Insect cell surface display libraries for selection of integrin-binding human antibodies

Background.

Monoclonal antibodies (mAbs) are one of the major therapeutics for a great variety of human diseases. The most prevalent applications are treatment of cancer and immunological diseases. Often, display libraries based on yeast or bacterial phage expression are used for the isolation of antibodies with desired specificities. These systems are highly suitable for the generation of large and diverse libraries. However, microbial systems only allow for the expression of antibody fragments instead of whole antibodies. Isolated fragments are then redesigned to reconstitute a whole antibody molecule required for therapy. This step is often crucial, since subsequent structural changes may lead to a loss in affinity and/or specificity.

So far, a suitable animal cell based display library platform does not exist. Baculoviruses are insect viruses and are widely used for the expression of glycosylated proteins. Fusion of a target protein to the viral coat protein leads to efficient surface display of the target protein on virions as well as on the surface of infected insect cells (Ernst et al., 1998; Grabherr & Ernst 2002; Oker-Blom et al., 2003; Maekelae et al., 2006). Selection of binders can be done by cell sorting via FACS (Ernst et al., 1998; Ojala et al., 2004). In terms of glycosylation, insect cells differ from mammalian cell systems. We found evidence that insect cell derived products imply a higher risk of causing allergies, when given as a therapeutic agent (Palmberger et al., 2014). Therefore, it is necessary to introduce glyco-engineering strategies. Selected glycosyltranferases will be co-expressed or down-regulated in order to mimic human glycosylation (Palmberger et al., 2012a). Thereby, baculovirus based protein expression serves to isolate as well as to produce human-like, full-length antibodies.

Aims and methods.

The goal of this project is to establish insect cell display libraries for selection of specific human antibodies. In combination with appropriate glyco-engineering, this cell system is able to produce correctly processed and stable antibodies (Palmberger et al., 2012b). The first scientific objective is the establishment of adequate and efficient protein surface display conditions using a model antibody (human HIV gp41 antibody 3D6). For providing a free N-terminus the baculovirus envelope protein gp64 has been shown to be a suitable fusion partner (Grabherr & Ernst 2002). For proper glycosylation the enzymes N-acetylglucosaminyltransferase II and 1,4-galactosyltransferase I will be introduced, and the baculovirus fucosyltransferase will be supressed by anti-sense RNA expression.

The glycan structure of the thus produced proteins will be analysed by mass spectrometry (WILSON). The second objective is to generate antibody surface display libraries, which will be done in collaboration with the CD-Laboratory for Antibody Engineering (OBINGER), providing a pool of human antibody heavy and light chain genes. Libraries will be selected by successive rounds of FACS sorting (BORTH) using avb3 integrin as a model target and clones binding to this integrin will be identified. Specificity of binding as well as binding to effector molecules (e.g. CD64) will be evaluated.

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