Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for...

Area of Science:

• Oncology
• Immunotherapy
• Protein Engineering

Background:

• T-cell engagers (TCEs) are promising immunotherapies but can cause off-tumor toxicity.
• Enhancing the therapeutic index of TCEs is crucial for clinical success.
• Tumor microenvironment proteases offer a potential mechanism for targeted activation.

Purpose of the Study:

• To engineer masked, precision-activated TCEs (XPAT proteins) for enhanced safety and efficacy.
• To evaluate the tumor-specific activation and therapeutic potential of XPAT proteins in vitro and in vivo.
• To assess the safety profile and translatability of XPAT technology across species.

Main Methods:

• Engineered XPAT proteins with protease-cleavable XTEN polypeptide masks targeting HER2 or EGFR and CD3.
• In vitro cytotoxicity assays to assess the efficacy of masked vs. unmasked TCEs.
• In vivo studies in animal models to evaluate antitumor activity, proteolytic stability, and safety.
• Analysis of XPAT protein cleavage in plasma samples from humans and non-human primates.

Main Results:

• XTEN masking provided up to 4-log-fold protection against cytotoxicity in vitro.
• HER2-XPAT protein demonstrated protease-dependent antitumor activity in vivo and was proteolytically stable in healthy tissues.
• HER2-XPAT protein showed a significant safety margin (>400-fold) in non-human primates compared to unmasked TCEs.
• Low and consistent cleavage of HER2-XPAT protein in human and non-human primate plasma supports translatability.
• EGFR-XPAT protein confirmed the utility of XPAT technology for targets expressed in healthy tissues.

Conclusions:

• XPAT technology enables precision activation of TCEs, enhancing their therapeutic index.
• Masked TCEs demonstrate potent, tumor-specific activity with improved safety profiles.
• XPAT proteins represent a promising platform for developing safer and more effective T-cell engager immunotherapies.