Homogeneous antibody-drug conjugates: DAR 2 anti-HER2 obtained by conjugation on isolated light chain followed by mAb assembly

Abstract

Despite advances in medical care, cancer remains a major threat to human health. Antibody-drug conjugates (ADCs) are a promising targeted therapy to overcome adverse side effects to normal tissues. In this field, the current challenge is obtaining homogeneous preparations of conjugates, where a defined number of drugs are conjugated to specific antibody sites. Site-directed cysteine-based conjugation is commonly used to obtain homogeneous ADC, but it is a time-consuming and expensive approach due to the need for extensive antibody engineering to identify the optimal conjugation sites and reductionoxidation protocols are specific for each antibody. There is thus a need for ADC platforms that offer homogeneity and direct applicability to the already approved antibody therapeutics. Here we describe a novel approach to derive homogeneous ADCs with drug-to-antibody ratio of 2 from any human immunoglobulin 1 (IgG 1), using trastuzumab as a model. The method is based on the production of heavy chains (HC) and light chains (LC) in two recombinant HEK293 independent cultures, so the original amino acid sequence is not altered. Isolated LC was effectively conjugated to a single drug-linker (vcMMAE) construct and mixed to isolated HC dimers, in order to obtain a correctly folded ADC. The relevance of the work was validated in terms of ADC homogeneity (HIC-HPLC, MS), purity (SEC-HPLC), isolated antigen recognition (ELISA) and biological activity (HER2-positive breast cancer cells cytotoxicity assays).

Site-selective modification strategies in antibody–drug conjugates

“…16). 248 Analysis via HIC revealed good recombination efficiency to reform the full antibody, while enzyme linked immunosorbent assay (ELISA) demonstrated retained binding affinity for the HER2 receptor, suggesting that this strategy could be possible for the modification of other therapeutic antibodies. Similar to traditional interchain cysteine modification, covalent linkage of the light and heavy chains is lost using this method.…”

New Technologies Bloom Together for Bettering Cancer Drug Conjugates

“…In addition, a recent publication presented an antibody self-assembly method. After generating antibody heavy chains and payloadconjugated light chains, the heavy and light chains could self-assemble into a correctly folded antibody to produce a homogenous ADC (Farr as et al, 2020).…”

The Chemistry of Creating Chemically Programmed Antibodies (cPAbs): Site-Specific Bioconjugation of Small Molecules

“…Both factors are technologically challenging and require efficient methods for producing completely homogeneous ADCs . Due to the challenges with producing homogeneous ADCs, previous food and drug administration (FDA) approved ADCs were developed and administered as heterogeneous mixtures due to the conventional and nonselective bioconjugation strategies used in their production resulting in a wide range of DARs. − However, in recent years, technological advancements facilitating site-specific conjugation with engineered or natural amino acids have led to continuous increases in homogeneity and optimal DAR. , Specific examples include the approval of Adcetris (brentuximab vedotin) by the FDA in 2011. Adcetris was developed by the introduction of cysteine-engineered residues into the antibody via site-directed mutagenesis providing free thiol groups for conjugation with thiol-specific maleimide linkers.…”

“…Adcetris was developed by the introduction of cysteine-engineered residues into the antibody via site-directed mutagenesis providing free thiol groups for conjugation with thiol-specific maleimide linkers. This technology generates almost homogeneous ADCs containing approximately 2 DAR, but additional antibody reduction/oxidation steps are required for the selective conjugation of the added cysteines. ,, Other examples include the recent approval of Trodelvy (sacituzumab govitecan; DAR 7.6) in 2020 and Zynlonta (loncastuximab tesirine; DAR 2.3) in 2021, which are conjugated via the hinge cysteine thiol to maleimide-bearing drug molecules as payload. Nonselective conjugation often leads to a population of subtly different bioconjugates, including antibodies with different average numbers of conjugated small molecules and/or conjugation to different physical locations of the antibody.…”