Mark Renshaw

Internalizing Antibodies to the C-Type Lectins, L-SIGN and DC-SIGN, Inhibit Viral Glycoprotein Binding and Deliver Antigen to Human Dendritic Cells for the Induction of T Cell Responses

The C-type lectin L-SIGN is expressed on liver and lymph node endothelial cells, where it serves as a receptor for a variety of carbohydrate ligands, including ICAM-3, Ebola, and HIV. To consider targeting liver/lymph node-specific ICAM-3-grabbing nonintegrin (L-SIGN) for therapeutic purposes in autoimmunity and infectious disease, we isolated and characterized Fabs that bind strongly to L-SIGN, but to a lesser degree or not at all to dendritic cell-specific ICAM-grabbing nonintegrin (DC-SIGN). Six Fabs with distinct relative affinities and epitope specificities were characterized. The Fabs and those selected for conversion to IgG were tested for their ability to block ligand (HIV gp120, Ebola gp, and ICAM-3) binding. Receptor internalization upon Fab binding was evaluated on primary human liver sinusoidal endothelial cells by flow cytometry and confirmed by confocal microscopy. Although all six Fabs internalized, three Fabs that showed the most complete blocking of HIVgp120 and ICAM-3 binding to L-SIGN also internalized most efficiently. Differences among the Fab panel in the ability to efficiently block Ebola gp compared with HIVgp120 suggested distinct binding sites. As a first step to consider the potential of these Abs for Ab-mediated Ag delivery, we evaluated specific peptide delivery to human dendritic cells. A durable human T cell response was induced when a tetanus toxide epitope embedded into a L-SIGN/DC-SIGN-cross-reactive Ab was targeted to dendritic cells. We believe that the isolated Abs may be useful for selective delivery of Ags to DC-SIGN- or L-SIGN-bearing APCs for the modulation of immune responses and for blocking viral infections.

A rationally designed agonist antibody fragment that functionally mimics thrombopoietin

By using rational design, antibody fragments (Fabs) that mimic thrombopoietin (TPO) were created. A peptide with cMpl receptor-binding capability was grafted into different complementarity-determining regions of a fully human Fab scaffold. Functional presentation of the peptide was optimized by using phage display and cell-based panning. Select antibodies and fragments containing two grafted peptides were assayed for their ability to stimulate the cMpl receptor in vitro. Several candidates demonstrated agonist activity in an in vitro cMpl receptor signaling reporter assay, including Fab59, which was estimated to be equipotent to TPO. Fab59 additionally was able to effectively stimulate platelet production in normal mice. These rationally designed mimetic Fabs may provide a therapeutic intervention for thrombocytopenia while avoiding the potential generation of neutralizing antibodies to endogenous TPO. Furthermore, this study demonstrates a method by which short-lived linear peptides with binding activity may be converted to more stable and potent agonists capable of activating cell surface receptors.

A step-wise approach significantly enhances protein yield of a rationally-designed agonist antibody fragment in E. coli

Fab59 is a rationally-designed antibody fragment (Fab) that mimics the activity of the cytokine thrombopoietin (TPO). Fab59 activity was initially detected directly from bacterial supernatants in a cell-based assay and was subsequently estimated to be equipotent to TPO using purified material. However, the expression of Fab59 was insufficient to support in vivo characterization of the Fab due to extremely low expression levels from its initial phage display expression vector. To boost expression, a new expression vector was designed and constructed, and Fab59 light chain codons were optimized for bacterial expression. However, from this a new challenge arose, in that the codon-optimized Fab59 was more toxic to Escherichia coli cells than parental Fab59. Co-expression of the bacterial chaperon protein Skp alleviated this toxicity. A two-step purification method was used to isolate monomeric Fab59 from the periplasm. Although Fab59 was prone to form aggregates during the purification process, buffer modification efficiently eliminated this problem. Overall, optimization of Fab59 expression and purification achieved a 100-fold increase in Fab59 production in E. coli relative to the starting yield. The yield of purified monomeric Fab59 from a shake flask reached up to 3.5mg/L, which was sufficient to support testing of the agonist activity of purified monomeric Fab59 in vivo. Even higher yields may be achieved by purification of Fab present in the culture media, as Skp most significantly increased accumulation of Fab59 in that location.

Abstract 1774: Development of potent trimeric DR4 agonist Atrimers with therapeutic potential

TRAIL death receptor DR4 is a promising therapeutic target in oncology with expression in a wide variety of tumors. DR4 agonists, including TRAIL and monoclonal antibodies, can induce p53-independent apoptosis and are currently being evaluated in clinical trials in combination with chemotherapy. We aimed to surpass currently available therapeutics by developing trimeric death receptor agonists with properties expected to significantly exceed that of recombinant human TRAIL. The agonists can mimic the natural trimer-trimer interaction of the native ligand/receptor, but do not cross-react with the decoy receptors. Potent DR4 agonist AtrimersTM were engineered using human tetranectin, a trimeric human serum protein of 60 kDa, as a scaffold. A panel of unique DR4 binders was selected from novel phage libraries displaying the C-type lectin domain (CTLD) of tetranectin containing randomized loop sequences. DR4 Atrimers have sub-nanomolar affinity to recombinant DR4-Fc and showed no detectable binding to recombinant forms of DR5 or the decoy receptors. In vitro, the DR4 Atrimers efficiently killed DR4-positive cancer cell lines with sub-nanomolar EC50, but did not kill DR4-negative cell lines. DR4 Atrimers induced cell death of DR4-expressing tumor cells through the caspase pathway, but did not kill primary human B cells and hepatocytes. Interestingly, DR4 Atrimers have differential activities on various cell lines, and also vary in their degree of internalization. While some Atrimers show potent killing of Colo-205 and are rapidly internalized, other agonist Atrimers did not show measurable internalization. These unique properties open the possibility of developing potent naked Atrimers with prolonged half-lives due to lack of internalization, as well as leveraging rapidly internalizing DR4 Atrimers for design of Atrimer-drug conjugates. Further characterization of DR4 Atrimers is ongoing in Colo-205 xenograft models. DR4 agonist AtrimersTM with their superior potency and expected improvement in tumor penetration (vs. antibodies) represent a novel class of targeted cancer therapeutics for efficient induction of apoptosis and provide a promising approach for the treatment of a broad range of cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1774. doi:10.1158/1538-7445.AM2011-1774