tailieunhanh - Methods in Molecular BiologyTM VOLUME 207 Edited by Martin Welschof Jürgen Krauss Recombinant Antibodies for Cancer Therapy Methods and Protocols

Twenty-five years ago, Georges Köhler and César Milstein invented a means of cloning individual antibodies, thus opening up the way for tremendous advances in the fields of cell biology and clinical diagnostics (1). However, in spite of their early promise, monoclonal antibodies (MAbs) were largely unsuccessful as therapeutic reagents resulting from insufficient activation of human effector functions and immune reactions against proteins of murine origin. These problems have recently been overcome to a large extent using genetic-engineering techniques to produce chimeric mouse/human and completely human antibodies. Such an approach is particularly suitable because of the domain structure of the antibody molecule (2), where functional domains carrying antigen-binding activities (Fabs. | M i t to A r V u M r y 7 --r BA 9 .kA 1 _ 1 _ r . J Me ithods k .inlMO lecular BiOlogyi k w Jr V LCr 07 . . VOLUME 207 1 Generation of Antibody Molecules Through Antibody Engineering Sergey M. Kipriyanov 1. Introduction Twenty-five years ago Georges Kohler and César Milstein invented a means of cloning individual antibodies thus opening up the way for tremendous advances in the fields of cell biology and clinical diagnostics 1 . However in spite of their early promise monoclonal antibodies MAbs were largely unsuccessful as therapeutic reagents resulting from insufficient activation of human effector functions and immune reactions against proteins of murine origin. These problems have recently been overcome to a large extent using genetic-engineering techniques to produce chimeric mouse human and completely human antibodies. Such an approach is particularly suitable because of the domain structure of the antibody molecule 2 where functional domains carrying antigen-binding activities Fabs or Fvs or effector functions Fc can be exchanged between antibodies see Fig. 1 . On the basis of sequence variation the residues in the variable domains V-region are assigned either to the hypervariable complementarity-determining regions CDR or to framework regions FR . It is possible to replace much of the rodent-derived sequence of an antibody with sequences derived from human immunoglobulins without loss of function. This new generation of chimeric and humanized antibodies represents an alternative to human hybridoma-derived antibodies and should be less immunogenic than their rodent counterparts. Furthermore genetically truncated versions of the antibody may be produced ranging in size from the smallest antigenbinding unit or Fv through Fab to F ab 2 fragments. More recently it has become possible to produce totally human recombinant antibodies derived either from antibody libraries 3 or single immune B cells 4 or from transgenic mice bearing human immunoglobulin loci 5 6 . .