Preparation of Well‐Defined Antibody–Drug Conjugates through Glycan Remodeling and Strain‐Promoted Azide–Alkyne Cycloadditions

Li, Xiuru; Fang, Tao; Boons, Geert‐Jan

doi:10.1002/ange.201402606

Cited by 41 publications

(23 citation statements)

References 39 publications

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“…Subsequent conjugation of a doxorubicin payload using strain-promoted azide−alkyne cycloaddition (SPAAC) resulted in an ADC with a drug-toantibody ratio (DAR) of 4. 15 Recently, SynAffix reported a strategy in which the N-glycan was partially deglycosylated by endoglycosidase S (EndoS), and fluorinated GalNAz derivatives ( Figure 1) were installed enzymatically using a mutant β1,4-galactosyltransferase (GalT(Y289L)), 13,16 followed by conjugation of monomethyl auristatin F to the antibody using SPAAC, yielding ADCs with a DAR of 2. 17 Herein we describe a one-step chemoenzymatic method to generate ADCs with a drug-to-antibody ratio of 4 using an alkynyl pyrrolobenzodiazepine (PBD) dimer payload (SG3364, Scheme 1) conjugated to an azide-modified GalNAc (GalNAz, Figure 1) using copper-catalyzed click chemistry (Figure 2A,C).…”

mentioning

confidence: 99%

Straightforward Glycoengineering Approach to Site-Specific Antibody–Pyrrolobenzodiazepine Conjugates

Thompson¹,

Ezeadi

Hutchinson

et al. 2016

ACS Med. Chem. Lett.

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Antibody−drug conjugates (ADCs) have become a powerful platform to deliver cytotoxic agents selectively to cancer cells. ADCs have traditionally been prepared by stochastic conjugation of a cytotoxic drug using an antibody's native cysteine or lysine residues. Through strategic selection of the mammalian expression host, we were able to introduce azide-functionalized glycans onto a homogeneously glycosylated anti-EphA2 monoclonal antibody in one step. Conjugation with an alkyne-bearing pyrrolobenzodiazepine dimer payload (SG3364) using copper-catalyzed click chemistry yielded a site-specific ADC with a drug-to-antibody ratio (DAR) of four. This ADC was compared with a glycoengineered DAR two site-specific ADC, and both were found to be highly potent against EphA2-positive human prostate cancer cells in both an in vitro cytotoxicity assay and a murine tumor xenograft model. KEYWORDS: Antibody−drug conjugates, pyrrolobenzodiazepine, SG3364, click chemistry, carbohydrate remodeling, site-specific conjugation A ntibody-drug conjugates (ADCs) are an important class of biologics, which combine the potency of cytotoxic drugs with the specificity of antibodies. To date, there are two clinically approved ADCs, Adcetris and Kadcyla, both of which are stochastically conjugated through either cysteine or lysine residues. 1,2 In addition to the knowledge gained through preclinical and clinical studies, advances in protein engineering and bioorthogonal chemistry have shifted the focus to generating site-specific ADCs, which yield homogeneous products that demonstrate improved in vivo properties. 3−6 Human immunoglobulins have a conserved glycosylation site in the CH2 domain of the heavy chain at position N297, making it an attractive target for generating site-specific antibody−drug conjugates. 7 However, since glycosylation is a heterogeneous post-translational modification, 8−10 remodeling of the glycan is often required before conjugation of the payload. Many reported strategies involve chemical drug conjugation following a multienzymatic process to install either natural or synthetic carbohydrate analogues, such as CMP-9-azido sialic acid and UDP-GalNAz derivatives (Figure 1), onto the antibody glycan. 11−13 Pan et al. described a multistep approach in which first the antibody glycan was remodeled to contain sialic acid residues, which were then chemically oxidized with sodium periodate and conjugated with an auristatin via oxime chemistry. 14 Boons et al.

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mentioning

confidence: 99%

Straightforward Glycoengineering Approach to Site-Specific Antibody–Pyrrolobenzodiazepine Conjugates

Thompson¹,

Ezeadi

Hutchinson

et al. 2016

ACS Med. Chem. Lett.

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“…This indicates the site specificity of DDA formation at the nucleobase level, and thus the number of drugs conjugated on one DNA is determined mainly by the G content, making it programmable to design DNA for DDA-based drug delivery. Even though the conjugation efficiency is not as high as some other reactions, such as click chemistry, 4 the use of intact DNA without any other modification makes this preparation extremely simple and cost-effective, which is promising for future production scale-up. In addition, adduct formation primarily on deoxyguanosine is speculated to cause drug fluorescence quenching in DDAs, given that deoxyguanosine can quench fluorescence of some proximal fluorophores through electron transfer.…”

Section: Resultsmentioning

confidence: 99%

“…2 In contrast, targeted therapy seeks to deliver drugs specifically to cancer cells. 3 Active targeting achieves this by using molecular targeting elements, including antibodies, aptamers, growth factors and vitamins, 4,5 which can specifically bind to overexpressed cognate receptors on target cancer cells. 6 Indeed, antibody-drug conjugates are of intensive interest to the pharmaceutical industry.…”

Section: Introductionmentioning

confidence: 99%

Nuclease-resistant synthetic drug-DNA adducts: programmable drug-DNA conjugation for targeted anticancer drug delivery

et al. 2015

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Targeted drug delivery is poised to improve cancer therapy, for which synthetic DNA can serve as targeting ligands (for example, aptamers) or drug nanocarriers. Inspired by natural DNA adducts, we report synthetic drug-DNA adducts (DDAs) for targeted anticancer drug delivery. Multiple copies of anthracycline drugs were site specifically (on deoxyguanosine) conjugated on each DNA, enabling programmable design of DNA and drugs for DDA preparation. DDAs were nuclease-resistant and stable for storage, yet gradually released drugs at physiological temperature. DDAs maintained DNA functionalities, including hybridizationmediated DNA nanoadduct formation and aptamer-mediated target recognition and targeted drug delivery into cancer cells. In a tumor xenograft mouse model, doxorubicin-aptamer adducts significantly inhibited target tumor growth while reducing the side effects. Using histopathological analysis and in situ immunohistochemical analysis of caspase-3 cleavage in mouse tumor and heart, DDAs were confirmed to have a potent antitumor efficacy while reducing tissue deformation and apoptosis in the heart, thus providing a new therapeutic avenue to prevent cardiomyopathy, the most dangerous side effect of doxorubicin leading to heart failure. Overall, DDAs are promising for scale-up production and clinical application in targeted anticancer drug delivery.

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“…Li et al exploited the very same approach as Zhou but introduced sialic acid derivatives, which have an azide functionality at the C-9 position, instead of normal sialic acid. The azide functionality was then reacted with various functionalities including a toxin to yield homogeneous antibody conjugates [103].…”

Section: Enzymatic and Chemo-enzymatic Modification Of Glycansmentioning

confidence: 99%

Antibody Conjugates: From Heterogeneous Populations to Defined Reagents

2015

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Monoclonal antibodies (mAbs) and their derivatives are currently the fastest growing class of therapeutics. Even if naked antibodies have proven their value as successful biopharmaceuticals, they suffer from some limitations. To overcome suboptimal therapeutic efficacy, immunoglobulins are conjugated with toxic payloads to form antibody drug conjugates (ADCs) and with chelating systems bearing therapeutic radioisotopes to form radioimmunoconjugates (RICs). Besides their therapeutic applications, antibody conjugates are also extensively used for many in vitro assays. A broad variety of methods to functionalize antibodies with various payloads are currently available. The decision as to which conjugation method to use strongly depends on the final purpose of the antibody conjugate. Classical conjugation via amino acid residues is still the most common method to produce antibody conjugates and is suitable for most in vitro applications. In recent years, however, it has become evident that antibody conjugates, which are generated via site-specific conjugation techniques, possess distinct advantages with regard to in vivo properties. Here, we give a comprehensive overview on existing and emerging strategies for the production of covalent and non-covalent antibody conjugates.

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Preparation of Well‐Defined Antibody–Drug Conjugates through Glycan Remodeling and Strain‐Promoted Azide–Alkyne Cycloadditions

Cited by 41 publications

References 39 publications

Straightforward Glycoengineering Approach to Site-Specific Antibody–Pyrrolobenzodiazepine Conjugates

Straightforward Glycoengineering Approach to Site-Specific Antibody–Pyrrolobenzodiazepine Conjugates

Nuclease-resistant synthetic drug-DNA adducts: programmable drug-DNA conjugation for targeted anticancer drug delivery

Antibody Conjugates: From Heterogeneous Populations to Defined Reagents

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