Summary The advent of immune checkpoint inhibitor (ICI) therapies has dramatically changed the treatment landscape for non-small cell lung cancer (NSCLC). However, most patients treated with ICIs are either non-responders or develop progressive disease (PD), despite an initial response to therapy. One of the mechanisms behind this, is the action of suppressive immune cell populations within the tumor microenvironment (TME) such as regulatory T cells (Tregs), which act to shield tumors from the patient's immune response. We plan to target and deplete these Tregs specifically within the TME through the C-C Chemokine G Protein-coupled Receptor 8 (CCR8), which is selectively upregulated in activated tumor-resident Tregs and is absent from essential effector T lymphocytes. Selective elimination of CCR8+ Tregs is anticipated to promote a more effective immune response against NSCLC tumors, while avoiding the dangerous autoimmune side effects associated with non-selective depletion. Targeting CCR8 and other G protein-coupled receptors (GPCRs) with antibody therapeutics has historically been a challenge. This is largely due to the intrinsic properties that make them poor antigens, which include their low abundance, poor immunogenicity, and conformational heterogeneity. To address this, Abilita Bio developed the Enabled Membrane Protein (EMP™) directed evolution technology, which generates enhanced GPCR variants with transformative improvements in biophysical properties, while preserving their structure and biological relevance. Using the EMP™ methodology, we evolved a conformationally stabilized version of CCR8, which was used as a protein antigen for llama immunization and the discovery of VHH single-domain antibody families that uniquely target the transmembrane core of the wild-type human receptor. Our goal is to leverage these antibodies' advantageous properties to construct a molecule with best-in-class potential. In the proposed Phase I SBIR research, we will complete a thorough in vitro characterization and optimization of antibody hits, select a lead molecule, and then perform in vivo efficacy studies in a novel CCR8-humanized mouse model of NSCLC in a head-to-head comparison with a competitor’s antibody to show superiority. We will accomplish this goal through the execution of the following scientific aims. Aim 1: we will analyze our diverse VHH hit collection for binding characteristics and biophysical stability in vitro, and then select the best molecules for further characterization. Aim 2: we will characterize antibody pharmacology and potency in mechanism of action studies to select a lead antibody for in vivo studies. In Aim 3: we will test in vivo efficacy of our lead antibody in a CCR8- humanized mouse model of NSCLC. This is, to our knowledge, the first in vivo disease model study for a CCR8 therapeutic program, where human CCR8 will be targeted in the context of a functional TME. Successful completion of this Phase I research will prov...