Georg Tiefenthaler

Targeted delivery of interferon-α to hepatitis B virus-infected cells using T-cell receptor-like antibodies.

During antiviral therapy, specific delivery of interferon‐α (IFNα) to infected cells may increase its antiviral efficacy, trigger a localized immune reaction, and reduce the side effects caused by systemic administration. Two T‐cell receptor‐like antibodies (TCR‐L) able to selectively bind hepatitis B virus (HBV)‐infected hepatocytes of chronic hepatitis B patients and recognize core (HBc18‐27) and surface (HBs183‐91) HBV epitopes associated with different human leukocyte antigen (HLA)‐A*02 alleles (A*02:01, A*02:02, A*02:07, A*02:11) were generated. Each antibody was genetically linked to two IFNα molecules to produce TCR‐L/IFNα fusion proteins. We demonstrate that the fusion proteins triggered an IFNα response preferentially on the hepatocytes presenting the correct HBV‐peptide HLA‐complex and that the mechanism of the targeted IFNα response was dependent on the specific binding of the fusion proteins to the HLA/HBV peptide complexes through the TCR‐like variable regions of the antibodies. Conclusion: TCR‐L antibodies can be used to target cytokines to HBV‐infected hepatocytes in vitro. Fusion of IFNα to TCR‐L decreased the intrinsic biological activity of IFNα but preserved the overall specificity of the protein for the cognate HBV peptide/HLA complexes. This induction of an effective IFNα response selectively in HBV‐infected cells might have a therapeutic advantage in comparison to the currently used native or pegylated IFNα. (HEPATOLOGY 2012;56:2027–2038)

A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood

We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits) yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal.

Candidate Biomarkers of Response to an Experimental Cancer Drug Identified through a Large-scale RNA Interference Genetic Screen

Purpose: A major impediment in the optimal selection of cancer patients for the most effective therapy is the lack of suitable biomarkers that foretell the response of a patient to a given drug. In the present study, we have used large-scale RNA interference–based genetic screens to find candidate biomarkers of resistance to a new acyl sulfonamide derivative, R3200. This compound inhibits the proliferation of tumor cells in vitro and in vivo, but its mechanism of action is unknown. Experimental Design: We used a large-scale RNA interference genetic screen to identify modulators of the efficacy of R3200. We searched for genes whose suppression in an in vitro cell system could cause resistance to the anticancer effects of R3200. Results: We report here that knockdown of either RBX1 or DDB1 causes resistance to the anticancer effects of R3200, raising the possibility that these two genes may have utility as biomarkers of response to this drug in a clinical setting. Interestingly, both RBX1 and DDB1 are part of an E3 ubiquitin ligase complex. Conclusions: We propose that suppression of the activity of a RBX1 and DDB1-containing E3 ligase complex leads to the stabilization of certain proteins, the increased abundance of which is in turn responsible for resistance to R3200. Moreover, our data suggest that RBX1 and DDB1 could potentially be developed into biomarkers of resistance to acyl sulfonamide–based cancer drugs. This will require clinical validation in a series of patients treated with R3200. (Clin Cancer Res 2009;15(18):5811–9)

Potential of Protein-based Anti-metastatic Therapy with Serpins and Inter α-Trypsin Inhibitors

In this review we summarize the principles of anti-metastatic therapy with selected serpin family proteins, such as pigment epithelial-derived factor (PEDF) and maspin, as well as inter α-trypsin inhibitor (IαIs) light chains (bikunin) and heavy chains (ITIHs). Case-by-case, antimetastatic activity may be dependent or independent of the protease-inhibitory activity of the corresponding proteins. We discuss the incidence of target deregulation in different tumor entities, mechanisms of deregulation, context-dependent functional issues as well as in vitro and in vivo target validation studies with transfected tumor cells or recombinant protein as anti-metastatic agents. Finally, we comment on possible clinical evaluation of these proteins in adjuvant therapy.