Ch14.18 antibody produced in CHO cells in relapsed or refractory Stage 4 neuroblastoma patients

Purpose: This study aimed to assess the safety, pharmacokinetic and activity profiles of the human-mouse chimeric monoclonal anti-disialoganglioside GD2 antibody ch14.18 produced in Chinese hamster ovary (CHO) cells (ch14.18/CHO).

Methods: Sixteen children with recurrent/refractory neuroblastoma (median age 7.6 y) were enrolled in this Phase 1 dose-finding study. Patients received ch14.18/CHO courses of 10, 20 or 30 mg/m²/day as an eight-hour infusion over five consecutive days. Three courses at the same dose level were allowed unless disease progressed. Clearance and biodistribution of radiolabelled ch14.18/CHO in Balb/c and A/J mice were analyzed.

Results: A total of 41 ch14.18/CHO courses were given (10 × 3 courses, 5 × 2 courses, 1 × 1 course). Side effects were similar in expectedness, frequency and magnitude to those reported for ch14.18/SP2/0. The dose level of 20 mg/m²/day was confirmed. Toxicity was reversible and no treatment-related deaths occurred. In children, the peak plasma concentration was 16.51 µg/ml ± 5.9 µg/ml and the half-life was 76.91 h ± 52.5 h. A partial response following ch14.18/CHO was observed in 2/7 patients with residual disease. In mice, the half-lives were 22.7 h ± 1.9h for ch14.18/CHO and 25.0 h ± 1.9 h for ch14.18/SP2/0. The biodistribution of ¹²⁵I-ch14.18/CHO in mice with neuroblastoma was identical to ¹²⁵I-ch14.18/SP2/0, indicating GD₂ targeting activity in vivo.

Ch14.18 produced in CHO cells showed an unchanged toxicity profile and pharmacokinetics in neuroblastoma patients compared with ch14.18 produced in SP2/0 cells, and evidence of clinical activity was observed. In mice, analysis of pharmacokinetics and biodistribution showed comparable results between ch14.18/CHO and ch14.18/SP2/0. Based on these results, ch14.18/CHO was accepted for prospective clinical evaluation.     

Tolerability,response and outcome of high-risk neuroblastoma patients treated with long-term infusion of anti-GD2 antibody ch14.18/CHO

Immunotherapy with short term infusion (STI) of monoclonal anti-GD₂ antibody (mAb) ch14.18 (4 × 25 mg/m²/d; 8–20 h) in combination with cytokines and 13-cis retinoic acid (RA) prolonged survival in high-risk neuroblastoma (NB) patients. Here, we investigated long-term infusion (LTI) of ch14.18 produced in Chinese hamster ovary cells (ch14.18/CHO; 10 × 10 mg/m²; 24 h) in combination with subcutaneous (s.c.) interleukin-2 (IL-2) in a single center program and report clinical response, toxicity and survival. Fifty-three high-risk NB patients received up to 6 cycles of 100 mg/m² ch14.18/CHO (d8–17) as LTI combined with 6 × 10⁶ IU/m² s.c. IL-2 (d1–5; 8–12) and 160 mg/m² oral RA (d19–32). Pain toxicity was documented with validated pain scores and intravenous (i.v.) morphine usage. Response was assessed in 37/53 evaluable patients following International Neuroblastoma Risk Group criteria. Progression-free (PFS) and overall survival (OS) was analyzed by the Kaplan-Meier method and compared to a matched historical control group from the database of AIEOP, the “Italian Pediatric Ematology and Oncology Association”. LTI of ch14.18/CHO showed acceptable toxicity profile indicated by low pain scores, reduced i.v. morphine usage and low frequency of Grade ≥3 adverse events that allowed outpatient treatment. We observed a best response rate of 40.5% (15/37; 5 CR, 10 PR), 4-year (4 y) PFS of 33.1% (observation 0.1- 4.9 y, mean: 2.2 y) and a 4 y OS of 47.7% (observation 0.27 – 5.20 y, mean: 3.6 y). Survival of the entire cohort (53/53) and the relapsed patients (29/53) was significantly improved compared to historical controls. LTI of ch14.18/CHO thus shows an acceptable toxicity profile, objective clinical responses and a strong signal of clinical efficacy in NB patients.     

Pharmacokinetics of human-mouse chimeric anti-GD2 mAb ch14.18 in a phase I trial in neuroblastoma patients

A comprehensive analysis of the pharmacokinetics of human-mouse chimeric anti-ganglioside GD2 antibody mAb ch 14.18 was performed during a phase I clinical trial of ten children with neuroblastoma and one adult with osteosarcoma. The patients received a total of 20 courses of ch 14.18 at dose levels from 10 mg/m² to 200 mg/m². The plasma clearance of ch 14.18 was biphasic. Following the first course of treatmentt1/2,α was 3.4±3.1 h andt1/2,β 66.6±27.4 h in 9/10 children. Thet1/2,β values were significantly less than those of 181±73 h previously reported in adult melanoma patients (P<-0.001), and 147.5 h in the adult osteosarcoma patient in our trial. The latter suggests different pharmacokinetics of mAb ch 14.18 in children and adults. After a second course of treatment, administered to 5/10 children,t1/2,β decreased significantly from 72.9±19.8 h to 31.7±18.4 h (P=0.015). We there-fore conclude that the elimination kinetics of mAbs ch 14.18 in children and adults are different, and furthermore that repeated administration of mAb ch 14.18 to children with neuroblastoma leads to accelerated antibody clearance.     

Pharmacokinetics of anti-ganglioside GD2 mAb 14G2a in a phase I trial in pediatric cancer patients

A phase I trial of a murine anti-ganglioside (GD2) monoclonal antibody (mAb) 14G2a was conducted in 14 neuroblastoma patients and 1 osteosarcoma patient to assess its safety, toxicity and pharmacokinetics in pediatric patients. The pharmacokinetics of mAb 14G2a were biphasic with at ₁ ^2/ of 2.8±2.8 h and at ₁ ^2/ of 18.3±11.8 h. In general,t ₁ ^2/ was dose-dependent with a level of significance ofP=0.036, and it reached a plateau at doses of 250 mg/m² or more. Overall the peak serum levels were dose-dependent atP<0.001. However, they demonstrated an abrupt increase between doses of 100 mg/m² and 250 mg/m². The latter two suggest a saturable mechanism for mAb elimination. In addition, peak serum concentrations were observed earlier at higher mAb doses, which indicates the achievement of a steady state. Thet ₁ ^2/ of mAb 14G2a in children appears to be shorter than in adults. Furthermore, 2 patients demonstrated a considerable decrease int ₁ ^2/ following retreatment with 14G2a. This was paralleled by high human anti-(mouse Ig) antibody levels. This study represents the first comprehensive analysis of murine mAb pharmacokinetics in children and will be useful in the future design of mAb therapy.This work was supported by grants from FDA, FD-R-000377 and NIH U10 CA 28439 and in part by a grant from the general Clinical Research Center program, MOI RR00827, of the National Center for Research Resources, National Institutes of Health. M. M. U.-F. and C.-S. H. were supported in part by a grant from the Children's Cancer Research Foundation, and R. A. R. was supported in part by NIH grant CA 42508     

Pharmacokinetics of human-mouse chimeric anti-GD2 mAb ch14.18 in a phase I trial in neuroblastoma patients

A comprehensive analysis of the pharmacokinetics of human-mouse chimeric anti-ganglioside GD2 antibody mAb ch 14.18 was performed during a phase I clinical trial of ten children with neuroblastoma and one adult with osteosarcoma. The patients received a total of 20 courses of ch 14.18 at dose levels from 10 mg/m² to 200 mg/m². The plasma clearance of ch 14.18 was biphasic. Following the first course of treatmentt1/2, was 3.4±3.1 h andt1/2, 66.6±27.4 h in 9/10 children. Thet1/2, values were significantly less than those of 181±73 h previously reported in adult melanoma patients (P<-0.001), and 147.5 h in the adult osteosarcoma patient in our trial. The latter suggests different pharmacokinetics of mAb ch 14.18 in children and adults. After a second course of treatment, administered to 5/10 children,t1/2, decreased significantly from 72.9±19.8 h to 31.7±18.4 h (P=0.015). We there-fore conclude that the elimination kinetics of mAbs ch 14.18 in children and adults are different, and furthermore that repeated administration of mAb ch 14.18 to children with neuroblastoma leads to accelerated antibody clearance.This work was supported by grants from FDA, FD-R-000377 and NIH U10 CA 28439, and in part by a grant from the General Clinical Research Center Program, MOI RR00827, of the National Center for Research Resources, National Institutes of Health. M.M.U.-F. and C.S.H. were in part supported by a grant from the Children's Cancer Research Foundation, and M.M.U.-F. was in part supported by a grant of the Kommission für Forschung und wissenschaftlichen Nachwuchs, Charité 95-010/9610766     

Pharmacokinetics and stability of the ch14.18–interleukin-2 fusion protein in mice

The fusion protein formed from ch14.18 and interleukin-2 (ch14.18-IL-2), shown to exhibit antitumor efficacy in mouse models, consists of IL-2 genetically linked to each heavy chain of the ch14.18 chimeric anti-GD2 monoclonal antibody. The purpose of this study was to determine the pharmacokinetics of ch14.18-IL-2 in mice and assess its stability in murine serum. Following i.v. injection, the fusion protein was found to have a terminal half-life of 4.1 h. Detection of IL-2 following injection of the ch14.18-IL-2 fusion protein showed a similar half-life, indicating that the fusion protein prolongs the circulatory half-life of IL-2. Detection of human IgG1 following injection of ch14.18-IL-2 showed a terminal half-life of 26.9 h. These data suggested that the native fusion protein is being altered in vivo, resulting in a somewhat rapid loss of detectable IL-2, despite prolonged circulation of its immunoglobulin components. In vitro incubation of the ch14.18-IL-2 fusion protein in pooled mouse serum at 37 degrees C for 48 h resulted in a loss of its IL-2 component, as detected in enzyme-linked immunosorbent assay systems and in proliferation assays. Polyacrylamide gel electrophoresis and Western blot analysis of the fusion protein incubated in mouse serum at 37 degrees C indicated that the ch14.18-IL-2 is cleaved, resulting in a loss of the 67-kDa band (representing the IL-2 linked to the IgG1 heavy chain) and the detection of a band of more than 50 kDa, slightly heavier than the IgG1 heavy chain itself. This suggests that the fusion protein is being cleaved in vitro within the IL-2 portion of the molecule. These studies show that (1) ch14.18-IL-2 prolongs the circulatory half-life of IL-2 (compared to that of soluble IL-2) and (2) the in vivo clearance of the fusion protein occurs more rapidly than the clearance of the ch14.18 antibody itself, possibly reflecting in vivo cleavage within the IL-2 portion of the molecule, resulting in loss of IL-2 activity.     

A phase-I trial of the epidermal growth factor receptor directed bispecific antibody MDX-447 without and with recombinant human granulocyte-colony stimulating factor in patients with advanced solid tumors

Introduction MDX-447 is a bispecific antibody directed against the epidermal growth factor receptor (EGFR) and the high affinity Fc receptor (FcγRI). Preclinical data suggest that co-administration of granulocyte-colony stimulating factor (G-CSF) may enhance the tumor cytotoxicity of bispecific antibodies. Methods In group 1, patients received MDX-447 intravenously (IV) weekly. Dose levels of MDX-447 evaluated in group 1 were 1, 3.5, 7, 10, 15, 20, 30, and 40 mg/m². In group 2, patients received MDX-447 IV weekly with G-CSF (3 mcg/kg/day) subcutaneously (days −3 to +2, 5–9, 12–16, etc.). Dose levels of MDX-447 evaluated in group 2 were 1, 3.5, 7, 10, and 15 mg/m². Results Sixty-four patients with advanced solid tumors were treated. Forty-one patients received MDX-447 alone (group 1); 23 patients received MDX-447 + G-CSF (group 2). Hypotension was the predominant dose-limiting toxicity (DLT) in both treatment groups, with seven patients experiencing ≥grade 3 events. MDX-447 half-life (T_1/2) ranged from 1.9 to 8.4 h, with no obvious differences between the two treatment groups. MDX-447 binding to neutrophils and peak levels of circulating tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) were higher in group 2. The MTD for MDX-447 alone was 30 mg/m². When G-CSF was given with MDX-447, treatment was not well tolerated and group 2 was closed early because of safety concerns, with the last patient being treated at the 7 mg/m² dose level. There were no objective complete or partial responses in either group. Conclusion MDX-447 alone was generally well tolerated, but did not achieve objective tumor responses. The MTD for MDX-447 alone was 30 mg/m² weekly. Co-administration of G-CSF with MDX-447 precluded meaningful dose escalation.     

Phase II trial of hu14.18-IL2 for patients with metastatic melanoma

Phase I testing of the hu14.18-IL2 immunocytokine in melanoma patients showed immune activation, reversible toxicities, and a maximal tolerated dose of 7.5?mg/m²/day. In this phase II study, 14 patients with measurable metastatic melanoma were scheduled to receive hu14.18-IL2 at 6?mg/m²/day as 4-h intravenous infusions on Days 1, 2, and 3 of each 28?day cycle. Patients with stable disease (SD) or regression following cycle 2 could receive two additional treatment cycles. The primary objective was to evaluate antitumor activity and response duration. Secondary objectives evaluated adverse events and immunologic activation. All patients received two cycles of treatment. One patient had a partial response (PR) [1 PR of 14 patients?=?response rate of 7.1?%; confidence interval, 0.2?C33.9?%], and 4 patients had SD and received cycles 3 and 4. The PR and SD responses lasted 3?C4?months. All toxicities were reversible and those resulting in dose reduction included grade 3 hypotension (2 patients) and grade 2 renal insufficiency with oliguria (1 patient). Patients had a peripheral blood lymphocytosis on Day 8 and increased C-reactive protein. While one PR in 14 patients met protocol criteria to proceed to stage 2 and enter 16 additional patients, we suspended stage 2 due to limited availability of hu14.18-IL2 at that time and the brief duration of PR and SD. We conclude that subsequent testing of hu14.18-IL2 should involve melanoma patients with minimal residual disease based on compelling preclinical data and the confirmed immune activation with some antitumor activity in this study.     

Eradication of established hepatic human neuroblastoma metastases in mice with severe combined immunodeficiency by antibody-targeted interleukin-2

 A major problem in the treatment of solid tumors is the eradication of established, disseminated metastases. Here we describe an effective treatment for established experimental hepatic metastases of human neuroblastoma in C. B.-17 scid/scid mice. This was accomplished with an antibody-cytokine fusion protein, combining the unique targeting ability of antibodies with the multifunctional activity of cytokines. An anti-(ganglioside GD2) antibody (ch14.18) fusion protein with interleukin-2 (ch14.18-IL2), constructed by fusion of a synthetic sequence coding for human interleukin-2 (IL-2) to the carboxyl end of the Cγ1 gene of ch14.18, was tested for its therapeutic efficacy against xenografted human neuroblastoma in vivo. The ch14.18-IL2 fusion protein markedly inhibited growth of established hepatic metastases in SCID (severe combined immunodeficiency) mice previously reconstituted with human lymphokine-activated killer cells. Animals treated with ch14.18-IL2 showed an absence of macroscopic liver metastasis. In contrast, treatment with combinations of ch14.18 and recombinant IL2 at dose levels equivalent to the fusion protein only reduced the tumor load. Survival times of SCID mice treated with the fusion protein were more than double that of control animals. These results demonstrate that an immunotherapeutic approach using a cytokine targeted by an antibody to tumor sites is highly effective in eradicating the growth of established tumor metastases. Received: 7 November 1995 / Accepted: 15 December 1995     

Functional Bioassays for Immune Monitoring of High-Risk Neuroblastoma Patients Treated with ch14.18/CHO Anti-GD2 Antibody

Effective treatment of high-risk neuroblastoma (NB) remains a major challenge in pediatric oncology. Human/mouse chimeric monoclonal anti-GD₂ antibody (mAb) ch14.18 is emerging as a treatment option to improve outcome. After establishing a production process in Chinese hamster ovary (CHO) cells, ch14.18/CHO was made available in Europe for clinical trials. Here, we describe validated functional bioassays for the purpose of immune monitoring of these trials and demonstrate GD₂-specific immune effector functions of ch14.18/CHO in treated patients. Two calcein-based bioassays for complement-dependent- (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were set up based on patient serum and immune cells tested against NB cells. For this purpose, we identified LA-N-1 NB cells as best suited within a panel of cell lines. Assay conditions were first established using serum and cells of healthy donors. We found an effector-to-target (E:T) cell ratio of 20∶1 for PBMC preparations as best suited for GD₂-specific ADCC analysis. A simplified method of effector cell preparation by lysis of erythrocytes was evaluated revealing equivalent results at an E:T ratio of 40∶1. Optimal results for CDC were found with a serum dilution at 1∶8. For validation, both within-assay and inter-assay precision were determined and coefficients of variation (CV) were below 20%. Sample quality following storage at room temperature (RT) showed that sodium-heparin-anticoagulated blood and serum are stable for 48 h and 96 h, respectively. Application of these bioassays to blood samples of three selected high-risk NB patients treated with ch14.18/CHO (100 mg/m²) revealed GD₂-specific increases in CDC (4.5–9.4 fold) and ADCC (4.6–6.0 fold) on day 8 compared to baseline, indicating assay applicability for the monitoring of multicenter clinical trials requiring sample shipment at RT for central lab analysis.     

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

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.     

Dose Escalation of Lobaplatin Concurrent with IMRT for the Treatment of Stage III-IVb NPC: A Phase I Clinical Trial

The maximum tolerated dose (MTD) of lobaplatin as a single agent chemotherapy concurrent with intensity-modulated radiotherapy (IMRT) in Asian population with nasopharyngeal carcinoma (NPC) remains unclear. From June 2016 to December 2017, 17 patients diagnosed with stage III-IVb NPC from an Asian population were prospectively enrolled. Patients were administered lobaplatin with 25-50?mg/m² escalation of dosage on day 1. Every 21?days (days 1, 22, and 43) during radiotherapy, cycles were repeated. We administered radiotherapy as 2.12-2.27 Gy per fraction with five daily fractions each week for 6 to 7 weeks. The evaluation of lobaplatin-related toxic effects was based on the Common Terminology Criteria for Adverse Events version 4.0. During the weekly treatment period, complete blood counts and biochemistry were performed. Dose-limiting toxicities (DLTs) were determined by the following events during any cycle in which lobaplatin was administered. Each dose group consisted of at least three cases. We proceeded to the subsequent dose group in the absence of DLT with a dose increment of 5 mg/m² until DLT occurred. Periods from 1 week prior to the chemotherapy initiation to 3 weeks after the last chemotherapy were defined as DLT observation periods. MTD was determined by the dose that was immediately below the dose that produced DLT. After analysis, DLT occurred in three patients, including a group with two of three patients in 45 mg/m² lobaplatin and another group with one of five patients in 40 mg/m² lobaplatin. No grade 3-4 toxicity was observed in patients treated with lobaplatin <40 mg/m². The tumor response rate at 12?weeks after treatment was 100%. In summary, lobaplatin concurrent with IMRT was active in stage III-IVb NPC, and the MTD for the lobaplatin as single-agent chemotherapy was 40 mg/m² when combined with IMRT in an Asian population. This trial is registered with ClinicalTrials.gov, number NCT03188497.     

Phase I study of bromohydrin pyrophosphate (BrHPP, IPH 1101), a Vγ9Vδ2 T lymphocyte agonist in patients with solid tumors

Purpose

Vγ9Vδ2 (γδ) T lymphocytes, a critical peripheral blood lymphocyte subset, are directly cytotoxic against many solid and hematologic tumor types. Vγ9Vδ2 T lymphocytes can be selectively expanded in vivo with BrHPP (IPH1101) and IL-2. The present phase I trial was conducted with the aim of determining the maximum-tolerated dose (MTD) and safety of IPH1101 combined with a low dose of IL-2 in patients with solid tumors.

Experimental design

A 1-h intravenous infusion of IPH11 was administered alone at cycle 1, combined with a low dose of SC IL-2 (1 MIU/M² d1 to d7) in the subsequent cycles (day 1 every 3 weeks). The dose of IPH1101 was escalated from 200 to 1,800 mg/m².

Results

As much as 28 patients with solid tumors underwent a total of 109 treatment cycles. Pharmacodynamics data demonstrate that γδ T lymphocyte amplification in humans requires the co-administration of IL-2 and is dependent on IPH 1101 dose. Dose-limiting toxicity occurred in two patients at a dose of 1,800 mg/m²: one grade 3 fever (1 patient) and one grade 3 hypotension (1 patient) suggesting cytokine release syndrome immediately following the first infusion. At lower doses the treatment was well tolerated; the most frequent adverse events were mild fever, chills and abdominal pain, without exacerbation in the IL-2 combined cycles.

Conclusion

IPH1101 in combination with SC low-dose IL-2 is safe, well tolerated and induces a potent γδ T lymphocyte expansion in patients. Its clinical activity will be evaluated in phase II clinical trials.     

Phase I trial of hydroxychloroquine with dose-intense temozolomide in patients with advanced solid tumors and melanoma

Blocking autophagy with hydroxychloroquine (HCQ) augments cell death associated with alkylating chemotherapy in preclinical models. This phase I study evaluated the maximum tolerated dose (MTD), safety, preliminary activity, pharmacokinetics, and pharmacodynamics of HCQ in combination with dose-intense temozolomide (TMZ) in patients with advanced solid malignancies. Forty patients (73% metastatic melanoma) were treated with oral HCQ 200 to 1200 mg daily with dose-intense oral TMZ 150 mg/m² daily for 7/14 d. This combination was well tolerated with no recurrent dose-limiting toxicities observed. An MTD was not reached for HCQ and the recommended phase II dose was HCQ 600 mg twice daily combined with dose-intense TMZ. Common toxicities included grade 2 fatigue (55%), anorexia (28%), nausea (48%), constipation (20%), and diarrhea (20%). Partial responses and stable disease were observed in 3/22 (14%) and 6/22 (27%) patients with metastatic melanoma. In the final dose cohort 2/6 patients with refractory BRAF wild-type melanoma had a near complete response, and prolonged stable disease, respectively. A significant accumulation in autophagic vacuoles (AV) in peripheral blood mononuclear cells was observed in response to combined therapy. Population pharmacokinetics (PK) modeling, individual PK simulations, and PK-pharmacodynamics (PD) analysis identified a threshold HCQ peak concentration that predicts therapy-associated AV accumulation. This study indicates that the combination of high-dose HCQ and dose-intense TMZ is safe and tolerable, and is associated with autophagy modulation in patients. Prolonged stable disease and responses suggest antitumor activity in melanoma patients, warranting further studies of this combination, or combinations of more potent autophagy inhibitors and chemotherapy in melanoma.     

Vaccination with anti-idiotype antibody ganglidiomab mediates a GD2-specific anti-neuroblastoma immune response

Purpose

Immunotherapy targeting disialoganglioside GD₂ emerges as an important treatment option for neuroblastoma, a pediatric malignancy characterized by poor outcome. Here, we report the induction of a GD₂-specific immune response with ganglidiomab, a new anti-idiotype antibody to anti-GD₂ antibodies of the 14.18 family.

Experimental design and results

Ganglidiomab was generated following immunization of Balb/c mice with 14G2a, and splenocytes were harvested to generate hybridoma cells. Clones were screened by ELISA for mouse antibody binding to hu14.18. One positive clone was selected to purify and characterize the secreted IgG protein (κ, IgG₁). This antibody bound to anti-GD₂ antibodies 14G2a, ch14.18/CHO, hu14.18, and to immunocytokines ch14.18-IL2 and hu14.18-IL2 as well as to NK-92 cells expressing scFv(ch14.18)-zeta receptor. Binding of these anti-GD₂ antibodies to the nominal antigen GD₂ as well as GD₂-specific lysis of neuroblastoma cells by NK-92-scFv(ch14.18)-zeta cells was competitively inhibited by ganglidiomab, proving GD₂ surrogate function and anti-idiotype characteristics. The dissociation constants of ganglidiomab from anti-GD₂ antibodies ranged from 10.8 ± 5.01 to 53.5 ± 1.92 nM as determined by Biacore analyses. The sequences of framework and complementarity-determining regions of ganglidiomab were identified. Finally, we demonstrated induction of a GD₂-specific humoral immune response after vaccination of mice with ganglidiomab effective in mediating GD₂-specific killing of neuroblastoma cells.

Conclusion

We generated and characterized a novel anti-idiotype antibody ganglidiomab and demonstrated activity against neuroblastoma.     

A phase I study of recombinant interferon gamma administered by s.c. injection three times per week in patients with solid tumours

Summary Human recombinant DNA interferon gamma (IFN-G), with a specific activity of 2×10⁶ IU/mg protein, was administered s.c. 3 days per week for 2 months to patients with solid tumors. The maximum tolerated dose (MTD) was 10×10⁶ IU/m² (5.0 mg/m²) per injection, and six patients were treated at the MTD. Two of these ceased treatment because of severe subjective toxity (headache, rigors and pyrexia) and three patients developed WHO grade 3 leucopenia. Subjective toxicity varied considerably between patients and some patients at low dose levels experienced severe constitutional symptoms whilst others treated at the MTD had few side effects. These differences were unrelated to pharmacokinetic parameters. Bioavailability of this IFN-G administered s.c. was very variable from one patient to another at the same dose level. We therefore counsel caution in using this IFN-G preparation s.c. in phase II studies.     

Primary antiphospholipid antibody syndrome—one further aspect of thrombophilia in overweight and obese patients with venous thromboembolism

Objective: Overweight and obesity are established risk factors for venous thromboembolism (VTE). We examined the difference in the frequency of primary antiphospholipid antibody syndrome (PAPS) in VTE patients according to their BMI. Design and Methods: We included 998 VTE patients treated at our institution between 2009 and 2011 in a retrospective data analysis. Thrombophilia screening including evaluation for APS (lupus anticoagulant, anti‐cardiolipin, and anti‐B2‐glycoprotein‐I IgG and IgM antibodies) was performed in all patients. Results: PAPS was diagnosed in 6.8% (24/355) of normal weight (BMI < 24 kg/m²) VTE patients, in 11.1% (50/452) of overweight (BMI 25–30 kg/m²) VTE patients, and in 15.7% (30/191) of obese (BMI > 31 kg/m²) VTE patients. The difference of PAPS occurrence between these groups was statistically significant (P = 0.001). PAPS patients demonstrated higher fibrinogen levels as compared to non‐PAPS patients (median 416.0 md/dl vs. 352.0 mg/dl, P = 0.001). Furthermore, fibrinogen levels increased significantly according to the body weight of patients (median normal weight patients 330.0 mg/dl vs. overweight patients 359.0 mg/dl vs. obese patients 415.0 mg/dl, P = 0.001). Conclusion: PAPS seems to be more frequent in overweight and obese patients. As PAPS patients showed significantly higher fibrinogen levels and as fibrinogen levels increased significantly according to the body weight of patients, an elevated inflammatory state in overweight and obese patients as a reason for the increased PAPS occurrence can be assumed.     

A Phase I Study of Vincristine,Irinotecan, Temozolomide and Bevacizumab (Vitb) in Pediatric Patients with Relapsed Solid Tumors

Background

To determine the maximum tolerated dose (MTD) and dose limiting toxicity (DLT) of irinotecan administered in combination with vincristine, temozolomide and bevacizumab in children with refractory solid tumors.

Methods

The study design included two dose levels (DL) of irinotecan given intravenously once daily for 5 consecutive days (DL1: 30 mg/m², and DL2: 50 mg/m²), combined with vincristine 1.5 mg/m² on days 1 and 8, temozolomide 100 mg/m² on days 1-5, and bevacizumab 15mg/kg on day 1, administered every 21 days for a maximum of 12 cycles.

Results

Thirteen patients were enrolled and 12 were evaluable for toxicity Dose limiting toxicity observed included grade 3 hyperbilirubinemia in 1 of 6 patients on DL1, and grade 3 colitis in 1 of 6 patients on DL2. DL 2 was the determined MTD. A total of 87 cycles were administered. Myelosuppression was mild. Grade 1-2 diarrhea occurred in the majority of cycles with grade 3 diarrhea occurring in only one cycle. Grade 2 hypertension developed in two patients. Severe hemorrhage, intestinal perforation, posterior leukoencephalopathy or growth plate abnormalities were not observed. Objective responses were noted in three Wilms tumor patients and one each of medulloblastoma and hepatocellular carcinoma. Five patients completed all 12 cycles of protocol therapy.

Conclusions

Irinotecan 50 mg/m²/day for 5 days was the MTD when combined with vincristine, temozolomide and bevacizumab administered on a 21 day schedule. Encouraging anti-tumor activity was noted.

Trial Registration

ClinicalTrials.gov; {"type":"clinical-trial","attrs":{"text":"NCT00993044","term_id":"NCT00993044"}}NCT00993044; http://clinicaltrials.gov/show/{"type":"clinical-trial","attrs":{"text":"NCT00993044","term_id":"NCT00993044"}}NCT00993044     

The role of neoadjuvant and adjuvant chemotherapy regimens consisting of different combinations of drugs in the treatment of advanced oral cancer

We performed a retrospective analysis on the effect of neoadjuvant chemotherapy with three cycles of methotrexate (100 mg/m² on day 1), cisplatin (90 mg/m² on day 1) and bleomycin (20 mg/m² on day 1–5) with 21 d gap between each cycle in 44 patients with advanced squamous cell carcinoma of the cheek, lip and tongue followed by surgery and adjuvant chemotherapy consisting of cisplatin (90 mg/m² on day 1), Mitomycin C (6 mg/m² on day 1) and 5-fluorouracil (1000 mg/m² 120 h continuous infusion from day 1) repeated every 3 weeks for three cycles. Following induction chemotherapy, complete response was observed in 11 out of 44 patients (25%), and a partial response in a further 28 patients (64%). The overall median survival of all patients was 29 months and those in stage III and stage IV were 30 and 15 months respectively (P<0.001). The median duration of the time to relapse in patients who responded to adjuvant chemotherapy was 28 months. The main toxic effect was vomiting followed by hematological toxicity. No treatment-related deaths occurred. The regimen showed a significant response, encouraging median survival and a good tolerability profile.     

Enhancement of antibody-dependent cytotoxicity with a chimeric anti-GD2 antibody

A mouse/human chimeric antibody (ch14.18) was developed that reacts with the disialoganglioside GD2 on the surface of tumor cells of neuroectodermal origin. ch14.18 has the constant regions of a human IgG1 antibody and was expressed in a murine hybridoma. This antibody was produced in tissue culture at concentrations up to 180 mg/liter of spent culture fluid. ch14.18 was characterized and compared to 14.G2a, a murine mAb against GD2 of IgG2a isotype derived from the same parental hybridoma as ch14.18. Scatchard plot analysis of data from saturation binding studies on M21 melanoma cells showed identical binding for ch14.18 and 14.G2a. Indirect immunofluorescence revealed the same staining pattern for ch14.18 and 14.G2a on different melanoma cell lines. Both antibodies were equally capable of targeting M21 xenografts in athymic nude mice. ch14.18- and 14.G2a-activated human C-mediated cytolysis of melanoma cell; however, ch14.18-mediated antibody-dependent cytotoxicity of human effector cells against melanoma cells 50- to 100-fold more efficiently than 14.G2a.