B-CLL Antibodies Comprised of Stereotypic VH1-69, D3-16, and JH3 Rearrangements Immunoprecipitate Cellular Protein(s).

Abstract

B-cell chronic lymphocytic leukemia (B-CLL) is typically due to a clonal expansion of B-lymphocytes that can be divided into two classes based on the mutation status of the variable region of the antibody produced by the leukemic clone. Mutated B-CLL (antibody with ≥ 2% mutation) is associated with longer survival than unmutated B-CLL. Segregation of patients based on antibody mutation suggests a functional role for antigen binding to antibody in this disease. Furthermore, many different subsets of B-CLL antibodies have very similar CDR3 sequences, suggesting some common antigen reactivity. Subset I contains rearrangements comprised of an unmutated sequence encoded by VH1-69, D3-16, and JH3 with a nearly identical HCDR3 region. Antibodies from this subset strongly bind cytoplasmic structures in HEp-2 cells by immunofluorescence and are polyreactive against multiple autoantigens (ssDNA, dsDNA, insulin) by ELISA. In contrast, subset VI, which comprises a mutated sequence encoded by VH3-21 and JH6 resulting in a nearly identical HCDR3 that differs from that of subset I, does not bind HEp-2 cells and is not polyreactive. Because the subset I B-CLL antibodies were reactive with HEp-2 cells, we prepared HEp-2 cell extracts in order to isolate the cellular antigen recognized by these antibodies. Antibodies from both subset I (patients CLL068 and 258) and VI (patient CLL412) were recombinantly overexpressed and purified as human IgG1s. Antibody was bound to protein G agarose beads and incubated with cell extracts overnight. Bound cellular antigen was immunoprecipitated, washed, eluted, run on SDS-PAGE, and stained with Coomassie Blue. Both CLL068 and 258 antibodies immunoprecipitated protein bands at about 45 and 200 kDa. These bands have been isolated and are undergoing microsequencing analysis. In contrast, CLL412 did not immunoprecipitate any protein bands under the same conditions. Thus, unmutated subset I CLL antibodies react with common cellular protein(s), whose identity may be useful in understanding B-CLL.