Generation and Reactivity Profiling of Functional Human Recombinant Monoclonal Antibodies

Mature B-cell lymphomas are a group of hematologic malignancies that differ in terms of pathophysiology, tissues involved, and biological profiles, as well as clinical presentation, prognosis, and outcome [1, 2]. However, a common feature in most lymphomas concerns the pronounced role of interactions between the malignant cells and elements of their microenvironment, especially (auto)antigens, throughout their natural history [3]. This highlights the critical role of the clonotypic B-cell receptor immunoglobulin (BcR IG) in lymphoma ontogeny and evolution given that it represents the most significant mediator of antigen-specific B-cell drive. Relevant studies have provided important insight into the BcR IG reactivity profiles of particular mature B-cell lymphomas, paving the way to functional studies that led to the identification of some of the relevant antigenic determinants [4-8]. Furthermore, inhibition of antigen interactions through the use of pharmacological agents that target the downstream signaling pathway of the BcR IG has displayed great efficacy and led to a therapeutic paradigm shift for mature B-cell lymphomas [9-11].Here, we describe a strategy to characterize the (auto)antigen reactivity of the BcR IG using the recombinant DNA technology. This approach allows the unbiased profiling of the human antibody repertoire, through the generation of recombinant monoclonal antibodies (rmAbs) derived from B cells of defined origin. The protocol starts with the isolation of total RNA from the B cells of interest, followed by reverse transcription-polymerase chain reaction (RT-PCR) for amplifying the clonotypic IG V(D)J gene rearrangements. Subsequently, the cDNA molecules corresponding to the BcR IG heavy and light chains are cloned into expression vectors for the in vitro production of rmAbs. Finally, the (auto)antigen reactivity profiling of the rmAbs is assessed using ELISA, flow cytometry, and immunohistochemistry. This strategy offers the potential to obtain crucial information regarding the antigenic stimuli that drive the development of mature B-cell lymphomas through the in-depth study of the reactivity profile of the clonotypic BcR IG.