Genetically Encoded Azide Containing Amino Acid in Mammalian Cells Enables Site-Specific Antibody–Drug Conjugates Using Click Cycloaddition Chemistry

VanBrunt, Michael Pete; Shanebeck, Kurt; Caldwell, Zachary; Johnson, Jeffrey; Thompson, Pamela; Martin, Tom; Dong, Huifang; Li, Gary; Xu, Hengyu; D’Hooge, François; Masterson, Luke A.; Bariola, Pauline A.; Tiberghien, Arnaud; Ezeadi, Ebele; Williams, David G.; Hartley, John A.; Howard, Philip W.; Grabstein, Kenneth H.; Bowen, Michael A.; Marelli, Marcello

doi:10.1021/acs.bioconjchem.5b00359

Cited by 122 publications

(101 citation statements)

References 74 publications

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“…Incorporation of AzK was accomplished by co-transfection of the antibodies with an orthogonal pylRS/tRNA pair derived from M. mazei in CHO cells (20, 22). In this expression system, incorporation of the nnAA competes with amber stop codon termination, leading to truncated species; therefore purified antibodies were analyzed by mass spectrometry and SDS-PAGE to confirm the AzK incorporation and full-length species (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The amber stop codon (TAG) was cloned into Fc positions using overlap extension PCR and InFusion® HD cloning (Takara 638911). Plasmids encoding the orthogonal pylRS and cognate tRNA for non-natural amino acid incorporation, pCEP4-pylRS and pOriP-9x-tRNA, respectively, were previously described (22). CHO cells (proprietary strain) were transiently co-transfected with the plasmid DNA of the antibody, pylRS, and tRNA at a 30:20:50 ratio using PEI max.…”

Section: Methodsmentioning

confidence: 99%

“…By incorporating the aliphatic nnAA N ϵ -((2-azidoethoxy)carbonyl)- l -lysine (AzK) into the Fc domain, we introduced a bio-orthogonal chemical handle (20). This azide group can then be covalently modified by an antidote containing a strained alkyne through strain promoted azide-alkyne cycloaddition (SPAAC), one variety of “click” chemistry (18, 22, 23). In this study, we have identified sites in the Fc domain to accommodate the nnAA AzK incorporation while maintaining native FcRn affinity, thus retaining natural half-life.…”

Section: Introductionmentioning

confidence: 99%

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An antidote approach to reduce risk and broaden utility of antibody-based therapeutics

Portnoff¹,

Gao²,

Borrok³

et al. 2017

Journal of Biological Chemistry

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Antibody therapeutics offer effective treatment options for a broad range of diseases. One of the greatest benefits of antibody therapeutics is their extraordinarily long serum half-life, allowing infrequent dosing with long-lasting effects. A characteristic of antibodies that drives long half-life is the ability to interact with the recycling receptor, FcRn, in a pH-dependent manner. The benefit of long half-life, however, carries with it liabilities. Although the positive effects of antibody therapeutics are long-lasting, any acute adverse events or chronic negative impacts, such as immunosuppression in the face of an infection, are also long-lasting. Therefore, we sought to develop antibodies with a chemical handle that alone would enjoy the long half-life of normal antibodies but, upon addition of a small-molecule antidote, would interact with the chemical handle and inhibit the antibody recycling mechanism, thus leading to rapid degradation and shortened half-life in vivo. Here we present a proof of concept study where we identify sites to incorporate a non-natural amino acid that can be chemically modified to modulate FcRn interaction in vitro and antibody half-life in vivo. This is an important first step in developing safer therapeutics, and the next step will be development of technology that can perform the modifying chemistry in vivo.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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An antidote approach to reduce risk and broaden utility of antibody-based therapeutics

Portnoff¹,

Gao²,

Borrok³

et al. 2017

Journal of Biological Chemistry

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“…The development of site-specifically labeled immunoconjugates is a rapidly evolving field, as illustrated by the emergence of a number of new studies during the writing of this work [64–66]. In the two installments of this review, we have covered a wide variety of approaches, each with distinct advantages and limitations (Fig.9).…”

Section: Moving Forwardmentioning

confidence: 99%

Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

et al. 2016

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Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly becoming a critical component of modern precision medicine. Yet despite this promise, the vast majority of these immunoconjugates are synthesized via the random coupling of amine-reactive bifunctional probes to lysines within the antibody, a process that can result in heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In an effort to circumvent these issues, the last 5 years have played witness to a great deal of research focused on the creation of effective strategies for the site-specific attachment of payloads to antibodies. These chemoselective modification methods yield immunoconjugates that are more homogenous and better defined than constructs created using traditional synthetic approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to exhibit superior in vivo behavior compared to their randomly modified cousins. The over-arching goal of this two-part review is to provide a broad yet detailed account of the various site-specific bioconjugation approaches that have been used to create immunoconjugates for positron emission tomography (PET), single photon emission computed tomography (SPECT), and fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on the modification of cysteine residues and the chemoenzymatic manipulation of glycans. In Part 2, we will detail two families of bioconjugation approaches that leverage biochemical tools to achieve site-specificity. First, we will discuss modification methods that employ peptide tags either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And second, we will examine bioconjugation strategies predicated on the incorporation of unnatural or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare the advantages and disadvantages of the modification strategies covered in both parts of the review and offer a brief discussion of the overall direction of the field.

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“…Engineered antibodies containing p-acetylphenylalanine residues are produced using either of the expression systems, and the carbonyl groups introduced react with alkoxyamine-functionalized linkers to provide oxime-conjugated ADCs. Other examples are p-azidomethyl-L -phenylalanine (Zimmerman et al, 2014) and N6-((2-azidoethoxy)carbonyl)-L -lysine (VanBrunt et al, 2015) (Fig. 5B).…”

Section: Non-natural Amino Acid Incorporation By Genetic Engineeringmentioning

confidence: 99%

Antibody-Drug Conjugates in Head and Neck Cancer

Kim¹

2017

Korean J Otorhinolaryngol-Head Neck Surg

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The antibody-drug conjugate (ADC), a humanized or human monoclonal antibody conjugated with highly cytotoxic small molecules (payloads) through chemical linkers, is a novel therapeutic format and has great potential to make a paradigm shift in cancer chemotherapy. This new antibody-based molecular platform enables selective delivery of a potent cytotoxic payload to target cancer cells, resulting in improved efficacy, reduced systemic toxicity, and preferable pharmacokinetics (PK)/ pharmacodynamics (PD) and biodistribution compared to traditional chemotherapy. Boosted by the successes of FDA-approved Adcetris ® and Kadcyla ® , this drug class has been rapidly growing along with about 60 ADCs currently in clinical trials. In this article, we briefly review molecular aspects of each component (the antibody, payload, and linker) of ADCs, and then mainly discuss traditional and new technologies of the conjugation and linker chemistries for successful construction of clinically effective ADCs. Current efforts in the conjugation and linker chemistries will provide greater insights into molecular design and strategies for clinically effective ADCs from medicinal chemistry and pharmacology standpoints. The development of site-specific conjugation methodologies for constructing homogeneous ADCs is an especially promising path to improving ADC design, which will open the way for novel cancer therapeutics.

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Genetically Encoded Azide Containing Amino Acid in Mammalian Cells Enables Site-Specific Antibody–Drug Conjugates Using Click Cycloaddition Chemistry

Cited by 122 publications

References 74 publications

An antidote approach to reduce risk and broaden utility of antibody-based therapeutics

An antidote approach to reduce risk and broaden utility of antibody-based therapeutics

Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

Antibody-Drug Conjugates in Head and Neck Cancer

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