Research ArticleIMMUNOLOGY

Supramolecular prodrug hydrogelator as an immune booster for checkpoint blocker–based immunotherapy

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Science Advances  29 Apr 2020:
Vol. 6, no. 18, eaaz8985
DOI: 10.1126/sciadv.aaz8985
  • Fig. 1 Schematic and characterization of in situ formed P-NT–aPD1 hydrogel.

    (A) Schematic illustration of localized CPT and aPD1 delivery using an in situ formed supramolecular hydrogel to attain bioresponsive drug release and tumor microenvironment regulation. (B) Representative transmission electron microscopy (TEM) images of diCPT-PLGLAG-iRGD nanotubes (P-NT). Scale bar, 100 nm. (C) The circular dichroism (CD) spectrum of the diCPT-PLGLAG-iRGD nanotubes solution. (D) Photographs of the sol-gel transition of P-NT upon the addition of phosphate-buffered saline (PBS). (E) Degradation profiles of diCPT-PLGLAG-iRGD in the presence or absence of glutathione (GSH) (10 mM). Data are given as means ± SD (n = 3). (F) In vitro cytotoxicity studies of free CPT and diCPT-PLGLAG-iRGD toward GL-261 brain cancer cells. IC50, median inhibitory concentration. (G) Inhibition of tumor spheroid growth was evaluated following treatment with free CPT or P-NT. Spheroids treated with drug-free Dulbecco’s modified Eagle’s medium were used as the blank control. Scale bar, 500 μm. (H) The degradation profiles of 200 μM diCPT-PLGLAG-iRGD solutions incubated in the presence or absence of matrix metalloproteinase 2 (MMP-2; 2 μg/ml). Data are given as means ± SD (n = 3). (I) Cumulative release profiles of CPT prodrugs (including diCPT-PLGLAG-iRGD and diCPT-PLG) and (J) aPD1 from P-NT–aPD1 hydrogels incubated in PBS with or without MMP-2. Data are given as means ± SD (n = 3). Photo credit: Feihu Wang, Johns Hopkins University.

  • Fig. 2 P-NT–aPD1 hydrogel enhances local retention and prolongs in vivo release of aPD1.

    (A) In vivo gel formation and retention after subcutaneous injection of P-NT solution in the back of C57BL/6 mice. (B) In vivo degradation profile of the P-NT hydrogel over time, as determined by the mass loss method. (C) Fluorescence IVIS imaging of the local retention and distribution of aPD1-Cy5.5 in mice, administered in solution form and with the P-NT hydrogel. Experiments were repeated three times. (D) Fluorescence imaging of tumor tissues and (E) tumor sections of GL-261 brain tumor–bearing mice after tumoral injections of free (CPT + aPD1) or P-NT–aPD1. Red, Cy5.5-labeled aPD1; blue, 4′,6-diamidino-2-phenylindole–stained nuclei. Scale bar, 200 μm. (F) Quantification of the in vivo retention profile of Cy5.5-aPD1 and (G) CPT. Statistical significance was calculated using a two-sided unpaired t test. Data are given as means ± SD (n = 3). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. Photo credit: Feihu Wang, Johns Hopkins University.

  • Fig. 3 Local delivery aPD1 by CPT prodrug hydrogel elicits a robust antitumor immunity.

    (A) Experimental schedule: GL-261 brain cancer cells were implanted into the right flank of mice on day 0. Mice were intratumorally injected on day 10 with P-NT, aPD1-loaded diC12-PLGLAG-iRGD (aPD1-L), or P-NT–aPD1 hydrogels, with diC12-PLGLAG-iRGD hydrogel (E-Gel) used as a drug-free control. Flow cytometric analysis was performed on lymphocytes extracted from the tumor on day 25. (B) Representative flow cytometric analysis images and (D) relative quantification of CD3+ T cell within the tumor by different treatment groups. (C) Representative flow cytometric images and (F) relative quantification of CD8+ T cell infiltration within the tumor by different treatment groups. (E) Quantification of CD4+ T cell that infiltration within the tumor in different treatment groups. (G) Representative flow cytometric analysis images and (H) relative quantification of Foxp3+CD4+ T cells (Tregs). (I) The percentages of PD-1–expressing CD8+ T cells and (J) PD-1–expressing CD4+ T cells after different treatments. (K) The percentage of PD-L1–expressing CD45 cells after different treatments. Statistical significance was calculated using a two-sided unpaired t test. Data are given as means ± SD (n = 3). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

  • Fig. 4 Intratumoral injection of P-NT–aPD1 elicits complete regression of established GL-261 brain tumors.

    (A) Experimental schedule: GL-261 brain cancer cells were implanted into the right flanks of mice on day 0. Mice were intratumorally (it.) injected on day 10 with free (CPT + aPD1), P-NT, aPD1-loaded diC12-PLGLAG-iRGD [aPD1(L)], or P-NT–aPD1 solutions. In the free (CPT + aPD1) group, treatment was administered three times (on days 10, 17, and 24). (B) The in vivo bioluminescence images of the GL-261 tumors on day 35 and (C) day 60. (D) Average tumor growth kinetics of different treatment groups; growth curves were plotted until the first mouse death. Data are given as means ± SD (n = 10 for P-NT–aPD1–treated group and n = 5 for other groups). (E) Survival curves corresponding to different treatment groups. (F) Quantification of CD3+ T cells and (G) CD8+ T cells infiltrating within the tumor between different treatment groups. (H) Ratios of the tumor-infiltrating CD8+ Teffs to Tregs in the tumors of different treatment groups. Statistical significance was calculated using a two-sided unpaired t test. Data are given as means ± SD (n = 3). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

  • Fig. 5 Intratumoral delivery of P-NT–aPD1 induces T cell memory against tumor.

    (A) Mice that considered long-term survival from all treatment groups were rechallenged on the opposite flank in an attempt to develop new tumors. (B) The in vivo bioluminescence imaging of the GL-261 tumors was observed on day 110 and (C) on day 130. (D) Survival curves for naive and rechallenged mice from different treatment groups. Statistical significance was calculated via the log-rank (Mantel-Cox) test. (E) The percentage of CD8+ Tcm cells and (F) CD8+ Tem cells in splenocytes of the naive and rechallenged mice. Statistical significance was calculated using a two-sided unpaired t test. Data are given as means ± SD (n = 3). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

  • Fig. 6 P-NT–aPD1 treatment induces systemic antitumor immune response.

    (A) Experimental scheme: Mice were implanted with GL-261 cells in the right back and left cortical surface, and then primary tumors were locally treated with P-NT–aPD1 on day 6. In vivo bioluminescence imaging of the tumors was observed at scheduled time points. (B) Tumors on the right flank were locally treated with P-NT–aPD1 hydrogel, while intracranial gliomas were designated as distant tumors and were left untreated. (n = 10 for P-NT–aPD1–treated group and n = 5 for saline group). (C) In vivo bioluminescence imaging of the GL-261 tumors in response to local P-NT–aPD1 hydrogel treatment. (D) Survival curves corresponding to saline and P-NT–aPD1–treated mice. Statistical significance was calculated via the log-rank (Mantel-Cox) test. (E) Quantification of CD8+ T cells infiltrating within the tumors of the two treatment groups. (F) Ratios of the tumor-infiltrating Teff to Treg in the tumors of the treatment groups. Statistical significance was calculated using a two-sided unpaired t test. Data are given as means ± SD (n = 3). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

Supplementary Materials

  • Supplementary Materials

    Supramolecular prodrug hydrogelator as an immune booster for checkpoint blocker–based immunotherapy

    Feihu Wang, Dongqing Xu, Hao Su, Weijie Zhang, Xuanrong Sun, Maya K. Monroe, Rami W. Chakroun, Zongyuan Wang, Wenbing Dai, Richard Oh, Han Wang, Qin Fan, Fengyi Wan, Honggang Cui

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