NO FULL TEXT AVAILABLE. Access is restricted indefinitely. ----- Kappa Myeloma Antigen (KMA) is a plasma membrane associated form of free immunoglobulin kappa light chain (FkLC) expressed on malignant B cells from patients with multiple myeloma (MM), Waldenstrom’s macroglobulinaema (WM) and non- Hodgkin’s lymphoma (Walker et al. 1985). KMA is recognized by the murine monoclonal antibody (mAb) mKap, and its human-mouse chimeric equivalent, cKap, which is currently undergoing clinical trials as a therapy for kappa type MM (Boux et al. 1983; Raison et al. 2005).
Earlier expression studies on KMA suggested that the antigen is not expressed by normal B cells in vivo. However, in vitro activation of tonsillar B cells induced expression of KMA on a subset of cells. Like their KMA expressing malignant counterparts, these were presumed to be FkLC secreting plasma cells or plasmablasts but, due to the lack of B cell lineage specific markers at the time, these cells were not phenotyped (Walker et al. 1985). Furthermore, given the extremely low frequency of plasmablasts and plasma cells in normal tissues, it was not possible to exclude the presence of a ‘normal’ KMA positive cell population in vivo.
The first section of this thesis expands upon this earlier work. By utilizing in vitro activation protocols on peripheral blood CD 19+ B cells, KMA expression was induced on a subset of cells. Phenotypic analysis revealed that the majority of KMA positive cells were CD27++ CD38+/- plasmablasts and CD38++ plasma cells. Analysis from normal human tissues found that a subset of plasma cells in the tonsils expressed the antigen. These cells co-expressed CD45, indicating that they are at an immature stage of plasma cell differentiation. In contrast, peripheral plasma cells, considered to be more fully mature cells in transit from secondary lymphoid organs to plasma cell niches in bone marrow or spleen, did not express KMA. This implies that KMA expression, in vivo, is limited to a small subset of immature plasma cells in secondary lymphoid organs such as the tonsils.
Despite cKap’s current assessment in clinical trials for the treatment of MM, very little is known about its molecular target KMA. Previous studies have showed that KMA is comprised of FkLC (Goodnow and Raison 1985); however it was never determined as to how FkLC is associated with the plasma membrane. Since FkLC is a secreted molecule, it was initially presumed that it associated with a proteinaceous ‘membrane receptor’ (Goodnow and Raison 1985). However membrane extraction studies, as described in the second part of this thesis, reveal that FkLC directly associates with the plasma membrane through a combination of hydrophobic and electrostatic forces to form KMA. Further investigations confirmed that FkLCs can bind directly to cellular and artificial membranes. Moreover, this binding is likely dependent on self-association processes, which suggest that KMA consists of aggregated, membrane associated FkLCs.
Lipid binding studies revealed that FkLCs associate specifically with saturated phosphocholine species such as sphingomyelin in membranes, and KMA expression was positively correlated with sphingomyelin expression in FkLC secreting cell lines.
The final section of this thesis examines how FkLCs might interact with saturated phosphocholine lipids. Molecular modeling of dimeric FkLC suggests they are able to weakly associate with phosphocholine in the conventional antigen binding pocket formed by the kLC variable domain (V-domain). Since FkLC aggregation is a feature of KMA, then the avidity effects of multi-valent binding likely increases the strength of the proposed FicLC-phosphocholine interaction. This hypothesis explains the observation of both electrostatic and hydrophobic interactions by FkLC, as KMA, with the plasma membrane - the electrostatic component, governed by single FkLC molecules interacting with the charged phosphocholine headgroups, and the hydrophobic component, due to selfassociation of adjacent FkLC molecules.
Finally, a model of KMA expression by FkLC secreting cells is proposed. FkLC is synthesized in the endoplasmic reticulum (ER) then transported to the golgi-apparatus and encapsulated into vesicles destined for secretion. There FkLCs interact with saturated phosphocholine lipids, such as sphingomyelin, and undergo aggregation resulting in stable association on the inner vesicular membrane. Fusion of the vesicle with the plasma membrane during exocytosis allows for membrane associated FkLC to become exposed on the extracellular face as KMA.