Bi-Specific Antibodies in Cancer Therapy
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BI-SPECIFIC ANTIBODIES IN CANCER THERAPY Hao Wang1,2, Yanjun Liu 1,2 , Lixin Wei1, and Yajun Guo1,2 1
Shanghai International Joint Cancer Institute The Second Military Medical University Shanghai, P.R. China 2 Sidney Kimmel Cancer Center San Diego, USA
1. PREFACE: NEW MAGIC BULLETS In the 1980s monoclonal antibodies promised an era of “magic” bullet therapies that could specifically kill tumor cells while leaving healthy tissues intact. But the need to covalently couple the killing agent (toxin, radioisotopes, etc.) limited their clinical potential. Now, new bispecific antibodies (BsAbs, or bi-functional antibodies) have been developed. The prototypic BsAb consists of two different specific binding sites, with one directed to the target antigen on the surface of tumor cells, and the other to a trigger molecule.
2. SCHEME OF BISPECIFIC ANTIBODIES In the early days of this field, BsAbs were prepared by chemical cross-linking of (mainly Fab fragments of) IgG molecules with different specificities (see Fig. 1) (Brennan et al., 1985; Nolan and O’Kennedy, 1990), or by fusion of two established hybridoma clones secreting antibodies of different specificties, yielding a quadroma (or hybrid hybridoma, tetradroma) (Milstein and Cuello, 1983), or by double transfectomas. However, Chemical modification of antibodies is inefficient and can lead to side reactions that damage the combining sites. In a quadroma it is difficult to separate the bispecific antibodies form the mixed population of heavy and light chains produced by the two parent hybridoma genomes, and even a large variety of mismatch (H/L chains) molecules may emerge. So, large-scale production of BsAb molecules with these methods has always result in batch-to batch variations and low yields. In addition, the rodent-derived Ig molecules usually have to be “humanized” to minimize host antibody response (human anti-mouse antibody response, HAMA) before applied in clinics. Cancer Gene Therapy: Past Achievements and Future Challenges, edited by Habib Kluwer Academic/Plenum Publishers, New York, 2000.
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Figure 1. Chemical linked bi-specific antibody The monospecific antiobdies are first digested and reduced, yied Fab´ fragments, and are then crosslinked by chemical method.
To address these problems, efforts have been made in improving purification
methods for hybrid hybridomas (Allard et al., 1993), for example, selective crosslinking
chemistries for bispecific (Brennan et al., 1985; Glennie et al., 1987; Carter et al., 1992) and the use of amphipatic helices (leusine-zipper) to drive association (Kostelny et al., 1992), etc. Recently, protein engineering has been adapted to design BsAbs with new formats, in which only the variable domains (Fv) of the antibodies were retained and linked by artificial linkers. These recombinant bispecific Fv fragments include bispecific single chain variable fragment of Ab (BsscFvs) (see Fig. 2) (George et al., 1994; Gruber et al., 1994; Mallender and Voss, 1994; De Jonge et al., 1995; Kurucz et al., 1995; Ma
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