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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.


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Immunoactivation with IL-2 and construct administration in vivo.(a) Total splenocyte counts in AJ mice after a five-day course of 200,000 IUe of wtIL-2 (blue), mutIL-2 (green) and OMCP-mutIL-2 (red). (b) NK cell expansion and activation after wtIL-2, mutIL-2, OMCP-mutIL-2, high dose wtIL-2, high dose mutIL-2 and wtIL-2/anti-IL-2 complexes measured by cell counts in the spleen (top) and KLRG1 upregulation (bottom). Expansion of splenocytes (c) and NK cells (d) in C57BL/6 mice treated with 750,000 IUe of cytokine or construct. All graphs represent an average cell count ± s.e.m. from 5–10 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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f4: Immunoactivation with IL-2 and construct administration in vivo.(a) Total splenocyte counts in AJ mice after a five-day course of 200,000 IUe of wtIL-2 (blue), mutIL-2 (green) and OMCP-mutIL-2 (red). (b) NK cell expansion and activation after wtIL-2, mutIL-2, OMCP-mutIL-2, high dose wtIL-2, high dose mutIL-2 and wtIL-2/anti-IL-2 complexes measured by cell counts in the spleen (top) and KLRG1 upregulation (bottom). Expansion of splenocytes (c) and NK cells (d) in C57BL/6 mice treated with 750,000 IUe of cytokine or construct. All graphs represent an average cell count ± s.e.m. from 5–10 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: When A/J mice received a regimen of 200,000 IUe of cytokine or fusion protein, given as 10 equal doses over 5 days, both wtIL-2 and OMCP-mutIL-2 increased splenocyte numbers compared with saline-treated controls (Fig. 4a). OMCP-mutIL-2 led to a substantial expansion and activation of NK cells as measured by cell number and surface KLRG1 levels (Fig. 4b). Remarkably, in OMCP-mutIL-2 treated mice, NK cells comprised close to half of all splenic lymphocytes, paralleling or even surpassing the total lymphocyte counts of saline or mutIL-2-treated mice (Fig. 4a versus b). NK cell expansion by 200,000 IUe of OMCP-mutIL-2 was greater than that seen with near toxic doses of wtIL-2 (750,000 IU), high dose mutIL-2 (3,500,000 IUe) or wtIL-2 complexed to anti-IL-2 antibody (clone MAB602)19 (Fig. 4b). Superior expansion of NK cells by OMCP-mutIL-2 was even possible at doses 2-fold lower than wtIL-2 (Supplementary Fig. 4a). Furthermore, a mutIL-2 fusion protein comprised of a different NKG2D ligand with a ∼500-fold lower affinity, ULBP3 (ref. 15), did not significantly expand NK cells but still offered superior activation compared with mutIL-2 alone (Supplementary Fig. 4a). No significant increase in CD4+Foxp3− or CD8+ T lymphocytes was evident in A/J mice after wtIL-2 or OMCP-mutIL-2 treatment (Supplementary Fig. 4b). Splenic DX5+CD3+ NKT cells, while lower in number than DX5+CD3− NK cells, did expand after OMCP-mutIL-2 treatment, albeit equivalent to wtIL-2 in the A/J strain of mice (Supplementary Fig. 4b).


Selective targeting of IL-2 to NKG2D bearing cells for improved immunotherapy
Immunoactivation with IL-2 and construct administration in vivo.(a) Total splenocyte counts in AJ mice after a five-day course of 200,000 IUe of wtIL-2 (blue), mutIL-2 (green) and OMCP-mutIL-2 (red). (b) NK cell expansion and activation after wtIL-2, mutIL-2, OMCP-mutIL-2, high dose wtIL-2, high dose mutIL-2 and wtIL-2/anti-IL-2 complexes measured by cell counts in the spleen (top) and KLRG1 upregulation (bottom). Expansion of splenocytes (c) and NK cells (d) in C57BL/6 mice treated with 750,000 IUe of cytokine or construct. All graphs represent an average cell count ± s.e.m. from 5–10 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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f4: Immunoactivation with IL-2 and construct administration in vivo.(a) Total splenocyte counts in AJ mice after a five-day course of 200,000 IUe of wtIL-2 (blue), mutIL-2 (green) and OMCP-mutIL-2 (red). (b) NK cell expansion and activation after wtIL-2, mutIL-2, OMCP-mutIL-2, high dose wtIL-2, high dose mutIL-2 and wtIL-2/anti-IL-2 complexes measured by cell counts in the spleen (top) and KLRG1 upregulation (bottom). Expansion of splenocytes (c) and NK cells (d) in C57BL/6 mice treated with 750,000 IUe of cytokine or construct. All graphs represent an average cell count ± s.e.m. from 5–10 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: When A/J mice received a regimen of 200,000 IUe of cytokine or fusion protein, given as 10 equal doses over 5 days, both wtIL-2 and OMCP-mutIL-2 increased splenocyte numbers compared with saline-treated controls (Fig. 4a). OMCP-mutIL-2 led to a substantial expansion and activation of NK cells as measured by cell number and surface KLRG1 levels (Fig. 4b). Remarkably, in OMCP-mutIL-2 treated mice, NK cells comprised close to half of all splenic lymphocytes, paralleling or even surpassing the total lymphocyte counts of saline or mutIL-2-treated mice (Fig. 4a versus b). NK cell expansion by 200,000 IUe of OMCP-mutIL-2 was greater than that seen with near toxic doses of wtIL-2 (750,000 IU), high dose mutIL-2 (3,500,000 IUe) or wtIL-2 complexed to anti-IL-2 antibody (clone MAB602)19 (Fig. 4b). Superior expansion of NK cells by OMCP-mutIL-2 was even possible at doses 2-fold lower than wtIL-2 (Supplementary Fig. 4a). Furthermore, a mutIL-2 fusion protein comprised of a different NKG2D ligand with a ∼500-fold lower affinity, ULBP3 (ref. 15), did not significantly expand NK cells but still offered superior activation compared with mutIL-2 alone (Supplementary Fig. 4a). No significant increase in CD4+Foxp3− or CD8+ T lymphocytes was evident in A/J mice after wtIL-2 or OMCP-mutIL-2 treatment (Supplementary Fig. 4b). Splenic DX5+CD3+ NKT cells, while lower in number than DX5+CD3− NK cells, did expand after OMCP-mutIL-2 treatment, albeit equivalent to wtIL-2 in the A/J strain of mice (Supplementary Fig. 4b).

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