Clinical Development of CV706 and CV787

CV706 and CV787 are novel therapeutic agents with a novel mechanism of action. As of this writing, a phase I/II clinical trial of CV706 for recurrent local prostate cancer has been completed and CV787 has entered a multicenter Phase I/II clinical trial for metastatic, hormonal refractory prostate cancer. A brief summary for CV706 trial result and factors impacting clinical efficacy and safety are discussed.

A. CV706 Phase I/II Trial for Locally Recurrent Prostate Cancer

A Phase I trial of CV706 was initiated in 1998 at the Brady Urological Institute of the Johns Hopkins Oncology Center under the direction of Jonathan Simons, MD, and Ted DeWeese, MD. The patient population consists of men with locally recurrent prostate cancer with rising PSA levels following definitive external beam irradiation. Men in this category are usually left untreated or receive androgen ablation therapy as serum PSA levels rise significantly above 10 ng/mL. On average, these men have a life expectancy of 3 years. The virus was administered under spinal anesthesia using the brachytherapy template and ultrasound 3D imaging using the MMS Terapac Plus 6.6 B3DTUI (Charlottesville, VA) treatment-planning software for implantation of radioactive seeds. Virus was initially administered with 0.1 mL aliquots from up to 40 brachytherapy needles. PSA levels were determined and biopsies obtained.

Systemic toxicity was minimal and limited to brief Grade 1 fever with or without an associated chill. These episodes were self-limited, responded to routine anti-pyretics, and no patient required antibiotics. This phenomenon is consistent with previously reported series using intratumoral injections of replication-competent adenovirus (Onyx-O15) [144, 145] and likely represents cytokine release (e.g., IL-1, IL-6, TNF-a) in response to the adenovirus [146, 147]. A transient non-clinically significant lymphopenia confined to the normal range was noted in a majority of patients (95% ) within 24 h of viral instillation. Full recovery of cell counts occurred within 4 to 7 days posttreatment. As with the transient fever, the timing of this decrement combined with the quick recovery are consistent with an acute-phase reaction mediated by transient cytokine release, as occurs with a variety of agents including bisphosphonates, and are not consistent with viral induced bone marrow suppression [148, 149]. Importantly, treatment with CV706 was not associated with significant hepatic or coagulation abnormalities. No patient experienced >Grade 2 elevation of liver transaminase levels and no patient had evidence of alteration in PT or PTT or a decrement in fibrinogen. This safety was evident even at the highest dose level of 1 x 1013 viral particles and with viral shedding into the blood as documented in this study. Taken together, these data reveal a high degree of safety and tolerability of CV706 when administered by intraprostatic injection.

The analysis of secondary study end points provided compelling evidence of CV706 activity. Serum PSA is well known to be a marker of both disease activity as well as disease burden and the use of serum PSA as a marker of therapeutic efficacy has become increasingly well defined. Several investigators have correlated declines in serum PSA of greater than 50% with prolongation of survival in men with hormone refractory prostate cancer [150, 151]. In addition, other investigators have found that a slowly rising PSA following definitive management with radiation or surgery is associated with an increased time to clinically evident metastatic disease when compared to patients with a more rapid PSA doubling time [152, 153],

Moreover, there was a statistically significant reduction in the PSA velocity following treatment with CV706, most pronounced for patients in dose levels 4 and 5, again suggestive of a dose-response relationship. In the final two dose levels, 50% of treated patients achieved a PSA partial remission (PR). It also appears that treatment with CV706 resulted in a prolongation of the time required for the serum PSA to double, suggesting a slowing of cancer growth within the prostate even among individuals not achieving a PR as defined by the protocol. It is well known that biopsy of the prostate results in significant elevations in serum PSA for 2 weeks [154, 155]. It is likely that while the design of this study, with frequent posttreatment biopsies, aided in the documentation of viral replication, these same invasive procedures prevented a full analysis of the PSA response to therapy with CV706. Thus, it is possible that significant reductions in serum PSA could have been obscured by these frequent prostatic manipulations. Despite this possibility, the evidence gathered on PSA levels following treatment with CV706 are encouraging and suggest that at the higher dose levels, a clinically meaningful treatment effect may be achievable.

This treatment effect at higher doses is associated with histologic evidence of viral replication. The viral inclusions seen on electron microscopy are consistent with viral replication in prostate epithelial cells. The positive staining for hexon protein seen on immuohistochemistry from day 4 biopsy materials is also confined to prostatic epithelial cells, is greatest in the highest dose levels, and, like the electron microscopy, is highly suggestive of intraprostatic replication of CV706 in these patients. Therefore, there is appropriate rationale for optimism given these findings in men treated with CV706, particularly at the high dose level. We believe these data to be significant and warrant CV706 evaluation in a Phase II study.

Importantly, we were able to rigorously document viral circulation in the blood following intraprostatic delivery of CV706 without significant associated clinical sequale. The quantitative PCR assay is very specific for CV706 and is capable of detecting 1300 copies per milliliter of plasma. These results confirm that a small but significant amount of the intraprostatically administered virus reached the circulation. The amount of virus released in the first "peak" varied between patients and did not appear to be related to the dose level or neutralizing antibody titer. The highest total amount of virus detected was in two patients (patients 12 and 14) with an estimate of less than 2% of the dose being detected. Circulating virus then became undetectable analogous to a virus "eclipse." A significant secondary "peak" of circulating CV706 was observed in most patients within 3 days of treatment, suggestive of viral replication in these patients. The appearance and size of the secondary "peak" seemed to correlate best inversely with the anti-Ad5 antibody titer at the time of treatment. These data are consistent with those derived from electron microscopy and immunohistochemistry, and are highly suggestive of CV706 replication in the human prostate.

Response of PSA to CV706 delivered directly into the prostate was not correlated with the presence of preexisting Ad5 neutralizing antibodies. As expected, following CV706 administration, most patients developed Ad5-neutralizing antibodies. However, development of neutralizing antibodies failed to correlate with response to treatment. Moreover, our data also reveal that the presence of preexisting anti-Ad5 antibodies was not correlated with treatment-related toxicity. These data extend the previously reported work on intratumoral delivery of adenovirus by revealing a lack of association between preexisting neutralizing antibodies and treatment efficacy and toxicity [118, 133],

While circulating anti-Ad5 antibody may significantly impact on the efficacy and toxicity of systemically administered adenovirus [75, 86], it is not clear that these antibodies have the same access to the tumor-bearing prostate and thus may have a limited impact on direct intratumoral injections [156].

In summary, these data reveal that CV706 is safe and not associated with irreversible serious short- or long-term side effects when delivered by intratumoral injection using a planned, stereotactic approach. These data also suggest that CV706 replicates selectively in prostatic epithelial cells, i.e., those prostate cells that make PSA, and does so in a time frame consistent with an adenovirus replication cycle. These data suggest that CV706 has significant biologic activity as evidenced by significant durable dose-related decreases in patient serum PSA. Indeed, three of five men treated with 1 x 1013 particles of CV706 experienced a PSA partial response. Thus, CV706 delivered by brachytherapy is an excellent candidate for the treatment of organ-confined prostate cancer.

B. Factors Impacting Clinical Efficacy and Safety

The pathogenesis of adenoviral infections is influenced by a large number of factors, some pertaining to the virus and others pertaining to the host defenses of the virus. Important issues for the virus include the route of infection, the size of the virus inoculum, the tropism of the virus for different-cell types, and whether the virus spreads directly from cell to cell or through extracellular fluid. Clearly, the vascularization of tumors, the leakiness of capillaries to virus, and the physical size of virus particle will affect intratumoral virus distribution. In the replication efficiency of the virus in prostate tumor cells, both the time of the replication cycle and the burst size are also important. Host defenses include mechanical defenses (epithelia, mucosal, liver Kupffer cells, or the blood-brain barrier), nonspecific immune defenses (interferons, recognition of infected cells by natural killer cells, release of cytokines, macrophage recruitment and activation, and triggering of complement and kinin cascades), and specific immune defenses (humoral immunity, mostly IgM and IgG but also IgA, IgD, and IgE, and finally cell-mediated immunity) [152],

In adenovirus-mediated prostate cancer therapy, the virus can be either injected directly into the tumor or administered by intravenous injection. In either case, the dose of virus is massive (10n-1015 particles) compared to natural, vaccine-induced adenovirus infections (10°-106 particles) [153, 157, 158], or the clinical trials with wild-type adenovirus (107-109 particles) [14], Very little is known about the human host response to large doses of adenoviruses [14, 159] and nothing is known about the human host response to using the intravenous route of administration of large doses of replicating adenoviruses. Liver toxicity of virion proteins may be limiting at these high doses.

Therapeutic antibody studies have indicated that antibodies do not effectively penetrate the core of a solid tumor; extravasation is limited to the tumor periphery. This suggests that the accessibility of replicating virus to antibody binding should be minimal following direct intratumoral injection [98, 131]. Cell-mediated immunity directed toward infected tumor cells may actually enhance the efficacy of replicating viruses in cancer patients if enough replication and spread occur initially. However, a systemically delivered replicating adenovirus is going to face several potential hurdles: (1) the nonspecific removal of adenovirus by liver Kupffer cells, (2) the inactivation of virus by preexisting circulating antibodies to adenovirus, (3) a limitation of viral replication mediated by a vigorous CTL response to virally infected cells, and (4) a limitation of the efficacy of repeat dosage by primary or secondary induction of humoral immunity.

Incorporation of the Ad5 genome into germ cells has been expressed as a concern but has not been found for any of the Ad5 gene therapy constructs. Indeed, adenovirus gene expression is characterized as transient in nature due to a lack of viral DNA integration. Virus shedding has been expressed as a concern but it has not been detected in any Ad5 clinical trial to date. In our clinical trial, virus replication was detected after 2-8 days but was undetectable after 2 weeks. It is difficult to estimate the increased cytolytic activity in humans of CV787 compared to CV706. However, replicating adenoviruses containing hepatitis B surface antigen (HbsAg), with and without the E3 region, have been tested in chimpanzees, a system permissive for infection by human adenoviruses [160]. In this study, the addition of the E3 region resulted in 10-to 100-fold increase in virus shedding and a 10- to 100-fold increase in titer to HBsAg. However, one should not lose sight of the fact that adenoviruses are ubiquitous. Twenty-three percent of normal healthy infants are seropositive for adenoviruses by 7 months of age [95] and CV787 is attenuated 10,000:1 compared to the wild-type virus. We believe the therapeutic use of CV787 will be safe; the major question is whether or not there is sufficient efficacy to be medically useful.

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