eIg-based bispecific T-cell engagers targeting EGFR: Format matters

ABSTRACT

Bispecific antibodies are molecules with versatile modes of action and applications for therapy. They are commonly developed as T-cell engagers (TCE), which simultaneously target an antigen expressed by tumor cells and CD3 expressed by T-cells, thereby inducing T-cell-mediated target cell killing. There is growing evidence that the molecular composition and valency for the target antigen influence the activity of TCEs. Here, the eIg platform technology was used to generate a set of bispecific TCEs targeting epidermal growth factor receptors (EGFR) and CD3. These molecules either included or lacked an Fc region and exhibited one binding site for CD3 and either one or two binding sites for EGFR (1 + 1 or 2 + 1 formats) utilizing different molecular arrangements of the binding sites. In total, 11 different TCE formats were analyzed for binding to target cells and T cells, T cell-mediated killing of tumor cells, and for the activation of T cells (release of cytokines and proliferation of T-cells). Bivalent binding to EGFR strongly increased binding and T cell-mediated killing. However, the molecular composition and position of the CD3-binding arm also affected target cell killing, cytokine release, and T-cell proliferation. Our findings support that screening of a panel of formats is beneficial to identify the most potent bispecific TCE, and that format matters.

KEYWORDS:

• Bispecific antibodies
• CD3
• EGFR
• T-cell retargeting
• valency

Acknowledgments

We would like to thank Nadine Heidel, Sabine Münkel, Beatrice Reiser, Vanessa Schmitt, Laura Walisch, and Alexandra Kraske for their excellent technical assistance.

Author contributions

L.K., A.K.S., S.K., N.A., and O.S. performed cloning, protein expression and purification, biochemical analysis, binding studies, and bioactivity assays. L.K., A.K.S., S.K., R.E.K., and O.S. analyzed and interpreted the data. L.K., N.A., R.E.K. and O.S. were responsible for experimental design and supervised the work. L.K., R.E.K. and O.S. wrote the manuscript. All authors read and approved the final manuscript.

Disclosure statement

R.E.K. and O.S. are named inventors on a patent application covering the eIg technology. No potential conflict of interest was reported by the other authors.

Supplementary data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2183540

Additional information

Competing interests: R.E.K. and O.S. are named inventors on a patent application covering the eIg technology. The data are available and can be sent upon request.

Additional information

Funding

The author(s) reported that there is no funding associated with the work featured in this article.