Lourdes Roque - Academia.edu (original) (raw)
Papers by Lourdes Roque
Molecular Immunology, Apr 1, 2011
Molecular Immunology, 2011
Antibody fragments Fv single chain (scFv) mono- and divalent (diabody), obtained by recombinant D... more Antibody fragments Fv single chain (scFv) mono- and divalent (diabody), obtained by recombinant DNA techniques from monoclonal antibody (mAb) anti-carcinoembryonic antigen (CEA) CB / ior-CEA.1. This Mab has very high affinity for CEA and is used in the diagnosis and monitoring of human colorectal tumors. As the original Mab, the scFv fragments monovalent and diabody exhibit high affinities for human CEA and recognize an epitope dependent of carbohydrate conservation. The scFv monovalent and diabody have affinity constants for CEA of (5.0 n 0.4) x 109 L mol-1 and (2.8 n 0.3) x 1010 L mol-1, respectively. These two fragments do not cross-react with cells and normal human tissues, except normal colonic mucosa where CEA is occasionally present. The fragments can be produced by expression in recombinant microorganisms, starting from the cloning of sequences for variable regions nucleicoscodificantes acids obtained from the hybridoma producing Mab CB / ior-CEA.1. Like the original Mab, th...
The present invention relates to the field ofthe present invention relates to the field of immuno... more The present invention relates to the field ofthe present invention relates to the field of immunology and more specifically with Immun immunology and more specifically to cancer immunotherapy, particularly with combinacioterapia cancer, particularly with immunotherapeutic combinations and treatment methods put immunotherapeutic and treatment methods to prevent tumor cell growth and / ara prevent tumor cell growth and / or remove said cells. The methods described eo eliminate these cells. The methods described herein are based on the effect Teran the present invention are based on the therapeutic effect obtained by the combination of idipeutico vaccines obtained by the combination of idiotypic vaccines whose active ingredient is a anotipicas antibody whose active ingredient is an anti-ganglioside antibody (Ab1) or recombinant versions of dtigangliosido (Ab1) or recombinant versions of said antibody with idiotypic vaccines whose prinicho antibody with idiotypic vaccines whose active i...
Immunobiology, 2011
Gangliosides containing the N-glycolyl (NGc) form of sialic acid are tumor-associated antigens an... more 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.
Hybridoma, 2007
Neonatal natural antibodies (NAbs) are characterized by their high degree of idiotypic cross reac... more 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.
Hybridoma and Hybridomics, 2003
Genetic engineering has provided several approaches to reduce immunogenicity of murine antibodies... more Genetic engineering has provided several approaches to reduce immunogenicity of murine antibodies. We described previously a new method based on the humanization of the linear epitopes presented to T cells. In brief, potential immunogenic epitopes in the variable region were identified and subjected to point mutations to make them human and/or to modify amphipatic motifs. The resulting recombinant antibody retained its antigen binding affinity and was less immunogenic in monkeys than their murine or chimeric predecessors are. The present study provides two new examples of this T-cell epitope humanization approach: ior-t1A murine monoclonal antibody (mMAb), which recognizes the human-CD6 molecule, and ior-C5 mMAb, which recognizes a novel glycoprotein expressed on the surface of malignant colorectal cells. Seven amino acids were substituted in ior-C5 and eleven residues in ior-t1A, by the corresponding residues from the highest homologous human sequences. Surprisingly, the homology between reshaped chimeric antibody variable region frameworks and human sequences was 80-90%. Experiments in monkeys showed that T1AhT and C5hT "detopes" antibodies were less immunogenic than their chimeric analogues while they retained 30-50% of antigen binding affinities. The proposed method might be of general applicability to reduce immunogenicity of chimeric antibodies with therapeutic potential.
Immunobiology, 2007
The heavy-chain variable regions (VH) from 14F7 MAb, an IgG1 antibody specific for GM3(NeuGc) gan... more The heavy-chain variable regions (VH) from 14F7 MAb, an IgG1 antibody specific for GM3(NeuGc) ganglioside, and its anti-idiotype, the 4G9 MAb, were cloned and sequenced. Comparison with previously reported sequences showed that VH 14F7 belongs to the J558(VHI) gene family and that it is highly mutated. VH 4G9 belongs to the Q52(VHII) gene family. The HCDR3 14F7 sequence contains three basic residues that could be involved in the binding to 4G9 MAb, which bears acidic residues in its HCDR3. Studies performed in the syngeneic model showed that 14F7 MAb requires both coupling to KLH and the use of Freund's adjuvant to induce an effective anti-idiotypic IgG (Ab2) response. In contrast, P3 MAb, a germline gene-encoded Ab1 that also recognizes the GM3(NeuGc) ganglioside through a basic motif in its H-CDRs, has been reported to be immunogenic in syngeneic mice, even when injected in saline. In addition, when Leghorn chickens were immunized with 14F7 or P3 MAbs emulsified in Freund...
Molecular Cancer Therapeutics, 2008
Gangliosides have been involved in multiple cellular processes such as growth, differentiation an... more Gangliosides have been involved in multiple cellular processes such as growth, differentiation and adhesion, and more recently as regulators of cell death signaling pathways. Some of these molecules can be considered as tumor-associated antigens, in particular, N-glycolyl sialic acid–containing gangliosides, which are promising candidates for cancer-targeted therapy because of their low expression in normal human tissues. In this study, we provided the molecular and cellular characterization of a novel cell death mechanism induced by the anti-NGcGM3 14F7 monoclonal antibody (mAb) in L1210 murine tumor cell line but not in mouse normal cells (B and CD4+ T lymphocytes) that expressed the antigen. Impairment of ganglioside synthesis in tumor cells abrogated the 14F7 mAb cytotoxic effect; however, exogenous reincorporation of the ganglioside did not restore tumor cell sensitivity to 14F7 mAb-induced cytotoxicity. 14F7 F(ab′)2 but not Fab fragments retained the cytotoxic capacity of the ...
Molecular Immunology, Apr 1, 2011
Molecular Immunology, 2011
Antibody fragments Fv single chain (scFv) mono- and divalent (diabody), obtained by recombinant D... more Antibody fragments Fv single chain (scFv) mono- and divalent (diabody), obtained by recombinant DNA techniques from monoclonal antibody (mAb) anti-carcinoembryonic antigen (CEA) CB / ior-CEA.1. This Mab has very high affinity for CEA and is used in the diagnosis and monitoring of human colorectal tumors. As the original Mab, the scFv fragments monovalent and diabody exhibit high affinities for human CEA and recognize an epitope dependent of carbohydrate conservation. The scFv monovalent and diabody have affinity constants for CEA of (5.0 n 0.4) x 109 L mol-1 and (2.8 n 0.3) x 1010 L mol-1, respectively. These two fragments do not cross-react with cells and normal human tissues, except normal colonic mucosa where CEA is occasionally present. The fragments can be produced by expression in recombinant microorganisms, starting from the cloning of sequences for variable regions nucleicoscodificantes acids obtained from the hybridoma producing Mab CB / ior-CEA.1. Like the original Mab, th...
The present invention relates to the field ofthe present invention relates to the field of immuno... more The present invention relates to the field ofthe present invention relates to the field of immunology and more specifically with Immun immunology and more specifically to cancer immunotherapy, particularly with combinacioterapia cancer, particularly with immunotherapeutic combinations and treatment methods put immunotherapeutic and treatment methods to prevent tumor cell growth and / ara prevent tumor cell growth and / or remove said cells. The methods described eo eliminate these cells. The methods described herein are based on the effect Teran the present invention are based on the therapeutic effect obtained by the combination of idipeutico vaccines obtained by the combination of idiotypic vaccines whose active ingredient is a anotipicas antibody whose active ingredient is an anti-ganglioside antibody (Ab1) or recombinant versions of dtigangliosido (Ab1) or recombinant versions of said antibody with idiotypic vaccines whose prinicho antibody with idiotypic vaccines whose active i...
Immunobiology, 2011
Gangliosides containing the N-glycolyl (NGc) form of sialic acid are tumor-associated antigens an... more 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.
Hybridoma, 2007
Neonatal natural antibodies (NAbs) are characterized by their high degree of idiotypic cross reac... more 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.
Hybridoma and Hybridomics, 2003
Genetic engineering has provided several approaches to reduce immunogenicity of murine antibodies... more Genetic engineering has provided several approaches to reduce immunogenicity of murine antibodies. We described previously a new method based on the humanization of the linear epitopes presented to T cells. In brief, potential immunogenic epitopes in the variable region were identified and subjected to point mutations to make them human and/or to modify amphipatic motifs. The resulting recombinant antibody retained its antigen binding affinity and was less immunogenic in monkeys than their murine or chimeric predecessors are. The present study provides two new examples of this T-cell epitope humanization approach: ior-t1A murine monoclonal antibody (mMAb), which recognizes the human-CD6 molecule, and ior-C5 mMAb, which recognizes a novel glycoprotein expressed on the surface of malignant colorectal cells. Seven amino acids were substituted in ior-C5 and eleven residues in ior-t1A, by the corresponding residues from the highest homologous human sequences. Surprisingly, the homology between reshaped chimeric antibody variable region frameworks and human sequences was 80-90%. Experiments in monkeys showed that T1AhT and C5hT "detopes" antibodies were less immunogenic than their chimeric analogues while they retained 30-50% of antigen binding affinities. The proposed method might be of general applicability to reduce immunogenicity of chimeric antibodies with therapeutic potential.
Immunobiology, 2007
The heavy-chain variable regions (VH) from 14F7 MAb, an IgG1 antibody specific for GM3(NeuGc) gan... more The heavy-chain variable regions (VH) from 14F7 MAb, an IgG1 antibody specific for GM3(NeuGc) ganglioside, and its anti-idiotype, the 4G9 MAb, were cloned and sequenced. Comparison with previously reported sequences showed that VH 14F7 belongs to the J558(VHI) gene family and that it is highly mutated. VH 4G9 belongs to the Q52(VHII) gene family. The HCDR3 14F7 sequence contains three basic residues that could be involved in the binding to 4G9 MAb, which bears acidic residues in its HCDR3. Studies performed in the syngeneic model showed that 14F7 MAb requires both coupling to KLH and the use of Freund's adjuvant to induce an effective anti-idiotypic IgG (Ab2) response. In contrast, P3 MAb, a germline gene-encoded Ab1 that also recognizes the GM3(NeuGc) ganglioside through a basic motif in its H-CDRs, has been reported to be immunogenic in syngeneic mice, even when injected in saline. In addition, when Leghorn chickens were immunized with 14F7 or P3 MAbs emulsified in Freund...
Molecular Cancer Therapeutics, 2008
Gangliosides have been involved in multiple cellular processes such as growth, differentiation an... more Gangliosides have been involved in multiple cellular processes such as growth, differentiation and adhesion, and more recently as regulators of cell death signaling pathways. Some of these molecules can be considered as tumor-associated antigens, in particular, N-glycolyl sialic acid–containing gangliosides, which are promising candidates for cancer-targeted therapy because of their low expression in normal human tissues. In this study, we provided the molecular and cellular characterization of a novel cell death mechanism induced by the anti-NGcGM3 14F7 monoclonal antibody (mAb) in L1210 murine tumor cell line but not in mouse normal cells (B and CD4+ T lymphocytes) that expressed the antigen. Impairment of ganglioside synthesis in tumor cells abrogated the 14F7 mAb cytotoxic effect; however, exogenous reincorporation of the ganglioside did not restore tumor cell sensitivity to 14F7 mAb-induced cytotoxicity. 14F7 F(ab′)2 but not Fab fragments retained the cytotoxic capacity of the ...