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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

In vitro evaluation of OMCP-mutIL-2.(a) In vitro flow cytometrically evaluated activation of A/J spleen-derived lymphocyte subsets after 36 h of culture in 100 IUe ml−1 of cytokines or OMCP-mutIL-2 construct. Graph demonstrating one representative histogram plot and median fluorescent intensity (MFI) ±s.e.m. across 5–7 separate experiments, containing one to two mice per group per experiment, as a graph in right upper corner. (b) In vitro activation of human peripheral blood lymphocyte subsets after 36 h of culture in 100 IUe ml−1 of cytokines or OMCP-mutIL-2 construct. Graph demonstrating one representative histogram plot and MFI±s.e.m. across 4–5 separate experiments as graph in right upper corner. Proliferation of C57BL/6 wild-type (c) or C57BL/6NKG2D−/− (d) splenic lymphocyte subsets after 5-day culture in 1,000 IUe ml−1 of cytokines or OMCP-mutIL-2. Graph demonstrating one representative histogram plot of four separate experiments. Composite MFI±s.e.m. shown as graph below the CFSE histogram plots. Comparison of MFI performed by ANOVA for multiple comparisons and unpaired t-test for each individual group. 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.
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f2: In vitro evaluation of OMCP-mutIL-2.(a) In vitro flow cytometrically evaluated activation of A/J spleen-derived lymphocyte subsets after 36 h of culture in 100 IUe ml−1 of cytokines or OMCP-mutIL-2 construct. Graph demonstrating one representative histogram plot and median fluorescent intensity (MFI) ±s.e.m. across 5–7 separate experiments, containing one to two mice per group per experiment, as a graph in right upper corner. (b) In vitro activation of human peripheral blood lymphocyte subsets after 36 h of culture in 100 IUe ml−1 of cytokines or OMCP-mutIL-2 construct. Graph demonstrating one representative histogram plot and MFI±s.e.m. across 4–5 separate experiments as graph in right upper corner. Proliferation of C57BL/6 wild-type (c) or C57BL/6NKG2D−/− (d) splenic lymphocyte subsets after 5-day culture in 1,000 IUe ml−1 of cytokines or OMCP-mutIL-2. Graph demonstrating one representative histogram plot of four separate experiments. Composite MFI±s.e.m. shown as graph below the CFSE histogram plots. Comparison of MFI performed by ANOVA for multiple comparisons and unpaired t-test for each individual group. 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.

Mentions: Based on this data we next set out to examine the efficacy of OMCP-mutIL-2 in activation of NK cells from two different strains of mice (A/J and C57BL/6) with poor and robust NK function, respectively16. Compared with wtIL-2 or mutIL-2, OMCP-mutIL-2 strongly upregulated CD69 on NK cells of both strains after a two-day co-culture with 100 IUe ml−1 of cytokine (Fig. 2a left panel; Supplementary Fig. 2a). Hundred fold higher concentrations of wtIL-2 or mutIL-2 induced similar increases in CD69 expression compared with OMCP-mutIL-2 (Supplementary Fig. 2b). CD8+ T cells demonstrated no upregulation of CD69 (Fig. 2a middle panel). This is consistent with the low surface expression of IL-2 receptors and NKG2D on unactivated, resting murine T lymphocytes. Activation of CD4+Foxp3+ Tregs, as measured by upregulation of ICOS, was evident after co-culture with wtIL-2, but not with mutIL-2 nor OMCP-mutIL-2 (Fig. 2a right panel). While CD69 upregulation is only a transient marker of lymphocyte activation, acquisition of cytotoxic mediators such as perforin has been described as a reliable measure of cytotoxicity17. To expand our murine observations we co-cultured freshly isolated human peripheral blood lymphocytes (PBLs) with 100 IUe ml−1 of OMCP-mutIL-2, wtIL-2 or mutIL-2 in a similar manner to murine splenocytes and evaluated intracellular perforin accumulation flow cytometrically. Consistent with murine CD69 data, human NK cells treated with OMCP-mutIL-2 had higher perforin levels than those treated with wild-type IL-2, mutIL-2 or saline (Fig. 2b left panel). Thus, exposure to OMCP-mutIL-2 results in preferential activation of human NK cells as well. Limited activation of CD8+ T cells was evident with any of the constructs (Fig. 2b middle panel) and human Tregs were preferentially activated by wtIL-2 but not mutIL-2 or OMCP-mutIL-2 similar to mice (Fig. 2b right panel).


Selective targeting of IL-2 to NKG2D bearing cells for improved immunotherapy
In vitro evaluation of OMCP-mutIL-2.(a) In vitro flow cytometrically evaluated activation of A/J spleen-derived lymphocyte subsets after 36 h of culture in 100 IUe ml−1 of cytokines or OMCP-mutIL-2 construct. Graph demonstrating one representative histogram plot and median fluorescent intensity (MFI) ±s.e.m. across 5–7 separate experiments, containing one to two mice per group per experiment, as a graph in right upper corner. (b) In vitro activation of human peripheral blood lymphocyte subsets after 36 h of culture in 100 IUe ml−1 of cytokines or OMCP-mutIL-2 construct. Graph demonstrating one representative histogram plot and MFI±s.e.m. across 4–5 separate experiments as graph in right upper corner. Proliferation of C57BL/6 wild-type (c) or C57BL/6NKG2D−/− (d) splenic lymphocyte subsets after 5-day culture in 1,000 IUe ml−1 of cytokines or OMCP-mutIL-2. Graph demonstrating one representative histogram plot of four separate experiments. Composite MFI±s.e.m. shown as graph below the CFSE histogram plots. Comparison of MFI performed by ANOVA for multiple comparisons and unpaired t-test for each individual group. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: In vitro evaluation of OMCP-mutIL-2.(a) In vitro flow cytometrically evaluated activation of A/J spleen-derived lymphocyte subsets after 36 h of culture in 100 IUe ml−1 of cytokines or OMCP-mutIL-2 construct. Graph demonstrating one representative histogram plot and median fluorescent intensity (MFI) ±s.e.m. across 5–7 separate experiments, containing one to two mice per group per experiment, as a graph in right upper corner. (b) In vitro activation of human peripheral blood lymphocyte subsets after 36 h of culture in 100 IUe ml−1 of cytokines or OMCP-mutIL-2 construct. Graph demonstrating one representative histogram plot and MFI±s.e.m. across 4–5 separate experiments as graph in right upper corner. Proliferation of C57BL/6 wild-type (c) or C57BL/6NKG2D−/− (d) splenic lymphocyte subsets after 5-day culture in 1,000 IUe ml−1 of cytokines or OMCP-mutIL-2. Graph demonstrating one representative histogram plot of four separate experiments. Composite MFI±s.e.m. shown as graph below the CFSE histogram plots. Comparison of MFI performed by ANOVA for multiple comparisons and unpaired t-test for each individual group. 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.
Mentions: Based on this data we next set out to examine the efficacy of OMCP-mutIL-2 in activation of NK cells from two different strains of mice (A/J and C57BL/6) with poor and robust NK function, respectively16. Compared with wtIL-2 or mutIL-2, OMCP-mutIL-2 strongly upregulated CD69 on NK cells of both strains after a two-day co-culture with 100 IUe ml−1 of cytokine (Fig. 2a left panel; Supplementary Fig. 2a). Hundred fold higher concentrations of wtIL-2 or mutIL-2 induced similar increases in CD69 expression compared with OMCP-mutIL-2 (Supplementary Fig. 2b). CD8+ T cells demonstrated no upregulation of CD69 (Fig. 2a middle panel). This is consistent with the low surface expression of IL-2 receptors and NKG2D on unactivated, resting murine T lymphocytes. Activation of CD4+Foxp3+ Tregs, as measured by upregulation of ICOS, was evident after co-culture with wtIL-2, but not with mutIL-2 nor OMCP-mutIL-2 (Fig. 2a right panel). While CD69 upregulation is only a transient marker of lymphocyte activation, acquisition of cytotoxic mediators such as perforin has been described as a reliable measure of cytotoxicity17. To expand our murine observations we co-cultured freshly isolated human peripheral blood lymphocytes (PBLs) with 100 IUe ml−1 of OMCP-mutIL-2, wtIL-2 or mutIL-2 in a similar manner to murine splenocytes and evaluated intracellular perforin accumulation flow cytometrically. Consistent with murine CD69 data, human NK cells treated with OMCP-mutIL-2 had higher perforin levels than those treated with wild-type IL-2, mutIL-2 or saline (Fig. 2b left panel). Thus, exposure to OMCP-mutIL-2 results in preferential activation of human NK cells as well. Limited activation of CD8+ T cells was evident with any of the constructs (Fig. 2b middle panel) and human Tregs were preferentially activated by wtIL-2 but not mutIL-2 or OMCP-mutIL-2 similar to mice (Fig. 2b right panel).

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