PROJECT SUMMARY
Notch 3 mutations are the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts
and leukoencephalopathy (CADASIL), a small vessel disease characterized by stroke and vascular
cognitive impairment. Transgenic mouse studies from our lab and others have shown that Notch 3 sig-
naling is critical for mural cell survival, and blood-brain barrier (BBB) integrity, and that Notch 3 knock-
out mice have increased susceptibility to hypoxia-induced stroke. Loss of mural cells (MCs), including
pericytes and vascular smooth muscle cells (SMCs), in the small penetrating arteries of the brain is a
hallmark of CADASIL and a major contributor to vascular dysfunction. Importantly, in both humans and
mouse models, loss-of-function (LOF) Notch 3 mutations cause MC death. Currently, there are no tar-
geted therapies available for CADASIL. We have also shown that antibodies can be used to modulate
Notch 3 signaling in mouse models of CADASIL. Specifically, we showed that a mouse monoclonal an-
tibody administered in a CADASIL mouse model can prevent MC loss. Our proposal will optimize a mo-
dality of treatment for CADASIL by humanizing a mouse monoclonal Notch 3 agonist antibody. Our pro-
ject goals are as follows. Aim 1. To generate a panel of agonist Notch3 human monoclonal antibodies
(humAbs). We propose to humanize a mouse monoclonal antibody capable of activating human Notch
3, via grafting of complementarity-determining regions (CDRs) into human framework regions (FRs).
Aim 2. To rank order the candidate antibodies using in vitro assays. We will screen candidate human-
ized antibodies for the preservation of specific Notch 3 antigen recognition using a sensitive in vitro
ELISA test detecting target engagement and activation of human Notch3 receptors via release of the
Notch 3 extracellular domain (N3ECD) biomarker. Aim 3. Prepare cell line for the production of lead
humAb. We will produce at least two humanized A13 (huA13) for preclinical in vivo testing by generat-
ing a cell line using Switchable Membrane Reporter (SwiMR) technology for antibody production. Aim
4. To test the preclinical efficacy of lead Notch 3 humAbs in mouse models of Notch 3 deficiency and
CADASIL. We will screen our humanized antibody candidates for biologic effects including Notch 3 acti-
vation in CNS vessels, crossing the blood-CNS barrier, normalization of N3ECD in blood, a target en-
gagement biomarker linked to Notch3 signaling in vivo, and rescue of mural cell loss. Aim 5. To evalu-
ate toxicity and pharmacokinetics. We will examine toxicity and pharmacokinetics antibody administra-
tion of increasing single-dose in mice.
