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Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency.

Hughes C, Sette A, Seed M, D'Acquisto F, Manzo A, Vincent TL, Lim NH, Nissim A - Arthritis Res. Ther. (2014)

Bottom Line: Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1.Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10).Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: We previously demonstrated that a single-chain fragment variable (scFv) specific to collagen type II (CII) posttranslationally modified by reactive oxygen species (ROS) can be used to target anti-inflammatory therapeutics specifically to inflamed arthritic joints. The objective of the present study was to demonstrate the superior efficacy of anti-inflammatory cytokines when targeted to inflamed arthritic joints by the anti-ROS modified CII (anti-ROS-CII) scFv in a mouse model of arthritis.

Methods: Viral interleukin-10 (vIL-10) was fused to anti-ROS-CII scFv (1-11E) with a matrix-metalloproteinase (MMP) cleavable linker to create 1-11E/vIL-10 fusion. Binding of 1-11E/vIL-10 to ROS-CII was determined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immune-staining of arthritic cartilage, whereas vIL-10 bioactivity was evaluated in vitro by using an MC-9 cell-proliferation assay. Specific in vivo localization and therapeutic efficacy of 1-11E/vIL-10 was tested in the mouse model of antigen-induced arthritis.

Results: 1-11E/vIL-10 bound specifically to ROS-CII and to damaged arthritic cartilage. Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1. When systemically administered to arthritic mice, 1-11E/vIL-10 localized specifically to the arthritic knee, with peak accumulation observed after 3 days. Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10).

Conclusions: Targeted delivery of anti-inflammatory cytokines potentiates their anti-arthritic action in a mouse model of arthritis. Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically.

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Bioassay of 1-11E/vIL-10. (A) ELISA showing increased binding of 1-11E/vIL-10 to ROS-CII (CII modified by glycation (Gly) or HOCl) compared with native CII (NT-CII). No binding to hen-egg lysozyme (HEL) was observed. In contrast, C7/vIL-10 bound only to HEL. (B) Western blot analysis showed that 1-11E/vIL10 bound to native CII (Lane 1), ROS modified CII (lane 2, glycated; lane 3, HOCl; lane 4, OH-; lane 5, peroxynitrate) but not to HEL (lane 6). (C). IL-10 bioassay of 1-11E/vIL10 and C7/vIL10. We added 10, 100, or 1,000 ng/ml of fusion protein (white, pattern, and black boxes, respectively) to IL-10-responsive MC-9 cells with (+) or without (-) previous MMP-1 digestion. As a positive control, 10, 100, or 1,000 ng/ml commercial recombinant vIL-10 (vIL-10) was used, whereas MMP-1 alone-treated cells were used as a negative control. After 3 days, cell growth was measured with the Cell Titer Glo assay. Significant enhanced cell growth of the fusion proteins was observed after MMP-1 digestion (P < 0.01 for 1,000 ng/ml). No significant difference in growth was seen between 1-11E/vIL-10, C7/vIL-10, and vIL-10 (P > 0.05).
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Figure 2: Bioassay of 1-11E/vIL-10. (A) ELISA showing increased binding of 1-11E/vIL-10 to ROS-CII (CII modified by glycation (Gly) or HOCl) compared with native CII (NT-CII). No binding to hen-egg lysozyme (HEL) was observed. In contrast, C7/vIL-10 bound only to HEL. (B) Western blot analysis showed that 1-11E/vIL10 bound to native CII (Lane 1), ROS modified CII (lane 2, glycated; lane 3, HOCl; lane 4, OH-; lane 5, peroxynitrate) but not to HEL (lane 6). (C). IL-10 bioassay of 1-11E/vIL10 and C7/vIL10. We added 10, 100, or 1,000 ng/ml of fusion protein (white, pattern, and black boxes, respectively) to IL-10-responsive MC-9 cells with (+) or without (-) previous MMP-1 digestion. As a positive control, 10, 100, or 1,000 ng/ml commercial recombinant vIL-10 (vIL-10) was used, whereas MMP-1 alone-treated cells were used as a negative control. After 3 days, cell growth was measured with the Cell Titer Glo assay. Significant enhanced cell growth of the fusion proteins was observed after MMP-1 digestion (P < 0.01 for 1,000 ng/ml). No significant difference in growth was seen between 1-11E/vIL-10, C7/vIL-10, and vIL-10 (P > 0.05).

Mentions: The antigen specificity of 1-11E/vIL-10 fusion proteins was determined with ELISA, by using native CII (NT CII), ROS-CII (CII modified by glycation (GLY) and HOCl (HOCl)), or control HEL as target antigens. 1-11E/vIL-10 had increased binding to both GLY and HOCl-derived ROS-CII (as previously demonstrated for 1-11E scFv [9]) and not to HEL (Figure 2A). Conversely, the C7/vIL-10 fusion protein was specific to HEL (Figure 2A).The specificity of the 1-11E/vIL-10 fusion to ROS-CII was further confirmed with Western blot. 1-11E/vIL-10 bound to all forms of CII, but not to HEL (Figure 2B). Binding pattern included binding to a range of CII fragments in the region of 25 to 100 kDa; and high-molecular-weight aggregates (higher than 250 kDa). In addition, 1-11E/vIL-10 bound to the electrophoretic band that corresponds to the intact native CII-chain polypeptide, although in ELISA, 1-11E/vIL-10 did not bind to native CII (Figure 2B).


Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency.

Hughes C, Sette A, Seed M, D'Acquisto F, Manzo A, Vincent TL, Lim NH, Nissim A - Arthritis Res. Ther. (2014)

Bioassay of 1-11E/vIL-10. (A) ELISA showing increased binding of 1-11E/vIL-10 to ROS-CII (CII modified by glycation (Gly) or HOCl) compared with native CII (NT-CII). No binding to hen-egg lysozyme (HEL) was observed. In contrast, C7/vIL-10 bound only to HEL. (B) Western blot analysis showed that 1-11E/vIL10 bound to native CII (Lane 1), ROS modified CII (lane 2, glycated; lane 3, HOCl; lane 4, OH-; lane 5, peroxynitrate) but not to HEL (lane 6). (C). IL-10 bioassay of 1-11E/vIL10 and C7/vIL10. We added 10, 100, or 1,000 ng/ml of fusion protein (white, pattern, and black boxes, respectively) to IL-10-responsive MC-9 cells with (+) or without (-) previous MMP-1 digestion. As a positive control, 10, 100, or 1,000 ng/ml commercial recombinant vIL-10 (vIL-10) was used, whereas MMP-1 alone-treated cells were used as a negative control. After 3 days, cell growth was measured with the Cell Titer Glo assay. Significant enhanced cell growth of the fusion proteins was observed after MMP-1 digestion (P < 0.01 for 1,000 ng/ml). No significant difference in growth was seen between 1-11E/vIL-10, C7/vIL-10, and vIL-10 (P > 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Bioassay of 1-11E/vIL-10. (A) ELISA showing increased binding of 1-11E/vIL-10 to ROS-CII (CII modified by glycation (Gly) or HOCl) compared with native CII (NT-CII). No binding to hen-egg lysozyme (HEL) was observed. In contrast, C7/vIL-10 bound only to HEL. (B) Western blot analysis showed that 1-11E/vIL10 bound to native CII (Lane 1), ROS modified CII (lane 2, glycated; lane 3, HOCl; lane 4, OH-; lane 5, peroxynitrate) but not to HEL (lane 6). (C). IL-10 bioassay of 1-11E/vIL10 and C7/vIL10. We added 10, 100, or 1,000 ng/ml of fusion protein (white, pattern, and black boxes, respectively) to IL-10-responsive MC-9 cells with (+) or without (-) previous MMP-1 digestion. As a positive control, 10, 100, or 1,000 ng/ml commercial recombinant vIL-10 (vIL-10) was used, whereas MMP-1 alone-treated cells were used as a negative control. After 3 days, cell growth was measured with the Cell Titer Glo assay. Significant enhanced cell growth of the fusion proteins was observed after MMP-1 digestion (P < 0.01 for 1,000 ng/ml). No significant difference in growth was seen between 1-11E/vIL-10, C7/vIL-10, and vIL-10 (P > 0.05).
Mentions: The antigen specificity of 1-11E/vIL-10 fusion proteins was determined with ELISA, by using native CII (NT CII), ROS-CII (CII modified by glycation (GLY) and HOCl (HOCl)), or control HEL as target antigens. 1-11E/vIL-10 had increased binding to both GLY and HOCl-derived ROS-CII (as previously demonstrated for 1-11E scFv [9]) and not to HEL (Figure 2A). Conversely, the C7/vIL-10 fusion protein was specific to HEL (Figure 2A).The specificity of the 1-11E/vIL-10 fusion to ROS-CII was further confirmed with Western blot. 1-11E/vIL-10 bound to all forms of CII, but not to HEL (Figure 2B). Binding pattern included binding to a range of CII fragments in the region of 25 to 100 kDa; and high-molecular-weight aggregates (higher than 250 kDa). In addition, 1-11E/vIL-10 bound to the electrophoretic band that corresponds to the intact native CII-chain polypeptide, although in ELISA, 1-11E/vIL-10 did not bind to native CII (Figure 2B).

Bottom Line: Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1.Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10).Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: We previously demonstrated that a single-chain fragment variable (scFv) specific to collagen type II (CII) posttranslationally modified by reactive oxygen species (ROS) can be used to target anti-inflammatory therapeutics specifically to inflamed arthritic joints. The objective of the present study was to demonstrate the superior efficacy of anti-inflammatory cytokines when targeted to inflamed arthritic joints by the anti-ROS modified CII (anti-ROS-CII) scFv in a mouse model of arthritis.

Methods: Viral interleukin-10 (vIL-10) was fused to anti-ROS-CII scFv (1-11E) with a matrix-metalloproteinase (MMP) cleavable linker to create 1-11E/vIL-10 fusion. Binding of 1-11E/vIL-10 to ROS-CII was determined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immune-staining of arthritic cartilage, whereas vIL-10 bioactivity was evaluated in vitro by using an MC-9 cell-proliferation assay. Specific in vivo localization and therapeutic efficacy of 1-11E/vIL-10 was tested in the mouse model of antigen-induced arthritis.

Results: 1-11E/vIL-10 bound specifically to ROS-CII and to damaged arthritic cartilage. Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1. When systemically administered to arthritic mice, 1-11E/vIL-10 localized specifically to the arthritic knee, with peak accumulation observed after 3 days. Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10).

Conclusions: Targeted delivery of anti-inflammatory cytokines potentiates their anti-arthritic action in a mouse model of arthritis. Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically.

Show MeSH
Related in: MedlinePlus