APOLLO'S IL-4 vs E.COLI

Increased Biological Stability of Human Cell Expressed IL-4

It has been proposed that glycosylation is important for secretion, solubility, resistance to proteolysis, immunogenicity, biological recognition, biological activity, in vivo stability and clearance of glycoproteins including cytokines and growth factors from the blood. Glycosylation of IL-4, known to be important for biological activity, can be diminished or completely absent in E. coli expressed proteins.

In vitro comparisons of the biological activity of human cell expressed cytokines, with cytokines derived from other sources revealed that human cell expression confers enhanced bioactivity. These data prompted us to compare the activity of our human cell expressed IL-4 with E. coli expressed IL-4.

Bioactivity of IL-4 was measured in a cell proliferation assay using a human factor-dependent cell line, TF-1. Comparison of IL-4 expressed in E. coli or human cells (Apollo Cytokine Research) showed that in an extended cell proliferation assay, Apollo's IL-4^hcx induced more cell proliferation after 7 days in culture, suggesting it has a greater biological activity and perhaps a greater half-life (Figure 1). The ED₅₀ of Apollo's IL-4^hcx was also lower (0.03 ng/ml) compared to E. coli (1 ng/ml).

Figure 1

When IL-4 was pre-incubated for 4 days in cell culture medium (RPMI 1640/10% FBS) at 37^oC before addition to the assay, Apollo's IL-4^hcx also induced a much higher level of cell proliferation, further demonstrating it has greater stability in cell culture conditions than E. coli expressed IL-4 (Figure 2).

Figure 2

The data from these preliminary studies demonstrate that human cell expressed IL-4, with human cell specific glycosylation, has greater biological activity and stability in cell culture than E. coli expressed IL-4.

This data has important implications for ex vivo procedures such as derivation of dendritic cells, where IL-4 in the culture medium must be replenished several times during the process of dendritic cell generation¹. As this requires up to 14 days in cell culture, the increased stability of human cell expressed proteins could facilitate fewer additions of cytokines. Derivation of dendritic cells using glycosylated human cell expressed cytokines and growth factors, which closely resemble the native proteins, may also enhance the activation and therapeutic efficacy of the cells in terms of their capacity to activate T-cells and their ability to take up, process and present antigens.

References:

1. Lutz (2004) Immunobiology 209 (79-87)

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