Humanized antibody structure

Fig. 2.1 Diagram showing the proportion of human (green) and murine (red) sequences in mouse, chimeric, humanized and human antibody structures, as exemplified by HuCAL...

Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, Hendrickson WA (1998) Structure of an HIV gpl20 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393 648-659... 

Fig. 61. Crystal structure of the FIIV-l neutralizing human antibody 2G12 bound to the oligomannoside MaiiyGlcNAc i present on the silent face of the gpl20 envelope glycoprotein (PDB 10P5).

Enbrel is a product now approved for medical use that is based upon this strategy. The product is an engineered hybrid protein consisting of the extracellular domain of the TNF p75 receptor fused directly to the Fc (constant) region of human IgG (see Box 13.2 for a discussion of antibody structure) The product is expressed in a CHO cell line from which it is excreted as a dimeric soluble protein of approximately 150 kDa. After purification and excipient addition (mannitol, sucrose and trometamol), the product is freeze-dried. It is indicated for the treatment of rheumatoid arthritis and is usually administered as a twice-weekly s.c. injection of 25 mg product reconstituted in WFI. Enbrel functions as a competitive inhibitor of TNF, a major pro-inflammatory cytokine. Binding of TNF to Enbrel prevents it from binding to its true cell surface receptors. The antibody Fc component of the hybrid protein confers an extended serum half-life on the product, increasing it by fivefold relative to the soluble TNF receptor portion alone. 

Recombinant DNA technology has provided an alternative (and successful) route of reducing the innate immunity of murine monoclonals. The genes for all human immunoglobulin sub-types have been cloned, and this has allowed generation of various hybrid antibody structures of reduced immunogenicity. 

The first and the second approaches have provided some positive results but, unfortunately, they depend upon the structure of the protein. Typical examples of sequence modifications to improve stability and pharmacokinetics are the preparation of humanized antibodies, where part of the mouse sequence is substituted by the human form, and the granulocyte colony-stimulating factor muteins, where up to seven amino acids are substituted. Examples of truncated sequence proteins with improved characteristic are the 7-36 analogues of glucagon-like peptides or the 1-29 sequence growth hormone-releasing factor [1, 2]. 

Elhott S, Chang D, Delorme E, Dunn C, Egrie J, Giffin J, Lorenzini T, Talbot C, Hesterberg L. Isolation and characterization of conformation sensitive antieryth-ropoietin monoclonal antibodies effect of disulfide bonds and carbohydrate on recombinant human erythropoietin structure. Blood 1996 87 2714-22. 

Following recent advances in antibody engineering, it is now possible to s)mthesize humanized antibodies [35]. Because of clinical applicability, antibody-based medicines are one of the biopharmaceutical categories that have attracted close attention in recent years [36]. IgG has been most frequently used for the prevention and treatment of various diseases. The CH2 region of IgG has A-linked carbohydrates (usually complex type double stranded chains) in diverse manners (O Scheme 14). It has recently been revealed that differences in the carbohydrate structure affect the effecter activity of antibodies such as antibody-dependent cellular cytotoxicity (ADCC) and their half-life in blood, inviting close attention from the viewpoint of improving the responses to antibody-based medicines [37,38,39]. 

Graille M, Stura EA, Corper AL, Sutton BJ, Taussig MJ, Charbonnier JB, Silverman GJ Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody structural basis for recognition of B-cell receptors and superantigen activity. Proc Natl Acad Sci USA 2000 97 5399-5404. 

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