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Selective targeting of IL-2 to NKG2D bearing cells for improved immunotherapy

View Article: PubMed Central - PubMed

ABSTRACT

Despite over 20 years of clinical use, IL-2 has not fulfilled expectations as a safe and effective form of tumour immunotherapy. Expression of the high affinity IL-2Rα chain on regulatory T cells mitigates the anti-tumour immune response and its expression on vascular endothelium is responsible for life threatening complications such as diffuse capillary leak and pulmonary oedema. Here we describe the development of a recombinant fusion protein comprised of a cowpox virus encoded NKG2D binding protein (OMCP) and a mutated form of IL-2 with poor affinity for IL-2Rα. This fusion protein (OMCP-mutIL-2) potently and selectively activates IL-2 signalling only on NKG2D-bearing cells, such as natural killer (NK) cells, without broadly activating IL-2Rα-bearing cells. OMCP-mutIL-2 provides superior tumour control in several mouse models of malignancy and is not limited by mouse strain-specific variability of NK function. In addition, OMCP-mutIL-2 lacks the toxicity and vascular complications associated with parental wild-type IL-2.

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Mechanism of OMCP-mutIL-2 Competition with Stromal Cells.(a) Serum levels of fluorochrome-labelled cytokine or fusion protein after injection of 1 × 106 IUe (i.v.) determined fluoroscopically according to a standard curve. (b) Decay in STAT5 phosphorylation after a 15-minute stimulation by 1,000 IUeml−1 (left) or 100 IUe ml−1 (right) of IL-2 or OMCP-mutIL-2 as determined flow cytometrically. Comparison performed by multiple unpaired t-tests at each individual time point. (c) Proposed model of competition between NK cells and stromal cells for IL-2. Width of arrow indicates proposed strength of IL-2 signaling. (d) Dose response in STAT5 phosphorylation of C57BL/6 NK cells in the presence of other splenocytes by wtIL-2 and OMCP-mutIL-2 as determined by flow cytometric staining. Comparison performed by multiple unpaired t-tests at each individual cytokine concentration. (e) STAT5 phosphorylation of wild-type or NKG2D−/− NK cells by wtIL-2 and OMCP-mutIL-2 in the presence of competing splenocytes. (f) STAT5 phosphorylation, as measured by fold change over saline-treated controls, of wild-type NK cells in the presence of competing splenocytes treated with saturating concentrations of rat anti-mouse CD25 (clone 3C7) or rat IgG isotype control. Comparison performed by unpaired t-test between groups as indicated by the lines above the graphs. ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; blue=wtIL-2, red=OMCP-mutIL-2, green=mutIL-2.
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f7: Mechanism of OMCP-mutIL-2 Competition with Stromal Cells.(a) Serum levels of fluorochrome-labelled cytokine or fusion protein after injection of 1 × 106 IUe (i.v.) determined fluoroscopically according to a standard curve. (b) Decay in STAT5 phosphorylation after a 15-minute stimulation by 1,000 IUeml−1 (left) or 100 IUe ml−1 (right) of IL-2 or OMCP-mutIL-2 as determined flow cytometrically. Comparison performed by multiple unpaired t-tests at each individual time point. (c) Proposed model of competition between NK cells and stromal cells for IL-2. Width of arrow indicates proposed strength of IL-2 signaling. (d) Dose response in STAT5 phosphorylation of C57BL/6 NK cells in the presence of other splenocytes by wtIL-2 and OMCP-mutIL-2 as determined by flow cytometric staining. Comparison performed by multiple unpaired t-tests at each individual cytokine concentration. (e) STAT5 phosphorylation of wild-type or NKG2D−/− NK cells by wtIL-2 and OMCP-mutIL-2 in the presence of competing splenocytes. (f) STAT5 phosphorylation, as measured by fold change over saline-treated controls, of wild-type NK cells in the presence of competing splenocytes treated with saturating concentrations of rat anti-mouse CD25 (clone 3C7) or rat IgG isotype control. Comparison performed by unpaired t-test between groups as indicated by the lines above the graphs. ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; blue=wtIL-2, red=OMCP-mutIL-2, green=mutIL-2.

Mentions: Antibody-IL-2 complexes improve cytokine activity by extending the duration of serum half-life2829. To investigate whether the linking of mutIL-2 to OMCP increased serum half-life we injected 500,000 IUe of fluorescently-labelled wtIL-2, mutIL-2 or OMCP-mutIL-2 intravenously and monitored serum levels over time with periodic blood draws. Whereas OMCP-mutIL-2 had a slightly higher serum concentration at early time points, all proteins were undetectable in the blood one hour post-injection (Fig. 7a). This is shorter than the described 11–14 h serum half-life of antibody-IL-2 conjugates28. Despite the injection of identical amounts of cytokine, lower levels were detected in C57BL/6 than A/J mice at all time points. Strain-specific differences in the clearance of IL-2 may explain why C57BL/6 mice tolerate and require higher doses of IL-2 for NK expansion. Nevertheless it is unlikely that prolonged circulation of the fusion protein was responsible for the increased activation of NK cells by OMCP-mutIL-2 in either strain.


Selective targeting of IL-2 to NKG2D bearing cells for improved immunotherapy
Mechanism of OMCP-mutIL-2 Competition with Stromal Cells.(a) Serum levels of fluorochrome-labelled cytokine or fusion protein after injection of 1 × 106 IUe (i.v.) determined fluoroscopically according to a standard curve. (b) Decay in STAT5 phosphorylation after a 15-minute stimulation by 1,000 IUeml−1 (left) or 100 IUe ml−1 (right) of IL-2 or OMCP-mutIL-2 as determined flow cytometrically. Comparison performed by multiple unpaired t-tests at each individual time point. (c) Proposed model of competition between NK cells and stromal cells for IL-2. Width of arrow indicates proposed strength of IL-2 signaling. (d) Dose response in STAT5 phosphorylation of C57BL/6 NK cells in the presence of other splenocytes by wtIL-2 and OMCP-mutIL-2 as determined by flow cytometric staining. Comparison performed by multiple unpaired t-tests at each individual cytokine concentration. (e) STAT5 phosphorylation of wild-type or NKG2D−/− NK cells by wtIL-2 and OMCP-mutIL-2 in the presence of competing splenocytes. (f) STAT5 phosphorylation, as measured by fold change over saline-treated controls, of wild-type NK cells in the presence of competing splenocytes treated with saturating concentrations of rat anti-mouse CD25 (clone 3C7) or rat IgG isotype control. Comparison performed by unpaired t-test between groups as indicated by the lines above the graphs. ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; blue=wtIL-2, red=OMCP-mutIL-2, green=mutIL-2.
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f7: Mechanism of OMCP-mutIL-2 Competition with Stromal Cells.(a) Serum levels of fluorochrome-labelled cytokine or fusion protein after injection of 1 × 106 IUe (i.v.) determined fluoroscopically according to a standard curve. (b) Decay in STAT5 phosphorylation after a 15-minute stimulation by 1,000 IUeml−1 (left) or 100 IUe ml−1 (right) of IL-2 or OMCP-mutIL-2 as determined flow cytometrically. Comparison performed by multiple unpaired t-tests at each individual time point. (c) Proposed model of competition between NK cells and stromal cells for IL-2. Width of arrow indicates proposed strength of IL-2 signaling. (d) Dose response in STAT5 phosphorylation of C57BL/6 NK cells in the presence of other splenocytes by wtIL-2 and OMCP-mutIL-2 as determined by flow cytometric staining. Comparison performed by multiple unpaired t-tests at each individual cytokine concentration. (e) STAT5 phosphorylation of wild-type or NKG2D−/− NK cells by wtIL-2 and OMCP-mutIL-2 in the presence of competing splenocytes. (f) STAT5 phosphorylation, as measured by fold change over saline-treated controls, of wild-type NK cells in the presence of competing splenocytes treated with saturating concentrations of rat anti-mouse CD25 (clone 3C7) or rat IgG isotype control. Comparison performed by unpaired t-test between groups as indicated by the lines above the graphs. ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; blue=wtIL-2, red=OMCP-mutIL-2, green=mutIL-2.
Mentions: Antibody-IL-2 complexes improve cytokine activity by extending the duration of serum half-life2829. To investigate whether the linking of mutIL-2 to OMCP increased serum half-life we injected 500,000 IUe of fluorescently-labelled wtIL-2, mutIL-2 or OMCP-mutIL-2 intravenously and monitored serum levels over time with periodic blood draws. Whereas OMCP-mutIL-2 had a slightly higher serum concentration at early time points, all proteins were undetectable in the blood one hour post-injection (Fig. 7a). This is shorter than the described 11–14 h serum half-life of antibody-IL-2 conjugates28. Despite the injection of identical amounts of cytokine, lower levels were detected in C57BL/6 than A/J mice at all time points. Strain-specific differences in the clearance of IL-2 may explain why C57BL/6 mice tolerate and require higher doses of IL-2 for NK expansion. Nevertheless it is unlikely that prolonged circulation of the fusion protein was responsible for the increased activation of NK cells by OMCP-mutIL-2 in either strain.

View Article: PubMed Central - PubMed

ABSTRACT

Despite over 20 years of clinical use, IL-2 has not fulfilled expectations as a safe and effective form of tumour immunotherapy. Expression of the high affinity IL-2R&alpha; chain on regulatory T cells mitigates the anti-tumour immune response and its expression on vascular endothelium is responsible for life threatening complications such as diffuse capillary leak and pulmonary oedema. Here we describe the development of a recombinant fusion protein comprised of a cowpox virus encoded NKG2D binding protein (OMCP) and a mutated form of IL-2 with poor affinity for IL-2R&alpha;. This fusion protein (OMCP-mutIL-2) potently and selectively activates IL-2 signalling only on NKG2D-bearing cells, such as natural killer (NK) cells, without broadly activating IL-2R&alpha;-bearing cells. OMCP-mutIL-2 provides superior tumour control in several mouse models of malignancy and is not limited by mouse strain-specific variability of NK function. In addition, OMCP-mutIL-2 lacks the toxicity and vascular complications associated with parental wild-type IL-2.

No MeSH data available.


Related in: MedlinePlus