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

Abstract

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.

T-Cell Engagers in Solid Cancers—Current Landscape and Future Directions

“…Conditional activation of T-cell engagers is another approach to enhance tumor selectivity and on-target/on-tumor efficacy while lessening off-tumor toxicity. Here, inactive T-cell engagers become activated upon entering a tumor by either leveraging tumor proteases or modulating the pH of the microenvironment [67]. In summary, this class of immune therapeutics holds great promise, especially in highly heterogeneous solid tumors, and commands the attention of anti-cancer drug developers.…”

The Masking Game: Design of Activatable Antibodies and Mimetics for Selective Therapeutics and Cell Control

“…Taking into account this criteria, several protein moieties have been N-terminally fused to antibodies, including endogenously derived inhibitory domains (i.e., LAP domain from pro-TGF-beta), 68 peptides able to recruit albumin, 69 and intrinsically disordered XTEN polypeptides. 70 The first two approaches enable 2- and 10-fold affinity decrease, respectively, and have not been reported in animals studies. Conversely, XTENylated protease-activated T-cell engagers (XPATs) show a significant increase in the maximal tolerated dose in cynomolgus monkeys and minimal effect on antibody pharmacokinetics.…”

“…(A) In the probody approach, extending the N-terminus with a mimotope that can be removed by proteolytic cleavage shows great versatility and decreases off-target binding, thereby enhancing antibody circulation time. − The graph schematically represents results from ref . (B) Epitope-mimetics and idiotypic masks enable good inactivation efficiency at the expense of transferability to to different antigen specificities. − (C) Approaches relying on the interaction with more conserved regions in the Fv could enable transferability to other antibody specificities and formats. , (D) Several strategies generally applicable to certain antibody formats have been developed relying on steric hindrance with variable degrees of inactivation efficiency. − (E) Masking via N-terminal coiled-coil domains is efficient and suggests high transferability to other antigen specificities . Adapted with permission from refs and .…”

“…A requirement for the steric mask is low immunogenicity. Taking into account this criteria, several protein moieties have been N-terminally fused to antibodies, including endogenously derived inhibitory domains (i.e., LAP domain from pro-TGF-beta), peptides able to recruit albumin, and intrinsically disordered XTEN polypeptides . The first two approaches enable 2- and 10-fold affinity decrease, respectively, and have not been reported in animals studies.…”

Activatable Antibody Mimetics for the Selective Delivery of Therapeutics

“…In our group we are developing new methods to reversibly mask antibodies with high transferability across antibody formats and specificities. Masking moieties used to provide sufficient hindrance with current approaches need to be very large, like in the case of XTENylated antibodies, where N-terminal elongations of 600-900 residues are required [3]. This increases considerably the size of the therapeutic, further limiting its diffusion into the tumor tissue.…”