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Anti-Tumor Effects

A PD-L1 tropism-expanded oncolytic adenovirus enhanced gene delivery efficiency and anti-tumor effects

Recombinant adenovirus serotype 5 (Ad5)-mediated virotherapy is a maturing technique in cancer treatment. However, the utility of adenovirus (Ad) has been limited by low expression of coxsackievirus and adenovirus receptor (CAR) in cancer cells resulting in poor infectivity of Ads. To overcome the problem, we aimed to develop a novel tropism-modified oncolytic adenovirus, ZD55-F-HI-sPD-1-EGFP, which contains the epitope of PD-1 (70-77aa) at the HI-loop of Ad fiber. Trimerization of Fiber-sPD-1 was confirmed by immunoblot analysis. ZD55-F-HI-sPD-1-EGFP shows a remarkable improvement in viral infection rate and gene transduction efficiency in the PD-L1-positive cancer cells. Competition assays with a PD-L1 protein reveals that cell internalization of ZD55-F-HI-sPD-1-EGFP is mediated by both CAR and PD-L1 at a high dose. The progeny virus production capacity showed that sPD-1 incorporated fiber-modified oncolytic Ad replication was not affected. Furthermore, treating with ZD55-F-HI-sPD-1-EGFP significantly increased viral infection rate and enhanced anti-tumor effect in vivo. This study demonstrates that the strategy to expand tropism of oncolytic Ad may significantly improve therapeutic profile for cancer treatment.

Oncolytic virotherapy is considered as one of the most promising cancer therapies, because of their ability to selectively replicate and lyse tumor cells, induce inflammatory response, release tumor-associated antigens to activate host’s adaptive anti-tumor immunity, and promote lymphocyte infiltration in tumors [1], [2], [3], [4], [5]. Adenovirus serotype 5 (Ad5) based oncolytic adenovirus are widely applied to pre-clinical and clinical trials [6], [7]. Ad5 infection of cells is initiated by the formation of a complex between the fiber protein knob domain of the virus and the host cell surface coxsackievirus and adenovirus receptor (CAR) [8], [9]. However, CAR is expressed at low levels in many types of cancer cells [10], [11], [12], [13], which limits the infectivity and oncolytic efficiency of Ad5-mediated oncolytic adenovirus [14], [15]. Therefore, the methods to increase infectivity of Ad5 are required to maximize the therapeutic efficacy of oncolytic Ad in cancers with CAR-deficient.
To broaden the native tropism of oncolytic Ad, several genetic modifications of Ads have been studied, which are mainly focused on fiber proteins. Considering that the tropism of infection often differed between adenovirus subgroups [16], fiber knob serotype switching is one of the main approaches to broaden the tropism of Ad5-based vectors. Numerous Ad5-based oncolytic vectors with chimeric fibers have been developed, such as replacement of Ad5 knob to Ad37 enhanced viral infectivity against glioma cell [17], and replacement of Ad5 knob to Ad35 knob enhanced CD46-dependent Ad gene transfer efficiency [18]. Chimeric fibers of Ad5/3 [19], [20] and Ad5/35 [21], [22] also have been developed as cancer therapies. In addition, insertion of a peptide to the C-terminus or HI-loop of the fiber protein is another main approach to expand the tropism of Ad5-based vectors. Incorporating the vesicular stomatitis virus glycoprotein (VSV-G) epitope onto the C terminus of the fiber knob facilitated the specificity binding of the virus to phosphatidylserine (PS) moieties on the cellular plasma membrane [23]. Addition a cationic polylysine sequence (pK7) to the fiber knob facilitated viral binding to negatively charged cell surface molecules, such as heparin sulfates and increased Ad infection to endothelial cells and fibroblasts [24]. Similarly, insertion a peptide of arginine-glycineaspartate (RGD) integrin recognition site to the C-terminus or HI-loop of the fiber protein signally increased Ad infection in wide range of cells such as fibroblasts, endothelial cells, and smooth muscle cells [25]. However, the current fiber protein modification aimed at increasing the Ad infectivity cannot satisfy all tumor cell types due to the diversity of tumors. Therefore, more fiber protein modification studies are needed to expand the tropism of adenovirus to invade different tumor cells. Structure of the complex of human PD-1 and its ligand PD-L1 showed that the 70–77 amino acid peptide (70 to 77, MSPSNQTD) of PD-1 plays a role in interacting with PD-L1 [26]. Therefore, we hypothesized that insertion short PD-1 (sPD-1, 70-77aa) to HI-loop of the fiber knob can expand the tropism of Ad to PD-L1 positive tumor cells.

Here, we constructed a tropism expanded oncolytic adenovirus (ZD55-F-HI-sPD-1-EGFP), which incorporated a short peptide of PD-1 (sPD-1, 70-77aa) in HI-loop of fiber knob and contained an exogenous reporter gene, EGFP. We verified that fiber trimer formation and viral replication ability of ZD55-F-HI-sPD-1-EGFP were not affected compared to the control virus ZD55-EGFP with wild-type fiber. ZD55-F-HI-sPD-1-EGFP increased viral infectivity, gene transduction efficiency and antitumoral efficacy, in comparison to control oncolytic adenovirus, ZD55-EGFP, in PD-L1-positive cancer cells. This is the first report to demonstrate sPD-1 epitope incorporated oncolytic Ad has expanded tropism that can improve viral infection and gene transduction efficiency in PD-L1-positive cancer cells and strengthened its therapeutic applications.

#PDL1, #OncolyticAdenovirus, #GeneDelivery, #AntiTumor, #CancerImmunotherapy, #PrecisionOncology, #ImmuneResponse, #AdenovirusEngineering, #TumorTargeting, #GeneTherapy, #CancerTreatment, #ViralTropism, #OncolyticVirus, #ImmuneCheckpoint, #PDL1Expression, #TumorLysis, #TargetedTherapy, #AdenoviralVectors, #TherapeuticGenes, #Oncology

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