Single chain antibody targeting of the ligand binding domain of the tyrosine kinase receptor anaplastic lymphoma kinase

Anaplastic lymphoma kinase (ALK) was initially identified as an abnormal translocation in anaplastic large cell lymphoma resulting in an oncogenic fusion protein. ALK gene codes for a tyrosine kinase receptor that belongs to the insulin receptor superfamily. Our lab has previously identified two growth factors, pleiotrophin and midkine, which bind to LBD and induce activation of the PI3-K and MAPK pathways resulting proliferative and mitogenic effects. Despite its limited presence in normal tissue, ALK and its ligands are overexpressed in human cancers including breast and pancreatic cancers as well as glioblastoma. Their importance of the ALK as a rate-limiting growth factor for glioblastoma has been shown. To better understand ALK signaling several groups developed antibodies targeting the extracellular domain of ALK and showed that they can inhibit signaling in vitro.; In light of the rate-limiting effects of ALK in glioblastoma tumor growth and the ability of antibodies to inhibit ALK signaling in vitro, we attempted to isolate an anti-ALK single chain antibody fragment (scFv) from a commercially available phage display library. Several panning attempts were made but none were successful. We subsequently obtained a human scFv targeting ALK LBD and showed its specificity and affinity of the scFv for the human ALK receptor. More importantly, we examined its effects on ALK-expressing cells and show that it can inhibit cell proliferation in vitro. We further report that the scFv inhibited tumor growth of glioblastoma xenografts in athymic nude mice. Collectively, these studies support a direct role of ALK in glioblastoma multiforme tumor growth and suggest that antibody targeting of the ALK LBD may have a potential use in cancer therapy. We also tested the ability of a mouse monoclonal anti-LBD antibody generated in our lab to inhibit the growth of tumor xenografts in nude mice. We found that it could inhibit the tumor growth of a human breast cancer, prostate cancer and glioblastoma cell lines.