Investigation Of The Antitumor Effects,immunotherapeutic Mechanism And Preclinical Safety Of A Novel Oncolytic Herpes Simplex Virus Carrying Genes Encoding For Humanized Scfv Against Human PD-1

Immune checkpoint inhibitor(ICI)therapy is one of the major breakthroughs in the field of cancer therapy in recent years.However,ICI therapy still cannot meet the clinical needs of cancer patients because its overall objective response rate in cancer patients remains unsatisfactory and the majority of cancer patients do not benefit from this therapy.Recent,oncolytic virus(OV)has become an emerging immunotherapeutic strategy in cancer immunotherapy due to its unique and multifaceted anti-tumor effects.Our previous study has demonstrated that an oncolytic HSV-1(OVH)integrated with a single-chain variable fragment(scFv)against mouse PD-1(aMPD-1)can effectively deliver aMPD-1 scFv into tumors to significantly reduce tumor burdens and prolong the survival of tumor-bearing mice.The "arming combination therapy" strategy incorporating immune checkpoint inhibitors into OVs as a single agent is significantly more effective than either OVs or immune checkpoint inhibitors monotherapy.This also indicates that OV is an ideal synergist for immune checkpoint inhibitors.The "arming combination therapy" strategy may effectively resolve the sore point of the low objective response rates of immune checkpoint inhibitors in cancer treatment.However,our previous proof-of-concept study demonstrated that a virus-delivered secreted PD-1 blockade agent just had the potential to treat cancers in mice,it was our top priority to construct an OV armed with a humanized antibody that recognizes hPD-1 for further clinical investigation.In order to translate the concept of "arming combination therapy" into clinical application,the following studies were conducted based on our previous findings.First,we investigated the efficacy of the combination therapy of OVH and anti-PD-1 antibody in treating large advanced tumors and discovered that CD8+T cells played a predominant role in the response to the combination therapy and had significantly better efficacy in treating large advanced tumors than either OVH or anti-PD-1 monotherapy.To this end,we first screened and obtained a humanized monoclonal antibody,hu17D5,that had higher activity than the commercially available anti-PD-1 antibodies Pembrolizumab(PEM)and Nivolumab(NIV).Based on the gene sequences of hu17D5,we then developed a single-chain antibody fragment(hPD-1scFv)over a full-length antibody as the gene for viral delivery.Next,we constructed a novel OV carrying the humanized hPD-1scFv gene in the backbone of the tumor-selective OV OVH.This new virus was named YST-OVH.Since hPD-1scFv was unable to function in immunodeficient and immunocompetent mouse tumor models and in vitro cell-killing assays,OVH and YSTOVH had comparable specific antitumor activity in these models and showed good tumor-suppressive effects against a variety of tumor types.We also demonstrated that YST-OVH-induced oncolysis can cause hPD-1scFv produced in tumor cells to be released into the serum.To evaluate the immunomodulating activity of YST-OVH,we evaluated the systemic efficacy of delivering the antibody hPD-1scFv to the THE with YST-OVH in immunocompetent and humanized PD-1(PD-1-HU)mouse models.We evaluated the antitumor effects of intravenous administration of YST-OVH against intravenous administration of OVH in an orthotopic tumor model and intratumoral administration of YST-OVH against intratumoral administration of OVH in a bilateral flank large advanced tumor model.Treatment of these mice with YST-OVH significantly inhibited the growth of orthotopic tumors,virus-injected tumors and distant flank tumors,increased the overall survival and prevented recurrence compared to mice receiving treatment with OVH or the vehicle control,indicating that YST-OVH treatment can activate the immune system of mice and produce effective systemic antitumor immunity.The results demonstrated the effectiveness of YST-OVH as a robust standalone treatment by integrating of OVH and PD-1 inhibitors,which was better than the corresponding single-agent treatments in treating large advanced tumors and improving mouse survival.To investigate the anti-tumor immune mechanism of YST-OVH,we performed immune profiling of tumors using CyTOF and revealed the enhanced antitumor effect of YST-OVH,which largely relied on CD8+ T cell activity by augmenting the tumor infiltration of effector CD8+T cells and establishment of memory CD8+T cells,reducing associated CD8+T cell exhaustion and transforming the immunosuppressive tumor microenvironment into a pro-inflammatory status.Furthermore,YST-OVH treatment modified the cancer-immune set point of tumors coupled to coexpression of CTLA-4 and TIM-3 on exhausted CD8+T cells and high levels of CTLA-4+KLRG1+Treg cells,which had immunosuppressive activity.These immunosuppressive signals may be important factors that restrict the anti-tumor efficacy of YST-OVH.A combination approach incorporating anti-CTLA-4 or anti-TIM-3 further improved efficacy,resulting in more significant tumor inhibition and longer survival,and more mice achieved complete response compared with monotherapy.It was also found that reversing the negative immune regulators in the tumor microenvironment could effectively increase tumor immunogenicity and activate adaptive anti-tumor immune response,and therefore further enhanced the YST-OVH-induced anti-tumor immunity.Moreover,to determine the safety profile of YST-OVH and further translate YSTOVH into the clinic,we evaluated the neurovirulence of intrathalamic(i.c.)injection of YST-OVH in rhesus macaques and the systemic toxicity of i.v.injection of YST-OVH in cynomolgus monkeys.This therapeutic strategy showed no neurotoxicity and was well tolerated in nonhuman primates.We finally tested the effectiveness of YST-OVH using a humanized mouse model.The benefit of intratumoral hPD-1scFv expression was also observed in humanized mice bearing human cancer cells.The therapeutic efficacy of YST-OVH was much better than OVH,suggesting that YST-OVH can effectively activate human immune cells and induce a tumor-specific immune response.In summary,our study demonstrated that localized delivery of PD-1 inhibitors by engineered YST-OVH was a highly effective and safe strategy for cancer immunotherapy.YST-OVH also synergized with CTLA-4 or TIM-3 blockade to enhance the immune response to tumors,which are unresponsive to OVs or immune checkpoint inhibitors monotherapy.These data provide a strong rationale for further clinical evaluation of this novel therapeutic approach in patients with non-inflamed solid tumors.