title: Targeting malignant B cells with an immunotoxin against ROR1.
abstract: The selective cell surface expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) has made ROR1 a novel and promising target for therapeutic monoclonal antibodies (mAbs). Four mouse mAbs generated by hybridoma technology exhibited specific binding to human ROR1. Epitope mapping studies showed that two mAbs (2A2 and 2D11) recognized N-terminal epitopes in the extracellular region of ROR1 and the other two (1A1 and 1A7) recognized C-terminal epitopes. A ROR1- immunotoxin (BT-1) consisting of truncated Pseudomonas exotoxin A (PE38) and the VH and VL fragments of 2A2-IgG was made recombinantly. Both 2A2-IgG and BT-1 showed dose-dependent and selective binding to primary CLL and MCL cells and MCL cell lines. Kinetic analyses revealed 0.12-nM (2A2-IgG) to 65-nM (BT-1) avidity/affinity to hROR1, depicting bivalent and monovalent interactions, respectively. After binding to cell surface ROR1, 2A2-IgG and BT-1 were partially internalized by primary CLL cells and MCL cell lines, and BT-1 induced profound apoptosis of ROR1-expressing MCL cell lines in vitro (EC 50 = 16 pM-16 nM), but did not affect ROR1-negative cell lines. Our data suggest that ROR1-immunotoxins such as BT-1 could serve as targeted therapeutic agents for ROR1-expressing B cell malignancies and other cancers.
authors.affiliation: Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA. baskars@mail.nih.gov
authors.forename: Sivasubramanian
authors.initials: S
authors.lastname: Baskar
authors.forename: Adrian
authors.initials: A
authors.lastname: Wiestner
authors.forename: Wyndham H
authors.initials: WH
authors.lastname: Wilson
authors.forename: Ira
authors.initials: I
authors.lastname: Pastan
authors.forename: Christoph
authors.initials: C
authors.lastname: Rader
mesh_terms: D036002:ADP Ribose Transferases / Q000627:therapeutic use*; D000818:Animals; D017209:Apoptosis; D001427:Bacterial Toxins / Q000627:therapeutic use*; D014408:Biomarkers, Tumor / Q000378:metabolism; D045744:Cell Line, Tumor; D005098:Exotoxins / Q000627:therapeutic use*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology; D000922:Immunotoxins / Q000627:therapeutic use*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000628:therapy*; D020522:Lymphoma, Mantle-Cell / Q000628:therapy*; D051379:Mice; D058990:Molecular Targeted Therapy / Q000379:methods*; D057050:Receptor Tyrosine Kinase-like Orphan Receptors / Q000037:antagonists & inhibitors* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use; D037521:Virulence Factors / Q000627:therapeutic use*; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D014408:Biomarkers, Tumor; D005098:Exotoxins; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D057050:Receptor Tyrosine Kinase-like Orphan Receptors; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C078716:ROR1 protein, human