Human antibodies and hybridomas最新文献

Hybridoma BSRF-S-97, secreting a human monoclonal antibody of IgG1 subclass reactive to the carcinoembryonic antigen, was generated by fusing the regional lymph node lymphocytes from a cervical cancer patient with RF-S1 human-mouse heteromyeloma fusion line. This monoclonal antibody was found specifically reactive to carinoembryonic antigen-producing cell lines, including those of cervical cancer (SKG-II), mucinous type ovarian cancer (RMUG-L), stomach cancer (MKN-45), and lung cancer (PC-10). The monoclonal antibody reactivity with pepsin- and periodate-treated carcinoembryonic antigen demonstrated that this monoclonal antibody recognizes the carbohydrate moiety of carcinoembryonic antigen specifically. Possibilities of the monoclonal antibody reaction with mucin and blood-group antigens were excluded by the comparative studies with a placental mucin-containing protein which reacted with carcinoembryonic antigen-specific rabbit polyclonal antibody. The monoclonal antibody conjugated with Pseudomonas exotoxin showed potent regression effects on the growth of the MKN-45 cell line in both the dish culture and xenografted nude mice, indicating potential usefulness of this human monoclonal antibody as a promising tumor targeting vehicle.

To examine the ability of normal and autoimmune individuals to produce circulating anti-TNF alpha antibodies, plasma samples from 10 RA patients, 10 SLE patients and 5 normal subjects were assessed for anti-TNF alpha antibody. While every individual tested demonstrated circulating IgM anti-TNF alpha antibody, IgG anti-TNF alpha autoantibody was seen predominantly in autoimmune patients. Only 1 of 5 normal individuals, but 15 of 20 autoimmune individuals had plasma IgG anti-TNF alpha antibodies. To examine the ability of normal and autoimmune individuals to produce anti-TNF alpha autoantibody from their circulating lymphocytes, EBV transformation was performed. Oligoclonal immortal cell lines were successfully established from 13 patients and each one secreted detectable IgM anti-TNF alpha autoantibody. Transformed cells from only 1 of 5 normal individuals secreted IgM anti-TNF alpha autoantibody. These results indicate a higher prevalence of anti-TNF alpha autoantibody production among autoimmune individuals although normal individuals are also capable of producing these autoantibodies.

A murine anti-human B-cell monoclonal antibody, Lym-1, has shown considerable promise for the treatment of human malignant lymphomas and has been utilized as a new radioimmunotherapy for refractory lymphoma. In order to enhance its clinical potential, a genetically engineered chimeric Lym-1 (chLym-1) with murine variable regions and human gamma 1 and kappa constant regions was constructed and expressed. The goal of this study was to generate a Lym-1 reagent with decreased immunogenicity and improved effector functions. Murine Lym-1 variable region cDNAs were isolated from the murine Lym-1 hybridoma cell line, fused to gamma 1 and kappa constant region cDNAs, and expressed in an insect cell expression system with the baculovirus transfer vector pAcUW31. The chLym-1 antibody expressed in this system was correctly processed and assembled into the expected immunoglobulin monomer. Chimeric Lym-1 bound to both target antigen-bearing Raji cells and a Lym-1 anti-idiotype antibody and had a similar binding affinity as murine Lym-1. The chimeric and murine versions of Lym-1 were assayed for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and to induce complement-mediated cytotoxicity (CMC) against Raji targets. Chimeric Lym-1 mediated a two-fold higher level of ADCC than murine Lym-1 and slightly lower levels of CMC than murine Lym-1. In addition, in Raji lymphoma-bearing nude mice, chLym-1 localized to the tumor with approximately equal uptake at 24 and 72 hours. Chimeric Lym-1, however, cleared from the blood of nontumor-bearing mice approximately 5 times faster than murine Lym-1 (20 h vs. 5 days), as expected for a xenogeneic protein. The improved in vitro and in vivo activities of this genetically engineered monoclonal antibody render it a new potential immunotherapeutic reagent for the treatment of human malignant lymphomas.

To increase the avidity of single-chain antibodies (scFv) for their antigen, we have fused them to core-streptavidin. The chimeric protein, expressed by the vector pSTE (plasmid for streptavidin-tagged expression) from Escherichia coli, can form tetrameric complexes, binds its antigen and contains four biotin binding sites per tetrameric complex. An additional cysteine inserted near the carboxy terminus further stabilised the complex. The scFv fusion protein tetramers could be enriched by affinity chromatography using the biotin analog 2-iminobiotin from periplasmic inclusion bodies after refolding. We have also shown that the scFv fusion protein could be used for direct detection of its antigen in ELISA when stained with biotinylated horseradish peroxidase. The affinity of the scFv-antibody complex was substantially increased by avidity effects due to the tetrameric structure. The biotin binding sites may be used for coupling other antibodies and molecules to form bispecific and bifunctional reagents.

The characteristic pathogenic autoantibodies in Goodpasture's syndrome (GPS) are directed to the noncollagenous domain (NC1) of basement membrane type IV collagen. To examine whether immunization with anti-NC1 antibodies could lead to GPS-like pathology, naive BALB/c mice were immunized intradermally with a mouse IgG anti-NC1 monoclonal antibody or IgG serum fraction derived from patients with GPS. Mice immunized with normal mouse or human IgG and nonimmunized mice served as controls. Anti-NC1 antibodies of IgG isotype were detected in the sera of mice injected with anti-NC1 antibodies, but not in the sera of control mice. The presence of circulating anti-NC1 antibodies coincided in some of the mice erythrocyturia or proteinuria and pathological changes in the kidneys. No pathologic alterations were seen in the control mice. The results show that specific idiotypic manipulation can induce anti-NC1 antibodies and pathological changes resembling human GPS.