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« –Targeted Monoclonal 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 (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 (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 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), (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). Otelixizumab reference standard at 12.1 mg/ml was used (Bangs Laboratories, Fishers, IN) per the manufacturer’s instructions. for all experiments. Otelixizumab ELISA. ELISA plates (96-well; Greiner Bio-One) Isolation of PBMC. Healthy volunteer blood were obtained from were coated with capture antibody (anti-otelixizumab mAb clone the on-site blood donation unit and appropriate informed consent was T54.1C9.A9) at 5 mg/ml in 50 mM bicarbonate buffer overnight at 4°C, obtained from all donors. Blood was withdrawn into bags containing washed 5withPBS1 0.05%Tween 20, and blocked with Superblock 1 IU/ml sodium heparin (MP Biomedicals, Santa Ana, CA) as anticoag- (Thermo Scientific). Assay samples and otelixizumab standards (pre- ulant. Heparinized blood was kept at room temperature and processed pared in culture supernatant) were added to the prepared plates for 2 within 2 hours of withdrawal. PBMC separation was performed by hours. After washing, 1 mg/ml detection antibody (biotinylated anti- density gradient centrifugation using 50-ml Leucosep tubes (Greiner otelixizumab mAb clone 6H12.B12.A3.G10) was added for 1 hour Bio-One, Kremsmünster, Upper Austria) containing Ficoll-Paque. followed by addition of a streptavidin–horseradish peroxidase conjugate Blood (25 ml per tube) was centrifuged for 20 minutes at 800g with (Invitrogen, Carlsbad, CA) at 1/10,000 dilution for 1 hour. A chromo- the brake off. PBMCs recovered from the interface were washed twice genic substrate tetramethylbenzidine (Sigma-Aldrich, St. Louis, MO) with Dulbecco’s phosphate-buffered saline (PBS; Gibco/Thermo was used and color allowed to develop for 10 minutes before reaction Fisher Scientific, Sunnyvale, CA) and finally resuspended in complete was stopped with 0.5 M sulfuric acid. A Molecular Devices SpectroMax medium for counting using the guava easyCyte flow cytometer (EMD plate reader provided readings at 450 nm, and otelixizumab concen- Millipore, Billerica, MA). Complete medium comprised RPMI 1640 trations were calculated from a four-parameter fit standard curve using (Gibco) 1 1 mM glutamine (Gibco), 100 IU/ml penicillin/100 mg/ml analytical software (Softmax Pro; Molecular Devices, Sunnyvale, CA). The lower limit of quantification for this assay was 2.5 ng/ml. In Vitro PK/PD Model. A TMDD model (Mager and Jusko, 2001) accounting for otelixizumab binding on both CD41 and CD81 lym- phocytes was proposed to describe in vitro experimental data, in- cluding the extension of two binding sites (Gibiansky and Gibiansky, 2010). The proposed model is illustrated in Fig. 2. Otelixizumab (here denoted by Cp) is administered in the central compartment, where it can bind to free CD3/TCR receptor complex on both CD41 and CD81 lymphocytes (FR4 and FR8, respectively) to form drug-receptor complexes (DR4 and DR8, respectively). The model is described through the following differential equations Fig. 1. Experimental design. Schematic of cell culture system. expressed as molar concentrations: PK/PD Model of Anti-CD3« mAb Activity In Vitro 201

Fig. 2. Mechanism-based PK/PD model for otelixizumab disposition and effect on CD3/TCR expression. Otelixizumab (here denoted by Cp)is administered in the central compartment, where it can bind to free receptors, CD3/TCR, on both CD4+ and CD8+ lymphocytes (FR4 and FR8, respectively) to form drug-receptor complexes (DR4 and DR8, respectively). The binding processes are governed by the second-order rate constants kon4 and kon8. Free receptors on both CD4+ and CD8+ lymphocytes are synthesized at a zero-order rate (ksyn4, ksyn8) and degraded at a first-order rate (kdeg4, kdeg8). Complexes, DR4 and DR8, may either dissociate (koff4, koff8) or be internalized and degraded, (kint4, kint8).

dCp=dt 5 InðtÞ=V-kon4 × Cp × FR4 1 koff 4 × DR4-kon8 × Cp× FR8 (FOCEI) method was used throughout the analysis, and LSODA 1 koff 8 × DR8 (1) algorithm was used to integrate differential equations (ADVAN13 subroutine with TOL 5 6). A nonparametric bootstrap method (n 5 1000) was used to study the uncertainty of all TMDD model parameter Downloaded from 5 × × 1 × 1 × dFR4=dt -kon4 Cp FR4 koff 4 DR4 ksyn4 kdeg4 FR4 (2) estimates. From the bootstrap empirical posterior distribution, rela- tive standard errors (RSE) were obtained for the parameters. dDR4=dt 51kon4 × Cp×FR4 koff 4 × DR4 kint4 × DR4 (3) The criteria for model selection included: 1) improved fitting of the diagnostic scatter plots (observations versus predictions, weighted residual versus predictions or time, individual weighted residual dFR8=dt 5 -kon8 × Cp × FR8 1 koff 8 × DR8 1 ksyn8 kdeg8 × FR8 (4) versus predictions or time), 2) convergence of the minimization, 3) jpet.aspetjournals.org residual standard errors on all the estimated parameters, 4) signifi- cant decrease in the objective function value (OFV). dDR8=dt 51kon8 × Cp × FR8 81 koff × DR8 kint8 × DR8 (5) with initial conditions given by Cp(0) 5 0, FR4(0) 5 ksyn4/kdeg4 5 BAS4, DR4(0) 5 0, FR8(0) 5 ksyn8/kdeg8 5 BAS8, DR8(0) 5 0. Results Assuming equal otelixizumab binding to CD41 and CD81 lym- Pharmacokinetics of Free Otelixizumab. To describe phocytes, the model can be simplified by imposing equal association pharmacological interactions in a static in vitro culture 5 5 5 at ASPET Journals on October 2, 2021 and dissociation rate constants (i.e., kon4 kon8 kon and koff4 system, the pharmacokinetics of otelixizumab was investi- koff8 5 koff). gated. Free otelixizumab concentration time-course profiles Otelixizumab molar concentration was converted into the observed concentration (ng/ml) using its molecular weight (150 kDa) and are shown in Fig. 3 from two donors at different initial drug appropriate scaling (apparent volume of distribution V). Receptor concentrations over a 48-hour incubation period. At initial dynamic measurements were expressed in MESF units assumed to be drug concentrations higher than 100 ng/ml, otelixizumab proportional to receptors molar concentrations. Otelixizumab bound concentration levels were relatively static over 48 hours. In to CD3 blocked the binding of all available anti-TCR and anti-CD3 contrast, there was an apparent initial concentration- detection antibodies that were tested. As a consequence, free and total dependent and time-dependent reduction in free otelixizumab receptors could be detected only where otelixizumab was not bound to concentrations observed with both donors at initial concen- CD3. This implied that total receptor measurements (MTR4 and trations lower than 100 ng/ml. At the lowest starting MTR8) were proportional to free receptors molar concentration (and concentrations (3 and 1 ng/ml), free otelixizumab concen- not to the sum of free and bound receptor concentrations): trations were below the limit of quantification for both 5 × 5 × donors. Some values were obtained by extrapolation of the MFR4 gFR FR4 MFR8 gFR FR8 (6) standard curve at early time points, but antibody was undetectable at later time points. Collectively, these obser- MDR4 5 g × DR4 MDR8 5 g × DR8 (7) DR DR vations suggest target-mediated elimination of otelixizumab 5 × 5 × MTR4 gTR FR4 MTR8 gTR FR8 (8) in this system. Pharmacodynamics of Free/Bound CD3 and Total where gFR, gDR, and gTR are conversion factors between variables and CD3 Expression. Next, the in vitro pharmacodynamic time the measured quantities MFR4, MFR8, MDR4, MDR8, MTR4, and courses of free and bound CD3« together with total CD3« MTR8. expression on CD41 and CD81 T lymphocytes in the PBMC The PK/PD model was fitted to the individual PK and PD (free, culture following incubation with otelixizumab were deter- engaged, and total CD3/TCR receptors) data simultaneously using mined. CD41 T lymphocytes pharmacodynamic time-course nonlinear mixed-effect statistical analysis. Model development in- profiles selected initial concentrations for donor 1 are shown in 1 cluded differences for the two donors in baseline values for CD4 and Fig. 4. Free CD3« and total CD3« expression followed similar CD81 T lymphocytes and different otelixizumab affinities for binding, time courses. There was a rapid decline in both free CD3« and respectively, on CD41 and CD81 T lymphocytes. Additive, pro- portional, and combined residual error models were tested for each total CD3« expression relative to preincubation levels. In both variable. The data fitting software used was NONMEM version 7.2 cases, the rate and extent of reduction appeared to increase (Beal et al., 2009) embedded in a wrapper program Perl-Speaks with higher initial concentrations, the nadirs occurring as Nonmem (PsN version 3.6.2) (Lindbom et al., 2004) in a Windows 7 early as 0.25 hours following the start of incubation and being environment. The first-order conditional estimation with interaction maintained for the whole 48-hour incubation period at initial 202 Page et al. Downloaded from jpet.aspetjournals.org

Fig. 3. In vitro pharmacokinetics of otelixizumab. Otelixizumab concentration versus time profiles after incubation with PBMCs from the two donors. Different symbols are used for different initial concentrations. Solid lines represent predictions from final TMDD model. Dashed horizontal lines indicate at ASPET Journals on October 2, 2021 the lower limit of quantification (LLOQ). Values below the LLOQ occur in the lowest curves for both subjects.

concentrations $1000 ng/ml. Similar results were obtained affinities for otelixizumab for CD3« on CD41 or CD81 lym- with both CD41 and CD81 cells and results from both donors phocytes were found to be similar and the model was were comparable (data not shown). simplified by assuming equal affinity for both lymphocyte Following washout of otelixizumab after 48 hours, there was subsets (i.e., kon4 5 kon8 5 kon and koff4 5 koff8 5 koff). a gradual but steady increase in free CD3« and re-expression The final model included combined residual error model for of total CD3«. Based on visual inspection of the data, the rate total CD3/TCR receptor measurements and free otelixizumab of re-expression of total CD3« appeared to be similar for all concentration, and an additive residual error model was adopted initial concentrations and independent of the degree of re- for free and engaged CD3/TCR receptors. All parameters were duction of CD3« expression induced by incubation with estimated with reasonable precision (,40%). Goodness-of fit otelixizumab. For low initial concentrations (,3 ng/ml) ex- diagnostic plots, including comparisons of observed and pression levels returned to preincubation levels by approxi- predicted values, as well as residual analysis, suggested that mately day 6 following wash out of otelixizumab. In contrast, although the model tended to underpredict the lowest free at the highest initial concentration (10,000 ng/ml) expression and total CD3/TCR observations, it provided a reasonable had recovered to only around 80% of preincubation levels by approximation for the data (Supplemental Data). The proposed the end of the experiment (day 14). Again, results were model captured the PK/PD time-course profiles reasonably well consistent for both CD41 and CD81 cells and comparable for both donors and all initial concentrations of otelixizumab between donors (data not shown). (Figs. 3 and 4). Characterization of the PK/PD–Time Relationship. The parameter estimates for the model are reported in Mechanistic, mathematical, TMDD PK/PD models were fitted Table 1 together with relative standard errors (RSE) based on simultaneously to the PK and PD (free, engaged, and total 1000 bootstrapped datasets. The binding constants were CD3/TCR receptors) data. In terms of the model iteration estimated as kon 5 51.5/nM per day and koff 5 4.64 per day, and adequacy, the most robust best fit model included suggesting high affinity of otelixizumab to human CD3«. different baseline levels for CD3/TCR on CD41 and CD81 Correspondingly, the derived equilibrium dissociation con- T lymphocytes (BAS4 and BAS8 respectively) for the two stant, KD (koff/kon) was 90 pM. donors. The final model did not include any between-subject Estimated internalization rates (kint4 5 1.26/day and kint8 5 variability on model parameters, so that the resulting analysis 1.29/day) were five times higher than degradation rates (kdeg4 5 can be considered as a naïve-pooling approach. The binding 0.273/day and kdeg8 5 0.275/day). PK/PD Model of Anti-CD3« mAb Activity In Vitro 203 Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021

Fig. 4. In vitro pharmacodynamics of otelixizumab. Free (A), engaged (B), and total (C) CD3/TCR receptor kinetic profiles on CD4+ cells from Donor 1 after incubation with otelixizumab for selected different initial concentrations. Open circles represent the available in vitro data and lines are predictions from the final TMDD model.

Discussion cell culture compositions attributable to cell death, the pro- posed model captured the concentration-dependent kinetic We established a static in vitro culture system to investigate profiles for free, bound, and total CD3« reasonably well. At and characterize otelixizumab pharmacology using human saturating concentrations, the engagement of target CD3« cells. We were able to study the dose range and time course molecules was found to be rapid, leading to full occupancy of with sufficient resolution to observe and quantify the concen- CD3« within minutes. At lower concentrations, binding of tration and time-dependent PK behavior consistent with antibody gradually achieved equilibrium. The estimated target-mediated disposition of the antibody. A mechanistic, parameters appeared reasonable and consistent with an mathematical model (TMDD model) used to describe this antibody of this type. According to the model, the affinity – phenomena was fitted to the PK/PD time data. The model constant for otelixizumab binding to CD3« is KD 5 90 pM. adequately described all the data and was sufficient to explain This finding is in line with observations made in a previous the nonlinear disposition of otelixizumab in this culture study (Wiczling et al., 2010), in which the estimated concen- system. The rate and degree of change of free, bound, and tration of otelixizumab producing 50% reduction in free CD3/ total CD3« expression was found to be determined by the TCR sites was in the range of 14–16 ng/ml (5 93.8–107.2 pM). concentration of otelixizumab. A moderate increase was However, Wiczling et al. estimated a nonlinear PK model observed in total CD3/TCR receptor profiles at the end of the (Michaelis-Menten elimination) with a Michaelis-Menten experiment. Although data described in this paper does not constant (Km 5 0.968 mg/ml) that is not in agreement with allow any conclusion regarding the cause of this modest our in vitro results. In fact, a Michaelis-Menten PK model can increase, it is possible that a small degree of cell death be considered still an approximation of a full TMDD model in (approximately 10% was observed during the 14-day culture) which the nonlinear elimination Km is related to the binding is a contributing factor. This was, however, not considered and internalization process (Km 5 (koff 1 kint)/kon) (Ma, a relevant feature to implement in the final model as it was 2012). In our specific case, since the estimated dissociation judged to be relatively minor compared with the degree of constant is faster than the internalization rate, the net CD3/TCR decreases achieved rapidly following otelixizumab result of the binding-internalization process is driven by the engagement. Although the use of a constant synthesis rate for affinity estimate (Km ∼ KD). Wiczling et al. suggested that CD3/TCR receptors does not account for possible changes in binding to CD3/TCR complexes did not affect otelixizumab 204 Page et al.

TABLE 1 Parameter estimates from the TMDD model

Parameter (Units) Description Value RSE [%] kon (/nM/day) Second-order rate constant for otelixizumab-CD3/TCR association 51.47 9.60 koff (/day) First-order rate constant for otelixizumab-CD3/TCR dissociation 4.64 28.88 kint4 (/day) First-order rate constant for internalization of otelixizumab-CD3/TCR 1.26 10.48 complexes on CD4+ cells kint8 (/day) First-order rate constant for internalization of otelixizumab-CD3/TCR 1.29 8.22 complexes on CD8+ cells Donor 1 BAS4 (pM) CD3/TCR receptor complex baseline on CD4+ cells for Donor 1 42.36 22.99 Donor 1 BAS8 (pM) CD3/TCR receptor complex baseline on CD8+ cells for Donor 1 34.47 23.06 Donor 2 BAS4 (pM) CD3/TCR receptor complex baseline on CD4+ cells for Donor 2 39.22 22.79 Donor 2 BAS8 (pM) CD3/TCR receptor complex baseline on CD8+ cells for Donor 2 33.32 22.69 kdeg4 (/day) First-order rate constant for degradation of CD3/TCR receptor 0.27 7.68 complex on CD4+ cells kdeg8 (/day) First-order rate constant for degradation of CD3/TCR receptor 0.28 7.83 complex on CD8+ cells V (ml) Apparent volume of Distribution 1.12 3.20 3 gFR (10 MESF/nM) Conversion factor from FR to MFR 4853.40 16.74 3 gDR (10 MESF/nM) Conversion factor from DR to MDR 5885.90 16.61 3 gTR (10 MESF/nM) Conversion factor from FR to MTR 760.88 16.85 Downloaded from sAD (Cp)(ng/ml) Additive residual error model for free Otelixizumab concentrations 0.924 38.56 sPR (Cp) (-) Proportional residual error model for free Otelixizumab 0.207 11.28 concentrations 3 sAD (FR) (10 MESF) Additive residual error model for free CD3/TCR receptors 12 5.65 3 sAD (DR) (10 MESF) Additive residual error model for engaged CD3/TCR receptors 20.4 9.39 3 sAD (TR) (10 MESF) Additive residual error model for total CD3/TCR receptors 2.11 33.27 sPR (TR)(-) Proportional residual error model for total CD3/TCR receptors 0.14 18.48 jpet.aspetjournals.org pharmacokinetics, and they considered the short terminal complex from the cell surface (Reinherz et al., 1982; Press half-life (t1/2 of approximately 0.5 days) to be related to the et al., 1988). absence of FcRn recycling. The data presented here suggest In contrast, despite considerable investigation, the degree, that the process of CD3/TCR internalization is stimulated as duration of action of otelixizumab binding to CD3«, CD3 a consequence of antibody binding, with an estimated in- expression, and its relationship to unbound antibody has only ternalization rate of drug-receptor complexes significantly been partially elucidated from clinical data alone. For practi- at ASPET Journals on October 2, 2021 faster than the free receptor degradation (kint 5 1.26–1.29 cal reasons, it has been difficult to study a sufficiently wide day21 . kdeg 5 0.27–0.28 day21), resulting in a mean dose range with an adequate blood sampling schedule follow- residence time of the free receptor of approximately five times ing treatment of otelixizumab in patients to determine higher than the one of the drug-receptor complexes. This whether the dynamics of antibody binding can be separated phenomenon is a common mechanism when natural ligand from the dynamics of the observed transient lymphopenia. binds to its corresponding receptor and has been described for Thus, attempts to quantify these relationships clinically have antigen binding to the T-cell receptor (Huang et al., 1999), and been largely empirical and simple (Wiczling et al., 2010). the B-cell receptor (Caballero et al., 2006), and for ligand Whereas the published models describe the data adequately, binding to the granulocyte-macrophage–colony-stimulating and may be useful for limited interpolation or extrapolation, factor receptor (Vainshtein et al., 2015). Following removal they lack the mechanistic detail to provide insight about of antibody from the culture, the rate of re-expression of CD3« the underlying pharmacological behavior of otelixizumab. was observed to be slower than otelixizumab-induced down- Understanding the in vivo interaction of otelixizumab to the regulation. Model estimates for internalization and degrada- CD3 target is challenging since observed data may not be tion rate constants for CD3/TCR receptors reflect these sufficient to estimate relevant system parameters. In this findings. At saturating concentrations, complete modulation case, the in vitro model can provide relevant information for was achieved within about 30 minutes, whereas full CD3/TCR the receptor synthesis (ksyn), degradation/internalization re-expression to pretreatment levels required about 8 days. rate (kdeg and kint), and binding affinity (KD) that can help These findings are in agreement with data obtained from to better inform any in vivo model in which other phenomena mouse models using surrogate anti-CD3 antibody fragments (mAb distribution, disposition, and systemic clearance (Mehta et al., 2010). These observations are supportive of the attributable to nonspecific target binding, together with target notion that CD3« reappearance requires new synthesis and expression, lymphocytes trafficking, etc.) would increase the assembly of complexes, known to be a slow process that takes complexity of the model structure, including identifiability issues days, as opposed to re-expression of transiently internalized for relevant system parameters. In general, the development of existing complexes, which has been described as a relatively mechanistic models from in vitro experiments offers a valuable rapid process (minutes to hour) (Menné et al., 2002). It should approach to addressing the complexity of in vivo systems, for be noted that the exact fate of otelixizumab was not in- example, by using the in vitro estimates as priors for the in vivo vestigated in this study. Several mechanisms are possible, models. with some studies demonstrating endocytosis of antibody Preclinical investigation of these phenomena in vivo is receptor complexes (Liu et al., 2000; Monjas et al., 2004; limited by the almost complete lack of crossreactivity of Kuhn et al., 2011), whereas others suggest shedding of the otelixizumab with animal CD3 and lack of suitable animal PK/PD Model of Anti-CD3« mAb Activity In Vitro 205 surrogate antibody. In conclusion, we have demonstrated using Lindbom L, Ribbing J, and Jonsson EN (2004) Perl-speaks-NONMEM (PsN)–a Perl – module for NONMEM related programming. Comput Methods Programs Biomed an in vitro cell culture system and a mechanistic model based 75:85–94. approach to data analysis, that we can successfully study and Liu H, Rhodes M, Wiest DL, and Vignali DA (2000) On the dynamics of TCR:CD3 complex cell surface expression and downmodulation. Immunity 13:665–675. quantify the complex antibody-receptor complex PK/PD inter- Ma P (2012) Theoretical considerations of target-mediated drug disposition models: actions that were hypothesized for otelixizumab. This will simplifications and approximations. Pharm Res 29:866–882. Mager DE and Jusko WJ (2001) General pharmacokinetic model for drugs exhibiting probably be applicable to other membrane-bound targets, target-mediated drug disposition. J Pharmacokinet Pharmacodyn 28:507–532. particularly for immune cell targets in which antibodies or Mehta DS, Christmas RA, Waldmann H, and Rosenzweig M (2010) Partial and transient modulation of the CD3-T-cell receptor complex, elicited by low-dose other protein therapeutics may cause altered cell trafficking. regimens of monoclonal anti-CD3, is sufficient to induce disease remission in non- The parameters estimated from such analysis may allow obese diabetic mice. 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Goodness of fit plots from final TMDD model for free otelixizumab, free, engaged and total CD3/TCR receptors. Top row: individual predictions versus observations. Bottom row: weigthed residuals versus individual predictions (loess regression line superimposed)"