Functional stability of a nucleic acid-hydrolyzing single chain antibody in various biochemical and biophysical environments 2015.05.27 Joungmin Lee

Antibody is… Nature Reviews Clinical Oncology Volume: 11, Pages:637–648 Year published:(2014)

Single chain variable fragment (scFv) scFv is recombinant antibody fragments, which commonly consist of a full variable (epitope-binding) region of an immunoglobulin heavy chain covalently linked to the corresponding variable region of an immunoglobulin light chain. scFv has multiple benefits over traditional monoclonal antibodies due to their greatly reduced size and ease of genetic manipulation. scFv form allow these molecules to penetrate more rapidly and evenly to tumors and other tissue in comparison to the whole antibodies. scFv can be produced rapidly and inexpensively in microorganisms, particularly E.coli.

Features of 3D8 scFv antibody Cell penetration Nucleic acid binding Nucleic acid hydrolysis Antiviral activity Cell Mol Life Sci. 2009 Jun;66(11-12):1985-97 J Biol Chem. 2006 Jun 2;281(22):15287-95 J Biol Chem. 2006 Jun 2;281(22):15287-95 Mol Biotechnol. 2015 Jun;57(6):506-12 Biochem Biophys Res Commun. 2010 May 14;395(4):484-9 Pharm Res. 2012 Apr;29(4):932-42 Antiviral Res. 2012 May;94(2):157-67 PLoS Pathog. 2014 Jun 26;10(6):e1004208 Appl Microbiol Biotechnol. 2015 Mar;99(6):2793-803

Abstract The protein 3D8 single-chain variable fragment (3D8 scFv) has potential anti-viral activity due to its ability to penetrate into cells and hydrolyze nucleic acids. Probiotic Lactobacillus paracasei engineered to secrete 3D8 scFv for oral administration was used to test the anti-viral effects of 3D8 scFv against gastrointestinal virus infections. We found that injection of 3D8 scFv into the intestinal lumen resulted in the penetration of 3D8 scFv into the intestinal villi and lamina propria. 3D8 scFv secreted from engineered L. paracasei retained its cell-penetrating and nucleic acid-hydrolyzing activities, which were previously shown with 3D8 scFv expressed in Escherichia coli. Pretreatment of RAW264.7 cells with 3D8 scFv purified from L. paracasei prevented apoptosis induction by murine norovirus infection and decreased messenger RNA (mRNA) expression of the viral capsid protein VP1. In a mouse model, oral administration of the engineered L. paracasei prior to murine norovirus infection reduced the expression level of mRNA encoding viral polymerase. Taken together, these results suggest that L. paracasei secreting 3D8 scFv provides a basis for the development of ingestible anti-viral probiotics active against gastrointestinal viral infection.

Purpose Observation of functional stability of 3D8 scFv by assessing DNA-hydrolyzing and –binding activity in various physical and chemical conditions in order to provide the basis for the formulation of 3D8 scFv to develop it as the anti-viral agent.

Figure 1. Confirmation of DNA-binding, -hydrolyzing activity depending on temperature Figure 2. Confirmation of DNA-binding, -hydrolyzing activity depending on pH A DNase I 3 4 5 6 7 8 9 10 11 12 pH B

Figure 3. Resistance of DNA-binding, -hydrolyzing activity against reducing condition Figure 4. Confirmation of durability against freezing-thawing and lyophilization A B Cycle lyophilized C 1 5 10 20 30 C

Figure 5. Observation of DNA-hydrolyzing activity of 3D8 scFv when pH condition has returned to pH 7 after incubation for 2 h, 12 h, 3 day, 1 week in each buffer condition A pH B 3 6 10 C

Figure 6. Observation of DNA-hydrolyzing activity of 3D8 scFv incubated for a 1 month at 37℃ and 23℃ (pH 7) A Control 1 week 2 week B

A B Figure 7. Determination of Unit 3.9 7.8 15.6 31.3 62.5 125 250 nM 1 U DNase I ⇒ 1U≒2μg ** 1 unit : the amount of 3D8 scFv required to produce a complete digest of 400 ng of linear DNA for 3 hr at 37°C

A B C Figure 8. Antiviral activity of 3D8 scFv depending on Unit 24 h Treatment H9N2 Infection (MOI 1) Harvest B C Mock Influenza A Influenza A + 3D8 scFv (×40)