Preparation And Functional Identification Of Monoclonal Antibody For Activated LM-1

In the past two decades, many cell adhesion molecules (CAMs) have been discovered, and their functions in cell morphology, locomotion, mitosis, cytokinesis, phagocytosis, and the maintenance of cell polarity have been studied. CAMs have important roles in different disease states such as cancer, thrombosis, and autoimmune diseases (e.g., rheumatoid arthritis and type-1 diabetes). Thus, researchers have been actively investigating the structure, function, and recycling mechanisms of some CAMs, as well as how to modulate them for controlling disease progression. In addition, CAMs have been implicated in pathogenic (i.e., virus and bacteria) infections.CAMs are glycoproteins found on the cell surface that act as receptors for cell-to-cell and cell-toextracellular matrix (ECM) adhesion. CAMs can be divided into four classes: integrins, cadherins, selectins, and the immunoglobulin superfamily. Some CAMs are internalized into the cytoplasm during the recycling process via the formation of clathrin-coated pits. Thus, the internalization process of CAMs can be exploited for targeting drugs into a compartment of a specific cell type (i.e., cancer and leukemic cells).In recent years, peptides, peptidomimetics, and proteins that bind to cell adhesion receptors have been investigated for targeting drugs, particles, and liposomes to specific cell-bearing cell adhesion receptors (i.e., integrin and immunoglobulin superfamily)LM-1 is one of the CAMs, which plays essential roles in leukocyte migration and adhesion in the gastrointestinal (GI) tract and surrounding tissue. They are expressed on monocytes, lymphocytes, eosinophils, basophils, and macrophages, but are not found on neutrophils. The primary ligands for LM-1 are the cytokine inducible cell adhesion molecules, vascular cell adhesion molecule (VCAM), and mucosal addressin cell adhesion molecule (MAdCAM). The role of LM-1 and their CAM ligands in leukocyte migration and adhesion during inflammatory processes is deeply studied. Interaction of LM-1 on the leukocyte cell surface with VCAM and/or MAdCAM expressed in the vascular endothelium of the affected tissues initiates attachment and rolling of the leukocyte along the blood vessel wall. This initial event is followed by chemokine activation of the LM-1 to a higher affinity state which allows firm adhesion of the leukocyte to the capillary wall, transmigration into the underlying tissue, and subsequent proliferation of inflammatory cells.Monoclonal antibodies directed against LM-1 or their cell adhesion molecule (CAM) ligands have been shown to be effective modulators in animal models for auto-inflammatory diseases such as asthma, rheumatoid arthritis (RA), and inflammatory bowel diseases (IBD). In addition to the their role in these inflammatory processes, binding of LM-1 to their ligands may also play important roles in stem cell adhesion to bone marrow stromal cells, and in tumor cell metastasis. The widespread implications for their involvement in disease processes has led dozens of pharmaceutical and biotech companies to actively pursue LM-1 antagonists as potential anti-inflammatory agents. But the only therapeutic Antibody for LM-1 recognizes both and non-activated LM-1 protein, which has side effect. So a therapeutic Antibody specificly recognizes activated LM-1 will have great advantage.Phage display, first introduced by G. Smith in 1985 , is a very effective way for producing large numbers (up to1010) of diverse peptides and proteins and isolating molecules that perform specific functions. This technique can also be used to study protein-ligand interactions, receptor and antibody-binding sites, and to improve or modify the affinity of proteins for their binding partners. Phage display involves the expression of proteins, including antibodies, or peptides on the surface of filamentous phage. DNA sequences of interest are inserted into a location in the genome of filamentous bacteriophage such that the encoded protein is expressed or"displayed"on the surface of filamentous phage as a fusion product to one of the phage coat proteins. Therefore, instead of having to genetically engineer proteins or peptide variants one-by-one and then express, purify, and analyze each variant, phage display libraries containing several billion variants can be constructed simultaneously. These libraries can then be easily used to select and purify specific phage particles bearing sequences with desired binding specificities from the nonbinding variants. And then, the functional protein can be expressed in the periplasmic space of E.coli.Above all, we would synthesis specificly anti-activated LM-1 monoclonal antibody by Phage display technology. We expressed and purified the mutational LM-1 protein(Q324T) in 293T cell, and made it as antigen. We detected that the positive multiclonal phage had been enriched remarkably after three cycles by Elisa. We selected 10 high potency phages from 190 monoclonal positive phages which were detected by Elisa one by one. Then we extracted phagemid from these phages, and infected the host strain HB2151, with IPTG-induced expression of scFV, purified the scFV by Ni2+ affinity chromatograph. Finally, we certificated that the scFV could specificly recognize activated LM-1 and had a very high titer by FACS and Flow Chamber System, whcih would be able to play very good role in 10ng/ul.We has laid a good foundation to further study of therapeutic antibodies for activated LM-1 with the success of screening specificly anti-activated LM-1 monoclonal antibody. Compared to the therapeutic antibody which can not distinguish between activated and non-activated LM-1, our monoclonal antibody has a distinct advantage of less side effects, as the targeted transport of drugs to the cells with activated LM-1 existing on the surface of the cell membrane, will have a very good application prospects.