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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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Tracking of 1-11E/vIL-10 to arthritic joints. One day after AIA induction in mice, 1 μg of Cy5.5-labeled fusion proteins was injected i.p. In vivo fluorescence images of the mice were taken to follow the tracking of the fusion proteins to the joint. (A) Representative fluorescence image taken 3 days after injection of 1-11E/vIL-10 (left) and C7/vIL-10 (right). (B) Quantification of the average fluorescence of the region of interest encompassing the knee joint, n = 3. (C)Ex vivo quantification of fluoresce of tissues after dissection of a single mouse 4 days after injection. (D) Representative fluorescence images of cryosections of the excised knee joints, showing the pericellular matrix of the chondrocytes in green (AlexaFluor-488-labeled secondary) and the fusion protein (Cy5.5) in red.
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Figure 4: Tracking of 1-11E/vIL-10 to arthritic joints. One day after AIA induction in mice, 1 μg of Cy5.5-labeled fusion proteins was injected i.p. In vivo fluorescence images of the mice were taken to follow the tracking of the fusion proteins to the joint. (A) Representative fluorescence image taken 3 days after injection of 1-11E/vIL-10 (left) and C7/vIL-10 (right). (B) Quantification of the average fluorescence of the region of interest encompassing the knee joint, n = 3. (C)Ex vivo quantification of fluoresce of tissues after dissection of a single mouse 4 days after injection. (D) Representative fluorescence images of cryosections of the excised knee joints, showing the pericellular matrix of the chondrocytes in green (AlexaFluor-488-labeled secondary) and the fusion protein (Cy5.5) in red.

Mentions: The targeting function of the 1-11E portion of the fusion protein to inflamed tissue was addressed by tagging the fusion protein with the Cy5.5 fluorophore and injecting i.p. into animals with AIA. Subsequent in vivo fluorescence imaging indicated that the 1-11E/vIL-10 fusion protein selectively tracked to the inflamed joint, compared with the contralateral uninflamed joint and the control C7/vIL-10 fusion protein (representative images taken 3 days after i.p. injection shown in Figure 4A).


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)

Tracking of 1-11E/vIL-10 to arthritic joints. One day after AIA induction in mice, 1 μg of Cy5.5-labeled fusion proteins was injected i.p. In vivo fluorescence images of the mice were taken to follow the tracking of the fusion proteins to the joint. (A) Representative fluorescence image taken 3 days after injection of 1-11E/vIL-10 (left) and C7/vIL-10 (right). (B) Quantification of the average fluorescence of the region of interest encompassing the knee joint, n = 3. (C)Ex vivo quantification of fluoresce of tissues after dissection of a single mouse 4 days after injection. (D) Representative fluorescence images of cryosections of the excised knee joints, showing the pericellular matrix of the chondrocytes in green (AlexaFluor-488-labeled secondary) and the fusion protein (Cy5.5) in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4225686&req=5

Figure 4: Tracking of 1-11E/vIL-10 to arthritic joints. One day after AIA induction in mice, 1 μg of Cy5.5-labeled fusion proteins was injected i.p. In vivo fluorescence images of the mice were taken to follow the tracking of the fusion proteins to the joint. (A) Representative fluorescence image taken 3 days after injection of 1-11E/vIL-10 (left) and C7/vIL-10 (right). (B) Quantification of the average fluorescence of the region of interest encompassing the knee joint, n = 3. (C)Ex vivo quantification of fluoresce of tissues after dissection of a single mouse 4 days after injection. (D) Representative fluorescence images of cryosections of the excised knee joints, showing the pericellular matrix of the chondrocytes in green (AlexaFluor-488-labeled secondary) and the fusion protein (Cy5.5) in red.
Mentions: The targeting function of the 1-11E portion of the fusion protein to inflamed tissue was addressed by tagging the fusion protein with the Cy5.5 fluorophore and injecting i.p. into animals with AIA. Subsequent in vivo fluorescence imaging indicated that the 1-11E/vIL-10 fusion protein selectively tracked to the inflamed joint, compared with the contralateral uninflamed joint and the control C7/vIL-10 fusion protein (representative images taken 3 days after i.p. injection shown in Figure 4A).

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