Therapeutic Antibody Detection

We dosed mice intravenously with various concentrations of a monospecific antibody targeting BACE1 or a bispecific antibody that is similar, but with the addition of a domain binding to the transferrin receptor (TfR1). The transferrin receptor is found in blood vessel endothelial cells where it acts as a carrier to transport iron. Antibodies binding to the transferrin receptor can hijack this mechanism to be transported across the blood-brain barrier.

When developing the methodology for whole-brain imaging of IV-dosed experimental therapeutic human antibodies, we tested multiple different secondary antibodies. The top panels show sagittal planes of intact brains stained with three different secondary antibodies. We determined that the Fcy antibody had better brain penetration and more even whole-brain staining. Importantly, all three secondary antibodies produced qualitatively similar patterns of immunoreactivity.

Our modified iDISCO+ protocol allows for imaging of an entire intact tissue, giving an unbiased snapshot of immunoreactivity throughout the brain. Consistent with the data from the hippocampus, we discovered that the monospecific antibody has negligible brain penetration, whereas the bispecific antibody is identified in both the vasculature and parenchyma throughout the brain. The bispecific BACE1 antibody is particularly enriched in a number of brain areas including the Pontine Gray, Medulla and Glomeruli in the Olfactory Bulb.

24 hours after dosing, using our modified iDISCO+ protocol, we cleared and stained perfusion-fixed brains using an anti-human IgG secondary antibody. We could not detect any immunoreactivity in the brains of mice injected with the monospecific antibody, with background signals identical to brains from non-infected mice. With the bispecific antibody, strong staining was observed in blood vessels, and importantly, in the brain parenchyma. In independent experiments, we saw similar patterns of staining in the hippocampus of IV-dosed mice stained with traditional slice IHC.

Traditional slice IHC experiments validated that there is enrichment of the IV-dosed bispecific antibody (50 and 125 mmol/kg) vs. the monospecific antibody (125 mmol/kg) in the dentate gyrus (DG) of the hippocampus (green panels and bar graph). This pattern is very similar to that seen in slices from non-dosed mice stained directly with an anti-BACE1 antibody (red staining).