Selection of functional human antibodies from retroviral display libraries.

Antibody library technology represents a powerful tool for the discovery and design of antibodies with high affinity and specificity for their targets. To extend the technique to the expression and selection of antibody libraries in an eukaryotic environment, we provide here a proof of concept that retroviruses can be engineered for the display and selection of variable single-chain fragment (scFv) libraries. A retroviral library displaying the repertoire obtained after a single round of selection of a human synthetic scFv phage display library on laminin was generated. For selection, antigen-bound virus was efficiently recovered by an overlay with cells permissive for infection. This approach allowed more than 10(3)-fold enrichment of antigen binders in a single selection cycle. After three selection cycles, several scFvs were recovered showing similar laminin-binding activities but improved expression levels in mammalian cells as compared with a laminin-specific scFv selected by the conventional phage display approach. Thus, translational problems that occur when phage-selected antibodies have to be transferred onto mammalian expression systems to exert their therapeutic potential can be avoided by the use of retroviral display libraries.

Chronic gene delivery of interferon-inducible protein 10 through replication-competent retrovirus vectors suppresses tumor growth.

Sustained maintenance of therapeutic levels of angiostatic proteins in tumor tissues continues to represent a major challenge to antiangiogenesis therapy of cancer. In this study, we tested the hypothesis of utilizing gene transfer via replication-competent retroviral (RCR) vectors for chronic protein delivery. We now show that bioactive human interferon-inducible protein-10 (IP10) can be secreted from a variety of mammalian cells upon transduction with RCR vectors carrying the human IP10 gene. The production of IP10 from RCR-transduced cells could be maintained for at least three months in culture. The level and duration of IP10 expression in vivo was sufficient to inhibit growth of subcutaneous (s.c.) tumors as well as metastatic lesions in mice. This tumor inhibition was correlated to a marked reduction in tumor vascularization and mitotic activity. By conducting immunohistological studies, we have been able to show that IP10 vector-affected tumors evidenced elevated levels of IL-12p35 mRNA, with no sign of changes in the local inflammatory response, however, as determined by macrophage infiltration and the expression of proinflammatory cytokines. We are addressing the feasibility of using RCR vector-based gene therapy as a more convenient alternative tool to chronically deliver antiangiogenic proteins for cancer therapy.

Replicating retroviral vectors mediating continuous production and secretion of therapeutic gene products from cancer cells.

The successful application of cancer gene therapy has been hampered by the low efficiency of in vivo gene delivery by currently used replication-defective vectors. Accordingly, considerable efforts are now being directed toward development and use of vectors capable of replicating in cancer cells. However, for replicating retroviruses, insertion of additional reading frames into the viral genome often resulted in the generation of unstable viruses. Here, we report a novel concept for the generation of replication-competent murine leukemia virus (MLV) vectors capable of mediating the secretion of soluble therapeutic proteins from infected cells. As a proof of principle, we inserted transgene regions encoding either a single-chain variable region fragment (scFv), here, the laminin-specific L36-scFv, or the T-cell-specific 7A5-scFv, or the cytokine GM-CSF into the MLV envelope (env) gene after +1 codon of the envelope (Env) protein, followed by a sequence specifying a furin protease cleavage site. The resulting viruses, termed L36-furin-A, 7A5-furin-A and GMCSF-furin-Mo, respectively, infected a variety of human cell lines, including HMEC-1 (endothelial), A301 (lymphoid), MDA-MB231 and MDA-MB468 (breast cancer) and HT1080 (fibrosarcoma) cells. Western blot analysis of conditioned culture medium from HT1080 cells infected by replicating L36-furin A, as an example, revealed that more than 90% of the Env fusion protein molecules were indeed intracellularly cleaved. After 5 days of infection, up to 3-4 mug/ml of soluble L36-scFv accumulated in the supernatant of HT1080 cells. The eukaryotically produced L36-scFv and 7A5-scFv were able to recognize their native antigens with high avidity, as assessed by ELISA and flow cytometry. Furthermore, the replicating viruses were genetically stable for more than 12 cell passages. In conclusion, a new generation of replication-competent retroviral vectors capable of mediating long-term and efficient secretion of therapeutic proteins suitable for cancer therapy was generated.