Expression of cytokines or growth factors and their subsequent binding to cell surface receptors in healthy individuals is a tightly regulated and coordinated process. The inappropriate expression of these circulating proteins can contribute to the onset or progression of a number of acute and chronic diseases.
One method for treating diseases that result from overexpressed cytokines or growth factors is to block their ability to bind to their cognate cell surface receptor. A common strategy to achieve this is to use mAbs that specifically target these proteins. An alternative strategy is to use a soluble version of the cognate receptor itself to bind to and block the action of the cytokine.
Soluble receptors are often expressed in mammalian cells as a hybrid or chimeric molecule consisting of the extracellular domain of the cell-surface receptor fused to the Fc portion of a human immunoglobulin (Ig). Such fusion proteins, when expressed, are designed to dimerize, in a manner analogous to the dimerization of the Ig heavy chain, to create a bivalent, soluble receptor capable of blocking the action of cytokines and growth factors. This approach is exemplified by the anti-inflammatory drug Enbrel®, which is a fusion of the extracellular domain of the TNF-a receptor with the Fc portion of human IgG1.
Low productivity levels and/or poor product quality beset the expression of some soluble receptor-fusion proteins when expressed under similar conditions as mAbs. Low expression levels will necessitate either increased manufacturing capacity or increased facilities time to deliver sufficient amounts of material to satisfy clinical or commercial demands. High levels of aggregate may necessitate the need to develop additional chromatography steps, which can greatly reduce the overall yield and drive up the overall process time and facilities costs.
Despite these problems, soluble receptors may remain an attractive alternative to mAbs. Cell surface receptors have evolved over time to bind with high specificity to their cognate cytokine or growth factor and are therefore already optimized for use as a blocking reagent.
Identifying and developing a therapeutically useful mAb is often a long and costly process (with additional time and costs if humanization of the mAb or other alterations are required). If the sequence information has been determined for the receptor, it may be relatively straightforward to design a soluble form of the receptor and have confidence that it will retain the desired binding characteristics. The speed and relative ease of designing soluble receptors enables more rapid, in vitro, proof-of-concept studies to be completed and can potentially accelerate the timeline for introducing the molecule into the clinic.
There may also be additional business drivers for choosing to produce a soluble receptor over a blocking mAb. Clearly, strategies for improving the expression and quality of these soluble receptor-fusion proteins may contribute to the success of bringing these molecules forward into the clinic.
A Problem with Misfolding
The low productivity of cell lines expressing soluble receptor-fusion proteins as well as the poor product quality of these proteins may be due to poor protein folding. While consideration may be given to the design of the soluble receptor-fusion protein at the primary amino acid level, secondary or higher-order structures may not be optimal for facilitating the proper folding or structural stability of the soluble receptor-fusion protein, with consequences to both expression and product quality.
The folding stability of proteins can be reflected in their thermal-melting profile as determined by differential-scanning calorimetry (DSC). A protein that exhibits its first thermal transition at temperatures that are well above typical cell culture temperatures (i.e., >37ºC) is likely to adopt a stable folding conformation and will be well expressed.
Most mAbs, for example, display their first thermal transition at 60ºC or greater. In contrast, proteins whose structures display a thermal transition at temperatures at or near the cell-culture temperature are less likely to adopt a stable folding conformation and as a result, are often poorly expressed and are prone to aggregation. Lowering the cell-culture temperature may promote more stable protein-folding conformations and should result in more efficient transit through the secretory pathway.