Novel ROR1 Antibody Is Able to Trigger Specific and Superior Complement-Dependent Cytotoxicity (CDC) on Primary CLL Cells

Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia of the elderly, remains largely incurable despite chemotherapy and small molecule inhibitors. Treatment targeting CD19 and CD20 has achieved important clinical benefits; however, these antigens are also present on normal B-cells rendering the patients immunodeficient and at a higher risk of infections. In view of this, our immunotherapy strategy is to produce therapeutic antibodies targeting the receptor-tyrosine-kinase-like orphan receptor 1 (ROR1), a protein present on the surface of malignant B-cells and cancer stem cell-like cells (CSC), but absent on most normal tissue.

In order to produce new ROR1 monoclonal antibodies (MAbs), we generated a rat hybridoma library and screened >150 anti-ROR1⁺clones. We then cloned 16 of our own novel antibodies as well as previously published clones: 4a5 and R12 to human IgG1, kappa constant regions. A total of 13 chimeric antibodies recognised human ROR1 on different cell types, out of which, clone SA1 showed superior complement-dependent cytotoxicity (CDC) than other ROR1 MAbs, including clones 4a5 and R12.

To determine the binding domain of our ROR1 MAbs, we generated stable cell lines expressing either full length-ROR1 or various forms of truncated ROR1 extracellular domains. Flow cytometry evaluation using these cells, and Epitope Mapping ELISA using a ROR1 peptide library, showed that our antibodies preferentially bind the Ig-like and Frizzled domains. Additionally, we fused clone SA1 to a mouse IgG2a, kappa constant regions in order to perform competition assays amongst our ROR1 MAbs and previously reported clones. We found that there was no epitope overlap amongst any of the tested clones. Moreover, by substituting single amino acids in a relevant region within the Ig-like domain, which we previously determined to be critical for SA1 binding, we found that this epitope is unique to SA1 and, notably is not shared by other clones previously reported in the literature; including clone D10, the prototype of Cirmtuzumab. Importantly, further characterisation of clone SA1 revealed that it can get internalised by both SKW 6.4 cells and CLL cells.

As a whole, we have generated and identified a novel anti-human ROR1 monoclonal antibody able to trigger specific CDC of ROR1⁺ cells. More than that, clone SA1 has a unique epitope and it is able to get internalised by both cell lines and CLL cells that endogenously express ROR1. Additional characterisation and functional analyses are ongoing in order to also develop this antibody as a drug-antibody conjugate (ADC). Importantly, effective targeting of ROR1 expressed on malignant cells and CSC cells but not healthy tissue would provide a safer and more effective treatment of CLL and, in turn, other ROR1⁺ malignancies.