CHTEOBIDIMA

Bioactive Proteins For
Drug Discovery

Multipass Membrane Proteins / Veriﬁed Functional Activities

Dedicate on immuno-oncology，Perfect with recombinant mAb development

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Bioactive Proteins For Drug Discovery

MEMBRANE PROTEINS & DRUG DEVELOPMENT

CHMembrane proteins (MPs) are the gatekeepers ofcells and play important roles in a variety ofcellular functions such as material transport,

signal transduction, and cell-to-cell recognition(Figure 1). Abnormal functions of MPs often leadTEto the occurrence of diseases. Therefore, MPs
account for more than 60% of all the current
FDA-approved drug targets and 90% of theantibody based drug targets. The process ofantibody drug development is very costly andlengthy. A good antigen can be the entry key toO

Figure 1. Membrane proteins with various
cellular functions: cell-to-cell recognition(upper left), material transport (upper right)
and signal transduction (bottom).

BI

the process（Figure 2).

Membrane proteins account for more than 60%
of all FDA-approved drug targets and 90% ofDIMA

antibody based drug targets.

Target Identiﬁcation&validation

MarketAntigen
Preparation

NDA/BLA

Lead Antibody
Discovery

Clinical Trials I, II，III

IND

Figure 2. The process of antibody drug development60%

In-vitro & In-vivo
functional evaluation

CMC & processdevelopment

Bioactive Proteins For Drug Discovery

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DIFFICULTIES & SOLUTIONS
cially for multi-pass transmembrane proteins..

Diﬃculties●●Membrane proteins tend to lose functions when removed from the membrane.
The expression level of membrane protein is usually low in host cells.

TE●CHDespite the importance, preparation of functional proteins for drug discovery is challenging, espe-

Cellular expression of recombinant membrane protein often results in protein aggregation andmisfolding due to the hydrophobic nature of transmembrane segments.

BISolutions

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Therefore, membrane proteins have been considered as“undruggable”targets in the past.

Extracellular Domain (ECD):
Pros:

Easy to be puriﬁed at high level

DIM
A

Cons:

May not have native conformation especiallyfor the multipass membrane proteins which
can cause loss of conformational epitopes.

Whole Cell Immunization:
Pros:

●
●
●

Native conformation & modiﬁcation

Don't need puriﬁcation of the target protein.
High immunogenicity

Cons:

The antigen contains complex components including lotsof cytoplasmic and nuclear proteins which can produce
non-speciﬁc antibodies against host cells but not thetarget protein.

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Bioactive Proteins For Drug Discovery

Full length membrane protein:
Pros:
●
●

Active protein with native conformation
One of the best antigens for multipass

membrane proteins
Cons:

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Hard to maintain native conformation in theform of soluble proteins because of thehydrophobic structures.

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DIMA Platforms for Membrane Proteins

DIMA Biotech invested heavily on new expression and extraction technologies for membrane pro-Otein production, and developed several platforms for diﬀerent proteins.
●For single transmembrane protein, we use the well-designed ECD fragment to mimic its function-●For full length multi-transmembrane proteins, such as GPCR and Claudin series proteins, we haveBI

al domain.

5 diﬀerent solutions to help with diﬀerent targets which include Membrane Nanoparticles(MNPs), Exosome (EXO), Virus-like particles (VLPs), Detergent and Synthetic Nanodisc. All plat-forms are based on mammalian expression systems and prepare full-length membrane proteinsDIMA

in soluble protein products with native conformation and activity. They can be used to develop

therapeutic antibodies for multi-pass transmembrane proteins, such as ion channels and transporters.

Figure 3. DIMA's multiple solutions for transmembrane proteinsBioactive Proteins For Drug Discovery

Advantages

Applications●

Mammalian cell expression system with●Native immunogens for therapeutic●Serum free medium culturing system to●

Native ligand and receptor identiﬁcation

Custom protein and large batch production available●In Vitro protein functional test
Cell based assaysBIAdvantages●

OECD proteins

Therapeutic drug In Vitro functional testCH●Function validated proteins●antibody drug developmentTE●authentic post-translational modiﬁcations
minimize host cell contamination

Wide Coverage●Over 1000 on-shelf products covering more●

Many ECD proteins have been used andthan 900 drug targets.

DIMA

validated by internal development projects.

Various Species

ECD proteins from diﬀerent species

including human, mouse, monkey anddog et al.

Function Validation

Ensure the puriﬁed protein can bindto the speciﬁc antibody or ligand.

Protein Biotinylation

Biotinylation is available to ﬁt diﬀerent
downstream application.

Multiple Tags

His, hFc, mFc, GST, S tag04

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Bioactive Proteins For Drug Discovery

Featured Protein Target

Selected Protein Targets For ECD Products

2B4

CCR8

CS1

IL18

PGLYRP14-1BB-L

CD123

CT83

IL2PSCA

ACPP

ADABP

ADAM15ADAM17ADAM28
ADAM8ADAM9ANXA2AXL

B7-1

B7-2

B7-H2

B7-H3

BAFF-R
BCMA

CD200
CD26

CD27

CD28

CD30

CD32a
CD33

CD38

CXCR7DLL3

DNAM1FAP

FLT1FOLR2GFAP
GITR

GITR Ligand

CD40

CD47

CD48GPRC5D
GUCY2C
HBEGF

CB1CB2CCR4PSMA

IL21R

IL4RA
IL5IL5RA
IL6R

RET

RNASE4
RNF43
ROR1

SIRPα

ITGA2KRAS
LAG3

LAMA4M-CSF
MDK

MDR1

Mesothelin

SLAMF1SLAMF3SLAMF5
STAB1

TENM4TIGIT
TIM3

TLR3

CD5

Her2

MMP13

TM4SF1

CD7

ICOS

MUC1

Trop2

CD56

CD70

CD74

HPSE

IFNAR1
IFNB1DIM
A

BTLA

CD160

CXCL4IL21

PRNP

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ACE2

CD155

CTLA-4

IL18BP

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ABCB5

CD138

CSF1R

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5T4

CD112

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4-1BB

CD93

CDH1

CLEC2DIGFBP7
IL17RAIL17RB

MMP9
NCL

NEFL

OX40
PD-1

PDL1

Featured Data (CTLA-4 Protein, Cat. No. PME100017)

Human CTLA-4 Protein, mFc-His Tag on SDS-PAGE under reducing condition.

TNFRSF10B
TweakR
TYRO3
VEGFA
VWF
ZP3

ELISA plate pre-coated by 2 μg/ml (100 μl/well) Human

B7-2, hFc tagged protein (PME100474) can bind Human

B7-1, hFc tagged protein (PME100473) can bind Human
CTLA4, mFc-His tagged protein (PME100017) in a linear
range of 0.048-2.094 ng/ml.

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range of 0.048-2.694 ng/ml.

ELISA plate pre-coated by 2 μg/ml (100 μl/well) HumanTE

CTLA4, mFc-His tagged protein (PME100017) in a linear

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Bioactive Proteins For Drug DiscoveryDIMA

HEK293 cell line transfected with irrelevant protein (B)

ELISA plate pre-coated by 2 μg/ml (100 μl/well) Human
CTLA-4, mFc-His tagged protein (PME100017) can bind

Anti-CTLA-4 Neutralizing antibody (BME100022) in a linear
range of 0.13-16.0 ng/ml.

and human B7-2 (A) were surface stained with Human
CTLA4, mFc-His tagged protein (PME100017) 1μg/ml
followed by Alexa 488-conjugated anti-mouse IgG
secondary antibody.

Flow cytometry analysis with 1ug/ml Human CTLA4 Protein, mFc-His tag(PME100017) on Expi293 cells transfected with human B7-1 (Blue histogram) or Expi293 transfected with irrelevant protein (Red histogram).

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Bioactive Proteins For Drug Discovery

Virus-like particle (VLPs)

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Full length multi-pass transmembrane protein

Virus-like particles (VLPs) are self-assembling multi-protein nanoparticles with similar structuralorganization and conformation as viruses but without viral genome. The size of the VLP is about

100-150nm. It is secreted from the surface of the cells that express target membrane proteins (MPs).

The puriﬁed VLPs have the target MPs inserted in a complete bilayer phospholipid membrane struc-TE

ture, mimic the natural membrane-penetrating state of the protein (Figure 4).

VLPs can be used for routine biochemical analysis, including ELISA, SPR aﬃnity analysis, phagedisplay screenings, protein labeling and cell binding experiments, Flow Virometry analysis, etc. It canalso be used as functional protein antigens to develop active antibodies with high druggable poten-DIMA

BIOtials because the target protein on VLP exhibits a state like its native state on the cell surface.

Figure 4. Virus-like particle (VLP) for the preparation of the full-length multipass transmembrane proteinsBioactive Proteins For Drug Discovery

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Featured VLPs ProductsTarget

Description

CCR4

Human CCR4 full length protein-VLP

FLP100015

Human CLDN6 full length protein-VLP

FLP100038

CLDN18.2
CLDN6

Human CLDN18.2 full length protein-VLP
Fluorescent Human CLDN18.2 Full Length Protein-VLP (EGFP)

FLP100006
FLP100017

CHCLDN18.2

Catalog No.

ELISA analysis of CLDN18.2 VLP（Cat: FLP100006）TE

ELISA plates were pre-coated with 0.5ug/per well puriﬁed human Claudin
18.2 VLP. Serial diluted Anti-Claudin18.2 monoclonal antibody (Zolbetuximab biosililar; IMAB362) solutions were added, washed, and incubatedwith secondary antibody before ELISA reading. From above data, theO

EC50 for IMAB362 binding with Claudin18.2 is 15.37ng/ml.

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Flow analysis of CLDN18.2 VLP（Cat: FLP100006）

A. CLDN18.2 VLP were stained only with Goat anti-human IgG Fc-PE
secondary antibody.

B. Negative control VLP were stained with anti-CLDN18.2 antibody

DIMA(Zolbetuximab, Cat# BME100075) at 1μg/mL, followed by Goat
anti-human IgG Fc-PE secondary antibody.

C. CLDN18.2 VLP were stained with anti-BCMA antibody at 1μg/mL,followed by Goat anti-human IgG Fc-PE secondary antibody.

D. CLDN18.2 VLP were stained with anti-CLDN18.2 antibody (Zolbetux-imab, Cat# BME100075) at 1μg/mL, followed by Goat anti-human IgG
Fc-PE secondary antibody.

ELISA analysis of CCR4 VLP（Cat: FLP100006）

ELISA plates were pre-coated with 0.5μg/per well puriﬁed human CCR4full length VLP. Serial diluted anti-CCR4 antibody (mogamulizumab,Cat# BME100086) solutions were added, washed, and incubated withsecondary antibody before ELISA reading. From above data, the EC50

for anti-CCR4 antibody binding with CCR4 full length VLP is 856.2ng/ml.

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Bioactive Proteins For Drug Discovery

Human CLDN18.2 full length protein-VLP (Cat#: FLP100006)

High Activity

Lyophilization testAccelerated Test(Lyophilized Powder)

Nanodisc

TECH

ELISA

High Stability

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Unlike other membrane scaﬀold protein (MSP) Nanodisc on the market, synthetic Nanodisc devel-oped by DIMA Bio can be prepared directly from the cells. The polymers used during this process havea dual function. It dissolves the cell membranes, like the detergent, and uses cellular phospholipids toBIform Nanodisc around the membrane proteins. The target protein embedded embedded within

DIMA

nanodiscs can then be puriﬁed (Figure 5).

Figure 5: Synthetic Nanodisc for the preparation of the full-length multipass transmembrane proteinsFeatured Nanodisc products
Description

GPRC5D

Human GPRC5D full length protein-synthetic nanodisc

FLP100011

CLDN18.2

Human CLDN18.2 full length protein-synthetic nanodisc

FLP100014

ADORA2A
CB1

CCR4

OR2H1
CD20

Human SSTR2 full length protein-synthetic nanodisc

Human ADORA2A full length protein-synthetic nanodisc

FLP100013

FLP100020

FLP100022

Human CCR4 full length protein-synthetic nanodisc

FLP100024

Human CD20 full length protein-synthetic nanodisc

FLP100027

Human CB1 full length protein-synthetic nanodisc

Human OR2H1 full length protein-synthetic nanodisc
Human CCR2 full length protein-synthetic nanodisc

DIMA

CCR2

FLP100008

Human TM4SF1 full length protein-synthetic nanodisc

BITM4SF1

Human CLDN6 full length protein-synthetic nanodisc

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SSTR2

Catalog No.

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Target

CLDN6

FLP100023
FLP100025
FLP100028

MDR-1

Human MDR-1 full length protein-synthetic nanodisc

FLP100029

GPR75

Human GPR75 full length protein-synthetic nanodisc

FLP100031

TRPA1

Human TRPA1 full length protein-synthetic nanodisc

FLP100033

Human F2RL1 full length protein-synthetic nanodisc

FLP100036

CD63

SLC25A4TSPAN33
F2RL1
CCR810CH

Bioactive Proteins For Drug Discovery

Human CD63 full length protein-synthetic nanodisc

Human SLC25A4 full length protein-synthetic nanodisc
Human TSPAN33 full length protein-synthetic nanodisc
Human CCR8 full length protein-synthetic nanodisc

FLP100030
FLP100032

FLP100035
FLP100037

Bioactive Proteins For Drug Discovery

MDR-1-synthetic nanodisc（12 transmembrane protein）

MDR-1-Nanodisc (Cat.NO.-

Flag Tag on SDS-PAGE

FLP100029) can bindCH

Human MDR-1-Nanodisc,anti-Flag monoclonal

antibody and the EC50 isTE

6.883ng/ml.

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GPRC5D -synthetic nanodisc（7 transmembrane protein）

Human GPRC5D-Nanodisc,
Flag Tag on SDS-PAGE

DIMA

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GPRC5D-Nanodisc (Cat.NO.FLP100011) can bindanti-GPRC5D monoclonal

antibody(DME100090) andthe EC50 is 32.86ng/ml.

Claudin6 -synthetic nanodisc（4 transmembrane protein）

Human CLDN6-Nanodisc,
Flag Tag on SDS-PAGE

CLDN6-Nanodisc (Cat.NO.FLP100008) can bindanti-CLDN6 monoclonal

antibody (BME100082) andthe EC50 is 66.99ng/ml.

Bioactive Proteins For Drug Discovery

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Human CD63 full length protein-synthetic nanodisc (Cat#: FLP100030)

High Activity

Lyophilization test

Accelerated Test
(Lyophilized Powder)

TECH

ELISA

High Stability

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Membrane-coated nanoparitcles (MNP)

Plasma membrane-coated nanoparticles (MNPs) leverage intact cell membranes and produce

BInanoparticles with overexpressed target proteins. MNPs have been used in many applications, including delivery of therapeutic agents and induction of immune responses et al., and show high biocompatibility, speciﬁcity, and low side eﬀects. DIMA biotech developed an optimized MNPs platform forthe full-length membrane protein productions using HEK293 based expression system. The nanoparticles were produced by extrusion of cellular membrane from host HEK293 cells with overexpressed

DIMAtarget proteins (Figure 6).

Figure 6. Membrane Nanoparticles (MNP) for the preparation of the full-length multipass transmembrane proteinsFeatured MNP Products
TargetDescription

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Bioactive Proteins For Drug Discovery

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CCR8

Human CCR8 full length protein-MNP

CB1

Human CB1 full length protein-MNP

CCR4

Human CLDN6 full length protein -MNP

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SSTR2

ADORA2A

FLP100004

FLP100009

Human SSTR2 full length protein-MNP

FLP100012

Human ADORA2A full length protein-MNP

FLP100019

Human CD20 full length protein-MNP

FLP100026

Human CCR4 full length protein-MNP

TM4SF1

Human TM4SF1 full length protein-MNP

CD63

Human CD63 full length protein-MNP

DIMA

CD20

FLP100001

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CLDN6

Catalog No.

FLP100016

FLP100021

FLP100034

ELISA analysis of CD63 MNP（Cat. No. FLP100034）

ELISA plates were pre-coated with 0.5μg/per
well puriﬁed human CD63 full length membrane nanoparticles (Cat. No. FLP100034).

Serial diluted anti-CD63 monoclonal antibody(Cat. No. DMC100425) solutions were added,
washed, and incubated with secondary

Western blot of CCR8 MNPs
(Cat. No. FLP100001)

antibody before ELISA reading. From above

data, the EC50 for anti-CD63 antibody binding
with CD63 MNP is 61.65ng/ml.

Bioactive Proteins For Drug Discovery

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Flow analysis of CD63 MNPs（Cat. No. FLP100034）

A. CD63 MNPs were stained only with Goat anti-human lgG 488
secondary antibody.

B. Negative Control MNPs were stained with anti-CD63 antibody (Cat.
No. DMC100425) at 2μg/mL, followed by Goat anti-human IgG 488

CHsecondary antibody.

C. CD63 MNPs were stained with anti-GPRC5D antibody at 2μg/mL,followed by Goat anti-human IgG 488 secondary antibody.

CD63 full length membrane nanoparticles samples were stained

D. with anti-CD63 antibody (Cat. No. DMC100425) at 2μg/mL, followed

TEby Goat anti-human IgG 488 secondary antibody.

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Flow analysis of CCR8 MNPs（Cat. No. FLP100001）

A. CCR8 MNPs were stained only with Goat anti-human lgG 488secondary antibody.

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B. Negative control MNPs were stained with anti-CCR8 antibody (Cat.
No. BME100063) at 2μg/mL, followed by Goat anti-human IgG 488
secondary antibody.

C. CCR8 MNPs were stained with anti-Claudin 18.2 antibody at 2μg/mL, followed by Goat anti-human IgG 488 secondary antibody.

DIMA

D. CCR8 MNPs were stained with anti-CCR8 antibody (BME100063) at
2μg/mL, followed by Goat anti-human IgG 488 secondary antibody.

Human CD63 full length protein-MNP (Cat#:FLP100034)

High Activity
ELISA

Lyophilization test

High Stability

Accelerated Test
(Lyophilized Powder)15

Bioactive Proteins For Drug Discovery

Exosomes（EXO）
Exosomes (EXO) are secreted membrane nanoparticles formed by fusion of multivesicular bodies(MVBs) with the plasma membrane. The size of exosomes is about 30-150nm. Exosomes can be

puriﬁed from in vitro cultures of cells. Like VLPs, EXO also shows outstanding immunogenicity andCH

have the membrane structure close to that of native plasma membrane. Overexpressed membraneproteins (MPs) were inserted in the host cell membrane and secreted out as Exosomes which can bepuriﬁed and used for a series of downstream studies. Unlike VLPs, the puriﬁed EXO has minimal

DIMABIOTE

cytotoxicity. However, not all MPs can be included in EXO and secreted into medium.

Figure 7: Exosomes (EXO) for the preparation of the full-length multipass transmembrane proteinsBioactive Proteins For Drug Discovery

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Featured EXO products
TargetDescription

GPRC5D

Human GPRC5D full length protein-exo

Human CD24 full length protein-exo

FLP100002
FLP100010

CHCD24Catalog No.

Figure 1. ELISA plates were pre-coated with 0.5 μg/per well puriﬁed

human CD24 exosome. Serial diluted Anti-CD24 monoclonal antibody

TEsolutions were added, washed, and incubated with secondary

antibody before ELISA reading. From above data, the EC50 is 69.61

DIMABIOng/ml.

Figure 2. Nanoparticle Tracking Analysis of CD24 exosomes

Figure 3. TEM image of CD24 exosomes

Detergent

Detergents are amphipathic molecules containing hydrophilic polar heads and non-polar hydrophobic tails. At certain concentration, detergent can form stable micelles with hydrophobic cores which
can be used to isolate the active membrane proteins (MPs) in soluble protein-detergent complexes.
Figure 6 demonstrates how to use detergent to isolate the full-length multipass transmembraneproteins, and to maintain the hydrophobic transmembrane structure of the membrane proteins.

Bioactive Proteins For Drug Discovery

OTECH

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DIMA

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Figure 8: Detergent for the preparation of the full-length multipass transmembrane proteins

Featured Detergent Products

Human Claudin-6 full-length protein（with 1% DDM）

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Bioactive Proteins For Drug Discovery

Custom Protein Service●●●●

1000+ membrane protein projects>90% success rate

HEK293 cell expression system

Multiple tags meeting diﬀerent requirements
Competitive price as low as $1,000TE●CHDIMA Biotech oﬀers custom protein service tailored to your needs.

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Workﬂow for recombinant protein production

Expression Plasmid
Constructions（1~2 weeks）

Protein Test
Expression & Validation(2~3 weeks)

Large Scale Production(Optional, 2~3 weeks)BI

Gene Synthesis(1~2 weeks)

SDS-page
ELISA data
FACS analysis

Protein Function
Evaluation

DIMA

Service process

Projectcommunication

Plasmidconstruction

Acceptingan orderFinal Deliverables:

Proteinpuriﬁcation
Proteinexpression

Deliverables

Puriﬁed protein powder with standard COA report including protein quantity, concentration before
lyophilization, purity by SDS-PAGE, etc.

DIMA Biotechnology LTDDIMA

BIOTECH

Dedicate on immuno-oncology，Perfect with recombinant mAb development

DIMA Biotechnology LTD

Fuchunjiang Rd No. 188, Gaoxin District, Suzhou+001-86-15307149771

Wuhan institute of Biotechnology B7, No.666 Gaoxin Road, Wuhan

info@dimabio. com

www.dimabio.com