A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies
Abstract
:1. Introduction
2. Results and Discussion
2.1. Construction of Mammalian Expression Vectors for the Production of scFv-Fc-scFv Antibodies
2.2. Production of scFv-Fc-scFvs Depends on the Employed scFv Domains
2.3. ScFv, Fc-scFv and scFv-Fc-scFv Antibodies Exclusively form Homodimers
Construct | scFv2 | ||
---|---|---|---|
Homodimer [kDa] | Molar mass [kDa] | ||
scFv-Fc | 103.8 | 112.4 | |
Fc-scFv | LA13-IIE3 | 107.7 | 115.1 |
scFv-Fc-scFv | LA13-IIE3 | 156.4 | 175.4 |
scFv-Fc-scFv | TOB4-B11 | 159.2 | 179.5 |
scFv-Fc-scFv | TOB5-D4 | 162.7 | 175.4 |
2.4. ScFv-Fc-scFv Antibodies Provide Dual Antigen Specificity
2.6. Avidity Effects of Tetravalent Antigen Binding by scFv-Fc-scFv Antibodies
Antibody construct | scFv- | Fc | -scFv | EC50 [M] * | Increase of CRP binding (fold) ** |
---|---|---|---|---|---|
scFv *** | LA13-IIE3 | - | - | 2 × 10−8 | 1 |
scFv-Fc | LA13-IIE3 | + | - | 4 × 10−10 | 50 |
Fc-scFv | - | + | LA13-IIE3 | 1 × 10−9 | 20 |
scFv-Fc-scFv | LA13-IIE3 | + | LA13-IIE3 | 4 × 10−11 | 500 |
scFv-Fc-scFv | LA13-IIE3 | + | TOB4-B11 | 5 × 10−11 | 400 |
scFv-Fc-scFv | LA13-IIE3 | + | TOB5-D4 | 4 × 10−10 | 50 |
2.7. Tetravalent scFv-Fc-scFv Antibodies Have Slower off-Rates Compared to Bivalent and Monovalent Antibody Formats
Antibody construct | kon | koff | Rmax | Chi2 | KD * | ||||
---|---|---|---|---|---|---|---|---|---|
scFv- | Fc | -scFv | [1/Ms] | [1/s] | [RU] | [M] | Ratio to scFv | Ratio to scFv-Fc | |
LA13-IIE3 ** | - | - | 1.6 × 105 | 1.6 × 10−3 | 160 | 16,6 | 1.0 × 10−8 | 1.0 | - |
LA13-IIE3 | + | - | 2.1 × 106 | 5.1 × 10−3 | 154 | 65,1 | 2.5 × 10−9 | 4.2 | 1.0 |
LA13-IIE3 | + | LA13-IIE3 | 4.3 × 106 | 3.1 × 10−4 | 216 | 65,4 | 7.2 × 10−11 | 141.9 | 34.1 |
LA13-IIE3 | + | TOB4-B11 | 1.2 × 106 | 6.3 × 10−4 | 131 | 116,0 | 5.2 × 10−10 | 19.8 | 4.8 |
LA13-IIE3 | + | TOB5-D4 | 1.6 × 106 | 7.8 × 10−4 | 157 | 131 | 5.0 × 10−10 | 20.8 | 5.0 |
Antibody construct | kon1 | koff1 | Rmax | Chi2 | ||
---|---|---|---|---|---|---|
scFv- | Fc | -scFv | [1/Ms] | [1/s] | [RU] | |
LA13-IIE3 | + | - | 9.5 × 105 | 6.5 × 10−3 | 178 | 6.93 |
LA13-IIE3 | + | LA13-IIE3 | 1.2 × 106 | 2.7 × 10−3 | 378 | 12.3 |
LA13-IIE3 | + | TOB4-B11 | 6.3 × 105 | 1.2 × 10−3 | 161 | 22.6 |
LA13-IIE3 | + | TOB5-D4 | 8.0 × 105 | 1.4 × 10−3 | 191 | 36.7 |
3. Experimental Section
3.1. Single Chain (sc)Fv Antibody Gene Fragments
3.2. Selection of scFvs against Human CRP by Phage Display
3.3. ScFv Production and Purification
3.4. Construction of pCMV2.2- and pCMV5.2-hIgG1Fc-XP Vectors and the scFv-Fc, Fc-scFv and scFv-Fc-scFv Antibody Constructs
3.5. Antigens
3.6. Cell Culture
3.7. Transient Transfection and Production in HEK293T Cells
3.8. Quantification of Antibodies by IgG/Fc Capture ELISA
3.9. Protein Purification with Protein A Magnetic Beads and by Protein A Filtration
3.10. SDS-PAGE and Silver Staining
3.11. Analytical Size Exclusion Chromatograpy (SEC)
3.12. Antigen ELISA
3.13. Affinity and Kinetics Measurements
4. Conclusions
Acknowledgments
References and Notes
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Pohl, S.C.; Schwarz, S.; Frenzel, A.; Schirrmann, T. A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies. Antibodies 2012, 1, 19-38. https://doi.org/10.3390/antib1010019
Pohl SC, Schwarz S, Frenzel A, Schirrmann T. A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies. Antibodies. 2012; 1(1):19-38. https://doi.org/10.3390/antib1010019
Chicago/Turabian StylePohl, Stefanie Claudia, Steffi Schwarz, André Frenzel, and Thomas Schirrmann. 2012. "A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies" Antibodies 1, no. 1: 19-38. https://doi.org/10.3390/antib1010019