Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: Deregulation of the tumor necrosis factor (TNF) plays an important role in the initiation and perpetuation of chronic inflammation and has been implicated in the development of various autoimmune diseases. Accordingly, TNF-inhibitors are successfully used for the treatment of several diseases, such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, total inhibition of TNF can cause severe side effects such as an increased risk of inflammation and reactivation of tuberculosis. This is likely due to the different actions of the two TNF receptors. Whereas TNFR1 predominantly promotes inflammatory signaling pathways, TNFR2 mediates immune modulatory functions and promotes tissue homeostasis and regeneration. Therefore, the specific blockage of TNFR1 signaling, either by direct inhibition with TNFR1-selective antagonists or by targeting soluble TNF, which predominantly activates TNFR1, may prevent the detrimental effects associated with total TNF-inhibitors and constitute a next-generation approach to interfere with TNF.
Abstract: Several autoimmune diseases are marked by a deficiency of soluble tumor necrosis factor (TNF). The TNF deficiency is caused in at least one autoimmune disease, multiple sclerosis, by an overabundance of TNF receptor 1 (TNFR1). Excess TNFR1 binds and inactivates TNF and this leaves less TNF bioavailable. This study sought to determine if expression of fresh or IL2-stimulated TNF receptors on Tregs cells, an important immunoregulatory cell involved in autoimmunity, is altered in type I diabetes. Standard fluorescence analysis was used to examine the levels of TNFR1 and TNFR2 on human Tregs in patients with type I diabetes (T1D) or controls. Fresh Tregs from T1D compared to control Tregs had identical levels of TNFR1. In marked contrast, Type 1 diabetic patients Treg cells had statistically elevated levels of TNFR2 compared to controls. Tregs stimulated with IL2 from both T1D and controls showed marked increase of TNFR2 expression in a dose-response manner, but the dose response increase in TNFR2 was significantly higher for T1D Treg cells. No IL2 dose-response was present for TNFR1 on either T1D or control Tregs exposed to IL2. A large study of serum for secreted levels of TNFR2 also revealed elevated circulating levels consistent with the elevated surface expression on Tregs. These findings suggest that abnormal regulation of TNFR2 expression with elevated cellular and secreted levels of TNFR2 is a characteristic of Type 1 diabetes. It is possible that the relative deficiency of TNF in type I diabetes, in contrast to multiple sclerosis, is caused by excess expression of TNFR such as TNFR2, a binding structure for inactivating TNF.
Antibodies2015, 4(1), 12-33; doi:10.3390/antib4010012 - published 26 January 2015 Show/Hide Abstract
Abstract: Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG) molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv) and antigen binding fragments (Fab), have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.