HER3 is a member of the EGF receptor family and elevated expression is associated with cancer progression and therapy resistance. HER3-specific T-cell engagers might be a suitable treatment option to circumvent the limited efficacy observed for HER3-blocking antibodies in clinical trials. In this study, we developed bispecific antibodies for T-cell retargeting to HER3-expressing tumor cells, utilizing either a single-chain diabody format (scDb) with one binding site for HER3 and one for CD3 on T-cells or a trivalent bispecific scDb-scFv fusion protein exhibiting an additional binding site for HER3. The scDb-scFv showed increased binding to HER3-expressing cancer cell lines compared to the scDb and consequently more effective T-cell activation and T-cell proliferation.
Fig:Biochemical characterization of scDb and scDb-scFv.(a) Composition and schematic illustration of scDb (1) and scDb-scFv (2). L, Igκ chain leader sequence. L1, G4S. L2, (G4S)3. L3, AAAGGS(G4S)GGGT, H, hexahistidyl-tag. (b) SDS PAGE analysis (12% PAA, 2 µg/lane, Coomassie blue staining) of (1) scDb and (2) scDb-scFv under reducing (R) and non-reducing (NR) condition. M, protein marker. (c) Size-exclusion chromatography by HPLC using a Tosoh TSKgel SuperSW mAb HR column. (d) Thermo stability determined by dynamic light scattering using the ZetaSizer Nano ZS (Malvern). Mean ± SD, n = 3.
Furthermore, the bivalent binding mode of the scDb-scFv for HER3 translated into more potent T-cell mediated cancer cell killing, and allowed to discriminate between moderate and low HER3-expressing target cells. Thus, our study demonstrated the applicability of HER3 for T-cell retargeting with bispecific antibodies, even at moderate expression levels, and the increased potency of an avidity-mediated specificity gain, potentially resulting in a wider safety window of bispecific T-cell engaging antibodies targeting HER3.
Aschmoneit, N., Steinlein, S., Kühl, L. et al. A scDb-based trivalent bispecific antibody for T-cell-mediated killing of HER3-expressing cancer cells. Sci Rep 11, 13880 (2021). https://doi.org/10.1038/s41598-021-93351-0