Affinity maturation of antibodies: optimized methods to generate high-quality ScFv libraries and isolate IgG candidates by high-throughput screening.

As a growing number of therapeutic antibodies are developed, robust methods to efficiently improve the affinity and/or specificity of antibody candidates are needed. Here we describe our powerful platform that combines scFv affinity maturation and IgG high-throughput screening. After creating diversity with our random mutagenesis technology (MutaGen™), the scFv libraries are fully cleaned using a fusion system introducing the beta-lactamase gene to select in-frame and stop codon free variants on the basis of ampicillin resistance. The high-quality scFv libraries thereby constructed are then selected on the target in vitro using phage display technology. Contrary to standard procedures, instead of producing a limited number of affinity matured scFv as IgG molecules, we developed a cloning system to directly transfer the entire pool of selected scFv into an IgG expression vector permitting rapid IgG small-scale production (96 wells) in mammalian cells. Our integrated process allows us to generate high-quality scFv libraries and test numerous IgG variants, increasing the chances to select the best therapeutic antibody candidate.

A cytoplasmic protein-protein interaction detection method based on reporter translation.

One approach to drug discovery involves the targeting of abnormal protein-protein interactions that lead to pathology. We present a new technology allowing the detection of such interactions within the cytoplasm in a yeast-based system. The interaction detection is based on the sequestration of a translation termination factor involved in stop codon recognition. This sequestration inhibits the activity of the factor, thereby permitting the translation of a reporter gene harboring a premature stop codon. This novel cytoplasmic protein-protein interaction (CPPI) detection system should prove to be useful in the characterization of proteins as well as in partner identification, interaction mapping, and drug discovery applications.

Improved gene delivery to B lymphocytes using a modified adenovirus vector targeting CD21.

Gene transfer by adenoviruses, which are widely used for gene therapy, may provide an alternative approach to treatment of several hematopoietic malignancies. However, a major limitation of adenovirus 5-based gene therapy lies in the natural tropism of the virus for the widely expressed hCAR receptor. The efficacy of adenoviral vectors could be improved if viral vectors that exhibit tissue-specific gene delivery were developed. For efficient gene transfer it is essential that every step from binding of virus to target cells to transgene expression is successfully accomplished. We developed a specific vector targeting the CD21 receptor, by inserting a CD21 binding sequence, derived from the EBV GP350/220 protein, into the HI loop of the HAdV5 fiber protein. This vector, HAdV5-CD21HIloop, binds specifically to CD21-positive cells and results in enhanced expression of the transgene in these cells and reduced expression in CD21-negative cells. Viral infection is highly correlated with the presence of CD21 receptors. Taken together, these results demonstrate that HAdV5-CD21HIloop is able to transduce CD21-positive cells specifically with reduced infection of nontarget cells. This is the result of the maintenance of the intracellular trafficking of the genetically modified adenovirus without vesicular retention, leading to enhanced nuclear transfer.

Abolition of hCAR-dependent cell tropism using fiber knobs of Atadenovirus serotypes.

Most adenoviral vectors use in gene therapy protocols derive from species C. However, expression of the primary receptor (human Coxsackievirus and Adenovirus receptor, hCAR) for these AdV is variable on cancer cells. In vivo targeting of a therapeutic gene to specific cells has then become a major issue in gene therapy. The Ad fiber protein largely determines viral tropism through interaction with specific receptors. Hereto, we constructed a set of HAdV5 vectors carrying chimeric fibers with knob domains from nonhuman AdV, namely from the FAdV-1 (Aviadenovirus), DAdV-1, and BAdV-4 (Atadenovirus). Correspondents viruses were produced using an established new HEK293 cell line, which express the HAdV2 fiber. Recombinant HAdV harboring chimeric fibers constituted of the N-terminal domain of HAdV2, and knob domain of bovine adenovirus type 4 (BAdV-4) demonstrated the greatest reduction in fiber-mediated gene transfer into human cells expressing the hCAR. Moreover, this vector infects with a better efficiency than vector with wild-type fiber, the Chinese Hamster Ovarian (CHO) and SKOV3 cell lines, both from ovarian origin, hamster and human, respectively. These studies support the concept that changing the fiber knob domain to ablate hCAR interaction should result in a de- or retargeted adenoviral vector. The adenoviral vector with the chimeric HAdV2/BAdV-4 fiber lacking hCAR interaction and with an ovarian cell tropism could be a nice candidate to elaborate vectors for ovarian tumor therapy.

Factors involved in the sensitivity of different hematopoietic cell lines to infection by subgroup C adenovirus: implication for gene therapy of human lymphocytic malignancies.

Gene transfer approaches using viruses such human adenovirus (HAdV) may provide an alternative treatment for diseases involving hematopoietic cells. Better understanding of the cellular mechanisms by which the HAdV introduces DNA into these cells should help in vector design. We examined HAdV intracellular delivery in several cell lines including B and T lymphocytes. We demonstrated that HAdV resistance in most B lymphocytes is the result of moderate HAdV uptake. In contrast, high levels of coxsackie and HAdV receptor (hCAR) are expressed on the surface of HSB2 (T cells), allowing efficient binding and uptake but no transgene expression, probably because of deficient endosomolysis and subsequent exocytose. This work demonstrates the existence of hCAR-dependent and -independent endocytic route in hematopoietic cells. Moreover, it precises the intracellular barriers to be overcome by HAdV in such cells to be infectious and gives previous information's to design new vectors for gene transfer.

A rapid and easy method for production and selection of recombinant adenovirus genomes.

Adenoviruses are used widely as vectors for gene therapy. Due to the large size of their genome there is a low frequency of unique restriction sites and many techniques have been described to construct recombinant viruses. Whatever the considered technique, the Escherichia coli strain BJ5183 is used to obtain recombinant adenovirus genomes in a plasmid, or to construct defective viral backbones which will be used to produce infectious viral particles by homologous recombination in HEK293 cells. Unfortunately BJ5183 bacteria do not produce a sufficient amount of plasmid DNA to allow for restriction analysis. Plasmids have to be transferred into another strain to detect the expected construction. It is reported now that the common E. coli strain, Top10F' can be used for the construction of recombinant adenovirus genomes. A plasmid carrying a kanamycin resistance gene and containing the two ends of the adenovirus genome was used. It permits modification by classical molecular biology techniques or homologous recombination at both ends of the genome. The remainder of the genome is introduced by homologous recombination in Top10F'. Several homologous recombination steps were successfully performed without the steps of extraction and introduction of plasmid DNA in another strain to check the plasmids obtained.