Production of monoclonal antibodies and their co-crystallization with intrinsically disordered protein tau

Adam Polák1, Stefana Njemoga1, Klaudia Mešková1, Michaela Škrabanová1, Katarína Tomková1, Ondrej Cehlár1, Ľubica Fialová1,2, Rostislav Škrabana1

 

1Institute of Neuroimmunology, Slovak Academy of Sciences; Bratislava, 845 10, Slovakia

2AXON Neuroscience R&D Services SE; Bratislava, 811 02, Slovakia

adam.polak@savba.sk

The lack of therapeutics to treat Alzheimer’s disease (AD) and its increasing worldwide prevalence are showcasing  the shortage of insight into the pathogenesis of the disease (Alzheimer’s Association report, 2023). Tau protein is responsible for the formation of insoluble neurofibrillary tangles, what is one of the main driver of the pathogenesis in AD. The study of its structure is limited by the fact that tau protein belongs to the family of intrinsically disordered proteins (IDPs). Moreover, tau protein is disordered almost in its full length and its conformation is depending on the binding with its binding partners.

Crystallization of proteins is one of the standard methods to obtain information about the structure of proteins. However, crystallization of IDPs is often unachievable with the standard methods and requires alternative approaches. Therefore, we utilise co-crystallization of tau protein with monoclonal antibodies, specifically its Fab (fragment antigen-binding) (fig. 1) (Skrabana et al., 2012, Sevcik et al., 2007). Monoclonal antibodies MN423 and DC11 recognize AD conformation of tau protein, therefore, their complexes with tau may enable to discover epitopes and structural motifs important for tau aggregation in tangles (Skrabana et al., 2012).

Main condition for successful crystallization is the production of Fabs in high quality as well as quantity. We developed CHO cells protein expression system consisting in universal cloning vector pCMV-3UTR and optimised polyethyleneimine DNA transfection, yielding up to 100 mg proteins per litre of growth medium (Meskova et al., 2023). To optimise the production yield of Fabs even further, we investigated the efficiency of production in different densities of cell cultures. Our results show that high-density cells are potentially more effective (fig. 2). We also performed transient Fab expression from pCMV-3UTR in  HEK293(EBNA) cells. HEK cells are human cells and thus proteins produced in HEK cells are the most comparable to human, most importantly mimicking human post-translation modifications.

We were able to obtain crystals of tau-Fab protein complexes with antibodies to discover tau structural motifs, presumably important in tau aggregation (fig. 1). Results obtained from structural analysis of tau protein will be applicable in the design of AD targeting therapeutics as well as for the development of AD-specific vaccine, both leading to improved management of the disease.

 

 

Figure 1. Co-crystallization of intrinsically disordered protein tau with Fabs: B) protein crystals of DC11, C) protein crystals of MN423Fab and dGAE tau isoform (complex), D) protein crystals of DC11Fab, DC25Fab and dGAE tau isoform (complex) (Meskova et al., 2023)

Figure 2. Comparison of production of monoclonal antibodies in CHO cells in high density (20x106 cells per ml of culture) and low density (5x106 cells per ml of culture) cell cultures.

1.         2023 Alzheimer's disease facts and figures. Alzheimers Dement. 2023 Apr;19(4):1598-1695. doi: 10.1002/alz.13016. Epub 2023 Mar 14. PMID: 36918389.

2.         Skrabana R, Cehlar O, Flachbartova Z, Kovac A, Sevcik J, Novak M. Crystallization and preliminary X-ray diffraction analysis of two peptides from Alzheimer PHF in complex with the MN423 antibody Fab fragment. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Oct 1;68(Pt 10):1186-90. doi: 10.1107/S1744309112033477. Epub 2012 Sep 25. PMID: 23027744; PMCID: PMC3497976.

3.         Sevcik J, Skrabana R, Dvorsky R, Csokova N, Iqbal K, Novak M. X-ray structure of the PHF core C-terminus: insight into the folding of the intrinsically disordered protein tau in Alzheimer's disease. FEBS Lett. 2007 Dec 22;581(30):5872-8. doi: 10.1016/j.febslet.2007.11.067. Epub 2007 Dec 3. PMID: 18061582.

4.         Meskova K, Martonova K, Hrasnova P, Sinska K, Skrabanova M, Fialova L, Njemoga S, Cehlar O, Parmar O, Kolenko P, Pevala V, Skrabana R. Cost-Effective Protein Production in CHO Cells Following Polyethylenimine-Mediated Gene Delivery Showcased by the Production and Crystallization of Antibody Fabs. Antibodies (Basel). 2023 Aug 4;12(3):51. doi: 10.3390/antib12030051. PMID: 37606435; PMCID: PMC10443350.

 

This project is supported by APVV-21-0479, VEGA 2/0141/23, 2/0125/23 and EU Grants ADDIT-CE, InterTau.