Design and Validation of Linkers for Site-Specific Preparation of Antibody-Drug Conjugates Carrying Multiple Drug Copies Per Cysteine Conjugation Site.

Amit Kumar,Changshou Gao,Nazzareno Dimasi,Shenlan Mao

doi:10.3390/ijms21186882

Amit Kumar, Changshou Gao + Show 2 more

Open Access

https://doi.org/10.3390/ijms21186882

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Abstract

First-generation cysteine-based site-specific antibody–drug conjugates (ADCs) are limited to one drug per cysteine. However, certain applications require a high drug to antibody ratio (DAR), such as when low-potency payloads are used. Higher drug load can be achieved using classical cysteine conjugation methods, but these result in heterogeneity, suboptimal efficacy and pharmacokinetics. Here, we describe the design, synthesis and validation of heterobifunctional linkers that can be used for the preparation of ADCs with a DAR of two, three and four in a site-specific manner per single cysteine conjugation site, resulting in site-specific ADCs with a DAR of four, six and eight. The designed linkers carry a sulfhydryl-specific iodoacetyl reactive group, and multiple cyclic diene moieties which can efficiently react with maleimide-carrying payloads through the Diels–Alder reaction. As a proof of concept, we synthesized site-specific DAR four, six and eight ADCs carrying tubulysin (AZ13601508) using engineered antibodies with a cysteine inserted after position 239 in the antibody CH2 domain. We evaluated and compared the in vitro cytotoxicity of ADCs obtained via the site-specific platform described herein, with ADCs prepared using classical cysteine conjugation. Our data validated a novel cysteine-based conjugation platform for the preparation of site-specific ADCs with high drug load for therapeutic applications.

Highlights

Antibody–drug conjugates (ADCs) are a class of therapeutic agents that are comprised of a monoclonal antibody tethered to a potent cytotoxic molecule through a chemical linker
The Food and Drug Administration (FDA) approval of several ADCs has provided tremendous impetus to research in this field
Reduced liquid chromatography mass spectrometry analysis, which was used to determine conjugation at the light or heavy chain and the drug to antibody ratio (DAR), was performed on an Agilent 1290 series uHPLC coupled to an Agilent 6230 TOF

Summary

Introduction

Antibody–drug conjugates (ADCs) are a class of therapeutic agents that are comprised of a monoclonal antibody tethered to a potent cytotoxic molecule through a chemical linker. Most ADCs approved or in clinical development employ traditional conjugation methods that are either lysine–amide coupling or cysteine–maleimide conjugation to tether the cytotoxic molecule to the antibody. These methods, though simple to use, produce heterogeneous conjugates complicating analytical characterization and batch reproducibility. SScchheemmaattiicc rreepprreesseennttaattiioonn ooff tthhee ssttrraatteeggyy ffoorr aattttaaiinniinngg hhiigghh--ddrruugg ttoo aannttiibbooddyy rraattiioo ((DDAARR)) aannttiibbooddyy––ddrruugg ccoonnjjuuggaatteess ((AADDCCss)) iinn aa ssiittee--ssppeecciifificc mmaannnneerr. IInn tthhee sseeccoonndd sstteepp ooff tthhee ttwwoo--sstteepp ccoonnjjuuggaattiioonn mmeetthhoodd,, tthhee aannttiibbooddyy––lliinnkkeerr ccoonnjjuuggaatteess wweerree rreeaacctteedd wwiitthh aa mmaalleeiimmiiddee ppaayyllooaadd. WWee cchhoossee AAZZ1133660011550088 ((AAZZ--11550088)),, aa ttuubbuullyyssiinn wwaarrhheeaadd aanndd mmaalleeiimmiiddee--bbaasseedd lliinnkkeerr ffoorr ddeemmoonnssttrraattiinngg tthhee pprrooooff ooff ccoonncceepptt ((FFiigguurree 33)) [[1177]]. Figure 5F.igIunrev5it.rIon vciytrtootcoyxtoictoitxyicaitsysaasysapyepreforfromrmeeddoonn (aa))HHERE2R22+2-e+x-perxespsrinesgsMinDgAM-MDBA-3-6M1 cBe-l3l 6li1necuelslinligne using site-spescitiefi-cspaellcyifidcaelrlyivdeedrivtreadsttruazstuumzuambabcocnonjujuggaatteess ccaarrrryyininggAAZ-Z15-01850b8) M(bD) AM-MDBA-3-6M1 Bce-l3l6l1ineceulslilnigne using site-spesciitfie-csaplelcyifidcaelrliyvdeedriNveIdPN22IP82c2o8ncjoungjuagtaetsescacarrryyiinng AAZZ--11550808c)(Hc)eHr2e1r+2T14+7DTC4e7lDl bCreealslt bcarnecaesrt cceallncer cell line usinlingesuitsein-sgpseitcei-fispceaclilfyicadlelyridveerdivetdratsrtaustzuuzummaabbccoonjjuuggaatetesscacraryrrinyginAgZA-1Z50-815as08thaesptahyelopadaydl)oHaedr2(d) Her2 1+ T47D1+CTe4l7lDbrCeealsltbcraeansctecracneclelrlcinelel luinseinugsisnigtes-istep-escpieficicfaiclalyllyddeerriviveedd NNIIPP222288ccoonnjujugagtaestecsarcrayrirnyginAgZ-AZ-1508

Synthesis

General Procedure for Linker Conjugation to the Antibody

General Procedure for Payload Conjugation to the Linker Antibody Construct

ADC Characterization

Conclusions