Oligopeptide-modified poly(beta-amino ester)s-coated AdNuPARmE1A: Boosting the efficacy of intravenously administered therapeutic adenoviruses

Abstract

Oncolytic adenoviruses are used as agents for the treatment of cancer. However, their potential is limited due to the high seroprevalence of anti-adenovirus neutralizing antibodies (nAbs) within the population and the rapid liver sequestration when systemically administered. To overcome these challenges, we explored using nanoparticle formulation to boost the efficacy of systemic oncolytic adenovirus administration. Methods: Adenovirus were conjugated with PEGylated oligopeptide-modified poly(β-amino ester)s (OM-pBAEs). The resulting coated viral formulation was characterized in terms of surface charge, size, aggregation state and morphology and tested for anti-adenovirus nAbs evasion and activity in cancer cells. In vivo pharmacokinetics, biodistribution, tumor targeting, and immunogenicity studies were performed. The antitumor efficacy of the oncolytic adenovirus AdNuPARmE1A coated with OM-pBAEs (SAG101) in the presence of nAbs was evaluated in pancreatic ductal adenocarcinoma (PDAC) mouse models. Toxicity of the coated formulation was analyzed in vivo in immunocompetent mice. Results: OM-pBAEs conjugated to adenovirus and generated discrete nanoparticles with a neutral charge and an optimal size. The polymeric coating with the reporter AdGFPLuc (CPEG) showed enhanced transduction and evasion of antibody neutralization in vitro. Moreover, systemic intravenous administration of the formulation showed improved blood circulation and reduced liver sequestration, substantially avoiding activation of nAb production. OM-pBAEs coating of the oncolytic adenovirus AdNuPARmE1A (SAG101) improved its oncolytic activity in vitro and enhanced antitumor efficacy in PDAC mouse models. The coated formulation protected virions from neutralization by nAbs, as antitumor efficacy was preserved in their presence but was completely lost in mice that received the non-formulated AdNuPARmE1A. Finally, coated-AdNuPARmE1A showed reduced toxicity when high doses of the formulation were administered. Conclusions: The developed technology represents a promising improvement for future clinical cancer therapy using oncolytic adenoviruses.

Keywords: oncolytic adenovirus; pancreatic cancer; poly(β-amino ester)s; polymer-coated viral vectors; systemic delivery.

Figure 1

Characteristics of the polymers and oncolytic adenovirus coating. A Synthesis steps and chemical structure of C6CR3 and C6PEGCR3. B Schematic representation of the OM-pBAEs-based coating technology for therapeutic oncolytic adenoviruses.

Figure 2

Biophysical characterization of OM-pBAE-adenovirus complexes. A Z-potential determination of viral particles complexed with increasing ratios of molecules of polymer/vp for C6CR3 and CPEG polymers, or (as a control) with the DMSO vehicle without polymer. B Size determination by DLS. Z-Average, mean number, and polydispersity index (PDI) are presented. C Size distribution determined by NTA of naked Ad, CPEGAd, or C6CR3Ad formulations. D Encapsulation capability of OM-pBAEs assessed by gel-retardation assay. E Transmission electron microscopy (TEM) images of negatively stained formulations. TEM image of naked Ad (a), C6CR3-coated Ad (b), and CPEG-coated Ad (c). Scale bar: 100 nm for a; 50 nm for b and 200 nm for c. F Single CPEGAd particle analysis by TEM tomography. TEM tomography frame (a), three-dimensional representation of the resulting tomography constructed using 3DMOD software. The red solid object represents the coating and the green structure represents the contour of the viral particle (b). G dSTORM characterization of fluorescently labeled CPEGAd complexes. Green signal from DyLight550 labeled viral particles and red light from Cy5-labeled pBAE molecules.

Figure 3

Transduction efficiency of CPEGAd in the presence of NAbs and CAR-independent cellular uptake. A Flow cytometry quantification of PANC-1 GFP-positive cells after infection with naked or CPEG-coated GFP-expressing reporter AdGFPLuc at MOI 50 in the presence or absence of nAbs. The positive symbol (+) represents pre-incubation of virus samples with 1 × 10⁴ dilution of commercial anti-Ad5 antibody (ab6982, Abcam). B Representative fluorescent images of A549 and MCF-7 cells transduced at MOI 100 of naked Ad or CPEGAd. The percentage of GFP-positive cells resulting from flow cytometry assay is indicated. C Infectivity of CPEG-coated AdGFPLuc in CAR+ (A549) and CAR- (MCF-7) cells. Five thousand cells/well were infected with naked Ad and CPEGAd at increasing MOIs ranging from 0 to 4500, and the percentage of GFP-positive cells was analyzed by flow cytometry. The MOI needed to achieve 15% of GFP-positive cells was determined and is shown for each condition. *P < 0.05, ** P < 0.01, *** P < 0.001

Figure 4

Circulation kinetics and biodistribution of CPEGAd in vivo. A qPCR quantification of virus genome copies (GCs) in blood at 2 or 10 min after intravenous administration of 1 × 10¹⁰ AdGFPLuc vp/animal in C57BL/6J mice of naked and CPEG-coated formulation (n = 5). B Luciferase activity quantification of protein extracts from liver, spleen, kidneys, lungs, and intestine of treated mice at five days after injection. C Bioluminescence in vivo imaging of mice at 5 days after injection. D Tumor-to-liver in vivo transduction study of CPEGAd complexes in PANC-1 tumor-bearing BALB/c Nu/Nu mice (n = 6) in each flank. Luciferase activity of protein extracts was quantified five days after the iv administration of 1 × 10¹⁰ vp/animal of naked AdGFPLuc and CPEGAd. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 5

Antitumoral efficacy of SAG101 (CPEG-coated AdNuPARmE1A) in vitro and in vivo. A-C Dose-response curves of PANC-1, MIA PaCa-2, or A549 tumoral cell lines treated with SAG101 or AdNu, with doses ranging from 1 to 20,000 vp/cell. Cell viability was quantified by MTT assay three days after infection. D IC₅₀ summary graph including the fold-change between coated and naked formulations. Results are shown as mean ± SEM of three independent experiments performed in triplicate for each condition. E, F Efficacy studies of SAG101 and AdNuPARmE1A in passively immunized PANC-1 and MIA PaCa-2 tumor-bearing mice (n = 8). Animals of the pre-immune groups (marked as + conditions) were passively-immunized by I.P. injection of neutralizing serum the day before the treatment. Eight animals per group were treated with 4 × 10¹⁰ vp/animal. Tumor progression data were compared between conditions using linear mixed effect in R v2.14.1 with the lme4 package. *P < 0.05, ** P < 0.01, *** P < 0.001

Figure 6

General toxicity profile of SAG101 after systemic administration in immunocompetent mice. A Percentage of body weight variation of BALB/c mice (n = 8, n = 3 for controls) after intravenous administration of two different doses of AdNu and SAG101 (low, 5 × 10¹⁰ vp; or high, 7.5 × 10¹⁰ vp); the same amount of vehicle was injected at a high virus dose. B Assessment of hepatotoxicity based on AST and ALT in the serum of treated mice at 7 days post-administration. Results are expressed as the mean ± SEM of n = 8 animals/group, or n = 3 for the control groups. *P < 0.05, ** P < 0.01. C Peripheral blood cell counting of neutrophils and monocytes in response to AdNu or SAG101 intravenous injection. D Platelet cells counts of BALB/c peripheral blood at the indicated time-points after treatment. Data show mean values ± SEM.

Figure 7

Immune response against OM-pBAE coated adenovirus in vitro and in vivo. A Neutralizing capacity of serums collected from naïve C57BL/6J mice after intravenous administration of two doses of naked Ad and CPEGAd (1 × 10¹⁰ vp/animal) were administered at days 1 and 14; and sera were collected at day 21 post-injection. Resulting sera were analyzed in order to determine the ND50 (dilution needed to neutralize 50% the infectivity in an in vitro neutralization assay). B IL-6 production by RAW264.7 macrophages in response to exposure to AdNu and SAG101. Cytokines were quantified from culture media collected 48 h post-infection of 1 × 10⁶ cells/well treated with 1000 vp/cell of naked AdNu, SAG101, the polymeric component without viral particles, or (as a control) the corresponding volume of DMSO as in the SAG101 sample. C In vivo cytokine release study in immunocompetent BALB/c mice in response to AdNuPARE1A and SAG101 intravenous injection. Serum samples were collected at 6 h and 72 h after intravenous administration of 5 × 10¹⁰ viral particles of each formulation and the respective amount of polymeric component without viral particles as a control. IL-6 levels in serum was quantified using a microbead-based ELISA kit (mouse cytokine 10-Plex Panel; Invitrogen) on the Luminex^TM 200^TM. Data show mean values ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001