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Display of a Full Length IgG Antibody on the Surface of Escherichia coli: Towards the Screening of an Antibody Library
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GRAPHICAL ABSTRACT
Randomized CDR3
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 - - 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,
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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
- n 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
- f 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 105 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
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ABSTRACT
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Discovery of new monoclonal antibody variants
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Development
- Animal immunization in
combination with hybridoma technology
- Phage display
(State of the art)
- Ribosome display
- Yeast display
- …
washing antibody fragment library displayed
- n phages
elution Amplification in E. coli new screening cycle binding directed evolution
Possible discrimination of the most potent binders due to mild elution conditions Size restriction for the peptide displayed on the surface PROBLEM PROBLEM SOLUTION Surface presentation on E. coli
INTRODUCTION
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Presentation of antibodies on the surface of E. coli via autodisplay
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Light chain Heavy chain
Signal peptide Passenger Linker ß-Barrel
A
A: Structure
- f
the autotransporter fusion precursor protein. One plasmid includes the gene for the fusion protein with the antibody’s heavy chain as passenger, another the light chain. B: Mechanism of translocation. Due to the mobility of the ß-barrel in the outer membrane, the heavy and the light chain are able to find each other, when co-expressed in
- ne cell.
INTRODUCTION
Encoding the heavy chain Encoding the light chain
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Proof of surface display by outer membrane preparation
6 200 150 120 100 85 70 60 50 40 30 Heavy chain Light chain kDa M 1 2 3 4 5 6 7 Prot K -
- + -
+ - +
200 150 120 100 85 70 60 50 40 30 Prot K -
- + -
+
- +
kDa M 1 2 3 4 5 6 7 Heavy chain Light chain
AIDA-1 autotransporter, T7 promoter EhaA autotransporter, araBAD promoter
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).
X
Prot K
Principle: Protease accessibility test
RESULTS AND DISCUSSION:
- 1. ANTI-CEA ANTIBODY AS PROOF OF PRINCIPLE
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Cells presenting an unrelated protein Cells presenting anti-CEA antibody, AIDA-1, T7 promoter Cells presenting anti-CEA antibody, EhaA, araBAD promoter Induction: 1h 30 °C Induction: 1h 30 °C + storage
- ver night
at 4 °C Induction: 22 h 23 °C
Principle: Staining for flow cytometric detection
RESULTS AND DISCUSSION:
- 1. ANTI-CEA ANTIBODY AS PROOF OF PRINCIPLE
Proof of surface display via flow cytometry
Comparison of different induction conditions
Fluorescence intensity Fluorescence intensity Fluorescence intensity
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0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
1 h, 30 °C 1h, 30 °C, storage over night 20h, 23 °C 1 h, 30 °C 1h, 30 °C, storage over night 20h, 23 °C
cell survival AIDA-1, T7 promoter EhaA, araBAD promoter
RESULTS AND DISCUSSION:
- 1. ANTI-CEA ANTIBODY AS PROOF OF PRINCIPLE
Cell survival after sorting by flow cytometry
Comparison of different induction conditions
Principle: 100 clones presenting the anti-CEA antibody on its surface were sorted on an agar plate and incubated
- ver night at 37 °C.
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Generation of an heavy chain antibody library
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Autodisplay- Vector for heavy chain
Induction
Library size: >105 Cells Sequencing: > 80% successful randomization
Ligation Electroporation
5‘ 3‘ 5‘ 5‘ 3‘
Randomized CDR3
3‘ 5‘
Klenow fragment
Restriction
RESULTS AND DISCUSSION:
- 2. ANTIBODY LIBRARY
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Library screening via flow cytometry
FITC Dylight
FSC P2 P1 Red Fluorescence Green Fluorescence FSC
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.
RESULTS AND DISCUSSION:
- 2. ANTIBODY LIBRARY
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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.
CONCLUSION
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