD-1 (pembrolizumab), anti-PD-L1 (atezolizumab), anti- CTLA-4 (ipilimumab)). Our team’s combined expertise in drug delivery, TNBC mouse models, and tumor immunology is sufficient to successfully complete this research. The outco...