Conjugation of Emtansine Onto Trastuzumab Promotes Aggregation of the Antibody–Drug Conjugate by Reducing Repulsive Electrostatic Interactions and Increasing Hydrophobic Interactions

Gandhi, Aditya; Randolph, Theodore W.; Carpenter, John F.

doi:10.1016/j.xphs.2019.01.029

Cited by 16 publications

(9 citation statements)

References 42 publications

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“…An increase in mAb labeling degree with hydrophobic moieties correlates with a rise in self-association, and this was shown for the conjugation to lysine residues 22,57 as well as to interchain cysteines. 40 It was also shown that increasing payload hydrophobicity levels represented by LogP correlated with an increase in selfinteraction behavior and higher tendency to generate aggregates 39 independently of their labeling degree.…”

Section: Self-interactionmentioning

confidence: 84%

“…Bioconjugation can alter the native protein charge by introducing an additional charge by attachment of a small molecule 35,57 or by modifying surface structures or residues by covalent crosslinking of the payload. 58 These effects are specific for individual conjugation techniques.…”

Section: Chargementioning

confidence: 99%

“…57,58 Complexity further increases because the position of these conjugating sites seems to affect the surface charge of the protein on a local 37 but also global level. 35,57,58 Boylan et al 58 observed a high number of charge variants with different pIs when a fluorophore was cross-linked to lysine residues of an Fcfragment. The authors concluded that the combination of the different loading species and the different conjugation sites were the root cause.…”

Section: Chargementioning

confidence: 99%

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Alteration of Physicochemical Properties for Antibody-Drug Conjugates and Their Impact on Stability

Buecheler

Winzer

Weber

et al. 2020

Journal of Pharmaceutical Sciences

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a b s t r a c tAntibody conjugates, in particular antibody-drug conjugates (ADCs), are a fast-growing area in research and in the pharmaceutical industry. The covalent attachment of an antibody to a chemical moiety can be an effective measure for drug targeting or can also positively impact pharmacokinetics of small molecular compounds by serum half-life extension. Stability, physicochemical properties, and degradation pathways of biotherapeutics or small molecule therapeutics are often not totally known and understood. However, ADCs represent a hybrid of small molecular and macromolecular components, and their properties are still not fully understood and described. This review discusses the alteration of the physicochemical properties of antibodies upon conjugation of chemical moieties to its surface and the resulting impact on ADC stability.

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Section: Self-interactionmentioning

confidence: 84%

Section: Chargementioning

confidence: 99%

Section: Chargementioning

confidence: 99%

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Alteration of Physicochemical Properties for Antibody-Drug Conjugates and Their Impact on Stability

Buecheler

Winzer

Weber

et al. 2020

Journal of Pharmaceutical Sciences

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“…Because it is known that more hydrophilic ADCs are, in general, less prone to undesired aggregation behavior, [30] we believe that compound 5 is able to address current issues associated with the hydrophobic linker used in trastuzumabemtansine. [31] Finally, we also wanted to apply our phosphonamidate building blocks to a more challenging thiol-containing molecule and aimed for the synthesis of an antibody-protein conjugate. We chose our previously established eGFP mutant C70M S147C, which carries only one addressable cysteine residue, and incubated it in a 3.5-fold excess with trastuzumab that was preactivated with 20 equivalents of 5 at a concentration of Figure 2.…”

Section: Resultsmentioning

confidence: 99%

N‐Hydroxysuccinimide‐Modified Ethynylphosphonamidates Enable the Synthesis of Configurationally Defined Protein Conjugates

et al. 2020

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Herein, the application of N‐hydroxysuccinimide‐modified phosphonamidate building blocks for the incorporation of cysteine‐selective ethynylphosphonamidates into lysine residues of proteins, followed by thiol addition with small molecules and proteins, is reported. It is demonstrated that the building blocks significantly lower undesired homo‐crosslinking side products that can occur with commonly applied succinimidyl 4‐(N‐maleimidomethyl)cyclohexane‐1‐carboxylate (SMCC) under physiological pH. The previously demonstrated stability of the phosphonamidate moiety additionally solves the problem of premature maleimide hydrolysis, which can hamper the efficiency of subsequent thiol addition. Furthermore, a method to separate the phosphonamidate enantiomers to be able to synthesize protein conjugates in a defined configuration has been developed. Finally, the building blocks are applied to the construction of functional antibody–drug conjugates, analogously to FDA‐approved, SMCC‐linked Kadcyla, and to the synthesis of a functional antibody–protein conjugate.

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“…To this point, a systematic study of trastuzumab, trastuzumab-MCC conjugate intermediate, and trastuzumab-DM1 found that both conjugates suffered decreased thermal stability and increased aggregation compared with trastuzumab [164]. Recently, the impact of drug conjugation on intra-and intermolecular interactions of trastuzumab-DM1 compared with trastuzumab was studied and the results confirmed that the lower colloidal stability and higher aggregation propensity for trastuzumab-DM1 are attributed to both reduced repulsive charge interaction and increased hydrophobicity [165]. Multiple publications have reported a more pronounced conjugation destabilizing effect on interchain cysteine conjugated ADCs and an inverse correlation between the drug load and stability [166][167][168][169][170].…”

Section: Conjugate Developability Formulations and Characteristicsmentioning

confidence: 95%

Antibody Conjugates-Recent Advances and Future Innovations

et al. 2020

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Monoclonal antibodies have evolved from research tools to powerful therapeutics in the past 30 years. Clinical success rates of antibodies have exceeded expectations, resulting in heavy investment in biologics discovery and development in addition to traditional small molecules across the industry. However, protein therapeutics cannot drug targets intracellularly and are limited to soluble and cell-surface antigens. Tremendous strides have been made in antibody discovery, protein engineering, formulation, and delivery devices. These advances continue to push the boundaries of biologics to enable antibody conjugates to take advantage of the target specificity and long half-life from an antibody, while delivering highly potent small molecule drugs. While the “magic bullet” concept produced the first wave of antibody conjugates, these entities were met with limited clinical success. This review summarizes the advances and challenges in the field to date with emphasis on antibody conjugation, linker-payload chemistry, novel payload classes, absorption, distribution, metabolism, and excretion (ADME), and product developability. We discuss lessons learned in the development of oncology antibody conjugates and look towards future innovations enabling other therapeutic indications.

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Conjugation of Emtansine Onto Trastuzumab Promotes Aggregation of the Antibody–Drug Conjugate by Reducing Repulsive Electrostatic Interactions and Increasing Hydrophobic Interactions

Cited by 16 publications

References 42 publications

Alteration of Physicochemical Properties for Antibody-Drug Conjugates and Their Impact on Stability

Alteration of Physicochemical Properties for Antibody-Drug Conjugates and Their Impact on Stability

N‐Hydroxysuccinimide‐Modified Ethynylphosphonamidates Enable the Synthesis of Configurationally Defined Protein Conjugates

Antibody Conjugates-Recent Advances and Future Innovations

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