Development of a therapeutic monoclonal antibody against circulating adipocyte fatty acid binding protein to treat ischemic stroke

Abstract

<h3>Background and Purpose</h3><p>Adipocyte fatty acid-binding protein (A-FABP) exacerbates cerebral ischemia injury by disrupting the blood-brain barrier (BBB) through inducing MMP-9 expression. Circulating A-FABP positively correlates with the infarct size of stroke patients. We hypothesize that targeting circulating A-FABP by a neutralizing antibody alleviates ischemic stroke outcome.</p><h3>Experimental Approach</h3><p>Monoclonal antibodies (mAbs) against A-FABP were generated using mouse hybridoma technique. Binding affinities of a generated mAb named 6H2 towards various FABPs were determined using Biacore. Molecular docking studies were performed to characterize the 6H2-A-FABP complex structure and epitope. The therapeutic potential and safety of 6H2 were evaluated in mice with transient middle cerebral artery occlusion (MCAO) and healthy mice, respectively.</p><h3>Key Results</h3><p>Replenishment of recombinant A-FABP exaggerated the stroke outcome in A-FABP-deficient mice. 6H2 exhibited nanomolar to picomolar affinities to human and mouse A-FABP, respectively, with minimal cross-reactivities with heart and epidermal FABPs. 6H2 effectively neutralized A-FABP-elicited JNK/c-Jun activation and reduced MMP-9 production in macrophages. Molecular docking suggested that 6H2 interacts with the “lid” of the fatty acid binding pocket of A-FABP, thus likely hindering the binding of its substrates. In mice with transient MCAO, 6H2 significantly attenuated BBB disruption, cerebral edema, infarction, neurological deficits, and decreased mortality associating with reduced cytokines and MMP-9 production. Chronic 6H2 treatment showed no obvious adverse effects in healthy mice.</p><h3>Conclusion and Implications</h3><p>These results establish the circulating A-FABP as a viable therapeutic target for ischemic stroke, and provide a highly promising antibody drug candidate with superb affinity and specificity.</p>