Construction And Research On The Antitumor Effects Of A Novel Oncolytic Herpes Simplex Virus Encoding A Monoclonal Antibody Against PD-1

BackgroundThe application of immune checkpoint blockade(ICB)is one of the most important breakthroughs in the history of tumor treatment.It has achieved inspiring clinical responses in a subset of patients with various tumor types.At present,the US Food and Drug Administration(FDA)has approved six antibodies against CTLA-4,PD-1 or PD-L1 for twelve cancer indications.Unfortunately,only about 10-40%of patients with certain tumors will benefit from checkpoint blockade therapy.The poor efficacy of PD-1/PD-L1 blockade is often attributed to limited infiltration of lymphocytes into the tumor microenvironment(TME).Oncolytic viruses(OVs)represent another novel group of promising immunotherapeutic agents for tumor treatment.By selectively lysing neoplastic cells resulting in immunogenic cancer cell death(ICD),OVs could cause the release of tumor-associated antigens(TAAs),danger-associated molecular patterns(DAMPs)and pathogen-associated molecular patterns(PAMPs),thus promote antigen presentation,recruit T cells,and lead to priming of tumor-specific T cells.OVs can also facilitate T cell infiltration through the induction of potent type Ⅰ interferon responses,which increase the production of chemokines.Based on the different action mechanisms of ICB and OVs5 we aim to explore a novel combinatorial strategy of them and whether it could acquire synergistic,durable clinical responses against a broad range of cancer types.MethodsWe used molecular cloning and homologous recombination techniques to construct a novel oncolytic herpes simplex virus 2,oncolytic HSV2-aPDl(oHSV2-aPD1),which carried an anti-human PD-1 monoclonal antibody encoding gene on basis of previously reported oHSV2.With Western blotting and co-culture experiments,we explored the combinative ability and biological function of the PD-1 monoclonal antibody expressed by oHSV2-aPDl infected cells.Through virus infection experiments and CCK8 cell viability assays,we compared the infectious and oncolytic activities of oHSV2-aPD1 and its parental virus oHSV2.Via mice B16R melanoma model,we tested the therapeutic effect of oHSV2-aPD 1 in vivo.Furthermore,with flow cytometry and RNA sequencing analysis,we assessed the influence of oHSV2-aPD1 application on tumor microenvironment and systemic immunity.Results(1)Here,we successfully constructed the novel oncolytic virus,oHSV2-aPDl,which encodes an anti-human PD-1 monoclonal antibody(anti-hPD1mAb).(2)oHSV2-aPDl-infected eukaryotic cells produced and secreted the right anti-hPD1mAb.And the antibody could recognize and bine the immune checkpoint PD-1,thus freeing and activating T cells,which was observed as an enhancement in IFN-γ and IL-2 production.(3)Introduction of the anti-hPD1mAb sequence into the oHSV2 viral backbone did not alter the oncolytic spectrum nor did it hinder the cytolytic activity of oHSV2-aPD1.(4)In the mouse B16R melanoma model,oHSV2-aPD1 effectively delayed tumor progression and prolonged the mice survival times.The therapeutic efficacy of oHSV2-aPDl also proved superior to unarmed oHSV2 treatment or PD-1 blockade alone and as effective as their combination(local application of oHSV2 plus systemic application of PD-1 blockade).(5)Additionally,localized oHSV2-aPD1 application induces a massive elevation of immune effector cells and downregulation of immunosuppressive cells both in the TME and in the peripheral immune system.(6)Compared with local immunity in the TME,systemic immunity had a better correlation with the therapeutic effect of immune therapy.ConclusionOur findings suggest that oHSV2-aPDl demonstrates enhanced antitumor activity and exerts a durable response in preclinical melanoma models,providing support for combinatorial strategies involving administration of OVs expressing a PD-1 inhibitor.