Effect of Linker Stereochemistry on the Activity of Indolinobenzodiazepine Containing Antibody–Drug Conjugates (ADCs)

Reid, Emily E.; Archer, Katie E.; Shizuka, Manami; Wilhelm, Alan; Yoder, Nicholas C.; Bai, Chen; Fishkin, Nathan; Harris, Luke; Maloney, Erin K.; Salomon, Paulin; Hong, Erica; Wu, Rui; Ab, Olga; Jin, Songqing; Lai, Katharine C.; Sikka, Surina; Chari, Ravi; Miller, Michael L.

doi:10.1021/acsmedchemlett.9b00240

Cited by 9 publications

(13 citation statements)

References 14 publications

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“…The preparation of target compound 1 involved chemical synthesis of protected tetrapeptide 4 from two peptide fragments 2 ( Denoel et al, 2014 ) and 3 ( Reid et al, 2019 ) followed by global deprotection and selective acylation of the amino group in the intermediate 5 with pentafluorophenyl ester of N-acetylglucosaminyl-(β1-4)-N-acetylmuramic acid. The activated ester was prepared from the free acid 6 using bis(pentafluorophenyl) carbonate following the procedure described for esterification of the analogous saccharide ( Jezek et al, 1990 ).…”

Section: Resultsmentioning

confidence: 99%

Chemical synthesis of peptidoglycan mimetic–disaccharide-tetrapeptide conjugate and its hydrolysis by bacteriophage T5, RB43 and RB49 L-alanyl-D-glutamate peptidases

Азев

Chulin

Молчанов

et al. 2021

PeerJ

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Background Endolysins of a number of bacteriophages, including coliphages T5, RB43, and RB49, target the peptidoglycans of the bacterial cell wall. The backbone of these bacterial peptidoglycans consist of alternating N-acetylglucosamine and N-acetylmuramic acid residues that is further “reinforced” by the peptide subunits. Because of the mesh-like structure and insolubility of peptidoglycans, the processes of the peptidoglycan binding and hydrolysis by enzymes cannot be studied by spectral methods. To overcome these issues we synthesized and analyzed here one of the simplest water soluble peptidoglycan mimetics. Methods A compound has been synthesized that mimics the peptidoglycan fragment of the bacterial cell wall, N-acetylglucosaminyl-β(1-4)-N-acetylmuramoyl-l-alanyl-γ-d-glutamyl-l-alanyl-d-alanine. NMR was used to study the degradation of this peptidoglycan mimetic by lytic l-alanoyl-d-glutamate peptidases of colibacteriophages T5, RB43, and RB49 (EndoT5, EndoRB43, and EndoRB49, respectively). Results The resulting glycopeptide mimetic was shown to interact with the studied enzymes. Its hydrolysis occurred through the bond between l-Ala and d-Glu. This artificial substrate mimetic was hydrolyzed by enzymes at different rates, which decreased outside the pH optimum. The EndoT5 demonstrated the lowest hydrolysis rate, whereas the EndoRB49-driven hydrolysis was the fastest one, and EndoRB43 displayed an intermediate potency. These observations are consistent with the hypothesis that EndoRB49 is characterized by the lowest selectivity, and hence the potentially broader spectrum of the peptidoglycan types subjected to hydrolysis, which was put forward in the previous study. We also show that to hydrolyze this glycopeptide mimetic, enzymes approach the glycopeptide near the methyl groups of all three alanines.

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Section: Resultsmentioning

confidence: 99%

Chemical synthesis of peptidoglycan mimetic–disaccharide-tetrapeptide conjugate and its hydrolysis by bacteriophage T5, RB43 and RB49 L-alanyl-D-glutamate peptidases

Азев

Chulin

Молчанов

et al. 2021

PeerJ

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“…However, it has been previously reported that the D-Ala-D-Ala linker anti-FRα-IGN ADC was about 90-fold less potent than the corresponding L-Ala-L-Ala linker ADC in a low FRα-antigen expressing H2110 cell line. 22 Presumably, poor lysosomal processing of D-Ala-D-Ala linker resulted in catabolite accumulation below the threshold level required for significant cytotoxic activity.…”

Section: ■ Discussionmentioning

confidence: 99%

“…21 In a previous study, an L-Ala-L-Ala peptide linker ADC with an IGN payload showed ∼90-fold greater cytotoxic potency in a low antigen cell line in vitro compared to a D-Ala-D-Ala peptide linker ADC. 22 In this study, with the goals of maximizing the rate of lysosomal activation of ADCs while limiting peptide length and ensuring plasma stability, we screened multiple dipeptide linkers for rates of protease cleavage and compared them to the linkers currently being evaluated in the clinic, Val-Cit, Val-Ala, Ala-Ala, and a noncleavable linker. Our study identified new Lamino acid dipeptide sequences that were rapidly and specifically cleaved by lysosomal proteases and cathepsins.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Several aliphatic tripeptide and tetrapeptide linkers were screened to map the substrate specificities of multiple cysteine and serine proteases, but not as anilino-peptide ADC linkers. , An ADC bearing a maytansinoid payload conjugated via a triglycyl peptide linker was reported to show greater cytotoxic activity in vitro in multiple cell lines, and improved efficacy in several in vivo tumor xenograft models compared to that with a noncleavable linker . In a previous study, an l -Ala- l -Ala peptide linker ADC with an IGN payload showed ∼90-fold greater cytotoxic potency in a low antigen cell line in vitro compared to a d -Ala- d -Ala peptide linker ADC …”

Section: Introductionmentioning

confidence: 99%

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Optimizing Lysosomal Activation of Antibody–Drug Conjugates (ADCs) by Incorporation of Novel Cleavable Dipeptide Linkers

et al. 2019

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Although peptide linkers are used in multiple clinical-stage ADCs, there are only few reports on optimizing peptide linkers for efficient lysosomal proteolysis and for stability in circulation. We screened multiple dipeptide linkers for efficiency of proteolysis and compared them to the dipeptide linkers currently being evaluated in the clinic: Val-Cit, Val-Ala, and Ala-Ala. Lead dipeptide linkers selected from the initial screen were incorporated into ADCs with indolinobenzodiazepine dimer (IGN) payloads to evaluate cellular processing, in vitro cytotoxic activity, plasma stability, and in vivo efficacy. ADCs with several dipeptide linkers bearing l-amino acids showed faster lysosomal processing in target cancer cells compared to the l-Ala-l-Ala linked ADC. These variances in linker processing rates did not result in different in vitro and in vivo activities among peptide linker ADCs, presumably due to accumulation of threshold cytotoxic catabolite levels for ADCs of several peptide linkers in the cell lines and xenografts tested. ADCs with l-amino acid dipeptide linkers exhibited superior in vitro cytotoxic potencies in multiple cell lines compared to an ADC with a d-Ala-d-Ala dipeptide linker and an ADC with a noncleavable linker. This work adds to the toolbox of stable, lysosomally cleavable peptide linkers for ADCs.

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“…In stereochemical studies, Reid et al . 24 and Salomon et al. 25 both indicated that ADCs containing an ( l , l ) dipeptide linker showed higher antitumor activity in vitro and in vivo than other amino acid configurations.…”

Section: Chemical Triggers Of the Linkermentioning

confidence: 99%

Antibody–drug conjugates: Recent advances in linker chemistry

Xiao²,

Xie³

et al. 2021

Acta Pharmaceutica Sinica B

159

114

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Antibody–drug conjugates (ADCs) are gradually revolutionizing clinical cancer therapy. The antibody–drug conjugate linker molecule determines both the efficacy and the adverse effects, and so has a major influence on the fate of ADCs. An ideal linker should be stable in the circulatory system and release the cytotoxic payload specifically in the tumor. However, existing linkers often release payloads nonspecifically and inevitably lead to off-target toxicity. This defect is becoming an increasingly important factor that restricts the development of ADCs. The pursuit of ADCs with optimal therapeutic windows has resulted in remarkable progress in the discovery and development of novel linkers. The present review summarizes the advance of the chemical trigger, linker‒antibody attachment and linker‒payload attachment over the last 5 years, and describes the ADMET properties of ADCs. This work also helps clarify future developmental directions for the linkers.

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Effect of Linker Stereochemistry on the Activity of Indolinobenzodiazepine Containing Antibody–Drug Conjugates (ADCs)

Cited by 9 publications

References 14 publications

Chemical synthesis of peptidoglycan mimetic–disaccharide-tetrapeptide conjugate and its hydrolysis by bacteriophage T5, RB43 and RB49 L-alanyl-D-glutamate peptidases

Chemical synthesis of peptidoglycan mimetic–disaccharide-tetrapeptide conjugate and its hydrolysis by bacteriophage T5, RB43 and RB49 L-alanyl-D-glutamate peptidases

Optimizing Lysosomal Activation of Antibody–Drug Conjugates (ADCs) by Incorporation of Novel Cleavable Dipeptide Linkers

Antibody–drug conjugates: Recent advances in linker chemistry

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