COMMUNICATION Monodisperse Polysarcosine-based Highly-loaded Antibody-Drug Conjugates

We report the synthesis of monodisperse (i.e. discrete) polysarcosine compounds and their use as a hydrophobicity masking entity for the construction of highly-loaded homogeneous βglucuronidase-responsive antibody-drug conjugates (ADCs). The highly hydrophilic drug-linker platform described herein improves drug-loading, physicochemical properties, pharmacokinetics and in vivo antitumor efficacy of the resulting conjugates. Antibody-drug conjugates (ADCs) represent an emerging class of oncology therapeutics, with 4 ADCs already on the market and more than 80 currently under clinical evaluation for various cancer indications.[1] Expected improvements in this field rely on the design of novel drug-linker technologies that strongly influence the physicochemical properties of the conjugates.[2] Key parameters such as (i) plasmatic stability of the drug-linker[3], (ii) Drug-Antibody-Ratio (DAR)[4], (iii) conjugation position on the antibody component[5], (iv) overall hydrophobicity[6] and homogeneity[7] of the conjugates dictates pharmacokinetics (PK) properties, efficacy and tolerability of ADCs. Within this framework, it has long been considered that a 2-to-4 cytotoxic payload per antibody ratio (DAR2-4) achieves the optimal balance among pharmacokinetics and in vivo potency.[4] Higher DAR species are traditionally known to hamper the therapeutic efficacy of ADCs because of the increased overall hydrophobicity of the conjugate that is conferred by the excessive number of highly hydrophobic drug cargo. In light of this finding, a great emphasis on site-specific bioconjugation technologies aiming to deliver homogeneous DAR2 or DAR4 conjugates is observed in the field and began to translate into the clinic.[8] These techniques require protein genetic re-engineering and/or the use of one or several coupling enzymes to graft the drug-linker payload to the antibody. As a consequence, their implementation is time-consuming, rather expensive and may prove to be difficult to transpose to largescale production. Recently, hydrophilic drug-linker architectures aiming to mask or minimize the apparent hydrophobicity of the payloads and overcome the DAR2-4 limitation have paved the way to a new generation of highly drug-loaded ADCs.[9] These innovations enable improved physicochemical properties, excellent PK profiles, decreased non-specific uptake, protection against payload metabolism, superior efficacy in low-target expressing tumors and allow the use of moderately potent drugs as ADC payloads.[1b,6,10] Furthermore, this approach offers the possibility to obtain homogeneous ADCs without tricky sitespecific conjugation technologies. Underlying all these observations, it is also admitted that a beneficial correlation exists between the overall hydrophilicity and tolerability of ADCs.[6,11] In the present work we envisioned the use of polysarcosine (PSAR) as a hydrophobicity masking entity that would be embedded into an ADC drug-linker platform. Thus, we herein report a novel generation of strongly hydrophilic PSARcontaining β-glucuronidase-responsive self-immolative druglinkers devoted to the preparation of highly-loaded homogeneous ADCs having improved physicochemical and pharmacological properties (Figure 1). In this pilot study, we designed drug-linkers that include the potent monomethyl auristatin E (MMAE) cytotoxin, a glucuronide trigger[12], a selfimmolative linker[13], an auto-hydrolyzable maleimide-based bioconjugation head[14] and a PSAR unit. With this design, we anticipated that the presence of both PSAR and glucuronide hydrophilic moieties would allow the construction of homogenous DAR8 ADCs, programmed for releasing MMAE within targeted cancer cells upon intracellular β-glucuronidase activation. Most of the strategies that have been employed to increase drug-linker hydrophilicity rely on the introduction of polyethylene glycol (PEG)[15], which is to-date the gold standard for improving physicochemical properties of therapeutic agents but is not exempt of several limitations (non-biodegradable backbone and reported cases of hypersensitivity or accelerated blood clearance).[16] Other approaches use hydrophilic stealth polymer carriers as drug-linker platforms, thus providing ADCs reaching DAR10-20.[10c,17] The main drawback of these approaches is the extreme polydispersity of the final ADCs, arising from the polydisperse nature of the polymer-linker and the heterogeneous coupling procedure to the antibody. Polysarcosine (PSAR) or poly(N-methylglycine), a polypeptoid based on the endogenous sarcosine aminoacid, is a [a] Dr. W. Viricel Mablink Bioscience SA, 14 rue Waldeck Rousseau, 69006 Lyon, France E-mail: w.viricel@mablink.com [b] Dr. G. Fournet, Prof. B. Joseph Université de Lyon, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires UMR CNRS 5246, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France [c] S. Beaumel, E. Perrial, Prof. C. Dumontet Université de Lyon, Centre de Recherche en Cancérologie de Lyon, INSERM 1052, CNRS 5286, 8 avenue Rockefeller, 69008 Lyon, France [d] Prof. S. Papot Université de Poitiers, Institut de Chimie des Milieux et Matériaux de Poitiers (IC2MP) UMR CNRS 7285, Groupe « Systèmes Moléculaires Programmés », 4 rue Michel-Brunet, TSA 51106, 86073 Poitiers, France [e] Prof. S. Papot Seekyo SA, 4 rue Carol Heitz, 86000 Poitiers, France Supporting information for this article is given via a link at the end of the document.