VHH antibody targeting the chemokine receptor CX3CR1 inhibits progression of atherosclerosis

Abstract

CX3CR1 has been identified as a highly attractive target for several therapeutic interventions. Despite this potential, no potent antagonists, either small molecule or monoclonal antibody, have been identified. Here we describe the lead finding and engineering approach that lead to the identification of BI 655088, a potent biotherapeutic antagonist to CX3CR1. BI 655088 is a potent CX3CR1 antagonist that, upon therapeutic dosing, significantly inhibits plaque progression in the standard mouse model of atherosclerosis. BI 655088 represents a novel and highly selective biotherapeutic that could reduce inflammation in the atherosclerotic plaque when added to standard of care treatment including statins, which could result in a significant decrease in atherothrombotic events in patients with existing cardiovascular disease.

Keywords: BI 655088; GPCR; VHH Antibodies; atherosclerosis; biophysical assessment; humanization; pharmacokinetic profile.

Figure 1.

Binding of bivalent family101 VHHs to Ba/F3/hCX3CR1 cells. Mean fluorescence per cell was measured by flow cytometry of various concentrations of Alexa Fluor labeled VHHs. IC50s were calculated to be <1 nM for all four constructs tested.

Figure 2.

Exemplar Chemotaxis Assay data showing the functional activity of BI 655088.

Figure 3.

Characterization of Target Engagement Biomarkers in hCX3CR1 KI Mouse Model. Free receptor levels, serum concentrations of BI 655088 and serum fractalkine levels in hCX3CR1 KI mice after receiving two doses at 30 mg/kg of BI 655088 or vehicle. Day 0 represents baseline concentrations for free receptor and serum fractalkine of hCX3CR1 KI mice dosed with vehicle control (Mean ± SD, *p < .0001 vs. Day 0 and Day 13). BI 655088 and Fractalkine levels were determined by immunoassay and free receptor levels assessed by FACS.

Figure 4.

Human CX3CR1 is expressed in the atherosclerotic plaques of hCX3CR1/ApoE KO mice (brachiocephalic artery) after feeding with an HF/HC diet (a). IHC was done using a human anti-CX3CR1 antibody and compared to a no-antibody negative control. Similar expression of CX3CR1 was also seen in the plaque from a cross-section of human coronary artery from an atherosclerotic patient (b).

Figure 5.

BI 665088 treatment significantly reduced the descending aorta plaque area (a) with a concomitant increase in serum fractalkine (b). Representative images of descending aortas after Oil Red O staining for plaque area (c).