APOLLO'S IL-12 hcx vs CHO-EXPRESSED IL-12

Human cell expressed IL-12 has enhanced pro-inflammatory activity

IL-12 is a potent pro-inflammatory cytokine produced in response to pathogenic organisms, and a key regulator of innate and adaptive immune response. In particular IL-12 stimulates T-cells and natural killer cells (NK), leading to production of interferon-gamma (IFN-γ) and induction of cell mediated immune responses [1].

Clinically, recombinant human (rh) IL-12 has been evaluated for its therapeutic efficacy in multiple clinical trials in cancer and chronic infections [2].

Currently, commercially available IL-12 is purified from non-human cells including insect and CHO. However, it is becoming apparent that human-specific post-translational modifications, in particular glycosylation, are important to human protein function.

Apollo has purified human cell-expressed rh IL-12 (IL-12 hcx) from modified human 293 cells.

IL-12 hcx has N-and O-linked glycans, and C-linked mannose structures. IL-12 hcx is a disulphide-linked heterodimeric protein consisting of an α-chain which contains 15-35% carbohydrate, and the β-chain contains up to 25% carbohydrate.

In order to assess the viability of IL-12 hcx as a superior cytokine, research was undertaken to compare the glycan structures and in vitro biological activities with CHO-expressed rh IL-12 (CHO IL-12) in human peripheral mononuclear cells.

Glycan Analysis

Both IL-12 and CHO IL-12 contain N and O-linked glycan structures. For N-lined structures, IL-12 hcx contains more sialylated and high mannose structures when compared to CHO IL-12. No differences were observed in O-linked structures. Both IL-12 hcx and CHO IL-12 express C-linked mannosylation.

Further glycan data can be found in the following poster:

• Download Poster: Human cell-expressed IL-12 has enhanced pro-inflammatory activity

Bioactivity Results

IL-12 hcx induced up to 3-fold more STAT4 (Figure 1a) and 2-fold more STAT5 (Figure 1b) activation than CHO IL-12 in lymphoblasts making it a more potent activator.

Figure 1a

Data are representative of 3 experiments

Figure 1b

Data are representative of 3 experiments

IL-12 hcx induces more IFN-γ production by lymphoblasts than CHO-expressed IL-12 (Figure 2). IL-12 hcx induced dose-dependent IFN-γ production by lymphoblasts. IL-12 induced IFN-γ production at concentrations as low as 0.5 ng/ml, whereas CHO IL-12 did not induce IFN-γ below 5ng/ml. IFN-γ induction by IL-12 hcx was significantly higher than CHO IL-12 at 0.5 - 7.5 ng/ml (p<0.001).

Figure 2

Data are representative of 3 experiments and expressed as ±s.d.

IL-12 hcx induces more proliferation of lymphoblasts than CHO-expressed IL-12 (Figure 3). It was shown to be 6-fold more active at inducing lymphoblast proliferation compared to CHO IL-12; ED50:80 ng/ml v 500 ng/ml.

Figure 3

Data are representative of 3 experiments

IL-12 hcx enhances the lytic activity of PBMC against K562 cells more than CHO-expressed IL-12 for all E:T ratios (Figure 4).

Figure 4

Data are representative of 3 experiments and expressed as ±s.d.

Conclusion

IL-12 hcx has greater biological activity compared to CHO-expressed IL-12. This has been demonstrated by:

• Increased activation of STAT molecules
• Increased production of IFN-γ
• Increased cell proliferation
• Enhanced cytolytic activity of PBMC

These effects may be attributed to the structural differences observed between human and non-human cell-expressed IL-12.

IL-12 hcx may provide unique benefits for the study of the role of IL-12 in disease and normal immunity.

Further reading:

• Download poster : Human cell-expressed IL-12 has enhanced pro-inflammatory activity
• Download brochure : Apollo's hcx proteins for inflammation research
• Download brochure : Apollo's hcx proteins for cancer research

View relevant products:

• » IL-12

References

1. Chehimi and Trinchieri (1994) J Clin. Immunology 14 (149-161)
2. Del Vecchio et al. (2007) Clin. Cancer Res. 16 (4677-4685)

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