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Therapeutic targeting of the RB1 pathway in retinoblastoma with the oncolytic adenovirus VCN-01

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Science Translational Medicine  23 Jan 2019:
Vol. 11, Issue 476, eaat9321
DOI: 10.1126/scitranslmed.aat9321
  • Fig. 1 Expression of targets for infection and replication of VCN-01 in retinoblastomas.

    (A) Gene expression values (represented in log2) of CXADR, ITGA5, ITGAV, E2F1, and RB1 in retinoblastoma patient tumors, cell lines, and fetal retinas are represented in colors, from lower (blue) to higher (red) expression. (B) Fold-change expression (relative to fetal retinas) of the five genes in patient tumors and cell lines. *P = 0.0081 and **P = 0.0000018 compared to fetal retinas (limma t test). (C) CAR, α5 integrin, αv integrin, E2F-1, and RB1 protein expression in all evaluable cases (n = 32, 23, 29, 32, and 27 cases, respectively) included in the IHC study. One section per sample was assessed. (D) Representative cases of CAR, αv integrin, E2F-1, and RB1 staining in human samples. Control (−) slides were treated with no primary antibody. Pictures show selected areas of the conserved retina and retinoblastoma. PS, photoreceptor segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve fiber layer.

  • Fig. 2 Oncolytic activity of VCN-01 in retinoblastoma and RB1−/− cells.

    (A) Concentration-dependent cytotoxicity of VCN-01 in patient-derived cultures established from chemorefractory tumors at Hospital Sant Joan de Deu (HSJD; Barcelona, Spain). Dots are means of six replicates, and best-fit curves were built in GraphPad Prism. (B) Cytotoxicity of VCN-01 in patient-derived cultures derived from naïve tumors. Dots are means of six replicates, and best-fit curves were built in GraphPad Prism. (C) Comparison of viral concentration inhibiting 50% of tumor cell proliferation (IC50 values) between cultures derived from chemorefractory and naïve tumors. Individual data (labeled dots) are represented. VCN-01–resistant HSJD-RBVS-1 cells were excluded from this analysis. (D) Cytotoxic effects of VCN-01 (100 MOI) in a model of large vitreous seeding of HSJD-RBT-1 cells. Scale bars, 100 μm. (E) Cell viability at day 12 in large vitreous seeding treated with 1.5, 6.25, and 100 MOI of VCN-01 relative to untreated controls. Mean ± SD from six replicates is shown. *P = 0.002 compared to control, ANOVA. (F) Antiproliferative activity of VCN-01 in RB1+/− and RB1−/− iPS cells at day 3 after virus infection. Mean ± SD of six replicates and best-fit curves is shown.

  • Fig. 3 Biodistribution of intravitreous adenoviruses in tumor-bearing mice and juvenile rabbits.

    (A) AdTLRGDK genomes in mouse tissues 48 hours after intravitreous injection of the maximum feasible dose (MFDmouse; 3 × 109 vp in 2 μl) in mice with or without intraocular HSJD-RBT-2 tumors (n = 6 mice per group). Individual data from injected and contralateral (Contral.) eyes, brain regions, and liver are represented. Optic nerves (O. Nerve) were pooled, obtaining one data per group. *P < 0.0001 compared to all tissues (ANOVA). (B) Luciferase signal (normalized to the background of untreated mice) quantified in mouse samples (n = 6 mice per group). *P < 0.0006 and **P = 0.0001 compared to all tissues (ANOVA). (C) VCN-01 genomes in rabbit blood after intravitreous injection of one MFDrabbit (6 × 1010 vp in 40 μl of undiluted good manufacturing practice (GMP)–grade product containing VCN-01; n = 24 rabbits). (D) VCN-01 genomes in intraocular content (rabbit vitreous humor, aqueous humor, and retinas) of injected and contralateral eyes (n = 6 samples at each time point). (E) VCN-01 genomes in rabbit ocular surface tissues and fluids contiguous to the injection site (tear film, cornea, and optic nerve) (n = 6 to 24 samples at each time point). (F) VCN-01 genomes in rabbit liver and brain regions (n = 6 samples at each time point). LOQ, limit of quantification; BLD, below limit of detection (values BLD are represented as 1). Dots represent data from individual animals.

  • Fig. 4 Efficacy of intravitreous VCN-01 in orthotopic retinoblastoma xenografts.

    (A) Ocular survival of eyes with Y79 xenografts treated with VCN-01 at the MFDmouse (3 × 109 vp in 2 μl; n = 16) or not treated (n = 18). (B) Representative image of the local activity of the virus in the treated (right) eye of a mouse with bilateral HSJD-RBT-7 intraocular xenografts. (C) Representative case of hematoxylin and eosin (H&E), human nuclei (hNu), E1A, and hyaluronic acid (HA) staining in bilateral HSJD-RBT-2 xenografts treated with one dose (MFDmouse) of VCN-01 in the right eye (Injected) and not treated in the left eye (Control). Samples were obtained 30 days after treatment. Control (−) slide was treated with no primary antibody. Scale bars, 100 μm. One section was assessed per sample. (D) Immunofluorescence of E1A and Ad5 staining in an HSJD-RBT-2 xenograft. (E) Ocular survival of eyes with Y79, HSJD-RBT-2, or HSJD-RBT-5 xenografts treated with two doses of VCN-01 at the MFDmouse and compared to controls (n = 6 each). (F) Ocular survival of eyes with HSJD-RBT-2 xenografts treated with standard-of-care chemotherapy (SoC chemo), intravitreous melphalan, or VCN-01 at three clinically feasible human-equivalent dosages. Data were pooled from three independent studies, in which median survival of controls was similar. (G) Representative H&E staining in a VCN-01–treated (at the MFDmouse dose level) survivor eye at day 120. (H) Frequency of brain metastases in animals bearing orthotopic HSJD-RBT-2 tumors after enucleation of the second eye or after surviving 120 days. Numbers at top of the columns indicate the percent positive samples for metastasis. *P = 0.0094, χ2 test and Bonferroni correction; all groups were compared to vehicle controls.

  • Fig. 5 First-in-human intravitreous injection of VCN-01.

    (A) Viral DNA in blood and aqueous humor. First injection was given at day 0. (B) Neutralizing antibodies in blood and aqueous humor during treatment. (C) Chronologic changes in fundoscopy of patients treated with VCN-01. Vitreous seeds in patient 2 (arrows). (D) Electroretinographic signals in treated eyes during treatment. Normal values of a healthy eye are shown for comparison. (E) H&E, CD4, CD8, EMA (plasma cells), and E1A staining in conserved fragments of the retina, peritumoral areas, viable tumor, and areas of necrosis and inflammation. Control (−) was treated without primary antibody. One section was assessed per sample.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/476/eaat9321/DC1

    Materials and Methods

    Fig. S1. mRNA and protein expression for VCN-01 infection and replication targets.

    Fig. S2. In vitro characterization of VCN-01 activity in retinoblastoma models.

    Fig. S3. AdTLRGDK genomes in mouse plasma after one intravitreous administration.

    Fig. S4. In vivo characterization of VCN-01 efficacy and replication.

    Fig. S5. Macrophage accumulation in the uvea/ciliary body and the inner surface of the retina in VCN-01–treated rabbits.

    Fig. S6. Human translation of VCN-01 for retinoblastoma treatment.

    Fig. S7. Histopathology of a VCN-01–treated human eye.

    Table S1. Gene expression in retinoblastoma primary tumors, retinoblastoma cell lines, and fetal retinas (provided as an Excel file).

    Table S2. Differential gene expression for fetal retinas and retinoblastomas (limma t test) (provided as an Excel file).

    Table S3. CAR, α5 integrin, and αv integrin expression scores (high, H; moderate, M; low, L; and negative, N) in tumors in relation to clinicopathologic characteristics of enucleated retinoblastoma patients.

    Table S4. E2F-1 and RB1 expression score (high, H; moderate, M; low, L; and negative, N) in tumors in relation to clinicopathologic characteristics of enucleated retinoblastoma patients.

    Table S5. Clinical details of patient-derived retinoblastoma cell models.

    Table S6. Ocular toxicity monitoring (edema, intraocular pressure, and slit-lamp imaging) in juvenile rabbits treated with VCN-01 (provided as an Excel file).

    Table S7. Clinical details for the first two patients of the clinical trial.

    Data file S1. Primary data (provided as an Excel file).

    References (5557)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. mRNA and protein expression for VCN-01 infection and replication targets.
    • Fig. S2. In vitro characterization of VCN-01 activity in retinoblastoma models.
    • Fig. S3. AdTLRGDK genomes in mouse plasma after one intravitreous administration.
    • Fig. S4. In vivo characterization of VCN-01 efficacy and replication.
    • Fig. S5. Macrophage accumulation in the uvea/ciliary body and the inner surface of the retina in VCN-01–treated rabbits.
    • Fig. S6. Human translation of VCN-01 for retinoblastoma treatment.
    • Fig. S7. Histopathology of a VCN-01–treated human eye.
    • Table S3. CAR, α5 integrin, and αv integrin expression scores (high, H; moderate, M; low, L; and negative, N) in tumors in relation to clinicopathologic characteristics of enucleated retinoblastoma patients.
    • Table S4. E2F-1 and RB1 expression score (high, H; moderate, M; low, L; and negative, N) in tumors in relation to clinicopathologic characteristics of enucleated retinoblastoma patients.
    • Table S5. Clinical details of patient-derived retinoblastoma cell models.
    • Table S7. Clinical details for the first two patients of the clinical trial.
    • References (5557)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Gene expression in retinoblastoma primary tumors, retinoblastoma cell lines, and fetal retinas (provided as an Excel file).
    • Table S2. Differential gene expression for fetal retinas and retinoblastomas (limma t test) (provided as an Excel file).
    • Table S6. Ocular toxicity monitoring (edema, intraocular pressure, and slit-lamp imaging) in juvenile rabbits treated with VCN-01 (provided as an Excel file).
    • Data file S1. Primary data (provided as an Excel file).

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