Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells
Abstract
:1. Introduction
2. Results
2.1. Generation of Anti-PD-L1/CD40L Bispecific Fusion Protein
2.2. Assessment of Anti-PD-L1 Activity of Anti-PD-L1/CD40L BsAbFP
2.3. Assessment of CD40L Activity of Anti-PD-L1/CD40L BsAbFP
2.4. Design of “Three-Cell-in-One” Dual Target Reporter Gene Bioassay for Anti-PD-L1/CD40L BsAbFP
2.5. Potency Assessment of Anti-PD-L1/CD40L BsAbFP by Dual Target Reporter Gene Bioassay
2.6. Characterization of PD-L1/CD40 Dual Target Reporter Bioassay
2.6.1. Assessment of the Role of Raji/NFkB-Luc Cells on the PD-L1/CD40L Dual Target Reporter Bioassay
2.6.2. Assessment of the Role of Jurkat/PD-1/NFAT-Luc Cells on the PD-L1/CD40L Dual Target Reporter Bioassay
2.7. Stability and Specificity Assessment of PD-L1/CD40 Dual Target Reporter Bioassay
2.8. Linearity Assessment of PD-L1/CD40 Dual Target Reporter Assay
2.9. Qualification of PD-L1/CD40L Dual Target Reporter Bioassay
2.10. Evaluation of Structure-Function Attributes Using Different Bioassays and Analytical Methods
3. Discussion
4. Materials and Methods
4.1. Cell Lines and Reagents
4.2. PD-1 Bioassay
4.3. CD40 Bioassay
4.4. PD-L1/CD40L Dual-Target Reporter Gene Bioassay
4.5. Size Exclusion Chromatography (SEC)
4.6. Peptide Mapping for PTM Measurements
5. Data Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
BsAb | Bispecific antibody |
BsAbFP | Bispecific antibody fusion protein |
mAb | Monoclonal antibody |
MOA | Mechanism of action |
APC | Antigen presenting cell |
PD-1 | Programmed cell death protein 1 |
PD-L1 | Programmed death-ligand 1 |
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Parameters | Acceptance Criteria | Results |
---|---|---|
Linearity Assay range | R2 ≥ 0.970 60–167% relative potency | R2 = 0.996 60–167% relative potency |
Accuracy | 80–125% across assay range 60–167% relative potency | Accuracy: 92.0–109.8% |
Precision | Repeatability: %GCV: ≤15% Intermediate precision: %GCV: ≤20% | Repeatability: %GCV = 7.7% Intermediate Precision: %GCV = 14.2% |
Specificity | No concentration-response to formulation buffer and structurally similar but functionally irrelevant antibody | No concentration-response for formulation buffer and irrelevant antibody |
Stability Indicating | Detects a decrease in potency of stressed samples | Demonstrated a decrease in potency of UV- and thermal-stressed samples |
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Pandey, M.S.; Wang, C.; Umlauf, S.; Lin, S. Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells. Int. J. Mol. Sci. 2021, 22, 11302. https://doi.org/10.3390/ijms222111302
Pandey MS, Wang C, Umlauf S, Lin S. Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells. International Journal of Molecular Sciences. 2021; 22(21):11302. https://doi.org/10.3390/ijms222111302
Chicago/Turabian StylePandey, Madhu S., Chunlei Wang, Scott Umlauf, and Shihua Lin. 2021. "Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells" International Journal of Molecular Sciences 22, no. 21: 11302. https://doi.org/10.3390/ijms222111302