Display of a Full Length IgG Antibody on the Surface of Escherichia - PowerPoint PPT Presentation

Display of a Full Length IgG Antibody on the Surface of Escherichia coli : Towards the Screening of an Antibody Library Carina Dilkaute 1, *, Wilhelmine Weckenbrock 1 and Joachim Jose 1 1 Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westfälische Wilhelms-Universität, Corrensstraße 48, 48151 Münster * Corresponding author: carina.dilkaute@uni-muenster.de 1

GRAPHICAL ABSTRACT Display of a Full Length IgG Antibody on the Surface of Escherichia coli : Towards the Screening of an Antibody Library Randomized CDR3 2

ABSTRACT Phage display is an often used technique to identify new antibody variants. Nevertheless, it is associated with some drawbacks as the possible discrimination of the most potent binders during biopanning, the incompatibility with flow cytometry or the size limitation of the protein displayed on the surface [1]. To circumvent these disadvantages, we presented a full-length antibody on the surface of Escherichia coli using the autodisplay technique [2,3]. As a proof of principle, the display of antibody T84.66, which is directed against carcinoembryonic antigen (CEA), was investigated. Based on this antibody a library was generated using a ligation-restriction strategy. The resulting library consists of up to 10 5 clones which could be analyzed via flow cytometry after incubation with a fluorescently labelled target protein. To examine the optimal conditions for the screening, two different autotransporters in combination with two promoters were investigated: the AIDA-1 autotransporter [2] under control of a T7 promoter and the EhaA-autotransporter [3] controlled by an araBAD promoter. Experiments with the T84.66 antibody revealed that the EhaA-araBAD combination suited better with regard to surface presentation and cell survival after sorting. These results indicate that it is possible to generate a full-length antibody library on the surface of E. coli. This library can be screened with the advantageous high-throughput screening system of flow cytometry. Keywords: antibody library; autodisplay; full-length antibody; surface display References: [1] Levin A. M., Weiss G.A.: Mol. BioSyst 2006 , 2: 49-57 [2] Jose J., Meyer T.F.: Microbiol. Mol. Biol. Rev., 2007, 71(4): 600-619 [3] Sichwart S. et al.: Food Technol. Biotechnol . 2015 , 53(3): 251-260 3

INTRODUCTION Discovery of new monoclonal antibody variants Development washing PROBLEM • Animal immunization in Possible combination with discrimination of the hybridoma technology most potent binders elution binding due to mild elution • Phage display conditions new screening (State of the art) cycle • Ribosome display Amplification in E. coli • Yeast display antibody fragment library displayed directed on phages evolution • … Size restriction for the peptide displayed on the SOLUTION surface Surface presentation on E. coli PROBLEM 4

INTRODUCTION Presentation of antibodies on the surface of E. coli via autodisplay A Light chain Heavy chain Signal Passenger Linker ß-Barrel peptide Encoding the Encoding the heavy chain light chain A: Structure of the autotransporter fusion B: Mechanism of translocation. Due to the mobility of the precursor protein. One plasmid includes the gene ß-barrel in the outer membrane, the heavy and the light for the fusion protein with the antibody’s heavy chain are able to find each other, when co-expressed in chain as passenger, another the light chain . one cell. 5

RESULTS AND DISCUSSION: 1. ANTI-CEA ANTIBODY AS PROOF OF PRINCIPLE Proof of surface display by outer membrane preparation AIDA-1 autotransporter, T7 promoter EhaA autotransporter, araBAD promoter kDa M 1 2 3 4 5 6 7 kDa M 1 2 3 4 5 6 7 200 200 150 150 120 120 Heavy chain 100 Heavy chain 100 85 Light chain 85 Light chain 70 70 60 Prot K 60 50 50 X 40 40 30 Principle: Protease 30 accessibility test Prot K - Prot K - - + - + - + - + - + - + SDS-PAGE analysis of outer membrane fractions from E. coli UT5600(DE3) cells without plasmid (1), from cells displaying the heavy chain (2,3), the light chain (4,5) or both chains (6,7) of the anti-CEA antibody. The exposure at the surface was confirmed by digestion with proteinase K, which cannot enter the cell (3,5,7). 6

RESULTS AND DISCUSSION: 1. ANTI-CEA ANTIBODY AS PROOF OF PRINCIPLE Proof of surface display via flow cytometry Comparison of different induction conditions Induction: Induction: Induction: 1h 1h 22 h 30 °C 30 °C 23 °C + storage over night at 4 °C Fluorescence intensity Fluorescence intensity Fluorescence intensity Cells presenting an unrelated protein Cells presenting anti-CEA antibody, AIDA-1, T7 promoter Principle: Cells presenting anti-CEA antibody, EhaA, araBAD promoter Staining for flow cytometric detection 7

RESULTS AND DISCUSSION: 1. ANTI-CEA ANTIBODY AS PROOF OF PRINCIPLE Cell survival after sorting by flow cytometry Comparison of different induction conditions 100% 90% Principle: 100 clones presenting the 80% anti-CEA antibody on its 70% surface were sorted on an cell survival agar plate and incubated 60% over night at 37 °C. 50% 40% 30% 20% 10% 0% 1 h, 30 °C 1h, 30 °C, 20h, 23 °C 1 h, 30 °C 1h, 30 °C, 20h, 23 °C storage over storage over night night AIDA-1, T7 promoter EhaA, araBAD promoter 8

RESULTS AND DISCUSSION: 2. ANTIBODY LIBRARY Generation of an heavy chain antibody library Randomized CDR3 3‘ Library size: >10 5 Cells 5‘ 5‘ Klenow fragment Sequencing: > 80% successful randomization 5‘ 3‘ 3‘ 5‘ Restriction Ligation Induction Electroporation Autodisplay- Vector for heavy chain 9

RESULTS AND DISCUSSION: 2. ANTIBODY LIBRARY Library screening via flow cytometry P1 P2 Dylight FSC FSC FITC Red Green Fluorescence Fluorescence The displayed heavy chain library was incubated with a Dylight633-labelled anti- human antibody and a FITC-labelled target protein. Afterwards the cells were analyzed by flow cytometry. Events with an increased fluorescence intensity for both dyes (Gate P1 and P2) should carry a target-binding antibody at their surface and were therefore sorted on an agar plate. 10

CONCLUSION A full-length antibody was displayed on the surface of E. coli. The combination of araBAD promoter and EhaA autotransporter was identified to be superior to an AIDA-1 autotransporter under control of a T7 promoter. To achieve the best combination of surface presentation and cell survival, an induction time of 22 hours at 23 °C was chosen. In further experiments, sorted variants from the library should be reanalyzed in order to identify new binding heavy chain variants. Furthermore, libraries consisting of heavy and light chain should be further investigated. 11

ACKNOWLEDGMENTS Thanks to Prof. Jose and all members of the working group. 12