Methods for site-specific drug conjugation to antibodies

Behrens, Christopher R.; Liu, Bin

doi:10.4161/mabs.26632

Cited by 146 publications

(129 citation statements)

References 55 publications

(54 reference statements)

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“…This effect is thought to be related to faster clearance of higher DAR species. Recent advances in conjugation chemistry have led to the production of ADCs with homogeneous drug loading and improved stability (53). These technologies use nonnatural amino acids, engineered cysteines, or transglutaminases to attach the warhead linker to specific sites on the antibody (54,55).…”

Section: Stabilitymentioning

confidence: 99%

Antibody Drug Conjugates: Nonclinical Safety Considerations

Hinrichs

Dixit

2015

AAPS J

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Abstract. Antibody drug conjugates (ADCs) are biopharmaceutical molecules consisting of a cytotoxic small molecule covalently linked to a targeted protein carrier via a stable cleavable or noncleavable linker. The process of conjugation yields a highly complex molecule with biochemical properties that are distinct from those of the unconjugated components. The impact of these biochemical differences on the safety and pharmacokinetic (PK) profile of the conjugate must be considered when determining the types of nonclinical safety studies required to support clinical development of ADCs. The hybrid nature of ADCs highlights the need for a science-based approach to safety assessment that incorporates relevant aspects of small and large molecule testing paradigms. This thinking is reflected in current regulatory guidelines, where sections pertaining to conjugates allow for a flexible approach to nonclinical safety testing. The aim of this article is to review regulatory expectations regarding early assessment of nonclinical safety considerations and discuss how recent advances in our understanding of ADCmediated toxicity can be used to guide the types of nonclinical safety studies needed to support ADC clinical development. The review will also explore nonclinical testing strategies that can be used to streamline ADC development by assessing the safety and efficacy of next generation ADC constructs using a rodent screen approach.

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Section: Stabilitymentioning

confidence: 99%

Antibody Drug Conjugates: Nonclinical Safety Considerations

Hinrichs

Dixit

2015

AAPS J

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“…The SpyTag/SpyCatcher system does not require reducing agents during the labeling reaction as in case of cysteine-maleimide conjugation. Thus, disulfide bonds present in the antibodies remain oxidized during labeling, and the need for removing the reducing agent postconjugation is eliminated [40]. Additionally, the SpyCatcher/SpyTag reaction takes place at room temperature in the same buffer in which the antibody was purified, and there was no need to change buffers or pH.…”

Section: Discussionmentioning

confidence: 99%

Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging

et al. 2018

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Purpose: Construction of antibody-based, molecular-targeted optical imaging probes requires the labeling of an antibody with a fluorophore. The most common method for doing this involves non-specifically conjugating a fluorophore to an antibody, resulting in poorly defined, heterogeneous imaging probes that often have suboptimal in vivo behavior. We tested a new strategy to site-specific label antibody-based imaging probes using the SpyCatcher/SpyTag protein ligase system. Procedures: We used the SpyCatcher/SpyTag protein ligase system to site specifically label nimotuzumab, an anti-EGFR antibody and an anti-HER3 diabody. To prevent the labeling from interfering with antigen binding, we introduced the SpyTag and SpyCatcher at the C-terminus of the antibody and diabody, respectively. Expression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. Sitespecific labeling of the antibody and diabody was performed in two steps. First, we labeled the SpyCatcher with IRDye800CW-Maleimide and the SpyTag with IRDye800CW-NHS. Second, we conjugated the IRDye800CW-SpyCatcher and the IRDye800CW-SpyTag to the antibody or diabody, respectively. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively. Results: Expression and binding properties of the C-terminal antibody-SpyTag and diabodySpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively. Site-specifically IRDye800CW-labeled imaging probes bound to their Md. Kausar Alam and Ayman El-Sayed contributed equally to this work. Electronic supplementary material The online version of this article (https:// doi.org/10.1007/s11307-018-1222-y) contains supplementary material, which is available to authorized users.Correspondence to: Humphrey Fonge; e-mail: humphrey.fonge@usask.ca, C. Geyer; e-mail: ron.geyer@usask.ca * The Author(s), 2018 immobilized targets, cells expressing these targets, and selectively accumulated in xenografts. Conclusions: These results highlight the ease and utility of using the modular SpyTag/ SpyCatcher protein ligase system for site-specific fluorescent labeling of protein-based imaging probes. Imaging probes labeled in this manner will be useful for optical imaging applications such as image-guided surger...

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“…52,53 Much work has been done on linkers, and there is now appreciation that ADCs with higher drug loads can cause more toxicity, with increased clearance rates for more heavily modified molecules. 54 Site of conjugation is also important in this regard with site-specific methods reducing heterogeneity, increasing stability and improving PK. 52,54 Cytotoxic drugs can be linked to the antibody with cleavable or noncleavable linkers.…”

Section: Arming Antibodies: Adcs and Beyondmentioning

confidence: 99%

New Horizons in Therapeutic Antibody Discovery: Opportunities and Challenges versus Small-Molecule Therapeutics

Smith¹

2015

SLAS Discovery

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Antibody drugs have become an increasingly significant component of the therapeutic landscape. Their success has been driven by some of their unique properties, in particular their very high specificity and selectivity, in contrast to the offtarget liabilities of small molecules (SMs). Antibodies can bring additional functionality to the table with their ability to interact with the immune system, and this can be further manipulated with advances in antibody engineering. This review summarizes what antibody therapeutics have achieved to date and what opportunities and challenges lie ahead. The target landscape for large molecules (LMs) versus SMs and some of the challenges for antibody drug development are discussed. Effective penetration of membrane barriers and intracellular targeting is one challenge, particularly across the highly resistant blood-brain barrier. The expanding pipeline of antibody-drug conjugates offers the potential to combine SM and LM modalities in a variety of creative ways, and antibodies also offer exciting potential to build bi-and multispecific molecules. The ability to pursue more challenging targets can also be further exploited but highlights the need for earlier screening in functional cell-based assays. I discuss how this might be addressed given the practical constraints imposed by high-throughput screening sample type and process differences in antibody primary screening.

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Methods for site-specific drug conjugation to antibodies

Cited by 146 publications

References 55 publications

Antibody Drug Conjugates: Nonclinical Safety Considerations

Antibody Drug Conjugates: Nonclinical Safety Considerations

Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging

New Horizons in Therapeutic Antibody Discovery: Opportunities and Challenges versus Small-Molecule Therapeutics

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