Optimization of an Antibody Light Chain Framework Enhances Expression, Biophysical Properties and Pharmacokinetics
Abstract
:1. Introduction
2. Materials and Methods
2.1. Antibody and Antigen Construction, Expression, and Purification
2.2. Biochemical Analytics
2.3. Pharmacokinetics
2.4. Statistics
3. Results
3.1. Design and Generation of Anti-oxMIF Antibody BaxM159 Variants
3.2. Framework Optimized BaxM159 Variants Have Unaltered Epitope Binding Properties
3.3. Framework Optimization of Anti-oxMIF Antibody BaxM159 Increases Expression
3.4. Framework Optimization of Anti-oxMIF Antibody BaxM159 Increases Stability and Decreases Aggregation Propensity
3.5. Framework Optimization of BaxM159 Does not Alter the In Vitro Functionality but Improves Its Pharmacokinetics
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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ID | ID and Name of BaxM159 Variant | Amino Acid at Positions 1–4 | Amino Acid at Position 66 | Amino Acid at Position 79 |
---|---|---|---|---|
1 | 1-DIQMAQ | DIQM | A | Q |
2 | 2-DIQMAQ-K | DIQM | A | Q |
3 | 3-EIVLAQ | EIVL | A | Q |
4 | 4-DIQMGQ | DIQM | G | Q |
5 | 5-DIQMAE | DIQM | A | E |
6 | 6-EIVLGQ | EIVL | G | Q |
7 | 7-EIVLAE | EIVL | A | E |
8 | 8-DIQMGE | DIQM | G | E |
9 | 9-EIVLGE | EIVL | G | E |
ID | ID and Name of BaxM159 Variant | ka (M−1s−1) | kd (s−1) | KD (M) |
---|---|---|---|---|
1 | 1-DIQMAQ | 1.1 × 105 | 2.9 × 10−4 | 2.6 × 10−9 |
2 | 2-DIQMAQ-K | 1.3 × 105 | 3.8 × 10−4 | 3.0 × 10−9 |
3 | 3-EIVLAQ | 2.0 × 105 | 3.1 × 10−4 | 1.5 × 10−9 |
4 | 4-DIQMGQ | 1.6 × 105 ± 8.7 × 104 | 5.6 × 10−4 ± 5.0 × 10−4 | 4.3 × 10−9 ± 2.6 × 10−9 |
5 | 5-DIQMAE | 1.6 × 105 | 4.0 × 10−4 | 2.5 × 10−9 |
6 | 6-EIVLGQ | 2.6 × 105 ± 1.2 × 105 | 6.4 × 10−4 ± 3.5 × 10−5 | 2.7 × 10−9 ± 1.1 × 10−9 |
7 | 7-EIVLAE | 1.8 × 105 ± 6.3 × 103 | 4.4 × 10−4 ± 1.5 × 10−4 | 2.4 × 10−9 ± 7.6 × 10−10 |
8 | 8-DIQMGE | 1.3 × 105 | 5.4 × 10−4 | 4.2 × 10−9 |
9 | 9-EIVLGE | 3.1 × 105 ± 6.5 × 104 | 4.4 × 10−4 ± 2.2 × 10−4 | 1.4 × 10−9 ± 4.1 × 10−10 |
Antibody | 2-DIQMAQ-K | 9-EIVLGE | ||||
---|---|---|---|---|---|---|
Dose (mg/kg) | 5 | 15 | 30 | 5 | 15 | 30 |
C0,α (mg/L) | n.d. | 6.0 ± 1.1 | 15.6 ± 3.1 | 29.7 ± 3.42 | 76.4 ± 7.3 | 149.1 ± 33.4 |
C0,β (mg/L) | n.d. | 3.7 ± 1.1 | 5.0 ± 2.5 | 14.0 ± 3.68 | 45.1 ± 7.9 | 55.2 ± 35.9 |
t½, β (h) | n.d. | 34.5 | 37.9 | 135.9 | 96.3 | 87.7 |
AUC(0–144h) iv (mg/L·h) | n.d. | 209.6 | 326.8 | 1873.6 | 5036.7 | 6611.3 |
Clearance CL (mL/h/kg) | n.d. | 72 ± 13 | 98 ± 16 | 3.5 ± 1.4 | 3.2 ± 1.4 | 3.6 ± 0.8 |
VD,ss (mL/kg) | n.d. | 3067 ± 710 | 3757 ± 957 | 170 ± 40 | 160 ± 23 | 190 ± 33 |
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Douillard, P.; Freissmuth, M.; Antoine, G.; Thiele, M.; Fleischanderl, D.; Matthiessen, P.; Voelkel, D.; Kerschbaumer, R.J.; Scheiflinger, F.; Sabarth, N. Optimization of an Antibody Light Chain Framework Enhances Expression, Biophysical Properties and Pharmacokinetics. Antibodies 2019, 8, 46. https://doi.org/10.3390/antib8030046
Douillard P, Freissmuth M, Antoine G, Thiele M, Fleischanderl D, Matthiessen P, Voelkel D, Kerschbaumer RJ, Scheiflinger F, Sabarth N. Optimization of an Antibody Light Chain Framework Enhances Expression, Biophysical Properties and Pharmacokinetics. Antibodies. 2019; 8(3):46. https://doi.org/10.3390/antib8030046
Chicago/Turabian StyleDouillard, Patrice, Michael Freissmuth, Gerhard Antoine, Michael Thiele, Daniel Fleischanderl, Peter Matthiessen, Dirk Voelkel, Randolf J. Kerschbaumer, Friedrich Scheiflinger, and Nicolas Sabarth. 2019. "Optimization of an Antibody Light Chain Framework Enhances Expression, Biophysical Properties and Pharmacokinetics" Antibodies 8, no. 3: 46. https://doi.org/10.3390/antib8030046