*4.4. The "Immune Heating" of the Tumor Predisposes to Combination Therapy*

The distant long-term protection, along with the dramatic changes to TME induced by R-337, suggested that the recombinant could render immunologically cold and CPIresistant tumors immunologically hot and possibly CPI-sensitive. LLC-1-HER2 tumors recapitulate tumors that are completely insensitive to anti-PD-1 (compare Figure 6B with Figure 4B), in agreement with the low immunogenicity of these tumors [61]. The experiment documented in Figure 6 was designed to ascertain whether R-335 and R-337 synergize with anti-PD-1. Mice were treated as in Figure 4, and additionally received anti-PD-1, administered i.p. (see Figure 6, panel A). It can be seen that, when combined with anti-PD-1 in a simultaneous regimen [62–65], R-335 displayed a tendency to increase efficacy (Figure 6B–E). Thus, in mice treated with R-335 alone, CR and PR occurred in 31 and 25% of mice, respectively, in agreement with data shown in Figure 4. In mice which received the combination therapy, CR and PR occurred in 41 and 35% of mice, i.e., 76% mice were protected, completely or partially. The Kaplan Meier survival curve is reported in Figure 6F. The mice which survived the primary tumor were fully protected from a challenge distant tumor (Figure 6G,H). The long-term protection was most likely based on the systemic immune response, documented as splenocyte and antibody reactivities to LLC-1-HER2 and wt-LLC-1 cells (Figure 6I,J).

To evaluate the efficacy of R-337 in combination with anti-PD-1, we decreased the overall amount of virus from five injections of 1 <sup>×</sup> <sup>10</sup><sup>8</sup> PFU each to three injections of 0.3 <sup>×</sup> <sup>10</sup><sup>8</sup> PFU each (in total, 0.9 <sup>×</sup> <sup>10</sup><sup>8</sup> vs 5 <sup>×</sup> <sup>10</sup><sup>8</sup> ) (Figure 6K). At this lower dosage, R-337 monotherapy induced CR in 36% mice and PR in 18%, with an overall response rate of about 55%. In the combination arm, 80% of mice exhibited CR, and 10% exhibited PR (Figure 6L–N). The difference between monotherapy and combination therapy was statistically significant with respect to tumor size (Figure 6O) and Kaplan Meier survival curve (Figure 6P). The surviving mice were fully protected from a distant challenge made of LLC-1-HER2 cells (Figure 6Q,R). At sacrifice, 80 days after primary tumor implantation, the mice treated with the combination therapy showed a tendency to increased splenocyte response (Figure 6S), and an increase in antibody response (Figure 6T). The results show that the R-337 and anti-PD-1 combination therapy was highly effective and are consistent with the view that the distant protection was immune-based.

**Figure 6.** Efficacy of R-335 of R-337 in combination with anti-PD1 antibodies on the growth of LLC-1-HER2 tumors. (**A**) Schedule of the treatments. The HER2-TG C57BL/6 mice were implanted with LLC-1-HER2 cells. At d 10 after implantation, when tumors reached the average volume of 70–100 mm<sup>3</sup> , mice received 5 i.t. injections of R-335, or R-335 plus i.p. injections of anti-PD-1, at 2–3 days intervals. The administration schedule of oHSV and anti-PD-1 treatments was according to [62–65]. At d 44, the mice which survived the primary tumor received a contralateral challenge LLC-1-HER2 tumor.

For the details, see Figure 4B–D Kinetics of tumor growth in mice treated with vehicle (**B**), R-335 alone (**C**), or R-335 plus anti-PD-1 combination therapy (**D**). Figures in panels indicate the number of mice exhibiting complete response (CR) or partial response (PR). (**E**) Volumes of the primary tumors at d 21 after implantation. Black (vehicle), blue (R-335) and open blue (combination) circles. (**F**) Kaplan-Meier survival curves of the three groups of mice. (**G**–**H**) Growth kinetics of contralateral challenge tumors in naïve mice (**G**), and in R-335 or combination arms (**H**). (**I**) Immune response in splenocytes harvested at sacrifice. Splenocytes were incubated with LLC-1-HER2 or LLC-1 cells. Activation was quantified as IFNγ secretion in the culture medium. (**J**) Serum antibody reactivity to LLC-1-HER2 or LLC-1 cells. (**K**) Schedule of the treatment with R-337 with or without combination with anti-PD-1 antibodies. The HER2-TG C57BL/6 mice were implanted with LLC-1-HER2 cells. At 10 d after tumor implantation, mice received 3 i.t injections of R-337 at 5 days interval, and, where indicated, i.p. injections of anti-PD-1 antibodies, as detailed in the drawing. At d 35, the mice which survived the primary tumor received a contralateral challenge LLC-1-HER2 tumor. (**L**–**T**) Kinetics of tumor growth (**L**–**N**), tumor size at d 21 (**O**), Kaplan Meier survival curves (**P**), growth curves of challenge tumors (**Q**–**R**), immune response in splenocytes (**S**), antibody reactivity to LLC-1-HER2 or LLC-1 cells (**T**). (**F**,**P**) Statistical significance was calculated by the Log-rank (Mantel-Cox) test. (E, I, J, O, S, T) Statistical significance was calculated by means of the ANOVA test and expressed as \* = *p*-value < 0.05; \*\* = *p*-value < 0.01; \*\*\* = *p*-value < 0.001; \*\*\*\* = *p*-value < 0.0001. Color codes: mice treated with vehicle, R-335, R-337 are indicated in black, blue or red, respectively. Full circles and continuous lines, monotherapies. Open circles and dotted lines, combination therapies.
