Transferrin Receptor 1 and its Targeting by Non-neutralizing Antibodies
The receptor for transferrin (Tf), otherwise known as transferrin receptor 1 (TfR1 or CD71), is a single pass type two transmembrane protein ubiquitously expressed in most vertebrate cells. TfR1 is responsible for facilitating the import of iron into cells from transferrin bound iron in circulation. In cells, release of iron from Tf occurs near pH 5.5 and requires clathrin-mediated endocytosis of the Tf-TfR1 complex to acidified endosomes, followed by its recycling to the cell surface without dissociation.
Since its discovery, high expression of TfR1 is typically associated with rapidly proliferating cells and a variety of tumor cell types. Because of its association with cancer, its cell surface accessibility, its facile entry into cells, and its critical role in the pathophysiology of cancer, TfR1 is an active target for anti-cancer therapies. Antibodies were the first vehicles designed to specifically target TfR1 with the aim of blocking Tf uptake into malignant cells and induce cytotoxicity.
Based on a panel of thirty-two mouse monoclonal antibodies developed, in the late 1980s, to recognize the extracellular domain of human TfR1, several were identified that did not appear to block binding of the receptor to Tf. One of these antibodies, otherwise referred to as 128.1, inspired the generation of a chimeric antibody-avidin fusion protein (ch128.1Av). This molecule was originally designed to act as a delivery vehicle for biotinylated agents into cancer cells. However, this molecule possesses a significant intrinsic anti-proliferative/pro-apoptotic activity against malignant B cells expressing TfR1, as did its parental chimeric antibody (ch128.1) although to a lesser extent. The detailed nature of the interactions of these antibodies (based on the murine monoclonal 128.1) with TfR1 is addressed in part by this thesis.
The experiments presented in this thesis are those from two published articles addressing the nature of the interactions between ch128.1 or ch128.1Av and TfR1, and the effects of these interactions on malignant B cells expressing the receptor. Special emphasis is placed on how these antibodies affect the recycling of TfR1 in order to reduce Tf uptake and induce lethal iron deprivation in malignant hematopoietic cells, as well as the transcriptional response of affected cells to insult by these antibodies.
While investigating the effect of a low pH (late endosome or lysosome like) environment on the stability of TfR1 in the presence of these antibodies, the unexpected observation was made of an intrinsic instability of TfR1 under these conditions. Thus, data that provide insights into the inducible proteolysis of the extracellular domain of TfR1 in a low pH environment (pH 3-5) are presented. These results show a distinct and consistent pattern of degradation of the TfR1 ectodomain selectively within this pH range, which is consistent with the enzymatic activity of a pH-dependent cysteine/serine protease.