؉ T lymphocytes are a mainstay of antitumoral immune responses, PTVs could be engineered for the transfer of specific tumor antigens provoking tailored antitumoral immunity. Therefore, PTVs can be used as safe and efficient alternatives to gene transfer vectors or live attenuated replicating vector platforms, avoiding genotoxicity or general toxicity in highly immunocompromised patients, respectively. Thereby, the potential for easy envelope exchange allows the circumventing of neutralizing antibodies, e.g., during repeated boost immunizations. V accination is the administration of one or more immunogens, the vaccine, into patients to trigger antigen-specific adaptive immune responses to prevent (prophylactic vaccination) or to treat (therapeutic vaccination) disease. Vaccines can be classified into several subtypes. Among these are live attenuated replicating vaccines, inactivated vaccines, subunit vaccines, DNA vaccines, or recombinant vector vaccines (for review, see reference 1). Wellknown live attenuated vaccines are, e.g., those against measles (2) or mumps (3). These vaccines replicate but have been attenuated to become apathogenic. The immune responses triggered by live attenuated vaccines are similar to those induced by the pathogenic form of the microbe (4, 5) and involve both the cellular and humoral arms of the immune system. However, attenuated replicating pathogens may carry an inherent risk of reversion to the parental virulent form by in vivo passaging during vaccination, as observed for the Sabin strain used as a polio vaccine (6), or may still be pathogenic in highly immunocompromised patients (7), depending on the respective degree of attenuation of the vaccine strains. On the other hand, inoculation of solely proteinaceous antigens (such as the hepatitis B virus vaccine [8]) or antigenencoding genes (as a DNA vaccine) is regarded as safe but relatively inefficient (9).As an alternative to such vaccines, the genes encoding an antigen can be transferred into cells and thereby presented to the immune system by using recombinant vaccine vectors. For that purpose, an attenuated vector is utilized as a carrier for the antigen-encoding sequences of another pathogen. Thereby, they are

To target oncolytic measles viruses (MV) to tumors, we exploit the binding specificity of designed ankyrin repeat proteins (DARPins). These DARPin-MVs have high tumor selectivity while maintaining excellent oncolytic potency. Stability, small size, and efficacy of DARPins allowed the generation of MVs simultaneously targeted to tumor marker HER2/neu and cancer stem cell (CSC) marker EpCAM. For optimization, the linker connecting both DARPins was varied in flexibility and length. Flexibility had no impact on fusion helper activity whereas length had. MVs with bispecific MV-H are genetically stable and revealed the desired double-target specificity. In vitro, the cytolytic activity of bispecific MVs was superior or comparable to mono-targeted viruses depending on the target cells. In vivo, therapeutic efficacy of the bispecific viruses was validated in an orthotopic ovarian carcinoma model revealing an effective reduction of tumor mass. Finally, the power of bispecific targeting was demonstrated on cocultures of different tumor cells thereby mimicking tumor heterogeneity in vitro, more closely reflecting real tumors. Here, bispecific excelled monospecific viruses in efficacy. DARPin-based targeting domains thus allow the generation of efficacious oncolytic viruses with double specificity, with the potential to handle intratumoral variation of antigen expression and to simultaneously target CSCs and the bulk tumor mass.

Measles virus (MeV) is naturally cytolytic by extensive cell-to-cell fusion. Vaccine-derived MeV is toxic for cancer cells and is clinically tested as oncolytic virus. To combine the potential of MeV with enhanced safety, different targeting strategies have been described. We generated a receptor-targeted MeV by using receptor-blind viral attachment protein genetically fused to designed ankyrin repeat protein (DARPin) binding domains specific for the epidermal growth factor receptor (EGFR). To reduce on-target toxicity for EGFR+ healthy cells, we used an engineered viral fusion protein activatable by tumor-associated matrix metalloproteases (MMPs) for additional protease targeting. The dual-targeted virus replicated exclusively on EGFR+/MMP+ tumor cells but was safe on healthy EGFR+ target cells, primary human keratinocytes. Nevertheless, glioblastoma and other tumor cells were efficiently killed by all targeted viruses, although replication and oncolysis were slower for protease-targeted MeV. In vivo, efficacy of EGFR-targeted MeV was virtually unimpaired, whereas also dual-targeted MeV showed significant intra-tumoral spread and efficacy and could be armed with a prodrug convertase. The use of DARPin-domains resulted in potent EGFR-targeted MeV and for the first time effective dual retargeting of an oncolytic virus, further enhancing tumor selectivity. Together with powerful cell-toxic genes, the application as highly tumor-specific platform is promising.