High-throughput, high-titer CHO production of miniproteins with high yields and low endotoxin levels, surpassing traditional E. coli production methods.
E ff icient In Vitro Evaluation Platform for ADC, TCE, and Autoimmune and Inflammation Molecules Wenyuan Cao, Jianxin Mao, Jianchang Cao, Zhuo Mao, Guoyun Zhu, Jiansheng Wu
WuXi Biologics, No.240 Hedan Road, Pudong New District, Shanghai, China (Contact: PS_Marketing@wuxibiologics.com)
In early stages of drug development, with massive amounts of data accumulating, how to screen out the most promising molecules is a highly challenging task. This poster presents our e ff icient and high-throughput in vitro evaluation methods based on literatures, patents, and our extensive project experience. Introduction Fig.1 One-Stop, Integrated In Vitro Platform for Therapeutic Antibody and ADC Evaluation Abstract The development of therapeutic antibodies and Antibody-drug conjugates (ADCs) requires high costs and long research and development cycles. An e ff icient in vitro evaluation and screening strategy can greatly ad- dress these issues. Besides regular protein and cell-based binding assays, our platform specialize in internal- ization, cytotoxicity and bystander killing, and plasma stability assays for ADCs, T cell killing, activation and cytokine release assays for T cell engagers (TCEs), and ligand competitive binding, reporter gene and prolif- eration assays for autoimmune and inflammation molecules. Moreover, 500+ custom cell lines have been generated for bioassays. Our in vitro evaluation platform will e ff iciently promote therapeutic development.
Case Study 3: TCEs In Vitro Characterization
Fig.4 RGA, T cell killing, cytokine production and T cell activation assays to charac- terize T cell engager molecular function
Reporter gene assay: High throughput screening for T cell activation
Cytokine Production
IFN-g, TNF-a, IL-2, IL-10, etc
T cell activation RGA
50
release (Tumor Cell TAAhi +PBMC)
IFN- γ
40
TCE1 TCE2
25
30
10 15 20
Ab1 Ab2 Isotype control
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10
0
0 5
10 -6
10 -4
10 -2
10 0
10 2
10 4
10 6
Abs (pM)
0.000001 0.0001 0.01
1
100
Abs (nM)
T Cell Activation CD25 & CD69 expression in CD3, CD4, CD8 T cells CD69 expression on CD4+ T Cell
T Cell Killing
T Cell Killing to Tumor Cell TAAhi
100 120
Molecular Assays
Cellular Assays
Immune Assays
100
20 40 60 80
0 20 40 60 80
Ab1 Ab2 hIgG4 control
· Mixed Lymphocyte Reaction · Ag-specific T cell proliferation · T/ γδ T/ Treg/ NK expansion/activation assay · γδ T/ NK degranulation assay · T/NK cytotoxicity assay · T/NK exhaustion and activation assay · APC activation assay
· Binding assays · Competition binding assays · Epitope binning assays · Dimerization assay · Receptor occupancy cy ng
· Reporter gene assay (30+) · Proliferation assays · Internalization assays s
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0.0001 0.01
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100 10000
Abs (pM)
· Apoptosis assays · Cell cycle analysis · Cell migration assay · Agglutination assay
Ab1 Ab2 Isotype control
0.000001 0.0001 0.01
1
100
Abs(nM)
Fig.5 Protein-based and cellular function high throughput screening methods, and proliferation & immune assays for autoimmune and inflammation molecules evaluation Case Study 4: Autoimmune & Inflammation Molecules
Biochemical Assays
Fc E ff ector Function
· Treg suppression assay · Cytokine release assays
· Enzymatic assays · Phosphorylation assays
· ADCC assay · CDC assay · ADCP assay y
Ligand Competitive Binding
Reporter Gene Assays
Proliferation & Immune Assay
Protein-based high throughput screening Autoimmune antagonist: such as ̶ IL-13R ē 1/IL-4R ē blocker Lebrikizumab
Cellular function screening Inflammation signaling
Proliferation assays Immune assays: T/NK cell activation, cytokine release assays, et al
p
g
0 10000 20000 30000 40000 50000 60000
Ab1, non-effective ligand blocker Ab2, effective ligand blocker
2.0
2×10 6 4×10 6 6×10 6 8×10 6 1×10 7
Case Study 1: Binding & Competition Assays Fig.2 Various methods for protein and cellular binding analysis
1.5
Ab1 Isotype control Ligand only Cell only
Ab1 Isotype control
1.0
Isotype Control
0.5
10 -4 10 -3 10 -2 10 -1 10 0 10 1 0
0.0
Various Methods
Dual Binding For dual functional molecules, e.g, bispecific antibody Three formats · Cell + Cell · Protein + Protein · Cell + Protein
0.0001 0.01
1
100 10000
0.01 0.1
1
10
100
Abs (nM)
Abs Con.(nM)
AbCon (nM)
Protein based · ELISA · SPR
Ligand competition Assay
IL-31 RGA Assay
Inhibition of IL-31-dependent cell proliferation
Cell based · Cell ELISA · Flow cytometry · FRET High throughput mode are available.
Fig.6 Immunogenicity & cell line development. (1) Ex Vivo immunogenicity assay by ELISPOT. (2) Cell line development mainly includes target protein overexpression and reporter gene cell line/pool generation. Case Study 5: Immunogenicity & Cell Line Development
Dual Binding: cell + cell FACS
40
BsAb1 BsAb2 anti-target A + anti-target B
Binding on a tumor cell line by FACS
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1000 1500 2000 2500
anti-Target A
20
hIgG1 isotype control anti-Target B
Ab1 Ab2
10
IFN- γ detection assay by enhanced ELISPOT
500+ custom cell lines generated for bioassays
0.0001 0.001 0.01 0.1 1 10 100 0
IFN- γ detection Assay
Target Protein Overexpression
0.00010.001 0.01 0.1 1 10 100 1000 0 500
Abs (nM)
Abs (nM)
in vitro immunogenicity: 34 donors
An Ɵ gen
High Throughput Screening: Multiple Cell Lines in ONE Well
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4000
** ** ** ** ****
An Ɵ gen-presen Ɵ ng cells
Cell-1
Cell-2
Cell-3
Cell line mixture
An Ɵ gen Fragment
3000
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30000
15000
3000
Cell-1 Cell-2
Cell-3
MHC II pep Ɵ de complex
2000
20000
10000
n.s. n.s.
n.s. n.s.
2000
Sample Blank Antigen A overexpression on CHOK1
10000
CD4 + T
5000
1000
TCR
1000
0
0
0
0.0001 0.01
1
100 10000
0.0001 0.01
1
100 10000
0.0001 0.01
1
100 10000
IFN- γ
SKOV3-luc-GFP tumor model by IVIS
0
[Abs]nM
[Abs]nM
[Abs]nM
Cell-4
Cell-5
Cell-6
Cell-4 Cell-5 Cell-6
Random integration & Lentiviral system
IFN- γ detec Ɵ on by ELISPOT
800
2500
Ab1 Ab2
3000
1000 1500 2000
BMK1 BMK2 hIgG4 Isotype Control hIgG1 Isotype Control
600
2000
400
WuXi Biologics has developed a comprehensive in vitro evaluation platform to facilitate therapeutic development. · For ADCs: internalization, cytotoxicity and bystander killing, and plasma stability assays; · For TCEs: T cell killing, activation, and cytokine release assays; · For autoimmune and inflammation molecules: ligand competitive binding, reporter gene, proliferation and immune assays. Conclusions We want to express our gratitude to the scientists in the CRO Services Department at WuXi Biologics for their invaluable support in the development of our bioassay platform. Additionally, we extend our appreciation to WuXi Biologics' legal, public relations, and marketing departments for their assistance in patent filings and the preparation and production of written materials. Acknowledgment
1000
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0 500
0
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0.0001 0.01
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0.0001 0.01
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[Abs]nM
[Abs]nM
[Abs]nM
Case Study 2: ADC In Vitro Characterization
Fig.3 Internalization, cytotoxicity and bystander killing, and serum/plasma stability assays for ADC high throughput screening and functional characterization
Internalization Analysis
Cytotoxicity and Bystander Killing
Serum/Plasma Stability
In vitro cytotoxicity assay
Method 1: pHrodo based
Serum/Plasma: human, cyno, mouse and rat
mAb screening by hFab-MMAE
Antigen
20 40 60 80 100
Ab1.hIgG1 Ab2.hIgG1 Ab3.hIgG1 Isotype.hIgG1 hFab-MMAE only
Internalization
Late endosome /lysosome
0.001 0.01 0.1 1 10 100 -20 0
Target cell
Incubation at 37°C for 0/1/4/7/14/21 days
Conc. (nM)
: anti-Fc F(ab)2-pHrodo
: Antibody or ADC
ADC Cytotoxicity
-20 0 20 40 60 80 100
Method 2: Acid quenching
Ab1.hIgG1-MMAE Ab3.hIgG1-MMAE Ab2.hIgG1-MMAE Isotype.hIgG1-MMAE
Antigen
Acid quench
About WuXi Biologics CRO Services
0.001 0.01 0.1 1 10 100
Conc. (nM)
· Antigen a ff inity purification · DAR value detection
· Released payload
Target cell
Bystander killing Cytotoxicity on Raji.luc (ONE-GLO)
Method 3: Lysosome tracking
WuXi Biologics is a leading global CRDMO, with the R represented by our premium-quality CRO Services. With expertise in diverse drug modalities, we deliver comprehensive research services, including antibody discovery, protein production and engineering, and in vitro/in vivo testing, to seamlessly advance our clients’ molecules from concept to CMC development.
Bound
Internalized
Multiple species: human, cyno, mouse, and rat. Aggregation can be assessed by SEC-UPLC with fluorescence detection. Antigen binding activity (by ELISA/FACS), total ADC & antibody quantification (by ELISA) can also be tested to evaluate ADC stability.
ADC (Raji-Luc+N87) DXd (Raji-Luc+N87) ADC (Raji-Luc only) DXd (Raji-Luc only)
150
100
50
Drug Development Expertise Empowering Research Services for Biologics For more information see us at https://www.wuxibiologics.com/cro-services
0.0001 0
0.01
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Concentration(nM)
In Vitro_Poster_202510_V2
To discuss this poster: PS_Marketing@wuxibiologics.com
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