Abstract 813: Generation of Activation-Specific Human Anti Mac-1 (αMβ2, CD 11b/CD18) Single-Chain Antibodies as Potential Diagnostic Tools and Therapeutic Agents
Introduction: The leukocyte integrin Mac-1 plays a pivotal role in inflammation and host defence. Upon leukocyte activation, Mac-1 undergoes a conformational change exposing interaction sites for multiple ligands.
Methods and Results: We aimed to generate single-chain antibodies (scFv) directed against activation-specific Mac-1 ligand binding sites. Using human scFv phage libraries, we developed subtractive strategies with depletion of phages binding to non-activated Mac-1 and selection of phages binding to activated Mac-1. Non-activated or PMA-activated monocytes as well as CHO cells transfected with native or activated (GFFKR-deletion in αM) Mac-1 were used for depletion/selection rounds. Three scFv clones demonstrated exclusive binding to activated Mac-1 as validated in flow cytometry (see figure⇓). Using alanine substitutions, the paratope could be identified within the scFv’s heavy chain HCDR3. The epitope was localized to Lys245-Arg261 of the αM I-domain. The potential therapeutic use was tested in adhesion assays under static and flow conditions demonstrating the selective blockade of activated monocytes only.
Conclusion: These single-chain antibodies promise to be useful for the diagnosis of monocyte activation in disease and promise to be beneficial as therapeutic agents that selectively block activated leukocytes.