FormalPara Key Points

Inotuzumab ozogamicin as a single-agent treatment resulted in promising response rates and acceptable tolerability in phase II trials for children with relapsed/refractory B-cell precursor acute lymphoblastic leukemia.

This is the first study to describe the population pharmacokinetics of inotuzumab ozogamicin in a pediatric population. The concentration–time profiles of inotuzumab ozogamicin in both adult and pediatric patients were well described by a two-compartment model with linear and time-dependent clearance components. Target-mediated drug clearance of inotuzumab ozogamicin declines more rapidly in children compared with adults.

Pediatric patients with B-cell precursor acute lymphoblastic leukemia receiving the recommended phase II dose achieve a desirable drug exposure at the end of the first cycle according to simulations. Additionally, the recommended phase II dose has been reported to be well tolerated in children with a satisfactory response rate. Therefore, clinically, no further dose adjustment is required.

1 Introduction

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in children [1, 2]. Contemporary treatment is associated with an 80–90% 5-year event-free survival rate and a 5-year overall survival rate of over 90% [3,4,5]. However, prognosis remains unsatisfactory in those who are refractory to first-line therapy or who relapse, with a 5-year overall survival rate of 50–60% at the first relapse and less than 30% in patients with second or further relapses [6,7,8]. Furthermore, despite the approval of immunotherapies such as blinatumomab [9] and chimeric antigen receptor T cells, 50% of patients treated with chimeric antigen receptor T cells relapse during an extended follow-up [10]. Moreover, replacing toxic therapy elements with equally effective but less toxic compounds is warranted as acute and long-term morbidity are burdensome for patients with ALL. Therefore, novel efficacious therapeutic agents for ALL in children are desirable.

Inotuzumab ozogamicin (InO) is an antibody-drug conjugate (ADC) consisting of a humanized, monoclonal immunoglobulin G4 antibody and a cytotoxic payload, N-acetyl-γ-calicheamicin dimethyl hydrazide, conjugated via an acid-labile linker [11,12,13]. The antibody of InO targets CD22, whereas calicheamicin is a DNA-binding cytotoxic agent with potent antitumor activity [11, 14]. CD22 is expressed on the surface of the majority of B-lymphocyte malignancies and on healthy B cells, but not on non-lymphoid hematopoietic cells [11, 15,16,17]. After InO binds to CD22, it is internalized into the slightly acidic lysosomal compartment and calicheamicin is released, then CD22 is re-expressed on the cell surface. Calicheamicin, after being released from InO, binds to the minor groove of double-helical DNA, cleaves the double-stranded DNA, and causes cell apoptosis [11, 18].

Inotuzumab ozogamicin was approved by the European Medicines Agency and the US Food and Drug Administration in 2017 for the treatment of adults with relapsed/refractory (R/R) B-cell precursor (BCP)-ALL. The approved adult dosing regimen is 1.8 mg/m2/cycle fractionated in three weekly administrations for the first cycle of 21 days, and in those achieving complete remission, followed by 1.5 mg/m2/cycle, for up to six cycles of 28 days. In the phase III INO-VATE trial conducted in adults with R/R CD22+ BCP-ALL, InO as a single agent was associated with significantly superior efficacy compared with standard intensive chemotherapy and acceptable toxicities [19]. In children with R/R BCP-ALL, the overall response rate in the single-agent phase II trials conducted in Europe and the USA spans from 81.5% (95% confidence interval 61.9–93.7) to 58.3% (90% confidence interval 46.5–69.3). Among responders, 82% and 67% minimal residual disease (MRD)-negativity rates were reported in respective trials [20, 21].

The pharmacokinetics of InO in adults has been well characterized by a two-compartment model with linear and time-dependent clearance components (representing target-mediated drug disposition) and several covariates (disease type, body size, baseline peripheral blast percentage, and concomitant rituximab treatment) that affect InO disposition have been identified [22, 23]. In the pediatric population, analyses of population pharmacokinetics are lacking. The aim of this analysis was to evaluate the population pharmacokinetics of InO, identify potential differences in InO disposition between pediatric and adult patients with R/R BCP-ALL, and to assess the pharmacokinetics at the pediatric recommended phase II dose (RP2D, the same as in  the approved adult dosing regimen) [24], using a dataset that includes pediatric (comprising the ITCC-059 phase IA and phase II single-agent trials) and adult trial data [23].

2 Methods

2.1 Study Design and Patients

This population pharmacokinetic (PK) analysis is based on clinical data from 11 studies in adult patients with R/R BCP-ALL or R/R B-cell non-Hodgkin lymphoma (NHL) [InO either as a single agent, or combined with rituximab, or with rituximab plus chemotherapy], and one study of single-agent InO in pediatric patients with R/R BCP-ALL (Trial ITCC-059 phase IA and phase II). Details on the studies conducted in adults were described by Garrett et al. [23]. Details on the ITCC-059 trial were published by Brivio et al. and Pennesi et al. [20, 24].

The inclusion criteria of trial ITCC-059 were: age ≥ 1 and < 18 years, diagnosis of R/R CD22+ BCP-ALL, and provision of informed consent. See Table 1 of the Electronic Supplementary Material (ESM) for the full inclusion and exclusion criteria. Inotuzumab ozogamicin was administered as an intravenous infusion over 60 minutes in 3-week cycles in a fractionated manner with dosing on a weekly basis. In the phase I part, two dose levels were explored; at dose level 1, InO was given at 1.4 mg/m2/cycle and 1.2 mg/m2/cycle once remission was achieved; at dose level 2, InO was given at 1.8 mg/m2/cycle and 1.5 mg/m2/cycle after remission. The latter was selected as the RP2D [24]. Each InO dosing regimen was fractioned in three doses/cycle on days 1, 8, and 15 (dose level 1: 0.6, 0.4, 0.4 mg/m2, and after remission 0.4, 0.4, 0.4 mg/m2; dose level 2: 0.8, 0.5, 0.5 mg/m2, and after remission 0.5, 0.5, 0.5 mg/m2). A maximum of six cycles was permitted, except for patients proceeding to a transplant, where a maximum of two to three cycles was used. Clinical characteristics of the children were a median age of 9 years (range 1–17 years) and a median white blood cell count of 3.33 × 109 (range 0.19–132 × 109, counts), 67.9% were male. Detailed pediatric patient characteristics were reported previously [20, 24].

All studies were approved by the independent ethics committee at each participating center and were conducted in accordance with the Declaration of Helsinki and the International Conference of Harmonization Guideline for Good Clinical Practice. The data from the adult patients were provided by Pfizer. ITCC-059 was sponsored by Erasmus MC and financed by Pfizer (EudraCT Number: 2016-000227-71).

2.2 PK Sampling and Bioanalytic Methods

Inotuzumab ozogamicin (as the parent drug) and unconjugated calicheamicin PK samples were collected and analyzed. The serum concentration of InO and unconjugated calicheamicin from children were quantified by validated high-performance liquid chromatography with tandem mass spectrometry. The lower limit of quantification (LLOQ) of the serum concentration of InO was 1.0 ng/mL; and 0.05 ng/mL for the serum concentration of unconjugated calicheamicin. The bioanalytical analysis method was designed for indirect measurement of N-acetyl-γ-calicheamicin dimethyl hydrazide conjugated to the antibody of InO. The same method was used for PK samples from adult patients with BCP-ALL, as described by Garrett et al. [23], where it measured the conjugated calicheamicin released from ADC and InO quantitation was based on the average drug-to-antibody ratio of the dosing formulation used to prepare calibration standards. A validated enzyme-linked immunosorbent assay method, designed to directly assess N-acetyl-γ-calicheamicin dimethyl hydrazide linked to the InO antibody, was used to measure the serum concentration of InO from adult patients with NHL [23]. The bioanalytical analysis methods were validated/revalidated by PPD (Richmond, VA, USA), and performed at laboratories designated by Pfizer.

In this study, the population PK analysis refers to InO concentrations because all unconjugated calicheamicin serum concentrations from pediatric trial participants were below the LLOQ. The majority of unconjugated calicheamicin serum concentrations were also below the LLOQ in prior adult studies [23]. Data in adults have shown that InO exhibits both linear-dependent and time-dependent clearance components [23]. In adults, the steady state was achieved by the fourth cycle and the linear clearance component predominates over the time-dependent component. Therefore, in children, PK samples were taken during cycles 1, 2, and 3, to better characterize both the linear-dependent and time-dependent clearance of InO. In total, six blood samples were collected per patient during cycle 1 on day 1 (pre-dose and 1 hour after dose), day 8 (pre-dose), day 15 (pre-dose and 1 hour after dose), and at the end of the cycle (day 22) for trough samples; five samples were collected in cycle 2 (trough samples collected on day 28 and without the pre-dose samples on day 1) and four samples were collected in cycle 3 (trough samples collected on day 28 and without the pre-dose samples on day 1 and without the day 15 1 hour after dose samples). Details of the sampling schedule for the pharmacokinetics of InO are reported in the Appendix (Table 2 of the ESM). No pediatric patients had treatment induced  anti-drug antibodies and only one had baseline positive anti-drug antibodies [20].

2.3 Model Development

The starting point for model development was a previously developed population PK model for adult patients with BCP-ALL and B-cell NHL based on data from 11 adult trials. This model consisted of a two-compartment model with linear (CL1, L/h) and time-dependent clearance (CLt, L/h) (Fig. 1). The two-compartment model structure aligns with the general modeling framework for the pharmacokinetics of monoclonal antibodies with target-mediated drug disposition [25, 26]. The linear InO clearance (CL1) is considered to represent the elimination of endogenous immunoglobulin G, mediated by the Fc receptors (in the skin, muscle, and liver) and salvaged by neonatal Fc receptor. The time-dependent clearance, described by \({CL}_{t}={CL}_{2}*{e}^{({-k}_{des}* Time)}\), relates to the target-mediated drug disposition, in which the capacity decreases over time as the tumor burden (and thus antigen expression) reduces. The differential equations used to describe the PK data were:

$$k_{10} =\frac{CL_{total}}{V_{1}},$$
$$Q = k_{12} *V_{1} = k_{21} *V_{2},$$
$$CL_{total} = CL_{1} + CL_{t} ,$$
$$\frac{{{\text{d}}A\left( 1 \right)}}{{{\text{d}}t}} = - k_{10} *A\left( 1 \right) - k_{12} *A\left( 1 \right) + k_{21} *A\left( 2 \right),$$
$$\frac{{{\text{d}}A\left( 2 \right)}}{{{\text{d}}t}} = k_{12} *A\left( 1 \right) - k_{21} *A\left( 2 \right),$$

where Vi represents the volume of the ith compartment, A(i) is the amount in the respective compartment, \({k}_{10}\) is the elimination rate constant from the central compartment, and Q is the intercompartmental clearance translated into the distribution rate constant (k12, k21).

Fig. 1
figure 1

Inotuzumab ozogamicin pharmacokinetic model structure. Total clearance (CLtotal) is the sum of linear clearance (CL1) and time-dependent clearance (CLt). CL2 initial value of time-dependent clearance, IV intravenous, kdes decay coefficient, Q intercompartment clearance, V1 volume of distribution in the central compartment, V2 volume of distribution in the peripheral compartment

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Covariates in the adult final model were baseline body surface area (BBSA, m2), disease type and/or analytical methods (ALL effect, NHL as the reference), and concomitant rituximab treatment (with rituximab as the reference) on CL1; BBSA on the volume of distribution in the central compartment (V1, L); BBSA on CL2; ALL effect (accounts for disease type (NHL/ALL) and/or different bioanalytical analysis methods) and baseline percentage of blasts in the peripheral blood (BLSTPB, %) on the decay coefficient (kdes) of CLt [23]. Interindividual variability (IIV) was modeled using the following equation:

$$P_{i} { } = { }P_{pop} { } \times { }e^{{\left( {\eta_{i} } \right)}} ,$$

where \({P}_{i}\) is the parameter estimate of the ith individual (empirical Bayes estimates/post hoc parameters), \({P}_{pop}\) represents the fixed population parameter estimate, and \({\eta }_{i}\) depicts the IIV of the ith individual, which is assumed to follow a normal distribution with a mean 0 and a variance ω2. Residual unexplained variability was described by two separate additive residual errors based on log-transformed data to take different bioanalytical methods used in different disease types into account. Last, the method 3 (M3) modeling approach was applied to handle InO concentration data that were below LLOQ [27, 28]. Observations below the LLOQ were retained in the analysis and treated as censored.

The first step in model building was to re-estimate the adult model including covariate effects on the pooled dataset and to estimate the separate residual error for a pediatric population to further account for variability between trials. Subsequently, specific covariates important for the pediatric population were further investigated. The included covariates relate to body size, age, and disease. The covariate–parameter relationships to be examined are shown in Table 1. Baseline covariates assessed in the model include replacing certain covariates presented in the adult model, namely, BBSA by body weight (kg), or lean body mass (LBM, kg) [29, 30], and BLSTPB by baseline absolute blast counts in peripheral blood (BLSTABL) on kdes. The InO serum concentration was measured in the central compartment (from blood), therefore BLSTABL is considered a better covariate than bone marrow blast for describing the target-mediated clearance component of InO in the bloodstream. Further testing included age (years), hepatic impairment (BHGRADE, National Cancer Institute Organ Dysfunction Working Group criteria for hepatic impairment [31]), baseline albumin (g/dL), baseline alanine aminotransferase (U/L) on CL1; age, ALL effect, BLSTABL on CL2; and age on kdes. Noteworthy, blast in peripheral blood (BLSTPB and BLSTABL) does not apply for patients with B-cell NHL. Therefore, missing blast-related covariates were not imputed for patients with B-cell NHL and the effects were only modeled in patients with BCP-ALL. In addition, age effect was also modeled solely for patients with BCP-ALL, as patients with NHL only consisted of adults.

Table 1 Covariates examined in a pediatric population pharmacokinetic analysis of inotuzumab ozogamicin
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2.4 Model Evaluation

Covariate selection was based on parameter precision, biological plausibility, and statistical significance. For hierarchically nested models, a drop of the objective function value ≥10.83, corresponding to p < 0.001 (\({x}^{2}\)-distribution with 1 degree of freedom), was used to determine a significant improvement of the fit. Model performance was evaluated by goodness-of-fit diagnostic plots, and prediction and variability corrected visual predictive checks (pvcVPC) [32]. The PK parameter estimates precision was assessed using the sampling importance resampling (SIR) procedure [33].

2.5 Model-Based Exposure Estimation

Cumulative area under the concentration–time curve (AUC) for cycle 1 was estimated using the maximum a posteriori Bayesian estimation with the POSTHOC option of NONMEM. The final model with actual trial dosing records was used to estimate InO exposure for each pediatric patient. Differences in cumulative AUC at the end of cycle 1 were compared between responders and non-responders and between positive/negative-MRD status among responders, to preliminarily evaluate the exposure–efficacy relationship. Cumulative AUC at the end of cycle 1 was selected as the exposure metric for statistical tests because of the positive association between the InO average concentration (calculated as the ratio of cumulative AUC over timeframe) and the efficacy endpoint in adults with R/R BCP-ALL [34]. The Wilcoxon rank-sum test and logistic regression with cumulative AUC and covariates included in the final model were used to examine the preliminarily exposure–efficacy relationship. Hematologic response was defined as patients with < 5% blasts in the bone marrow and no circulating blasts or extramedullary disease. Minimal residual disease negativity was defined as MRD < 1 × 10-4 with real-time quantitative-polymerase chain reaction or < 0.01% with flow cytometry when the polymerase chain reaction was negative but the quantitative range was > 1 × 10-4. The relationship between pharmacodynamic parameters and the response was analyzed before and published by our group [20].

2.6 Model-Based Simulations

Simulations of the final InO population PK model were performed to simulate the expected concentration–time profile in adult and pediatric patients with BCP-ALL at a fixed dosing regimen (the approved dosing regimen for adult R/R BCP-ALL and the pediatric RP2D: 1.8 mg/m2/cycle fractionated in three weekly administrations for the first cycle of 21 days, followed by 1.5 mg/m2/cycle for up to five cycles of 28 days). The simulations were employed to assess the PK endpoints in adult and pediatric patients, such as the cumulative AUC and terminal half-life. In addition, to evaluate whether similar InO exposure can be achieved without a loading dose, the final PK model was used for simulation in adult and pediatric patients with BCP-ALL at the above-mentioned fixed dosing regimen without a loading dose on day 1 in the first cycle (1.5 mg/m2/cycle fractionated evenly in three weekly administrations for the first cycle of 21 days, followed by 1.5 mg/m2/cycle for up to five cycles of 28 days).

2.7 Software

Nonlinear mixed-effects modeling was performed using NONMEM (version 7.5.0; ICON Development Solutions, Ellicott City, MD, USA) and Pearl-speaks-NONMEM (PsN, version 5.3.0) with stochastic approximation expectation maximization (SAEM) and importance sampling (IMP) expectation maximization as an estimation method [35,36,37]. Parameter precision was obtained by SIR as implemented in PsN [33]. Pirana (version 2.9.9) was used as a graphical user interface for NONMEM [38]. R (version 4.2.1) was used for data handling and visualization.

3 Results

3.1 Population PK Analysis Dataset and Patient Characteristics

The dataset included 8924 serum InO PK observations from 818 patients; 5609 observations were from 531 adult patients with NHL, 2752 observations from 234 adult patients with BCP-ALL, and 563 observations from 53 pediatric patients with BCP-ALL (13 treated at dose level 1 and 40 at dose level 2). Among children, ten patients received three cycles, 20 received two cycles, and 23 received one cycle only. A total of 3394 observations (38.03%) were below the LLOQ; whereas only two observations were below the LLOQ in pediatric patients with BCP-ALL. Patient baseline characteristics and covariates are summarized in Table 2 and in Table 3 of the ESM.

Table 2 Summary of patient baseline covariates
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3.2 InO Population PK model

After re-estimating the PK parameters and covariate effects of the adult InO population PK model [23] based on observations from both adult and pediatric patients, there is a difference in the distribution of empirical Bayes estimate of IIV (\(\eta\)) on kdes between pediatric and adult patients (Fig. 2a). The difference in IIV distribution and the negative trend across age categories (Fig. 2b) demonstrate that the model did not appropriately account for the age effect on kdes. Further model development steps to examine the potential pediatric population relevant differences in our analysis and the corresponding changes in model fit are shown in Table 4 of the ESM. The results of the model development include: (i) inclusion of a separate residual error on the pediatric population; (ii) replacing BBSA by LBM to represent the body size effect and replacing percentage of blast in peripheral blood by absolute counts (BLSTPB by BLSTABL), which both did not significantly affect the model fit, yet aided in the clinical interpretation; (iii) introduction of age on kdes and ALL effect on CL2, which improved the model fit associated with a p-value < 0.001. The inclusion of age effect on kdes reflects the difference in the decline rate of the target-mediated drug clearance (CLt) across age. In patients with NHL, a higher initial value of CLt was found compared with patients with ALL, indicated by the inclusion of the ALL effect on CL2. No further covariate inclusion significantly improved the model performance. Inotuzumab ozogamicin PK parameter estimates and the final population PK model are summarized in Table 3.

Fig. 2
figure 2

Distribution of interindividual variability on the decay coefficient (kdes) from the previously developed adult model after re-estimation versus (a) adult and children population and (b) age categories, the red solid line is the reference line (y = 0)

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Table 3 Result of final InO population PK model and parameter estimates
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Compared to the adult patients with BCP-ALL in the previous population PK analysis [23], a similar effect magnitude of body size and blasts in peripheral blood were identified in pediatric patients using the pooled data in our analysis. A larger body size was associated with a higher CL1, V1, and CL2. Lean body mass is considered to be more relevant to organs contributing to the endogenous immunoglobulin G antibody clearance [26]; therefore, LBM is used to represent body size instead of BBSA. Compared with patients with a median LBM (52.7 kg), in individuals with a low LBM (37.5 kg; tenth percentile), CL1 decreased by 29.0%, V1 by 29.1%, and CL2 by 21.4%, leading to higher InO exposures (e.g., cumulative AUC, given the same overall dose). Whereas for patients with a high LBM (69.0 kg; 90th percentile), CL1 increased by 31.3%, V1 by 30.9%, and CL2 by 21.0%, leading to lower exposures. An increase in peripheral blood blasts count was related to a decrease in kdes; hence a decrease in the decline rate of CLt. However, considering the magnitude of the BLSTABL effect and the rapid decline in CLt (reduce by > 50% within 1 week for patients with BCP-ALL with typical covariate values), BLSTABL is not deemed a clinically relevant effect on InO disposition over the treatment duration, as described by Garrett et al. [23].

For patients with BCP-ALL, in addition to the covariates identified in the adult model, an additional age effect on kdes was observed in our analysis. Increasing age was correlated with a decrease in kdes (Fig. 3), reflecting that the target-mediated drug clearance (CLt)declined more rapidly in children compared with adults. Relative to the time for CLt to reduce by 50% at the age of 60 years (158 hours, for patients with BCP-ALL with the median BLSTABL value), the corresponding times required at age 1, 12, 18, and 30 years were 47.0 hours, 98.2 hours, 111 hours, and 129 hours, respectively. It is noteworthy that CLt is one of the components contributing to the total clearance of InO, where the contribution of CLt reduced over time. Its contribution decreased by 90% after 3.12 weeks (age 60 years), 0.93 weeks (age 1 year), 1.94 weeks (age 12 years), and 2.19 weeks (age 18 years). Therefore, the linear clearance component predominates after the first few weeks of treatment, indicating the effect of age on kdes is not considered clinically meaningful.

Fig. 3
figure 3

Distribution of decay coefficient of the time-dependent clearance versus age in patients with B-cell precursor acute lymphoblastic leukemia. The blue dotted lines are the LOESS

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3.3 Model Evaluation

Goodness-of-fit diagnostic plots of the final model showed no indication of model misspecification in both children (Fig. 4) and adults (Fig. 1 of the ESM). Furthermore, the pvcVPC indicated the observed data were aligned with simulated predictions from the final model for pediatric patients (Fig. 5) and adult patients (Fig. 2 of the ESM). Pharmacokinetic parameter estimates precision was verified through the stable estimates and confidence interval from the SIR procedure (Table 4). In addition, the distribution of the empirical Bayes estimate of IIV (\(\eta\)) on kdes was centered around 0 against age after the inclusion of age in the model (Fig. 6), suggesting that the final model appropriately describes the PK difference across ages. Last, IIV distribution on PK parameters against other covariates (Fig. 3 of the ESM) also indicates the final model properly addressed the PK variability associated with the covariates.

Fig. 4
figure 4

Goodness-of-fit diagnostic plots of the final model for pediatric patients with B-cell precursor acute lymphoblastic leukemia (ALL). Log observed inotuzumab ozogamicin concentration versus a population prediction and b individual prediction. The solid lines show the reference line (y = x). c Scatter plots of conditional weighted residuals against population prediction and d time after each dose

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Fig. 5
figure 5

Prediction-corrected and variability-corrected visual predictive checks in a pediatric B-cell precursor acute lymphoblastic leukemia population. Black circles represent the observed data. The black lines show the median (solid) and the 10th and 90th percentiles (dash) of the observed data. The shaded regions show the 95% confidence interval of the median (red) and the 10th and 90th percentiles (blue) of the simulated concentration (N = 1000). hrs hours

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Table 4 Final model-based inotuzumab ozogamicin exposure estimation
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Fig. 6
figure 6

Distribution of interindividual variability on the decay coefficient (kdes) from the final model versus a the adult and children population and b age categories, the red solid line is the reference line (y = 0)

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3.4 Model-Based Exposure Estimation

Estimated by the final population PK model with the clinical trial dosing records, in pediatric trial participants, the median cumulative AUC at the end of cycle 1 was significantly higher in responders (n = 42) compared with non-responders (n = 10) [26.1 vs 10.1 ng*h/mL, p < 0.001; Fig. 7, Table 4]. Among responders, the median cumulative AUC at the end of cycle 1 was slightly higher in MRD-negative patients when compared with MRD-positive patients (26.4 vs 21.8 ng*h/mL, p = 0.235; Fig. 7, Table 4). A multi-covariate (cumulative AUC, LBM, age, and BLSTABL) logistic regression analysis indicates a statistically significant positive association between cumulative AUC and overall response at the end of the first cycle (p = 0.005, data not shown). Comparisons at later cycles were not considered, as receiving additional cycles might be dependent on treatment responses after cycle 1 and the interpretation limited because of the small sample size. Finally, it is worth noting that responders exhibited a faster rate of decline in CLt as compared with non-responders. This distinction is substantiated by a higher kdes value caused by a higher variability median (0.168 vs −0.724; p < 0.001) in responders. This might explain the difference in cumulative AUC between responders and non-responders.

Fig. 7
figure 7

Estimation of inotuzumab ozogamicin exposure in pediatric patients with B-cell precursor acute lymphoblastic leukemia using the final model and dosing record in the trial. (a) Cumulative area under the concentration–time curve (AUC) at the end of cycle 1 for non-responders and responders. (b) Cumulative AUC at the end of each cycle 1 for minimal residual disease (MRD)-positive and MRD-negative patients

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3.5 Model-Based Simulations

A fixed dosing regimen at the RP2D was used for the final model-based simulation. For adult and pediatric patients with BCP-ALL following the fixed dosing scheme, the simulated concentration–time profiles are generally overlapped and the steady state was fully achieved by the fourth cycle (Fig. 8). The terminal beta half-life of the adult and pediatric patients with BCP-ALL was 285 hours (11.9 days) and 423 hours (17.6 days), respectively. The predicted median cumulative AUC at the end of each cycle (Fig. 7, Table 5) showed a 30–35% higher exposure in pediatric patients compared with adults. A 9% lower predicted median cumulative AUC was reached in patients who received the fixed dosing regimen without the loading dose on day 1 in the first cycle (Table 5). The simulation results stratified by age strata are presented in the Appendix (Table 5 and Fig. 4 of the ESM).

Fig. 8
figure 8

Simulation of inotuzumab ozogamicin (InO) in adult and pediatric patients with B-cell precursor acute lymphoblastic leukemia (ALL). a Simulated concentration–time profile for four cycles. Green lines denote InO exposure in pediatric patients and red lines denote InO exposure in adults. Medians are shown with dashed lines, the 10th and 90th percentiles are shown with solid lines. b Cumulative area under the concentration–time curve (AUC) at the end of each cycle

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Table 5 Final model-based inotuzumab ozogamicin exposure simulation
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4 Discussion

This study is the first to describe the population pharmacokinetics of InO in a pediatric population by analyzing the pooled PK data from adult patients with B-cell NHL and BCP-ALL and pediatric patients with BCP-ALL. The concentration–time profile of InO in both adult and pediatric patients were well described by the final population PK model. The structure of the model aligns with the PK analyses of similar therapeutic molecules; a two-compartment PK model was reported in most population PK analyses for monoclonal antibodies [25, 26]. The time-dependent/time-varying clearance component has been applied to other B-cell-targeting antibodies and to ADCs, for example., rituximab and gemtuzumab ozogamicin [39, 40]. The time-dependent clearance component of InO reflects the decline in the target-mediated clearance pathway, which is related to the tumor burden reduction over time, supported by the fact that complete remissions were achieved in majority of pediatric patients by the end of the first cycle [20], a time when time-dependent clearance decreased by >95% in pediatric patients.

A high correlation was observed between BBSA and LBM in the dataset; therefore, replacing BBSA by LBM did not significantly change the model as expected, and it might be plausible to use BSA and LBM interchangeably in the model. However, considering the organs contributing the most (skin, muscle, and liver) to endogenous immunoglobulin G antibody clearance [26], LBM might be a more representative and relevant covariate to represent body size. Therefore, without significantly affecting the model fit, LBM replaced BBSA in the covariate model in order to enhance the generalizability for future studies in specific subgroups. The trend and size of the LBM effect on InO disposition are consistent with the influence of BBSA reported in the adult model [23]; hence, supporting the current BSA-based dosing strategy for pediatric patients with R/R BCP-ALL.

The inclusion of age effect on kdes implies that the target-mediated drug clearance (CLt, related to tumor burden) declines more rapidly in children compared with adults. The age effect might indicate that in pediatric patients with BCP-ALL, tumor cells were depleted faster, while tumor cells were more persistent in adult patients with BCP-ALL. The discrepancy in the decline rate of target-mediated clearance might suggest a difference in the rate of intracellular calicheamicin accumulation or in the sensitivity to this cytotoxic agent. This might be explained by a different role played by drug efflux pumps (such as P-glycoprotein and multidrug resistance-associated protein) in the unresponsiveness to InO [16]. While in adult patients with BCP-ALL drug efflux pumps have a notable role in resistance [16, 41], their involvement is less likely in drug resistance in pediatric BCP-ALL [16, 42, 43]. Other possible reasons for a different time-dependent clearance kinetics between children and adults might reside in the CD22 expression. In children, most trials with InO reported baseline percentages of CD22+ blasts in peripheral blood in the 90% range or higher [20, 21], while in adults this percentage might be lower [19]. Nevertheless, the percentage of CD22+ positive blasts in peripheral blood is only an approximation of the total quantity of CD22 receptors in the central compartment, which is dependent also on the CD22+ density on the blast surface as well as the absolute value of blasts and mature B cells (also expressing CD22) [44]. Another mechanism that is known to reduce the cytotoxic effect of calicheamicin-based ADCs is the overexpression of genes for BCL-2 family members [45]. No correlation between response and BCL-2 gene expression was observed in the phase II of trial ITCC-059, despite being tested on a small scale [20]. Nevertheless, a synergetic effect between InO, the BCL-2 inhibitor Venetocalx, and dexamethasone was observed in a small, murine, patient-derived, xenograft ALL model [46]. Whether or not differences in the BCL-2 family might correlate with a faster decline in the leukemic burden of patients with ALL is not known, but for example, BCL2/MYC rearrangements are associated with a poor prognosis in ALL and are usually present only in adults and adolescents [47].

It is worth mentioning that phase III trials conducted in adults with ALL treated with InO also reported an 80% overall response rate [19], similar to what is observed in children. From other calicheamicin-loaded ADCs such as gemtuzumab ozogamicin in children, the PK data were reported by Buckwalter et al. [48]. Interestingly, in this case, the PK profile in children seems to overlap closely with the profile seen in adults. Similarly to InO, a higher mean exposure (AUC) was observed in younger patients (infants and children below 11 years of age), treated at 9 mg/m2 compared with adults in period one (134, 184, and 123 mg*h/L, respectively), despite a wide variability shown by the high standard deviation [48].

Estimated from pediatric patients with BCP-ALL following the dosing scheme in the phase IA and phase II part of the ITCC-059 Trial, the cumulative AUC was positively associated with overall response and significantly higher among responders at the end of the first cycle. This is in agreement with the exposure–response analysis for efficacy of InO in adult patients with R/R BCP-ALL [34], where InO exposure (average concentration, as the ratio of cumulative AUC over time) is significantly and positively correlated with achieving remission and MRD negativity. There has been limited information regarding the relationship between InO exposure and clinical effectiveness in children. Therefore, the cumulative AUC value estimated from responding pediatric trial participants could be considered as a preliminary reference of an effective InO exposure in pediatric BCP-ALL. From the simulation in patients with BCP-ALL following the RP2D, a 30–35% higher cumulative AUC in pediatric patients compared with adult patients with BCP-ALL was found. However, the simulation results demonstrated that the effective InO exposure level was reached in pediatric patients with BCP-ALL following the RP2D; moreover, the RP2D was well tolerated with a satisfactory response rate [20, 24]. Therefore, clinically, no further dose adjustment is required for pediatric patients with BCP-ALL.

A limitation of the study is the potential influence on InO exposure estimation due to higher shrinkage on kdes, which might result in a biased interpretation of the p-value. However, the contribution of CLt reduced rapidly. Therefore, the effect of the high shrinkage on kdes on clearance estimates is considered mild. Another limitation relates to the lack of prior knowledge on therapeutic monitoring and the causal inference of the exposure and efficacy relationship of InO in children. It is commonly reported that the exposure–response relationship of monoclonal antibodies is, at least partially, confounded by general disease risk factors or the underlying immune system [49]. Ergo, it was uncertain whether the InO exposure in pediatric patients with BCP-ALL following a lower dosing regimen (i.e., without a loading dose) is sufficient. A reduction in the InO dose could not only be beneficial from a pharmacoeconomic point of view but also for safety concerns, as in adults, sinusoidal obstruction syndrome was associated with a higher InO exposure [34]. At present, several trials are evaluating combinations of InO, often at a dose inferior to the RP2D of trial ITCC-059, combined with low-intensity chemotherapy and immunotherapies (e.g., blinatumomab), for example, by the MD Anderson Center and the Children’s Oncology Group [50, 51]. More studies are required to unravel the exposure–response and exposure–safety correlations of InO in pediatrics.

5 Conclusions

The PK profile of InO in pediatric patients with R/R BCP-ALL was well described by our model using pediatric and adult data. In patients with BCP-ALL, compared to the adult model [23], a similar body size effect on InO clearance and distribution and blasts in peripheral blood effect on kdes were identified, whereas the additional age effect may provide further physiological insights into the difference between adult and pediatric BCP-ALL. Despite the difference in simulated InO exposure between adult and pediatric patients, children receiving the RP2D achieved a desirable cumulative AUC at the end of the first cycle; additionally, the RP2D has been reported to be well tolerated in pediatrics [20, 24]. Therefore, no dose adjustment is required in pediatric patients with R/R BCP-ALL for clinical reasons.