title: Evaluation of assay interference and interpretation of CXCR4 receptor occupancy results in a preclinical study with MEDI3185, a fully human antibody to CXCR4.
abstract: BACKGROUND
  Receptor occupancy (RO) assays provide a means to measure the direct interaction of therapeutics with their cell surface targets. Free receptor assays quantify cell-surface receptors not bound by a therapeutic while total receptor assays quantify the amount of target on the cell surface.
  
  
  METHODS
  We developed both a flow cytometry-based free RO assay to detect free surface CXCR4, and a total surface CXCR4 assay. In an effort to evaluate potential displacement interference, we performed in vitro experiments to compare on-cell affinity with the IC50 values from in vitro and in vivo from the free CXCR4 assay. We determined free and total surface CXCR4 on circulating blood cells in cynomolgus monkeys dosed with MEDI3185, a fully human monoclonal antibody to CXCR4.
  
  
  RESULTS
  We devised an approach to evaluate displacement interference during assay development and showed that our free assay demonstrated little to no displacement interference. After dosing cynomolgus monkeys with MEDI3185, we observed dose-dependence in the magnitude and duration of receptor occupancy and found CXCR4 to increase on lymphocytes, monocytes, and granulocytes. In a multiple dose study, we observed time points where surface CXCR4 appeared fully occupied but MEDI3185 was not detectable in serum. These paradoxical results represented a type of assay interference, and by comparing pharmacokinetic, ADA and total CXCR4 results, the most likely reason for the free CXCR4 results was the emergence of neutralizing anti-drug antibodies (ADA). The total CXCR4 assay was unaffected by ADA and provided a reliable marker of target modulation in both in vivo studies.
authors.affiliation: Clinical Pharmacology & DMPK, Medimmune, LLC, Mountain View, California, 94043.
authors.forename: Martin
authors.initials: M
authors.lastname: Schwickart
authors.affiliation: Clinical Pharmacology & DMPK, Medimmune, LLC, Mountain View, California, 94043.
authors.forename: Carlos
authors.initials: C
authors.lastname: Chavez
authors.affiliation: Clinical Pharmacology & DMPK, Medimmune, LLC, Mountain View, California, 94043.
authors.forename: Simon
authors.initials: S
authors.lastname: Henderson
authors.affiliation: Clinical Pharmacology & DMPK, Medimmune, LLC, Mountain View, California, 94043.
authors.forename: Inna
authors.initials: I
authors.lastname: Vainshtein
authors.affiliation: Clinical Pharmacology & DMPK, Medimmune, LLC, Mountain View, California, 94043.
authors.forename: Nathan
authors.initials: N
authors.lastname: Standifer
authors.affiliation: Clinical Pharmacology & DMPK, Medimmune, LLC, Mountain View, California, 94043.
authors.forename: Christopher
authors.initials: C
authors.lastname: DelNagro
authors.affiliation: Clinical Pharmacology & DMPK, Medimmune, LLC, Mountain View, California, 94043.
authors.forename: Freshta
authors.initials: F
authors.lastname: Mehrzai
authors.affiliation: Clinical Pharmacology & DMPK, Medimmune, LLC, Mountain View, California, 94043.
authors.forename: Amy
authors.initials: A
authors.lastname: Schneider
authors.affiliation: Clinical Pharmacology & DMPK, Medimmune, LLC, Mountain View, California, 94043.
authors.forename: Lorin
authors.initials: L
authors.lastname: Roskos
authors.affiliation: Clinical Pharmacology & DMPK, Medimmune, LLC, Mountain View, California, 94043.
authors.forename: Meina
authors.initials: M
authors.lastname: Liang
mesh_terms: D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000008:administration & dosage* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000008:administration & dosage* / Q000276:immunology; D005434:Flow Cytometry; D006098:Granulocytes / Q000187:drug effects / Q000276:immunology; D006801:Humans; D016756:Immunoglobulins, Intravenous / Q000008:administration & dosage; D008214:Lymphocytes / Q000187:drug effects / Q000276:immunology; D008252:Macaca fascicularis / Q000276:immunology; D009000:Monocytes / Q000187:drug effects / Q000276:immunology; D019718:Receptors, CXCR4 / Q000276:immunology / Q000627:therapeutic use*
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; C527276:CXCR4 protein, human; D016756:Immunoglobulins, Intravenous; C000612952:MEDI3185; D019718:Receptors, CXCR4
keywords: MEDI3185; antidrug antibodies; antitherapeutic antibodies; biopharmaceutical; flow cytometry; pharmacodynamic biomarker; receptor occupancy