Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33
Abstract
:1. Introduction
2. Results
2.1. Nanobodies Were Generated against Recombinant Human CD33
2.2. In Vitro Characterization of the Anti-CD33 Nanobodies
2.3. Biodistribution of 99mTc-Labeled Nbs
3. Discussion
4. Material and Methods
4.1. Anti-CD33 Nanobodies Generation, Selection, and Expression
4.2. THP-1 Maintenance and Preparation
4.3. Cell-Binding Assay
4.4. Leukemogenic Activity Assay
4.5. Surface Plasmon Resonance—Affinity Determination and Epitope Binning
4.6. Thermal Stability
4.7. Preparation of 99mTc Labeled Nbs
4.8. Animal Model
4.9. In Vivo Biodistribution of Radiolabeled Nbs
4.10. Statistical Analysis
5. Conclusions
Author Contributions
Conflicts of Interest
References
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Nanobody | ka (M−1·s−1) | kd (s−1) | KD (M) | Tm (°C) |
---|---|---|---|---|
Nb_7 | 1.1 × 106 | 6.1 × 10−3 | 5.5 × 10−9 | 54.47 ± 0.05 |
Nb_12 | 5.4 × 105 | 2.1 × 10−3 | 3.9 × 10−9 | 58.00 ± 0.23 |
Nb_16 | 1.3 × 104 | 3.4 × 10−3 | 2.6 × 10−7 | 57.80 ± 0.03 |
Nb_21 | 1.3 × 106 | 9.1 × 10−3 | 7.0 × 10−9 | 52.67 ± 0.04 |
Nb_22 | 1.5 × 106 | 1.9 × 10−3 | 1.3 × 10−8 | 58.22 ± 0.03 |
Nb_87 | 9.3 × 106 | 7.7 × 10−3 | 8.1 × 10−8 | 67.80 ± 0.08 |
αBabA Nb_19 * | 2.8 × 106 | 1.2 × 10−3 | 0.4 × 10−9 | 61.75 ± 0.14 |
Nanobody | Nb_7 | Nb_12 | Nb_16 | Nb_21 | Nb_22 | Nb_87 |
---|---|---|---|---|---|---|
Nb_7 | competitive | non-competitive | non-competitive | competitive | competitive | competitive |
Nb_12 | competitive | competitive | non-competitive | non-competitive | non-competitive | non-competitive |
Nb_16 | competitive | competitive | competitive | non-competitive | non-competitive | non-competitive |
Nb_21 | competitive | competitive | competitive | competitive | competitive | competitive |
Nb_22 | competitive | competitive | competitive | competitive | competitive | competitive |
Nb_87 | competitive | competitive | competitive | competitive | competitive | competitive |
Nb_7 | Nb_12 | Nb_16 | Nb_21 | Nb_87 | ctrl_Nb | |
---|---|---|---|---|---|---|
Blood | 0.70 ± 0.18 | 0.46 ± 0.06 | 0.62 ± 0.07 | 0.73 ± 0.14 | 0.78 ± 0.07 | 0.57 ± 0.09 |
Heart | 0.32 ± 0.05 | 0.23 ± 0.15 | 0.31 ± 0.04 | 0.50 ± 0.13 | 0.37 ± 0.03 | 0.25 ± 0.06 |
Lungs | 0.94 ± 0.24 | 0.80 ± 0.31 | 0.81 ± 0.08 | 1.40 ± 0.20 | 1.23 ± 0.07 | 0.78 ± 0.04 |
Liver | 0.92 ± 0.07 | 1.22 ± 0.38 | 1.02 ± 0.34 | 3.42 ± 0.65 | 1.50 ± 0.35 | 0.57 ± 0.05 |
Spleen | 0.39 ± 0.06 | 0.50 ± 0.53 | 0.36 ± 0.11 | 2.28 ± 0.82 | 0.44 ± 0.02 | 0.28 ± 0.01 |
Stomach | 0.39 ± 0.15 | 0.57 ± 0.52 | 0.38 ± 0.07 | 0.48 ± 0.10 | 0.37 ± 0.11 | 1.02 ± 0.63 |
Intestines | 0.48 ± 0.10 | 0.36 ± 0.09 | 0.46 ± 0.19 | 0.97 ± 0.24 | 0.58 ± 0.12 | 0.37 ± 0.13 |
Kidneys | 232.04 ± 15.83 | 177.47 ± 12.14 | 211.16 ± 64.88 | 197.04 ± 25.96 | 249.11 ± 37.07 | 163.68 ± 4.78 |
Muscle | 0.15 ± 0.03 | 0.16 ± 0.06 | 0.16 ± 0.04 | 0.16 ± 0.04 | 0.27 ± 0.13 | 0.12 ± 0.01 |
Bone | 0.15 ± 0.03 | 0.17 ± 0.05 | 0.13 ± 0.02 | 0.49 ± 0.31 | 0.22 ± 0.00 | 0.11 ± 0.00 |
Tumor | 2.53 ± 0.69 | 1.91 ± 0.64 | 0.70 ± 0.13 | 2.28 ± 0.56 | 2.34 ± 0.79 | 0.16 ± 0.04 |
Tumor/Blood | 3.66 ± 0.94 | 4.15 ± 0.69 | 1.12 ± 0.09 | 3.11 ± 0.46 | 3.02 ± 1.08 | 0.27 ± 0.03 |
Tumor/Muscle | 17.57 ± 5.13 | 12.56 ± 4.43 | 4.39 ± 0.88 | 14.03 ± 3.08 | 9.20 ± 3.51 | 1.31 ± 0.28 |
Tumor/Bone | 16.37 ± 1.95 | 11.42 ± 3.60 | 5.36 ± 1.12 | 5.35 ± 1.66 | 10.64 ± 3.68 | 1.46 ± 0.43 |
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Romão, E.; Krasniqi, A.; Maes, L.; Vandenbrande, C.; Sterckx, Y.G.-J.; Stijlemans, B.; Vincke, C.; Devoogdt, N.; Muyldermans, S. Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33. Int. J. Mol. Sci. 2020, 21, 310. https://doi.org/10.3390/ijms21010310
Romão E, Krasniqi A, Maes L, Vandenbrande C, Sterckx YG-J, Stijlemans B, Vincke C, Devoogdt N, Muyldermans S. Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33. International Journal of Molecular Sciences. 2020; 21(1):310. https://doi.org/10.3390/ijms21010310
Chicago/Turabian StyleRomão, Ema, Ahmet Krasniqi, Laila Maes, Camille Vandenbrande, Yann G.-J. Sterckx, Benoit Stijlemans, Cécile Vincke, Nick Devoogdt, and Serge Muyldermans. 2020. "Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33" International Journal of Molecular Sciences 21, no. 1: 310. https://doi.org/10.3390/ijms21010310