Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
A Flow Cytometric Receptor Occupancy Assay Demonstrates Dose-Dependent Blockade of B7RP-1 by AMG 557 on Circulating B Cells from SLE Subjects.
Sullivan1, Barbara A., Green2, Cherie L., Zhang1, Ming, Abbott1, Christina, Belouski1, Shelley, Thomas1, John, Gorski1, Kevin
Assessment of RO (receptor occupancy) is useful in drug development to confirm physical target coverage and support dose selection when combined with PK modeling. We describe a B7-related protein-1 (B7RP-1, B7h, ICOSL) FACS RO assay from initial experiments in SLE mouse models to validation and application to whole blood samples from subjects treated with a fully human IgG2 anti-B7RP-1 clinical candidate (AMG 557). B7RP-1 blockade in SLE is supported by the role of ICOS:B7RP-1 engagement in germinal center formation, B cell class switch recombination and antibody affinity maturation. Establishment of a correlation of B7RP-1 occupancy and efficacy with in vivo studies, including a murine lupus model, provided a rationale for incorporation of RO in an AMG 557 clinical trial.
Preclinical: NZB/W F1 mice were injected with 8, 1.5, or 0.3 mg/kg of 1B7-V2, 8 mg/kg mouse IgG1 isotype control or PBS once weekly for 23 weeks. RO was measured 3 months after starting treatment and just prior to the next dose; serum anti-dsDNA antibody levels were measured by ELISA as one measure of disease at 6 months. Clinical: Whole blood was collected from subjects in a Phase 1a single dose-escalating study of AMG 557. A four-color flow cytometric whole blood assay was constructed and validated to measure B7RP-1 on human circulating B cells. Antibody reagents directed against B7RP-1 were generated in-house according to standard procedures in mice and rats; all other antibodies were commercially available (Becton Dickinson, Inc, Immunotech S.A.).
B7RP-1 RO on circulating B cells correlated with efficacy in the NZB/NZW F1 mouse model of lupus. For the clinical assay, two monoclonal antibodies from two immunization campaigns (mouse and rat) were chosen: one that competed with AMG 557 for B7RP-1 (Ab 1.64.1) and one that did not (Ab 3.7.1). These antibodies were fluorescently labeled for use in a 4-color FACS assay. The assay was validated by initially demonstrating selective inhibition of 1.64.1 binding to B cells in vitro (by AMG 557), followed by analysis of specimen stability and biological variability from ten healthy volunteers across three sequential visits. The RO assay was next implemented in the Phase 1a single dose-escalating study of AMG 557. The RO data from the clinical trial showed a reversible, dose-dependent coverage of B7RP-1 by AMG 557 in this single dose study. Moreover, measurement of receptor levels with antibody 3.7.1 indicated a dose-dependent transient rise in total B7RP-1 on circulating B cells in the treated subjects.
The RO assay demonstrated coverage of B7RP-1 on circulating B cells from SLE subjects after single dose administration of AMG 557 in a dose-dependent and reversible manner. Total receptor levels on the B cells increased in association with increasing occupancy in a dose dependent manner in keeping with preclinical observations in mouse studies. The relationship of receptor occupancy and pharmacodynamic effects in mouse models provides an important consideration for interpreting the RO results from the Phase 1 study and for guiding dose selection in future clinical trials of AMG 557.
To cite this abstract, please use the following information:
Sullivan, Barbara A., Green, Cherie L., Zhang, Ming, Abbott, Christina, Belouski, Shelley, Thomas, John, et al; A Flow Cytometric Receptor Occupancy Assay Demonstrates Dose-Dependent Blockade of B7RP-1 by AMG 557 on Circulating B Cells from SLE Subjects. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1141