Pharmacokinetics and pharmacodynamics of ch14.18/CHO in relapsed/refractory high-risk neuroblastoma patients treated by long-term infusion in combination with IL-2

Abstract

Ch14.18 manufactured in Chinese hamster ovary (CHO) cells is currently being evaluated in clinical trials. Short-term infusion (STI) (8-20 h/day; 4-5 days) of 100 mg/m2 ch14.18/CHO (dinutiximab β) per cycle in combination with cytokines is standard treatment of neuroblastoma (NB) patients. As pain is a limiting factor, we investigated a novel delivery method by continuous long-term infusion (LTI) of 100 mg/m2 over 10 days. 53 NB patients were treated with 5-6 cycles of 6 × 106 IU/m2 subcutaneous interleukin-2 (d 1-5, 8-12), LTI of 100 mg/m2 ch14.18/CHO (d 8-18) and 160 mg/m2 oral 13-cis-retinoic acid (d 22-35). Human anti-chimeric antibody (HACA), antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity were determined. With LTI, we observed a maximum concentration of ch14.18/CHO (Cmax) of 12.56 ± 0.68 µg/ml and a terminal half-life time (t1/2 β) of 32.7 ± 16.2 d. The clearance values for LTI and STI of 0.54 ± 0.13 and 0.41 ± 0.29 L/d m2 and area under the serum concentration-time curve (AUC) values of 189.6 ± 41.4 and 284.8 ± 156.8 µg×d/ml, respectively, were not significantly different. Importantly, we detected ch14.18/CHO trough concentration of ≥ 1 µg/ml at time points preceding subsequent antibody infusions after cycle 1, allowing a persistent activation of antibody effector mechanisms over the entire treatment period of 6 months. HACA responses were observed in 10/53 (19%) patients, similar to STI (21%), indicating LTI had no effect on the immunogenicity of ch14.18/CHO. In conclusion, LTI of ch14.18/CHO induced effector mechanisms over the entire treatment period, and may therefore emerge as the preferred delivery method of anti-GD2 immunotherapy to NB patients.

Keywords: Ch14.18/CHO; HACA; immunotherapy; neuroblastoma; pharmacokinetics.

Figures

Figure 1.

Sensitivity and precision optimization of the ch14.18/CHO ELISA method. Sensitivity and precision of the ch14.18 ELISA method were improved following a two-step procedure (A and B). First, the optimal minimal sample dilution factor was evaluated using the reported ELISA protocol (A). Standard samples (1.0, 0.5, 0.25, 0.13, 0.06, 0.03 and 0 μg/ml) were diluted 1:70 (closed triangles), 1:100 (open triangles), 1:200 (closed squares) and 1:400 (open squares) and analyzed by ELISA. Results are presented as mean OD of three replicates ± SD from two representative experiments. Second, the optimal minimal sample dilution factor of 1:70 was used to further improve the precision by distinct modifications of sample-, wash- and blocking-buffers as described in “Materials and Methods” (B). Standard samples (1.0, 0.5, 0.25, 0.13, 0.06, 0.03 and 0 μg/ml) were diluted 1:70 and analyzed by “high sensitivity” ELISA. Results are presented as mean OD of three replicates ± SD from two representative experiments. Optimization of the buffer composition resulted in an improved linear fit of the standard curve (R2 = 0.998, closed circles), compared to the previously reported ELISA protocol (R2 = 0.991, closed triangles). (C) LOD for the “high sensitivity” ELISA method was determined using 11 samples containing serum of a healthy donor without ch14.18/CHO (“zero analyte”) analyzed on the same plate. The solid line indicates the LOD of 58 ng/ml, calculated as follows:mean + 3× SD. When error bars are not visible they are covered by the symbol.

Figure 2.

Within- and inter-assay precision of ch14.18/CHO “high sensitivity” ELISA and stability of low ch14.18/CHO concentrations in serum. For reliable and reproducible detection of ch14.18/CHO concentrations with the “high sensitivity” ELISA, within- (A) and inter- (B) assay precision analyses were performed. Two samples containing known concentrations of ch14.18/CHO (0.6 μg/ml, open circles and 0.3 μg/ml, closed circles) were prepared in human serum and analyzed as described in the “Materials and Methods” section. CVs for both concentrations were calculated according to the formula:SD/mean × 100% and were found to be in the range of ± 20% (indicated by the dashed line). Mean values of the respective data sets are also indicated (solid line). To determine stability of low ch14.18/CHO concentrations in serum, samples of two defined concentrations (0.6 μg/ml (open circles) and 0.3 μg/ml (closed circles)) were prepared. Aliquots were subjected to either eight freeze-thaw cycles (C) or storage at RT for up to 168 h (7d) (D) and analyzed using the “high sensitivity” ch14.18/CHO-ELISA. When error bars are not visible they are covered by the symbol. Data represent mean ch14.18/CHO concentrations of at least three replicates ± SD from two representative experiments.

Figure 3.

Serum levels of ch14.18/CHO of cycle 1 in neuroblastoma patients. Samples collected from patients treated with (A) continuous infusion of ch14.18/CHO over 10 days (10× 10 mg/m2/d; LTI; 13/53) or (B) 8 h infusion over five days (5× 20 mg/m2/d; STI; 8/16) were evaluated using the triple-ELISA strategy as described in the Materials and Methods section. Ch14.18/CHO levels were analyzed prior to start (d 8), during (d 8-13 for STI and d 8-18 for LTI) and after the end of Ab infusion (d 14-40 for STI and d 19-43 for LTI). The intravenous administration of ch14.18/CHO is indicated as a solid line for LTI and as a dashed line for STI treatment regimen. Data represent concentration time curves of each patient (gray lines) and the mean (bold black line) for cycle 1.

Figure 4.

Ch14.18/CHO concentration-time profiles of LTI- and STI treated patients. Serum concentrations of ch14.18/CHO were determined with triple ELISA strategy and concentration-time fits were obtained with the two-compartment model. Results of two representative subjects of the LTI- (A) (left LTI-1, right LTI-2) and STI treatment regimen (B) (left STI-1, right STI-4) are shown. The population means and inter-individual variabilities (SD) of five pharmacokinetic parameters were estimated using a two-compartment model (CL, Vdss, MRT, t1/2 α, and t1/2 β; Table 2) and the AUCs were calculated as AUC = dose/CL (Table 1). The clearance values obtained from the LTI and STI data were not statistically different (Table 2). Consequently, also no significant difference between the AUCs (189.6 ± 41.4 µg×d/ml for LTI and 284.8 ± 156.8 µg×d/ml STI) were found. Similarly, the terminal half-lives (t1/2 β) observed with STI and LTI were not different. Gender-specific and age related analysis of all parameters in patients treated by LTI or STI did not reveal significant differences.

Figure 5.

Effect of HACA on ch14.18/CHO concentration-time curves. Serum samples collected from 37/53 patients treated with ch14.18/CHO LTI were evaluated using the triple-ELISA strategy as described in “Materials and Methods” prior to start (d 1-8), during (d 8-18; indicated by the gray field) and after the end of Ab infusion (d 19-35) in every treatment cycle. Ch14.18/CHO levels in the circulation of HACA-negative patients (n = 30) (A) were compared to HACA-low (n = 4) (B) and HACA-high responders (n = 3) (C). Data are shown as mean values ± SEM of experiments performed in triplicate; t-test or Mann-Whitney Rank Sum test; §P < 0.05 vs. day 18, cycle 1; *P < 0.001 vs. baseline. When error bars are not visible they are covered by the symbol. Solid lines indicate the trend increase in Cmax over time and the 1 µg/ml ch14.18/CHO level. Numbers indicate the Cmax levels in cycles 1-5.

Figure 6.

Ch14.18/CHO-mediated ADCC and impact of HACA response. (A) Induction of GD2-specific ch14.18/CHO-mediated ADCC in 49 patients treated with the LTI regimen was determined in every cycle seven days after the start of Ab infusion (d 8) (black column) and compared to baseline ADCC (d 1) of the respective cycle (white column) using the calcein-AM-based cytotoxicity assay as described in “Materials and Methods.” Data are shown as mean values ± SEM of experiments performed in six replicates. Mann-Whitney Rank Sum test or one-way ANOVA, followed by appropriate post hoc comparison test; *P < 0.05 vs. baseline (prior to the first Ab infusion, d 1, cycle 1); §P < 0.05 vs. d 1 of the respective cycle. (B) The effect of HACA on ch14.18/CHO-mediated GD2-specific ADCC against NB cells was evaluated in 3/53 HACA-high responders (gray circles) and 5/53 HACA-low responders (white circles) and compared with 41/53 HACA-negative patients (black circles). The solid lines in black, white and gray indicate cycle-specific median values of ADCC activity in HACA-negative-, HACA-low- and HACA-high responders, respectively, and the solid thin line in black indicates baseline ADCC prior to the first Ab infusion (d 1, cycle 1). Data are shown as patient-specific ADCC (percentage values), Mann-Whitney Rank Sum test; *P < 0.05 vs. baseline. (C) The effect of HACA response on ADCC was determined at time points prior to subsequent treatment cycles (d 1 of cycles 2, 3, 4 and 5; ch14.18/CHO trough levels) and compared to baseline ADCC on day 1 of cycle 1 prior to the first Ab administration (Fig. 5C). The solid line indicates baseline ADCC activity prior to the first Ab infusion (d 1, cycle 1). Data are shown as mean values ± SEM of experiments performed in six replicates. Differences between the groups were not significant, Mann-Whitney Rank Sum test or one-way ANOVA, followed by appropriate post hoc comparison test.

Figure 7.

Ch14.18/CHO-mediated CDC and impact of HACA response. (A) Induction of a GD2-specific ch14.18/CHO-mediated CDC in 53/53 patients treated with the LTI regimen. CDC was determined in every cycle seven days after the start of Ab infusion (d 8) (black column) and compared to baseline (prior to the first Ab infusion, d 1, cycle 1) or day 1 of the respective cycle (white column) using the calcein-AM-based cytotoxicity assay as described in “Materials and Methods.” The solid line indicates baseline CDC activity prior to the first Ab infusion (d 1, cycle 1). Data are shown as mean values ± SEM of experiments performed in six replicates, Mann-Whitney Rank Sum test or one-way ANOVA, followed by appropriate post hoc comparison test; *P < 0.05 vs. baseline; §P < 0.05 vs. d 1 of the respective cycle. (B) The effect of HACA response on ch14.18/CHO-mediated GD2-specific CDC against NB cells was evaluated in serum samples of 4/53 HACA-high responders (gray circles) and 6/53 HACA-low responders (white circles) and compared with 43/53 HACA-negative patients (black circles). The solid lines in black, white and gray indicate cycle-specific median values of CDC activity in HACA-negative-, HACA-low- and HACA-high responders, respectively, and the solid thin line indicates baseline CDC prior to the first Ab infusion (d 1, cycle 1). Data are shown as CDC mean values ± SEM of experiments performed in six replicates, Mann-Whitney Rank Sum test or one-way ANOVA, followed by appropriate post hoc comparison test; *P < 0.05 vs. baseline; §P < 0.05 vs. HACA-negative patients of the respective cycle; #P < 0.05 vs. HACA-low responders of the respective cycle. (C) The effect of HACA response on CDC activity in patient serum was determined in HACA-positive patients at time points prior to subsequent treatment cycles (d 1 of cycles 2, 3, 4 and 5; ch14.18/CHO trough levels) and compared to HACA-negative patients and baseline CDC on day 1 of cycle 1 prior to the first Ab administration. Results show two cohorts of HACA-positive patients (HACA-low responders (white columns, n=6) and HACA-high responders (gray columns, n=4)) and HACA-negative patients (black columns, n=43). The solid line indicates baseline CDC activity prior to the first Ab infusion (d 1, cycle 1). Data are shown as mean values ± SEM of experiments performed in six replicates. Mann-Whitney Rank Sum test or one-way ANOVA, followed by appropriate post hoc comparison test; *P < 0.05 vs. baseline; #P < 0.05 vs. HACA-negative patients of the respective cycle.