Page 139 - ebook

Evaluation of novel anti-human CD133 human antibody developed
using phage display in human synthetic scFv library

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Sang-In Park , Sungjin Lee , Do-Hyung Kim , Hae Li Ko , Deuk-ki Lee , Younghyeon Kim , Seok-Ho Go , Jin Won Park 1,*,**
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1.Division of Research Program, Scripps Korea Antibody Institute, Chuncheon-si, Gangwon-do, 24341, Republic of Korea
2.Department of Systems Immunology, College of Biomedical science, Kangwon National University, Chuncheon 24341, Republic of Korea
ABSTRACT RESULTS (cont.) RESULTS (cont.)
CD133 is colon cancer stem cell surface marker and highly [ Polyclonal ELISA ] [ IgG Flow Cytometry ]
expressed in refractory colorectal cancers. We developed
novel anti-human CD133 antibody using phage display
technics in human synthetic scFv library. In this study, novel
CD133 human antibody evaluated recombinant CD133 antigen
and live cell surface binding affinity.
Antibody screening were performed 3 times by phage
display using as antigen recombinant human CD133 produced
from 293 cell. A scFv was converted to IgG form through gene
cloning. Antibody binding affinities to CD133 were analyzed by • In polyclonal ELISA, the 3 rd order optical density at 340 nm
ELISA, flow cytometry. of CD133/BSA ratio compared to 1 st order increased by 28
times from 0.8 to 22.4.
CD133 antibody screenings were performed three times
with phage display, and the final titers were 1.08x108 CFU/ml.
Each 400 colonies picked up and confirming by ELISA. We [ Monoclonal ELISA ] • Six lead antibodies confirmed cell surface CD133 binding to
selected 15 effective scFv antibodies with individual the HT29 cell line. Among them, antibodies #1, #4, and #12
sequences, among them, picked 6 lead scFv antibodies with showed high binding affinity through FACS.
flow cytometry analysis. After human IgG form conversions,
three CD133 antibodies were showed high affinities to HT29
cell line high CD133 expressed cell line on flow cytometry [ Confocal Microscope Imaging ]
analysis.
We developed three novel anti-human CD133 human
antibody that showed high affinity on high CD133 expressed
cell line.

MATERIALS AND METHODS
• Representatively, #1 antibody was showed binding of cell
▪ Materials surface CD133 on HT29 cell line through PE fluorescence
• Synthetic human scFv library was observed only on the cell surface in confocal image.
• Recombinant human CD133
• HT29 human colon cancer cell line
▪ Phage display [ Cytotoxicity ]
• Antibody screening repeat 3times
• 10ug/ml recombinant protein coated on epoxy bead • Four hundred of the colonies from the 3rd output were picked
▪ ELISA and monoclonal ELISA was performed.
• 30ng recombinant protein coated on immune plate for
O/N • As a result of DNA sequencing by selecting colonies with OD
• Primary antibody 1:1000 at 340 nm of CD133/BSA ratio greater than 20 times, 15
• Secondary antibody 1:5000 independent sequences were obtained.
• Compared of CD133 to BSA ratio at OD 340nm
▪ IgG conversion
• One vector system (pdCMV based) [ scFv Flow Cytometry ] • When 6-lead antibody was dose-dependently treated with
• Antibody production on ExpiCHO cell #1 #3 #4 #7 #8 HT29 cell line, cytotoxicity was not observed for 72 hours.
• Purification using protein A bead
▪ Flow cytometry analysis
• HT29 human colon cell line using CD133 high FUTURE PLAN
expression cells #11 #12 #15 #16 #18
Six lead antibodies showed non-cytotoxicity in single
RESULTS treatment. However, we confirmed that high affinity on cell
#21 #33 #41 #44 #45 surface CD133 binding of HT29 human colon cancer cell
[ Page Display ] line that high expressed CD133. These antibodies will
develop cancer therapeutic antibody using ADCC or ADC
• We selected 6 lead scFv colonies fallowing flow cytometry target to CD133-expressing cancer cells or cancer stem
analysis on CD133 high expressed HT29 cell line. cells specific drug.

[ Human IgG conversion ] CONCLUSION
We developed three novel anti-human CD133 human
antibody that showed high affinity on high CD133
expressed cell line.
• The phage display for cd133 was repeated three times by
coating 1ug/ml of recombinant CD133 on epoxy beads FOUNDING
using synthetic human scFv library.
• In the phage display, all input titers were fixed to 4*10 12 • After 6 scFv colonies were converted to IgG form, antibodies This work was supported by grant (NRF-2021R1F1A1060734)
cfu/ml, and the output titers were 1.56*10 8 cfu/ml in the 1st were produced in expiCHO cells and purified. IgG conversion from the Basic Science Research Program through the
order, 2.03*10 7 cfu/ml in the 2 nd order, and 1.08*10 8 cfu/ml and antibody production were confirmed by reducing or non- National Research Foundation of Korea (NRF), the Ministry of
in the 3rd order. reducing PAGE. Science, Republic of Korea