Phage display-derived peptides as tool for single B cell populations targeting in polyclonal CLL patients

Background. Chronic lymphocy7c leukemia (CLL) is a neoplas7c disease of mature B cells that express a func7onal Immunoglobulin B-cell Receptor (IgBCR) on the cell surface. Taking advantage of our already reported methodological background (Mimmi et al. Leukemia 2016), the present study is aimed at the iden7fica7on of pep7de ligands of CLL IgBCRs expressed by B-cell subpopula7ons in the course of the disease. Materials and Methods. Within a cohort of 50 CLL pa7ents, we focused on CLL1, a pa7ent who progressed from Binet stage A to C within 12 months observa7on. We collected tumour B-cells from peripheral blood, before and aer Binet stage switching. The VDJ sequence of IgBCRs was PCR-amplified and cloned into expression vectors for sequencing and produc7on of recombinant IgBCRs. Purified IgBCRs have been used as bait for screening a phage display pep7de library. Results. We detected dis7nct B-cell subpopula7ons in CLL1 pa7ent switching from Binet stage A to stage C. At month 1 (Binet stage A), a tumour sub-popula7on with un-mutated VDJ sequence (VH1-69) was 60% and a sub-popula7on with mutated VDJ sequence (VH4-4) was 40% of total B cells. At month 6 (Binet stage A), the un-mutated VH1-69 subpopula7on was 50% and four sub-popula7ons with mutated VDJ sequence (10% VH3-15; 20% VH3-21; 10% VH5-12; 10% VH4-59*01) appeared. At month 12 (Binet stage C), the unmutated VH 1-69 sub-popula7on was 80%, the mutated VH3-15, VH3-21, VH3-30, and VH4-59*01 subpopula7ons were undetected, and two new mutated subpopula7ons (10% VH5-10; 10% VH4-59*08) appeared. Conclusions. By IgBCR sequencing, we monitored CLL sub-popula7ons in a single pa7ent from indolent to the aggressive stage of the disease. We observed that the aggressiveness of disease correlated with the progressive expansion of an un-mutated VDJ sub- popula7on over mutated VDJ sub-popula7ons. We are selec7ng specific IgBCRs pep7de binders, the unique tools able to discriminate the single B cell popula7ons among total white blood cells. In this regards, B cell clone-specific pep7des will be used to sort the single B cell popula7ons (especially the un-mutated one) by flow citometry in order to analyse the profile of leukemic cells (in terms of proteome, trascriptome, miRNA etc.), for understanding the causes of aggressiveness and drug-resistance.