Jacquelyn A. Hank

Antitumor Activity of Hu14.18-IL2 in Patients With Relapsed/Refractory Neuroblastoma: A Children's Oncology Group (COG) Phase II Study

PURPOSE The hu14.18-IL2 fusion protein consists of interleukin-2 molecularly linked to a humanized monoclonal antibody that recognizes the GD2 disialoganglioside expressed on neuroblastoma cells. This phase II study assessed the antitumor activity of hu14.18-IL2 in two strata of patients with recurrent or refractory neuroblastoma. PATIENTS AND METHODS Hu14.18-IL2 was given intravenously (12 mg/m(2)/daily) for 3 days every 4 weeks for patients with disease measurable by standard radiographic criteria (stratum 1) and for patients with disease evaluable only by [(123)I]metaiodobenzylguanidine (MIBG) scintigraphy and/or bone marrow (BM) histology (stratum 2). Response was established by independent radiology review as well as BM histology and immunocytology, and durability was assessed by repeat evaluation after more than 3 weeks. RESULTS Thirty-nine patients were enrolled (36 evaluable). No responses were seen in stratum 1 (n = 13). Of 23 evaluable patients in stratum 2, five patients (21.7%) responded; all had a complete response (CR) of 9, 13, 20, 30, and 35+ months duration. Grade 3 and 4 nonhematologic toxicities included capillary leak, hypoxia, pain, rash, allergic reaction, elevated transaminases, and hyperbilirubinemia. Two patients required dopamine for hypotension, and one patient required ventilatory support for hypoxia. Most toxicities were reversible within a few days of completing a treatment course and were expected based on phase I results. CONCLUSION Patients with disease evaluable only by MIBG and/or BM histology had a 21.7% CR rate to hu14.8-IL2, whereas patients with bulky disease did not respond. Hu14.18-IL2 warrants further testing in children with nonbulky high-risk neuroblastoma.

A Phase I Clinical Trial of the hu14.18-IL2 (EMD 273063) as a Treatment for Children with Refractory or Recurrent Neuroblastoma and Melanoma: a Study of the Children’s Oncology Group

Purpose: Evaluate the clinical safety, toxicity, immune activation/modulation, and maximal tolerated dose of hu14.18-IL2 (EMD 273063) in pediatric patients with recurrent/refractory neuroblastoma and other GD2-positive solid tumors. Experimental Design: Twenty-seven pediatric patients with recurrent/refractory neuroblastoma and one with melanoma were treated with a humanized anti-GD2 monoclonal antibody linked to human interleukin 2 (IL-2). Cohorts of patients received hu14.18-IL2, administered i.v. over 4 hours for three consecutive days, at varying doses. Patients with stable disease, partial, or complete responses were eligible to receive up to three additional courses of therapy. Results: Most of the clinical toxicities were anticipated and similar to those reported with IL-2 and anti-GD2 monoclonal antibody therapy and to those noted in the initial phase I study of hu14.18-IL2 in adults with metastatic melanoma. The maximal tolerated dose was determined to be 12 mg/m2/d, with agent-related dose-limiting toxicities of hypotension, allergic reaction, blurred vision, neutropenia, thrombocytopenia, and leukopenia. Three patients developed dose-limiting toxicity during course 1; seven patients in courses 2 to 4. Two patients required dopamine for hypotension. There were no treatment-related deaths, and all toxicity was reversible. Treatment with hu14.18-IL2 led to immune activation/modulation as evidenced by elevated serum levels of soluble IL-2 receptor α (sIL2Rα) and lymphocytosis. The median half-life of hu14.18-IL2 was 3.1 hours. There were no measurable complete or partial responses to hu14.18-IL2 in this study; however, three patients did show evidence of antitumor activity. Conclusion: Hu14.18-IL2 (EMD 273063) can be administered safely with reversible toxicities in pediatric patients at doses that induce immune activation. A phase II clinical trial of hu14.18-IL2, administered at a dose of 12 mg/m2/d × 3 days repeated every 28 days, will be done in pediatric patients with recurrent/refractory neuroblastoma.

Phase I Clinical Trial of the Immunocytokine EMD 273063 in Melanoma Patients

PURPOSE To evaluate the safety, toxicity, in vivo immunologic activation, and maximum-tolerated dose (MTD) of EMD 273063 (hu14.18-IL-2) in patients with metastatic melanoma. PATIENTS AND METHODS Thirty-three patients were treated with EMD 273063, a humanized anti-GD2 monoclonal antibody (mAb) linked to interleukin-2 (IL-2). EMD 273063 was given as a 4-hour intravenous infusion on days 1, 2, and 3 of week 1. Patients with stabilization or regression of disease could receive a second course of treatment at week 5. Dose levels evaluated were 0.8, 1.6, 3.2, 4.8, 6.0, and 7.5 mg/m2/d. RESULTS Nineteen of 33 patients completed course 1 with stable disease and went on to receive course 2. Eight patients had stable disease on completion of course 2. Grade 3 adverse events included hypophosphatemia (11 patients), hyperglycemia (three patients), hypotension (two patients), thrombocytopenia (one patient), hypoxia (three patients), elevated hepatic transaminases (two patients), and hyperbilirubinemia (one patient). Opioids were required for treatment-associated arthralgias and/or myalgias during 17 of 52 treatment courses. No grade 4 adverse events were observed. Dose-limiting toxicities at the MTD included hypoxia, hypotension, and elevations in AST/ALT. Grade 3 toxicities were anticipated based on prior studies of IL-2 or anti-GD2 mAbs, and all resolved. Immune activation was induced, as measured by lymphocytosis, increased peripheral-blood natural killer activity, and cell numbers, and increased serum levels of the soluble alpha chain of the IL-2 receptor complex. CONCLUSION Treatment with the immunocytokine EMD 273063 induced immune activation and was associated with reversible clinical toxicities at the MTD of 7.5 mg/m2/d in melanoma patients.

Genotypes of NK cell KIR receptors, their ligands, and Fcγ receptors in the response of neuroblastoma patients to Hu14.18-IL2 immunotherapy.

Response to immunocytokine (IC) therapy is dependent on natural killer cells in murine neuroblastoma (NBL) models. Furthermore, killer immunoglobulin-like receptor (KIR)/KIR-ligand mismatch is associated with improved outcome to autologous stem cell transplant for NBL. Additionally, clinical antitumor response to monoclonal antibodies has been associated with specific polymorphic-FcγR alleles. Relapsed/refractory NBL patients received the hu14.18-IL2 IC (humanized anti-GD2 monoclonal antibody linked to human IL2) in a Childrens Oncology Group phase II trial. In this report, these patients were genotyped for KIR, HLA, and FcR alleles to determine whether KIR receptor-ligand mismatch or specific FcγR alleles were associated with antitumor response. DNA samples were available for 38 of 39 patients enrolled: 24 were found to have autologous KIR/KIR-ligand mismatch; 14 were matched. Of the 24 mismatched patients, 7 experienced either complete response or improvement of their disease after IC therapy. There was no response or comparable improvement of disease in patients who were matched. Thus KIR/KIR-ligand mismatch was associated with response/improvement to IC (P = 0.03). There was a trend toward patients with the FcγR2A 131-H/H genotype showing a higher response rate than other FcγR2A genotypes (P = 0.06). These analyses indicate that response or improvement of relapsed/refractory NBL patients after IC treatment is associated with autologous KIR/KIR-ligand mismatch, consistent with a role for natural killer cells in this clinical response.

TCGF Production for Cloning and Growth of Functional Human T Lymphocytes

In an effort to increase the potency of T cell growth factor (TCGF), several variables were examined for their effects on the production of TCGF. The following manipulations enhanced the potency of TCGF: first, the removal of adherent cells and addition of indomethacin to the producing cultures; second, irradiation with 1000 rads of the cells used to produce TCGF; and, third, the addition of Epstein‐Barr virus transformed lymphoblastoid (LCL) cells. It was also noted that the addition of irradiated feeder cells increased the efficiency of limiting dilution cloning.

A Phase I/IB trial of murine monoclonal anti-GD2 antibody 14.G2a plus interleukin-2 in children with refractory neuroblastoma

The murine monoclonal antibody (MoAb) 14.G2a recognizes GD2, a disialoganglioside expressed in tumors of neuroectodermal origin, and facilitates antibody dependent cellular cytotoxicity (ADCC) in vitro. When given in vivo, interleukin‐2 (IL‐2) can increase ADCC by enhancing the activity and number of circulating lymphocytes.

Phase I Study of ch14.18 With Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-2 in Children With Neuroblastoma After Autologous Bone Marrow Transplantation or Stem-Cell Rescue: A Report From the Children's Oncology Group

PURPOSE Recurrence of high-risk neuroblastoma is common despite multimodality therapy. ch14.18, a chimeric human/murine anti-G(D2) antibody, lyses neuroblastoma cells. This study determined the maximum tolerable dose (MTD) and toxicity of ch14.18 given in combination with interleukin-2 (IL-2) after high-dose chemotherapy (HDC)/stem-cell rescue (SCR). Biologic correlates including ch14.18 levels, soluble IL-2 receptor levels, and human antichimeric antibody (HACA) activity were evaluated. PATIENTS AND METHODS Patients were given ch14.18 for 4 days at 28-day intervals. Patients received IL-2 during the second and fourth courses of ch14.18 and granulocyte-macrophage colony-stimulating factor (GM-CSF) during the first, third, and fifth courses. The MTD was determined based on toxicities occurring with the second course. After the determination of the MTD, additional patients were treated to confirm the MTD and to clarify appropriate supportive care. RESULTS Twenty-five patients were enrolled. The MTD of ch14.18 was determined to be 25 mg/m(2)/d for 4 days given concurrently with 4.5 x 10(6) U/m(2)/d of IL-2 for 4 days. IL-2 was also given at a dose of 3 x 10(6) U/m(2)/d for 4 days starting 1 week before ch14.18. Two patients experienced dose-limiting toxicity due to ch14.18 and IL-2. Common toxicities included pain, fever, nausea, emesis, diarrhea, urticaria, mild elevation of hepatic transaminases, capillary leak syndrome, and hypotension. No death attributable to toxicity of therapy occurred. No additional toxicity was seen when cis-retinoic acid (cis-RA) was given between courses of ch14.18. No patient treated at the MTD developed HACA. CONCLUSION ch14.18 in combination with IL-2 was tolerable in the early post-HDC/SCR period. cis-RA can be administered safely between courses of ch14.18 and cytokines.

NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Cancer Immunotherapy

Natural killer (NK) cells play a major role in cancer immunotherapies that involve tumor-antigen targeting by monoclonal antibodies (mAbs). NK cells express a variety of activating and inhibitory receptors that serve to regulate the function and activity of the cells. In the context of targeting cells, NK cells can be “specifically activated” through certain Fc receptors that are expressed on their cell surface. NK cells can express FcγRIIIA and/or FcγRIIC, which can bind to the Fc portion of immunoglobulins, transmitting activating signals within NK cells. Once activated through Fc receptors by antibodies bound to target cells, NK cells are able to lyse target cells without priming, and secrete cytokines like interferon gamma to recruit adaptive immune cells. This antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells is utilized in the treatment of various cancers overexpressing unique antigens, such as neuroblastoma, breast cancer, B cell lymphoma, and others. NK cells also express a family of receptors called killer immunoglobulin-like receptors (KIRs), which regulate the function and response of NK cells toward target cells through their interaction with their cognate ligands that are expressed on tumor cells. Genetic polymorphisms in KIR and KIR-ligands, as well as FcγRs may influence NK cell responsiveness in conjunction with mAb immunotherapies. This review focuses on current therapeutic mAbs, different strategies to augment the anti-tumor efficacy of ADCC, and genotypic factors that may influence patient responses to antibody-dependent immunotherapies.

Enhanced activity of hu14.18-IL2 immunocytokine against murine NXS2 neuroblastoma when combined with interleukin 2 therapy.

Established s.c. NXS2 murine neuroblastoma tumors exhibited transient resolution after suboptimal therapy using the hu14.18-IL2 immunocytokine (IC). The hu14.18-IL2 IC is a fusion protein that has linked a molecule of interleukin 2 (IL-2) to the COOH terminus of each of the IgG heavy chains on the humanized anti-GD2 monoclonal antibody hu14.18. To induce more potent and longer lasting in vivo antitumor effects, we tested hu14.18-IL2 IC in a regimen combining it with constant infusion IL-2 in NXS2 tumor-bearing mice. The addition of the constant infusion IL-2 augmented the antitumor response induced by treatment with the hu14.18-IL2 IC in animals with experimentally induced hepatic metastases and in animals bearing localized s.c. tumors. The combined treatment induced prolonged tumor eradication in most animals bearing s.c. tumors and involved both natural killer cells and T cells. The enhanced ability of this combined treatment to prevent tumor recurrence was not observed when a larger dose of hu14.18-IL2 IC, similar in IL-2 content to the IC plus systemic IL-2 regimen, was tested as single-agent therapy. Animals showing prolonged tumor eradication of established tumors after the combined hu14.18-IL2 plus IL-2 regimen exhibited a protective T-cell-dependent antitumor memory response against NXS2 rechallenge.

Anti-renal-cell carcinoma chimeric antibody G250 facilitates antibody-dependent cellular cytotoxicity with in vitro and in vivo interleukin-2-activated effectors

Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy, whereas treatment with biologics has achieved limited success. Although monoclonal antibodies able to recognize human RCC have been identified, most induce little complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity (ADCC), and thus are of limited potential as therapeutic modalities in their natural conformation. We evaluated a human/ mouse chimeric derivative of the previously described G250 murine monoclonal antibody (mAb), reactive with RCC, to identify a reagent for potential immunotherapy. This chimeric antibody (ch-G250) is composed of the murine variable region from the G250 mAb, which recognizes a tumor-associated antigen expressed on 95% of primary and 86% of metastatic renal cell carcinomas. The constant region of the ch-G250 is comprised of the human IgG1 isotype domains. This chimeric antibody does not bind to normal renal tissue or other normal human tissues, with the exception of gastric mucosal cells and large bile-duct epithelium. Clinical radiolocalization studies have demonstrated the relative tumor-targeting potential of this radiolabeled antibody. This ch-G250 antibody facilitated potent ADCC against several RCC lines when using in vitro and in vivo interleukin-2 (IL-2)-activated peripheral blood mononuclear cells obtained from healthy control donors and patients with cancer, respectively. This lymphocyte-mediated ADCC was specific for RCC cells recognized by the ch-G250 antibody. Using flow cytometry, we found that the level of ADCC was directly related to the degree of binding of ch-G250 to the renal cell target. These in vitro data suggest that this antibody may improve efficacy of IL-2 therapy by targeting cytokine-activated effector cells directly to the tumor and facilitating in vivo ADCC. Clinical studies combining this chimeric antibody with IL-2 treatment will be needed to test the antitumor effects of this ADCC effect in vivo.