Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2018/10/17/jpet.115.224899.DC1 1521-0103/355/2/199–205$25.00 http://dx.doi.org/10.1124/jpet.115.224899 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 355:199–205, November 2015 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics Temporal Pharmacokinetic/Pharmacodynamic Interaction between Human CD3« Antigen–Targeted Monoclonal Antibody Otelixizumab and CD3« Binding and Expression in Human Peripheral Blood Mononuclear Cell Static Culture s Kevin R. Page, Enrica Mezzalana, Alexander J. MacDonald, Stefano Zamuner, Giuseppe De Nicolao, and Andre van Maurik GlaxoSmithKline, Stevenage, United Kingdom (K.R.P., A.J.M., S.Z., A.vM.); University of Pavia, Pavia PV, Italy (E.M., G.D.N.) Received April 22, 2015; accepted August 17, 2015 Downloaded from ABSTRACT Otelixizumab is a monoclonal antibody (mAb) directed to human a shorter apparent half-life at low concentration. Correspondingly, CD3«, a protein forming part of the CD3/T-cell receptor (TCR) a rapid, otelixizumab concentration–, and time-dependent re- complex on T lymphocytes. This study investigated the temporal duction in CD3/TCR expression was observed. These combined interaction between varying concentrations of otelixizumab, bind- observations were consistent with the phenomenon known as jpet.aspetjournals.org ing to human CD3 antigen, and expression of CD3/TCR complexes target-mediated drug disposition (TMDD). A mechanistic, mathe- on lymphocytes in vitro, free from the confounding influence of matical pharmacokinetic/pharmacodynamic (PK/PD) model was changing lymphocyte frequencies observed in vivo. A static in vitro then used to characterize the free otelixizumab-CD3 expression- culture system was established in which primary human peripheral time relationship. CD3/TCR modulation induced by otelixizumab blood mononuclear cells (PBMCs) were incubated over an was found to be relatively fast compared with the re-expression extended time course with titrated concentrations of otelixizumab. rate of CD3/TCR complexes following otelixizumab removal from At each time point, free, bound, and total CD3/TCR expression on supernatants. In summary, the CD3/TCR receptor has been shown at ASPET Journals on October 2, 2021 both CD41 and CD81 T cells and the amount of free otelixizumab to have a major role in determining otelixizumab disposition. A antibody in the supernatant were measured. The pharmacokinetics mechanistic PK/PD model successfully captured the PK and PD in of free otelixizumab in the culture supernatants was saturable, with vitro data, confirming TMDD by otelixizumab. Introduction determined largely empirically, or when animal disease or in vitro models were used for extrapolation, the ability to adjust Monoclonal antibodies (mAb) directed against human lym- for animal-human or in vitro–in vivo differences was limited. phocyte antigen CD3« have been investigated clinically in The following pharmacology has been observed clinically with a number of autoimmune diseases such as type 1 diabetes anti-CD3 mAbs: 1) Binding leads to apparent partial T-cell (Herold et al., 2002; Keymeulen et al., 2005, Hagopian et al., activation, resulting in release of proinflammatory cytokines, 2013), ulcerative colitis (Sandborn et al., 2010), multiple and a temporary disruption of normal T-cell trafficking ’ sclerosis (Weinshenker et al., 1991), and Crohn s disease (Chatenoud et al., 1982; Waldron Smith et al., 1997; Smith (van der Woude et al., 2010). One such anti-CD3 mAb, et al., 1998; -Lynch et al., 2012) and 2) internalization of the muromonab-CD3 (tradename Orthoclone OKT3), is approved resultant mAb/CD3/T-cell receptor (TCR) complex causes loss for the treatment of steroid-resistant acute rejection of of the complex from the T-cell surface membrane and degra- allogeneic renal, heart, and liver transplants. Despite encour- dation and elimination of the antibody (Reinherz et al., 1982; aging results from clinical investigations in autoimmune Press et al., 1988; Liu et al., 2000; Monjas et al., 2004), diseases, especially in new-onset type 1 diabetes, there has a phenomenon known as target-mediated drug disposition been little progress in understanding the relationship be- (TMDD) (Levy, 1994). Whereas some quantification of these tween anti-CD3 mAb target engagement and its putative clinical observations has been possible (Wiczling et al., 2010), mechanisms of action. Owing to this lack of pharmacologi- it has been difficult separating and quantifying these individ- cal understanding, dosage regimens appear to have been ual pharmacological components at a mechanistic level owing to the close temporal relationship between them. dx.doi.org/10.1124/jpet.115.224899. In an attempt to overcome the limitations of studying anti- s This article has supplemental material available at jpet.aspetjournals.org. CD3 mAb pharmacology in vivo, a static in vitro culture ABBREVIATIONS: ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; KD, binding affinity; mAb, monoclonal antibody; MESF, mean equivalent surface fluorescence; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PD, pharmacodynamic; PerCP-Cy5.5, peridinin-chlorophyll-cyanine 5.5; PK, pharmacokinetic; SSC, side scatter; TCR, T-cell receptor; TMDD, target- mediated drug disposition. 199 200 Page et al. system was established to investigate the time course of the streptomycin (Gibco) and 5% heat-inactivated human AB serum interaction between anti-CD3 mAb, CD3, and CD3/TCR (Gemini Bio-Products, West Sacramento, CA). complex, free from the confounding influence of changes in Time Course of PBMC Treatment with Otelixizumab. Freshly  6 number of circulating T lymphocytes, observed as transient isolated PBMC from two healthy donors (1 10 /ml) were cultured in 1 peripheral lymphopenia in vivo. A mechanistic nonlinear 24-well plates (Costar/Corning, Corning, NY) in RPMI 5% AB serum with titrated concentrations of otelixizumab (0, 1, 3, 10, 30, 100, 300, 1000, mixed effect pharmacokinetic/pharmacodynamic (PK/PD) 3000, and 10,000 ng/ml), 16 wells for each concentration. At time points model was then used to analyze and describe the complex 0.25, 0.5, 1, 2, 4, 8, 16 hours and 1, 2, 3, 4, 6, 8, 10, 12, and 14 days the pharmacological interactions in this static in vitro system. The cell suspension was removed from one well for each donor transferred in vitro pharmacokinetics was investigated in which primary to fluorescence-activated cell sorting (FACS) tubes and centrifuged human peripheral blood mononuclear cells (PBMCs) were 5 minutes at 1350 rpm. The supernatant was removed and stored for used; CD31 T lymphocytes in PBMCs is typically in the range subsequent ELISA at –20°C and the cells were resuspended in 4 ml FACS of 50–70%. PBMCs were cultured over an extended time buffer and split between two tubes for FACS staining. For CD3/TCR re- 6 course with titrated concentrations of otelixizumab. At each expression PBMC from each donor were cultured at 1  10 /ml (10 ml) 1 time point, free, bound, and total CD3/TCR expression on both in RPMI 5% AB serum with the same titrated concentrations of CD41 and CD81 T cells and the amount of free antibody in otelixizumab. After 48 hours cells were removed from wells and trans- ferred to 50-ml tubes and centrifuged at 1350 rpm for 5 minutes. Cells the supernatant were measured so that PK could be related to were resuspended in 50 ml PBS and centrifuged again. After resuspen- PD effects. To investigate the kinetics of CD3/TCR re- sion in fresh RPMI 1 5% AB serum, counting cells were adjusted to 106/ml expression, cells were washed on day 2 to remove exogenous and 1 ml/well was added to 24-well plates, eight replicate wells for each Downloaded from otelixizumab and thereby allow the rate of CD3/TCR complex population. One well of each replicatewasharvestedandthecellsstained re-expression to be monitored. for FACS as for other cells at 8 hours, and 1, 2, 4, 6, 8, 10, and 12 days. Quantification of CD3/TCR Modulation by Flow Cytometry. At the indicated time points the cells were removed, split between two Materials and Methods tubes, and washed twice with FACS buffer (PBS 1 1% FCS 1 sodium azide 0.1%). After blocking of Fc receptors for 5 minutes with TruStain Experimental Design. PBMCs isolated from healthy volunteer FcX (BioLegend, Sacramento, CA) cells were surface-stained with jpet.aspetjournals.org bloods were cultured in the presence of a titration of otelixizumab over the following murine antibodies: Tube A: CD4-PerCP-Cy5.5 (clone an extended time course (Fig. 1). Flow cytometry was used to measure RPA-T4), CD8-APC (clone RPA-T8), CD3-FITC (clone SK7), and anti- free and bound CD3« and TCR at the indicated time points. Human IgG Fc-PE (clone HP6017); and Tube B: CD4-PerCP-Cy5.5 Simultaneously, an enzyme-linked immunosorbent assay (ELISA) (clone RPA-T4), CD8-APC (clone RPA-T8), and TCRab-PE (clone was used to measure the amount of free antibody in the supernatant of IP26) (all BioLegend) for 30 minutes at room temperature protected these cultures. In addition, some cells were washed to remove from light. After washing twice with 3 ml FACS buffer the cells were exogenous antibody after 2 days and measurement of CD3/TCR re- resuspended in 300 ml Cytofix (BD Biosciences, San Jose, CA). expression examined at the indicated time points postwash. Lymphocytes were gated on forward scatter versus side scatter and at ASPET Journals on October 2, 2021 Antibodies. Otelixizumab (also known as ChAglyCD3) is an 5000 CD4 events were acquired. The mean fluorescence intensity data aglycosylated nonmitogenic recombinant antibody (human g1) di- for phycoerythrin and fluorescein isothiocyanate fluorescence for rected against CD3« chain, a protein forming part of the CD3/T-cell gated CD4 and CD8 cells were converted to mean equivalent surface receptor complex (TCR) on T lymphocytes (Routledge et al., 1991; Bolt fluorescence (MESF) values using Quantum Simply Cellular beads et al., 1993).