Mesothelin-targeted nanobody-drug conjugates to treat pancreatic cancer

Development of targeted therapies that can penetrate the dense stroma of the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) is essential to improve outcomes for pancreatic cancer patients. We have discovered a camel VHH nanobody (A101) using phage display, which effectively binds to mesothelin (MSLN), a cancer antigen that is expressed in PDAC. Using the A101 VHH nanobody with an MMAE payload, we generated nanobody-drug conjugates (NDCs) that can be advantageous compared to existing biologic therapies by enhancing tumor penetration through the PDAC TME and improving pharmacokinetic distribution, while minimizing systemic toxicity. The structure of the A101 nanobody was computationally predicted and molecular dynamic simulations were run to understand its interaction with MSLN. Site-specific modifications were engineered and small linker-MMAE molecules were synthesized, then bioconjugated to the modified A101 nanobody using maleimide chemistry. The chemical linkers used in these constructs were valine-citrulline, glycine-proline, and AB (a novel small molecule). The NDCs were tested for binding to MSLN via bio-layer interferometry assays. Internalization and cell viability were also assessed using mouse and human PDAC cell lines. Finally, in vivo activity was assayed in MSLN+ and MSLN knockout (MLSN-KO) subcutaneous mouse PDAC tumor models to determine efficacy and specificity of each therapeutic. We found that modified A101 nanobody retains binding to human (K D = 18 nM) and mouse (K D = 5.6 nM) MSLN. A101 NDCs displayed a statistically significant difference in internalization into MSLN+ as compared to isogenic MSLN-KO cells. Additionally, the NDCs efficiently killed PDAC cell lines in vitro (IC 50 = 20-200 nanomolar). In mice, the A101-AB-MMAE NDC (at 20 mg/kg, 3x per week) displayed the most promising inhibition of tumor growth in MSLN+ tumors and had the highest specificity compared to other A101-NDCs tested, showing limited efficacy in analogous MSLN-KO tumors. We have synthesized A101-based NDCs with high-affinity MSLN binding and promising anti-tumor activity in PDAC cell lines. Our preliminary animal studies suggest that the A101-AB-MMAE conjugate, which has a novel exclusively intracellular cleavable linker, has superior efficacy and specificity compared to other A101-based NDCs tested with known linkers. Future studies will explore A101-AB-MMAE activity in orthotopic humanized MSLN mouse models of PDAC to assess safety, biodistribution, and anti-tumor efficacy.