The assay design used for measurement of therapeutic antibody concentrations can affect pharmacokinetic parameters: Case studies

Abstract

To interpret pharmacokinetic (PK) data of biotherapeutics, it is critical to understand which drug species is being measured by the PK assay. For therapeutic antibodies, it is generally accepted that "free" circulating antibodies are the pharmacologically active form needed to determine the PK/pharmacodynamic (PD) relationship, safety margin calculations, and dose projections from animals to humans and the eventual characterization of the exposure in the clinic. However, "total" drug may be important in evaluating the dynamic interaction between the drug and the target, as well as the total drug exposure. In the absence of or with low amounts of soluble ligand/shed receptor, total and free drug species are often equivalent and their detection is less sensitive to assay formats or reagent choices. In contrast, in the presence of a significant amount of ligand, assay design and characterization of assay reagents are critical to understanding the PK profiles. Here, we present case studies where different assay formats affected measured PK profiles and data interpretation. The results from reagent characterizations provide a potential explanation for the observed discrepancies and highlight the importance of reagent characterization in understanding which drug species are being measured to accurately interpret PK parameters.

Figure 1. Pharmacokinetic Parameters of Evaluable Subjects for ocrelizumab (monoclonal anti-drug CDR (MAC) Assay and polyclonal anti-drug CDR (PAC) Assay (A). Averaged concentration-time profiles of rituximab and ocrelizumab following IV infusion of 500 mg (B) or 1000 mg (C) on Day 1 and Day 15 to human patients during phase I/II, randomized, placebo controlled, double blinded, multicenter ACTION clinical study. Averaged concentration-time profiles of ocrelizumab (PAC and MAC assay) in comparison with rituximab PK profile (PAC assay) following IV infusion of 1000 mg on Day 1 and Day 15 to human patients from the ACTION study (D).

Figure 2. Representative concentration-time profiles of ocrelizumab (A) and v114 (B) in samples collected during NHL clinical study. Concentrations were determined by ELISA based polyclonal anti-drug CDR (PAC) and monoclonal anti-drug CDR (MAC) assays. Both of the v114 assay formats (MAC and PAC) showed remarkably similar PK profiles as compared with the two ocrelizumab assay formats.

Figure 3. (A) CD20 Interference was tested in the rituximab PAC, ocrelizumab MAC and ocrelizumab PAC PK assays using samples with various molar ratios of recombinant CD20 and either rituximab or ocrelizumab. CD20 interference was only observed in the ocrelizumab MAC assay. Polyclonal anti-drug CDR (PAC) assay; monoclonal anti-drug CDR (MAC) assay; PK – pharmacokinetic. (B) CD20 interference was tested with v114 in both the PAC and MAC versions of the human PK assay. Both v114 assay formats showed remarkably similar PK profiles; thus, CD20 does not interfere in the v114 MAC or PAC assay formats.

Figure 4. (A) Evaluation of the ability of the drug molecules (ocrelizumab/ v114) to bind to the CD20 on the surface of WIL2-S cells in the presence of antibodies used as assay reagents in the PK measurements. No anti-CD20 binding = 100% Stained, complete inhibition of anti-CD20 binding = Unstained, partial interference with anti-CD20 binding = Partially stained. (B) FACS characterization of the reagents used in the ocrelizumab PK assay. At higher concentrations both 8A3 and 2B3 mAbs inhibit ocrelizumab from binding to CD20 on WIL2-S cells. The polyclonal antibody only inhibits CD20 binding at the highest concentration (260 nM). This data indicates that the mAbs and CD20 are binding to the same epitope of ocrelizumab. If ocrelizumab is bound to soluble CD20 in serum the MAC assay will be unable to detect this complex resulting in a “free” PK assay. (C) FACS characterization of the reagents used in the v114 PK assay. Neither 7G7 nor 2D3 mAbs inhibit v114 from binding to CD20 on WIL2-S cells. The polyclonal antibody inhibits v114 binding most significantly at the highest concentration (1000 nM). This data suggests that both the MAC and PAC assay should measure both free and complexed drug equally (i.e., “total” drug).

Figure 5.

Red highlight, thrombin cleavage site; Blue highlight, His-tag; Green highlight, Flag-tag.