| 摘要 |
In accordance with the present invention, it has been discovered that a class of anti-dsDNA antibodies, e.g., mAb 3E10, binds membranes of fixed human renal tubular cells, penetrates live murine renal tubular cells in vivo, and localizes in the cell nucleus. mAb 3E10 binds both dsDNA and an extracellular matrix protein expressed in high endothelial venules (i.e., HP8/HEVIN). Previous studies showed both shared and distinct binding determinants in the variable heavy region of mAb 3E10 for DNA and HP8/HEVIN. To independently assess the requirement of HP8/HEVIN and DNA in cellular penetration, site directed mutants of mAb 3E10 VH and Vk were investigated for the ability to penetrate kidney cell lines. The results showed that residues required for binding DNA but not HP8/HEVIN were necessary for antibody penetration, indicating that cellular penetration required the presence of DNA or binding of antibody to a membrane determinant precisely resembling DNA. Antibody Fab penetrates cells, indicating that neither the Fc nor multivalent antibody binding are necessary for cellular penetration. Antibody synthesized in the cytoplasm as a result of deleting heavy and light chain signal peptides is not translocated to the nucleus, indicating the need for a membrane mediated pathway or for post-translational modifications of the antibody. The present invention demonstrates the usefulness of using molecular mutants and/or functional fragments thereof to study the cellular pathways of autoantibody penetration and nuclear localization. |
