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This figure demonstrates the relationship between prior treatment with chimeric or humanized 14.18 K322A antibody and A) the area under the concentration curve for the antibody (AUC); B elimination half-life of the antibody (T1/2 Beta); and C) the highest observed concentration of the antibody (Cmax). Although the data in panels A and B suggest that prior exposure to ch14.18 or hu14.18K322A tends to increase the AUC and the elimination half-life, there is no significant difference between prior treatment groups using the Kruskal-Wallis non-parametric test. The data in panel C do not suggest an association between prior exposure and the peak serum concentration of the hu14.18K322A antibody.

Box and whisker plot showing absolute NK cells per mm3 in samples. NK cells were identified by flow cytometry as CD14 negative, CD56 positive, CD3 negative and percentage of total WBC was computed from flow cytometry results. Absolute WBC count was obtained using a Sysmex{trade mark, serif} KX-21 blood cell analyzer, and final absolute NK count was obtained by multiplying percent NK by absolute WBC. Sample identifiers and associated N are: DONOR (N=10) blood sample drawn at or before time of first NK cell donation; CS1_PRE (N=13), Patient blood drawn prior to first treatment course; CS1_14 (N=9); Patient on day +14 of first treatment course; CS1_21 (N=14), Patient on day +21 of first treatment course; CS2_NK_07 (N=11), Patient on day +7 following NK cell administration in second treatment course; CS2_NK_14 (N=11), Patient on day +14 following NK cell administration in second treatment course; CS3_14 (N=7), Patient on day +14 of third treatment course; CS3_21 (N=9), Patient on day +21 of third treatment course; CS4_NK_07 (N=9), Patient on day +7 following NK cell administration in fourth treatment course; CS4_NK_14 (N=10), Patient on day +14 following NK cell administration in fourth treatment course; CS5_14 (N=3), Patient on day +14 of fifth treatment course; CS5_21 (N=8), Patient on day +21 of fifth treatment course; CS6_PRE (N=1), Patient blood drawn prior to initiation of 6th treatment course; CS6_NK_07 (N=7), Patient on day +7 following NK cell administration in sixth treatment course; CS6_NK_14 (N=7), Patient on day +14 following NK cell administration in sixth treatment course. Central Point (ï�¯) - median; Box - Inner quartiles (25th percentile to 75th percentile) of data; Whiskers - non-outlier min and max data; Outliers (ï,¡) - data points outside of median {plus minus} inner quartile range; Extreme Outliers (*) - data points outside of median {plus minus} 1.5 X inner quartile range.

Human anti-human antibody (HAHA) level and peak hu14.18K322A serum level by course and prior antibody therapy.

Purpose: Anti-GD2 mAbs, acting via antibody-dependent cell-mediated cytotoxicity, may enhance the effects of chemotherapy. This pilot trial investigated a fixed dose of a unique anti-GD2 mAb, hu14.18K322A, combined with chemotherapy, cytokines, and haploidentical natural killer (NK) cells.Experimental Design: Children with recurrent/refractory neuroblastoma received up to six courses of hu14.18K322A (40 mg/m2/dose, days 2–5), GM-CSF, and IL2 with chemotherapy: cyclophosphamide/topotecan (courses 1,2), irinotecan/temozolomide (courses 3,4), and ifosfamide/carboplatin/etoposide (courses 5,6). Parentally derived NK cells were administered with courses 2, 4, and 6. Serum for pharmacokinetic studies of hu14.18K322A, soluble IL2 receptor alpha (sIL2Rα) levels, and human antihuman antibodies (HAHA) were obtained.Results: Thirteen heavily pretreated patients (9 with prior anti-GD2 therapy) completed 65 courses. One patient developed an unacceptable toxicity (grade 4 thrombocytopenia >35 days). Four patients discontinued treatment for adverse events (hu14.18K322A allergic reaction, viral infection, surgical death, second malignancy). Common toxicities included grade 3/4 myelosuppression (13/13 patients) and grade 1/2 pain (13/13 patients). Eleven patients received 29 NK-cell infusions. The response rate was 61.5% (4 complete responses, 1 very good partial response, 3 partial responses) and five had stable disease. The median time to progression was 274 days (range, 239–568 days); 10 of 13 patients (77%) survived 1 year. Hu14.18K322A pharmacokinetics was not affected by chemotherapy or HAHA. All patients had increased sIL2Rα levels, indicating immune activation.Conclusions: Chemotherapy plus hu14.18K322A, cytokines, and NK cells is feasible and resulted in clinically meaningful responses in patients with refractory/recurrent neuroblastoma. Further studies of this approach are warranted in patients with relapsed and newly diagnosed neuroblastoma. Clin Cancer Res; 23(21); 6441–9. ©2017 AACR.

The left panel plots the calculated area under the concentration vs time curve (AUC) of hu14.18K322A and the right panel plots the highest observed serum concentration of hu14.18K322A (Cmax) against the pre-treatment concentration of HAHA antibodies, reported in units of optical density. There is no clear association between the concentration of HAHA at the start of treatment and the AUC of hu14.18K322A and the peak concentration of hu14.18K322A using the Spearman test.

Plot of ADCC assay reports comparing 6 lots of hu14.18K322A in comparison to dinutuximab (unituxin). M21 cells, expressing the GD2 cell surface epitope, served as the target cells. Engineered Jurkat cells, with a cell-surface FcyRIIIa (V158) receptor, served as the effector cells. Crosslinking of the two cells by either hu14.18K322A or dinutuximab led to gene transcription through NFAT (nuclear factor of activated T-cells) with subsequent NFAT response element (RE) driving expression of a firefly luciferase reporter protein. The luciferase reporter substrate was converted to a luminescent product and quantified as relative luminescence units (RLU) on a plate reader.

Tables of antibody infusion times and Curie Scores

Purpose:We sought to evaluate whether combining a humanized antidisialoganglioside mAb (hu14.18K322A) with induction chemotherapy improves early responses and outcomes in children with newly diagnosed high-risk neuroblastoma.Patients and Methods:We conducted a prospective nonrandomized, single-arm, two-stage, phase II clinical trial. Six courses of induction chemotherapy were coadministered with hu14.18K322A and followed with granulocyte–macrophage colony-stimulating factor (GM-CSF) and low-dose IL2. Consolidation was performed with a busulfan/melphalan preparative regimen. An additional course of hu14.18K322A was administered with parent-derived natural killer cells, when available, during consolidation. Hu14.18K322A, GM-CSF, IL2, and isotretinoin were then administered. Secondary outcomes included reduced tumor volume and semiquantitative 123I-metaiodobenzylguanidine scoring [i.e., Curie scores (CS)] at the end of induction.Results:Forty-two patients received hu14.18K322A and induction chemotherapy. This regimen was well tolerated, with continuous-infusion narcotics adjusted to patient tolerance. Partial responses (PR) or better after the first two chemoimmunotherapy courses occurred in 32 patients [76.2%; 95% confidence interval (CI), 60.6–88.0]. This was accompanied by primary tumor volume reductions (median, –76%; range, –100% to 5%). Of 35 patients with stage IV disease who completed induction, 31 had end-of-induction CSs of 2 or less. No patients experienced progression during induction. Two-year event-free survival (EFS) was 85.7% (95% CI, 70.9–93.3).Conclusions:Adding hu14.18K322A to induction chemotherapy produced early PR or better in most patients, reduced tumor volumes, improved CSs at the end of induction, and yielded an encouraging 2-year EFS. These results, if validated in a larger study, may change the standard of care for children with high-risk neuroblastoma.

Individual pharmacokinetic parameters in 3 subpopulations (no prior 14.18 mAb, prior dinutuximab mAb, prior hu14.18K322A mAb).

ADCC EC50 values of hu14.18K322A and dinutuximab.

Donor characteristics, dose and chimerism of NK cells infused.

Purpose: We sought to evaluate whether combining a humanized antidisialoganglioside mAb (hu14.18K322A) with induction chemotherapy improves early responses and outcomes in children with newly diagnosed high-risk neuroblastoma. Patients and Methods: We conducted a prospective nonrandomized, single-arm, two-stage, phase II clinical trial. Six courses of induction chemotherapy were coadministered with hu14.18K322A and followed with granulocyte–macrophage colony-stimulating factor (GM-CSF) and low-dose IL2. Consolidation was performed with a busulfan/melphalan preparative regimen. An additional course of hu14.18K322A was administered with parent-derived natural killer cells, when available, during consolidation. Hu14.18K322A, GM-CSF, IL2, and isotretinoin were then administered. Secondary outcomes included reduced tumor volume and semiquantitative 123I-metaiodobenzylguanidine scoring [i.e., Curie scores (CS)] at the end of induction. Results: Forty-two patients received hu14.18K322A and induction chemotherapy. This regimen was well tolerated, with continuous-infusion narcotics adjusted to patient tolerance. Partial responses (PR) or better after the first two chemoimmunotherapy courses occurred in 32 patients [76.2%; 95% confidence interval (CI), 60.6–88.0]. This was accompanied by primary tumor volume reductions (median, –76%; range, –100% to 5%). Of 35 patients with stage IV disease who completed induction, 31 had end-of-induction CSs of 2 or less. No patients experienced progression during induction. Two-year event-free survival (EFS) was 85.7% (95% CI, 70.9–93.3). Conclusions: Adding hu14.18K322A to induction chemotherapy produced early PR or better in most patients, reduced tumor volumes, improved CSs at the end of induction, and yielded an encouraging 2-year EFS. These results, if validated in a larger study, may change the standard of care for children with high-risk neuroblastoma.

Purpose: Anti-GD2 mAbs, acting via antibody-dependent cell-mediated cytotoxicity, may enhance the effects of chemotherapy. This pilot trial investigated a fixed dose of a unique anti-GD2 mAb, hu14.18K322A, combined with chemotherapy, cytokines, and haploidentical natural killer (NK) cells. Experimental Design: Children with recurrent/refractory neuroblastoma received up to six courses of hu14.18K322A (40 mg/m2/dose, days 2–5), GM-CSF, and IL2 with chemotherapy: cyclophosphamide/topotecan (courses 1,2), irinotecan/temozolomide (courses 3,4), and ifosfamide/carboplatin/etoposide (courses 5,6). Parentally derived NK cells were administered with courses 2, 4, and 6. Serum for pharmacokinetic studies of hu14.18K322A, soluble IL2 receptor alpha (sIL2Rα) levels, and human antihuman antibodies (HAHA) were obtained. Results: Thirteen heavily pretreated patients (9 with prior anti-GD2 therapy) completed 65 courses. One patient developed an unacceptable toxicity (grade 4 thrombocytopenia >35 days). Four patients discontinued treatment for adverse events (hu14.18K322A allergic reaction, viral infection, surgical death, second malignancy). Common toxicities included grade 3/4 myelosuppression (13/13 patients) and grade 1/2 pain (13/13 patients). Eleven patients received 29 NK-cell infusions. The response rate was 61.5% (4 complete responses, 1 very good partial response, 3 partial responses) and five had stable disease. The median time to progression was 274 days (range, 239–568 days); 10 of 13 patients (77%) survived 1 year. Hu14.18K322A pharmacokinetics was not affected by chemotherapy or HAHA. All patients had increased sIL2Rα levels, indicating immune activation. Conclusions: Chemotherapy plus hu14.18K322A, cytokines, and NK cells is feasible and resulted in clinically meaningful responses in patients with refractory/recurrent neuroblastoma. Further studies of this approach are warranted in patients with relapsed and newly diagnosed neuroblastoma. Clin Cancer Res; 23(21); 6441–9. ©2017 AACR.

Purpose: To determine the maximum-tolerated dose (MTD), dose-limiting toxicities (DLT), pharmacokinetics, and pharmacodynamics of sorafenib, bevacizumab, and low-dose oral cyclophosphamide in children and young adults with recurrent/refractory solid tumors. Experimental Design: Sorafenib dose was escalated from 90 to 110 mg/m2 twice daily with fixed doses of bevacizumab at 5 mg/kg every 3 weeks and cyclophosphamide at 50 mg/m2 daily. Once sorafenib's MTD was established, bevacizumab dose was escalated. Each course was of 21 days. Pharmacokinetics and pharmacodynamics studies were conducted during the first course. Results: Nineteen patients (11 males; median age, 9.2 years) received a median of four courses (range, 1–23). DLTs during course 1 included grade 3 rash (two), increased lipase (one), anorexia (one), and thrombus (one). With an additional 71 courses of therapy, the most common toxicities ≥ grade 3 included neutropenia (nine), lymphopenia (nine), and rashes (four). Five of 17 evaluable patients had partial tumor responses, and five had disease stabilization (>2 courses). Median day 1 cyclophosphamide apparent oral clearance was 3.13 L/h/m2. Median day 1 sorafenib apparent oral clearance was 44 and 39 mL/min/m2 at the 2 dose levels evaluated, and steady-state concentrations ranged from 1.64 to 4.8 mg/L. Inhibition of serum VEGF receptor 2 (VEGFR2) was inversely correlated with sorafenib steady-state concentrations (P = 0.019). Conclusion: The recommended phase II doses are sorafenib, 90 mg/m2 twice daily; bevacizumab, 15 mg/kg q3 weeks; and cyclophosphamide, 50 mg/m2 once daily. This regimen is feasible with promising evidence of antitumor activity that warrants further investigation. Clin Cancer Res; 19(1); 236–46. ©2012 AACR.