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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.


Related in: MedlinePlus

Treg Activation and Expansion with IL-2 and construct administration in vivo.(a) Splenic CD4+Foxp3+ Treg expansion and activation as measured by cell counts in the spleen (top) and ICOS, CD25, GITR and KI67 expression (bottom) in A/J and C57BL/6 (b) mice. (c) In vitro suppression of C57BL/6CD45.1+ congenic T cell proliferation by CD4+Foxp3+ Tregs isolated from splenocytes of C57BL/6CD45.2+Foxp3+GFP+ mice treated with saline (black), wtIL-2 (blue), mutIL-2 (green) and OMCP-mutIL-2 (red) in vivo. Data representative of four separate in vitro experiments with comparison performed by unpaired t-test to saline-treated control at each ratio indicated and confirmed by ANOVA. (d) NK/Treg ratio in the spleen of A/J or C57BL/6 (e) mice treated with saline (black), wtIL-2 (blue), mutIL-2 (green) and OMCP-mutIL-2 (red) in vivo. All graphs in a,b,d,e represent an average cell count±s.e.m. from 5 to 10 mice per group performed as 4–7 separate experiments for each strain of mice. Comparison performed by unpaired t-test between groups as indicated by the lines. ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; black=saline; blue=wtIL-2, red=OMCP-mutIL-2, green=mutIL-2; turkoise wtIL-2 complexed to anti-IL-2.
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f5: Treg Activation and Expansion with IL-2 and construct administration in vivo.(a) Splenic CD4+Foxp3+ Treg expansion and activation as measured by cell counts in the spleen (top) and ICOS, CD25, GITR and KI67 expression (bottom) in A/J and C57BL/6 (b) mice. (c) In vitro suppression of C57BL/6CD45.1+ congenic T cell proliferation by CD4+Foxp3+ Tregs isolated from splenocytes of C57BL/6CD45.2+Foxp3+GFP+ mice treated with saline (black), wtIL-2 (blue), mutIL-2 (green) and OMCP-mutIL-2 (red) in vivo. Data representative of four separate in vitro experiments with comparison performed by unpaired t-test to saline-treated control at each ratio indicated and confirmed by ANOVA. (d) NK/Treg ratio in the spleen of A/J or C57BL/6 (e) mice treated with saline (black), wtIL-2 (blue), mutIL-2 (green) and OMCP-mutIL-2 (red) in vivo. All graphs in a,b,d,e represent an average cell count±s.e.m. from 5 to 10 mice per group performed as 4–7 separate experiments for each strain of mice. Comparison performed by unpaired t-test between groups as indicated by the lines. ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; black=saline; blue=wtIL-2, red=OMCP-mutIL-2, green=mutIL-2; turkoise wtIL-2 complexed to anti-IL-2.

Mentions: Preferential activation of IL2Rα-expressing Tregs has been one of the main challenges to IL-2 cytokine immunotherapy8. Consistent with this notion wtIL-2 led to a significant expansion and activation of CD4+Foxp3+Tregs as measured by total cell number and expression of ICOS, GITR, CD25 and KI-67 in A/J mice (Fig. 5a). Expansion of this cell population was also evident when wtIL-2 was complexed to anti-IL-2 antibodies (Fig. 5a)19. Similar results were evident for the C57BL/6 strain except for the ameliorated Treg expansion when wtIL-2 was complexed to anti-IL-2 antibodies (Fig. 5b). However, for both strains of mice no expansion or activation of Tregs was evident after treatment with OMCP-mutIL-2 compared mutIL-2 or saline-treated controls (Fig. 5a,b).


Selective targeting of IL-2 to NKG2D bearing cells for improved immunotherapy
Treg Activation and Expansion with IL-2 and construct administration in vivo.(a) Splenic CD4+Foxp3+ Treg expansion and activation as measured by cell counts in the spleen (top) and ICOS, CD25, GITR and KI67 expression (bottom) in A/J and C57BL/6 (b) mice. (c) In vitro suppression of C57BL/6CD45.1+ congenic T cell proliferation by CD4+Foxp3+ Tregs isolated from splenocytes of C57BL/6CD45.2+Foxp3+GFP+ mice treated with saline (black), wtIL-2 (blue), mutIL-2 (green) and OMCP-mutIL-2 (red) in vivo. Data representative of four separate in vitro experiments with comparison performed by unpaired t-test to saline-treated control at each ratio indicated and confirmed by ANOVA. (d) NK/Treg ratio in the spleen of A/J or C57BL/6 (e) mice treated with saline (black), wtIL-2 (blue), mutIL-2 (green) and OMCP-mutIL-2 (red) in vivo. All graphs in a,b,d,e represent an average cell count±s.e.m. from 5 to 10 mice per group performed as 4–7 separate experiments for each strain of mice. Comparison performed by unpaired t-test between groups as indicated by the lines. ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; black=saline; blue=wtIL-2, red=OMCP-mutIL-2, green=mutIL-2; turkoise wtIL-2 complexed to anti-IL-2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036003&req=5

f5: Treg Activation and Expansion with IL-2 and construct administration in vivo.(a) Splenic CD4+Foxp3+ Treg expansion and activation as measured by cell counts in the spleen (top) and ICOS, CD25, GITR and KI67 expression (bottom) in A/J and C57BL/6 (b) mice. (c) In vitro suppression of C57BL/6CD45.1+ congenic T cell proliferation by CD4+Foxp3+ Tregs isolated from splenocytes of C57BL/6CD45.2+Foxp3+GFP+ mice treated with saline (black), wtIL-2 (blue), mutIL-2 (green) and OMCP-mutIL-2 (red) in vivo. Data representative of four separate in vitro experiments with comparison performed by unpaired t-test to saline-treated control at each ratio indicated and confirmed by ANOVA. (d) NK/Treg ratio in the spleen of A/J or C57BL/6 (e) mice treated with saline (black), wtIL-2 (blue), mutIL-2 (green) and OMCP-mutIL-2 (red) in vivo. All graphs in a,b,d,e represent an average cell count±s.e.m. from 5 to 10 mice per group performed as 4–7 separate experiments for each strain of mice. Comparison performed by unpaired t-test between groups as indicated by the lines. ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; black=saline; blue=wtIL-2, red=OMCP-mutIL-2, green=mutIL-2; turkoise wtIL-2 complexed to anti-IL-2.
Mentions: Preferential activation of IL2Rα-expressing Tregs has been one of the main challenges to IL-2 cytokine immunotherapy8. Consistent with this notion wtIL-2 led to a significant expansion and activation of CD4+Foxp3+Tregs as measured by total cell number and expression of ICOS, GITR, CD25 and KI-67 in A/J mice (Fig. 5a). Expansion of this cell population was also evident when wtIL-2 was complexed to anti-IL-2 antibodies (Fig. 5a)19. Similar results were evident for the C57BL/6 strain except for the ameliorated Treg expansion when wtIL-2 was complexed to anti-IL-2 antibodies (Fig. 5b). However, for both strains of mice no expansion or activation of Tregs was evident after treatment with OMCP-mutIL-2 compared mutIL-2 or saline-treated controls (Fig. 5a,b).

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