Synthesis and Analysis of Novel Antibody-Drug Conjugates (ADCs)

Abstract

Antibody-Drug Conjugates (ADCs) are a family of targeted therapeutic agents for the treatment of cancer. They consist of potent cytotoxic small molecules or biological toxins (i.e., the payload) and highly selective antibodies joined through cleavable or noncleavable linkers, thus combining the unique and selective targeting capability of antibodies with the cell-killing ability of cytotoxic agents. There are eight ADCs containing small molecule payloads and two with biological toxins currently approved for use, and many more are in the pipeline in the clinical and pre-clinical setting. The majority of ADCs utilise anti-microtubule agents such as the auristatins (e.g., MMAE) or maytansines (e.g. DM1) as payloads, although in the past decade interest has grown in the use of DNA-interactive agents (e.g., the calicheamicins, PBD dimers, topoisomerase inhibitors or duocarmycin-type analogues) resulting in three of the approved ADCs (i.e., two with calicheamicin and one with deruxtecan). Also, there are many new families of DNA-interactive agents being studied for use as ADC payloads such as the pyridinobenzodiazepines (PDDs). One example of a PDD payload, FGX2-62, is a sequence-selective DNA mono-alkylating agent capable of binding to unique DNA sequences and inhibiting the interaction of transcription factors which can be lethal to tumour cells, hence the significant cytotoxicity of compounds of this type. The major goal of this project was to synthesize and evaluate a novel ADC based on a combination of the commercially-available CD20-targeting antibody rituximab and the DNA-interactive FGX2-62 payload joined through a Valine-Alanine linker (i.e., the FGX11- 38 construct). To achieve this goal, extensive analytical characterisation and quality control methods were developed based on Polymeric Reversed Phase Chromatography (PLRP), Hydrophobicity Interaction Chromatography (HIC), Size Exclusion Chromatography (SEC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE). In particular, a novel UPLC-based hydrophobicity model was developed to evaluate payloads and linker-payloads at an early stage of their development, to allow their hydrophobic characteristics (important for efficient conjugation and efficacy of the ADC) to be evaluated so that the information can be fed back into the design stage. This methodology has been published and should be useful to researchers in the ADC area. The novel Rituximab-(FGX11-38) ADC produced was purified and characterized using the analytical methods previously developed in the project. Although a number of batches with different average DARs were produced, the version with DAR = 2.8 was evaluated for in vitro cytotoxicity in cell lines with high (Raji), medium (JVM2) and low (Jurkat) expression of the CD20 antigen. As anticipated, the Rituximab-(FGX11-38) ADC had potent and selective cytotoxic activity in the high-expressing CD20-cell line while being several orders of magnitude less cytotoxic in the low-expressing cell line. A RituximabMMAE ADC was also synthesised for comparative purposes, and this had cytotoxicity in the high-expressing cell line broadly consistent with other Rituximab-MMAE ADCs reported in the literature. Furthermore, the Rituximab-(FGX11-38) ADC was more potent than the Rituximab-MMAE ADC in the Raji, JVM2 and Jurkat cell lines by 64-, 129- and 18-fold, respectively, highlighting the greater cytotoxicity of DNA covalent modifying payloads compared to tubulin inhibitors. Although, time and resources did not permit progression of the Rituximab-(FGX11-38) to in vivo studies and beyond, it is anticipated that this ADC, if efficacious in vivo, could have a useful role in treating patients who have become resistant to rituximab alone.

Date of Award	1 Feb 2021
Original language	English
Awarding Institution	King's College London
Supervisor	David Thurston (Supervisor) & Miraz Rahman (Supervisor)