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).

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Background Monoclonal antibodies (mAbs) and their derivatives have become one of the most important classes of therapeutic drugs. Their multiple applications increased the interest for understanding their complex structure. In vivo, animal cells are able to fold mAbs correctly (Song et al, J Biosci Bioeng 110:135-40, 2010), whereas previous in vitro approaches were scarce and mostly unsuccessful. Results In this work, we compared in vitro assembly characteristics of trastuzumab, produced either by A) physical separation and refolding of its sub-units or B) direct joining of individually produced heavy and light chains. Native and denatured structures of trastuzumab were determined by SEC-HPLC, HIC-HPLC and SDS-PAGE. Conclusions Our results demonstrate the requirement of correctly folded HC, forming disulfide-bonded dimers, in order to form a fully functional mAb. Otherwise, the unfolded HC tend to precipitate. We were able to assemble trastuzumab in this fashion by only mixing them to LC in pH-buffered conditions, while monomeric HC structure was too unstable to render a functional mAb. This approach has been used in the generation of homogeneous ADC, with results pending to be published.

Cysteine‐based conjugation is one of the main antibody conjugation strategies for generating both heterogeneous and homogeneous Antibody Drug Conjugates (ADCs), being the reduction of the antibody a crucial step in these processes. In this work we have analysed the reduction conditions for the site‐directed conjugation of an anti‐HER2 (Human Epidermal Receptor 2) Trastuzumab_cys114 antibody to the cytotoxic drug vcMMAE (valine‐citrulline monomethyl auristatin E), with an aimed average DAR (Drug‐Antibody Ratio) of 2. Initial reduction was found to have a direct impact on the availability of free thiols for the conjugation: increasing of reducing agent concentration (until a molar excess of 50x) in the reduction step resulted in a lower proportion of naked antibody in the final ADC product and allowed us to obtain an ADC close to the DAR of interest. This work shows that reduction conditions must be adjusted in order to obtain the desired homogeneous ADC product.