Rebuilding the Cell-Free System and Their Applications for R&D of Biologics

Takashi Ebihara, Ph.D.
COO
GeneFrontier Corporation

PEGS Boston
2-6 May 2022

Totally constructive, molecular based system-Customize expression toolbox for your research-

Only necessary molecules
for transcription/translation

Open theBlack box!

<Ex, Membranewith Nanodisc; artificial membrane-like structure>
Non-naturalprotein
AA introduction>✓ Rebuilt cell-free system✓ Tunable for your biologics✓ Simple, Fast
✓ Suited for High throughput system✓ Very low contaminants

2-Having good productivity-• Reaction at 37ºC for 4 h• 0.5 µL of reaction mix/ lane
• stained with Oriole (Bio-Rad) andanalyzed with an image analyzer (LAS)

Design of DNA template is important.
Manual is Free to download from our Web site.

✓ Good expression for many proteins, small to large.
✓ Good purity with simple purification.
✓ Good productivity, ~g/L.

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-Improve process from Days to HoursPUREfrex® Synthesis

orUse forapplication
Mix solution
I, II and III

Addition oftemplate DNA

Incubation for 2-4 hours(Purification, if necessary)

Total: 2-4 hoursm L～

E. coli Expression
Transformation andovernight cultivation

Induction andexpression

Total: 3-4 days

Purification

Use forapplication

L scale4

-Expression of scFv and FabSDS-PAGE

Activity+/For correct folding
Murakami et al. (2019) Sci. Rep. vol.9, p.671. (Supplementary Information)✓ Active scFv and Fab (derived from Herceptin) can be synthesized.
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-For HTS of mAb-

✓ Active Fabs are expressed/screened in HT manner.
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-For HTS of mAbiBody’s Ecobody Technology

Collecting B cells fromblood and tumor tissue
Active FabProtein

Selection of desired
single B cells

Ecobody is…
・Human/Rabbit derived・mAb in “Only 2” days!
・Fully in vitro screening
・No culture

Single-cell RT-PCR
amplification of clonal
antibody genes

Cell-freeExpression

Drug Discovery
Diagnosis
Reagent
AntibodyEvaluation

iBody’s Technology with two patents
1. High Expression with SKIK-tag
2. High Binding Activity with LZ-tag

Ojima-Kato et al. (2017) Sci. Rep., 7, 13979.
https://www.ibody.co.jp/en/✓ Active Fab is expressed/screened in HT manner.
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-For display technologySDNC system is…
・for VHH/cyclic peptide
・with High diversity
・Fully in vitro screening

Evaluation・ELISA・SPR(BIAcore)・BLI(Octet)
・Cell based assay
https://www.epsilon-mol.co.jp/eng/✓ PUREfrex is applied for cDNA display based screening.
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-Application for BiTEanti-CD3-scFv/CD19-scFv

VL

VH

VH
VL

✓ 0.6mg/mL active BiTE was produced under optimized condition.
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-Application for IgGWith a littlecustomization

Only 2 days for IgG!!
Murakami et al. (2019) Sci. Rep. vol.9, p.671.

✓ Full size IgG can be synthesized.
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-Application for complex molecule-Jensen et al. (2021) Protein Expr. Purif., 186, 105910.

✓ Functional insulin analogues can be synthesized.
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-Application for glycosylation; CFPS-IVGCell-freeProtein
Synthesis(CFPS)

(kDa)
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10UDP-/ Enzyme AUDP-/ Enzyme B

CMP-/ Enzyme C-+-++-+++

in vitro Glycosylation (IVG)

Glycosylated
ProteinApplicable to glycosylation of target proteins.
Various reaction system can be designed.
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-Application for glycosylation; CFPS-IVG-

Solubilized membrane protein, hADRB2, are also
glycosylated by multi-step IVG
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Ribosome display system using PUREfrex◆ Advanced screening systemfor Biologics• mAb (scFv / Fab)
• VHH
• Cyclic peptide◆ High Selection Efficiency• Completely molecular based system• >1012 diversity

Licensed technology under JP4931135 etc.

Zimmermann I. et al. (2018) eLife, 7, e34317.

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-Broad applications, yet to come!-

Uyeda et al. (2022) J. Biosci & Bioeng. vol.133, p181.

Kattan et al. (2021) ACS. Synth. Biol. vol.10, p.2447.

Dwidar et al. (2019) J. Am. Chem. Soc. vol.141, p.11103.

Furukawa et al. (2022) RSC. Chem. Biol. vol.3, p.231.

Nakai et al. (2022) Anal. Chem. vol.94, p.3831.

Levy et al. (2021) ACS. Synth. Biol. vol.10, p.609.
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Contact information

Booth# 1218
For reagent use for expression and screening of biologics
https://purefrex.genefrontier.com/

For screening service / collaboration / technology transfer
for generation of new biologics
Takashi Ebihara, Ph.D., COO, GeneFrontier
E-mail: ebihara@genefrontier.com