Multiplex LC-MS/MS Assays for Clinical Bioanalysis of MEDI4276, an Antibody-Drug Conjugate of Tubulysin Analogue Attached via Cleavable Linker to a Biparatopic Humanized Antibody against HER-2.

Morse Faria,Michael Waldron,Meina Liang,Anton Rosenbaum,Moucun Yuan,Brandon Lam,Marlking Peay,William Mylott,Eric Ma

doi:10.3390/antib8010011

Abstract

Bioanalysis of complex biotherapeutics, such as antibody-drug conjugates (ADCs), is challenging and requires multiple assays to describe their pharmacokinetic (PK) profiles. To enable exposure-safety and exposure-efficacy analyses, as well as to understand the metabolism of ADC therapeutics, three bioanalytical methods are typically employed: Total Antibody, Antibody Conjugated Toxin or Total ADC and Unconjugated Toxin. MEDI4276 is an ADC comprised of biparatopic humanized antibody attached via a protease-cleavable peptide-based maleimidocaproyl linker to a tubulysin toxin (AZ13599185) with an approximate average drug-antibody ratio of 4. The conjugated payload of MEDI4276 can undergo ester hydrolysis to produce the conjugated payload AZ13687308, leading to the formation of MEDI1498 (de-acetylated MEDI4276). In this report, we describe the development, validation and application of three novel multiplex bioanalytical methods. The first ligand-binding liquid chromatography coupled with tandem mass spectrometry (LBA-LC-MS/MS) method was developed and validated for simultaneous measurement of total antibody and total ADC (antibody-conjugated AZ13599185) from MEDI4276. The second LBA-LC-MS/MS assay quantified total ADC (antibody-conjugated AZ13687308) from MEDI1498. The third multiplex LC-MS/MS assay was used for simultaneous quantification of unconjugated AZ13599185 and AZ13687308. Additional stability experiments confirmed that quantification of the released warhead in the presence of high concentrations of MEDI4276 was acceptable. Subsequently, the assays were employed in support of a first-in-human clinical trial (NCT02576548).

Highlights

Antibody-drug conjugates (ADCs) are a class of biotherapeutic drugs that use antibodies targeting tumor-associated antigens as means to deliver covalently attached small-molecule toxins into cancerAntibodies 2019, 8, 11; doi:10.3390/antib8010011 www.mdpi.com/journal/antibodiesAntibodies 2019, 8, 11 cells [1]
ADCs are comprised of three major components: Monoclonal antibody for selective targeting, a cytotoxic drug and a linker molecule that conjugates the drug to the monoclonal antibody
MEDI4276 consists of the tubulysin payload conjugated to a biparatopic anti-human epidermal growth factor receptor 2 (HER-2) antibody

Summary

Introduction

Antibody-drug conjugates (ADCs) are a class of biotherapeutic drugs that use antibodies targeting tumor-associated antigens as means to deliver covalently attached small-molecule toxins into cancerAntibodies 2019, 8, 11; doi:10.3390/antib8010011 www.mdpi.com/journal/antibodiesAntibodies 2019, 8, 11 cells [1]. Antibody-drug conjugates (ADCs) are a class of biotherapeutic drugs that use antibodies targeting tumor-associated antigens as means to deliver covalently attached small-molecule toxins into cancer. ADCs are comprised of three major components: Monoclonal antibody (mAb) for selective targeting, a cytotoxic drug ( referred to as warhead) and a linker molecule that conjugates the drug to the monoclonal antibody. The warhead and the linker molecule are referred to as the payload. Upon binding to target antigens expressed on cancer cells, ADCs are internalized into endosomes/lysosomes where the cytotoxic drug is released following cleavage of the linker. The released cytotoxic payload binds to its molecular target (e.g., tubulin, DNA, or topoisomerase 1) resulting in cell cycle arrest and apoptosis [1]

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Results

Conclusion