Citation: Huang, S.; Segués, A.; Waterfall, M.; Wright, D.; Vayssiere, C.; van Duijnhoven, S.M.J.; van Elsas, A.; Sijts, A.J.A.M.; Zaiss, D.M. Shortened Hinge Design of Fab x sdAb-Fc Bispecific Antibodies Enhances Redirected T-Cell Killing of Tumor Cells. Biomolecules 2022, 12, 1331. https://doi.org/10.3390/ biom12101331 Academic Editor: Kenichi Suda Received: 25 August 2022 Accepted: 16 September 2022 Published: 21 September 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biomolecules Article Shortened Hinge Design of Fab x sdAb-Fc Bispecific Antibodies Enhances Redirected T-Cell Killing of Tumor Cells Shuyu Huang 1,2,† , Aina Segués 1,2,† , Martin Waterfall 1 , David Wright 1 , Charlotte Vayssiere 1 , Sander M. J. van Duijnhoven 3 , Andrea van Elsas 4 , Alice J. A. M. Sijts 2 and Dietmar M. Zaiss 1,5,6,7, * 1 Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FL, UK 2 Faculty of Veterinary Medicine, Department of Infectious Diseases and Immunology, Utrecht University, 3584 CS Utrecht, The Netherlands 3 ImmunoPrecise Antibodies Ltd., 5349 AB Oss, The Netherlands 4 Third Rock Ventures, San Francisco, CA 94158, USA 5 Department of Immune Medicine, University Regensburg, 93053 Regensburg, Germany 6 Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Regensburg, 93053 Regensburg, Germany 7 Institute of Pathology, University Regensburg, 93053 Regensburg, Germany * Correspondence: dietmar.zaiss@ukr.de † These authors contributed equally to this work. Abstract: T cell engager (TCE) antibodies have emerged as promising cancer therapeutics that link cytotoxic T-cells to tumor cells by simultaneously binding to CD3E on T-cells and to a tumor- associated antigen (TAA) expressed by tumor cells. We previously reported a novel bispecific format, the IgG-like Fab x sdAb-Fc (also known as half-IG_VH-h-CH2-CH3), combining a conventional antigen-binding fragment (Fab) with a single domain antibody (sdAb). Here, we evaluated this Fab x sdAb-Fc format as a T-cell redirecting bispecific antibody (TbsAbs) by targeting mEGFR on tumor cells and mCD3E on T cells. We focused our attention specifically on the hinge design of the sdAb arm of the bispecific antibody. Our data show that a TbsAb with a shorter hinge of 23 amino acids (TbsAb.short) showed a significantly better T cell redirected tumor cell elimination than the TbsAb with a longer, classical antibody hinge of 39 amino acids (TbsAb.long). Moreover, the TbsAb.short form mediated better T cell-tumor cell aggregation and increased CD69 and CD25 expression levels on T cells more than the TbsAb.long form. Taken together, our results indicate that already minor changes in the hinge design of TbsAbs can have significant impact on the anti-tumor activity of TbsAbs and may provide a new means to improve their potency. Keywords: bispecific antibody; cancer immunotherapy; mCD3E; mEGFR; hinge 1. Introduction T-cell engager antibodies (TCEs) redirect cytotoxic T-cells to tumor cells by simul- taneously binding to a component of the TCR complex (commonly CD3E) and a tumor associated antigen (TAA) on tumor cells [1]. Due to the clinical success of the bispecific T-cell engager (BiTE) blinatumomab, approved by the FDA in 2014 [2,3], the majority of bispecific antibodies (BsAbs) in clinical development are currently TCEs [4]. TCEs can further be classified into two broad classes according to their formats: IgG-like or fragment- based TCEs. Currently, the IgG-like T cell redirecting bispecific antibodies (TbsAbs) being the most widely used form, largely due to their longer in vivo serum half-life due to the presence of an Fc region [5]. Although the concept of BsAbs has a long history, due to challenges in BsAb manu- facturing, they only began to stimulate the interest of pharmaceutical companies in the past decade. The production of IgG-like BsAbs requires the correct assembly of antibody’s Biomolecules 2022, 12, 1331. https://doi.org/10.3390/biom12101331 https://www.mdpi.com/journal/biomolecules