Arming oncolytic Coxsackievirus B3 with Neoleukin-2/15 enhances growth inhibition of colorectal carcinomas and induces T cell activation

Figures

• 

  Figure 1. Systematic evaluation of transgene insertion sites in the CVB3 PD-H genome (A) Schematic overview of GFP transgene insertion sites in the PD-H genome. The PD-H genome is shown with red crosses indicating regions unsuitable for transgene insertion due to constraints of the viral replication cycle. Below, all tested insertion sites are depicted, ranging from upstream of VP4 to downstream of the 3D polymerase, including the respective protease CS. (B) Nucleotide and aa sequences of wild-type (light gray) and artificial (dark gray) 3Cpro-CS with embedded GFP (green). Nucleotide substitutions introduced into the artificial CS are indicated in red. Viral aa are shown in light blue. The cleavage positions are indicated by brown arrows according to van Kuppeveld et al.50 (C) Nucleotide and aa sequences of wild-type (light gray) and artificial (dark gray) 2Apro-CS with embedded GFP (green). Nucleotide substitutions within the artificial CS are shown in red; viral aa are shown in light blue. The cleavage positions are indicated by brown arrows according to Höfling et al.32 (D) GFP expression (upper images) and cell lysis (lower images, light microscopy) after transfection of various PD-GFP…

• 

  Figure 2. Transgene size is negatively correlated with PD-H generation and influences stability (A) Schematic overview of PD-H constructs carrying transgenes of varying sizes. Shorter inserts (90, 180, 357, and 534 bp) were generated by 3′ truncation of the GFP cDNA, whereas longer inserts (894, 1,071, and 1,428 bp) were generated using 3′-truncated luciferase cDNAs. The construct PD-GFP contains the GFP cDNA (714 bp). The total viral genome sizes of each construct and percentage increase of the genome compared to PD-H are indicated. Transgenes are flanked at the N terminus with a 2Apro-CS and at the C terminus end with a mutated 2Apro-CS. (B) Assessment of virus-induced cell lysis 72 h post-transfection of HEK293T cells with 2.5 μg DNA in a six-well plate. Treatment with all constructs resulted in cell lysis, as documented by light microscopy. (C) Viral titers measured 72 h post-transfection by TCID50 assay on HeLa cells. (D) Stability of engineered viruses up to P10. HeLa (left), CaCo-2 (middle), Colon-26 (right) cells were infected with PD-90, PD-180, PD-357, PD-534, PD-GFP, or PD-1071 at MOI 0.1. Virus-containing supernatants were transferred every 48 h to fresh cells at the same MOI.…

• 

  Figure 3. Increasing transgene size negatively affects viral growth and cytotoxicity (A) Virus replication. HeLa, CaCo-2 and Colon-26 cells were infected with PD-H, PD-90, PD-180, PD-357, PD-534, PD-GFP, and PD-1071 at MOI 0.1. Viral titers were determined by TCID50 assay on HeLa cells; Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; n.s., not significant. (B) Viral cytotoxicity. HeLa, CaCo-2, and Colon-26 cells were infected with the indicated viruses and MOIs. Cell viability was assessed by XTT assay at 24 and 48 h post-infection and normalized to untreated controls (100%). Comparisons were performed between PD-H carrying a transgene and parental PD-H at each MOI; Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data are shown as mean (SD) (n = 3). The two-tailed unpaired t test was used for statistical analysis.

• 

  Figure 4. Characterization of PD-Neo-2/15 and PD-Neo-2/15-His (A) Schematic representation of PD-Neo-2/15, PD-Neo-2/15-His and PD-GFPtrunc constructs. Nucleotide and amino acid sequences of native (light gray) and artificial (dark gray) 2Apro-CS with embedded transgenes (dark red, light red, green). Nucleotide substitutions within the sequence encoding for the artificial 2Apro-CS are shown as red letters. Cleavage positions are indicated by brown arrows according to van Kuppeveld et al.50 (B) Virus growth kinetics in Colon-26 cells. Cells were infected with PD-H, PD-Neo-2/15, PD-Neo-2/15-His or PD-GFPtrunc at MOI 0.1. Virus titers were measured at indicated time points using the TCID50 method on HeLa cells. Significance: PD-H vs. PD-Neo-2/15: ∗p < 0.05, ∗∗p < 0.01; PD-H vs. PD-Neo-2/15-His: #p < 0.05, ##p < 0.01; PD-H vs. PD-GFPtrunc: †p < 0.05. (C) Cell viability of Colon-26 cells following infection. Cells were infected with PD-H, PD-Neo-2/15, PD-Neo-2/15-His, or PD-GFPtrunc at the indicated MOIs. Viability was assessed by XTT assay at 24 h (top) and 48 h (bottom) post-infection and normalized to untreated controls (100%); Significance PD with transgene vs. PD-H: ∗p < 0.05, ∗∗p < 0.01.…

• 

  Figure 5. Induction of T cell proliferation by Neo-2/15 (A) IL-2Rβγ bioassay confirming Neo-2/15 functionality. HEK293T cells were infected with PD-H or PD-Neo-2/15 at MOI 0.1 or 0.01. After 48 h, supernatants were collected, centrifuged, and analyzed in an IL-2Rβγ bioassay. Heat-inactivated samples (30 min, 60°C) retained activity, consistent with the heat stability of Neo-2/15; Significance: ∗∗∗∗p < 0.0001. (B) Proliferation of CD4+ and CD8+ T cells in response to IL-2 or Neo-2/15. Cells were stimulated with anti-CD3 and anti-CD28 or left unstimulated for 48 h and subsequently cultured for 5 days with no additive (control), 12 ng/mL recombinant IL-2, or indicated volumes of heat-inactivated viral supernatant containing Neo-2/15. Supernatant was derived from HEK293T-cells infected with PD-Neo-2/15 at MOI 0.1 for 48 h. Proliferation was assessed by CFSE dilution and flow cytometry; Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (untreated vs. treated); #p < 0.05, ##p < 0.01 (IL-2 vs. Neo-2/15). (C) Proliferation of stimulated CD4+ and CD8+ T cells in response to Neo-2/15. Cells were stimulated with anti-CD3 and anti-CD28 in the presence of 50 μL heat-inactivated viral…

• 

  Figure 6. In vivo oncolytic efficacy and transgene stability of PD-Neo-2/15 in the Colon-26 syngeneic mouse tumor model (A) Experimental timeline. A total of 5 × 105 Colon-26 cells were injected subcutaneously (s.c.) into the right flank of Balb/C mice. Five days later, tumors were treated intratumorally (i.t.) on three consecutive days (days 5–7) with 5 × 106 PFU of PD-H, PD-Neo-2/15, PD-GFPtrunc, or PBS (n = 5 per group). Tumors and organs were collected at the indicated time point. (B) Tumor growth curves. Dashed lines indicate virus injections. (C) Individual tumor volumes corresponding to (B), shown per animal. (D) Increase in tumor volume, calculated as the ratio of tumor volume at day 15 to day 5; Significance: ∗∗p < 0.01. (E) Tumor weights at experimental endpoint (day 15); Significance: ∗p < 0.05. (F) Tumor-adjusted body weight on day 15. Tumor mass was subtracted from total body weight and normalized to baseline (day 0); Significance: ∗p < 0.05. (G) Biodistribution of viruses in tumor, heart, and pancreas on day 15. Viral titers in tumor and heart were determined by TCID50 assay; viral genome copy numbers in pancreas were assessed by qRT-PCR. Data are shown per animal. (H)…