RESUMEN

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/genética

RESUMEN

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)→R 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/inmunología

RESUMEN

Detailed information on the immunological relevance of α-type anti-idiotypic antibodies is lacking after more than 30 years since Jerne postulated his Idiotypic Network Theory. The B7Y33 mutant is a mouse-human chimeric version of the B7 MAb, a polyreactive α-type anti-idiotypic antibody, generated against an anti-GM2 ganglioside IgM Ab1 antibody. It retained the unusual self-binding activity and multispecificity of the parental murine antibody, being able to recognize several anti-ganglioside IgM antibodies as well as non-immunoglobulin antigens. Previous work with the murine B7 MAb suggested that this antibody might have immunoregulatory properties, and therefore we investigated the possible interaction of B7Y33 with immune cells. We found that B7Y33 binds to human and murine B lymphocytes. Inhibition assays using flow cytometry indicated that this antibody is capable of binding the Fc γ receptor II (FcγRII). The recognition of FcγRII-expressing K562, Raji and Daudi human cell lines, together with the capability of inhibiting the binding of an anti-human FcγRII antibody to these cells, suggest that B7Y33 interacts with both the FcγRIIa and FcγRIIb isoforms. We evaluated the contribution to the binding of different surface-exposed residues at the top of the heavy chain variable region (VH) CDR loops through the construction of mutants with substitutions in the three conventional VH CDRs (HCDRs) and the "HCDR4", located in the framework 3 (HFR3). In addition, we assessed the involvement of the Fc region by performing key mutations in the CH2 domain. Furthermore, chimeric hybrid molecules were obtained by combining the B7Y33 heavy chain with unrelated light chains. Our results indicate that the multispecificity and self-binding properties of B7Y33 are not linked to its recognition of B lineage cells, and that this phenomenon occurs in a non-classical way with the participation of both the variable and constant regions of the antibody. Two possible models for this interaction are proposed, with B7Y33 binding to two FcγRIIb molecules through the Fc and Fv regions, or simultaneously to FcγRIIb and another unknown antigen on B cells. The FcγRIIb has recently received great attention as an attractive target for therapies directed to B lymphocytes. The recognition of peripheral B lymphocytes from B cell chronic lymphocytic leukemia (B-CLL) patients by B7Y33 suggests its potential application for the treatment of B cell malignancies.

Asunto(s)

Anticuerpos Antiidiotipos/inmunología , Especificidad de Anticuerpos/inmunología , Linfocitos B/inmunología , Receptores de IgG/inmunología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos/genética , Línea Celular , Separación Celular , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Receptores de IgG/genética

RESUMEN

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 FcgammaR binding capacity than the unmutated chimeric molecule T3q, as well as a reduced ability to induce T-cell proliferation, proinflammatory cytokine release (TNFalpha 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.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Complejo CD3/inmunología , Hibridomas/inmunología , Animales , Especificidad de Anticuerpos/inmunología , Proliferación Celular , Receptores ErbB/inmunología , Vectores Genéticos/inmunología , Humanos , Ratones , Linfocitos T/inmunología

RESUMEN

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.

Asunto(s)

Gangliósido G(M3)/análogos & derivados , Inmunohistoquímica/métodos , Neoplasias Renales/metabolismo , Tumor de Wilms/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Membrana Celular/metabolismo , Membrana Celular/patología , Terapia Combinada , Células Epiteliales/metabolismo , Células Epiteliales/patología , Técnica del Anticuerpo Fluorescente Indirecta , Gangliósido G(M3)/metabolismo , Humanos , Neoplasias Renales/patología , Neoplasias Renales/terapia , Ratones , Estudios Retrospectivos , Células del Estroma/metabolismo , Células del Estroma/patología , Tumor de Wilms/patología , Tumor de Wilms/terapia

RESUMEN

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 V(H)J558 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.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Autoanticuerpos/inmunología , Idiotipos de Inmunoglobulinas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Reacciones Cruzadas , Bases de Datos Factuales , Epítopos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/aislamiento & purificación , Datos de Secuencia Molecular , Mutación , Filogenia

RESUMEN

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.

Asunto(s)

Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/inmunología , Gangliósidos/inmunología , Animales , Anticuerpos Antiidiotipos/química , Anticuerpos Monoclonales/química , Reacciones Antígeno-Anticuerpo , Sitios de Unión , Células Cultivadas , Femenino , Gangliósidos/química , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología

RESUMEN

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.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Gangliósido G(M2)/inmunología , Gangliósido G(M3)/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/genética , Secuencia de Bases , Línea Celular Tumoral , Cadenas Pesadas de Inmunoglobulina/genética , Idiotipos de Inmunoglobulinas/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Datos de Secuencia Molecular , Hipermutación Somática de Inmunoglobulina