Browsing by Author "Diebolder, Philipp"

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    Generation of "LYmph Node Derived Antibody Libraries" (LYNDAL) : a concept for recovering human monoclonal antibodies with therapeutic potential
    (2014) Diebolder, Philipp; Kontermann, Roland E. (Prof. Dr.)
    The development of efficient strategies for generating human monoclonal antibodies with therapeutic potential remains a major challenge in the antibody technology field. In present thesis, an applicable approach has been developed for recovering such antibodies from antigen-encountered, human B cell repertoires. As the source for variable antibody genes, immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 head and neck cancer patients were employed for cloning individual antibody libraries. Sequence analysis of “Lymph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences that represent all known variable gene families and most functional germline sequences. To demonstrate the feasibility of presented approach, test selections against distinct therapeutic targets have been performed including the viral glycoprotein B of herpes simplex virus type 1 (HSV-1) and human epidermal growth factor receptor (EGFR) being frequently overexpressed in head and neck cancer. Panning of LYNDAL from donors with target-specific IgG serum titers delivered 34 anti-gB-1 and seven anti EGFR single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as result of natural affinity maturation. Target specificity was confirmed by binding antiviral scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains and by binding EGFR specific scFvs to various EGFR-overexpressing cancer cell lines. The majority of analyzed scFvs bound to the targets with nanomolar affinity as determined on recombinant proteins as well as on protein variants in their natural context. Therapeutic potential of LYNDAL antibodies was evaluated by functional in vitro assays testing either the virus neutralizing capacity or the potential for mediating tumor cell growth inhibition. From eight scFvs with HSV-neutralizing capacity, the most potent antibody neutralized 50% HSV-2 at 4.5 nM as dimeric (scFv)2. One of the EGFR-specific antibodies showed auspicious anti-proliferative effects on tumor cells. Using SKOV-3 tumor cells, 50% of the EGF-induced cell growth promoting effect was inhibited at 6.2 nM of bivalent scFv Fc. In conclusion, the LYNDAL approach is useful for recovering fully human antibodies with therapeutic potential and is expected to be extendable to others than the here evaluated targets.