Purpose: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal forms of cancer with a mortality rate of 76% at 1 year after diagnosis. Alternatively spliced Tissue Factor (asTF) is the secreted isoform of Tissue Factor. asTF drives the progression of various solid tumors including PDAC, by promoting tumor proliferation, angiogenesis, metastatic disease and recruitment of monocytes to tumor stroma. These effects occur in response to asTF’s interactions with β1 integrins. Our previous studies have shown that inhibition of asTF:β1 through the use of an asTF specific rabbit monoclonal antibody, RabMab1, suppresses cell migration in vitro and tumor growth in vivo when co-implanted with Pt45.P1 cells, a human PDAC cell line, in athymic nude mice. Based on these previous studies, targeting asTF is potentially of therapeutic benefit in PDAC. To this end, we generated a chimeric RabMab1 that possesses a human IgG1 constant domain, hereafter referred to as RabMab1 hIgG1. To facilitate pre-clinical studies employing a dosing regimen most likely to yield efficacious RabMab1 systemic exposure, and to derive a dose response relationship, we characterized anti-tumor activity and pharmacokinetic (PK) properties of RabMab1 hIgG1.
Methods: For co-implantation studies, RabMab1 hIgG1 (5 mg/kg in PBS) was incubated with 1x106 Pt45.P1 cells. The cell/antibody mixture was then injected into the pancreases of 6-week old athymic nude mice. Tumors were harvested after 45 days of growth. For PK studies, RabMab1 hIgG1 (6 mg/kg in PBS) was administered to mice (N=24, age 6-8 weeks; body weight, 18-20 g,) via tail vein injection. Terminal blood draws were performed over a course of 17 days post injection (2 mice per time point). Plasma was isolated and immediately frozen. To determine plasma concentration of RabMab1 hIgG1, an enzyme-linked immunosorbent assay (ELISA) specific for human IgG Fc fragment (Abcam ab195215) was used. For the ELISA, plasma was thawed slowly, diluted 1:10000, and RabMab1 hIgG1 concentration determined by interpolating a standard curve created using serial dilutions of RabMab1 hIgG1 in mouse plasma.
The resulting plasma concentration – time profile was analyzed using Phoenix WinNonlin6.2.1 to obtain PK parameters.
Results: Tumors arising from cells pretreated with PBS had an average weight of 412.1 mg (±149.4 mg), whereas those from cells pretreated with hIgG1 Fc fragment control had an average weight of 356.2 mg (±181.1 mg). RabMab1 hIgG1 pre-treatment reduced tumor growth significantly, leading to an average tumor weight of 197.8 mg (±108.4 mg). Graphical representation of these results is in Fig. 1.
The PK profile for RabMab1 IgG1 is shown in Figure 2. As typically observed for IgG antibodies, RabMab1 IgG1 exhibited a biphasic PK profile comprising of rapid distribution followed by a prolonged elimination phase. The PK data are summarized in Table 1.
Summary of major parameters obtained from the PK analysis of RabMab1 hIgG1 is as follows: plasma half-life for distribution of RabMab1 hIgG1 was 0.81 hr elimination of RabMab1 hIgG1 was 280 hr, Cmax was 101.65 µg/ml, the mean residence time (MRT0-∞) was 404 hr, and steady state volume of distribution (Vss) was 63 ml/kg. Plasma concentration profile of RabMab1 hIgG1 is in Fig. 2.
Conclusion: Data obtained from co-implantation studies demonstrate that RabMab1 hIgG1 suppresses the growth of human PDAC cells. Similar to currently FDA approved immunotherapeutics, RabMab1 hIgG1 has favorable PK properties including sustained systemic persistence (long plasma half-life/MRT), limited non-specific tissue distribution (low volume of distribution), and extensive systemic exposure reflected in Cmax and AUC values relative to effective drug concentrations derived in vitro for the parental RabMab1. We are currently designing comprehensive dose optimization studies to discern the maximal therapeutic activity of RabMab1 IgG1 in orthotopic models of PDAC.
Clayton Lewis– Postdoctoral Fellow, University of Cincinnati, Cincinnati, Ohio
Pankaj Desai– Professor, James L. WInkle College of Pharmacy, University of Cincinnati, Cincinnati, Ohio
Vladimir Bogdanov– Associate Professor , Director, Hemostasis Research Program, College of Medicine, University of Cincinnati, CIncinnati, Ohio