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1.
Cancer Immunol Immunother ; 58(3): 351-60, 2009 Mar.
Article in English | MEDLINE | ID: mdl-18677479

ABSTRACT

Major histocompatibility complex (MHC) class II molecules have been considered as a good target molecule for use in immunotherapy, because of the high expression in some lymphoma and leukaemia cells and, also, because of their restricted expression on human cells (monocytes, dendritic, B lymphocytes, thymic epithelial cells, and some cytokine-activated cells, such as T lymphocytes). We have obtained a human IgM monoclonal antibody directed against human leukocyte antigen (HLA) class II molecules, using transgenic mice carrying human Ig genes. The antibody BH1 (IgM/kappa isotype) recognises HLA-class II on the surface of tumour cells from patients suffering from haematological malignancies, such as chronic and acute lymphocytic leukaemias, non-Hodgkin lymphomas and myeloid leukaemias. Interestingly, functional studies revealed that BH1 mAb recognises and kills very efficiently tumour cells from several leukaemia patients in the presence of human serum as a source of complement. These results suggest that this human IgM monoclonal antibody against HLA-class II could be considered as a potential agent in the treatment of several malignancies.


Subject(s)
Antibodies, Monoclonal/chemistry , Hematologic Neoplasms/immunology , Histocompatibility Antigens Class II/chemistry , Immunoglobulin M/chemistry , Immunotherapy/methods , Leukemia/immunology , Animals , Female , Hematologic Neoplasms/metabolism , Humans , Immunoglobulin M/metabolism , K562 Cells , Leukemia/therapy , Male , Mice , Mice, Transgenic , Prostatic Neoplasms/immunology , Uterine Cervical Neoplasms/immunology
2.
Mol Immunol ; 43(11): 1827-35, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16343622

ABSTRACT

In recent years, mice carrying human IG transgenes are being generated for the production of human monoclonal antibodies as an alternative approach to the conventional use of mouse or chimeric-humanized antibodies. Theoretically, the size of the repertoire of human antibodies that these mice could produce would be critically dependent on the number of human V genes introduced in the transgene. This could be the case for BABkappa and BABkappa,lambda transgenic mice, which carry several genes from the human IGK (BABkappa), and IGK and IGL (BABkappa,lambda) loci, but only five human IGHV genes and the entire IGHD-IGHJ cluster linked to two human IGHC (IGHM-IGHD) genes. We analyzed the expressed human IG genes in 30 IgM-secreting hybridomas generated from transgenic mice immunized either with soluble proteins (human IgM coupled to KLH) or with cells (human PBMC, tumour cell lines or rat cells transfected with human CD69). The results show that all hybridoma cells analyzed rearranged exclusively the IGHV1-2 gene, in contrast with naive spleen B cells that used three out of the five IGHV genes present in the transgene. The configuration of the rearranged CDR3 region revealed a much higher heterogeneity in the heavy chains. A variety of IGHJ and IGHD genes were used in hybridomas, and somatic mutations were also seen in some hybrids. Regarding the rearranged light chains genes, it was a much higher variety in the use of V and J genes in both, kappa and lambda chains, than in the heavy chain, and also in the level of mutation. The results indicate that only one IGHV gene is sufficient to generate a wide repertoire of antigen specific antibody responses. Thus, efforts aimed at the generation of new transgenic mice should focus more on the integrity of the D/J region and on the DNA regions regulating somatic hypermutation, rather than on the number of V genes present in the transgene.


Subject(s)
Antibody Formation/immunology , Antibody Specificity/immunology , Antigens/immunology , Gene Rearrangement, B-Lymphocyte/immunology , Genes, Immunoglobulin Heavy Chain/genetics , Genes, Immunoglobulin Heavy Chain/immunology , Immunoglobulin Variable Region/genetics , Animals , Humans , Hybridomas/metabolism , Immunoglobulin Variable Region/immunology , Mice , Mice, Transgenic , Mutation/genetics , Sequence Analysis, DNA , Spleen/cytology , Transgenes/genetics
3.
J Immunol Methods ; 282(1-2): 147-58, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14604548

ABSTRACT

The CD69 antigen is the earliest activation marker expressed on leukocyte surfaces after stimulation and it has been correlated with disease state in a variety of inflammatory and autoimmune diseases. We were interested in the generation of a human monoclonal antibody (mAb) against the CD69 antigen. To do this, mice carrying human Ig transgenes (on an inactivated endogenous immunoglobulin H and Igkappa background) were immunized with rat cells transfected with the human CD69 molecule. From over 2000 hybridoma clones generated in different fusions, we were able to obtain a human monoclonal antibody, hAIM-29, which specifically recognizes human CD69 on the surface of activated-human leukocytes. We demonstrate that the antibody is specific for the human CD69 molecule, as shown by double staining with mouse anti-human CD69 antibodies, ELISA, immunoblot and immunoprecipitation studies. Results of additional experiments show that hAIM-29 activates intracellular calcium influx without Ig cross-linking and enhances phorbol myristate acetate-induced cell proliferation in a manner similar to other mouse anti-CD69 antibodies. This report is the first to describe the isolation and characterization of a novel human mAb, hAIM-29, which may have therapeutic potential in diseases associated with the presence of activated cells expressing CD69 antigen.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/immunology , Animals , Antigens, CD/analysis , Antigens, Differentiation, T-Lymphocyte/analysis , Calcium/metabolism , Cell Line, Tumor , Humans , Lectins, C-Type , Lymphocyte Activation , Mice , Mice, Transgenic , Rats
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