Mario Otto

Phase I Trial of a Novel Anti-GD2 Monoclonal Antibody, Hu14.18K322A, Designed to Decrease Toxicity in Children With Refractory or Recurrent Neuroblastoma

PURPOSE The addition of immunotherapy, including a combination of anti-GD2 monoclonal antibody (mAb), ch14.18, and cytokines, improves outcome for patients with high-risk neuroblastoma. However, this therapy is limited by ch14.18-related toxicities that may be partially mediated by complement activation. We report the results of a phase I trial to determine the maximum-tolerated dose (MTD), safety profile, and pharmacokinetics of hu14.18K322A, a humanized anti-GD2 mAb with a single point mutation (K322A) that reduces complement-dependent lysis. PATIENTS AND METHODS Eligible patients with refractory or recurrent neuroblastoma received escalating doses of hu14.18K322A ranging from 2 to 70 mg/m(2) per day for 4 consecutive days every 28 days (one course). RESULTS Thirty-eight patients (23 males; median age, 7.2 years) received a median of two courses (range, one to 15). Dose-limiting grade 3 or 4 toxicities occurred in four of 36 evaluable patients and were characterized by cough, asthenia, sensory neuropathy, anorexia, serum sickness, and hypertensive encephalopathy. The most common non-dose-limiting grade 3 or 4 toxicities during course one were pain (68%) and fever (21%). Six of 31 patients evaluable for response by iodine-123 metaiodobenzylguanidine score had objective responses (four complete responses; two partial responses). The first-course pharmacokinetics of hu14.18K322A were best described by a two-compartment linear model. Median hu14.18K322A α (initial phase) and β (terminal phase) half-lives were 1.74 and 21.1 days, respectively. CONCLUSION The MTD, and recommended phase II dose, of hu14.18K322A is 60 mg/m(2) per day for 4 days. Adverse effects, predominately pain, were manageable and improved with subsequent courses.

Anti-GD2 with an FC point mutation reduces complement fixation and decreases antibody-induced allodynia

&NA; Monoclonal antibodies against GD2 ganglioside, such as ch14.18, the human–mouse chimeric antibody, have been shown to be effective for the treatment of neuroblastoma. However, treatment is associated with generalized, relatively opiate‐resistant pain. We investigated if a point mutation in ch14.18 antibody (hu14.18K332A) to limit complement‐dependent cytotoxicity (CDC) would ameliorate the pain behavior, while preserving antibody‐dependent cellular cytotoxicity (ADCC). In vitro, CDC and ADCC were measured using europium‐TDA assay. In vivo, allodynia was evaluated by measuring thresholds to von Frey filaments applied to the hindpaws after injection of either ch14.18 or hu14.18K332 into wild type rats or rats with deficient complement factor 6. Other rats were pretreated with complement factor C5a receptor antagonist and tested following ch14.18 injection. The mutation reduces the antibodys ability to activate complement, while maintaining its ADCC capabilities. Injection of hu14.18K322 (1 or 3 mg/kg) produced faster resolving allodynia than that engendered by ch14.18 (1 mg/kg). Injection of ch14.18 (1 mg/kg) into rats with C6 complement deficiency further reduced antibody‐induced allodynia, while pre‐treatment with complement factor C5a receptor antagonist completely abolished ch14.18‐induced allodynia. These findings showed that mutant hu14.18 K322 elicited less allodynia than ch14.18 and that ch14.18‐elicited allodynia is due to activation of the complement cascade: in part, to formation of membrane attack complex, but more importantly to release of complement factor C5a. Development of immunotherapeutic agents with decreased complement‐dependent lysis while maintaining cellular cytotoxicity may offer treatment options with reduced adverse side effects, thereby allowing dose escalation of therapeutic antibodies.

Combination Immunotherapy with Clinical-Scale Enriched Human γδ T cells, hu14.18 Antibody, and the Immunocytokine Fc-IL7 in Disseminated Neuroblastoma

Purpose: To evaluate a combined cellular and humoral immunotherapy regimen in a mouse model of disseminated human neuroblastoma. We tested combinations of clinical-grade, isolated human γδ T cells with the humanized anti-GD2 antibody hu14.18 and a novel fusion cytokine, Fc-IL7. Experimental Design: γδ T cells were large-scale enriched from leukapheresis product obtained from granulocyte colony-stimulating factor–mobilized donors. γδ T cell cytotoxicity was tested in a europium-TDA release assay. The effect of Fc-IL7 on γδ T-cell survival in vitro was assessed by flow cytometry. NOD.CB17-Prkdcscid/J mice received 1 × 106 NB-1691 neuroblastoma cells via the tail vein 5 to 6 days before therapy began. Treatment, for five consecutive weeks, consisted of injections of 1 × 106 γδ T cells weekly, 1 × 106 γδ T cells weekly, and 20 μg hu14.18 antibody four times per week, or 1 × 106 γδ T cells weekly with 20 μg hu14.18 antibody four times per week, and 20 μg Fc-IL7 once weekly. Results: The natural cytotoxicity of γδ T cells to NB-1691 cells in vitro was dramatically enhanced by hu14.18 antibody. Fc-IL7 effectively kept cultured γδ T cells viable. Combination therapy with γδ T cells and hu14.18 antibody significantly enhanced survival (P = 0.001), as did treatment with γδ T cells, hu14.18 antibody, and Fc-IL7 (P = 0.005). Inclusion of Fc-IL7 offered an additional survival benefit (P = 0.04). Conclusions: We have shown a new and promising immunotherapy regimen for neuroblastoma that requires clinical evaluation. Our approach might also serve as a therapeutic model for other malignancies.

Human γδ T Cells From G-csf-mobilized Donors Retain Strong Tumoricidal Activity and Produce Immunomodulatory Cytokines After Clinical-scale Isolation

Human γδ T cells are a small fraction of T cells that have been shown to exert major histocompatibility (MHC)-unrestricted natural cytotoxicity against a variety of solid tumors and some subsets of leukemias and lymphomas. They are also involved in the immune response to certain bacterial, viral, and parasitic infections and expand significantly in CMV- or HSV-infected organ allografts. They are able to mediate antibody-dependent cytotoxicity and are not alloreactive, which makes them attractive candidates for cell-based immunotherapy. However, their frequency in peripheral blood is low and ex vivo expansion of γδ T cells is labor-extensive, does not always yield cells with full innate cytotoxic power, and has the potential for microbial contamination. Therefore, the authors developed a clinical-scale, automated cell purification method for the efficient enrichment of γδ T cells from leukapheresis products. Six leukapheresis products were purified for γδ T cells using a single-step immunomagnetic method. Purity and phenotype were assessed by flow cytometry. A standard Europium release assay was performed to determine the cytotoxic capacity of the cells. Cytokine production was measured using a multiplex sandwich immunoassay. The mean percentage of γδ T cells in the final product was 91%, with an average recovery of 63%. The cells showed a high co-expression of CD8, CD56, CD28, and CD11b/CD18. In some products an unusually high proportion of Vγ9Vδ1 T cells was found. The isolated cells were cytotoxic against the neuroblastoma cell line NB1691 and the erythroleukemic line K562 in vitro. They were able to produce a variety of immunomodulatory cytokines such as IFNγ, TNFα, and MIP-1β, but also GM-CSF and G-CSF when co-incubated in culture with and without various stimuli. In summary, the authors describe a rapid, automated, and efficient method for the large-scale enrichment of human γδ T cells. The cytotoxic properties of the cells were preserved. This method yields sufficient purified γδ T cells for use in adoptive immunotherapy as well as laboratory investigations and animal studies.

Leading Neuroblastoma Cells To Die by Multiple Premeditated Attacks from a Multifunctionalized Nanoconstruct

To conquer complex and devastating diseases such as cancer, more coordinated and combined attack strategies are needed. We suggest that these can be beautifully achieved by using nanoconstruct design. We present an example showing that neuroblastoma cells are selectively killed by a nanoconstruct that specifically targets neuroblastoma cells, pushes cells to the vulnerable phase of the cell cycle, and greatly enhances radiation-induced cell death. The success of this multipronged attack approach launched by cell-embedded nanoconstructs demonstrates the power and flexibility of nanotechnology in treating cancer, a difficult task for a small molecule.

Linker-free conjugation and specific cell targeting of antibody functionalized iron-oxide nanoparticles

Specific targeting is a key step to realize the full potential of iron oxide nanoparticles in biomedical applications, especially tumor-associated diagnosis and therapy. Here, we developed anti-GD2 antibody conjugated iron oxide nanoparticles for highly efficient neuroblastoma cell targeting. The antibody conjugation was achieved through an easy, linker-free method based on catechol reactions. The targeting efficiency and specificity of the antibody-conjugated nanoparticles to GD2-positive neuroblastoma cells were confirmed by flow cytometry, fluorescence microscopy, Prussian blue staining and transmission electron microscopy. These detailed studies indicated that the receptor-recognition capability of the antibody was fully retained after conjugation and the conjugated nanoparticles quickly attached to GD2-positive cells within four hours. Interestingly, longer treatment (12 h) led the cell membrane-bound nanoparticles to be internalized into cytosol, either by directly penetrating the cell membrane or escaping from the endosomes. Last but importantly, the uniquely designed functional surfaces of the nanoparticles allow easy conjugation of other bioactive molecules.

Interactions of Iron Oxide Nanoparticles with the Immune System: Challenges and Opportunities for their Use in Nano-oncology

Iron oxide (IO) nanoparticles hold great promise as diagnostic and therapeutic agents in oncology. Their intrinsic physical properties make IO nanoparticles particularly interesting for simultaneous drug delivery, molecular imaging, and applications such as localized hyperthermia. Multiple non-targeted IO nanoparticle preparations have entered clinical trials, but more exciting, new tumortargeted IO nanoparticle preparations are currently being tested in preclinical settings. This paper will analyze the challenges faced by this new theranostic modality, with a specific focus on the interactions of IO nanoparticles with the innate and adaptive immune systems, and their effect on nanoparticle biodistribution and tumor targeting. Next, we will review the critical need for innovative surface chemistry solutions and strategies to overcome the immune interactions that prevent existing tumor-targeted IO preparations from entering clinical trials. Finally, we will provide an outlook for the future role of IO nanoparticles in oncology, which have the promise of becoming significant contributors to improved diagnosis and treatment of cancer patients.

High specificity targeting and detection of human neuroblastoma using multifunctional anti-GD2 iron-oxide nanoparticles

AIM To develop biocompatible, tumor-specific multifunctional iron-oxide nanoconstructs targeting neuroblastoma, an aggressive pediatric malignancy. MATERIALS & METHODS Clinical-grade humanized monoclonal antibody (hu14.18K322A), designed to target GD2 antigen on neuroblastoma with reduced nonspecific immune interactions, was conjugated to hydroxyethyl starch-coated iron-oxide nanoparticles. Targeting capability in vitro and in vivo was assessed by immunofluorescence, electron microscopy, analytical spectrophotometry, histochemistry and magnetic resonance R2* relaxometry. RESULTS The biocompatible nanoconstructs demonstrated high tumor specificity in vitro and in vivo, and low background uptake in a mouse flank xenograft model. Specific accumulation in tumors enabled particle visualization and quantification by magnetic resonance R2* mapping. CONCLUSION Our findings support the further development toward clinical application of this anti-GD2 iron-oxide nanoconstruct as diagnostic and therapeutic scaffold for neuroblastoma and potentially other GD2-positive malignancies.

Enhancing both CT imaging and natural killer cell-mediated cancer cell killing by a GD2-targeting nanoconstruct

Although nanomaterials have been widely investigated for drug delivery, imaging and immunotherapy, their potential roles in triggering innate cellular immune responses while simultaneously serving as imaging enhancer remain unexplored. In this work, gold nanoparticles (GNPs) conjugated to the tumor-targeting anti-GD2 antibody hu14.18K322A, namely HGNPs, were designed and synthesized to specifically enhance computerized tomography (CT) imaging contrast and to stimulate the attack of neuroblastoma and melanoma cells by natural killer (NK) cells. The HGNPs specifically targeted GD2-positive neuroblastoma (NB1691) and melanoma (M21) cells, with an enhancement of CT contrast images of the HGNP-labeled cell pellets by 5.27- and 7.66-fold, respectively, compared to images of unlabeled cell pellets. The HGNPs also triggered NK-mediated antibody-dependent cellular cytotoxicity (ADCC) in NB1691 and M21 cells with a two-fold higher efficacy compared to that elicited by hu14.18K322A alone, with no adverse effect to GD2-negative PC-3 cells. These results suggest that HGNPs are promising theranostic agents for neuroblastoma and melanoma cancers.

NK Cell-based Immunotherapies in Pediatric Oncology

The past decade has seen several anticancer immunotherapeutic strategies transition from “promising preclinical models” to treatments with proven clinical activity or benefit. In 2013, the journal Science selected the field of Cancer Immunotherapy as the overall number-1 breakthrough for the year in all of scientific research. In the setting of cancer immunotherapy for adult malignancies, many of these immunotherapy strategies have relied on the cancer patient’s endogenous antitumor T-cell response. Although much promising research in pediatric oncology is similarly focused on T-cell reactivity, several pediatric malignancies themselves, or the chemo-radiotherapy used to achieve initial responses, can be associated with profound immune suppression, particularly of the T-cell system. A separate component of the immune system, also able to mediate antitumor effects and less suppressed by conventional cancer treatment, is the NK-cell system. In recent years, several distinct immunotherapeutic approaches that rely on the activity of NK cells have moved from preclinical development into clinical testing, and some have shown clear antitumor benefit. This review provides an overview of NK cell-based immunotherapy efforts that are directed toward childhood malignancies, with an emphasis on protocols that are already in clinical testing.