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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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Cytokine-mediated tumour immunotherapy.(a) In vivo cytotoxicity for YAC-1 lymphoblast cell line after intravenous injection of tumour into A/J mice treated with 200,000 IUe of cytokine or OMCP-mutIL-2 construct. Comparison performed by unpaired t-test between groups as indicated by the lines above the graphs. (b) In vitro lysis of YAC-1 lymphoma by bulk A/J splenocytes after 200,000 IUe of cytokine or OMCP-mutIL-2 construct treatment. (c) LLC flank growth after cytokine treatment of wild-type C57BL/6 mice. Treatment with 750,000 IUe of cytokine or fusion construct was started on day 5-post tumour injection, after palpable tumors were evident, and continued through day 10 in 10 equal doses of 75,000 IUe/dose. Raw data are shown in Supplementary Data 1 and represents 6 mice in the saline group and 5 in the wtIL-2, mutIL-2 and OMCP-mutIL-2 group. (d) LLC tumour growth in C57BL/6 mice depleted of NK1.1 cells or (e) mutant mice deficient in NKG2D. Data representative of 5 mice per group for both NK1.1 depleted and NKG2D deficient mice. (f) LLC tumour growth in C57BL/6Rag1−/− mice. Data representative of 5 mice per group. Comparison performed by multiple unpaired t-tests at each individual time point to saline-treated controls for all tumour growth experiments. (g) Survival of C57BL/6 mice treated with 750,000 IUe of cytokine 5 days after injection of 1 × 105 LLC intravenously. Data extrapolated from groups consisting of 7 mice in the wtIL-2 and mutIL-2 treated groups and 8 mice from the saline and OMCP-mutIL-2 treated groups. Cytokines or OMCP-mutIL-2 fusion protein was given as ten doses on days 5–10 post tumor injection with A/J mice receiving a total of 200,000 IUe and C57BL/6 mice receiving 750,000 IUe. Kaplan–Meier survival graphs were compared by Log-rank (Mantel–Cox) test. 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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f8: Cytokine-mediated tumour immunotherapy.(a) In vivo cytotoxicity for YAC-1 lymphoblast cell line after intravenous injection of tumour into A/J mice treated with 200,000 IUe of cytokine or OMCP-mutIL-2 construct. Comparison performed by unpaired t-test between groups as indicated by the lines above the graphs. (b) In vitro lysis of YAC-1 lymphoma by bulk A/J splenocytes after 200,000 IUe of cytokine or OMCP-mutIL-2 construct treatment. (c) LLC flank growth after cytokine treatment of wild-type C57BL/6 mice. Treatment with 750,000 IUe of cytokine or fusion construct was started on day 5-post tumour injection, after palpable tumors were evident, and continued through day 10 in 10 equal doses of 75,000 IUe/dose. Raw data are shown in Supplementary Data 1 and represents 6 mice in the saline group and 5 in the wtIL-2, mutIL-2 and OMCP-mutIL-2 group. (d) LLC tumour growth in C57BL/6 mice depleted of NK1.1 cells or (e) mutant mice deficient in NKG2D. Data representative of 5 mice per group for both NK1.1 depleted and NKG2D deficient mice. (f) LLC tumour growth in C57BL/6Rag1−/− mice. Data representative of 5 mice per group. Comparison performed by multiple unpaired t-tests at each individual time point to saline-treated controls for all tumour growth experiments. (g) Survival of C57BL/6 mice treated with 750,000 IUe of cytokine 5 days after injection of 1 × 105 LLC intravenously. Data extrapolated from groups consisting of 7 mice in the wtIL-2 and mutIL-2 treated groups and 8 mice from the saline and OMCP-mutIL-2 treated groups. Cytokines or OMCP-mutIL-2 fusion protein was given as ten doses on days 5–10 post tumor injection with A/J mice receiving a total of 200,000 IUe and C57BL/6 mice receiving 750,000 IUe. Kaplan–Meier survival graphs were compared by Log-rank (Mantel–Cox) test. 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: NK cells form the primary barrier for expansion of some malignancies, such as lymphoma and lung cancer16323334. To evaluate lymphoma clearance we intravenously injected cytokine or fusion protein-treated A/J mice with the A/J-derived CFSE-labelled YAC-1 lymphoblast cell line and evaluated their lungs 6 h later. Near complete clearance of YAC-1 was evident in OMCP-mutIL-2 treated mice while a significant number of viable lymphoblasts remained in wtIL-2, mutIL-2 and saline-treated mice (Fig. 8a and Supplementary Data 1). Similarly bulk splenocytes of A/J mice treated with OMCP-mutIL-2 lysed YAC-1 more efficiently than those treated with wtIL-2, mutIL-2 or saline (Fig. 8b). Similar results were obtained with LM-2 lung cancer targets (Supplementary Fig. 6a).


Selective targeting of IL-2 to NKG2D bearing cells for improved immunotherapy
Cytokine-mediated tumour immunotherapy.(a) In vivo cytotoxicity for YAC-1 lymphoblast cell line after intravenous injection of tumour into A/J mice treated with 200,000 IUe of cytokine or OMCP-mutIL-2 construct. Comparison performed by unpaired t-test between groups as indicated by the lines above the graphs. (b) In vitro lysis of YAC-1 lymphoma by bulk A/J splenocytes after 200,000 IUe of cytokine or OMCP-mutIL-2 construct treatment. (c) LLC flank growth after cytokine treatment of wild-type C57BL/6 mice. Treatment with 750,000 IUe of cytokine or fusion construct was started on day 5-post tumour injection, after palpable tumors were evident, and continued through day 10 in 10 equal doses of 75,000 IUe/dose. Raw data are shown in Supplementary Data 1 and represents 6 mice in the saline group and 5 in the wtIL-2, mutIL-2 and OMCP-mutIL-2 group. (d) LLC tumour growth in C57BL/6 mice depleted of NK1.1 cells or (e) mutant mice deficient in NKG2D. Data representative of 5 mice per group for both NK1.1 depleted and NKG2D deficient mice. (f) LLC tumour growth in C57BL/6Rag1−/− mice. Data representative of 5 mice per group. Comparison performed by multiple unpaired t-tests at each individual time point to saline-treated controls for all tumour growth experiments. (g) Survival of C57BL/6 mice treated with 750,000 IUe of cytokine 5 days after injection of 1 × 105 LLC intravenously. Data extrapolated from groups consisting of 7 mice in the wtIL-2 and mutIL-2 treated groups and 8 mice from the saline and OMCP-mutIL-2 treated groups. Cytokines or OMCP-mutIL-2 fusion protein was given as ten doses on days 5–10 post tumor injection with A/J mice receiving a total of 200,000 IUe and C57BL/6 mice receiving 750,000 IUe. Kaplan–Meier survival graphs were compared by Log-rank (Mantel–Cox) test. 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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f8: Cytokine-mediated tumour immunotherapy.(a) In vivo cytotoxicity for YAC-1 lymphoblast cell line after intravenous injection of tumour into A/J mice treated with 200,000 IUe of cytokine or OMCP-mutIL-2 construct. Comparison performed by unpaired t-test between groups as indicated by the lines above the graphs. (b) In vitro lysis of YAC-1 lymphoma by bulk A/J splenocytes after 200,000 IUe of cytokine or OMCP-mutIL-2 construct treatment. (c) LLC flank growth after cytokine treatment of wild-type C57BL/6 mice. Treatment with 750,000 IUe of cytokine or fusion construct was started on day 5-post tumour injection, after palpable tumors were evident, and continued through day 10 in 10 equal doses of 75,000 IUe/dose. Raw data are shown in Supplementary Data 1 and represents 6 mice in the saline group and 5 in the wtIL-2, mutIL-2 and OMCP-mutIL-2 group. (d) LLC tumour growth in C57BL/6 mice depleted of NK1.1 cells or (e) mutant mice deficient in NKG2D. Data representative of 5 mice per group for both NK1.1 depleted and NKG2D deficient mice. (f) LLC tumour growth in C57BL/6Rag1−/− mice. Data representative of 5 mice per group. Comparison performed by multiple unpaired t-tests at each individual time point to saline-treated controls for all tumour growth experiments. (g) Survival of C57BL/6 mice treated with 750,000 IUe of cytokine 5 days after injection of 1 × 105 LLC intravenously. Data extrapolated from groups consisting of 7 mice in the wtIL-2 and mutIL-2 treated groups and 8 mice from the saline and OMCP-mutIL-2 treated groups. Cytokines or OMCP-mutIL-2 fusion protein was given as ten doses on days 5–10 post tumor injection with A/J mice receiving a total of 200,000 IUe and C57BL/6 mice receiving 750,000 IUe. Kaplan–Meier survival graphs were compared by Log-rank (Mantel–Cox) test. 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: NK cells form the primary barrier for expansion of some malignancies, such as lymphoma and lung cancer16323334. To evaluate lymphoma clearance we intravenously injected cytokine or fusion protein-treated A/J mice with the A/J-derived CFSE-labelled YAC-1 lymphoblast cell line and evaluated their lungs 6 h later. Near complete clearance of YAC-1 was evident in OMCP-mutIL-2 treated mice while a significant number of viable lymphoblasts remained in wtIL-2, mutIL-2 and saline-treated mice (Fig. 8a and Supplementary Data 1). Similarly bulk splenocytes of A/J mice treated with OMCP-mutIL-2 lysed YAC-1 more efficiently than those treated with wtIL-2, mutIL-2 or saline (Fig. 8b). Similar results were obtained with LM-2 lung cancer targets (Supplementary Fig. 6a).

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