ganglioside--gd2 and Liver-Neoplasms

Immunocytokines (ICKs) targeting cytokines to the tumor environment using antibodies directed against a tumor-associated antigen often have a higher therapeutic index than the corresponding unconjugated cytokines. Various ICKs displaying significant antitumoral effects in several murine tumor models have already been developed, and some of them, in particular interleukin (IL)-2-based ICKs, are in Phase II clinical trials. Although sharing common biological activities with IL-2 in vitro, IL-15 is now considered as having a better potential in antitumor immunotherapeutical strategies and has been shown to be less toxic than IL-2 in preclinical studies. We previously developed the fusion protein RLI, linking a soluble form of human IL-15Rα-sushi+ domain to human IL-15. RLI showed better biological activities than IL-15 in vitro as well as higher antitumoral effects in vivo in murine and human cancer models. Here, we investigated, in the context of an ICK, the effect of associating RLI with an antibody targeting the GD2 ganglioside, a validated tumoral target expressed on many neurectodermal tumors. Anti-GD2-RLI fully retained the cytokine potential of RLI and the antibody effector functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity). It displayed strong antitumor activities in two syngeneic cancer models in immunocompetent mice (subcutaneous EL4 and metastatic NXS2). Its therapeutic potency was higher than those of RLI and anti-GD2 alone or in combination. We suggest that this is related to its bifunctional (cytokine and antibody) nature.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Antibody-Dependent Cell Cytotoxicity; Antigens, Neoplasm; Cell Line, Tumor; Cell Proliferation; Female; Gangliosides; Humans; Immunotherapy; Interleukin-15; Interleukin-15 Receptor alpha Subunit; Liver Neoplasms; Lymphoma, T-Cell; Male; Mice; Mice, Inbred A; Mice, Inbred C57BL; Neoplasm Metastasis; Neuroblastoma; Protein Binding; Recombinant Fusion Proteins

Early and correct diagnosis of local tumor recurrence, occurrence of metastases, and therapy response are essential in patients with neuroblastoma stage IV. The aim of the study was to evaluate the diagnostic value of metaiodobenzylguanidine (mIBG) and a chimeric GD2 antibody in the follow-up of patients with neuroblastoma. In a prospective study, mIBG (N = 31 scans) and immunoscintigraphy were compared with a chimeric antiganglioside antibody, ch14.18 (MAb) (N = 31 scans), labeled with technetium Tc 99m in the follow-up of 18 patients with stage IV neuroblastoma. The findings were compared with histologic findings, other imaging examinations, and clinical changes over the course of 4 to 6 years. For the diagnosis of local tumor recurrences, sensitivity was 80% for MAb and 70% for mIBG. Specificity was 93% for MAb and 72% for mIBG. The MAb was superior for the detection of skeletal metastases, with a sensitivity of 82% compared with 72% for mIBG. Specificity was 100% for both techniques. Also, for soft tissue/lymph node metastases, sensitivity for MAb was higher (50%) than for mIBG (31%). Specificity was 100% for each technique. In sequential studies, metastases were detected earlier with MAb (mean: 2.3 m for skeletal metastases, 3.6 m for soft tissue metastases) than with mIBG. After therapy, tumor uptake was visualized longer with mIBG (mean 6.3 m) than with MAb. The chimeric antibody ch14.18 is likely to be valuable for follow-up examinations and for assessment of therapy response because of earlier detection of new metastases.

Topics: 3-Iodobenzylguanidine; Adolescent; Antibodies, Monoclonal; Bone Neoplasms; Child; Child, Preschool; False Negative Reactions; Female; Fluorescent Antibody Technique; Follow-Up Studies; Gangliosides; Humans; Liver Neoplasms; Male; Neoplasm Recurrence, Local; Neoplasm Staging; Neuroblastoma; Radionuclide Imaging; Radiopharmaceuticals; Recombinant Fusion Proteins; Soft Tissue Neoplasms; Technetium

Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody-IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell-dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell-deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell-stimulating agents, such as poly I:C or recombinant mouse interferon-gamma. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.

Topics: Animals; Antibodies; Bone Marrow Neoplasms; Female; Gangliosides; Histocompatibility Antigens Class I; Immunotherapy; Interleukin-2; Killer Cells, Natural; Liver Neoplasms; Mice; Mice, SCID; Neuroblastoma; Recombinant Fusion Proteins; Tumor Cells, Cultured

After 10 years of clinical trials in patients with advanced cancer, monoclonal antibodies (mAbs) against cell surface antigens have not lived up to their initial promise. One such cell surface antigen is the ganglioside GD2. GD2 is richly expressed at the cell surfaces of human neuroblastomas, sarcomas, and melanomas. We have described a murine lymphoma (EL4) that is syngeneic in C57BL/6 mice and expresses GD2, a mAb against GD2 (mAb 3F8), and we have prepared a conjugate vaccine (GD2-keyhole limpet hemocyanin plus immunological adjuvant QS-21) that consistently induces antibodies against GD2. We demonstrate here, for the first time in a syngeneic murine model, that passively administered and vaccine-induced antiganglioside antibodies prevent outgrowth of micrometastases, and we use this model to establish some of the parameters of this protection. The level of protection was proportional to antibody titer. Treatment regimens resulting in the highest titer antibodies induced the most protection, and protection was demonstrated even when immunization was initiated after tumor challenge. Treatment with 3F8 1, 2, or 4 days after i.v. tumor challenge was highly protective, but waiting until 7 or 10 days after challenge resulted in minimal protection. The results were similar whether number of liver metastases or survival was used as the end point. These results suggest that unmodified mAbs or antibody-inducing vaccines against GD2 (and possibly other cancer cell surface antigens) should be used exclusively in the adjuvant setting, where circulating tumor cells and micrometastases are the primary targets.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; Cancer Vaccines; Gangliosides; Hemocyanins; Liver Neoplasms; Lymphoma; Mice; Mice, Inbred C57BL; Neoplasm Proteins; Neoplasm Transplantation

Antibody-cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody-interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the Cgamma1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumor-specific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumor-specific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.

Topics: Animals; Antibodies; Base Sequence; Cell Line; DNA Primers; ErbB Receptors; Gangliosides; Humans; Immunotherapy; Immunotoxins; Interleukin-2; Killer Cells, Lymphokine-Activated; Liver Neoplasms; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Mice, SCID; Molecular Sequence Data; Polymerase Chain Reaction; Recombinant Fusion Proteins; Skin Neoplasms; Survival Rate; Time Factors; Transplantation, Heterologous

The inability of current therapy to prevent metastases arising from uveal melanoma often results in patient mortality. With the goal of developing a treatment for metastasis, gangliosides were studied as potential tumor-associated antigens. Our report describes the production of a metastatic liver variant (MH) from a human uveal melanoma cell line (SP6.5). Cells were injected into nude mouse spleens and liver metastases collected 2 months later. After 21 days of in vitro subculture, the cells were re-injected into normal nude mice spleen; 10 cycles (MH10) were performed. Gangliosides were extracted, purified, chromatographed on HPTLC plates and sprayed with a resorcinol-HCl reagent, the sialic acid spots being quantified by densitometry. Gangliosides were analyzed in each metastatic liver variant and compared with the SP6.5 s.c. tumor. The results showed a significant increase in GM3 and a significant decrease in GD3 and GD2 in the last metastatic variants obtained (MH5, MH8, MH9 and MH1O) compared with the primary s.c. tumor, SP6.5. Such evolution in the ganglioside pattern was maintained throughout the progression of the different liver variants. Our results indicate that precursor ganglioside GM3 and gangliosides GD3 and GD2 could be associated with neoplastic evolution of malignancy of human uveal melanoma in nude mice.

Topics: Animals; G(M3) Ganglioside; Gangliosides; Humans; Liver Neoplasms; Melanoma; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Tumor Cells, Cultured; Uveal Neoplasms

A genetically engineered fusion protein consisting of a human/mouse chimeric anti-ganglioside GD2 antibody (ch14.18) and recombinant human interleukin 2 (rhIL-2) was tested for its ability to target rhIL-2 to tumor sites and stimulate immune effector cells sufficiently to achieve effective tumor cell lysis in vivo. The ch14.18-IL-2 fusion protein proved more effective than equivalent doses of rhIL-2 in suppressing dissemination and growth of human neuroblastoma in an experimental hepatic metastases model of scid (severe combined immunodeficiency) mice reconstituted with human lymphokine-activated killer cells. The ch14.18-IL-2 fusion protein was also more proficient than equivalent doses of rhIL-2 in prolonging the life-span of these animals. This recombinant antibody-cytokine fusion protein may prove useful for future treatment of GD2-expressing human tumors in an adjuvant setting.

Topics: Animals; Antibodies; Cytotoxicity, Immunologic; Gangliosides; Humans; Immunotherapy, Adoptive; Interleukin-2; Killer Cells, Lymphokine-Activated; Liver Neoplasms; Mice; Mice, SCID; Neuroblastoma; Recombinant Fusion Proteins; Transplantation, Heterologous

In vivo and in vitro studies were performed to determine: (a) if human uveal melanoma cells expressed GD2 and GD3 gangliosides; (b) if anti-GD2 monoclonal antibodies would inhibit the propensity of human uveal melanoma cells to localize in the liver following intravenous injection; and (c) if anti-GD2 monoclonal antibody would reduce the spontaneous metastasis of primary intraocular melanomas in nude mice. The results showed that all three of the human uveal melanoma cell lines tested expressed GD2 and GD3 gangliosides in vitro and in vivo. The human uveal melanoma cell lines preferentially localized in the liver and entered the hepatic parenchyma following spontaneous metastasis from the eyes of nude mice. In vivo administration of anti-GD2 monoclonal antibody produced a sharp reduction in the number of uveal melanoma cells that disseminated to the liver following either intravenous injection or by spontaneous metastasis from primary intraocular melanomas. Collectively, the results demonstrate that uveal melanoma cells display a propensity to localize in the liver after entering the bloodstream; however, this localization can be significantly inhibited by in vivo administration of anti-ganglioside antibodies. The expression of GD2 and GD3 surface gangliosides on uveal melanomas and the capacity of anti-ganglioside antibodies to inhibit metastasis formation in mouse models of ocular and cutaneous melanomas raise the possibility of implementing anti-ganglioside antibodies as potential therapeutic agents for the management of uveal melanoma.

Topics: Animals; Antibodies, Monoclonal; Disease Models, Animal; Female; Flow Cytometry; Gangliosides; Humans; Immunotherapy; Liver Neoplasms; Melanoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Skin Neoplasms; Tumor Cells, Cultured; Uveal Neoplasms