MONOCLONAL-ANTIBODIES THAT BLOCK THE ACTIVITY OF LEUKOCYTE FUNCTION-ASSOCIATED ANTIGEN-1 RECOGNIZE 3 DISCRETE EPITOPES IN THE INSERTED DOMAIN OF CD11A

The epitopes recognized by eight independently isolated monoclonal ant ibodies to the alpha chain of human and murine leukocyte function-asso ciated antigen 1 (LFA-1), all able to inhibit receptor function, were identified. Initial localization of epitopes was accomplished using ch imeric proteins constructed by splicing fragments of cDNAs encoding th e alpha subunit of LFA-1 (CD11a) and the alpha subunit of the closely related leukocyte integrin, Mac-1 (CD11b). Antibody binding to CD11a/C D11b chimeras, expressed in the 293 human kidney cell line, demonstrat ed that the epitopes recognized by six monoclonal antibodies to human CD11a were located in a similar to 200-amino acid sequence found in al l beta(2)-integrin alpha subunits, termed the inserted (I) domain. Thr ee distinct epitopes within the I domain (IdeA, IdeB, and IdeC) were i dentified using a series of mutants in which sequences from murine CD1 1a were substituted into human CD11a. A series of mutants incorporatin g single amino acid substitutions was used to identify individual amin o acids essential for antibody binding. The location of these residues accounts for the binding specificity of LFA-1-blocking antibodies and identifies particular conserved sequences (residues 126-150) in the I domain of CD11a and homologous sequences in other beta(2)-integrin al pha subunits that may be important for ligand binding.