Effect of Linker-Drug Properties and Conjugation Site on the Physical Stability of ADCs

Mills, Brittney J.; Kruger, Terra M.; Bruncko, Milan; Zhang, Xinxin; Jameel, Feroz

doi:10.1016/j.xphs.2020.01.029

Cited by 16 publications

(6 citation statements)

References 48 publications

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“…It is known that the formation of mAb aggregates is affected by stress-inducing aggregation, the biopharmaceutical formulation, and mAb structure ( 11 , 34 , 35 ). The impact of ADC-specific characteristics, such as the structures of payload and linker, DAR, and conjugation site, on the aggregation of ADCs have also been well studied recently ( 36 – 39 ). On the other hand, studies evaluating the characteristics (e.g., FcγR-activation property) of ADC aggregates are still limited.…”

Section: Discussionmentioning

confidence: 99%

Fcγ Receptor-Dependent Internalization and Off-Target Cytotoxicity of Antibody-Drug Conjugate Aggregates

et al. 2021

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Purpose Antibody-drug conjugates (ADCs), which are monoclonal antibodies (mAbs) conjugated with highly toxic payloads, achieve high tumor killing efficacy due to the specific delivery of payloads in accordance with mAbs’ function. On the other hand, the conjugation of payloads often increases the hydrophobicity of mAbs, resulting in reduced stability and increased aggregation. It is considered that mAb aggregates have potential risk for activating Fcγ receptors (FcγRs) on immune cells, and are internalized into cells via FcγRs. Based on the mechanism of action of ADCs, the internalization of ADCs into target-negative cells may cause the off-target toxicity. However, the impacts of aggregation on the safety of ADCs including off-target cytotoxicity have been unclear. In this study, we investigated the cytotoxicity of ADC aggregates in target-negative cells. Methods The ADC aggregates were generated by stirring stress or thermal stress. The off-target cytotoxicity of ADC aggregates was evaluated in several target-negative cell lines, and FcγR-activation properties of ADC aggregates were characterized using a reporter cell assay. Results Aggregation of ADCs enhanced the off-target cytotoxicity in several target-negative cell lines compared with non-stressed ADCs. Notably, ADC aggregates with FcγR-activation properties showed dramatically enhanced cytotoxicity in FcγR-expressing cells. The FcγR-mediated off-target cytotoxicity of ADC aggregates was reduced by using a FcγR-blocking antibody or Fc-engineering for silencing Fc-mediated effector functions. Conclusions These results indicated that FcγRs play an important role for internalization of ADC aggregates into non-target cells, and the aggregation of ADCs increases the potential risk for off-target toxicity.

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Section: Discussionmentioning

confidence: 99%

Fcγ Receptor-Dependent Internalization and Off-Target Cytotoxicity of Antibody-Drug Conjugate Aggregates

et al. 2021

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“…Linkers can also affect the physicochemical properties of the ADC, such as the tendency to aggregate, which can impact ADC efficacy, hepatotoxicity, and immunogenicity [ 64 , 65 ]. When hydrophilic glucuronide-based drug linkers were compared to dipeptide linkers during the development of ADCs employing camptothecin cytotoxins, the more hydrophilic glucuronide drug linkers led to less antibody aggregation than the dipeptide drug linkers [ 66 ].…”

Section: Linker Chemistrymentioning

confidence: 99%

Considerations for the Nonclinical Safety Evaluation of Antibody–Drug Conjugates

Fisher

2021

Antibodies

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The targeted delivery of drugs by means of linking them to antibodies (Abs) to form antibody–drug conjugates (ADCs) has become an important approach in oncology and could potentially be used in other therapeutic areas. Targeted therapy is aimed at improving clinical efficacy while minimizing adverse reactions. The nonclinical safety assessment of ADCs presents several unique challenges involving the need to examine a complex molecule, each component of which can contribute to the effects observed, in appropriate animal models. Some considerations for the nonclinical safety evaluation of ADCs based on a literature review of ADCs in clinical development (currently or previously) are discussed.

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“…Similarly, it needs to be considered that the inherent properties of the antibody, together with the conjugation chemistry, the linker, and the cytotoxic molecule used, strongly influence the druglike properties of the resulting conjugates and their stability. 5 It is known that reactive thiols present in plasma molecules, for example, in glutathione or Cys-34 in albumin, may react with the conjugates and reduce their stability in vivo. 6,7 In the context of artificial enzymes, for example, it is well known that the engineering of proximal amino acids in the active site can affect their activity significiantly.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Ideally, ADCs should remain intact in the bloodstream and efficiently release their payload in the target cell for maximum efficacy and minimal toxicity. Similarly, it needs to be considered that the inherent properties of the antibody, together with the conjugation chemistry, the linker, and the cytotoxic molecule used, strongly influence the drug-like properties of the resulting conjugates and their stability . It is known that reactive thiols present in plasma molecules, for example, in glutathione or Cys-34 in albumin, may react with the conjugates and reduce their stability in vivo. , In the context of artificial enzymes, for example, it is well known that the engineering of proximal amino acids in the active site can affect their activity significiantly. , Similarly, it is known that the chemical and steric environment of the conjugation site is known to modulate both the conjugation and deconjugation properties of the linkers and thus, in the latter case, their stability and efficacy. , Vollmar et al reported that modulation of the p K a of the thiol of a cysteine residue could affect the stability of ADCs.…”

Section: Introductionmentioning

confidence: 99%

Single Mutation on Trastuzumab Modulates the Stability of Antibody–Drug Conjugates Built Using Acetal-Based Linkers and Thiol-Maleimide Chemistry

et al. 2022

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Antibody-drug conjugates (ADCs) are a class of targeted therapeutics used to selectively kill cancer cells. It is important that they remain intact in the bloodstream and release their payload in the target cancer cell for maximum efficacy and minimum toxicity. The development of effective ADCs requires the study of factors that can alter the stability of these therapeutics at the atomic level. Here, we present a general strategy that combines synthesis, bioconjugation, linker technology, site-directed mutagenesis, and modeling to investigate the influence of the site and microenvironment of the trastuzumab antibody on the stability of the conjugation and linkers. Trastuzumab is widely used to produce targeted ADCs because it can target with high specificity a receptor that is overexpressed in certain breast cancer cells (HER2). We show that the chemical environment of the conjugation site of trastuzumab plays a key role in the stability of linkers featuring acid-sensitive groups such as acetals. More specifically, Lys-207, located near the reactive Cys-205 of a thiomab variant of the antibody, may act as an acid catalyst and promote the hydrolysis of acetals. Mutation of Lys-207 into an alanine or using a longer linker that separates this residue from the acetal group stabilizes the conjugates. Analogously, Lys-207 promotes the beneficial hydrolysis of the succinimide ring when maleimide reagents are used for conjugation, thus stabilizing the subsequent ADCs by impairing the undesired retro-Michael reactions. This work provides new insights for the design of novel ADCs with improved stability properties.

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Effect of Linker-Drug Properties and Conjugation Site on the Physical Stability of ADCs

Cited by 16 publications

References 48 publications

Fcγ Receptor-Dependent Internalization and Off-Target Cytotoxicity of Antibody-Drug Conjugate Aggregates

Fcγ Receptor-Dependent Internalization and Off-Target Cytotoxicity of Antibody-Drug Conjugate Aggregates

Considerations for the Nonclinical Safety Evaluation of Antibody–Drug Conjugates

Single Mutation on Trastuzumab Modulates the Stability of Antibody–Drug Conjugates Built Using Acetal-Based Linkers and Thiol-Maleimide Chemistry

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