Related Experiment Video
Updated: Jan 14, 2026
Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody
Published on: May 16, 2020
Structure-guided design and engineering of a biparatopic antibody targeting CCR2 in triple negative breast cancer
Timothy S Little1, Yuyan Wang1, Ameya P Chaudhari1
1Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Abstract:
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.
Insights
A novel biparatopic antibody targeting two sites on the CCR2 receptor effectively inhibits cancer progression. This antibody enhances anti-tumor macrophage polarization and reduces tumor growth in preclinical models.
Area of Science:
- Immunology
- Oncology
- Structural Biology
Background:
- Chemokine C-C motif receptor 2 (CCR2) signaling, driven by its ligand C-C motif chemokine ligand 2 (CCL2), is crucial for cancer progression by recruiting immunosuppressive cells.
- Current CCR2-targeting antibodies primarily block the N-terminal domain (NTD), the main CCL2 binding site, but CCR2 has other extracellular loops involved in signaling.
Purpose of the Study:
- To develop and evaluate a biparatopic antibody targeting two distinct epitopes on CCR2 (NTD and extracellular loop 3) for enhanced inhibition of the CCR2/CCL2 axis.
- To assess the therapeutic potential of this biparatopic antibody in modulating the tumor microenvironment and reducing tumor growth.
Main Methods:
- Computational modeling (AlphaFold, HADDOCK) and experimental validation (ELISA, surface plasmon resonance) of antibody-target interactions.
- In vitro functional assays assessing macrophage polarization (M1/M2 ratios) in response to biparatopic antibody treatment.
- In vivo efficacy studies using a murine model of triple-negative breast cancer.
Main Results:
- The biparatopic antibody exhibited superior binding affinity compared to monospecific constructs.
- Treatment with the biparatopic antibody significantly enhanced pro-inflammatory macrophage polarization, improving M1/M2 ratios by up to 70-fold.
- The biparatopic antibody demonstrated significant tumor growth reduction in a triple-negative breast cancer mouse model.
Conclusions:
- Simultaneous blockade of two distinct CCR2 epitopes via a biparatopic antibody is a potent strategy to inhibit the CCR2/CCL2 signaling axis.
- This biparatopic antibody effectively reshapes macrophage polarization towards an anti-tumor phenotype.
- The developed biparatopic antibody shows promising therapeutic potential for cancer treatment, particularly in triple-negative breast cancer.
