Monoclonal antibodies against ROR1 induce apoptosis of chronic lymphocytic leukemia (CLL) cells

Abstract

ROR1 is a receptor tyrosine kinase (RTK) recently identified to be overexpressed at the gene and protein levels in chronic lymphocytic leukemia (CLL). Monoclonal antibodies (MAbs) against RTKs have been successfully applied for therapy of solid tumors. We generated five MAbs against the Ig (n ¼ 1), cysteine-rich (CRD) (n ¼ 2) and kringle (KNG) (n ¼ 2) domains, respectively, of the extracellular part of ROR1. All CLL patients (n ¼ 20) expressed ROR1 on the surface of the leukemic cells. A significantly higher frequency of ROR1 expression was found in patients with progressive versus non-progressive disease, and in those with unmutated versus mutated IgVH genes. All five MAbs alone induced apoptosis in the absence of complement or added effector cells (Annexin-V and MTT, as well as cleavage of poly-(ADP ribose)-polymerase, caspase-8 and caspase-9) of CLL cells but not of normal B cells. Most effective were MAbs against CRD and KNG, significantly superior to rituximab (Po0.005). Cross-linking of anti-ROR1 MAbs using the F(ab 0 ) 2 fragments of anti-Fc antibodies significantly augmented apoptosis. Two of the MAbs induced complement-dependent cytotoxicity (CDC) similar to that of rituximab and one anti-ROR1 MAb (KNG) (IgG1) showed killing activity by antibody-dependent cellular cytotoxicity. The identified ROR1 epitopes may provide a basis for generating human ROR1 MAbs for therapy.

High-level ROR1 associates with accelerated disease progression in chronic lymphocytic leukemia

“…20 Patients were considered to have progressive disease if they had worsening disease-related anemia (hemoglobin ,10 g/dL), thrombocytopenia (,100 3 10 9 /L), lymphadenopathy, or doubling of the blood lymphocyte count in the preceding 3-month period; patients were considered nonprogressive if they lacked these criteria. However, 8 of the 10 CLL patients in the progressive disease group had previously received treatment, whereas none of the patients in the nonprogressive group had received therapy.…”

Crosstalk between ROR1 and BCR pathways defines novel treatment strategies in mantle cell lymphoma

“…[22][23][24] ROR1 mAb 2A2 was active in blocking Wnt5a binding to ROR1 and its downstream signaling in CLL. 30,31 We observed that ROR1 2A2 was effective when used at similar concentrations as other ROR1 mAbs (5-10 mg/mL) 19,23 in ROR1 Figure 2C-D).…”

“…[22][23][24] ROR1 mAb 2A2 was active in blocking Wnt5a binding to ROR1 and its downstream signaling in CLL. 30,31 We observed that ROR1 2A2 was effective when used at similar concentrations as other ROR1 mAbs (5-10 mg/mL) 19,23 in ROR1 Figure 2C-D). Moreover, ROR1 2A2 cytotoxicity in Jeko-1 and Mino cells could be partially Hyper-CVAD, hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone; ND, not determined; R-Benda, rituximab and bendamustine; R-CHOP, rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone; R-Fludarabine, rituximab and fludarabine.…”

“…19 Furthermore, high ROR1 levels on B-ALL or CLL cells can sustain prosurvival signaling through activation of MEK/ERK and AKT pathways, whereas targeting ROR1 expression efficiently induced apoptosis in malignant cells, suggesting a critical role for this molecule in maintaining cancer cell survival. [20][21][22][23][24] ROR1 monoclonal antibody (mAb) cirmtuzumab has shown excellent preclinical efficacy in directly inducing apoptosis in ROR1…”

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

“…As a model antibody, we chose to use variable regions of a murine mAb (2A2) 28 that is specific for the cancer cell-associated surface molecule ROR1. 29,30 L11 cells were transposed using DNA mixes of HC/LC/TP, and were either selected by simultaneous addition of hygromycin and puromycin 24 hours after electroporation and cultured under selective pressure for 5 days, or were subcultured without antibiotic selection for the same amount of time. Cells were then double-stained for antibody surface expression using PE-coupled, Fc-specific anti-human IgG, and for antigen-binding using soluble, strep-tagged ROR1 that was detected using a fluorophore-labeled strep-tag-specific antibody.…”