RATIONAL DESIGN AND ENGINEERING OF A BIPARATOPIC ANTIBODY TARGETING CCR2 IN TRIPLE NEGATIVE BREAST CANCER

Chemokine signaling plays a pivotal role in cancer progression, shaping the tumormicroenvironment and influencing disease outcomes. Among these, interactions between thechemokine CCR2 receptor and its ligand CCL2 has been shown to be a key driver of cancerprogression by recruiting immune-suppressive cells to the tumor microenvironment. MostCCR2-targeting antibodies block the N-terminal domain (NTD) of the receptor as this is theprimary binding site of its ligand CCL2. However, CCR2 contains three additional extracellularloops that also participate in binding interactions, thus mediating downstream signaling. Here,we developed a biparatopic antibody that targets two distinct, non-overlapping epitopes onCCR2: the NTD and the extracellular loop 3 (ECL3). We hypothesized that simultaneouslyblocking two distinct epitopes on CCR2 would more effectively inhibit the CCR2/CCL2signaling axis, thus curtailing tumor progression. Binding interactions were modeled usingAlphaFold and HADDOCK and validated by ELISA and surface plasmon resonance assays.When compared to monospecific constructs, the biparatopic antibody demonstrated strongerbinding affinity to a CCR2-mimicking peptide. Functional studies showed that treatment with thebiparatopic antibody enhanced macrophage polarization to a pro-inflammatory (anti-tumor) state,with up to 70-fold improvement in M1/M2 ratios in bone marrow-derived macrophages relativeto single-epitope blockers for as long as 24 hours after treatment. In a murine model of triple-iiinegative breast cancer, the biparatopic antibody demonstrated a significant reduction in tumorgrowth. These findings highlight its potential as a therapeutic agent for reshaping macrophagepolarization and inhibiting tumor progression.