Retargeted oncolytic adenovirus displaying a single variable domain of camelid heavy-chain-only antibody in a fiber protein

摘要

Conditionally replicative adenoviruses are promising agents for oncolytic virotherapy. Various approaches have been attempted to retarget adenoviruses to tumor-specific antigens to circumvent deficiency of receptor for adenoviral binding and to provide an additional level of tumor specificity. Functional incorporation of highly specific targeting molecules into the viral capsid can potentially retarget adenoviral infection. However, conventional antibodies are not compatible with the cytoplasmic adenovirus capsid synthesis. The goal of this study was to evaluate the utility of single variable domains derived from heavy chain camelid antibodies for retargeting of adenovirus infection. We have combined transcriptional targeting using a tumor-specific promoter with transductional targeting through viral capsid incorporation of antihuman carcinoembryonic antigen single variable domains. Obtained data demonstrated that employment of a single variable domain genetically incorporated into an adenovirus fiber increased specificity of infection and efficacy of replication of single variable domain-targeted oncolytic adenovirus. The double targeting, both transcriptional through the C-X-C chemokine receptor type 4 promoter and transductional using the single variable domain, is a promising means to improve the therapeutic index for these advanced generation conditionally replicative adenoviruses. A successful strategy to transductional retargeting of oncolytic adenovirus infection has not been shown before and therefore we believe this is the first employment of transductional targeting using single variable domains derived from heavy chain camelid antibodies to enhance specificity of conditionally replicative adenoviruses. prs.rt("abs_end"); Introduction A growing body of evidence demonstrates the promise of gene therapy and oncolytic virotherapy in preclinical and clinical studies. Among the oncolytic viruses, adenoviruses were the first and most frequently used vectors in clinical trials. 1 Recently, the first oncolytic adenovirus H101 has been approved for the treatment of head and neck carcinoma in combination with chemotherapy. 2 In most trials with adenovirus vectors, only mild adverse events were seen and no dose-limiting toxicity was reported. However, the antitumor efficacy of these Ad vectors was only modest, therefore, improvement of the efficacy of the treatment is needed. 3 Oncolytic conditionally replicative adenoviruses (CRAds) are novel therapeutic agents for targeted-therapy based on the cytolytic effect of replicating viruses which results in tumor cell death. Replicative specificity of the virus is the basis for the targeted action of CRAds and results in tumor-specific cytotoxicity. This tumor selectivity is established via the use of tumor specific promoters (TSPs) to achieve conditional replication. In this regard, TSPs restrict viral replication by replacing the native viral E1a promoter. The use of TSPs in CRAd designs has therefore represented the principle means to limit viral replication to tumor cells. Despite controlled replication via the above mentioned TSP approach, off-target infection and replication may provide the basis for limiting toxicity. 4 Thus, additional levels of control of virus infection and replication are therefore desirable. Another strategy to achieve CRAd specificity is controlling adenovirus infection at the level of target cell attachment. Transductional targeting is the strategy of altering viral binding to achieve target cell specific binding. 5 Importantly, such an approach could potentially synergize with transcriptional targeting. In this regard, adenoviral tropism is dictated by the interaction of the adenoviral fiber with the native receptor—coxsackievirus and adenovirus receptor (CAR). 6 Various approaches have been attempted to retarget adenovirus serotype 5 (Ad5) towards cancer cells, including the use of adaptor molecules and genetic modifications of the adenovirus capsid. 7 , 8 Although the adapter approach has proven the feasibility of retargeting adenovirus vectors, a “single-unit” molecule is preferred for the purpose of therapeutic use. Thus, genetic modification of the fiber potentially provides a way to redirect the adenovirus to an alternative cellular receptor. The goal of this study was to design, develop, and characterize a clinically relevant oncolytic virotherapy agent. A central facet of our approach is to confer the CRAd capacity to specifically infect tumor-associated targets in a CAR-independent manner. The camelid family heavy-chain-only antibodies possess ideal characteristics for a CRAd retargeting strategy including cytosolic stability to allow functional incorporation into the CRAd capsid as well as compatibility with phage biopanning selection to allow tumor cell specificity. 9 In addition, engineered single domain antibo