Immunotherapy is revolutionizing cancer treatment, and checkpoint inhibitors have improved overall survival outcomes in a number of tumor types, especially ‘inflamed’ tumors with high mutational burden and infiltrating T-cells1. T-cell engagers (TCEs) have emerged as an option for the treatment of ‘immunologically cold’ tumors that are unresponsive to other immune therapies, however their clinical use in patients with solid tumors has been limited by on-target off-tumor effects, specifically immune activation in healthy tissues that ultimately results in cytokine release syndrome and a narrow therapeutic window1-5.
Here, we describe XPAT® proteins, bispecific TCEs with protease-releasable masking technology (Pro-XTENTM technology), that represent a novel and customizable platform for precise tumor targeting with TCEs. XPAT proteins are made up of a potent TCE core comprised of 2 domains, each flanked by a polypeptide mask designed to be cleaved by proteases that are dysregulated in the tumor microenvironment, but tightly regulated in healthy tissues and systemic circulation (Figure 1). The use of XTEN® masks provides a novel approach to extend the half-life of the TCEs while minimizing the risk of systemic exposure to the unmasked TCE (uTCE), intended to limit off-tumor side effects that have limited the therapeutic potential of TCEs in solid tumors. We demonstrated the effectiveness of the XPAT platform in preclinical models using two well-validated tumor targets, HER2 and EGFR. Both XPAT proteins contain bispecific TCE cores targeting the tumor-associated antigen (TAA) and CD3.
FIGURE 1
XPAT proteins are designed to exploit the dysregulated protease activity present in tumors versus healthy tissues and expand the therapeutic index of TCEs through preferential unmasking in the tumor microenvironment.
In vitro, a fully masked HER2-XPAT protein demonstrated up to a 4-log-fold difference in cytotoxicity versus the uTCE counterpart. In vivo, a HER2-XPAT protein exhibited protease-dependent tumor growth inhibition and T-cell activation that were similar to results seen with an equimolar amount of uTCE. In mice dosed with XPAT protein, approximately 24% of the concentration in tumors was detected as uTCE, while no measurable uTCE concentrations were detected in healthy tissues, highlighting the ability of XPAT proteins to precisely target tumors for unmasking and subsequent immune activation and cell killing.
In non-human primates (NHPs), a HER2-XPAT protein was tolerated at >400-fold higher maximum concentrations than the uTCE. The uTCE exhibited a half-life of approximately 2 hours in NHPs, and induced significant cytokine release at low doses (0.2 mg/kg). The HER2-XPAT protein exhibited a longer half-life (approximately 3 days), but despite the much higher doses tested and tolerated (up to 42 mg/kg), there were no gross or microscopic findings in HER2 expressing tissues, minimal to no cytokine induction at even the highest doses, and no detectable uTCE in circulation. The robust safety margin observed in NHPs where there is no tumor present and therefore no dysregulated protease activity emphasizes the reduced risk of off-tumor toxicities with the fully masked XPAT protein as compared to uTCE. Further, HER2-XPAT protein cleavage in plasma samples was low and consistent between cancer patients, healthy subjects, patients with inflammatory disease, and NHPs with or without inflammation, supporting the translatability of XPAT protein stability to patients.
An EGFR-XPAT protein prototype further highlights the utility and flexibility of the XPAT platform for extending the therapeutic window of TCEs, even for TAAs that are highly expressed in healthy tissue. The EGFR-XPAT protein prototype demonstrated robust protease-dependent tumor growth inhibition in mice, similar to that seen with the uTCE, but with a >200-fold higher tolerated maximum concentration than the uTCE in NHPs.
Although the results are promising, further studies are needed to validate the safety and efficacy of XPAT proteins in patients with solid tumors. A clinical study evaluating the safety and pharmacokinetics of HER2-XPAT protein has been initiated (ClinicalTrials.org identifier NCT05356741), and additional XPAT proteins are in development to address the unmet need for targeted immunotherapy across different solid tumors.
References
- Goebeler, M.-E. & Bargou, R. C. T cell-engaging therapies—BiTEs and beyond. Nat. Rev. Clin. Oncol. 17, 418–434 (2020).
- Lowe, K. L. et al. Novel TCR-based biologics: mobilising T cells to warm ‘cold’ tumours. Cancer Treat. Rev. 77, 35–43 (2019).
- Ochoa de Olza, M., Navarro Rodrigo, B., Zimmermann, S. & Coukos, G. Turning up the heat on non-immunoreactive tumours: opportunities for clinical development. Lancet Oncol. 21, e419–e430 (2020).
- Klinger, M., Benjamin, J., Kischel, R., Stienen, S. & Zugmaier, G. Harnessing T cells to fight cancer with BiTE antibody constructs—past developments and future directions. Immunol. Rev. 270, 193–208 (2016).
- Vafa, O. & Trinklein, N. D. Perspective: designing T-cell engagers with better therapeutic windows. Front. Oncol. 10, 446 (2020).
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