Case Study 2: In Vitro Characterization of ADCs
In vitro characterization of ADCs encompasses essential assays, including internalization, cytotoxicity and bystander killing, and plasma stability tests. These assays provide critical insights into the functionality and stability of ADCs.
Internalization
Cytotoxicity and Bystander Killing
Serum Stability
In vitro cytotoxicity assay
Serum/Plasma: human, cyno, mouse and rat
pHrodo
Antigen
mAb screening by hFab-MMAE
20 40 60 80 100 20 40 60 80 100
20 40 60 80 100 20 40 60 80 100
Ab7.mIgG1K Ab8.mIgG1K Ab9.mIgG2aK Isotype.mIgG1 mFab-MMAE only Ab7.mIgG1K Ab8.mIgG1K Ab9.mIgG2aK Isotype.mIgG1 mFab-MMAE only
Ab7.hIgG1 Ab8.hIgG1 Ab9.hIgG1 Isotype.hIgG1 hFab-MMAE only Ab7.hIgG1 Ab8.hIgG1 Ab9.hIgG1 Isotype.hIgG1 hFab-MMAE only
Late endosome/ lysosome
Target cell
Incubation at 37C for 0/1/4/7/14/21 days
0.001 0.01 0.1 1 10 100 -20 0 0.001 0.01 0.1 1 10 100 -20 0
0.001 0.01 0.1 1 10 100 -20 0 Antibody or ADC 0.001 0.01 0.1 1 10 100 -20 0
anti-FC F(ab)2-pHrodo
Conc. (nM) Conc. (nM)
Conc. (nM) Conc. (nM)
ADC Cytotoxicity
20 40 60 80 100 20 40 60 80 100
20 40 60 80 100 20 40 60 80 100
Acid quenching
Ab7.hIgG1 Ab8.hIgG1 Ab9.hIgG1 Ab7.hIgG1 Ab8.hIgG1 Ab9.hIgG1
Antigen
Ab7.hIgG1-MMAE Ab9.hIgG1-MMAE Ab8.hIgG1-MMAE Isotype.hIgG1-MMAE Ab7.hIgG1-MMAE Ab9.hIgG1-MMAE Ab8.hIgG1-MMAE Isotype.hIgG1-MMAE
Acid quench
Isotype.hIgG1 hFab-ZAP only Isotype.hIgG1 hFab-ZAP only
0.001 0.01 0.1 1 10 100 -20 0 0.001 0.01 0.1 1 10 100 -20 0
0.001 0.01 0.1 1 10 100 -20 0 0.001 0.01 0.1 1 10 100 -20 0
• Antigen affinity purification • DAR value detection
Released payload
Target cell
Conc. (nM) Conc. (nM)
Conc. (nM) Conc. (nM)
Lysosome tracking Bound Binding
• Multiple species: human, cyno, mouse and rat • Free-payload release and drug-to-antibody ratio (DAR) can be assessed by LC-MS • Aggregation can be assessed by SEC-UPLC with fluorescence detection • Antigen binding activity (by ELISA/FACS), total ADC and antibody quantification (by ELISA/LC-MS) can be tested to evaluate ADC stability
Internalized Internalization
Bystander killing
Cytotoxicity on Raji.luc (ONE-GLO)
150
100
ADC (Raji-Luc+N87) DXd (Raji-Luc+N87) ADC (Raji-Luc only) DXd (Raji-Luc only)
50
0
0.0001 0.01
1
100
10000
Conc. (nM)
Case Study 3: In Vitro Characterization of TCEs
In vitro characterization of TCEs includes assays for T cell killing, activation, and cytokine release, providing a comprehensive evaluation of TCE efficacy.
T Cell Killing
Cytokine Production
T Cell Activation
IFN- g release (Tumor Cell TAAhi + PBMC)
T cell killing to tumor cell TAAhi
Real-time T cell killing assay
CD69 expression on CD4+ T Cell
100 120
0 20 40 60 80 100 0.000001 0.0001 0.01
10 15 20 25
Isotype -10nM
20 40 60 80
Ab1 Ab2 hlgG4 control
Ab1 Ab2 Isotype control
Ab1 Ab2 Isotype control
TCE-10nM
0 5
-20 0
hours 0
24
48
72
96
1
100
0.0001 0.01
1
100 10000
0.000001 0.0001 0.01
1
100
Abs (nM)
Abs (nM)
Abs (pM)
Case Study 4: In Vitro Characterization of Autoimmune and Inflammation Molecules
Ligand Competitive Binding
Reporter Gene Assays
Proliferation & Immune Assays
• High-throughput assays • Autoimmune antagonist such as IL-13Rα1/IL-4Rα blocker
• Cellular function screening • Inflammation signaling
• Proliferation assays • Immune assays: T/NK cell activation, cytokine release assays, et al.
Ligand competition assay
IL-31 RGA assay
Inhibition of IL-31-dependent cell proliferation
2 x 10 6 4 x 10 6 6 x 10 6 8 x 10 6 1 x 10 7
2.0
10000 20000 30000 40000 50000 60000 0.0
Ab1, non-eecive ligand blocker Ab2, eecive ligand blocker Isotype control
1.5
Ab1 Isotype control Ligand only Cell only
Ab1 Isotype control
1.0
0.5
0.0
0
0.0001 0.01
1
100 10000
0.01 0.1
1
10 100
10 -3 10 -2 10 -1 10 0
10 1
10 -4
Abs (nM)
Abs (nM)
Abs (nM)
About WuXi Biologics
WuXi Biologics is a leading contract research, development, and manufacturing organization (CRDMO) that provides end-to-end capabilities to healthcare organizations worldwide. With operations in China, the United States, Ireland, Germany, and Singapore, we enable our partners to effectively and efficiently bring biologics and vaccines to patients worldwide through our comprehensive and high-quality drug development model.
The world’s leading global single-source platform from concept to commercialization
wuxibiologics.com | PS_BD@wuxibiologics.com
12-3-2024
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