Transductional Targeting For Conditionally Replicating Adenoviruses

Although nonreplicating first-generation Ad vectors have provided high in vitro and in vivo transduction rates and good safety data, clinical cancer trials have suggested that the single-agent antitumor effect may not be sufficient for all treatment approaches (18). Viruses that replicate and spread specifically inside the tumor have been suggested as a way to improve tumor penetration with an additional benefit of local amplification of effect. To this end, conditionally replicating adenoviruses (CRAds) have been explored. These viruses are genetically modified to take advantage of tumor-specific changes that allow preferential replication of the virus in target cells (19,90-94). The viral replication cycle causes oncolysis of the cell, resulting in the release of newly generated virions and subsequent infection of neighboring cells. Thus, the antitumor effect is not delivered with a transgene but by replication of the virus per se. In theory, the oncolytic process continues as long as target cells for the virus persist. There are two main ways to control viral replication. One method is the control of replication regulators, such as the viral early genes, with TSPs. The other method involves introduction of deletions in the viral genome that require specific cellular factors to compensate the effects of these deletions. Further, both approaches can be combined with the potential for increased specificity. Therefore, CRAds are by definition transcription-ally targeted agents.

Various promoters have been used to control viral replication (95-101), reviewed in (19). Typically, the TSP is placed to control expression of E1A, the crucial regulator of Ad replication, sometimes combined with other genes such as E1B or E4. An interesting concept is targeting CRAds to tumor vasculature (102). However, this strategy is more challenging to study preclinically, as animal models are unavailable -endothelial cells derive from the host in xenograft systems and murine cells do not support replication of human Ads. To further increase the oncolytic effect, transgenes for cyto-kines or prodrug-activating enzymes have been included in CRAds (103,104). This approach may also allow noninvasive imaging and abrogation of virus replication in case of toxicity.

Heretofore, two approaches have been used for creation of deletion-type CRAds. The first was 0NYX-015 (initially reported as dl1520), which has two mutations in the gene coding for the E1B 55-kd protein (105,106). The purpose of this protein is binding and inactivation of p53 in infected cells, for induction of S-phase, which is required for virus replication. Thus, this virus should only replicate in cells with an aberrant p53-p14ARF pathway, a common feature in human tumors (107). Although this is still subject of debate, initial studies suggested that this agent replicates more effectively in tumor than in normal cells (108-111). Unfortunately, the function of E1B55kD is not limited to p53 binding, which causes inefficient replication compared with wild-type adeno-virus (105,106,112).

The second group of deletion-mutant CRAds have a 24-bp deletion in the constant region (CR) 2 of E1A (113,114). This domain of the E1A protein is responsible for binding the retinoblastoma tumor suppressor/cell cycle regulator protein (Rb), thereby allowing Ad to induce S-phase entry. Therefore, viruses with this type of deletion have reduced ability to overcome the G1-S checkpoint and replicate efficiently only in cells where this interaction is not necessary (e.g., tumor cells defective in the Rb-p16 pathway). Appropriately, this pathway seems to be inactive in most all human tumors (115). It has been shown that replication of CR2-deleted viruses is attenuated in nonproliferating normal cells (113,114). Importantly, abrogation of replication was also demonstrated when Rb was reintroduced into otherwise permissive cells (113). Ads with mutations in both CR 1 and 2 of E1A have also been found to replicate selectively in tumor cells, although increase in selectivity in comparison to just CR2-deleted CRAds has not yet been demonstrated (116-119).

Like Ad vectors, most published CRAds rely on CAR for entry into cells. Unfortunately, CAR levels are variable in many types of clinical cancers (10-13,31-36) (14,15,17). Nevertheless, even CRAds with wild-type tropism have shown evidence of clinical utility (120). These initial successes suggest that if efficiency of infection and specificity of replication of these agents could be enhanced, further improvements in clinical efficacy could be gained. This is corroborated by demonstration of the close association between infectivity and oncolytic potency (54,121,122). Consequently, infectivity-enhanced CRAds have been constructed, with impressive preclinical efficacy. Ad5-A24RGD features an RGD-4C modification of the fiber (123), and displays similar oncolytic potency to wild-type virus in ovarian cancer cells (124). Further, this virus is able to replicate in ovarian cancer primary cell spheroids and results in significantly prolonged survival in an aggressive orthotopic ovarian cancer model (124).

A A24-based agent featuring the serotype 3 knob (Ad5/3-A24) was created (125). This agent demonstrated dramatic antitumor efficacy in ovarian cancer cell lines, primary tumor specimens, and orthotopic animal models of ovarian cancer. The first TSP-controlled infectivity-enhanced CRAd has recently been constructed and tested on ovarian and pancreatic cancer substrates [Masato Yamamoto, submitted; (126)]. Rep-licative specificity was achieved with a Cox-2 promoter controlling expression of E1A, whereas the fiber was modified with RGD-4C.

A major problem in assessing CRAd efficacy and safety preclinically, is the lack of an appropriate animal model. Human serotype Ads or CRAds do not replicate productively in commonly used animal models. Therefore, meaningful safety data are difficult to obtain, and efficacy data may be skewed due to deficient immune responses in xenograft models. Further, evaluation of host-virus interactions and their modulation has not been possible. This problem could be partially alleviated if a syngeneic CRAds could be developed for existing animal models of cancer (129).

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