RESUMO
BACKGROUND: The aberrant expression of N-glycolyl GM3 ganglioside (NeuGcGM3) in patients with sarcomas was reevaluated by assessing the relation of this molecule with some clinicopathological features and overall survival (OS) of patients. METHODS: Fifty formalin-fixed and paraffin-embedded specimens from patients diagnosed with sarcomas were included. For the evaluation of NeuGcGM3, the 14F7 monoclonal antibody followed by a peroxidase avidin-biotin system was used. Clinicopathological features were obtained from patient records. Survival rates were estimated by the Kaplan-Meier method and compared with the log-rank test. For multivariate analyses, the Cox regression model was used to identify independent prognostic factors for OS. RESULTS: The majority of samples had high levels of NeuGcGM3 expression (66.0%) that showed statistical correlation with age (p = 0.014), TNM stage (p = 0.022), histological grade (p = 0.013) and proliferation rates (p = 0.012). In addition, a tendency for association with tumor depth (p = 0.070) was evidenced. In univariate survival analysis, TNM stage (p = 0.000), occurrence of metastasis (p = 0.000) and expression of NeuGcGM3 (p = 0.034) were significant prognostic factors for OS, while a tendency for association was evidenced for histological grade (p = 0.091). Among these variables, only the presence of metastasis (p = 0.001) was an independent prognostic factor on multivariate analysis. CONCLUSIONS: The present research suggests the evaluation of NeuGcGM3 expression as a complementary prognostic factor in sarcoma, although our results need to be validated in a larger series and prospective studies. Moreover, our results could support the use of this molecule as a target for immunotherapy.
Assuntos
Gangliosídeo G(M3)/análogos & derivados , Regulação Neoplásica da Expressão Gênica , Sarcoma/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células , Feminino , Gangliosídeo G(M3)/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Prognóstico , Estudos Prospectivos , Sarcoma/mortalidade , Análise de Sobrevida , Adulto JovemRESUMO
Antitumor strategies based on positive modulation of the immune system currently represent therapeutic options with prominent acceptance for cancer patients' treatment due to its selectivity and higher tolerance compared to chemotherapy. Racotumomab is an anti-idiotype (anti-Id) monoclonal antibody (mAb) directed to NeuGc-containing gangliosides such as NeuGcGM3, a widely reported tumor-specific neoantigen in many human cancers. Racotumomab has been approved in Latin American countries as an active immunotherapy for advanced non-small cell lung cancer (NSCLC) treatment. In this work, we evaluated the induction of Ab-dependent cell-mediated cytotoxicity (ADCC) in NSCLC patients included in a phase III clinical trial, in response to vaccination with racotumomab. The development of anti-NeuGcGM3 antibodies (Abs) in serum samples of immunized patients was first evaluated using the NeuGcGM3-expressing X63 cells, showing that racotumomab vaccination developed antigen-specific Abs that are able to recognize NeuGcGM3 expressed in tumor cell membranes. ADCC response against NeuGcGM3-expressing X63 (target) was observed in racotumomab-treated- but not in control group patients. When target cells were depleted of gangliosides by treatment with a glucosylceramide synthase inhibitor, we observed a significant reduction of the ADCC activity developed by sera from racotumomab-vaccinated patients, suggesting a target-specific response. Our data demonstrate that anti-NeuGcGM3 Abs induced by racotumomab vaccination are able to mediate an antigen-specific ADCC response against tumor cells in NSCLC patients.
Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Carcinoma Pulmonar de Células não Pequenas/terapia , Gangliosídeo G(M3)/análogos & derivados , Imunoterapia Ativa , Neoplasias Pulmonares/terapia , Anticorpos Monoclonais Murinos , Apoptose , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Gangliosídeo G(M3)/imunologia , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Células Tumorais CultivadasRESUMO
This study aimed to investigate the immunogenicity of a cancer vaccine consisting of the NeuGcGM3 ganglioside combined with the outer membrane protein complex of Neisseria meningitides to form very small size particles. The vaccine is administered together with Montanide ISA51, as adjuvant treatment for breast cancer patients. After surgical resection and standard first-line chemo/radiotherapy, breast cancer patients in stage II-III were enrolled in a phase III clinical trial and allocated into 2 strata, according to the number of positive lymph nodes [stratum I (0-3); stratum II (≥4)]. Subsequently, patients were randomly assigned to receive the vaccine or placebo. The treatment consisted of 5 vaccine doses (200 µg) every 2 weeks and thereafter monthly reimmunizations to complete 15 doses. The vaccine was well-tolerated and high titers of immunoglobulin M and immunoglobulin G anti-NeuGcGM3 antibodies were similarly detected in each stratum. Hyperimmune sera were able to specifically recognize and kill the NeuGcGM3-expressing L1210 tumor cell line, and these functional capacities were significantly associated with a better clinical outcome in patients of stratum II. Besides, postimmune sera had the capacity to revert in vitro the immunosuppression induced by NeuGcGM3, as measured by the prevention of CD4 downmodulation on human T lymphocytes. Vaccination had no impact on the frequency of regulatory T cells or circulating NK cells. This study demonstrated, for the first time, the immunogenicity of the NeuGcGM3/VSSP/Montanide ISA 51 vaccine in the adjuvant setting and describes the functionality of induced anti-NeuGcGM3 antibodies as potential surrogate biomarkers of clinical benefit.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Neoplasias da Mama/terapia , Vacinas Anticâncer/imunologia , Gangliosídeo G(M3)/análogos & derivados , Neisseria meningitidis/genética , Adjuvantes Imunológicos/administração & dosagem , Anticorpos/sangue , Apoptose , Biomarcadores Farmacológicos/sangue , Neoplasias da Mama/imunologia , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Feminino , Gangliosídeo G(M3)/genética , Humanos , Imunidade Humoral , Manitol/administração & dosagem , Manitol/análogos & derivados , Pessoa de Meia-Idade , Neisseria meningitidis/metabolismo , Estadiamento de Neoplasias , Ácidos Oleicos/administração & dosagem , Resultado do TratamentoRESUMO
Interaction between epidermal growth factor receptor (EGFR) signaling with GM3 ganglioside expression has been previously described. However, little is known about EGFR and NeuGcGM3 co-expression in cancer patients and their therapeutic implications. In this paper, we evaluate the co-expression of EGFR and NeuGcGM3 ganglioside in tumors from 92 patients and in two spontaneous lung metastasis models of mice (Lewis lung carcinoma (3LL-D122) in C57BL/6 and mammary carcinoma (4T1) in BALB/c). As results, co-expression of EGFR and NeuGcGM3 ganglioside was frequently observed in 63 of 92 patients (68 %), independently of histological subtype. Moreover, EGFR is co-expressed with NeuGcGM3 ganglioside in the metastasis of 3LL-D122 and 4T1 murine models. Such dual expression appears to be therapeutically relevant, since combined therapy with mAbs against these two molecules synergistically increase the survival of mice treated. Overall, our results suggest that NeuGcGM3 and EGFR may coordinately contribute to the tumor cell biology and that therapeutic combinations against these two targets might be a valid strategy to explore.
Assuntos
Carcinoma Pulmonar de Lewis/genética , Receptores ErbB/genética , Gangliosídeo G(M3)/análogos & derivados , Neoplasias Mamárias Animais/genética , Animais , Anticorpos Monoclonais/administração & dosagem , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Receptores ErbB/biossíntese , Feminino , Gangliosídeo G(M3)/biossíntese , Gangliosídeo G(M3)/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias Mamárias Animais/patologia , Camundongos , Metástase NeoplásicaRESUMO
INTRODUCTION: Racotumomab (originally known as 1E10 mAb) is an anti-idiotype murine IgG1 directed to membrane glycoconjugates expressed in aggressive solid tumors. It was developed as a mirror image of the idiotype of another antibody against N-glycolyl-containing molecules, such as the NeuGcGM3 ganglioside. After a successful phase II/III study, racotumomab formulated in alum was conditionally approved in Latin American countries as maintenance therapy for advanced non-small cell lung cancer. AREAS COVERED: This review analyzes the biology of the target antigen, summarizes preclinical studies and discusses clinical trials in adults and the pediatric experience with racotumomab. EXPERT OPINION: Proper patient selection and combination with chemotherapy, radiotherapy or checkpoint inhibitors appear to be critical issues to maximize the effects of racotumomab vaccination in lung cancer. In a recent phase I clinical trial in children with relapsed or resistant neuroectodermal malignancies, racotumomab was well tolerated and immunogenic, and its evaluation as immunotherapy for high-risk neuroblastoma is warranted.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Animais , Anticorpos Anti-Idiotípicos/uso terapêutico , Anticorpos Monoclonais Murinos , Criança , Gangliosídeo G(M3)/análogos & derivados , Gangliosídeo G(M3)/imunologia , HumanosRESUMO
While not naturally expressed in normal human tissues, N-glycolylated (NeuGc) gangliosides are overexpressed in several tumors and have immunosuppressive capacity, which contributes to cancer progression. Naturally occurring antibodies against NeuGcGM3 exist in healthy donors that specifically recognize and kill tumor cells expressing the antigen by complement-dependent and -independent mechanisms, the latter resembling an oncotic necrosis-type of cell death. Both the levels of anti-NeuGcGM3 antibodies in the sera of healthy donors and the percentage of donors with these natural antibodies decrease with age. Our work has shown that anti-NeuGcGM3 antibodies are not detected in the sera of non-small cell lung cancer (NSCLC) patients, compared to age- and sex-matched healthy donors, which have anti-NeuGcGM3. Interestingly, the level of serum total IgM, but not IgG, was significantly lower in cancer patients than in healthy donors. Screening of immortalized mouse splenic and peritoneal-derived hybridomas showed that peritoneal B-1 cells secrete anti-NeuGcGM3 with tumor cytotoxic capacity. Defects in the natural surveillance against tumor antigens could increase the risk of elderly donors developing cancer and affect the capacity of cancer patients to effectively fight against tumor cells.
Assuntos
Autoanticorpos/imunologia , Subpopulações de Linfócitos B/imunologia , Gangliosídeo G(M3)/análogos & derivados , Hibridomas/imunologia , Vigilância Imunológica/imunologia , Animais , Autoanticorpos/sangue , Subpopulações de Linfócitos B/metabolismo , Linhagem Celular Tumoral , Cães , Feminino , Gangliosídeo G(M3)/sangue , Gangliosídeo G(M3)/imunologia , Cavalos , Humanos , Hibridomas/metabolismo , Camundongos , Camundongos Endogâmicos BALB CRESUMO
INTRODUCTION: The identification of molecules expressed selectively on the surface of retinoblastoma cells would allow applying targeted therapies. The Ganglioside, N-Glycolyl-GM3 (NeuGc-GM3), is an attractive candidate, as it has been detected in other paediatric neuroectodermic tumours, and it is not expressed in human normal tissues. The 14F7 antibody recognizes specifically the ganglioside NeuGc-GM3. PURPOSE: To characterize the expression of NeuGc-GM3 in retinoblastoma cell lines and in retinoblastoma tumours using the 14F7 monoclonal antibody. METHODS: We studied WERI-Rb1 and Y79 cell lines, 24 retinoblastoma primary tumours from unilateral and bilateral cases and two bone marrow biopsies from metastatic retinoblastoma. Tumours were classified into three groups: non-invasive (n = 13), invasive (n = 9) and metastatic (n = 2). Three eyes enucleated because of non-tumoural conditions were used as controls. Cell lines and tumour sections were studied by immunohistochemistry using the 14F7 antibody. NeuGc-GM3 expression was evaluated by analysing the percentage of positive tumoural cells and the staining intensity. These parameters were analysed comparatively among the three groups. RESULTS: Both retinoblastoma cell lines showed immunoreactivity to NeuGc-GM3 but WERI-Rb1 presented higher intensity than Y79. All the tumours studied showed strong immunoreactivity to NeuGc-GM3 with no significant differences among groups. In both bone marrow specimens, NeuGc-GM3 immunoreactivity was observed in retinoblastoma cells. In bilaterally enucleated cases, NeuGc-GM3 immunoreactivity was not altered before and after chemotherapy. Non-tumoural retinas were negative. CONCLUSIONS: NeuGc-GM3 is highly expressed in retinoblastoma cell lines, tumours and metastatic cells to the bone marrow, and it is not detectable in control eyes. There were no significant differences in the immunoreactivity to 14F7 among tumours from different disease stages. Its immunoreactivity did not change after chemotherapy.
Assuntos
Autoantígenos/análise , Gangliosídeo G(M3)/análogos & derivados , Neoplasias da Retina/química , Retinoblastoma/química , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Gangliosídeo G(M3)/análise , Gangliosídeo G(M3)/imunologia , Humanos , Técnicas ImunoenzimáticasRESUMO
Racotumomab-alum vaccine is an anti-idiotypic vaccine able to mimic the tumor-associated antigen NeuGcGM3. Different Phase I clinical trials and compassionate use studies demonstrated its low toxicity and capacity to induce a strong anti-NeuGcGM3 response, able to bind and directly kill tumor cells expressing the antigen. A Phase II/III randomized double-blind clinical trial in advanced non-small cell lung cancer patients showed a significant improvement in overall survival and progression-free survival for racotumomab-alum versus placebo. Patients who developed anti-NeuGcGM3 antibodies capable of binding and killing NeuGcGM3 expressing tumor cells showed significantly longer median survival times. The impact of using racotumomab-alum as switch maintenance followed by second-line therapy is currently being explored in a new randomized, multinational Phase III study.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen/administração & dosagem , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Adjuvantes Imunológicos/efeitos adversos , Compostos de Alúmen/efeitos adversos , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Murinos , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/efeitos adversos , Gangliosídeo G(M3)/análogos & derivados , Gangliosídeo G(M3)/antagonistas & inibidores , Gangliosídeo G(M3)/imunologia , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto , Análise de Sobrevida , Resultado do TratamentoRESUMO
N-glycolylated gangliosides are not naturally expressed in healthy human tissues but are overexpressed in several tumors. We demonstrate the existence of antibodies that bind (N-glycolylneuraminyl)-lactosylceramide (NeuGcGM3) and are detectable in the sera of 65 from the 100 donors (65%) tested by ELISA. From those 65 NeuGcGM3 antibody-positive donors, 35 had antibodies that were able to recognize and kill NeuGcGM3-expressing tumor cells by a complement-mediated mechanism. After complement inactivation, 11 of the 35 positive sera showed a direct cytotoxic effect on the tumor cells. This complement-independent cytotoxicity was dependent on the presence of antigen on the membrane and resembles an oncotic necrosis cell death. Both the levels of anti-NeuGcGM3 antibodies in the sera as well as the percentage of healthy donors with this immunity decreased with the age of the donor. In contrast to age and gender-matched healthy donors, we could only detect low reactivity against NeuGcGM3 in the sera of six out of 53 non-small cell lung cancer patients. These results suggest the existence of antibodies against NeuGcGM3 with antitumor immune surveillance functions, reinforcing the importance of N-glycolylated gangliosides as antitumor targets.
Assuntos
Anticorpos/imunologia , Anticorpos/farmacologia , Antineoplásicos/farmacologia , Gangliosídeo G(M3)/análogos & derivados , Animais , Anticorpos/toxicidade , Antineoplásicos/toxicidade , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Gangliosídeo G(M3)/imunologia , Gangliosídeo G(M3)/metabolismo , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Camundongos , Necrose , Neoplasias/imunologia , Neoplasias/metabolismoRESUMO
The structurally related gangliosides N-glycolyl GM3 and N-acetyl GM3 are potential targets for tumor immunotherapy. 14F7 is a monoclonal antibody able to discriminate the tumor-specific antigen N-glycolyl GM3 from the closely related N-acetyl GM3 on the basis of the presence of a single additional hydroxyl group in the former. A combinatorial phage display strategy, based on the screening of a large library followed by refined mutagenesis, allowed a thorough exploration of the binding chemistry of this unique antibody. Three essential features of the heavy chain variable region were identified: two aromatic rings (in positions 33 and 100D) contributing to the binding site architecture and an arginine residue (position 98) critical for recognition. Directed evolution of 14F7 resulted in novel variants that cross-react with the tumor-associated antigen N-acetyl GM3 and display recurrent replacements: the substitution W33Q and the appearance of additional arginine residues at several positions of CDR H1. Successful conversion of such engineered variable regions into whole cross-reactive anti-GM3 immunoglobulins validated our phage-based approach to study and modify the lead antibody 14F7. The resulting family of closely related antibodies offers new tools to study the mechanisms of cell death induced by antibodies targeting gangliosides. In vitro directed evolution was useful to overcome the technical limitations to obtain anti-ganglioside antibodies. The case of 14F7 illustrates the power of combining library screening with focused site-directed randomization for a comprehensive scanning of protein interactions.
Assuntos
Anticorpos Monoclonais/imunologia , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Gangliosídeo G(M3)/análogos & derivados , Engenharia de Proteínas , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Reações Antígeno-Anticorpo , Gangliosídeo G(M3)/química , Gangliosídeo G(M3)/imunologia , Biblioteca Gênica , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-DirigidaRESUMO
The majority of the most effective monoclonal antibodies (mAbs) currently in the clinics bind to cancer or immune cells. Classic mechanisms of cell killing by therapeutic mAbs include antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and induction of apoptosis by engagement of specific cell ligands. A few reports have described mAbs whose cytotoxic activity is Fc-independent and that do not induce the morphological and biochemical changes associated with the apoptosis-type of cell death. Even fewer works describe mAbs able to directly induce membrane lesions. Here, we discuss the available data on those molecules and their cell killing activity, with particular attention to the case of a mAb specific for the tumor-associated N-glycolyl (Neu5Gc)-GM3 ganglioside (GM3(Neu5Gc)). Some similarities are found in the cell death pathways triggered by these mAbs, but data are not abundant. We conclude that the usefulness of mAbs with a direct cytotoxic activity for immunotherapeutic strategies deserves deeper research.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos , Membrana Celular/patologia , Gangliosídeo G(M3)/imunologia , Neoplasias/terapia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/uso terapêutico , Antígenos de Neoplasias/imunologia , Membrana Celular/imunologia , Gangliosídeo G(M3)/análogos & derivados , Humanos , Imunoterapia , Camundongos , Neoplasias/imunologiaRESUMO
The N-glycolylated ganglioside NeuGc-GM3 has been described in solid tumors such as breast carcinoma, nonsmall cell lung cancer, and melanoma, but is usually not detected in normal human cells. Our aim was to evaluate the presence of NeuGc-GM3 in pediatric neuroectodermal tumors by immunohistochemistry. Twenty-seven archival cases of neuroblastoma and Ewing sarcoma family of tumors (ESFT) were analyzed. Formalin-fixed, paraffin-embedded tumor samples were cut into 5 µm sections. The monoclonal antibody 14F7, a mouse IgG1 that specifically recognizes NeuGc-GM3, and a peroxidase-labeled polymer conjugated to secondary antibodies were used. Presence of NeuGc-GM3 was evident in 23 of 27 cases (85%), with an average of about 70% of positive tumors cells. Immunoreactivity was moderate to intense in most tumors, showing a diffuse cytoplasmic and membranous staining, although cases of ESFT demonstrated a fine granular cytoplasmic pattern. No significant differences were observed between neuroblastoma with and without NMYC oncogene amplification, suggesting that expression of NeuGc-GM3 is preserved in more aggressive cancers. Until now, the expression of N-glycolylated gangliosides in pediatric neuroectodermal tumors has not been investigated. The present study evidenced the expression of NeuGc-GM3 in a high proportion of neuroectodermal tumors, suggesting its potential utility as a specific target of immunotherapy.
Assuntos
Gangliosídeo G(M3)/análogos & derivados , Tumores Neuroectodérmicos/química , Adolescente , Vacinas Anticâncer/imunologia , Criança , Pré-Escolar , Gangliosídeo G(M3)/análise , Gangliosídeo G(M3)/imunologia , Regulação Neoplásica da Expressão Gênica , Genes myc , Humanos , Imuno-Histoquímica , Lactente , Antígeno Ki-67/metabolismo , Tumores Neuroectodérmicos/tratamento farmacológico , Tumores Neuroectodérmicos/patologiaRESUMO
Gangliosides containing the N-glycolyl (NGc) form of sialic acid are tumor-associated antigens and promising candidates for cancer therapy. We previously generated the murine 14F7 monoclonal antibody (mAb), specific for the N-glycolyl-GM3 ganglioside (NGcGM3), which induced an oncosis-like type of cell death on malignant cell lines expressing this antigen and recognized breast carcinoma by immunoscintigraphy in cancer patients. As humanization is expected to enhance its use for human cancer therapy, herein we describe the design and generation of two humanized versions of the 14F7 mAb by disrupting potential human T cell epitopes on its variable region. No differences in antigen reactivity or cytotoxic properties were detected among the variants tested and with respect to the chimeric counterpart. Humanized 14F7 genes were transfected into the NGcGM3-expressing NS0 cell line. Therefore, in the industrial scaling-up of the transfectoma in serum-free medium, cell viability was lost due to the cytotoxic effect of the secreted antibody. This shortcoming was solved by knocking down the CMP-N-acetylneuraminic acid hydroxylase enzyme, thus impairing the synthesis of NGc-glycoconjugates. Humanized 14F7 mAb is of potential value for the therapy of NGcGM3-expressing tumors.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antígenos de Neoplasias/imunologia , Neoplasias da Mama/tratamento farmacológico , Gangliosídeo G(M3)/imunologia , Imunoterapia , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Animais , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Citotoxicidade Imunológica/genética , Epitopos de Linfócito T/genética , Feminino , Gangliosídeo G(M3)/análogos & derivados , Humanos , Hibridomas , Camundongos , Oxigenases de Função Mista/genética , Terapia de Alvo Molecular , Mutagênese Sítio-Dirigida , Engenharia de Proteínas , RNA Interferente Pequeno/genéticaRESUMO
Gangliosides are membrane-associated glycosphingolipids. N-Acetyl GM3 and N-glycolyl GM3 are two tumor-associated antigens expressed in cancer tissues such as melanoma and mammalian cancer. In order to use these antigens in GM3-based vaccines for patients with early stage cancer, the synthetic version is recommended to avoid the risk of animal virus transmission from the source. However, the isolation of natural gangliosides is of comparative value for the structural characterization. The structures of N-acetyl and N-glycolyl GM3 extracted from dog and horse erythrocytes were evaluated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nuclear magnetic resonance techniques; additionally, the natural N-acetyl ganglioside was compared to a synthetic one. In addition to the main compound with C24:0 fatty acid chain, a minor component with an additional unsaturation in the ceramide chain was detected, in both the dog and the horse gangliosides. This paper shows spectroscopic evidence of the aforementioned compounds.
Assuntos
Gangliosídeo G(M3)/química , Gangliosídeos/química , Animais , Ceramidas/química , Cães , Eritrócitos/química , Gangliosídeo G(M3)/análogos & derivados , Gangliosídeo G(M3)/isolamento & purificação , Gangliosídeos/isolamento & purificação , Cavalos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
1E10 is a murine anti-idiotypic mAb specific for an idiotypic mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In melanoma, breast, and lung cancer patients, this anti-idiotypic Ab was able to induce a specific Ab response against N-glycosylated gangliosides, attractive targets for cancer immunotherapy as these glycolipids are not naturally expressed in humans. A clinical study with nonsmall cell lung cancer patients showed encouraging clinical benefits. Immunological studies performed in 20 of these patients suggested a correlation between the induction of Abs against NeuGcGM3 and longer survival times. The induced anti-NeuGcGM3 Abs recognized and directly killed tumor cells expressing the Ag, by a mechanism independent of complement activation. In the present work, we show that this cytotoxicity differs from apoptosis because it is temperature independent, no chromatin condensation or caspase 3 induction are detected, and the DNA fragmentation induced has a different pattern than the one characteristic for apoptosis. It is a very quick process and involves cytosqeleton reorganization. The Abs induce cellular swelling and the formation of big membrane lesions that allow the leakage of cytoplasm and the loss of the cell membrane integrity. All of these characteristics resemble a process of oncotic necrosis. To our knowledge, this is the first report of the active induction in cancer patients of NeuGcGM3-specific Abs able to induce complement independent oncotic necrosis to tumor cells. These results contribute to reinforcing the therapeutic potential of anti-idiotypic vaccines and the importance of NeuGcGM3 ganglioside as antitumor target.
Assuntos
Anticorpos Antineoplásicos/fisiologia , Vacinas Anticâncer/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/terapia , Gangliosídeo G(M3)/análogos & derivados , Gangliosídeo G(M3)/imunologia , Idiótipos de Imunoglobulinas/fisiologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Animais , Anticorpos Antineoplásicos/administração & dosagem , Anticorpos Antineoplásicos/biossíntese , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/terapia , Carcinoma Pulmonar de Lewis/ultraestrutura , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/ultraestrutura , Morte Celular/imunologia , Linhagem Celular Tumoral , Cães , Cavalos , Humanos , Imunoglobulina G/biossíntese , Idiótipos de Imunoglobulinas/administração & dosagem , Imunoglobulina M/biossíntese , Leucemia L1210/imunologia , Leucemia L1210/patologia , Leucemia L1210/terapia , Neoplasias Pulmonares/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Plasmocitoma/imunologia , Plasmocitoma/patologia , Plasmocitoma/terapiaRESUMO
Active specific immunotherapy is a promising field in cancer research. N-glycolyl (NGc) gangliosides, and particularly NGcGM3, have received attention as a privileged target for cancer therapy. Many clinical trials have been performed with the anti-NGc-containing gangliosides anti-idiotype monoclonal antibody racotumomab (formerly known as 1E10) and the conjugated NGcGM3/VSSP vaccine for immunotherapy of melanoma, breast, and lung cancer. The present paper examines the role of NGc-gangliosides in tumor biology as well as the available preclinical and clinical data on these vaccine products. A brief discussion on the relevance of prioritization of cancer antigens in vaccine development is also included.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer , Gangliosídeo G(M3)/análogos & derivados , Imunoterapia , Neoplasias/imunologia , Animais , Anticorpos Anti-Idiotípicos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Ensaios Clínicos como Assunto , Gangliosídeo G(M3)/imunologia , Humanos , Neoplasias/terapiaRESUMO
OBJECTIVE: The target concept means not only an aberrant expression of a particular molecule in tumour tissues but also evidence of a clear therapeutic advantage, as a consequence of immune-intervention, in an antigen-positive relevant tumour model. Since we reported the presence of NGcGM3 ganglioside in human breast tumours years ago and though Phase I clinical trials of a ganglioside containing vaccine have been conducted, a definitive direct validation of this peculiar molecule as target for cancer immunotherapy has remained unperformed. METHODS: Two animal models were used: leghorn chickens and C57BL/6 mice. The murine 3LL-D122 cell line, the derived subcutaneous tumours and metastatic lung lesions were processed for gangliosides identification. Active immunotherapy experiments in the 3LL-D122 spontaneous lung metastasis model were performed with NGcGM3/VSSP vaccine prepared by conjugation of NGcGM3 with the outer membrane proteins of Neisseria meningitides. RESULTS: The 3LL-D122 Lewis lung carcinoma results were consistent with an increased expression of NGcGM3 from primary tumours to metastatic lesions, as observed in human breast cancer samples. Both vaccines, prepared with synthetic or natural-source-derived ganglioside, showed similar anti-tumour and immunogenicity profiles. Finally, a clear involvement of NK1.1(+) cells and CD8(+) T cells in the anti-metastatic effect elicited by the vaccine was manifested. CONCLUSIONS: While 'proof of concept' Phase II and III clinical trials with the NGcGM3/VSSP vaccine in cancer patients are currently ongoing these results reasonably sustain the validation of this peculiar ganglioside as a novel target for cancer immunotherapy.
Assuntos
Vacinas Anticâncer/uso terapêutico , Gangliosídeo G(M3)/análogos & derivados , Gangliosídeo G(M3)/imunologia , Imunoterapia/métodos , Neoplasias/terapia , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Carcinoma Pulmonar de Lewis/imunologia , Eritrócitos/imunologia , Feminino , Citometria de Fluxo , Gangliosídeo G(M3)/metabolismo , Cavalos/imunologia , Humanos , Imuno-Histoquímica , Pulmão/patologia , Neoplasias Pulmonares/secundário , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Neisseria meningitidis/imunologia , Metástase Neoplásica/prevenção & controle , Transplante de NeoplasiasRESUMO
Gangliosides are glycolipids present on the cell surface. The N-glycolylated ganglioside NeuGc-GM3 has been described in some neoplasms, such as breast carcinoma and melanoma, but is usually not detected in normal human cells. Our aim was to evaluate the presence of NeuGc-GM3 in Wilms tumor by immunohistochemistry. Postchemotherapy tumors were grouped into different histologic subtypes considering the main preserved component. Formalin-fixed, paraffin-embedded tumor samples were cut into 5-microm sections. The monoclonal antibody 14F7, a mouse IgG1 that specifically recognizes NeuGc-GM3, and a peroxidase-labeled polymer conjugated to secondary antibodies were used. Sections from breast carcinoma were employed as positive controls. Presence of NeuGc-GM3 was evident in 22 of 25 (88%) cases. The staining was stronger in the epithelial component, with a membrane pattern and cytoplasmic diffusion. The stromal component expressed cytoplasmic NeuGc-GM3 in cells with rhabdomyoblastic differentiation. Tubules of adjacent renal tissue were also positive, but no expression of NeuGc-GM3 was detected in nontumoral fetal kidney. Until now, the expression of N-glycolylated gangliosides in pediatric solid tumors has not been investigated. The present study evidenced the expression of NeuGc-GM3 in a high proportion of Wilms tumors, suggesting its potential utility as a specific target of immunotherapy.
Assuntos
Gangliosídeo G(M3)/análogos & derivados , Imuno-Histoquímica/métodos , Neoplasias Renais/metabolismo , Tumor de Wilms/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Membrana Celular/metabolismo , Membrana Celular/patologia , Terapia Combinada , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Técnica Indireta de Fluorescência para Anticorpo , Gangliosídeo G(M3)/metabolismo , Humanos , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Camundongos , Estudos Retrospectivos , Células Estromais/metabolismo , Células Estromais/patologia , Tumor de Wilms/patologia , Tumor de Wilms/terapiaRESUMO
N-Glycolylated (NeuGc) gangliosides are tumor-specific antigens and as such represent attractive targets for cancer immunotherapy. The chimeric antibody chP3 selectively recognizes a broad variety of NeuGc gangliosides, showing no cross-reactivity to the highly similar N-acetylated (NeuAc) gangliosides that are common cellular antigens in humans. Here, we report the crystal structure of the chP3 Fab and its computer-docking model with the trisaccharide NeuGcalpha3Galbeta4Glcbeta, which represents the carbohydrate moiety of the tumor-antigen NeuGc-GM3. The interaction involves only the heavy chain of the chP3 antibody. The modelled complex is consistent with all available experimental data and shows good surface complementarity. The negatively charged sialic acid residue NeuGc is buried in a pocket flanked by two arginine residues, VH Arg31 and VH Arg100A. We have further investigated the interaction of chP3 with its anti-idiotypic antibody, 1E10 (also known as Racotumomab), currently in clinical trials as a cancer vaccine. While many of the chP3 residues predicted to interact with the NeuGc ganglioside also feature prominently in the modelled complex of chP3 and 1E10, we do not observe structural mimicry. Rather, we suspect that the anti-idiotype 1E10 may serve as an imprint of the structural characteristics of the chP3 idiotype and, consequently, give rise to antibodies with P3-like properties upon immunization.
Assuntos
Anticorpos Monoclonais/química , Gangliosídeo G(M3)/análogos & derivados , Fragmentos Fab das Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/química , Modelos Moleculares , Proteínas Recombinantes de Fusão/química , Animais , Anticorpos Anti-Idiotípicos/química , Vacinas Anticâncer/química , Cristalografia por Raios X , Gangliosídeo G(M3)/química , Humanos , Estrutura Terciária de ProteínaRESUMO
1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id(-)Ag(+) Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id(+)Ag(+) and Id(-)Ag(+) fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id(-)Ag(+) fraction. Both Id(+)Ag(+) and Id(-)Ag(+) Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.