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

Authors
R. Newman, H. Jiang, K. Liddell, L. Crofts, R. Simpson, D. Marks

Introduction
• IL-12 is a potent pro-inflammatory cytokine that consists of a heterodimer composed of two disulphide-linked subunits, p35 and p40 [1].

• Produced by peripheral blood mononuclear cells, IL-12 plays a central role in the initiation and control of cell mediated immune responses through its effects on NK cells and T lymphocytes [2].

• IL-12 activates the Jak/STAT pathway via the IL-12 receptor, inducing IFN-? production. IL-12 enhances the lytic activity of NK and lymphokine-activated killer cells, and induces the proliferation of activated T and NK- cells p40 [1].

• Clinically, recombinant human (rh) IL-12 has been evaluated for its therapeutic efficacy in multiple clinical trials in cancer and chronic infections [3].

• Currently, rhIL-12 is produced in non-human cell systems including insect and CHO. However, it is becoming apparent that human-specific post-translational modifications, in particular glycosylation, are important to human protein function.

Aim
We have purified human cell-expressed rhIL-12 (IL-12 hcx) from modified human 293 cells. Our aim was to compare the glycan structures and in vitro biological activities of IL-12 hcx to that of CHO-expressed IL-12 (CHO IL-12) in human peripheral mononuclear cells.

Methods
Characterisation of IL-12
Purified IL-12A and CHO IL-12 were subjected to enzymatic treatment for the analysis of glycan structures using LCMS. N- and O-linked structures were assigned from the acquired data using GlycosidIQ (www.glycosuite.com). The C-mannosylated tryptophan was identified by MS/MS of the peptides in a trypsin-V8 digest of IL-12.

PBMC isolation and activation
Peripheral blood mononuclear cells (PBMC) were purified from healthy donors by density centrifugation using Lymphoprep (Axis-Shield). Cells were activated with 10 µg/ml phytohemagglutin (PHA; Sigma-Aldrich) for a total of 4 days, during which time the cells became lymphoblasts. On day 3, the cultures were split and 10 ng/ml rhIL-2 (R&D Systems) was added to promote lymphoblast proliferation. On day 4, the lymphoblasts were harvested, washed and prepared for IL-12 treatment.

Phosphorylated STAT4 and STAT5 immunoblotting
Lymphoblasts were serum starved for 4 hrs prior to stimulation with 50 ng/ml IL-12 at 37ºC/5% CO₂. Lysates were prepared with 1x passive lysis buffer (Promega), subjected to western blotting and probed with antibodies to phospho-STAT4 (pY693) and total STAT4 (BD Biosciences, Santa Cruz Biotechnologies) or phospho-STAT5 (pY694) and total STAT5 (Cell Signalling). Western blots were analysed with Fuji Film LAS-3000 and quantification of bands by densitometry was performed with MultiGauge software.

IFN-? detection by ELISA
2x10⁵ lymphoblasts were treated with IL-12 (0 –10 ng/ml) for 2 days at 37ºC/5% CO₂. Supernatants were collected and IFN-? concentrations measured using the IFN-? DuoSet ELISA Development kit (R&D Systems). Data is expressed as mean ± s.d. Statistical significance was evaluated using one way ANOVA using GraphPad Prism 5.

Proliferation assays
4x10⁴ lymphoblasts were treated with serial dilutions (0 – 50 ng/ml) of IL-12 for 2 days at 37ºC/5% CO₂. Proliferation was measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) and ED₅₀s were determined.

Cytolytic assay: Calcein-AM release
PBMC were stimulated with 1 ng/ml IL-12 for 3 days at 37ºC/5% CO₂ for use as effector cells. K562 cells were labelled with 15 µM Calcein-AM (Molecular Probes) and used as target cells. Effector and target cells were added together in 96-well u-bottom plates and incubated for 4 hrs at 37ºC/5% CO₂ at various effector (E):target (T) ratios. 75 µl of supernatant was then transferred into a new plate and Calcein-AM release was measured using a FLUOstar OPTIMA plate reader at 485nm/520nm (BMG LABTECH). Specific lysis was calculated as: [(test release – spontaneous release)/(maximum release – spontaneous release)] x 100%.

Results and Discussion
Glycan Analysis of IL-12 hcx
Both IL-12 hcx 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 (Tables 1a and b). No differences were observed in O-linked structures (Table 2). Both IL-12 hcx and CHO IL-12 express C-linked mannosylation (data not shown).

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.

Data are representative of 3 experiments

Data are representative of 3 experiments

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

Data are representative of 3 experiments and expressed as mean ± 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.

Data are representative of 3 experiments

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

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

Summary and Conclusions
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-?
• 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.

References:
1. Gately et al. (1998) Ann. Rev. Immunology, 16, 495-521
2. Chehimi and Trinchieri (1994) J Clin. Immunology, 14, 149-161
3. Del Vecchio et al. (2007) Clin. Cancer Res., 16, 4677-4685

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