Clinical Immunogenicity of rHuPH20, a Hyaluronidase Enabling Subcutaneous Drug Administration.

Recombinant human PH20 hyaluronidase (rHuPH20) is used to facilitate dispersion of subcutaneously delivered fluids and drugs. This report summarizes rHuPH20 immunogenicity findings from clinical trials where rHuPH20 was co-administered with SC human immunoglobulin, trastuzumab, rituximab, or insulin. Plasma samples were obtained from evaluable subjects participating in ten different clinical trials as well as from healthy plasma donors. A bridging immunoassay and a modified hyaluronidase activity assay were used to determine rHuPH20-reactive antibody titers and neutralizing antibodies, respectively. rHuPH20-binding antibody populations from selected subjects with positive titers were affinity-purified and subjected to further characterization such as cross-reactivity with endogenous PH20. Among individual trials, the prevalence of pre-existing rHuPH20-reactive antibodies varied between 3 and 12%, excepting the primary immunodeficiency (PID) studies. Incidence of treatment-induced rHuPH20 antibodies was 2 to 18%, with the highest titers (81,920) observed in PID. No neutralizing antibodies were observed. Within most trials, the kinetics of antibody responses were comparable between pre-existing and treatment-induced antibody responses, although responses classified as persistent were more common in subjects with pre-existing titers. There was no association between antibody positivity and either local or systemic adverse events. Pre-existing and treatment-induced antibody populations were of similar immunoglobulin isotypes and cross-reacted to endogenous PH20 to similar extents. No cross-reactivity to PH20 paralogs was detected. rHuPH20 induces only modest immunogenicity which has no association with adverse events. In addition, antibodies purified from baseline-positive individuals are qualitatively similar to those purified from individuals developing rHuPH20-reactive antibodies following exposure to the enzyme.

Recombinant adenovirus-p21 attenuates proliferative responses associated with excessive scarring.

Excessive cutaneous scarring is an important clinical disorder resulting in adverse tissue growth and function as well as undesirable cosmetic appearance. p21WAF-1/Cip-1 is a cyclin-dependent kinase inhibitor that blocks cell cycle progression and inhibits cell proliferation. We used a recombinant adenovirus containing the human p21WAF-1/Cip-1 cDNA (rAd-p21) to evaluate proliferative responses in skin models. In vitro dose-response studies using primary human dermal fibroblasts resulted in a dose-dependent expression of p21WAF-1/Cip-1 protein and a 3- to 80-fold reduction in cell proliferation as measured by 5-bromodeoxyuridine incorporation. Further, rAd-p21 reduced type I procollagen production when compared to control virus. A rat polyvinyl alcohol sponge model was used to determine rAd-p21 effects on granulation tissue formation in vivo. Sponges pretreated with a granulation tissue stimulator, rAd-PDGF-B and subsequently rAd-p21 on a second injection, showed a p21WAF-1/Cip-1 specific dose-dependent decrease in percent granulation fill as the rAd-p21 dose increased (p < 0.001). Immunohistochemistry identified human p21WAF-1/Cip-1 expression in sponges treated with rAd-p21 5 days postinjection. Additionally, 5-bromodeoxyuridine and Ki67 staining in sponges treated with rAd-p21 showed a significant decrease in proliferation when compared to rAd-platelet-derived growth factor-B alone or vehicle control groups (p < 0.01). These data support the utility of p21WAF-1/Cip-1 in targeting hyperproliferative disorders of the skin.

Characterization of hemodynamic events following intravascular infusion of recombinant adenovirus reveals possible solutions for mitigating cardiovascular responses.

Intravascular administration of recombinant adenovirus (rAd) in cancer patients has been well tolerated. However, dose-limiting hemodynamic responses associated with suppression of cardiac output have been observed at doses of 7.5 x 10(13) particles. While analysis of hemodynamic responses induced by small-molecule pharmaceuticals is well established, little is known about the cardiovascular effects of rAd. Telemetric cardiovascular (CV) monitoring in mice was utilized to measure hemodynamic events following intravascular rAd administration. Electrocardiogram analysis revealed a block in the SA node 3-4 min postinfusion, resulting in secondary pacemaking initiated at the AV node. This was associated with acute bradycardia, reduced blood pressure, and hypothermia followed by gradual recovery. Adenovirus-primed murine sera with high neutralizing antibody (nAb) titers could inhibit CV responses, whereas human sera with equivalent nAb titers induced by natural infection were, surprisingly, not inhibitory. Interestingly, repeat dosing within 2-4 h of the primary injection resulted in desensitization, resembling tachyphylaxis, for subsequent CV responses. Last, depletion of Kupffer cells prior to rAd infusion precluded induction of CV responses. These inhibitory effects suggest that rAd interactions with certain cells of the reticular endothelial system are associated with induction of CV responses. Significantly, these studies may provide insight into management of acute adverse effects following rAd systemic delivery, enabling a broadening of therapeutic index.

Efficacy of adenoviral p53 delivery with SCH58500 in the intracranial 9l and RG2 models.

Malignant gliomas remain incurable entities that provide fertile ground for experimental therapy. The observation that impaired p53 expression is present in a proportion of these tumors suggests that reconstitution of this ability may impart some degree of tumor control. In this investigation, the 9L and RG2 intracranial rodent tumor models are utilized to assess SCH58500, an adenoviral p53 delivery system. The RG2 tumors demonstrate a greater propensity for transfection with this vector in vitro than the 9L tumors. In vivo, little tumor transfection beyond the immediate area of the needle tract used for direct SCH58500 injection was observed in either tumor type. Intracarotid injection resulted in no tumor transfection. Even at high concentrations of SCH58500 or control virus, injections resulted in no apparent toxicity in terms of weight gain, eating habits or activity in normal animals. The intratumoral administration of SCH58500 enhances the survival of animals with established 9L tumors. Both SCH58500 and its control viral construct not containing the p53 gene enhance survival in animals with RG2 tumors. None of the injected viral constructs caused an alteration in the markers used to detect the character of the white cell infiltrate in either of these tumors. Utilization of SCH58500 provides measurable efficacy in these preclinical brain tumor models without significant toxicity.

Assessment of p53 gene transfer and biological activities in a clinical study of adenovirus-p53 gene therapy for recurrent ovarian cancer.

A cohort study was designed to evaluate the efficiency of gene transfer and whether biological activity from the expressed therapeutic gene resulted after administration of a recombinant adenovirus containing the human wild-type p53 (p53(wt)) gene (rAd-p53 SCH 58500). The cohort study was conducted in five trial subjects with recurrent ovarian cancer. Each trial subject received multiple cycles of rAd-p53 SCH 58500, each cycle comprised of doses of 7.5 x 10(13) particles on each of five consecutive days. Subjects were treated with rAd-p53 SCH 58500 alone during Cycle 1 and in combination with gemcitabine during the subsequent cycles. Both tumor biopsies and peritoneal aspirates were collected and evaluated for gene transfer and evidence of the biological activities of the expressed p53(wt) gene. Using quantitative PCR and RT-PCR, and in situ PCR, gene transfer and expression were documented in tumor biopsies (four of five patients) collected from Cycle 1. Furthermore, upregulation of p21/WAF1, bax and mdm-2, and downregulation of survivin were observed in these same tumor biopsy samples, suggesting that intraperitoneal administration of rAd-p53 SCH 58500 leads to detectable p53 biological activity in target tumor tissue. In addition, gene transfer and its expression were observed in cells obtained from peritoneal aspirates. These fluids were mainly comprised of polymorphonuclear neutrophils, indicating that successful gene transfer can be achieved by multiple cycle intraperitoneal administration of recombinant adenovirus.