Ror1 Targeting Monoclonal Antibodies Induced Apoptosis of Chronic Lymphocytic Leukemia Cells– A Potential Novel Therapeutic Approach

A peptide-based mouse monoclonal antibody generation method was used for producing MAbs against the three extracellular domains of Ror1. Twenty CLL patients (10 with progressive and 10 with non-progressive disease) were enrolled in this study. Flow cytometry was used for surface staining of Ror1. Annexin V and propidium iodide (flow cytometry) as well as PARP cleavage (Western blot) was used for detection of apoptosis.

Six monoclonal antibodies of different isotypes (IgG, IgM) were produced against Ror1. The frequency of Ror1 positive cells were in the range of 24–89%. Patients with progressive disease had a significantly higher number of Ror1 positive cells as compared to those with non-progressive disease as well as in patients with unmutated compared to mutated IgVH genes. All six antibodies alone induced apoptosis (Annexin V and PARP cleavage) of CLL cells. A higher frequency of apoptotic CLL cells was induced by the antibodies against the CRD region (ligand binding site for Wnt proteins) as well as one antibody against the kringle domain (also a binding region for regulatory proteins) . Apoptosis induced by these three antibodies alone was significantly higher than that induced by Rituximab (p<0.001). Apoptosis induction by all antibodies was statistically significantly augmented by crosslinking of the Ror1 antibodies with anti-Fc F(ab^’)₂ fragment antibodies. None of the antibodies induced apoptosis of PBMC of healthy individuals and normal PBMC did not express Ror1.

Monoclonal antibodies alone against the RTK, Ror1, were shown to selectively kill CLL cells. Development of MAb targeting Ror1 might be a novel therapeutic approach complementary to existing therapies.

No relevant conflicts of interest to declare.