Abstract 3376: HER2-XPAT and EGFR-XPAT: Pro-drug T-cell engagers (TCEs) engineered to address on-target, off-tumor toxicity with potent efficacy in vitro and in vivo and large safety margins in NHP
Authors: Cattaruzza Fiore , Nazeer Ayesha , Lange Zachary , Hammond Mikhail , Koski Caitlin , Henkensiefken Angela , Derynck Mika K. , Irving Bryan , Schellenberger Volker 
Published: September 2020

Background: TCEs have proven to be effective in creating durable remissions in hematologic malignancies but have been challenging in solid tumors due to on-target, off-tumor toxicity. To circumvent the toxicity, many have tried step-up, fractionated dosing or complex formats, but these have largely been unsuccessful due to toxicity and/or enhanced immunogenicity. To address this challenge, Amunix has developed a conditionally active TCE, the XPAT or XTENylated Protease-Activated T Cell Engager, that exploits the protease activity present in tumors vs. healthy tissue, enabling expansion of the therapeutic index (TI). The core of the XPAT consists of 2 tandem scFVs targeting CD3 and a tumor antigen. Two unstructured and modular polypeptide masks (XTEN) are attached to the core that sterically reduce target engagement and extend T1/2. Protease cleavage sites at the base of these XTEN polypeptide masks are then proteolytically released in the tumor microenvironment to unleash a highly potent TCE with a short T1/2, further improving the TI.

Methods: HER2-XPAT and EGFR-XPATs were each purified from E.coli expressed from a single plasmid. The activity of both the prodrugs (XPATs) and their protease-activated counterparts (PATs) were characterized for cytotoxicity in vitro and in huPBMC-transduced tumor-bearing mice. Pilot toxicity studies were conducted in NHPs (cyno).

Results: Both protease-activated (PATs) forms of EGFR-XPAT and HER2-XPAT demonstrated potent in vitro T cell directed cytotoxicity against tumor cells (EC50s 1-2pM), while the masked XPAT provided 3,000-14,000-fold protection against target cell killing. Intact EGFR-XPAT demonstrated dose-dependent complete tumor responses (CRs) in HT-29 CRC xenografts, with HER2-XPAT demonstrating similar efficacy and CRs in both BT-474 and SK-OV-3 tumors. In cynos, 1 mg/kg of EGFR-XPAT was the MTD and 1.5 mg/kg exceeded the MTD, while the activated PAT was lethal at 0.132mg/kg/day by continuous infusion (with single dose 1 hour bolus of 0.066ug/kg). This indicated a near 2 log protection or more in predicted Cmax of XPAT vs PAT. A HER2-XPAT has been dose escalated up to 21 mg/kg in cynos and was well-tolerated, with the MTD still unreached. At doses starting from 2.5mg/kg of a HER2-XPAT, lymphocyte margination and laboratory abnormalities were observed, indicating evidence of biologic activity well below the tolerated 21 mg/kg dose. An estimated T1/2 of ~4 days was observed.

Conclusions: EGFR-XPAT and HER2-XPAT are novel T cell engagers with protease-cleavable XTEN masks with preclinical evidence of potent anti-tumor activity and a wide margin of protection in cynos. With the prior XTEN clinical data of low immunogenicity, the XPAT TCEs provide a promising solution to overcome On-target, Off-tumor toxicity. Additional PD, safety and efficacy data will be presented.

Citation Format: Fiore Cattaruzza, Ayesha Nazeer, Zachary Lange, Mikhail Hammond, Caitlin Koski, Angela Henkensiefken, Mika K. Derynck, Bryan Irving, Volker Schellenberger. HER2-XPAT and EGFR-XPAT: Pro-drug T-cell engagers (TCEs) engineered to address on-target, off-tumor toxicity with potent efficacy in vitro and in vivo and large safety margins in NHP [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3376.