Antibodies, Volume 4, Issue 2 (June 2015) – 4 articles , Pages 71-140

Anti-tumor necrosis factor (TNF) monoclonal antibodies and TNF receptor ectodomain fusion protein are in clinical use to neutralize circulating TNF and ameliorate symptoms of many autoimmune diseases and pathological conditions with chronic inflammation. In this paper we present data to prove that reverse signaling, elicited by agonist molecules interacting with the membrane-bound TNF of myeloid cells, significantly contributes to the therapeutic effect of these anti-TNF medicines. Interaction of agonist monoclonals with cell surface TNF significantly attenuates the expression of pro-inflammatory cytokines and induces changes in the production of extracellular and intracellular signaling molecules. This phenomenon is not dependent on the Fc portion of antibodies as Fab constructs are as efficient as full antibody molecules. Full article

The development of in vitro antibody selection technologies has allowed overcoming some limitations inherent to the hybridoma technology. In most cases, large repertoires of antibody genes have been assembled to create highly diversified libraries allowing the isolation of antibodies recognizing virtually any antigen. However, these universal libraries might not allow the isolation of antibodies with specific structural properties or particular amino acid contents that are rarely found in natural repertoires. Purpose-oriented libraries specially designed to incorporate desired characteristics have been successfully used. However, the workload required for library construction has limited the attractiveness of this approach compared to the use of large universal libraries. We have developed an approach to capture synthetic or natural diversity into the complementarity determining regions 3 (CDR3) of human antibody repertoires using Type IIS restriction enzymes. In this way, we generated several libraries either biased in amino acid content or towards long CDRH3 loops. The latter were successfully used to identify antibodies inhibiting the enzymatic activity of horseradish peroxidase, whereas libraries enriched in histidines allowed for the isolation of antibodies binding to human Fc in a pH-dependent manner. These libraries indicate that tailored diversification of CDR3 is sufficient to generate purpose-oriented libraries and isolate antibodies with uncommon properties. Full article

We have previously described ProxiMAX, a technology that enables the fabrication of precise, combinatorial gene libraries via codon-by-codon saturation mutagenesis. ProxiMAX was originally performed using manual, enzymatic transfer of codons via blunt-end ligation. Here we present Colibra™: an automated, proprietary version of ProxiMAX used specifically for antibody library generation, in which double-codon hexamers are transferred during the saturation cycling process. The reduction in process complexity, resulting library quality and an unprecedented saturation of up to 24 contiguous codons are described. Utility of the method is demonstrated via fabrication of complementarity determining regions (CDR) in antibody fragment libraries and next generation sequencing (NGS) analysis of their quality and diversity. Full article

There is considerable interest in the characterization of novel tumor-associated antigens that lend themselves to antibody-mediated pharmacodelivery strategies. Delta-like 1 homolog protein (DLK1), which exists both as transmembrane protein and in soluble form, shows a restricted pattern of expression in healthy organs, while being overexpressed in some tumors. We have generated a human antibody specific to DLK1 using phage display technology. This reagent was used for a comprehensive characterization of DLK1 expression in freshly frozen sections of normal human adult tissues and of xenografted human tumors. DLK1 was virtually undetectable in most organs, except for placenta which was weakly positive. By contrast, DLK1 exhibited a moderate-to-strong expression in 8/9 tumor types tested. Our analysis shed light on previous conflicting reports on DLK1 expression in health and disease. The study suggests that DLK1 may be considered as a target for antibody-mediated pharmacodelivery strategies, in view of the protein’s limited expression in normal tissues and its abundance in the interstitium of neoplastic lesions. Full article