Search

The immunogenicity of immunoglobulin idiotypes in syngeneic systems is a rather rare phenomenon. Very few studies have attempted to determine the mechanisms underlying the anti-idiotypic response that certain autologous idiotypes can elicit. Furthermore, the studies addressing a possible physiological role for such behaviors are even less. In the present article, the results of the characterization of a highly immunogenic idiotype are reviewed and compared with some related works on the subject. We finally propose a possible immunoregulatory role for idiotypic immunogenicity.

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-glycolilated 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 cytosqueleton 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. [ABSTRACT FROM AUTHOR]

IOR-T3, a mouse monoclonal antibody specific for human CD3, has been successfully used in the treatment of acute transplant rejection due to its potential as T-cell immunosuppressant. In the present work we report the construction of a human IgG1 chimeric variant of IOR-T3, named T3q. In order to reduce the T-cell activating capacity of the newly obtained chimeric molecule, the two leucine residues at positions 234 and 235 of the CH2 region were replaced by alanines, obtaining the T3q(Ala/Ala) molecule. In vitro evaluation of T3q and T3q(Ala/ Ala) showed that there were no differences in the recognition of human CD3 in comparison with murine IOR-T3. However, the Fc-mutated version T3q(Ala/Ala) displayed a much weaker FcγR binding capacity than the unmutated chimeric molecule T3q, as well as a reduced ability to induce T-cell proliferation, proinflammatory cytokine release (TNFα and IL-6), and early activation surface marker expression (CD25 and CD69). We also found that, unlike treatment with T3q, the reduction in T-cell proliferation was less marked on CD8+ cells compared to the CD4+ cells after treatment with T3q(Ala/Ala). These properties make T3q(Ala/Ala) an attractive clinical alternative as an immunoregulatory agent endowed with reduced toxicity. [ABSTRACT FROM AUTHOR]

Abstract: 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 NeuGcα3Galβ4Glcβ, 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. [Copyright &y& Elsevier]

Abstract: The heavy chain of anti-N-glycolyl-ganglioside P3 mAb plays the main role in its binding properties. At least one hybrid idiotype consisting on the P3 VH and an unrelated VL domain retains antigen recognition. Moreover, the unusual immunogenic properties of P3 idiotype could be modified by single mutations of H-CDR residues. Here, we show that DNA gene gun immunization with the P3 VH combined with an unrelated VL domain or with itself (VH dimer, VHD) is enough for inducing anti-idiotypic antibodies, independently of antigen recognition by the resulting molecule. The scFv fragment of P3 mAb was also able to induce an anti-idiotypic response. For both the P3 and the P3 anti-idiotypic 1E10 mAbs, heavy chains dominate the induction of antibodies against the respective idiotypes. [Copyright &y& Elsevier]

Neonatal natural antibodies (NAbs) are characterized by their high degree of idiotypic cross reactivity, together with some restrictions in the genetic mechanisms of variable region diversity. We report here the immunogenetic analysis of two anti-idiotype antibodies (B7 and 34B7 monoclonal antibodies [MAbs]), which are also polyreactive as NAbs. Evidence of a process of somatic mutations were found for heavy and light chain variable regions of both antibodies. A phylogenetic analysis of the VHJ558 family showed that the immunoglobulin cross-reactivity displayed by B7 and 34B7 MAbs is not restricted to a particular subgroup of this family. Moreover, we identified amino acid motifs in the CDR H1 and H2 of B7 and 34B7 MAbs that are also present in high proportion in immunoglobulin cross-reactive antibodies (ICRA) reported in the Kabat database. We propose that these regions are involved in ICRA activity. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: This report is focused on the molecular basis for the interaction of a monoclonal antibody (mAb) and its anti-idiotypic mAb. P3 mAb (Ab1) recognizes N-glycolyl-gangliosides, and 1E10 mAb is one of its anti-idiotypic mAbs (Ab2). Chimeric versions of both antibodies retained their specificity. Charged residues in their H-CDRs, particularly H-CDR3, were considered to play a major role in their binding and immunogenic properties. P3 mAb has the unusual property of generating a strong antibody response in syngeneic mice, even when it is administered in saline. We selected phagotopes from a 12mer peptide library displayed on filamentous phage to characterize amino acid motifs recognized by these antibodies. The peptides were enriched in charged amino acids similar to those present in P3 and 1E10 mAb H-CDR3. We also report the construction of four mutants of the P3 antibody, where arginine residues in the heavy chain CDRs were substituted by serine residues, and the characterization of their interaction with 1E10 mAb and GM3(NeuGc) ganglioside, as well as their immunogenic properties in Balb/c mice. H-CDR1 R31 residue appears to have a central role in P3 mAb reactivity and antigenicity. H-CDR3 R100a residue seems to be more involved in the immunogenicity of the P3 idiotype. [Copyright &y& Elsevier]

The epidermal growth factor receptor (EGFR) is considered one of the main targets for the treatment of epithelial tumors. Growing evidence shows the essential role of this receptor in disseminating metastasis. The Center of Molecular Immunology (CIM) has pioneered the development of this new therapeutic concept, generating different anti-EGFR agents that are currently under preclinical and clinical evaluation. One of these agents is the h-R3 monoclonal antibody (MAb h-R3), used to treat patients with metastatic tumors. The mechanisms by which the anti-EGFR MAbs exert their anti-metastatic effect is not yet clear, since studies have been made in athymic mice transplanted with human tumors, where the role of T cells cannot be characterized. We obtained here for the first time a MAb that can recognize the extracellular domain of the murine EGFR, named 7A7. It allowed us to confirm the potent anti-metastatic effect of anti-EGFR MAbs in immunocompetent mice. The EGFR signaling inhibition, mediated by anti-proliferative, anti-migratory and pro-apoptotic effects contributes to its anti-metastatic effect. Also, these results suggest the induction of cytotoxic responses through the Fc region of 7A7 could increase its antitumor effect. This was also the first report of a MAb that depends on CD4+ and CD8+ T cell functions to exert its anti-metastatic effects. [ABSTRACT FROM AUTHOR]

El receptor del factor de crecimiento epidérmico (EGFR, del inglés epidermal growth factor receptor) es considerado uno de los blancos principales para el tratamiento de los tumores de origen epitelial. Se han acumulado evidencias de que este receptor puede desempeñar un papel importante en la diseminación metastásica. El Centro de Inmunología Molecular (CIM) ha sido pionero en el desarrollo de este nuevo concepto terapéutico, y ha generado diferentes agentes anti-EGFR que se encuentran en fases de evaluación preclínica y clínica. Un ejemplo de ello es el anticuerpo monoclonal (AcM) h R3, utilizado para el tratamiento de pacientes con tumores metastásicos. Los mecanismos asociados al efecto antimetastásico de los AcM anti-EGFR no se han dilucidado totalmente, ya que los estudios han sido en ratones atímicos trasplantados con tumores humanos, donde no se puede analizar la participación de las células T. Tras esta investigación se obtuvo por primera vez un AcM capaz de reconocer el dominio extracelular del EGFR murino. Este AcM nos permitió confirmar el potente efecto antimetastásico de los AcMs anti-EGFR en ratones inmunocompetentes. Se demostró que la inhibición de la señalización por el EGFR contribuye a este fenómeno, mediante efectos antiproliferativos, antimigratorios y proapoptóticos, y sugiere que la inducción de respuestas citotóxicas por la región constante de este AcM es un mecanismo que puede incrementar su potencial antitumoral. Este estudio constituye el primer reporte sobre la dependencia de un AcM anti-EGFR de la función de las células T-CD4+ y CD8+ para ejercer su efecto antimetastásico. [ABSTRACT FROM AUTHOR]

P3 mAb is a murine IgM that specifically recognizes N-glycolyl (NeuGc)-gangliosides and sulfatides. It also reacts with antigens expressed in human breast tumors and melanoma. In syngeneic model, P3 mAb is able to elicit a strong anti-idiotypic (Ab2) antibody response, even in absence of adjuvants or carrier proteins. Here we reported the construction of the human IgG1 chimeric P3 antibody, and the evaluation of the maintenance of the main properties of the murine P3 mAb. Chimeric P3 antibody specifically reacted with GM3(NeuGc) and GM2(NeuGc) gangliosides, and not with their acetylated variants. Also, it recognized the anti-idiotypic 1E10 mAb. Upon immunization of Balb/c mice with chimeric P3 antibody, we were able to demonstrate the immunodominance of its variable region. The anti-idiotypic response induced by this antibody was very strong and in three out of four mice was even significantly higher than the anti-isotypic response, despite the fact that 70% of the chimeric molecule is xenogenic with respect to the animal model. In chickens, the immunogenicity of chimeric P3 antibody idiotype was also evaluated. [ABSTRACT FROM AUTHOR]

We developed a phase I clinical trial for patients with stage III/IV breast carcinoma treated with the anti-idiotype mAb 1E10 (Ab2) specific to an Ab1 mAb (P3) able to react specifically with N-glycolyl-containing gangliosides and with antigens expressed on human melanoma and breast carcinoma cells. In animal models this Ab2 mAb induced Ab3 antibody responses sharing immunochemically-defined idiotopes with the Ab1, but unable to recognize N-glycolyl-containing gangliosides. The treatment of tumor-bearing mice with 1E10 mAb induced a strong antitumor activity. In a previous clinical trial carried out with anti-id mAb 1E10 in patie nts with advanced malignant melanoma, the vaccine proved to be safe and immunologically effective, with most patients being able to generate a specific immune response against 1E10 and NeuGc-GM3 ganglioside. The purpose of the present study was to evaluate the toxicity, the immune response to 1E10 anti- id vaccine and to test two levels of doses of the vaccine preparation. Ten patients of breast carcinoma, distributed in two groups of five, received six doses of 1 and 2 mg respectively, of aluminum hydroxide precipitated 1E10 anti-id mAb, intradermally into multiple sites at two weeks intervals. In eight of the nine patients who received at least four doses of the anti- id vaccine strong specific responses were observed both against 1E10 mAb and the NeuGc-GM3 ganglioside (Ab3Id+Ag+). Non-classical Ab1' antibodies (Id-Ag+) were also elicited by 1E10 mAb vaccine treatment. The comparative analyses of the responses indicated that there were not differences between the two levels of dose tested in relation to toxic ity and immunogenicity. No evidences of serious or unexpected effects were observed. The results of this clinical trial showed that 1E10 anti-id vaccine was well tolerated and immunologically active, capable of inducing a specific immune response against 1E10 and NeuGc-GM3 ganglioside in most breast cancer patients. [ABSTRACT FROM AUTHOR]

Transforming Growth Factor alpha (TGFα) is an endogenous growth factor that is associated with the loss of growth regulation of the transformed phenotype and its action is by the interaction with the epidermal growth factor (EGF) receptor. Also this molecule is over-expressed by carcinomas as a tumor-associated antigen. In this regard the principal goal of this work is to development an active immunoteraphy capable to block the autocrine loop TGFα-EGFR and/or to elicit a specific cellular response against tumor that over-expressed this growth factor. To our knowledge TGFα has not been used in active specific cancer immunotherapy. A new vaccine containing a fusion protein between human TGFα (hTGFα) and P64k from Neisseria meningitidis, was generated and characterized in a mouse model using different adjuvants. Interestingly a good correlation between anti-hTGFα serum antibody titers, mainly IgG, and antibody secreting B cells in the lymph nodes of immunized mice was seen. The isotype profile of hTGFα-specific antibody responses was similar when Freund adjuvant complete or incomplete was used showing increase levels of IgG2a and IgG2b immunoglobulins respect to aluminum hydroxide. Noteworthy, those anti-hTGFα antibodies recognize the hTGFα precursor in cell line expressing this growth factor. For 4 peptide represent the different loops of hTGFα tested, serum from immunized mice mainly recognize the C-loop and C-terminal region. That region is important in the TGFα/EGF-R binding. Not crossreactivity against human or murine epidermal growth factor (EGF) was found. [ABSTRACT FROM AUTHOR]