Mild Method for Succinimide Hydrolysis on ADCs: Impact on ADC Potency, Stability, Exposure, and Efficacy

Tumey, L. Nathan; Charati, Manoj B.; He, Tao; Sousa, Eric; Ma, Dangshe; Han, Xiaogang; Clark, Tracey; Casavant, Jeff; Loganzo, Frank; Barletta, Frank; Lucas, Judy; Graziani, Edmund I.

doi:10.1021/bc500357n

Cited by 153 publications

(179 citation statements)

References 18 publications

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“…We were interested whether mouse Ces1C can catalyze hydrolysis of the VC-PABC linker in the context of maleimide-coupled conjugates. Analysis of maleimide-linked ADC metabolites after in vivo or in vitro plasma incubation can be challenging due to the heterogeneous nature of conventional conjugates, as well as maleimide ring opening or maleimide decoupling that can occur in plasma environment (7,26,27). We therefore utilized purified mouse Ces1C in the absence of plasma to examine its ability to cleave the maleimide-caproyl-VC-PABC-Aur0101 (mc-VC-PABCAur0101) linker-payload chemically coupled to the native cysteines of the C16 antibody ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Molecular Basis of Valine-Citrulline-PABC Linker Instability in Site-Specific ADCs and Its Mitigation by Linker Design

Dorywalska

Dushin

Moine

et al. 2016

Molecular Cancer Therapeutics

149

163

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The degree of stability of antibody-drug linkers in systemic circulation, and the rate of their intracellular processing within target cancer cells are among the key factors determining the efficacy of antibody-drug conjugates (ADC) in vivo. Previous studies demonstrated the susceptibility of cleavable linkers, as well as auristatin-based payloads, to enzymatic cleavage in rodent plasma. Here, we identify Carboxylesterase 1C as the enzyme responsible for the extracellular hydrolysis of valine-citrulline-paminocarbamate (VC-PABC)-based linkers in mouse plasma. We further show that the activity of Carboxylesterase 1C towards VC-PABC-based linkers, and consequently the stability of ADCs in mouse plasma, can be effectively modulated by small chemical modifications to the linker. While the introduced modifications can protect the VC-PABC-based linkers from extracellular cleavage, they do not significantly alter the intracellular linker processing by the lysosomal protease Cathepsin B. The distinct substrate preference of the serum Carboxylesterase 1C offers the opportunity to modulate the extracellular stability of cleavable ADCs without diminishing the intracellular payload release required for ADC efficacy. Mol Cancer Ther; 15(5); 958-70. Ó2016 AACR.

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Section: Resultsmentioning

confidence: 99%

Molecular Basis of Valine-Citrulline-PABC Linker Instability in Site-Specific ADCs and Its Mitigation by Linker Design

Dorywalska

Dushin

Moine

et al. 2016

Molecular Cancer Therapeutics

149

163

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“…Low-resolution mass spectra (LRMS) were recorded on a ThermoScientific Advantage LCQ Linear ion-trap electrospray or a Waters LCMS consisting of a 2767 Sample manager, 2525 pump, 2996 UV-detector, and a Micromass ZQ with an Xbridge C18 3.5 μm column (ESI). 3,4,6-Tri-O-acetyl-2-azido-2-deoxy-α-D-galactose-1-phosphate (9). Compound 9 was prepared from D-galactosamine according to the procedure described for D-glucosamine by Linhardt et al 46 The resulting tributylammonium uridine-5′-monophosphate was dissolved in dry DMF (25 mL) under an argon atmosphere.…”

Section: ■ Experimental Proceduresmentioning

confidence: 99%

Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody–Drug Conjugates

Geel¹,

Wijdeven²,

Heesbeen³

et al. 2015

Bioconjugate Chem.

208

190

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A robust, generally applicable, nongenetic technology is presented to convert monoclonal antibodies into stable and homogeneous ADCs. Starting from a native (nonengineered) mAb, a chemoenzymatic protocol allows for the highly controlled attachment of any given payload to the N-glycan residing at asparagine-297, based on a two-stage process: first, enzymatic remodeling (trimming and tagging with azide), followed by ligation of the payload based on copper-free click chemistry. The technology, termed GlycoConnect, is applicable to any IgG isotype irrespective of glycosylation profile. Application to trastuzumab and maytansine, both components of the marketed ADC Kadcyla, demonstrate a favorable in vitro and in vivo efficacy for GlycoConnect ADC. Moreover, the superiority of the native glycan as attachment site was demonstrated by in vivo comparison to a range of trastuzumab-based glycosylation mutants. A side-by-side comparison of the copper-free click probes bicyclononyne (BCN) and a dibenzoannulated cyclooctyne (DBCO) showed a surprising difference in conjugation efficiency in favor of BCN, which could be even further enhanced by introduction of electron-withdrawing fluoride substitutions onto the azide. The resulting mAb-conjugates were in all cases found to be highly stable, which in combination with the demonstrated efficacy warrants ADCs with a superior therapeutic index.

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“…41,51,52 Most Nalkyl thiosuccinimide ADCs require prolonged hydrolysis (>16 h) at high pH (>8) 41,51 which is not desirable from a process view and can lead to deamidation and loss of payload.…”

Section: 1242mentioning

confidence: 99%

“…10,41,48 N-Alkyl succinimide conjugates can be stabilised against retro-Michael reactions by hydrolysis to succinamic acid derivatives, [49][50][51] which resulted in ADCs with improved pharmacokinetics and in vivo efficacy. 41,51,52 Most Nalkyl thiosuccinimide ADCs require prolonged hydrolysis (>16 h) at high pH (>8) 41,51 which is not desirable from a process view and can lead to deamidation and loss of payload.…”

Section: 35-39mentioning

confidence: 99%

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)

et al. 2017

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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...

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Mild Method for Succinimide Hydrolysis on ADCs: Impact on ADC Potency, Stability, Exposure, and Efficacy

Cited by 153 publications

References 18 publications

Molecular Basis of Valine-Citrulline-PABC Linker Instability in Site-Specific ADCs and Its Mitigation by Linker Design

Molecular Basis of Valine-Citrulline-PABC Linker Instability in Site-Specific ADCs and Its Mitigation by Linker Design

Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody–Drug Conjugates

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)

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