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Determination of Antibody-Drug Conjugate Released Payload Species Using Directed in Vitro Assays and Mass
Andrew J Bessire1,2,3, T Eric Ballard1,2,3, Manoj Charati1,2,3
1Pharmacokinetics, Dynamics and Metabolism, ∥Worldwide Medicinal Chemistry, and ¶Pharmaceutical Sciences Chemical R&D, Pfizer Worldwide R&D , Groton, Connecticut 06340, United States.
Abstract:
Antibody-drug conjugates (ADC) are currently an active area of research, focused primarily on oncology therapeutics, but also to a limited extent on other areas such as infectious disease. The success of this type of targeted drug delivery is dependent upon many factors, one of which is the performance of the linker in releasing an active drug moiety under the appropriate conditions. As a tool in the development of linker/payload chemistry, we have developed an in vitro method for the identification of payload species released from ADCs in the presence of lysosomal enzymes. This method utilizes commercially available human liver S9 fraction as the source of these enzymes, and this has certain advantages over lysosomal fractions or purified enzymes. This article describes the characterization and performance of this assay with multiple ADCs composed of known and novel linkers and payloads. Additionally, we report the observation of incomplete degradation of mAb protein chains by lysosomal enzymes in vitro, believed to be the first report of this phenomenon involving an ADC therapeutic.
Insights
We developed an in vitro assay using human liver S9 fraction to study payload release from antibody-drug conjugates (ADCs). This method also revealed incomplete antibody degradation, a novel finding for ADC therapeutics.
Area of Science:
- Biochemistry
- Pharmacology
- Drug Delivery
Background:
- Antibody-drug conjugates (ADCs) are crucial targeted therapeutics, particularly in oncology.
- ADC efficacy relies heavily on linker stability and appropriate drug release.
- Understanding linker-payload interactions is key for ADC development.
Purpose of the Study:
- To establish an in vitro assay for identifying payload species released from ADCs.
- To characterize the performance of this assay using various ADCs.
- To investigate the degradation of antibody components within ADCs.
Main Methods:
- Utilized commercially available human liver S9 fraction as a source of lysosomal enzymes.
- Developed and validated an in vitro assay for payload release identification.
- Tested the assay with multiple ADCs featuring known and novel linker/payload combinations.
Main Results:
- Successfully identified payload species released from ADCs in vitro.
- Demonstrated the assay's effectiveness with diverse ADC constructs.
- Observed incomplete degradation of antibody protein chains by lysosomal enzymes, a novel finding in ADC research.
Conclusions:
- The developed in vitro assay is a valuable tool for ADC linker/payload chemistry development.
- Human liver S9 fraction provides a practical enzyme source for ADC metabolism studies.
- Incomplete antibody degradation in ADCs is a previously unreported phenomenon requiring further investigation.
