RESUMEN
The GM3(Neu5Gc) ganglioside represents a tumor-specific antigen that is considered a promising target for cancer immunotherapy. We previously demonstrated that the humanized antibody 14F7hT, specific for this ganglioside, exhibited significant antitumor effects in preclinical hematological tumor models. As this antibody recognizes human tumor tissues from several origins, we addressed its potential effect on different tumor types. The use of cell lines for testing GM3(Neu5Gc)-targeting strategies, in particular for human malignancies, is complicated by the absence in humans of functional cytidine monophospho-N-acetyl-neuraminic acid hydroxylase (CMAH), the enzyme required for Neu5Gc sialic acid biosynthesis. Quantitative flow cytometry revealed the absence of surface GM3(Neu5Gc) in several human but also mouse cell lines, in the last case due to low expression of the enzyme. Hypoxia-induced expression of this ganglioside on human SKOV3 cells was observed upon culture in Neu5Gc-containing medium without evidence for CMAH-independent biosynthesis. However, only transfection of the mouse Cmah gene into human SKOV3 and mouse 3LL cells induced a stable expression of GM3(Neu5Gc) on the cancer cell surface, resulting in effective models to evaluate the antitumor responses by 14F7hT in vitro and in vivo. This antibody exerted antibody-dependent cell-mediated cytotoxicity (ADCC) and in vivo antitumor effects on these Cmah-transfected non-hematological tumors from both mouse and human origin. These results contribute to validate GM3(Neu5Gc) as a relevant target for cancer immunotherapy and reinforces the value of 14F7hT as a novel anti-cancer drug.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos/farmacología , Gangliósido G(M3)/inmunología , Oxigenasas de Función Mixta/inmunología , Neoplasias/tratamiento farmacológico , Animales , Antígenos de Neoplasias/inmunología , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Oxigenasas de Función Mixta/química , Neoplasias/inmunología , Neoplasias/patología , Células Tumorales CultivadasRESUMEN
Numerous molecules have been considered as targets for cancer immunotherapy because of their levels of expression on tumor cells, their putative importance for tumor biology, and relative immunogenicity. In this review we focus on the ganglioside GM3(Neu5Gc), a glycosphingolipid present on the outer side of the plasma membrane of vertebrate cells. The reasons for selecting GM3(Neu5Gc) as a tumor-specific antigen and its use as a target for cancer immunotherapy are discussed, together with the development of antitumor therapies focused on this target by the Center of Molecular Immunology (CIM, Cuba).
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/inmunología , Gangliósido G(M3)/inmunología , Neoplasias/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales de Origen Murino , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Secuencia de Carbohidratos , Modelos Animales de Enfermedad , Gangliósido G(M3)/antagonistas & inhibidores , Gangliósido G(M3)/química , Humanos , Inmunoterapia/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos , Carcinoma de Pulmón de Células no Pequeñas/terapia , Gangliósido G(M3)/análogos & derivados , Inmunoterapia Activa , Neoplasias Pulmonares/terapia , Anticuerpos Monoclonales de Origen Murino , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Gangliósido G(M3)/inmunología , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Células Tumorales CultivadasRESUMEN
The expression of N-glycolyl-monosialodihexosyl-ganglioside (NGcGM3) in humans is restricted to cancer cells; therefore, it is a tumor antigen. There are measurable quantities of circulating anti-NGcGM3 antibodies (aNGcGM3 Abs) in human serum. Interestingly, some people have circulating Ag-specific immunoglobulins G (IgGs) that are capable of complement mediated cytotoxicity against NGcGM3 positive cells, which is relevant for tumor surveillance. In light of the chemical nature of Ag, we postulated it as a candidate ligand for CD1d. Furthermore, we hypothesize that the immune mechanism involved in the generation of these Abs entails cross talk between B lymphocytes (Bc) and invariant natural killer T cells (iNKT). Combining cellular techniques, such as flow cytometry and biochemical assays, we demonstrated that CD1d binds to NGcGM3 and that human Bc present NGcGM3 in a CD1d context according to two alternative strategies. We also showed that paraformaldehyde treatment of cells expressing CD1d affects the presentation. Finally, by co-culturing primary human Bc with iNKT and measuring Ki-67 expression, we detected a reproducible increment in the proliferation of the iNKT population when Ag was on the medium. Our findings identify a novel, endogenous, human CD1d ligand, which is sufficiently competent to stimulate iNKT. We postulate that CD1d-restricted Bc presentation of NGcGM3 drives effective iNKT activation, an immunological mechanism that has not been previously described for humans, which may contribute to understanding aNGcGM3 occurrence.
Asunto(s)
Antígenos CD1d/inmunología , Linfocitos B/inmunología , Comunicación Celular/inmunología , Gangliósido G(M3)/inmunología , Células T Asesinas Naturales/inmunología , Adulto , Presentación de Antígeno/inmunología , Antígenos CD1d/metabolismo , Linfocitos B/metabolismo , Línea Celular , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Citometría de Flujo , Gangliósido G(M3)/metabolismo , Humanos , Ligandos , Activación de Linfocitos/inmunología , Células T Asesinas Naturales/metabolismo , Tonsila Palatina/citología , Unión Proteica/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Vacunas contra el Cáncer/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Anticuerpos Antiidiotipos/uso terapéutico , Anticuerpos Monoclonales de Origen Murino , Niño , Gangliósido G(M3)/análogos & derivados , Gangliósido G(M3)/inmunología , HumanosRESUMEN
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.
Asunto(s)
Autoanticuerpos/inmunología , Subgrupos de Linfocitos B/inmunología , Gangliósido G(M3)/análogos & derivados , Hibridomas/inmunología , Vigilancia Inmunológica/inmunología , Animales , Autoanticuerpos/sangre , Subgrupos de Linfocitos B/metabolismo , Línea Celular Tumoral , Perros , Femenino , Gangliósido G(M3)/sangre , Gangliósido G(M3)/inmunología , Caballos , Humanos , Hibridomas/metabolismo , Ratones , Ratones Endogámicos BALB CRESUMEN
Gangliosides are sialic acid-bearing glycosphingolipids expressed on all mammalian cell membranes, and participate in several cellular processes. During malignant transformation their expression changes, both at the quantitative and qualitative levels. Of particular interest is the overexpression by tumor cells of Neu5Gc-gangliosides, which are absent, or detected in trace amounts, in human normal cells. The GM3(Neu5Gc) ganglioside in particular has been detected in many human tumors, and it is considered one of the few tumor specific antigen. We previously demonstrated that a humanized antibody specific for this molecule, named 14F7hT, retained the binding and cytotoxic properties of the mouse antibody. In this work, we confirm that 14F7hT exerts a non-apoptotic cell death mechanism in vitro and shows its potent in vivo antitumor activity on a solid mouse myeloma model. Also, we demonstrate, in contrast to the murine counterpart, the capacity of this antibody to induce antibody-dependent cell-mediated cytotoxicity using human effector cells, which increases its potential for the treatment of GM3(Neu5Gc)-expressing human tumors.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Gangliósido G(M3)/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Especificidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Gangliósido G(M3)/inmunología , Humanos , Isoinjertos , Ratones , Carga Tumoral/efectos de los fármacos , Carga Tumoral/inmunologíaRESUMEN
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.
Asunto(s)
Autoantígenos/análisis , Gangliósido G(M3)/análogos & derivados , Neoplasias de la Retina/química , Retinoblastoma/química , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Gangliósido G(M3)/análisis , Gangliósido G(M3)/inmunología , Humanos , Técnicas para InmunoenzimasRESUMEN
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.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Vacunas contra el Cáncer/inmunología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Adyuvantes Inmunológicos/efectos adversos , Compuestos de Alumbre/efectos adversos , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales de Origen Murino , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/efectos adversos , Gangliósido G(M3)/análogos & derivados , Gangliósido G(M3)/antagonistas & inhibidores , Gangliósido G(M3)/inmunología , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Humans, in contrast to other mammals, do not synthesize N-glycolyl-neuraminic acid (Neu5Gc) due to a deletion in the gene (cmah) encoding the enzyme responsible for this conversion, the cytidine monophospho-N-acetyl-neuraminic acid hydroxylase (CMP-Neu5Ac hydroxylase). The detection of considerable amounts of Neu5Gc-sialoconjugates, in particular gangliosides, in human malignancies makes these antigens attractive targets for immunotherapy, in particular with monoclonal antibodies (mAbs). We have previously described a GM3(Neu5Gc) ganglioside-specific mAb, named 14F7, with the ability to kill tumor cells in a complement-independent manner. Silencing the cmah gene in GM3(Neu5Gc)-expressing L1210 mouse lymphocytic leukemia B cells caused the abrogation of this cytotoxic effect. We now show that cmah-silenced L1210 cells (cmah-kd) express a high level of GM3(Neu5Ac) and have an impaired ability for anchorage-independent cell growth and tumor development in vivo. No evidences of increased immunogenicity of the cmah-kd cell line were found. These results provide new evidences on the role of GM3(Neu5Gc), or Neu5Gc-sialoconjugates in general, in tumor biology. As an important tool in this study, we used the humanized version (here referred to as 7C1 mAb) of a recently described, rationally-designed mutant of 14F7 mAb that is able to bind to both GM3(Neu5Gc) and GM3(Neu5Ac). In contrast to its parental antibody, the humanized 14F7 (14F7hT) mAb, 7C1 mAb was able to kill not only GM3(Neu5Gc)-expressing L1210 wild type cells, but also GM3(Neu5Ac)-expressing cmah-kd cells, which endorses this antibody as a potential agent for cancer immunotherapy.
Asunto(s)
Carcinogénesis/metabolismo , Gangliósido G(M3)/metabolismo , Leucemia Linfoide/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Línea Celular Tumoral , Femenino , Gangliósido G(M3)/inmunología , Células HEK293 , Humanos , Leucemia Linfoide/inmunología , Ratones , Ratones Endogámicos DBA , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismoRESUMEN
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.
Asunto(s)
Anticuerpos/inmunología , Anticuerpos/farmacología , Antineoplásicos/farmacología , Gangliósido G(M3)/análogos & derivados , Animales , Anticuerpos/toxicidad , Antineoplásicos/toxicidad , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Gangliósido G(M3)/inmunología , Gangliósido G(M3)/metabolismo , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Masculino , Ratones , Necrosis , Neoplasias/inmunología , Neoplasias/metabolismoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Sitios de Unión de Anticuerpos/genética , Sitios de Unión de Anticuerpos/inmunología , Gangliósido G(M3)/análogos & derivados , Ingeniería de Proteínas , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Reacciones Antígeno-Anticuerpo , Gangliósido G(M3)/química , Gangliósido G(M3)/inmunología , Biblioteca de Genes , Modelos Moleculares , Estructura Molecular , Mutagénesis Sitio-DirigidaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos , Membrana Celular/patología , Gangliósido G(M3)/inmunología , Neoplasias/terapia , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Anticuerpos Antineoplásicos/uso terapéutico , Antígenos de Neoplasias/inmunología , Membrana Celular/inmunología , Gangliósido G(M3)/análogos & derivados , Humanos , Inmunoterapia , Ratones , Neoplasias/inmunologíaRESUMEN
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.
Asunto(s)
Gangliósido G(M3)/análogos & derivados , Tumores Neuroectodérmicos/química , Adolescente , Vacunas contra el Cáncer/inmunología , Niño , Preescolar , Gangliósido G(M3)/análisis , Gangliósido G(M3)/inmunología , Regulación Neoplásica de la Expresión Génica , Genes myc , Humanos , Inmunohistoquímica , Lactante , Antígeno Ki-67/metabolismo , Tumores Neuroectodérmicos/tratamiento farmacológico , Tumores Neuroectodérmicos/patologíaRESUMEN
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.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antígenos de Neoplasias/inmunología , Neoplasias de la Mama/tratamiento farmacológico , Gangliósido G(M3)/inmunología , Inmunoterapia , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Animales , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/metabolismo , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Citotoxicidad Inmunológica/genética , Epítopos de Linfocito T/genética , Femenino , Gangliósido G(M3)/análogos & derivados , Humanos , Hibridomas , Ratones , Oxigenasas de Función Mixta/genética , Terapia Molecular Dirigida , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , ARN Interferente Pequeño/genéticaRESUMEN
Gangliosides are sialic acid-containing glycosphingolipids present in the plasma membrane of most mammalian cells. In humans, the expression of the N-glycolylated (Neu5Gc) variant of the sialic acid has been associated with malignant transformation, constituting therefore an attractive target for cancer immunotherapy. P3 monoclonal antibody (mAb) recognizes Neu5Gc-containing gangliosides, as well as sulfatides. Heavy chain CDR3 (H-CDR3) arginine residues have been shown to be crucial for ganglioside recognition, but less important for anti-idiotypic antibody binding. Here, we describe the effect on antibody reactivity of different mutations involving a single H-CDR3 acid residue. Substitution of glutamate 99 (Kabat numbering) by arginine, aspartate or serine residues resulted in no differences in anti-idiotype binding. However, the first mutation caused increased reactivity with the antigen, including a cytotoxic effect of the antibody on ganglioside-expressing cells previously unseen for the wild type antibody. Another antibody that recognizes N-glycolyl-GM3 ganglioside (GM3(Neu5Gc)), but not other glycolipids, named 14F7, exhibits also an arginine-enriched H-CDR3 and a complement-independent cell death activity. Unlike 14F7 mAb, the cytotoxicity of the P3 E(99)âR mutant antibody did not exclusively depend on ganglioside expression on tumor cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Gangliósido G(M2)/inmunología , Gangliósido G(M3)/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Mutación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Idiotipos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ratones , Datos de Secuencia Molecular , Unión Proteica/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Antineoplásicos/fisiología , Vacunas contra el Cáncer/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/terapia , Gangliósido G(M3)/análogos & derivados , Gangliósido G(M3)/inmunología , Idiotipos de Inmunoglobulinas/fisiología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/terapia , Animales , Anticuerpos Antineoplásicos/administración & dosificación , Anticuerpos Antineoplásicos/biosíntesis , Vacunas contra el Cáncer/inmunología , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/terapia , Carcinoma Pulmonar de Lewis/ultraestructura , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/ultraestructura , Muerte Celular/inmunología , Línea Celular Tumoral , Perros , Caballos , Humanos , Inmunoglobulina G/biosíntesis , Idiotipos de Inmunoglobulinas/administración & dosificación , Inmunoglobulina M/biosíntesis , Leucemia L1210/inmunología , Leucemia L1210/patología , Leucemia L1210/terapia , Neoplasias Pulmonares/ultraestructura , Ratones , Ratones Endogámicos BALB C , Plasmacitoma/inmunología , Plasmacitoma/patología , Plasmacitoma/terapiaRESUMEN
The interaction between cancer vaccine adjuvants and myeloid-derived suppressor cells (MDSCs) is currently poorly understood. Very small size proteoliposomes (VSSP) are a nanoparticulated adjuvant under investigation in clinical trials in patients with renal carcinoma, breast cancer, prostate cancer, and cervical intraepithelial neoplasia grade III. We found that VSSP adjuvant induced a significant splenomegaly due to accumulation of CD11b(+)Gr-1(+) cells. However, VSSP-derived MDSCs showed a reduced capacity to suppress both allogeneic and Ag-specific CTL response compared with that of tumor-induced MDSCs. Moreover, splenic MDSCs isolated from tumor-bearing mice treated with VSSP were phenotypically more similar to those isolated from VSSP-treated tumor-free mice and much less suppressive than tumor-induced MDSCs, both in vitro and in vivo. Furthermore, different from dendritic cell vaccination, inoculation of VSSP-based vaccine in EG.7-OVA tumor-bearing mice was sufficient to avoid tumor-induced tolerance and stimulate an immune response against OVA Ag, similar to that observed in tumor-free mice. This effect correlated with an accelerated differentiation of MDSCs into mature APCs that was promoted by VSSP. VSSP used as a cancer vaccine adjuvant might thus improve antitumor efficacy not only by stimulating a potent immune response against tumor Ags but also by reducing tumor-induced immunosuppression.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Antineoplásicos/administración & dosificación , Vacunas contra el Cáncer/administración & dosificación , Linfoma/prevención & control , Células Mieloides/inmunología , Nanopartículas/administración & dosificación , Proteolípidos/administración & dosificación , Sarcoma Experimental/prevención & control , Animales , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Femenino , Gangliósido G(M3)/administración & dosificación , Gangliósido G(M3)/inmunología , Inhibidores de Crecimiento/administración & dosificación , Linfoma/inmunología , Linfoma/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Mieloides/patología , Neisseria meningitidis/inmunología , Proteolípidos/inmunología , Sarcoma Experimental/inmunología , Sarcoma Experimental/patologíaRESUMEN
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.
Asunto(s)
Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer , Gangliósido G(M3)/análogos & derivados , Inmunoterapia , Neoplasias/inmunología , Animales , Anticuerpos Antiidiotipos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Ensayos Clínicos como Asunto , Gangliósido G(M3)/inmunología , Humanos , Neoplasias/terapiaRESUMEN
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.