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Engineered collagen-binding serum albumin as a drug conjugate carrier for cancer therapy

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Science Advances  14 Aug 2019:
Vol. 5, no. 8, eaaw6081
DOI: 10.1126/sciadv.aaw6081
  • Fig. 1 Synthesis and characterization of Dox-CBD-SA.

    (A) Schematic of CBD-SA mediated drug delivery. (B) Synthesis scheme of Dox-CBD-SA. (C) Affinities [dissociation constant (Kd) values are shown] of CBD-SA and SA against collagen type I and collagen type III were measured by enzyme-linked immunosorbent assay (ELISA). N.D., not determined due to low signal. Graphs with [concentrations] versus [signals] are shown in fig. S2. Two experimental replicates. (D) Dox conjugation ratio per protein is presented. Values were calculated on the basis of the results of bicinchoninic acid assay protein quantification assay (proteins) and absorbance at 495 nm (Dox) (mean ± SD of three experimental replicates). (E) Dox release kinetics from Dox-CBD-SA under three different pH conditions was evaluated by fluorescence (excitation at 495 nm, emission at 590 nm) (n = 3, mean ± SD; two experimental replicates). (F) MMTV-PyMT cells were seeded and incubated overnight. Dox, Dox-SA, or Dox-CBD-SA was added (red). Cells were also stained with LysoTracker (green). Scale bars, 20 μm. Representative pictures are presented. Two experimental replicates. (G and H) Cytotoxicity of Dox variants against MMTV-PyMT cells or MC38 cells in vitro (n = 6, mean ± SEM). Two experimental replicates. IC50, half maximal inhibitory concentration.

  • Fig. 2 Dox-CBD-SA shows comparable plasma pharmacokinetics with Dox-SA and higher tumor accumulation than aldoxorubicin and Dox-SA.

    (A) Aldoxorubicin, Dox-SA, or Dox-CBD-SA (5 mg/kg on a Dox basis) was administered to tumor-free FVB mice via tail vein injection. Blood plasma was collected at the indicated time points. Plasma concentration of Dox was measured by fluorescence (mean ± SEM; n = 4 for aldoxorubicin, n = 5 for Dox-SA and Dox-CBD-SA). (B) Plasma half-lives of Dox were calculated using two-phase exponential decay: MFI (t) = Ae−αt + Be−βt. t½, α, fast clearance half-life; t½, β, slow clearance half-life (mean ± SEM; n = 4 for aldoxorubicin, n = 5 for Dox-SA and Dox-CBD-SA). (C) MMTV-PyMT tumor-bearing mice were treated with aldoxorubicin, Dox-SA, or Dox-CBD-SA (4.16 mg/kg on a Dox basis). At the indicated time points, tumors were harvested, and the amount of Dox within the tumors was quantified (mean ± SEM; n = 5 for 2 hours, n = 7 for 24 hours per group). (D) DyLight 488–labeled SA (100 μg) or equimolar amounts of DyLight 488–labeled CBD-SA were injected intravenously to MMTV-PyMT tumor-bearing mice. One hour after injection, tumors were harvested and fluorescence was analyzed by confocal microscopy. Tissues were also stained with 4′,6-diamidino-2-phenylindole (DAPI) and anti-CD31 antibody. Scale bars, 100 μm. Representative images of three tumors each. Two experimental replicates. Statistical analyses were done using analysis of variance (ANOVA) with Tukey’s test. *P < 0.05; **P < 0.01; N.S., not significant.

  • Fig. 3 Dox-CBD-SA shows enhanced antitumor efficacy and infiltration of lymphocytes into tumor in the MMTV-PyMT breast cancer model.

    (A) MMTV-PyMT cells (5 × 105) were inoculated into FVB mice on day 0. Aldoxorubicin, Dox-SA, or Dox-CBD-SA (5 mg/kg on a Dox basis) was injected intravenously on day 7. Graphs depict tumor volume until the first mouse died (mean ± SEM). (B) Survival rate. (C to F) Individual tumor growth curves. CR indicates complete response frequency. Three experimental replicates. (G to L) MMTV-PyMT cells (5 × 105) were inoculated on day 0. Aldoxorubicin, Dox-SA, or Dox-CBD-SA (5 mg/kg on a Dox basis) was injected intravenously on day 7. Lymphocytes within tumors were extracted on day 14, followed by flow cytometric analysis. (G to I) Graphs depict the number of (G) CD45+CD8+CD3+ T cells, (H) CD45+CD4+CD3+ T cells, and (I) CD45+NK1.1+CD3 NK cells per tumor weight (in milligrams). Bars represent mean ± SEM. (J to L) Graph shows [CD45+CD8+CD3+ T cells per tumor weight (mg)] (J), [CD45+CD4+CD3+ T cells per tumor weight (mg)] (K), or [CD45+NK1.1+CD3 NK cells per tumor weight (mg)] (L) versus [tumor weight]. Two experimental replicates. Statistical analyses were done using (A, H, and I) ANOVA with Tukey’s test or (G) Kruskal-Wallis test followed by Dunn’s test or (B) log-rank (Mantel-Cox) test. *P < 0.05; **P < 0.01.

  • Fig. 4 Dox-CBD-SA treatment shows reduced toxicity.

    Aldoxorubicin or Dox-CBD-SA (20 mg/kg on a Dox basis) was administered to tumor-free FVB mice via tail vein injection on day 0. (A to D) Plasma cytokine concentrations on day 3. (E) Red blood cell (RBC) counts on day 6. (F) White blood cell (WBC) counts on day 3. (G) Spleen weights on day 16. Data represent mean ± SEM. Two experimental replicates. Statistical analyses were done using ANOVA with Tukey’s test. *P < 0.05; **P < 0.01.

  • Fig. 5 Dox-CBD-SA treatment completely eradicates established MC38 tumor in combination with anti-PD-1 CPI.

    MC38 cells (5 × 105) were inoculated on day 0. Mice were injected intravenously with aldoxorubicin or Dox-CBD-SA (5 mg/kg on a Dox basis) on days 6, 9, and 12. αPD-1 was also injected intraperitoneally on days 10 and 13. (A) The experimental schedule. (B) Graphs depict tumor volume until the first mouse died (mean ± SEM). (C) Survival rate. (D to G) Individual tumor growth curves. (H) On day 60, Dox-CBD-SA + αPD-1–treated survivors were rechallenged by subcutaneous injection of 5 × 105 MC38 cells. Naïve mice were also challenged with the same amounts of cells as a control group. The number of mice that developed palpable tumors is shown. Two experimental replicates. Statistical analyses were done using log-rank (Mantel-Cox) test for survival curves. *P < 0.05; **P < 0.01.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaaw6081/DC1

    Fig. S1. Confirmation of CBD fusion to SA by MALDI-TOF MS analysis.

    Fig. S2. Binding affinities of CBD-SA to collagen type I and type III.

    Fig. S3. SDS-PAGE analysis of mouse SA and CBD-SA conjugated with Dox.

    Fig. S4. Hydrodynamic sizes.

    Fig. S5. The binding interface between collagen type III and A3 domain of VWF.

    Fig. S6. In vitro release kinetics of Dox from Dox-SA.

    Fig. S7. Plasma pharmacokinetics of DyLight 800–labeled SA and CBD-SA.

    Fig. S8. Changes of hematological values in mice receiving aldoxorubicin or Dox-CBD-SA (20 mg/kg).

    Fig. S9. Histological analysis of major organs after Dox-CBD-SA treatment.

    Fig. S10. MC38 tumor rechallenge and body weight changes of MC38 tumor-bearing mice during the treatment.

    Table S1. Amino acid sequence of CBD-SA.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Confirmation of CBD fusion to SA by MALDI-TOF MS analysis.
    • Fig. S2. Binding affinities of CBD-SA to collagen type I and type III.
    • Fig. S3. SDS-PAGE analysis of mouse SA and CBD-SA conjugated with Dox.
    • Fig. S4. Hydrodynamic sizes.
    • Fig. S5. The binding interface between collagen type III and A3 domain of VWF.
    • Fig. S6. In vitro release kinetics of Dox from Dox-SA.
    • Fig. S7. Plasma pharmacokinetics of DyLight 800–labeled SA and CBD-SA.
    • Fig. S8. Changes of hematological values in mice receiving aldoxorubicin or Dox-CBD-SA (20 mg/kg).
    • Fig. S9. Histological analysis of major organs after Dox-CBD-SA treatment.
    • Fig. S10. MC38 tumor rechallenge and body weight changes of MC38 tumor-bearing mice during the treatment.
    • Table S1. Amino acid sequence of CBD-SA.

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