Received: 12 December 2018 | Revised: 4 February 2019 | Accepted: 14 February 2019 DOI: 10.1002/jcp.28453 ORIGINAL RESEARCH ARTICLE Novel trastuzumab‐DM1 conjugate: Synthesis and bio‐evaluation Mehri Abedi 1 | Reza Ahangari Cohan 2 | Fereidoun Mahboudi 1 | Mohammad Ali Faramarzi 3 | Ramin Fazel 4 | Narges Damavandi 4 | Mehdi Shafiee Ardestani 5 | Fatemeh Davami 1 1 Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran 2 Department of Pilot Nanobiotechnology, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran 3 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy & Biotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran 4 Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran 5 Department of Radiopharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Correspondence Mehdi Shafiee Ardestani, Department of Radiopharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 3183815194, Iran. Email: shafieeardestani@tums.ac.ir; Fatemeh Davami, Biotechnology Research Center, Pasteur Institute of Iran, Tehran 1316943551, Iran. Email: f_davami@pasteur.ac.ir Abstract Antibody‐drug conjugates are now of considerable interest and are recommended for the treatment of cancers. Linkers are having a crucial role in potency and efficacy of these drugs. Herein, for the first time, we have used a water‐soluble poly‐ethylene glycol based linker (succinimidyl‐[(N‐maleimido propionamido)‐diethyleneglycol] [SM (PEG)2]) for lysine amide coupling of DM1 drug to trastuzumab considering evaluation of the effect of using a hydrophilic linker on physicochemical and biological properties of the resulting conjugate in comparison to the conjugate containing succinimidyl 4‐(N‐maleimidomethyl) cyclohexane‐1‐carboxylate (SMCC) linker, which has a relative hydrophobic nature. The physicochemical properties of synthesized conjugates were investigated in terms of drug to antibody ratio, size variants and free drug quantities. In vitro biological activity of trastuzumab‐DM1 conjugates was assessed on breast cancer cell lines expressing different levels of HER2 using binding affinity, antiproliferative, apoptosis, and antibody‐dependent cell‐ mediated cytotoxicity (ADCC) assays. Synthesized conjugate containing hydrophilic linker, showed higher drug to antibody ratio, no aggregated form and higher cellular toxicity in comparison to SMCC bearing conjugate. Binding affinity and ADCC potential of conjugates was not affected upon the usage of hydrophilic linker. In conclusion, application of SM(PEG)2 for coupling of DM1 to trastuzumab enhance desirable characteristics of the resulting conjugate. KEYWORDS antibody‐drug conjugate, hydrophilic linker, trastuzumab, trastuzumab‐DM1 conjugates, TSPD2 1 | INTRODUCTION The application of chemotherapeutic agents as traditional cancer therapy is clinically restricted due to their narrow therapeutic indices, which, in turn, leads to severe systemic adverse effects in patients suffering from malignancies (Burris, Tibbitts, Holden, Sliwkowski, & Phillips, 2011; Verstappen, Heimans, Hoekman, & Postma, 2003). Antibody‐drug conjugates (ADCs) bring together the cytotoxic effect of chemotherapy and the specificity of monoclonal antibodies (mAbs). The anticancer activity of chemical component of such biopharmaceuticals is conducted by specific targeting of tumor‐ associated antigens by mAbs, providing a higher potency in killing cancer cells while leaving normal tissues unaffected (Burris et al., 2011; Chari, 1998, 2007; Ducry & Stump, 2009; Junutula et al., 2008; Lambert, 2005; Nolting, 2013). ADCs are developed by conjugation of specific mAbs with a chemotherapy agent through various linkers, producing ADCs with a defined drug to antibody ratio (DAR). Since many of the cytotoxic agents that are being used in the development J Cell Physiol. 2019;1–8. wileyonlinelibrary.com/journal/jcp © 2019 Wiley Periodicals, Inc. | 1