Abstract 583: AVA3996, a novel pre|CISION™ medicine, targeted to the tumor microenvironment via fibroblast activation protein (FAP) mediated cleavage
Authors: Jones David H. , Houée Marine , Buist Hanna , Marco Sergi , Braconi Chiara , Bell Neil , Wilson Francis X. , McLaughlin Fiona 
Published: June 2023

Abstract
AVA3996 is a therapeutic product based on proprietary pre|CISION™ technology which incorporates a substrate that is sensitive to cleavage by FAP. The pre|CISION™ substrate can be utilized in a drug conjugate linker or to generate chemotherapeutics that are only activated in the tumor microenvironment. AVA3996 consists of a proteasome inhibitor molecule covalently bonded to a peptide containing a cleaving sequence (D-Ala-L-Pro), which is designed to be susceptible to hydrolysis by Fibroblast Activation Protein α (FAP) but is resistant to hydrolysis by both closely related and wider mammalian peptidases. FAP, a post-prolyl endopeptidase, is overexpressed on the surface of activated fibroblastic cells which are abundant in the supporting stroma of over 90% of malignant epithelial cancers.
Proteasome inhibitors are a first line of treatment for certain hematologic indications such as multiple myeloma. One consequence of proteasome inhibition is inhibition of the NFκB pathway due to reduced cleavage of the inhibitor IκBα; this pathway is activated in many tumors and represents an attractive target for a range of indications.
However, clinical utility of proteasome inhibitors is limited by severe dose-limiting toxicities, including peripheral neuropathy. AVA3996 has the potential to deliver elevated, effective levels of proteasome inhibitor directly to the solid tumor microenvironment while reducing systemic exposure and hence associated toxicities.
We have demonstrated that the pre|CISION™ substrate is exquisitely sensitive to FAP and is not cleaved by related proteases. The active AVA3996 warhead should therefore be released primarily following FAP cleavage in the tumor microenvironment.
Cancer cell lines were assessed for their sensitivity to AVA3996 in vitro: activity was typically 100-fold less than the active warhead or Bortezomib alone. Upon co-incubation with soluble FAP, potency of AVA3996 increased to similar levels as seen for the warhead alone demonstrating the masking effect of the pre|CISION™ substrate. A DRF/MTD study established the maximum tolerated dose in rats was around 6-fold higher for AVA3996 compared to warhead alone. This provides further evidence that AVA3996 can be dosed at higher levels than proteasome inhibitor alone, with the potential for greater tumor targeting and hence reduced systemic toxicity.
Ongoing in vivo and co-culture studies aim to further validate the efficacy and tolerability profile of AVA3996 to direct future development of this drug. In addition, the data supports wider utility of the pre|CISION™ platform to target therapeutics to the tumor while reducing systemic dose-limiting toxicities.
Citation Format: David H. Jones, Marine Houée, Hanna Buist, Sergi Marco, Chiara Braconi, Neil Bell, Francis X. Wilson, Fiona McLaughlin. AVA3996, a novel pre|CISION™ medicine, targeted to the tumor microenvironment via fibroblast activation protein (FAP) mediated cleavage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 583.