German Edition: DOI: 10.1002/ange.201411507 Antibody Modification International Edition: DOI: 10.1002/anie.201411507 AVersatile Approach for the Site-Specific Modification of Recombinant Antibodies Using a Combination of Enzyme-Mediated Bioconjugation and Click Chemistry** Karen Alt,* Brett M. Paterson, Erik Westein, Stacey E. Rudd, Stan S. Poniger, Shweta Jagdale, Katie Ardipradja, Timothy U. Connell, Guy Y. Krippner, Ashish K. N. Nair, Xiaowei Wang, Henri J. Tochon-Danguy, PaulS. Donnelly,* Karlheinz Peter, and ChristophE. Hagemeyer* Abstract: A unique two-step modular system for site-specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclo- octyne functional groups. The second step of this modular approach involves the azide–alkyne cycloaddition click reac- tion. The versatility of the two-step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules. The attachment of small molecules, nanoparticles, and imaging agents to recombinant proteins such as antibodies offers exciting possibilities in the quest for better diagnostics and therapeutics. [1] Conventional strategies include reacting electrophilic groups such as isothiocyanates and carboxylic acids with the amine group of lysine, [2] or Michael addition with the thiol present in cysteine. [3] Under certain conditions, reactions involving sulfhydryl groups are reversible in vivo [4] and non-specific conjugation to lysine can compromise protein function. [2, 5] In contrast, site-specific conjugation provides homogeneity and improved outcomes in the reten- tion of biological function. [6] Orthogonal groups for site- specific conjugation can be introduced into proteins by genetic modification using amber codons and feeding the host organism amino acids with orthogonal groups. [7, 8] This approach requires special plasmids, expression systems, and expensive cell media supplements, and in some cases overall yields are low. [9] Enzymatic protein modification is emerging as an attractive alternative as it can generate high yields while proceeding under mild conditions. Among other approaches, [10, 11] the Sortase A (SrtA) enzyme from Staph- ylococcus aureus has been extensively used for protein engineering [12, 13] and antibody modification. [14–18] SrtA recog- nizes substrate proteins bearing a short recognition motif (LPXTG) and cleaves the peptide between threonine and glycine forming a new bond with nucleophiles containing N- terminal glycine residues. [19] In most cases the reaction does not interfere with function; however, to obtain high yields, a high molar excess of SrtA and the nucleophile over the LPXTG substrate is required, which is one of the main shortcomings of this approach. Herein we present the use of SrtA for the site-specific incorporation of orthogonal alkyne functional groups into single-chain antibodies (scFvs) to enable further modification by cycloaddition click reactions. The Cu I -catalyzed azide–alkyne cycloaddition (CuAAC), a variant of the Huisgen azide–alkyne cycloaddition, produ- ces 1,4-substituted triazoles from the reaction of azides and terminal alkynes with essentially perfect regioselectivity. [20, 21] In instances where it is necessary to avoid the use of Cu I as a catalyst, the strain-promoted azide–alkyne cycloaddition (SPAAC) reaction of azides with strained cyclooctynes is a bioorthogonal alternative (Figure 1). [22] [*] Dr. K. Alt, S. Jagdale, K. Ardipradja, Dr. G.Y. Krippner, Prof. C. E. Hagemeyer [+] Vascular Biotechnology, Baker IDI Melbourne (Australia) E-mail: karen.alt@bakeridi.edu.au christoph.hagemeyer@bakeridi.edu.au Dr. B. M. Paterson, S. E. Rudd, T. U. Connell, Prof. P. S. Donnelly School of Chemistry/Bio21 Institute University of Melbourne (Australia) E-mail: pauld@unimelb.edu.au Dr. E. Westein, A. K. N. Nair, Dr. X. Wang, Prof. K. Peter [+] Atherothrombosis and Vascular Biology, Baker IDI Melbourne (Australia) S. S. Poniger, Prof. H. J. Tochon-Danguy Department of Molecular Imaging and Therapy, Austin Health Melbourne (Australia) [ + ] These authors contributed equally to this work. [**] This work was funded by the National Health and Medical Research Council (NHMRC), Grants 1029249, 1017670 and 1011418 as well as the Australian Research Council (P.S.D.). K.Al. is supported by the German Research Foundation (Al 1521/1-1). B.M.P. is sup- ported by a Victorian Postdoctoral Research Fellowship funded by the Victorian Government. K.Ar. is supported by the NHMRC and the National Heart Foundation (586740). P.S.D. is an Australian Research Council Future Fellow. K.P. is a Principal Research Fellow of the NHMRC. C.E.H. is a National Heart Foundation Career Development Fellow. This research was undertaken using equip- ment provided by Monash Biomedical Imaging, Monash University as part of the Victorian Biomedical Imaging Capability (Victorian Government). The work was also supported in part by the Victorian Government’s Operational Infrastructure Support Program, Victo- ria’s Science Agenda Strategic Project Fund, and the PET Solid Target Laboratory, an ANSTO-Austin-LICR Cyclotron Partnership. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201411507. A ngewandte Chemi e 1 Angew. Chem. Int. Ed. 2015, 54,1–6  2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! Ü Ü