Higher affinity human D MoAb prepared by light-chain shuffling and selected by phage display.

Publication Type:

Journal Article


Transfusion, Volume 42, Issue 1, p.59-65 (2002)


Amino Acid Sequence, Animals, Antibodies, Monoclonal, Antibody Affinity, Bacteriophages, Blood Grouping and Crossmatching, Gene Library, Gene Rearrangement, B-Lymphocyte, Light Chain, Humans, Hybridomas, Immunoglobulin Fab Fragments, Immunoglobulin kappa-Chains, Immunosorbent Techniques, Isoantibodies, Mice, Molecular Sequence Data, Recombinant Fusion Proteins, Rho(D) Immune Globulin, Sequence Alignment, Sequence Analysis, DNA, Sequence Homology, Amino Acid


<p><b>BACKGROUND: </b>In blood banks, D MoAbs are routinely used to phenotype donors and patients. However, most D MoAbs do not agglutinate RBCs that weakly express D. The use of higher affinity MoAbs could overcome this problem. In this work, an attempt has been made to increase the affinity of the human clone 43F10, an IgG anti-D, by light (L)-chain shuffling followed by selection using phage display.</p><p><b>STUDY DESIGN AND METHODS: </b>PBMNCs of three polyimmunized individuals were used to construct the kappa L-chain repertoire that was recombined with the 43F10 heavy chain in a phagemid vector system (pComb3H, Scripps Institute, La Jolla, CA). L-chain-shuffled 43F10-F(ab) phages were selected on intact RBCs and characterized by ELISA, indirect agglutination, and sequence analysis.</p><p><b>RESULTS: </b>L-chain shuffling combined with phage display permitted the selection of a 43F10 MoAb variant (p3.17) with improved reactivity with weak D RBCs in agglutination assays. Nucleic acid sequence analysis showed that p3.17 and wild-type (wt) 43F10 L chains are encoded by different VL segments of the Vk1 family and different J segments, thus showing a relatively low degree of homology (86.4%).</p><p><b>CONCLUSION: </b>The use of a variant such as p3.17 could permit a further increase of the potency of existing anti-D reagents. The low homology between p3.17 and wt 43F10 sequences further exemplifies the predominant role of the heavy chain in determining the specificity of the anti-D.</p>

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