*4.1. Tracking Viral Replication and Spread*

Initially, reporter genes were inserted for tracking of MeV replication. Carcinoembryonic antigen (CEA) and β-human chorionic gonadotropin (HCG) were selected, which can be measured in routine clinical laboratory testing [12]. Encoding the sodium iodide symporter, NIS, yielding MV-NIS, allowed for γ-camera imaging of iodine-123 (123I) or 99mtechnetium uptake and also radiotherapy with <sup>131</sup>I [66]. In later studies, MV-NIS was used for advanced imaging techniques, such as pinhole micro-single photon emission computed tomography/computed tomography (SPECT/CT) [67] and contrast-enhanced CT [68]. Recently, a recombinant MeV variant encoding a fluorescent reporter gene was used for intravital imaging of viral spread at single-cell resolution by two-photon microscopy [69].

Data from preclinical studies with MV-NIS have also been used to develop mathematical models of oncolytic virotherapy and its combinations. This has been devised as a means to rationalize testing of distinct dosing and scheduling regimens [70–73].

**Figure 3.** Engineering of oncolytic measles virus. Top: Targeting for increased tumor specificity can be achieved on the entry or post-entry level. For entry targeting, the viral attachment protein H can be mutated to ablate natural tropism and redirected by fusing targeting moieties such as antibody single-chain variable fragments to H (red). Post-entry targeting is achieved via target sites for microRNAs (gray) introduced into viral genes which are differentially expressed in malignant compared to healthy tissues, leading to degradation of the respective viral RNAs in normal cells. Middle: Viruses equipped with reporter genes encoding, e.g.**,** fluorescent proteins (light green) can be used to track viral spread. To increase therapeutic efficacy, viruses can be armed with additional genes encoding radiosensitizers, prodrug convertases, or immunomodulators. Bottom: Shielding against neutralizing antibodies can be achieved by exchanging the viral envelope proteins for the counterparts from a related virus. **Figure 3.** Engineering of oncolytic measles virus. Top: Targeting for increased tumor specificity can be achieved on the entry or post-entry level. For entry targeting, the viral attachment protein H can be mutated to ablate natural tropism and redirected by fusing targeting moieties such as antibody single-chain variable fragments to H (red). Post-entry targeting is achieved via target sites for microRNAs (gray) introduced into viral genes which are differentially expressed in malignant compared to healthy tissues, leading to degradation of the respective viral RNAs in normal cells. Middle: Viruses equipped with reporter genes encoding, e.g., fluorescent proteins (light green) can be used to track viral spread. To increase therapeutic efficacy, viruses can be armed with additional genes encoding radiosensitizers, prodrug convertases, or immunomodulators. Bottom: Shielding against neutralizing antibodies can be achieved by exchanging the viral envelope proteins for the counterparts from a related virus.

*4.1. Tracking Viral Replication and Spread*  Initially, reporter genes were inserted for tracking of MeV replication. Carcinoembryonic antigen (CEA) and β-human chorionic gonadotropin (HCG) were selected, which can be measured in routine clinical laboratory testing [12]. Encoding the sodium iodide symporter, NIS, yielding MV-NIS, allowed for γ-camera imaging of iodine-123 (123I) or 99m-technetium uptake and also radiotherapy with 131I [66]. In later studies, MV-NIS was used for advanced imaging techniques, such as pinhole micro-single photon emission Valuable information was gained by employing viruses with reporter genes in clinical trials. After intraperitoneal administration of MV-CEA, dose-dependent increases in CEA levels were measured in peritoneal fluid and serum [74]. After intraperitoneal administration of MV-NIS, <sup>123</sup>I SPECT/CT scans were positive in three of 13 ovarian cancer patients, confirming viral gene expression at the tumor site. Scans were positive in eight of 31 multiple myeloma patients receiving MV-NIS i.v. [75]. In both studies, positive scans were associated with higher virus doses.

computed tomography/computed tomography (SPECT/CT) [67] and contrast-enhanced

#### CT [68]. Recently, a recombinant MeV variant encoding a fluorescent reporter gene was *4.2. Retargeting MeV*

used for intravital imaging of viral spread at single-cell resolution by two-photon microscopy [69]. Data from preclinical studies with MV-NIS have also been used to develop mathematical models of oncolytic virotherapy and its combinations. This has been devised as a means to rationalize testing of distinct dosing and scheduling regimens [70–73]. Valuable information was gained by employing viruses with reporter genes in clinical trials. After intraperitoneal administration of MV-CEA, dose-dependent increases in Virus engineering has not only enabled tracking viral spread, but also modifying its tropism to increase tumor specificity. Retargeting of MeV was accomplished by mutating the intrinsic receptor binding sites and fusing antibody single-chain variable fragments (scFv) to the C-terminus of the viral hemagglutinin [76]. Using this strategy, oncolytic MeV were targeted to the myeloma surface antigen CD38 [13], to CD20 for targeting of B cell malignancies [77], folate receptor (FR)-α for treatment of ovarian cancer [78], and EGFRvIII expressed in glioblastoma [79], among others (reviewed in [18]). A range of different

targeting moieties beyond scFv has been employed, such as the cytokine interleukin (IL)- 13 [80] or the urokinase plasminogen activator [81] for direction of viral tropism to their respective receptors. Successful targeting has also been achieved using integrin-binding peptides [82], DARPins [83] and cystine knot proteins [84]. Viral tropism can be redirected to specific cell populations within the tumor, including tumor-initiating cells [85], the tumor stroma [86], and vasculature [81].

A sophisticated means of viral entry targeting employs proteases expressed within the tumor microenvironment. The MeV fusion protein encompasses a furin cleavage site and requires proteolytic processing for activity. Replacing the furin cleavage site with sequences recognized by matrix metalloproteinases or the urokinase-type plasminogen activator can increase tumor specificity [87,88].

Tumor targeting on the post-entry level was achieved using microRNA target sites inserted into the untranslated regions (UTRs) of viral genes [89]. This concept exploits downregulation of specific microRNAs in malignant vs. benign cells, leading to virus restriction in healthy tissue while spread within tumor tissue is unimpaired.

Proof-of-concept was also obtained for using riboswitches to control oncolytic MeV. Insertion of a ligand-activated ribozyme into the UTR of the MeV fusion gene enabled regulation of MeV infectivity and spread by addition of the cognate small molecule [90]. Recently, a photocontrollable MeV variant was reported which harbors a split L protein for control of viral replication by blue light illumination [91].
