Structure-guided 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 tumor microenvironment and influencing disease outcomes. Among these, interactions between the chemokine CCR2 receptor and its ligand CCL2 has been shown to be a key driver of cancer progression by recruiting immune-suppressive cells to the tumor microenvironment. Most CCR2-targeting antibodies block the N-terminal domain (NTD) of the receptor as this is the primary binding site of its ligand CCL2. However, CCR2 contains three additional extracellular loops that also participate in binding interactions, thus mediating downstream signaling. Here, we developed a biparatopic antibody that targets two distinct, non-overlapping epitopes on CCR2: the NTD and the extracellular loop 3 (ECL3). We hypothesized that simultaneously blocking two distinct epitopes on CCR2 would more effectively inhibit the CCR2/CCL2 signaling axis, thus curtailing tumor progression. Binding interactions were modeled using AlphaFold and HADDOCK and validated by ELISA and surface plasmon resonance assays. When compared to monospecific constructs, the biparatopic antibody demonstrated stronger binding affinity to a CCR2-mimicking peptide. Functional studies showed that treatment with the biparatopic 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 relative to single-epitope blockers. In a murine model of triple-negative breast cancer, the biparatopic antibody demonstrated a significant reduction in tumor growth. These findings highlight its potential as a therapeutic agent for reshaping macrophage polarization and inhibiting tumor progression.