Efalizumab (rhuMAb CD11a, hu1124) is a full-length, IgG1 kappa isotype antibody composed of two identical kappa light chains consisting of 214 amino acid residues, and two gamma heavy chains consisting of 451 residues. Each light chain is covalently coupled through a disulphide link to a heavy chain. The two heavy chains are covalently coupled to each other via inter-chain dis-ulphide bonds consistent with the structure of human
Fig. 5.2. Humanized monoclonal antibody efalizumab. CDR complementarity determining regions, FR framework regions, VH variable part of the heavy chain, VL variable part of the light chain
IgG1. The molecular weight of intact efalizumab is 148,841 daltons.
Originally developed as a murine anti-CD11a monoclonal antibody (MHM24; Hildreth and August 1985), efalizumab has been prepared by substituting human DNA sequences using genetic engineering methods to reduce immunogenicity. In detail, complementarity determining regions (CDRs) from the murine antibody MHM24 were grafted into consensus human IgG1 heavy and light chain sequences. This results in a "humanized" mAb (HuIgG1) in which the CDRs of the murine antibody - important for specific antigen recognition - are preserved.
Previous studies on murine MHM24 have shown that, similar to other anti-CD11a antibodies, it is able to inhibit T-cell function.
The consensus sequences for the human heavy chain subgroup III (Vh-CH1) and the light chain subgroup k 1 were used as the framework for the humanization (Fig. 5.2). Several humanized variants were made and screened for binding as Fabs. To construct the first Fab variant of humanized MHM24, all six CDR residues were transferred from the murine antibody to the human framework.
Further variants were constructed by targeted exchange of either framework residues, or residues within CDRs using the first variant (Fab-1), as a tem a plate. For that purpose, both light and heavy chains were completely sequenced for each variant. Plasmids containing the sequences were then transformed to Escherichia coli for protein expression.
All variants were tested for CD11a binding in the Jurkat cell assay. VL and VH domains of the variant with optimal binding characteristics were then transferred to human IgG1 constant domains, giving the full-length intact humanized antibody (Werther et al. 1996).
Several in vitro assays were performed to compare efalizumab with its parent murine antibody MHM24, including the keratinocyte cell-adhesion assay and the mixed lymphocyte response assay (MLR).
The results showed that, in these assays, efalizumab worked as well as MHM24. In addition, the apparent Kd values, as determined by saturation binding using peripheral blood mononuclear cells (PBMCs) of two human donors, were similar for both MHM24 and efalizumab (0.16±0.01 nM and 0.13±0.02 nM vs 0.11 ± 0.08 nM and 0.18± 0.03 nM, respectively).
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