IF a recombinant GPNMB-targeted immunoenzyme — consisting of purified ColG from Clostridium histolyticum site-specifically conjugated to an anti-GPNMB single-chain variable fragment (scFv) derived from the GPNMB vaccination epitope described by Suda et al. — is administered at 0.5 mg/kg via weekly tail vein injection for 8 weeks to 24-month-old male and female C57BL/6J mice, co-administered with bi-weekly subcutaneous GPNMB peptide vaccine (50 μg with alum adjuvant) and a low-dose systemic neutral protease (thermolysin, 0.1 mg/kg IV, co-injected with the immunoenzyme to address the laminin scaffold that ColG cannot degrade),

THEN a ≥40% reduction in p16INK4a+/CD31+ senescent endothelial cells in the thoracic aorta (measured by immunofluorescence), a ≥35% reduction in en face aortic plaque area (Oil Red O), and a ≥50% reduction in collagen IV immunostaining intensity in the aortic basement membrane will be observed versus vaccine-only controls, with vascular hemorrhage or aneurysm occurring in fewer than 5% of animals (versus >20% predicted for non-targeted systemic ColG),

BECAUSE the following causal chain:

1. Senescent vascular endothelial cells in aged mice selectively upregulate GPNMB on their luminal surface, co-expressing p16INK4a, creating a surface-accessible, high-density antigenic target that distinguishes these cells from healthy neighboring endothelium (Suda et al., Nature Aging, 2021 — referenced in Evidence Set, Section 5). The GPNMB-scFv moiety of the immunoenzyme binds these cells preferentially, concentrating enzymatic activity in the immediate pericellular microenvironment surrounding senescent cells.

2. ColG (class I collagenase) preferentially cleaves the triple-helical regions of type IV collagen in the vascular basement membrane, with higher catalytic efficiency against fibrillar collagens but documented activity against the interrupted helical domains of type IV collagen (Matsushita et al., Journal of Biological Chemistry, 2001 — referenced in Evidence Set, Section 3). Critically, by tethering ColG to an anti-GPNMB scFv, enzyme activity is geometrically restricted to the pericellular space of antigen-positive cells, preventing off-target basement membrane degradation in GPNMB-negative vessels — the mechanism responsible for hemorrhage in non-targeted systemic collagenase models (Rosenberg et al. — referenced in Evidence Set, Section 4).

3. ColG alone is insufficient to fully breach the basement membrane because it lacks proteolytic activity against the laminin meshwork, which remains structurally intact after collagen IV hydrolysis, as demonstrated in pancreatic islet isolation protocols requiring co-administration of neutral protease (thermolysin) alongside ColG to achieve full pericellular ECM dissolution (McCarthy et al., Transplantation Proceedings, 2011 — referenced in Evidence Set, Section 3). Co-administration of thermolysin at sub-hemorrhagic dose (0.1 mg/kg, well below collagenase do...

SENS category: GlycoSENS