A scDb-based trivalent bispecific antibody for T-cell-mediated killing of HER3-expressing cancer cells

Abstract

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. 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.

Fc-comprising scDb-based trivalent, bispecific T-cell engagers for selective killing of HER3-expressing cancer cells independent of cytokine release

“…Human anti-HER3 antibody 3–43 34 and a humanized version of anti-CD3 UCHT1 27 were used to generate the different trivalent, bispecific antibodies. Genes encoding the different polypeptide chains (see figure 1 ) were cloned into the pSecTagAL1 vector (a modified version of pSecTagA (Invitrogen, Thermo Fisher Scientific, V90020)) and were produced in transiently transfected HEK293-6E cells (NRC Biotechnology Research Institute, Canada) using polyethylenimine (linear, 25 kDa, Sigma-Aldrich, 764604) as described previously.…”

“…T-cell proliferation PBMCs were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) at 625 nM/1×10 6 cells/mL to analyze the proliferative effect on T-cells. 27 MCF-7 cells (2×10 4 cells/well) were incubated with a serial dilution of the trivalent, bispecific antibodies for 15 min at RT and 2×10 5 CFSE-labeled PBMCs/well were added subsequently. Following an incubation for 6 days at 37°C and 5% CO 2 , fluorescence-conjugated antibodies directed against cell surface markers were used to label immune cells of interest and their proliferation was determined Open access by multicolor flow cytometry analysis using MACSQuant Analyzer 10 (Miltenyi Biotec) as described previously.…”

“…Following an incubation for 6 days at 37°C and 5% CO 2 , fluorescence-conjugated antibodies directed against cell surface markers were used to label immune cells of interest and their proliferation was determined Open access by multicolor flow cytometry analysis using MACSQuant Analyzer 10 (Miltenyi Biotec) as described previously. 27…”

“…[53][54][55] Our results are in accordance with recent findings for an Fc-less trivalent, bispecific scDb-scFv fusion protein directed against HER3 and CD3 that, compared with a bivalent bispecific scDb, mediated a strongly increased cytotoxicity toward medium HER3-expressing target cells, which correlated with increased target cell binding, while cytokine release was unaffected. 27 Thus, cytotoxicity mediated by the best (1-2)+1 scDb/scFv-Fc molecule was only reduced 3-fold to 13-fold to that of the scDb-scFv fusion protein described in the previous study, for example, with an EC 50 value of 3 nM for the (1-2)+1 scDb/scFv-Fc and 1 nM for scDb-scFv for MCF-7 and 3 nM vs 41 nM for LIM1215, respectively. 27 Of note, only medium HER3-expressing cells were lysed by the (1-2)+1 scDb/scFv-Fc molecule, while low HER3-expressing cells remained basically unaffected.…”

“… 17 24–26 We have recently demonstrated that a small trivalent, bispecific single-chain diabody (scDb)-scFv showed superior binding to target expressing tumor cells translating into more efficient target cell killing by peripheral blood mononuclear cells (PBMCs). 27 Small bispecific formats such as BiTEs, 28 dual-affinity re-targeting antibodies (DARTs) 29 and scDb 30 have been reported to mediate tight contacts between target cell and T-cells due to their small size and the short distance between the two binding sites, resulting in efficient T-cell activation. However, their pharmacokinetic properties are characterized by a very short serum half-life and continuous infusion is necessary.…”