Purpose: The basis of the antitumor immunotherapy, of which the purpose is the stimulation of the immune system. We have used two of the Pathogen Associated Molecular Patterns: unmethylated CpG oligonucleotide, a ligand of Toll-like receptor 9 (TLR9), and lipopolysaccharide (LPS) which is recognized by TLR4, combined with an agonistic OX40 receptor-specific monoclonal antibody (anti-OX40), which is expressed by activated regulatory T-cells (and by other activated T-cell populations as well). The objective of this study was to prove the effectiveness of the aforementioned compounds in an animal model, on a bladder cancer cell line. Methods: We have instilled MB49 cells subcutaneously, to the left musculus biceps femoris. We have created three observation groups, each containing ten mice. After eleven days, all treated mice bearing the size of 5–8 mm (in diameter) tumor were administered CpG + anti-OX40 or LPS + anti-OX40 three times with a three-day lap between each treatment. Mice in the control group did not receive any treatment. Results: All the specimens from the control and LPS + anti-OX40 groups have died by the sixtieth day of the observation period, however, five mice from the CpG + anti-OX40 group were still alive. The experiment lasted until the last surviving mouse died, which occurred on the 357th day after tumor implantation. Discussion: The treatment with LPS did not make anti-OX40 more potent and did not increase the survival times. However, CpG + anti-OX40 has shown increased antitumor activity compared to the other two groups.

Effective cancer immunotherapy often requires coordinated activation of innate and adaptive immune responses. While the previous studies have primarily focused on whole oncolytic viruses, synthetic or engineered viral nucleic acids, or inactivated viral particles, the therapeutic potential of purified, naturally derived viral nucleic acids has remained largely unexplored. Here, we performed a preclinical evaluation of naturally derived viral RNA- and DNA-based combination immunotherapy in the murine Renca renal adenocarcinoma model. BALB/c mice-bearing subcutaneous tumors received intratumoral treatment with CpG plus anti-OX40, CpG plus 3p-hpRNA, 3p-hpRNA alone, influenza virus RNA (IV-RNA) plus anti-OX40, or porcine parvovirus DNA (PPV1-DNA) plus anti-OX40. Several treatment regimens were associated with improved survival and delayed tumor progression compared with PBS-treated controls. The greatest survival benefit was observed with CpG plus anti-OX40 (70% survival at day 38), followed by IV-RNA plus anti-OX40 (60%) and 3p-hpRNA alone (50%). IV-RNA combined with anti-OX40 demonstrated therapeutic activity comparable to that observed with CpG plus anti-OX40. In addition, treated tumors exhibited well-demarcated morphology and a markedly lower incidence of metastasis (13–38%) than PBS-treated controls (78%), consistent with improved local tumor control. Overall, these findings provide preclinical evidence that naturally derived viral nucleic acids may represent promising components of combination cancer immunotherapy. Although the underlying mechanisms remain to be elucidated, the observed therapeutic activity and reduced incidence of metastasis support further investigation of naturally derived viral nucleic acids in preclinical cancer immunotherapy.

The interplay of regulatory T cells (Tregs) within the tumour microenvironment presents a significant challenge in anticancer immunotherapy. This study investigates the potential of Treg blockade to enhance the efficiency of effector T cells. Two distinct treatment cocktails were examined: 3p-hpRNA (5′ triphosphate hairpin RNA) combined with unmethylated CpG oligonucleotide (CpG); CpG in combination with OX40 receptor-specific monoclonal antibody (anti-OX40). Treatment efficacy was assessed using a murine model of kidney adenocarcinoma. Renca cells (renal cortical cells with adenocarcinoma) were subcutaneously engrafted in 30 BALB/c mice, then animals were allocated into three treatment groups: Group 1: CpG+anti-OX40, Group 2: CpG+3p-hpRNA, Group 3: untreated control. Treatment efficacy was evaluated based on tumour growth, the occurrence of metastases and overall survival. On day 28 post-implantation, experiments had to be terminated due to tumour progression. Although comparisons of survival times and primary tumour sizes thus became inconsequential, histological examinations provided valuable insights. We observed distinct variations in primary tumour characteristics among the different groups: Groups 1 and 2 displayed demarcations, while Group 3 exhibited diffuse tumours with necrosis. Lung metastases were evident in 70% of untreated mice, whereas none were observed in either of the treated groups. Our findings instil confidence in the potential efficacy of the treatments, thereby laying a solid foundation for future investigations.