Id-peptides as new tools for diagnosis and treatment of B-cell Chronic Lymphocytic Leukemia (B-CLL)

Introduction: Several features of the B-cell receptor (BCR) complex have emerged as major markers for prognostic classification of B-cell chronic lymphocytic leukemia (B-CLL). In particular, characterizing BCR signaling profiles may help to identify signaling markers useful for patient stratification, disease monitoring and therapeutic targeting in B-CLL. In this regard, we used random peptides libraries (RPLs) to identify peptide ligands of the Ig-BCR of neoplastic B-cells, and to determine the epitope diversity recognized by the Ig-BCR. Material and method: The full-length Ig gene transcripts (VDJ) from a B-CLL human sample were amplified by two nested PCRs using a combination of forward primers specific for the respective VH, Vκ or Vλ leader regions and a reverse primer specific for the respective constant region. The PCR products were sequenced and analysed by IgBlast to identify the IgV mutation status. The IgVH and IgVL PCR products were cloned into eukaryotic expression vectors and co-expressed in 293T cells in order to produce the corresponding IgBCR. The quality of the recombinant IgBCR was assessed by SDS-PAGE under non-reducing and reducing conditions. A flow cytometry analysis showed that the Id-peptides specifically recognized the respective target cells. Results and discussion: We developed a methodology that combines the IgBCR variable genes repertoire analysis and the IgBCR production. We identified small bioactive peptides, "Id-peptides", that bind to the idiotypic determinants of Ig-BCR of the to B-CLL. This approach will allow us to: 1) identify of a pool of Id-peptides for B-CLL shared by a substantial number of B-CLL patients; 2) understand the general differences in antigenic reactivity between mutated and unmutated IgBCR, 3) understand the correlation between IgBCR signal transduction in leukemic cells and the clinical course of the disease. Conclusion: The described strategy is able to provide us of (i) patient-specific and tumor-specific Ig-BCR that will be used for screening RPLs (ii) monitoring the CLL clinical progression by recognizing the circulating B-CLL in lymph nodes and bone marrow and (ii) prognostic information based on the IgVH genetic status of each patient.