RESUMO
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).
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/imunologia , Gangliosídeo G(M3)/imunologia , Neoplasias/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Murinos , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Sequência de Carboidratos , Modelos Animais de Doenças , Gangliosídeo G(M3)/antagonistas & inibidores , Gangliosídeo G(M3)/química , Humanos , Imunoterapia/métodos , Neoplasias/tratamento farmacológico , 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 synthesis of gangliosides GM3 and GD3 is carried out by the successive addition of sialic acid residues on lactosylceramide (LacCer) by the Golgi located sialyltransferases Sial-T1 and Sial-T2, respectively. CHO-K1 cells lack Sial-T2 and only express GM3. Here we show that the activity of Sial-T1 was near 2.5-fold higher in homogenates of CHO-K1 cells transfected to express Sial-T2 (CHO-K1(Sial-T2)) than in untransfected cells. The appearance of Sial-T1 enzyme or gene transcription activators or the stabilization of the Sial-T1 protein were discarded as possible causes of the activation. Sial-T2 lacking the catalytic domain failed to promote Sial-T1 activation. Since Gal-T1, Sial-T1 and Sial-T2 form a multienzyme complex, we propose that transformation of formed GM3 into GD3 and GT3 by Sial-T2 in the complex leaves Sial-T1 unoccupied, enabled for new rounds of LacCer utilization, which results in its apparent activation.
Assuntos
Antígenos CD/química , Gangliosídeo G(M3)/química , Gangliosídeos/química , Glicolipídeos/química , Glicosiltransferases/metabolismo , Lactosilceramidas/química , Animais , Células CHO , Domínio Catalítico , Cricetinae , Cricetulus , Glicosilação , Complexo de Golgi/metabolismo , Estrutura Terciária de Proteína , Transcrição Gênica , Ativação TranscricionalRESUMO
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
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
We generated the 1E10 gamma-type anti-idiotype mAb (Ab2) specific to an Ab1 mAb able to react specifically with N-glycolyl-containing gangliosides and with Ags expressed on human melanoma and breast carcinoma cells. This Ab2 mAb induced an Ab response in animal models sharing immunochemically defined idiotopes with the Ab1. The treatment of tumor-bearing mice with 1E10 mAb induced a strong antitumor activity. A clinical trial was conducted in 20 patients with advanced malignant melanoma. Patients were treated with six intradermal injections of aluminum hydroxide-precipitated 1E10 anti-Id mAb given at 2-wk intervals. Sixteen of the 17 patients who received at least four doses of the anti-Id vaccine develop Ab3 Abs capable of inhibiting Ab2 binding to Ab1 (Ab3Id+). In contrast to the incapacity of 1E10 mAb to generate Ab3 Abs with the same antigenic specificity as the Ab1 mAb in mice, a very specific and strong Ab3 response against N-glycolyl-containing gangliosides was induced in 16 patients (Ab3Ag+). No evidence of serious or unexpected adverse effects has been observed in this clinical trial. 1E10 anti-Id vaccine was safe, well tolerated, and immunologically effective, with most patients being able to generate a specific immune response against 1E10 and Neu-glycolyl-GM(3) ganglioside.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Gangliosídeo G(M3)/imunologia , Melanoma/imunologia , Ácido N-Acetilneuramínico/imunologia , Adulto , Idoso , Anticorpos Anti-Idiotípicos/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Especificidade de Anticorpos , Antígenos de Neoplasias/química , Ligação Competitiva , Vacinas Anticâncer/uso terapêutico , Feminino , Gangliosídeo G(M3)/química , Humanos , Imuno-Histoquímica , Cinética , Masculino , Melanoma/terapia , Pessoa de Meia-IdadeRESUMO
Three methods (using GM3 quantities ranging from a few milligrams to grams) have been developed to prepare, in high yield, the three derivatives of ganglioside GM3 [alpha-Neu5Ac-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-ceramide]: deacetyl-GM3 [alpha-Neu-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-ceramide], lyso-GM3 [alpha-Neu5Ac-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-sphingosine], and deacetyl-lyso-GM3 [alpha-Neu-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-sphingosine]. This is the first report of the preparation of lyso-GM3 by a one-pot reaction. We can now define the optimal conditions for the different preparations. Preparation of deacetyl-GM3: alkaline reagent, 2 M KOH in water; GM3 concentration, 33 mg/ml; reaction temperature, 90 degrees C; reaction time, 3.5 h; nitrogen atmosphere. Preparation of deacetyl-lyso-GM3: alkaline reagent, 8 M KOH in water; GM3 concentration, 10 mg/ml; reaction temperature, 90 degrees C; reaction time, 18 h; nitrogen atmosphere. Preparation of lyso-GM(3): alkaline reagent, 1 M sodium tert-butoxide in methanol; GM3 concentration, 10 mg/ml; reaction temperature, 80 degrees C; reaction time, 18 h; anhydrous conditions. The percentage yield of deacetyl-GM3 was 70;-75%, that of deacetyl-lyso-GM3 100%, and of lyso-GM3 36;-40%.Deacetyl-GM3, deacetyl-lyso-GM3, and lyso-GM3 were purified by column chromatography, and chemical structures were confirmed by electron spray-mass spectrometry.
Assuntos
Gangliosídeo G(M3)/análogos & derivados , Gangliosídeo G(M3)/química , Cromatografia Líquida de Alta Pressão , Colorimetria , Gangliosídeo G(M3)/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Hidróxidos , Cinética , Ácido N-Acetilneuramínico/química , Ácidos Neuramínicos/química , Compostos de Potássio , Espectrometria de Massas por Ionização por Electrospray , Esfingosina/química , TrítioRESUMO
The sialic acid of gangliosides not containing GalNAc (i.e., GM3, GD3) is readily hydrolyzed either enzymatically by sialidases or chemically in acid conditions. On the other hand, in gangliosides having the sialic acid on the internal galactose residue linked to GalNAc (i.e., GM1, GM2) the Neu5Ac is largely resistant to acid or enzymatic hydrolysis. In the present work GM1 (NH4+), GM1(H+), and several de-acetylated derivatives in the sialic acid and in both sialic acid and N-acetylgalactosamine moieties were prepared. Studies by counterion exchange with DEAE-Sephadex A-25 and Dowex 50WX8, acid-base titration, and acid or enzymatic hydrolysis with sialidases were performed on these derivatives. Our results provide cumulative evidence supporting that a hydrogen bonding interaction between the hydrogen atom of un-ionized carboxyl group in Neu5Ac and the oxygen atom of the carbonyl group in GalNAc reduces the dissociation of the Neu5Ac carboxyl group and impairs its enzymatic and acid hydrolysis. In addition, our results suggest that the enzymatic hydrolysis of the ionized form of sialic acid in GM1(Na+) and GM1(NH4+) is impaired by a second hydrogen bonding interaction between the proton of the acetamide group in GalNAc and the carbonyl moiety of the carboxyl group of the Neu5Ac.