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Targeting inflammatory sites through collagen affinity enhances the therapeutic efficacy of anti-inflammatory antibodies

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Science Advances  06 Nov 2019:
Vol. 5, no. 11, eaay1971
DOI: 10.1126/sciadv.aay1971
  • Fig. 1 CBP conjugation provided collagen affinity to α-TNF.

    (A) Unmodified α-TNF and CBP–α-TNF binding affinities to types I, II, and III human collagen are analyzed by ELISA (n = 3, means + SD). OD450–570, optical density at 450 nm with subtraction of optical density at 570 nm. (B) Blocking activity of unmodified α-TNF and CBP–α-TNF against the binding between TNF-α and TNF receptor 2 was analyzed by ELISA (n = 3, means ± SD). (C) Representative images of human RA specimen probed with either unmodified α-TNF or CBP–α-TNF, anti-CD31 antibody, and anti–type I collagen antibody. Scale bars, 200 μm. (D) Representative images of human osteoarthritis specimen probed with either unmodified α-TNF or CBP–α-TNF and anti–type II collagen antibody. Scale bars, 500 μm.

  • Fig. 2 CBP–α-TNF accumulated in the inflamed paw.

    Arthritis (CAIA) was induced selectively in the right hind paw by passive immunization of anticollagen antibodies, followed by subcutaneous injection of LPS at the right hind footpad and phosphate-buffered saline at the left hind footpad. On the day following LPS injection, Cy7-labeled CBP–α-TNF and Cy7-labeled α-TNF were intravenously injected into naïve and CAIA mice. Representative images of accumulation in arthritic or nonarthritic paws of mice injected with CBP–α-TNF (A) and α-TNF (B) are shown. (C) Changes in radiant efficiency ratio of the arthritic paw (right hind) to the nonarthritic paw (left hind) in naïve and CAIA mice (n = 3 to 4, means ± SD). *P < 0.05 compared with the area under the radiant efficiency ratio-time curve from 0 to 72 hours of each treatment group (Tukey’s multiple comparison test). (D) Representative histology images of joints in CBP–α-TNF–injected CAIA mouse [left, hematoxylin and eosin (H&E) staining; right, immunohistochemistry staining against anti-rat immunoglobulin G (IgG)]. Scale bars, 200 μm.

  • Fig. 3 CBP–α-TNF suppressed arthritis development more effectively than unmodified α-TNF.

    Arthritis (CAIA) was induced by passive immunization of anticollagen antibodies, followed by intraperitoneal injection of LPS. On the day of LPS injection, control IgG, unmodified α-TNF, or CBP–α-TNF was injected intravenously into the arthritic mice. (A) Arthritis scores represent the means + SE from six mice. *P < 0.05, compared with control (Dunnett’s multiple comparison test). #P < 0.05 compared with the scores on day 8 of each treatment group (Tukey’s multiple comparison test). (B) Representative H&E image of joints on day 8 in each treatment group. Scale bars, 200 μm. (C) The severity of synovial hyperplasia and bone resorption was scored 0 to 4 as described in Materials and Methods. *P < 0.05 compared with control (Dunnett’s multiple comparison test).

  • Fig. 4 Localization in lung and efficacy of CBP–α–TGF-β in the BLM-induced pulmonary fibrosis model.

    Mice were intranasally instilled with 50 μg of BLM sulfate on day 0. (A) Cy7-labeled CBP–α–TGF-β or Cy7-labeled α–TGF-β was intravenously injected into naïve mice or BLM-injected mice on day 7. The lung was harvested 4 hours after the fluorescence injection, and fluorescence intensity was measured. (B and C) Control IgG, unmodified α–TGF-β, or CBP–α–TGF-β at a dose of 50 μg per mouse three times a week from day 7. On day 21, the left lung lobe was fixed and provided to histological analysis. (B) Representative image of Masson’s trichrome staining of the lung on day 21 in each treatment group. Scale bars, 200 μm (C) Lung fibrosis was scored 0 to 8 by Ashcroft scoring as described in Materials and Methods. *P < 0.05 compared with the scores of each treatment group (Tukey’s multiple comparison test).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaay1971/DC1

    Fig. S1. The molecular weight of α-TNF was increased by CBP conjugation.

    Fig. S2. The target and sequence specificity of CBP.

    Fig. S3. Distribution of CBP–α-TNF to organs and tissues in the arthritis model.

    Fig. S4. CBP–α-TNF reduces macrophages and neutrophils within the paws.

    Fig. S5. Effect of subcutaneous injection of CBP–α-TNF in the arthritis model.

    Fig. S6. Short-term safety study of CBP–α-TNF in the arthritis model.

    Fig. S7. CBP conjugation provided collagen affinity to α–TGF-β.

    Data file S1. Original data.

  • Supplementary Materials

    The PDFset includes:

    • Fig. S1. The molecular weight of α-TNF was increased by CBP conjugation.
    • Fig. S2. The target and sequence specificity of CBP.
    • Fig. S3. Distribution of CBP–α-TNF to organs and tissues in the arthritis model.
    • Fig. S4. CBP–α-TNF reduces macrophages and neutrophils within the paws.
    • Fig. S5. Effect of subcutaneous injection of CBP–α-TNF in the arthritis model.
    • Fig. S6. Short-term safety study of CBP–α-TNF in the arthritis model.
    • Fig. S7. CBP conjugation provided collagen affinity to α–TGF-β.

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    Other Supplementary Material for this manuscript includes the following:

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