Impact of Drug Conjugation and Loading on Target Antigen Binding and Cytotoxicity in Cysteine Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) consist of a target-specific antibody that is covalently conjugated to a drug via a linker. ADCs are designed to deliver cytotoxic drugs (payloads), specifically to cancer cells, while minimizing systemic toxicity. Conventional cysteine conjugation typically results in the formation of ADC molecules containing a heterogeneous mixture of 2, 4, 6, and 8 drug-loaded species. The drug-to-antibody ratio (DAR) of the mixture represents the weighted average of these species. In this report, we have investigated the impact of the hydrophobicity of payloads and the overall drug loading on the in vitro binding and cytotoxicity of ADC species. Several ADCs were prepared by conventional cysteine conjugation using different payloads. ADC species with different DAR values were purified from the ADC mixture and characterized by standard analytical techniques. These ADC species were evaluated for target antigen binding using an immunoassay, enzyme-linked immunosorbent assay (ELISA). The potency was assessed using a cell-based cytotoxicity assay. These structure-function studies lead to a better understanding of factors that impact the in vitro target binding and cytotoxicity of ADC species. ADC species containing hydrophobic payloads with high DAR were found to have lower target binding by ELISA compared to that of the unconjugated antibody or the heterogeneous reference ADC with DAR ∼4. Under similar assay conditions, the ADCs conjugated to hydrophilic payloads did not show a significant impact on the target binding. The cytotoxic potency of ADC species increased with increasing level of drug loading in the cell-based cytotoxicity assay.

Conjugations to Endogenous Cysteine Residues.

Interchain disulfide bonds of antibodies can be reduced by agents such as TCEP or DTT to form reactive cysteine residues. These endogenous cysteines are used for conjugation to biologically active drugs either directly or via linkers to prepare antibody drug conjugates (ADCs). The anti-notch 3 ADC described here is being evaluated in the early clinical development program as a potential treatment for a variety of cancers. The ADC is composed of an IgG1 mAb that is conjugated by endogenous cysteines to a cytotoxic microtubulin inhibitor via a maleimide-containing linker. The endogenous cysteine residues are produced by partial reduction of the mAb with TCEP reducing agent. The conjugation results in the formation of a mixture of 2, 4, 6, and 8 loaded ADC species. In addition to the desired product, several product-related impurities such as aggregates are generated during the conjugation reaction. The product- and process-related impurities are separated from the monomeric ADC by column chromatography and ultrafiltration-diafiltration techniques. The temperature of TCEP reduction step has an impact on the level of aggregates produced in the reaction. The temperature also impacts the isomeric composition of the 4 loaded ADC species.