Abstract
PD-L1 is a 40 kDa trans-membrane protein of B7 family and an important T cell regulator. Binding of PD-L1 and PD-1 inhibits proliferation and activation of T cell results cell exhaustion. This phenomenon can be reversed by blocking PD-L1/PD-1 interactions with single chain variables fragment (scFv) fusion proteins and by direct inhibition of tumor cells with drug conjugates. The human phage-displayed scFv library was utilized to generate scFv against the PD-L1 antigen by affinity bio-panning. The positive clones were selected by continuous transfection of bacterial cells and sequence analysis. The binding affinity and specificity of the scFv and antibody fragments were determined by using surface plasmon resonance biosensor, western blot analysis, and immunofluorescence assay. After three rounds of panning selection, about 30% of clones have a binding affinity with targeted PD-L1 antigen. Eight positive clones with accurate sequences were isolated and analyzed for binding affinity with PD-L1 antigen. Three of those with accurate sequences and binding affinity were selected for the recombinant formation and soluble expression by Escherichia coli host machinery. The highly positive recombinant clones with the exact orientation of FR and CDR domains were developed and can be used as a drug carrier tools in ADC formation or direct inhibition of immune checkpoint in cancer immunotherapy. The conjugate achieved its initial potency and need efficient improvement to enhance direct tumor suppression and bio-therapeutics strategies enrichment.
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Abbreviations
- ADC:
-
Antibody-drug conjugate
- ATCC:
-
American Type Culture Collection
- CDR:
-
Complementarity determining regions
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- ECD:
-
Extracellular domain
- ELISA:
-
Enzyme-linked immune-sorbent assay
- FITC:
-
Fluorescein isothiocyanate
- FR:
-
Framework region
- IL:
-
Interleukin
- IPTG:
-
Isopropyl β-d-1-thiogalactopyranoside
- Ni–NTA:
-
Nickel–nitrilotriacetic acid
- PCR:
-
Polymerase chain reaction
- PD-1:
-
Programmed cell death protein-1
- PD-L1:
-
Programmed cell death ligand-1
- PEG:
-
Polyethylene glycol
- scFv:
-
Single chain variable fragment
- SDS-PAGE:
-
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
- TMB:
-
3,3′,5,5′-Tetramethylbenzidine
- VH:
-
Heavy variable region of Ig
- VL:
-
Light variable region of Ig
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant Nos. 81872784 and 81430081).
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Kalim, M., Liang, K., Khan, M.S.I. et al. Efficient development and expression of scFv recombinant proteins against PD-L1 surface domain and potency in cancer therapy. Cytotechnology 71, 705–722 (2019). https://doi.org/10.1007/s10616-019-00316-3
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DOI: https://doi.org/10.1007/s10616-019-00316-3