RESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Gangliosídeo G(M2)/imunologia , Gangliosídeo G(M3)/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Mutação , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Camundongos , Dados de Sequência Molecular , Ligação Proteica/imunologiaRESUMO
P3 mAb is an IgM monoclonal antibody specific for N-glycolyl-containing gangliosides. The immunogenicity of the P3 idiotype has been previously described by immunizing syngeneic BALB/c mice with the purified murine IgM or the mouse-human chimeric IgG antibody. In the present work we study the antibody response against the idiotype of P3 mAb through immunization with DNA. We used small immune proteins (SIP) consisting on the idiotype in the scFv format, covalently linked to gamma1CH3, the self-dimerizing domain of murine IgG1. SIPs were previously shown to be appropriate to induce specific anti-idiotypic responses. By gene gun immunization, a polyspecific response was occasionally generated, particularly with the P3 idiotype. A single shot of DNA was sufficient to induce a strong and long-lasting anti-P3 idiotype response. In addition, by delivery of the same DNA construct with a recombinant adeno-associated virus the unique immunogenicity of the P3 idiotype was demonstrated. The requirement of T cells in the anti-P3 idiotype response was indicated by the lack of P3-specific anti-idiotypic antibodies following immunization of both, allogeneic C57BL/6 and athymic BALB/c mice.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Gangliosídeo G(M2)/imunologia , Gangliosídeo G(M3)/imunologia , Idiótipos de Imunoglobulinas/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Anti-Idiotípicos/genética , Anticorpos Monoclonais/genética , Especificidade de Anticorpos , Biolística , DNA/imunologia , Feminino , Gangliosídeo G(M2)/genética , Gangliosídeo G(M3)/genética , Humanos , Imunização , Idiótipos de Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
The antibody heavy chain is generally more important than the light chain for the interaction with the antigen, although many reports demonstrate the influence of the light chain in the antibody binding properties. The heavy chains of anti-N-glycolyl-ganglioside P3 mAb and anti-idiotypic 1E10 mAb display complementary charged residues in their H-CDRs, particularly in H-CDR3. A basic residue in P3 mAb H-CDR1 was shown to be crucial for the interaction with the antigen and 1E10 mAb. The immunogenetic features of three other P3 mAb anti-idiotypic mAbs are now analyzed. One of them bears the same heavy chain as 1E10 mAb and a different light chain, but differs in its binding to P3 mAb mutants where H-CDR basic residues were replaced and in the binding to 1E10-specific phagotopes. Chimeric hybrid antibodies with P3 and 1E10 mAb heavy chains and unrelated light chains were obtained to further determine the importance of heavy chains in P3 and 1E10 mAb binding properties. One of the P3 heavy chain hybrid antibodies retained the specificity of P3 mAb with slight affinity differences. The heavy chains appear to play the main role in these mAb interactions, with the light chains modulating the affinity to their ligands.
Assuntos
Anticorpos Monoclonais/imunologia , Gangliosídeo G(M2)/imunologia , Gangliosídeo G(M3)/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/genética , Sequência de Bases , Linhagem Celular Tumoral , Cadeias Pesadas de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Hipermutação Somática de ImunoglobulinaRESUMO
We have previously generated a murine anti-idiotype (Ab2) monoclonal antibody (mAb) to a murine Ab1 mAb, named P3, which selectively binds Neu-glycolyl (NeuGc)-sialic acid on several monosialo- and disialogangliosides, and also reacts with sulfatides and antigens expressed in human melanoma and breast tumors. This Ab2 mAb, designated as 1E10, induced anti-anti-idiotype antibodies (Ab3) in mice and cancer patients. These Ab3 generated by 1E10 mAb were characterized by bearing P3 mAb idiotopes (Ab3, Id +). But when the specificity of these Ab3 antibodies was tested, no specific humoral response against NeuGc-containing gangliosides was detected in sera from immunized mice. However, hyperimmune sera from melanoma and breast cancer patients vaccinated with this Ab2 mAb were able to react specifically with these gangliosides. The different expression of NeuGc-containing gangliosides in the normal tissues of mice and humans could explain these results. In order to demonstrate these findings in other animal species with a different NeuGc-sialic acid expression, we performed similar studies in monkeys and chickens. In monkeys, as in most mammals, NeuGc-containing gangliosides are self-antigens. In contrast, chickens, like humans, lack the expression of these antigens in normal tissues. Here we report that the antibody response against NeuGc-containing gangliosides induced by immunization with 1E10 mAb was completely different in both species. No specific antibody response against these gangliosides was detected in hyperimmune monkey sera. In contrast, a strong and specific Ab3 response against GM3(NeuGc) and GM2(NeuGc) gangliosides (Ab3, Ag+) was generated in chickens due to the administration of 1E10 mAb.
Assuntos
Anticorpos Monoclonais/imunologia , Gangliosídeo G(M2)/imunologia , Gangliosídeo G(M3)/imunologia , Imunização , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Galinhas/imunologia , Citometria de Fluxo , Humanos , Macaca/imunologia , Especificidade da EspécieRESUMO
The most important link between the immune network theory and clinically useful therapies so far is the use of human intravenous immunoglobulin (IVIg) in the treatment of autoimmune diseases. Although still controversial, one of the main mechanisms that has been postulated for the in vivo effects of IVIg, is the selection of immune repertoires through idiotypic interactions. We describe here anti-idiotype IgG monoclonal antibodies (MAbs), which were obtained by immunization of syngeneic mice (Balb/c) with an anti-ganglioside antibody. These anti-idiotype MAbs show multiple idiotypic connections and share some of the properties of the IVIg pool. The antiidiotype (Ab2) MAbs B7 and 34B7 showed heterogeneous binding with the idiotypes of several anti-ganglioside antibodies, MAbs obtained from splenocytes of nonimmunized newborn mice, F(ab')2 fragments of IgG human myeloma proteins, and nonimmunoglobulin antigens. The recognition pattern of the B7 MAb to the idiotypes of human immunoglobulins was also studied using a phage display library obtained from the variable region genes of an asymptomatic AIDS patient and also F(ab')2 fragments obtained from an IVIg pool of healthy human donors. We also demonstrated that these MAbs produced some of the in vitro effects reported for the human IVIg pool, such as the inhibition of cell proliferation of human B and T cell lines and of normal human lymphocytes activated with different mitogens. Another striking property of the MAb B7 was its ability to induce a dose-dependent specific antibody T-cell response in vivo in syngeneic mice. Both anti-idiotype MAbs showed anti-metastatic effect in vivo when injected intravenously to mice inoculated with MB16-F10 melanoma cells. The antimetastatic effect of the antiidiotype MAbs was not observed in athymic mice inoculated with the same tumor. This kind of antibody can become an interesting tool for further exploration of the role of idiotypic network connections in the regulation of the immune system and to study the effects of interventions on network connectivity in experimental autoimmune disease, using a reagent better chemically defined than the IVIg pool.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Idiótipos de Imunoglobulinas/imunologia , Imunoglobulinas Intravenosas/imunologia , Animais , Anticorpos Anti-Idiotípicos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Doenças Autoimunes/imunologia , Doenças Autoimunes/terapia , Linfócitos B/citologia , Linfócitos B/imunologia , Divisão Celular , Linhagem Celular , Reações Cruzadas , Gangliosídeo G(M2)/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Técnicas In Vitro , Melanoma Experimental/imunologia , Melanoma Experimental/secundário , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Purified GM1 and GM2 gangliosides incorporated into liposomes were injected subcutaneously in BALB/c mice every 3-4 days after pretreatment of the animals with low-dose cyclophosphamide. Serum samples were collected at different intervals and tests by ELISA for the presence of anti-ganglioside antibodies. Four doses (50 micrograms each) were sufficient to raise a measurable primary type of response to GM1, while nine doses were required to obtain measurable IgM antibody titers to GM2. Three monoclonal antibodies (MAbs) wer generated by fusing splenocytes with mouse myeloma cells. The specificity of MAbs was determined by ELISA and HPTLC-immunostaining using a panel of purified glycolipids. The MAb designated E1 showed a high degree of specificity because it reacted only with N-acetyl GM2. Monoclonal antibody A3 reacted predominantly with GM2 and GM1, but also reacted moderately with the GM3 ganglioside. The epitope recognized by this MAb is suggested to be the trisaccharide sequence GalNAc beta 1-4(NeuAc alpha 2-3)Gal. The third MAb (F6) reacted strongly with GM1 but a weak reactivity was also observed with GD1b as well as with asialo-GM1, indicating that the terminal tetrasaccharide Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal- structure is probably involved in antigenic recognition. Formalin-fixed and paraffin-embedded tissue sections were stained with the E1 and A3 MAbs, using the avidin-biotin complex (ABC) technique. Strong immunoreactivity for E1 appeared in the tumor cells of five primary lung carcinomas and in five malignant melanomas. No immunoreactivity was demonstrated in the parenchyma of a lung without malignancy, or in a metastasis from a colon carcinoma.