Monoclonal antibody bispecific antibodies

In a comparative study of disulfide reducing agents, it was determined that use of the relatively strong reductants DTT and TCEP required only 3.25 and 2.75 mole equivalents per mole equivalent of antibody molecule to achieve the reduction of two interchain disulfide bonds between the heavy chains of a monoclonal IgG (Sun et al., 2005). This limited reduction strategy retains intact bispecific antibody molecules while providing discrete sites for conjugation to thiols. 

Polyclonal antibodies can react with many epitopes, whereas MAbs are restricted to one epitope on proteins that do not have repeating sequences.24 By definition, polyclonal immunoassays are generally much more sensitive but less specific than monoclonal assays. Bispecific or hybrid antibodies can be used to increase the affinity. Bispecific antibodies are formed by the fusion of two previously established hybridomas to produce antibodies displaying the binding characteristics of both of the antibodies in one molecule.25... 

Bispecific monoclonal antibodies (BsMAb) which combine the specificity of two different antibodies within one molecule and cross-link an effector cell or a toxic molecule with the target cell (Section 8.5.2). 

Another approach to selectively inducing tumour cell kilhng is by the use of bispecific monoclonal antibodies (BsMAb).They combine the specificity of two separate antibodies within one molecule and cross-hnk an effector killer cell or a toxic molecule with the target cell to be destroyed [75]. There are three major approaches for creating BsMAbs.They can be obtained by chemical cross-linking of two MAbs, by fusing two hybridomas [76], or by genetic... 

Staerz, U.D., and M.J. Bevan, Hybrid hybridoma producing a bispecific monoclonal antibody that can focus effector T-cell activity. Proc Natl Acad Sci USA, 1986. 83(5) 1453-7. 

Mezzanzanica, D., S. Canevari, S. Menard, S.M. Pnpa, E. Tagliabue, A. Lanzavecchia, and M.I. Colnaghi, Human ovarian carcinoma lysis by cytotoxic T cells targeted by bispecific monoclonal antibodies analysis of the antibody components. Int J Cancer, 1988. 41(4) 609-15. 

Kroesen, B.J., A. ter Haar, H. Spakman, P. Willemse, D.T. Sleijfer, E.G. de Vries, N.H. Mulder, H.H. Berendsen, PC. Limburg, T.H. The, et al.. Local antitumour treatment in carcinoma patients with bispecific-monoclonal-antibody-redirected T cells. Cancer Immunol Immunother, 1993.37(6) 400-7. 

Bivalent and Bispecific Monoclonal Antibodies in Cancer Therapy... 

Bivalent and bispecific monoclonal antibodies have many practical applications, including immunodiagnosis and immunotherapy. Bivalency can allow antibodies to bind... 

Because many potentially useful monoclonal antibodies do not possess the appropriate isotype and so are unable to activate human complement and/or trigger Fc-yR on human cells, treatment strategies are needed. One such strategy is the application of bispecific monoclonal antibodies that exploit the specificity of monoclonal antibody and ensure activation of cellular cytotoxic mechanisms (Fanger et al., 1993). 

Bispecific monoclonal antibodies are artificially developed antibodies with antigenbinding sites physically linked to different specificities. It is thought that bispecific monoclonal antibodies activate the cellular immune response by crosslinking immune cells to tumor cells, thus circumventing the proper structures for tumor cell-immune cell interactions (Koelemij et al., 1999). These antibodies are effective in low concentrations in vivo. For example, Kufer et al. (1996) have combined the anti-CD3 specificity directed against T cells in a bispecific monoclonal antibody, with the specificity against the tumor-associated 17-1A antigen. This antibody could be a major improvement, for example, in the therapy for disseminated micrometastatic tumor cells. 

Bispecific monoclonal antibodies are being evaluated in phase I and II studies in a variety of malignant diseases in the fields of hematooncology and solid tumors. It is likely that in the next decade immunotherapy using bispecific monoclonal antibodies will have a place, complementary to the current modalities such as surgery, chemotherapy, hormone therapy, and radiation, in the treatment of malignancies. 

The earliest bispecific antibodies were obtained by dimerization after mild oxidation of a mixture of Fab fragments of polyclonal antibodies of two different specificities. Bispecific antibodies were also obtained by crosslinking two monoclonal antibodies using succinimidyl-3-2-pyridyldithiol-propionate, resulting in heteroaggregates of two monoclonal antibodies (Staerz et al., 1985). Although this is an easy method with a high yield,... 

The most commonly used technique to produce bispecific antibodies from two monoclonal antibodies is by fusing two hybridoma cell lines by conventional cell fusion procedure (Staerz and Bevan, 1986). These cells produce all possible combinations of the heavy and light chains of both antibodies, including the desired bispecific antibody. A limitation is that only part of the antibodies is the desired bispecific monoclonal antibody therefore, further purification is necessary (Van Ravenswaay et al., 1993). 

Recently, Sen et al. (2001) have described methods for generating highly effective HER-2-specific cytotoxic T cells by arming activated T cells with anti-CD3 X anti-HER-2 bispecific antibody. In this method OKT3 and 9184 anti-HER-2 monoclonal antibodies were conjugated and used to arm T cells that were subsequently tested for binding, cytotoxicity, and cytokine secretion assays. Armed T cells aggregate and selectively kill HER-2-positive breast cancer cells (MCF-7). 

Brennan, M., Davison, P. F., and Paulus, H. 1985. Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments. Science 229 81-83. 

There are several relatively new therapeutic modalities for the treatment of SLE. Trying to eliminate pathogenic anti-dsDNAs, Ferguson etal. developed an antigen-based heteropolymer (AHP) (F3). AHP is a bispecific dsDNA x monoclonal antibody (mAb) complex (dsDNA x anti-CRl mAb) that enables the use of the unique immune complex-binding and clearing capacity of the complement receptor (CR1) on primate erythrocytes. In vitro studies of AHP show a substantial reduction (>90%) of anti-dsDNA titer (F20). In vivo studies in two rhesus monkeys indicate that the erythrocyte-bound antibodies are rapidly cleared from the circulation (F3). 

Manzke, O., Tesch, H., Diehl, V., and Bohlen, H. (1997). Single-step purification of bispecific monoclonal antibodies for immunotherapeutic use by hydrophobic interaction chromatography. J. Immunol. Methods 208, 65-73. 

Tarditi, L., Camagna, M., Parisi, A., Vassarotto, C., DeMonte, L. B., Letarte, M., Malavasi, F., and Mariani, M. (1992). Selective high-performance liquid chromatographic purification of bispecific monoclonal antibodies. J. Chromatogr. 599, 13-20. 

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