Use of a next generation maleimide in combination with THIOMAB™ antibody technology delivers a highly stable, potent and near homogeneous THIOMAB™ antibody-drug conjugate (TDC)

Abstract

Herein we demonstrate that conjugation of a next generation maleimide (NGM) to engineered cysteines in a THIOMAB™ antibody delivers a THIOMAB™ antibody-drug conjugate (TDC) with a drug loading of ca. 2. This TDC is highly stable in blood serum conditions, selective and potent towards HER2 expressing cell lines and meets the current criteria for optimised antibody-drug conjugates (ADCs).Antibody-drug conjugates (ADCs) have emerged as targeted therapeutics against cancer by combining the selectivity of the antibody component with the potency of cytotoxic drugs. There are two FDA-approved ADCs already on the market (Adcetris™ and Kadcyla™) and over 40 additional ADCs currently undergoing clinical trials. 1-4Classical methods of conjugation for producing ADCs include lysine modication 5 and cysteine modication via reduction of solvent accessible disulde bonds followed by reaction with maleimides.6 It has been recognised that both methods generate heterogeneous mixtures of ADCs with different drug loadings and varied sites of attachment, resulting in sub-optimal therapeutic indices and higher clearance rates.7-13 Formation of higher drug-to-antibody ratio species (DAR > 4) have narrower therapeutic indices. 1,7,9,12,14 The latter method also results in the loss of structural disulde bonds which can lead to poor stability. 7,9,14The next generation of conjugation methods has aimed to reduce heterogeneity by focusing on site-selectivity. Chemoenzymatic methods have been used for ADC synthesis such as glycan modication, 15-17 glutamine amidation 18 and cysteine oxidation to aldehyde tags.19 Site-selective disulde modica-tion with small molecules has been achieved using nextgeneration maleimides (NGMs), 20-27 pyridazinediones (PDs)28-32 and bis-sulfones. 33,34 Site-specic reactivity has also been accomplished through incorporation of non-natural aminoacids into proteins for bioorthogonal conjugation. 10,35-39The THIOMAB™ antibody technology platform uses sitedirected mutagenesis to incorporate cysteines into the antibody. Conjugation to a classical maleimide bearing the drug of choice affords a THIOMAB™ antibody-drug conjugate (TDC) with full control over site reactivity and a precise drug-toantibody ratio (DAR).12,40-42 The technology also avoids issues with some chemoenzymatic methods that can result in over oxidation of aminoacids 43 and aldehyde tag conversion to unreactive gem-diols.44 TDCs have been demonstrated to improve therapeutic indices and pharmacodynamics over ADCs prepared by classical cysteine and lysine modication. 11,12,42Maleimides are rapid thiol-selective conjugation reagents.45,46 However, the succinimide conjugates thus formed can undergo retro-Michael reactions and scavenging with albumin, 47 resulting in premature drug release during blood circulation.10,41,48 N-Alkyl succinimide conjugates can be stabilised against retro-Michael reactions by hydrolysis to succinamic acid derivatives, 49-51 which resulted in ADCs with improved pharmacokinetics and in vivo efficacy. 41,51,52 Most Nalkyl t...

Bioconjugation with Maleimides: A Useful Tool for Chemical Biology

“…In proteins, bromomaleimides demonstrate high levels of chemoselectivity towards thiol functionalization over nitrogen alkylation, as exemplified by the exclusive modification of a cysteine residue in Grb2 mutants (L111C) featuring eight solvent exposed lysine residues . The effectiveness of this Michael acceptor has been further exemplified by enabling access to highly homogenous THIOMAB trastuzumab constructs (DAR=2) via single cysteine functionalization . The resulting constructs promptly underwent stabilization through the hydrolysis of the thiomaleimide ring (<1 h, pH 8), most likely due to the presence of local cationic charges near to the bioconjugation site .…”

“…Eur.J. 2019,25,[43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59] www.chemeurj.org Review diverging froma liphatic maleimides.T he simple rational behind this novel strategy relied on the presence of an adjacent basic amino group to promote intramolecular base catalyzed hydrolysis. [25] To study this, the authors preparedasmall subset of maleimides containing af ree amine in the N-alkyl chain, allowing the distance between the two nitrogen atoms to increase alongside with the side-chain length.…”

“…[44] The effectiveness of this Michael acceptorh as been further exemplified by enabling accesst oh ighly homogenous THIOMAB trastuzumab constructs (DAR = 2) via single cysteine functionalization. [45] The resulting constructs promptly underwent stabilization through the hydrolysis of the thiomaleimide ring (< 1h,p H8), most likely due to the presence of local cationic charges near to the bioconjugation site. [34] In comparison to classical maleimides, bromomaleimides provided access of well-definedA DC constructs with superior stability,a st hiosuccinimide-based constructs requirel ongers tabilization procedures at higherp H values( > 16 h, pH > 8).…”

“…Eur.J. 2019,25,[43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59] www.chemeurj.org Given the reversible nature of 5MP-based bioconjugations, they have been showcased in protein pull-down strategies (Scheme 22 C). As olid support featuring primary amines in its surfacew as condensed with a5 MP precursor 101 to form immobilized 5MP 102.T hen, the first protein of interest (IlvN) was quantitatively immobilizedi nt he solids upport (< 10 min), while the remaining unreacted 5MP sites were capped with BME, ahead of incubation with ilvB (4 8C, 1h).…”

“…Scheme6.Intramolecular acid catalysis for rapid thiosuccinimide hydrolysis.Scheme7.The impact of acetal containingmaleimide in plasma stability.Chem. Eur.J 2019,25,[43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59]. www.chemeurj.org2019 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim 48 Review…”