AbstractsBiology & Animal Science

Chronic lymphocytic leukemia (CLL) is characterized by accumulation of malignant B cells in blood, bone marrow, spleen and lymph nodes. It is the most common leukemia in the Western world. Major progress has been made in recent years to prolong the survival and improve the well-being of the patients. Despite advances, CLL is still a disease with no cure. New therapeutic strategies, based on new targets and novel drugs, are needed. Receptor tyrosine kinase-like orphan receptor (ROR1) belongs to one of the twenty families of receptor tyrosine kinases (RTKs). RTKs are involved in important cellular processes including proliferation, differentiation, survival, signaling and migration. Many RTKs are deregulated in various types of cancer and may act as a therapeutic target. ROR1 is highly expressed in CLL and other malignancies but not in normal adult cells. ROR1 has been shown to be a survival factor for CLL cells. The main goal of the study was to develop novel ROR1 targeting therapeutics for CLL. In the first study, five monoclonal antibodies (mAbs) directed against different domains of the extracellular part of ROR1 were produced and their apoptotic activity was analysed. All five mAbs recognized surface bound ROR1 and induced selective apoptosis of CLL cells but not of normal B cells. Antibodies alone against CRD and KNG domains of ROR1 were the most effective in inducing apoptosis of CLL cells without the need of complement or immune effector cells. KNG and CRD mAbs induced higher cytotoxicity than rituximab in vitro. Crosslinking of ROR1 mAbs with F(ab)2 fragments significantly enhanced apoptosis. Two of the mAbs also induced complement dependent cytotoxicity (CDC) similar to rituximab and one KNG mAb (IgG1) induced antibody-dependent cellular cytotoxicity (ADCC). In the second study, different ROR1 isoforms as well as the phosphorylation pattern in CLL cells were analysed. Two major ROR1 bands with the size of 105 and 130 kDa were identified which may correspond to unglycosylated (immature) and glycosylated (mature) ROR1 respectively. The 105 kDa band was significantly higher expressed in non-progressive than in progressive CLL patients. Two other ROR1 bands with the size of 260 kDa and 64 kDa were also noted which might represent dimerized ROR1 and truncated ROR1, respectively. The 64 kDa band was localized to the nucleus indicating a possible role as a transcription factor. ROR1 isoforms of 64, 105, 130 and 260 kDa were all phosphorylated at tyrosine and serine residues. The phosphorylation intensity for the 130 kDa ROR1 isoform was significantly higher in progressive than in non-progressive CLL patients. ROR1 mAbs against the CRD and KNG domains induced dephosphorylation of ROR1 and subsequent apoptosis of CLL cells. In the third study, the effect of ROR1 mAbs on PI3K/AKT/mTOR signaling was analyzed. Anti-CRD ROR1 mAb induced apoptosis of CLL cells and reduced phosphorylation levels of ROR1 as well as of SRC, PI3K, AKT, mTOR, and CREB. An anti-ROR1 mAb against the Ig-like domain did not induce…