Maturation, Selection and Production in CHO Cells

Antibody Generation System

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Antibody Production - Hybridoma Technique -

2

Disadvantages:

• laborious • time consuming • Murine formats • Humanization required

Chinese Hamster Ovary Cells

• Research • Diagnostic • Therapy

Fc

CH2

CH3

VL

CL CH1

VH

1. Immunization and Killing of mice

2. Cell fusion to Hybridoma cells and Selection

3. Cloning and reformatting for Production

Our Approach 1. 2. 3.

Diverse Antibodies Maturation

by Enzyme

3

Virus, magnetic beads and FACS for selection

Advantages:

• Animal Friendly • Fast • Up-Scaling • Diverse Formats • Human Antibodies

Antibody Formats

Camelid Antibody Human Antibody

Single-Chain Fragment Variable domain (scFv)

Single Domain Antibody, Nanobody

small antibody construct

scFv425

BBa_K929101

scFv anti-EGFR domain 3

4

Fc

CH2

CH3

VL

CL

CH1

VH

CH2

CH3 Fc

VHH VHH

Human IgG1 CH region

Anti-GFP

BBa_K929107

Nanobody anti-GFP/YFP

advanced antibody construct

Fc

Antibody Constructs

Small Antibody Construct

Anti-GFP

Fc

BBa_K929107

Signal TMD

transmembrane domain of B-cell receptor

scFv425

BBa_K929101

TMD TEV

Signal peptide transmembrane domain

of B-cell receptor

TEV protease cleavage site

5

Advanced Antibody Construct

Signal

Signal peptide

TEV

TEV protease cleavage site

Antibody Constructs

BBa_K929107

anti-GFP Nanobody

transmembrane domain of B-cell receptor

LoxP site LoxP site

Cre

Cre recombinase • allows specific recombination between two recognition sites

6

Favorite Biobrick

LoxP TMD LoxP TEV Signal Fc Anti-GFP

Antibody Constructs

scFv425 Signal

BBa_K929101

Small Antibody Construct

Transmembrane domain TEV

Advanced Antibody Construct

eYFP

Enhanced YFP ( BBa_E0030)

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Anti-GFP

Fc

BBa_K929107

Signal TMD TEV LoxP LoxP mCherry

mCherry

Antibody Results

Transient transfection of CHO cells with advanced antibody construct (BBa_K929107) Transient transfection with smaller antibody construct (BBa_K929101)

Stable transfection of CHO cells with advanced antibody construct (BBa_K929107)

Stable transfection with smaller antibody construct (BBa_K929101) both by application of Flp-In system

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Antibody Results

9

membrane localization of advanced antibody construct BBa_K929107

proof of functionality of Cre recombinase

Antibody

Cre

Mutation Module

• enzyme: AID (activation-induced cytidine deaminase) • deaminates cytidines in DNA U:G mismatches point mutations

cytidine

C A T A A T

uridine

AID

10

Mutation Module

Cytidine deaminase motif APOBEC-like C-terminal NES

enzyme: AID (activation-induced cytidine deaminase)

NLS

pSB1C3 Modified AID

11

Mutation Module Biobrick Design

Cytidine deaminase motif APOBEC-like C-terminal NES

enzyme: AID(activation-induced cytidine deaminase)

NLS eGFP

pSB1C3 Modified AID + eGFP

Favorite Biobrick

12 nuclear localization of modified AID+eGFP

co-transfection of AID and advanced antibody construct

Mutation Rate

13

mutation rate:

result (each 5000 bp sequenced):

comparison of mutation rates between wt and modified AID

0

0.0005

0.001

0.0015

0.002

0.0025

without AID wt AID mod. AID mod. AID eGFP

mu

tati

on

/se

qu

ence

d n

ucl

eoti

des

3. retransformation into E. coli, sequencing

1. co-transfection of antibody construct & AID -incubation for several days

2. lysis of CHO cells & plasmid preparation

Selection Module Approaches

Fluorescent activated cell sorting

(FACS) Magnetic Beads Viral selection

Magnetic

Beads G

FP Laser Photomultiplier

+ - -

Cell suspension + labeled antigen

electrical wire

-

-

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Viral Selection Principle

15

antibiotic resistance

+ Neo-

mycin

left

ITR

right

ITR CMV

Selection of Anti-GFP nanobody

no unspecific infection by rAAV – negative control

Target cell line infected with rAAV – positive control

16 Assembled with ViralBrick

Universal Virus Modification

sortase

Virus with sortase motif is completely assembled Expression of EGFR-D3 (~25 kDa)

D3 -

17

Modeling

wt CHO

with AID

Infected

wt CHO

Infected

mutated CHO

𝑘𝑑𝑒𝑎𝑡ℎ

𝑘𝑑𝑒𝑎𝑡ℎ

𝑘𝑙𝑜𝑠𝑠

𝑘𝑙𝑜𝑠𝑠

𝑘𝑚𝑢𝑡

𝑘𝑑𝑖𝑣

𝑘𝑑𝑖𝑣

𝑘𝑑𝑖𝑣𝐴𝑖𝑛𝑓

𝑘𝑑𝑖𝑣𝐴𝑖𝑛𝑓

𝑘𝑖𝑛𝑓𝐶

𝑘𝑖𝑛𝑓𝑀

𝑘𝑑𝑒𝑎𝑡ℎ𝐴𝑖𝑛𝑓

𝑘𝑑𝑒𝑎𝑡ℎ𝐴𝑖𝑛𝑓

18

Mutated

CHO

with AID

Modeling - Deterministic

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Modeling - Stochastic

20

initial virus concentration initial wt cell concentration

% s

ucc

essf

ul e

xper

imen

ts

Summary – Antibody System

Chinese Hamster Ovary Cells

1.

Antibody library

2.

Maturation by Enzyme

21

3.

Virus, magnetic bead and FACS for selection

Membrane localization

Stable and transient transfection of both AB constructs

Summary – Antibody System

Chinese Hamster Ovary Cells 22

Transient transfection of AID

Co-transfection of AID and advanced construct

Determination of mutation rates for wt and modified AID

Functionality of Cre recombinase

Expression of EGFR D3

Preparation and control tests on the viral construct Initial tests on FACS and magnetic beads

Potsdam Standard – RFC 91

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digestion with Apa I and Sph I

RFP expression cassette

Sph I Apa I

Gene of Interest

Gene of Interest

Gene of Interest

PCR with Phosphorothioate Primers

Knock out of Phosphorothioate with iodine/ethanol solution at 70°C

Gene of Interest

s

s s

s s s

s s s

s s s

PLICing

Blanusa, M., et al. (2010) Anal. Biochem., 406:141

E X S P

E X S P

1.

SocialBricks

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The concept of SocialBricks is to modularize human practice activities in order to provide adapted versions of activities to various target audiences.

People

Politics

Science meets Politics

Minister of Education and Research Prof. Dr. Schavan (CDU)

25

Parliamentary State Secretary for Education and Research Dr. Braun (CDU)

… and 6 others

Science meets People

3 scientific conferences in Germany Mobile street lab

Survey people about Synthetic Biology Street lab for the Day of Synthetic Biology

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• Biosafety level 1 organisms and plasmids only

• Our laboratory follows all the federal laws

Software Safety Attribution

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• All lab experiments were

done by us!

• We have to thank for material and lab access.

• For details, see our Wiki or poster

Sponsoring

Thank you for your attention

Q uestions? ? ?

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