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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.

No MeSH data available.


Mechanism of OMCP-mutIL-2 Signalling in NKG2D-Expressing Cells.(a) STAT5 phosphorylation in isolated NK cells from splenocytes of A/J (left) or C57BL/6 mice (right) by increasing doses of cytokine. (b) Canonical IL-2 and NKG2D signaling pathways. (c) Jak1 phosphorylation of Ky1.1 NK cell line in vitro (d) Vav phosphorylation in freshly isolated splenic C57BL/6 NK cells. Representative of two separate experiments with full blots demonstrated in Supplementary Fig. 7. (e) Construction and phenotype of BWZ.36 LacZ reporter cell line (top) and expression of NKG2D (bottom left) and CD132 (bottom right) with parental cell shown in black, functional NKG2D/DAP12 WT in green and NKG2D/mutant Y2F DAP12 in blue. Phenotype is representative of two separate experiments. (f) β-galactosidase expression by BWZ.36 cell line bearing functional NKG2D/DAP12 (left panel), NKG2D/mutant Y2F DAP12 (middle), or parental BWZ. 36 cell line not expressing NKG2D/DAP12. Data are representative of three separate experiments with comparison performed by ANOVA for multiple comparisons or unpaired t-test between individual groups as indicated by the lines above the graphs.
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f6: Mechanism of OMCP-mutIL-2 Signalling in NKG2D-Expressing Cells.(a) STAT5 phosphorylation in isolated NK cells from splenocytes of A/J (left) or C57BL/6 mice (right) by increasing doses of cytokine. (b) Canonical IL-2 and NKG2D signaling pathways. (c) Jak1 phosphorylation of Ky1.1 NK cell line in vitro (d) Vav phosphorylation in freshly isolated splenic C57BL/6 NK cells. Representative of two separate experiments with full blots demonstrated in Supplementary Fig. 7. (e) Construction and phenotype of BWZ.36 LacZ reporter cell line (top) and expression of NKG2D (bottom left) and CD132 (bottom right) with parental cell shown in black, functional NKG2D/DAP12 WT in green and NKG2D/mutant Y2F DAP12 in blue. Phenotype is representative of two separate experiments. (f) β-galactosidase expression by BWZ.36 cell line bearing functional NKG2D/DAP12 (left panel), NKG2D/mutant Y2F DAP12 (middle), or parental BWZ. 36 cell line not expressing NKG2D/DAP12. Data are representative of three separate experiments with comparison performed by ANOVA for multiple comparisons or unpaired t-test between individual groups as indicated by the lines above the graphs.

Mentions: To evaluate IL-2 signalling we next quantitated STAT5 phosphorylation of NK cells ex vivo. Lower levels of STAT5 phosphorylation were evident in A/J compared with C57BL/6 NK cells at all concentrations tested (Fig. 6a) suggesting that lymphocyte dysfunction of A/J mice may partially result from inefficient IL-2 signal transduction. Interestingly in such purified NK cell preparations wtIL-2 and OMCP-mutIL-2 demonstrated identical dose-dependent STAT5 phosphorylation (Fig. 6a). In the absence of NKG2D, OMCP-mutIL-2 failed to increase STAT5 phosphorylation over mutIL-2 alone (Fig. 6a). Taken together, these data suggest that IL-2Rα engagement is important for peak IL-2 signalling even in resting NK cells, and that NKG2D-binding may substitute for IL-2Rα-interaction in IL-2-mediated signal transduction. Such data, however, opened the possibility that the superior efficacy of OMCP-mutIL-2 over mutIL-2 alone was due to concomitant signalling through NKG2D, since activation of this receptor can act as a primary or co-stimulatory factor for NK cells20.


Selective targeting of IL-2 to NKG2D bearing cells for improved immunotherapy
Mechanism of OMCP-mutIL-2 Signalling in NKG2D-Expressing Cells.(a) STAT5 phosphorylation in isolated NK cells from splenocytes of A/J (left) or C57BL/6 mice (right) by increasing doses of cytokine. (b) Canonical IL-2 and NKG2D signaling pathways. (c) Jak1 phosphorylation of Ky1.1 NK cell line in vitro (d) Vav phosphorylation in freshly isolated splenic C57BL/6 NK cells. Representative of two separate experiments with full blots demonstrated in Supplementary Fig. 7. (e) Construction and phenotype of BWZ.36 LacZ reporter cell line (top) and expression of NKG2D (bottom left) and CD132 (bottom right) with parental cell shown in black, functional NKG2D/DAP12 WT in green and NKG2D/mutant Y2F DAP12 in blue. Phenotype is representative of two separate experiments. (f) β-galactosidase expression by BWZ.36 cell line bearing functional NKG2D/DAP12 (left panel), NKG2D/mutant Y2F DAP12 (middle), or parental BWZ. 36 cell line not expressing NKG2D/DAP12. Data are representative of three separate experiments with comparison performed by ANOVA for multiple comparisons or unpaired t-test between individual groups as indicated by the lines above the graphs.
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f6: Mechanism of OMCP-mutIL-2 Signalling in NKG2D-Expressing Cells.(a) STAT5 phosphorylation in isolated NK cells from splenocytes of A/J (left) or C57BL/6 mice (right) by increasing doses of cytokine. (b) Canonical IL-2 and NKG2D signaling pathways. (c) Jak1 phosphorylation of Ky1.1 NK cell line in vitro (d) Vav phosphorylation in freshly isolated splenic C57BL/6 NK cells. Representative of two separate experiments with full blots demonstrated in Supplementary Fig. 7. (e) Construction and phenotype of BWZ.36 LacZ reporter cell line (top) and expression of NKG2D (bottom left) and CD132 (bottom right) with parental cell shown in black, functional NKG2D/DAP12 WT in green and NKG2D/mutant Y2F DAP12 in blue. Phenotype is representative of two separate experiments. (f) β-galactosidase expression by BWZ.36 cell line bearing functional NKG2D/DAP12 (left panel), NKG2D/mutant Y2F DAP12 (middle), or parental BWZ. 36 cell line not expressing NKG2D/DAP12. Data are representative of three separate experiments with comparison performed by ANOVA for multiple comparisons or unpaired t-test between individual groups as indicated by the lines above the graphs.
Mentions: To evaluate IL-2 signalling we next quantitated STAT5 phosphorylation of NK cells ex vivo. Lower levels of STAT5 phosphorylation were evident in A/J compared with C57BL/6 NK cells at all concentrations tested (Fig. 6a) suggesting that lymphocyte dysfunction of A/J mice may partially result from inefficient IL-2 signal transduction. Interestingly in such purified NK cell preparations wtIL-2 and OMCP-mutIL-2 demonstrated identical dose-dependent STAT5 phosphorylation (Fig. 6a). In the absence of NKG2D, OMCP-mutIL-2 failed to increase STAT5 phosphorylation over mutIL-2 alone (Fig. 6a). Taken together, these data suggest that IL-2Rα engagement is important for peak IL-2 signalling even in resting NK cells, and that NKG2D-binding may substitute for IL-2Rα-interaction in IL-2-mediated signal transduction. Such data, however, opened the possibility that the superior efficacy of OMCP-mutIL-2 over mutIL-2 alone was due to concomitant signalling through NKG2D, since activation of this receptor can act as a primary or co-stimulatory factor for NK cells20.

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.

No MeSH data available.