EuroMAbNet: European Monoclonal Antibodies Network

Members

Egon Ogris

Name of the laboratory

Members of the laboratory

Scientific Supervisor

Univ.-Prof. Dr. Egon Ogris egon.ogris@meduniwien.ac.at

Facility Head

Priv.-Doz. Dr. Stefan Schüchner stefan.schuechner@meduniwien.ac.at

Technicians

Alexander Stadler, BSc monoclonals@maxperutzlabs.ac.at

David Drescher, MSc amonoclonals@maxperutzlabs.ac.at

Patricia Odermatt; MSc monoclonals@maxperutzlabs.ac.at

Laboratory activity

Monoclonal antibodies are indispensable tools in basic life science research but have also transformed the shape of modern medicine allowing the precise treatment of many medical conditions from cancer to autoimmune diseases, neurodegenerative diseases, allergies or viral infections. While in vitro discovery methods like phage or yeast display together with improved library construction technologies as well as single B-cell antibody isolation methods have expanded the toolkit repertoire for monoclonal antibody discovery, the hybridoma fusion technology following the original approach by Koehler and Milstein still represents one of the most frequently used methods for the generation of research monoclonal antibodies.

The Max Perutz Labs Monoclonal Antibody facility provides researchers at the Vienna BioCenter with a service for tailor-made mouse monoclonal antibodies that is also open for scientists from other academic institutes as well as non-academic commercial users. Our service starts with the design of the immunogens based on combined protein sequence, structure and post-transcriptional modification information. We perform western blot screening of immune sera with wild-type and knock-out cell lysates to ensure faithful identification of antibodies that specifically recognize the full-length target protein in the context of the whole cellular proteome. Additional ELISA screening is performed when required, e.g., for post-translational modification specific antibodies. Our emphasis on comprehensive validation in a variety of techniques, including immunocytochemistry, immunoprecipitation, or flow cytometry and performed in close collaboration with our customers ensures optimal selection of mice for splenocyte fusion as well as identification of the best clones for monoclonalization.

Besides our service for custom tailored antibodies, we have also generated numerous monoclonal antibodies for the general scientific community, amongst them several CRISPR/Cas9 monoclonal antibodies including the signature SpCas9 antibody, clone 7A9-3A3, epitope tag specific antibodies, e.g., myc-tag clone 4A6, or T2A/P2A-tag clone 3H4, as well as point-mutant/variant specific monoclonal antibodies like progerin clone 13A4, or ApoE4 clone 4E4.

Research Activities

Protein phosphatase 2A

Our research focuses on the regulation of protein phosphatase 2A (PP2A) biogenesis and its role in cellular signaling. PP2A represents a family of trimeric enzymes that are assembled from a large pool of distinct yet often homologous subunits. Holoenzyme assembly is therefore a tightly controlled process, orchestrated by PP2A chaperones and post-translational modifications to ensure precise substrate specificity. Over the last years, we have generated a portfolio of PP2A subunit/regulator/PTM-specific monoclonal antibodies that are key to study the molecular mechanisms of holoenzyme activation and regulation. A particularly important modification for the assembly and activation of certain tumor-suppressive PP2A holoenzymes is the carboxy-methylesterification of the C-terminal leucine within the absolutely conserved TPDYFL sequence of the PP2A catalytic (PP2Ac) subunit. PP2A and two related phosphatases, PP4 and PP6, share the YFL motif and all three are methylated by the same methyltransferase, LCMT1. Our unique, thoroughly validated PP2A/PP4/PP6 antibodies are specific either for the methylated or the non-methylated state of each phosphatase, and allow the precise analysis of this crucial PTM for the regulation of each of the three phosphatases. Furthermore, we have shown that carboxy-methylated PP2Ac can serve as a novel marker for metastatic prostate cancer detected by our methyl-PP2Ac specific clone 7C10-C5 (Rasool et al., 2023).

Antibody validation

A second and related focus of our lab is on antibody validation. In a community-service effort we extensively validated a series of commercially available PP2A and epitope tag specific antibodies. Antibodies that were advertised as being specific for phosphorylated tyrosine 307 of PP2Ac did not recognize their intended target but were hampered by carboxy-terminal methylation instead, suggesting that a model of PP2A inactivation in cancer cells by Y307 phosphorylation needs to be revisited (Frohner et al., 2020a). In a second publication, we showed that many commercial PP2Ac antibodies that had been raised against C-terminal peptides preferentially detect non-modified PP2Ac (Frohner et al., 2020b). Thus, these antibodies have a bias toward certain PP2A trimeric complexes and should therefore not be used to assess general PP2A phosphatase activity and holoenzyme composition. Finally, our analysis of myc-tag specific antibodies revealed that several of the tested antibodies, including the most cited clone, 9E10, show severe context-sensitivity for myc-tag recognition (Schüchner et al., 2020), highlighting the need for continuing antibody validation efforts by academic researchers and commercial antibody providers alike.

Publications (2021-present)

• Rasool RU, O'Connor CM, Das CK, Alhusayan M, Verma BK, Islam S, Frohner IE, Deng Q, Mitchell-Velasquez E, Sangodkar J, Ahmed A, Linauer S, Mudrak I, Rainey J, Zawacki KP, Suhan TK, Callahan CG, Rebernick R, Natesan R, Siddiqui J, Sauter G, Thomas D, Wang S, Taylor DJ, Simon R, Cieslik M, Chinnaiyan AM, Busino L, Ogris E, Narla G, Asangani IA. Loss of LCMT1 and biased protein phosphatase 2A heterotrimerization drive prostate cancer progression and therapy resistance. Nat Commun. 2023 Aug 29;14(1):5253. doi: 10.1038/s41467-023-40760-6. PMID: 37644036.
• Balay SD, Hochstoeger T, Vilceanu A, Malkemper EP, Snider W, Dürnberger G, Mechtler K, Schuechner S, Ogris E, Nordmann GC, Ushakova L, Nimpf S, Keays DA. The expression, localisation and interactome of pigeon CRY2. Sci Rep. 2021 Oct 13;11(1):20293. doi: 10.1038/s41598-021-99207-x. PMID: 34645873.