Engineering Antibody Molecules
Advances in PCR techniques and the increase of the antibody V region sequences in the database have boosted developments in the field of antibody engineering. The V region genes can be amplified from hybridomas (1 ), preimmunized donors (2 ), naive donors
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3 Engineering Antibody Molecules Rakesh Verma and Ekaterini Boleti 1. Cloning of V Region Genes Advances in PCR techniques and the increase of the antibody V region sequences in the database have boosted developments in the field of antibody engineering. The V region genes can be amplified from hybridomas (1), preimmunized donors (2), naive donors (3), or from the cells expressing antibodies. A number of strategies have been used to amplify the V region sequences and a large number of primers have been described that amplify the V region of human and other species based on the database of V region sequences. The following types of primers are commonly used. 1. Primers specific for leader sequences and constant region of the gene. 2. Degenerate primers designed to complement the 5v and 3v ends of the conserved sequences of V region. 3. Panels of oligonucleotides specific for families of the V region genes.
2. Antibody Molecules There are two main classes of recombinant antibodies. The first is based on the intact immunoglobulin molecule (Fig. 1) and is designed to reduce the immunogenicity of the murine molecule. Thus, both chimeric molecules, which consist of the murine V regions and human constant regions, have been developed (4–7) as well as humanized antibodies in which just the CDRs are of rodent origin (8,9). The second class of molecules consists of fragments of antibody molecules. These include fragments that are accessible through proteolysis, such as Fab, Fabv, and F(abv)2, as well as other fragments, such as Fv-based molecules (Fig. 2). These molecules include sFv (single-chain Fv) (10,11), and the dsFv (disulfide-stabilized Fv) (Fig. 3) (12). From: Methods in Molecular Medicine, Vol. 40: Diagnostic and Therapeutic Antibodies Edited by: A. J. T. George and C. E. Urch © Humana Press Inc., Totowa, NJ
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Verma and Boleti
Fig. 1. Reduction of immunogenicity. This illustrates the most commonly used strategies to make mAb that are less immunogenic in patients. Regions with murine sequences are shown in black, those with human in gray. On the left is the parental murine mAb. In chimeric mAb the variable domains are of murine origin, the rest is human. In humanized antibodies the entire sequence is human with the exception of the residues that constitute the antigen binding site (derived from the CDRs of the mAb).
2.1. Chimeric and Humanized Antibodies The ability to clone V region genes has allowed generation of novel constructs based on the IgG molecule. The first class of such molecules was designed to reduce the immunogenicity of rodent antibodies in humans, thus preventing the induction of human antimouse antibody (HAMA), as described in Chapter 1. Other constructs have been designed to reduce the size of the molecule, remove the Fc portion, and add novel effector functions. The first generation of antibody molecules designed to reduce the immunogenicity were chimeric molecules. These consist of the variable region domains (VH and VL) from the parental rodent mAb, but the constant region domain
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