*4.1. Efficacy against LLC-1-HER2 Primary Tumors*

Early studies from our laboratory indicated that retargeted oHSVs are highly effective in nude mice, a property which only accounted for direct oncolytic effects [41,56]. The key question arose as to how effective the retargeted oHSVs are in immunocompetent mice, in particular in eliciting the innate response to the virus, the innate and the adaptive long-term immunity to the tumor. To address this question here we provide the first description of the efficacy of R-335 and R-337 and review previously described efficacy data on 1st generation recombinants R-LM113, R-115 and R-123. A list of the most significant

preclinical studies carried out in our laboratory is reported in Table 2. As discussed above, the HER2-retargeted oHSVs are strictly dependent on (human) HER2 to carry out infection, a feature that required an ad hoc immunocompetent murine model. Preliminarily, we screened a number of murine tumor cell lines and found that LLC-1 (Lewis lung carcinoma-1) cells enabled the highest HSV replication [55]. The ad hoc model consists of LLC-1 cells made transgenic for HER2 (LLC-1-HER2) and of the syngeneic C57Bl/6 mice transgenic and tolerant (TG) to HER2. Mouse tolerance to HER2 was critical to prevent that the immune response to the tumor was mainly driven by the allogeneic HER2 [55]. With respect to antitumor activity, the 1st generation R-115 protected 60% of mice, of which 16% exhibited a complete response (CR) and 44% a partial response (PR) [55]. The experimental design in anti-tumor efficacy experiments is illustrated in Figure 4, panel A. Briefly, mice were implanted with subcutaneous tumors; the recombinants were administered intratumorally (i.t.) to well-developed tumors. The mice that survived the primary tumor received a second challenge tumor, which was untreated. The R-335 and R-337 recombinants were administered i.t. to well-developed tumors as five injections of 1 <sup>×</sup> <sup>10</sup><sup>8</sup> PFU each, every other day (Figure 4A). The antitumor activity of R-335 was similar to that of R-115, while that of R-337 was higher. In particular, R-335 protected 60% of mice, 30% of which exhibited a complete response (CR) (Figure 4B–E). R-337 protected 100% of the mice, 80% of which exhibited CR (Figure 4B–E). The Kaplan Meier survival curve shows highly statically significant differences between each virus and the control, and between the two viruses (Figure 4F). The superior efficacy R-337 relative to R-335 should be interpreted in light of the fact the only genotypic difference between the two viruses resides in mIL-12, which is a heterodimer in R-335 and a fusion form in R-337. The results clearly indicate that a significant contribution to the control of primary tumor growth is immune mediated.



**Figure 4.** Efficacy of R-335 and R-337 monotherapy on the growth of LLC-1-HER2 tumors. (**A**) Schedule of treatments. The six-to-eight weeks old HER2-transgenic/tolerant (HER2-TG) C57BL/6 mice were subcutaneously implanted in the left flank with 5 <sup>×</sup> <sup>10</sup><sup>5</sup> LLC-1-HER2 cells in 100 <sup>µ</sup>L of PBS [55]. 10 d later, when the tumor volumes averaged 70–100 mm<sup>3</sup> , mice received 5 intratumoral injections of R-335, R-337 (1 <sup>×</sup> <sup>10</sup><sup>8</sup> PFU per injection, diluted in 50 <sup>µ</sup>L PBS) or vehicle (50 <sup>µ</sup>L PBS), at 2–3 day intervals. At d 33, the mice which survived the primary tumor received a contralateral challenge LLC-1-HER2 tumor of 5 <sup>×</sup> <sup>10</sup><sup>5</sup> cell per mouse. Tumor volume was calculated using the formula: largest diameter x (smallest diameter) 2 × 0.5. Mice were sacrificed when tumor volumes exceed 1000–2000 mm<sup>3</sup> , ulceration occurred, or animals exhibited distress or pain. (**B**–**D**) Kinetics of tumor growth in mice treated with vehicle (**B**), R-335 (**C**) or R-337 (**D**). The numbers reported in each panel indicate the numbers of mice which were completely cured from tumors (complete response, CR), or which showed a delay/reduction in tumor growth (partial response, PR). The mice were scored PR when the tumor volume was <50% smaller than the mean size of the tumors in the vehicle group. (**E**) Volumes of the primary tumors at d 21 after implantation. Black (vehicle), blue (R-335) and red (R-337) circles. (**F**) Kaplan-Meier survival curves of the three groups of mice. (**G**,**H**) Kinetics of growth of contralateral challenge tumor in naïve mice (**G**), and in the R-335 or R-337 (**H**) arms. (**I**) Immune response in splenocytes harvested at sacrifice. To isolate splenocytes, spleens were smashed through a 70 µm cell strainer in PBS, red blood cells were lysed with ACK buffer, and samples were resuspended in medium (RPMI 1640 containing 10% heat inactivated FBS, 1% penicillin/streptomycin). Splenocytes (1 <sup>×</sup> <sup>10</sup><sup>6</sup> cell/well) were incubated with 1 <sup>×</sup> <sup>10</sup><sup>5</sup> LLC-1-HER2 or LLC-1 cells in 0.5 mL medium, and cocultured for 48 h. The amount of secreted IFNγ (quantified by ELISA) was a measure of the splenic anti-LLC-1 and anti- LLC-1-HER2 immune response [55]. (**J**,**K**) Antibody reactivity in sera harvested at sacrifice to LLC-1-HER2 or LLC-1 cells (**J**), and to HSV-1-infected cells (**K**), as determined by cell enzyme-linked immunosorbent assay (CELISA).

Wt-LLC-1 and LLC-1-HER2 single cell preparations were reacted with mouse serum, diluted 1:150 in flow cytometry buffer (PBS + 2% FBS), in ice for 1 h, washed with flow cytometry buffer and incubated with anti-mouse PE (1:400). Data were acquired on BD C6 Accuri. For CELISA assay, RS cells were infected with HSV-1 at 3 PFU/cell for 24 h, then they were fixed with paraformaldehyde, reacted with mouse serum diluted 1:60, or with the anti-gD monoclonal antibody HD1 (green) diluted 1:400 (positive control), followed by anti-mouse peroxidase. Peroxidase substrate o-phenylenediamine dihydrochloride was added and plates were read at 490 nm as detailed [55]. (F) Statistical significance was calculated by the Log-rank (Mantel-Cox) test. (**E**,**I**–**K**) Statistical significance was calculated by means of the One Way ANOVA test and expressed as \* = *p*-value < 0.05; \*\* = *p*-value < 0.01; \*\*\* = *p*-value < 0.001; \*\*\*\* = *p*-value < 0.0001. Color code: mice which received Vehicle, R-335 or R-337 are indicated in black, blue or red, respectively.
