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 = 1), cysteine-rich (CRD) (n = 2) and kringle (KNG) (n = 2) domains, respectively, of the extracellular part of ROR1. All CLL patients (n = 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 (P < 0.005). Cross-linking of anti-ROR1 MAbs using the F(ab')(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.
Subject(s)
Antibodies, Monoclonal/pharmacology , Apoptosis/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Receptor Tyrosine Kinase-like Orphan Receptors/immunology , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Animals , Antibody Formation , Antibody-Dependent Cell Cytotoxicity , Humans , Immunization , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Mice , Mice, Inbred BALB C , Peptide Fragments/immunology , Tumor Cells, CulturedABSTRACT
Dendritic cells function as the main cellular population responsible for professional antigen presentation and hence for induction of primary immune responses. Although they are present in virtually every tissue, nevertheless their number is usually so low that it makes their isolation for studies very difficult. In this study, we purified dendritic cells from mouse spleen by a three-step enrichment method and evaluated morphological and cytochemical characteristics of isolated cells. We showed that isolated dendritic cells from mouse spleen had all lobulated nuclei with multiple cytoplasmic projections and their morphological features changed after an overnight incubation. It was also shown that typical dendritic cells lacked both Myeloperoxidase (MPO) and Non Specific Esterase (NSE) activity. In conclusion, for reaching a reasonable purity in isolation of dendritic cells from lymphoid tissues, many enrichment steps should be taken, and for determining the purity of isolated cells, we recommend that a combination of morphological and cytochemical studies be used.
