1. GPCR over-expression cell lines
GPCRs are typically difficult to be expressed in cells and purified in active forms. Lacking good quality immunogens has led to very limited success in generating antibodies against GPCRs and remains a bottleneck for GPCR target validation research and monoclonal antibody based drug discovery. Over expression of GPCRs in yeast, insect cells, bacteria and wheat germ have all been explored. The drawback of these alternative systems is obvious: GPCRs proteins are not expressed at high level or folded well in those systems for lacking necessary co-receptors, membrane structures and post-translational modifications in mammalian cells, all of which required for protein stability, correct folding and appropriate function of GPCRs.

Multispan uses a proprietary technology to enable expression of GPCR proteins in mammalian cells at high levels. We have been able to obtain 0.4–2 x e6 GPCR molecules expressed per mammalian cell surface for multiple GPCRs in multiple cell lines. An example of CXCR4 expression in 293 human cell line is shown in Figure 1. High expressing cells can be further enriched and cloned with flow-cytometry and used as immunogens for antibody generation and membrane preparations for binding assays. The robustness of our GPCR expression technology has also made development of cell based assays for “difficult” GPCRs possible, such as Free Fatty Acid (FFA), Lysophospholipid (LPA and S1P), Prostanoid, Metabotropic Glutamate receptor families.

Figure 1. CXCR4 was cloned into Multispan proprietary expression vectors and transfected into human 293 cells. Expression of each GPCR was detected by FITC fluorescence-tagged antibody against the expression tag fused to the GPCR gene. High Geo Mean reflects high expression. Similar results have been observed in 3 independent experiments.

2. GPCR monoclonal antibodies
Peptides and protein fragments are commonly used as antigens for GPCR antibody generation and have yielded very little success. Since these antigens do not represent native forms of the GPCRs, any antibodies derived from these approaches tend to have high non-specificity and do not alter functionality of the GPCR targets.

Multispan takes the approach of using live mammalian cells highly expressing GPCRS to immunize animals directly which has yielded robust immune response specifically against GPCR immunogen in it native form (Figure 2). Our technology platform is ideally suited for generating antibodies that recognize natural conformations of epitopes with high affinity, specificity, and functional activity that pure protein and peptide-based antigen approach can otherwise can not achieve.

Figure 2: Mouse immune sera were collected after 3 immunizations with CXCR4 transfected Cf2 cells as immunogen. The polycloncal antibody was tested against CXCR4 transfected CHO cells with CHO parental cells as controls in ELISA assay. Two immune serum samples, showed clearly increased specific antibody against CXCR4 protein at 1:10,000 dilution.

3. GPCR assay development
Multispan has also developed a robust technology platform for generating recombinant cell lines selected for optimal functional read-out for high-throughput screening for small molecule compounds or antibody drug leads. We produce cell lines according to the small G-protein (Gs, Gq, Gi or G15/16) that a specific GPCR is coupled to upon ligand binding and a reporter assay system that is most suitable to obtain the best signal-to-noise ratio. We have so far engineered 160 cell-based assays for different GPCRs using Ca⁺⁺ and cAMP as second messenger readout or NFAT or CRE driven luciferase as reporter activity readout.