Antibody-drug conjugates (ADCs) have great potential as targeted cancer therapeutics. In 2011, the ADC brentuximab vedotin was approved for treating anaplastic large cell lymphoma and Hodgkin’s lymphoma. And in early 2012 it was reported that trastuzumab emtansine increased median survival time among patients with a type of breast cancer that overexpresses the HER2 protein. Brentuximab vedotin and trastuzumab emtansine are both full monoclonal antibodies, and in the wake of their success, new and established companies are attempting to make ADCs with a variety of antibody backbones, including antibody fragments.
While monoclonal antibody expression must be done in mammalian cells, several types of antibody fragments can be produced in microbial cells (mainly bacteria or yeast).
Recombinant protein production in microbial systems tends to be faster and cheaper than in mammalian systems. Because bacteria and yeast are unicellular, they have a robust cellular structure that makes them amenable to culturing. In contrast, mammalian cells, which are derived from multicellular organisms, are not adapted to survive outside the body and are thus sensitive to external conditions like shear stress and osmotic shock. The doubling time of bacteria and yeast is every 20 minutes to 2 hours versus every 24 hours to 2 days for mammalian cells. The cost for bacterial cell media is 90% lower than for mammalian Chinese hamster ovary (CHO) media. Producing a stable recombinant protein production system takes approximately 4 to 6 months in mammalian cells versus 1 month in the bacteria Escherichia coli.
Expression of antibody fragments does not always work well in mammalian cells. For example, the product can get stuck in the endoplasmic reticulum, which precludes secretion and isolation. In these cases, expression may be more effective in microbial systems, such as the following.
Gram-negative bacteria, particularly Escherichia coli, have been extensively studied for recombinant antibody production. Fab (antigen-binding) fragments have been successfully expressed in E. coli, as have fusion proteins containing the Fc fragment. However, production of antibody fragments in E. coli can be problematic because of unreliable protein expression levels and difficulties in consistently secreting the active antibody proteins. Secreting antibody fragments into the medium is a prerequisite for easy downstream processing. In addition, gram-negative bacteria contain endotoxins that are toxic to humans. The process of removing endotoxins complicates protein purification and can lead to decreased yield.
Unlike gram-negative bacteria, gram-positive bacteria more efficiently secrete recombinant proteins into culture medium. Additionally, gram-positive bacteria do not contain endotoxins. Successful production of antibody fragments has been done in Lactobacillus zeae, Bacillus subtilis, Streptomyces lividans, Staphylococcus carnosus, and Bacillus megaterium (Joosten 2003 et al.; Jordan et al., 2009). Recently, a strain of Corynebacterium glutamicum has been engineered to express correctly folded, active recombinant proteins directly into the extracellular fermentation broth (Date et al., 2006). C. glutamicum is being explored for its ability to express antibody fragments.
The yeast Pichia pastoris has been used to produce recombinant antibodies and antibody fragments. Although products expressed using P. pastoris have potential for commercial applications, they are not generally regarded as safe (GRAS) by regulatory agencies, which limits their use in human therapeutics (Joosten et al., 2003).
Baker’s yeast Saccharomyces cerevisiae has been used to produce and secrete Fab fragments (Joosten et al., 2003). Schizosaccharomyces pombe has been used to express scFv antibody fragments. And the nonconventional yeasts Yarrowia lipolytica and Kluyveromyces lactis have produced functional and soluble anti-Ras scFv.
ADCs are changing the landscape of targeted therapeutics. Current antibody-based therapeutics are produced in mammalian cell lines. However, microbial cell lines, which are relatively easy and inexpensive to culture, are being explored for their use in producing antibody fragments.