Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B
Abstract
:1. Introduction
2. Results and Discussion
2.1. Libraries Used for Monoclonal Antibody Generation
Library | Immunizing Antigen | Vector | Library Size | mAbs |
---|---|---|---|---|
Human donor libraries | BoNT/A-E Toxoid | pYD2 | >107 | 1B10.1, 1B22, 2B23, 2B25.1, 4B19, B6.1 |
Mouse B1 * | BoNT/B1 Okra | pYD4 | 4.0 × 107 | 16B3, 18A6, 18D10, 18E5, 18F2, 19A9, 19D22, 19G6 |
Mouse B LC | BoNT/B1 LC | pYD4 | 5.0 × 107 | 31A5, 31E2, 31G2, 31H3, 34E8 |
2.2. Characterization of Monoclonal Antibodies
scFv | mAb Origin | Epitope-Group | Affinity KD × 10−9 M (± SD) | Subserotype cross Reactivity | |||
---|---|---|---|---|---|---|---|
B LC | B1 | B2 | B3 | B4 | |||
B6.1 | Human | I-3 | 0.24 (±0.14) | ++++ | +++ | +++ | +++ |
1B10.1 | Human | I-1 | 14.33 (±3.73) | ++ | ++ | + | +++ |
1B22 | Human | I-1 | 1.05 (±0.38) | ++++ | ++++ | ++++ | ++++ |
2B23 | Human | II | 0.60 (±0.22) | ++++ | ++++ | ++++ | ++++ |
2B25.1 | Human | I-1 | 0.24 (±0.13) | ++++ | ++++ | ++++ | ++++ |
4B19 | Human | II | 0.54 (±0.23) | ++++ | +++ | +++ | +++ |
16B3 | Mouse | I-1 | 0.84 (±0.22) | ++++ | ++++ | ++++ | ++++ |
18A6 | Mouse | I-3 | 4.21 (±0.36) | +++ | ++ | ++ | ++ |
18A7 | Mouse | I-2 | 13.5 (±3.02) | + | + | ++ | + |
18D10 | Mouse | I-2 | 7.53 (±1.72) | +++ | +++ | +++ | +++ |
18E5 | Mouse | I-1 | 1.00 (±0.17) | +++ | +++ | +++ | +++ |
18F2 | Mouse | III | 1.19 (±0.16) | + | +++ | ++ | - |
19A9 | Mouse | II | 3.16 (±0.17) | +++ | ++++ | +++ | ++++ |
19D22 | Mouse | I-1 | 9.31 (±1.98) | +++ | +++ | +++ | +++ |
31A5 | Mouse | I-1 | 1.75 (±0.13) | - | - | - | - |
31E2 | Mouse | III | 17.0 (±2.46) | - | - | - | - |
34E8 | Mouse | I-2 | 1.08 (±0.17) | - | - | - | - |
31G2 | Mouse | III | 3.34 (±0.37) | - | - | - | - |
31H3 | Mouse | I-2 | 1.39 (±0.34) | +++ | +++ | +++ | +++ |
2.3. Inhibition of BoNT LC Endopeptidase Activity
mAb | Format | IC50 (×10−9 M) a | KD for BoNT/B1LC on Yeast (×10−9M) b | Epitope Group |
---|---|---|---|---|
1B10.1 | IgG | 2.09 (2.04–2.14) | 118 b (69.0–197) | 1 |
16B3 | scFv | 5.21 (3.04–7.39) | 0.84 (0.40–1.28) | 1 |
18E5 | scFv | 7.01 (6.09–7.93) | 1.00 (0.66–1.34) | 1 |
19D22 | scFv | 5.47 (2.48–8.46) | 9.31 (5.35–13.3) | 1 |
18D10 | scFv | 59.0 (54.8–63.3) | 7.53 (4.09–11.0) | 2 |
2.4. Defining the mAb Epitopes
Epitope | mAb | Amino Acids Affecting mAb Binding |
---|---|---|
Epitope cluster I group 1 | 16B3 | G119, D120, Q177, N178, H179, R183, G185, I189 |
18E5 | Y117, G119, R121, R122, I132, A133, Q177, H179 | |
1B10.1 | R121, H179, D244 | |
2B25.1 | Y117, G119, E127, C308, I309 | |
1B22 | E127, P312, I309, I314, N315 | |
31A5 | G188, G185, P124, V135, H236, A133, I309 | |
19D22 | E22, A26, R31, P55 | |
Epitope cluster I group 2 | 18A7 | R31, P55, T501 |
18D10 | R31, P55, T501 | |
31H3 | R31, R48, P55, F58 | |
Epitope cluster I group3 | B6.1 | N81, K84 |
18A6 | N303, L306, V307, K320, D332, E334, K336, S338 | |
Epitope cluster II | 4B19 | N6, N8, N10, D11, P12 |
19A9 | T392, I393, N396, S400, K402, D403, R409, Q411, V416 | |
Epitope cluster III | 18F2 | K285, V341, E342K, D345, L347 |
31G2 | V341, S343, K346, K349 | |
31E2 | L238, F295, K326, G237, K346, K349 |
2.5. The Epitope of the Inhibitory mAb 1B10.1 Includes Active Site Residues
Mutation of BoNT/B1 LC | Mean KD a (±SD) | Ratio Mutant/Wild Type | ΔΔG | |
---|---|---|---|---|
R121A | No binding - ≥5.0 | - | ≥5.0 | |
R122A | 89.57 (±2.31) | 0.54 | −0.36 | |
V123A | 106.25 (±4.45) | 0.64 | −0.26 | |
P124A | 105.90 (±8.06) | 0.64 | −0.26 | |
L125A | 105.95 (±1.20) | 0.64 | −0.26 | |
I132A | 99.38 (±2.86) | 0.60 | −0.30 | |
Q177A | 127.10 (±6.51) | 0.76 | −0.16 | |
N178A | 110.55 (±9.12) | 0.66 | −0.24 | |
H179A | 14621.50 (±3178) | 87.92 | 2.61 | |
F180A | 90.55 (±15.63) | 0.54 | −0.35 | |
R183A | 100.25 (±23.8) | 0.60 | −0.29 | |
E184A | 89.29 (±52.3) | 0.54 | −0.36 | |
F186A | 97.24 (±25.4) | 0.58 | −0.31 | |
V243A | 99.78 (±18.3) | 0.60 | −0.30 | |
D244A | 1349.37 (±523) | 8.11 | 1.22 | |
D245A | 123.65 (±20.7) | 0.74 | −0.17 | |
Q264A | 127.75 (±21.6) | 0.77 | −0.15 | |
K290A | 154.20 (±74.8) | 0.93 | −0.04 | |
Q293A | 114.88 (±22.1) | 0.69 | −0.22 | |
N294A | 126.05 (±26.8) | 0.76 | −0.16 | |
G297A | 127.70 (± 34.2) | 0.77 | −0.15 | |
I298A | 97.71 (±11.7) | 0.59 | −0.31 | |
R301A | 93.56 (±20.4) | 0.56 | −0.33 | |
D512A | 119.30 (±50.9) | 0.72 | −0.19 | |
N514A | 145.10 (±26.6) | 0.87 | −0.08 | |
V515A | 115.80 (±20.1) | 0.70 | −0.21 | |
D516A | 274.90 (±42.8) | 1.65 | 0.29 | |
Wild Type | 166.30 (±84.7) | - | - |
3. Experimental Section
3.1. Ethics Section
3.2. Oligonucleotides for Library Construction
3.3. Strains, Media, Antibodies, and Toxin
3.4. Protein Expression and Purification
3.5. Mouse Immunization and Spleen Harvest
3.6. Yeast Displayed scFv Library Construction and Library Sorting
3.7. Measurement of Yeast Displayed scFv KD
3.8. Classification of mAbs Based on Overlap of Epitopes
3.9. MAb Inhibition of Synaptobrevin-2 Cleavage by BoNT/B LC
3.10. Determination of IC50 Values
3.11. Random Mutation Library Construction and Sorting
3.12. Site-Directed Mutagenesis
3.13. Fine Epitope Mapping of mAb 1B10.1
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
AAALAC/I | Assessment and Accreditation of Laboratory Animal Care, International |
BoNTs | Botulinum neurotoxins |
mAb | Monoclonal antibody |
HC | Heavy chain |
LC | Light chain |
HC | BoNT binding domain |
HN | BoNT translocation domain |
KD | Equilibrium binding constant |
CHO | Chinese hamster ovary |
IgG | Immunoglobulin G |
Fab | Antigen binding fragment of immunoglobulin with variable domain and first constant domain |
scFv | Single-chain variable fragment |
FACS | Fluorescent activated cell sorting |
sb2 | Synaptobrevin-2 |
SNAP 25 | Synaptosomal-associated protein, 25kDa |
Vh | Heavy chain variable region |
Vk | Kappa chain variable region |
Wt | wild type |
IMAC | Immobilized metal affinity chromatography |
PCR | polymerase chain reaction |
PBS | Phosphate buffered saline |
SD-CAA | Selective growth dextrose casamino acids media |
SG-CAA | Selective growth galactose casamino acids media |
SDS-PAGE | SDS-polyacrylamide gel electrophoresis |
MFI | Mean fluorescence intensity |
FRET | Fluorescence Resonance Energy Transfer |
YsCsY | Yellow fluorescent protein (YFP)-synaptobevin-2-cyan fluorescent protein (CFP)-synaptobevin-2-YFP |
PE | Phycoerythrin |
IC50 | the half maximal inhibitory concentration |
ΔΔG | Change of Gibbs free energy |
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Fan, Y.; Dong, J.; Lou, J.; Wen, W.; Conrad, F.; Geren, I.N.; Garcia-Rodriguez, C.; Smith, T.J.; Smith, L.A.; Ho, M.; et al. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B. Toxins 2015, 7, 3405-3423. https://doi.org/10.3390/toxins7093405
Fan Y, Dong J, Lou J, Wen W, Conrad F, Geren IN, Garcia-Rodriguez C, Smith TJ, Smith LA, Ho M, et al. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B. Toxins. 2015; 7(9):3405-3423. https://doi.org/10.3390/toxins7093405
Chicago/Turabian StyleFan, Yongfeng, Jianbo Dong, Jianlong Lou, Weihua Wen, Fraser Conrad, Isin N. Geren, Consuelo Garcia-Rodriguez, Theresa J. Smith, Leonard A. Smith, Mengfei Ho, and et al. 2015. "Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B" Toxins 7, no. 9: 3405-3423. https://doi.org/10.3390/toxins7093405
APA StyleFan, Y., Dong, J., Lou, J., Wen, W., Conrad, F., Geren, I. N., Garcia-Rodriguez, C., Smith, T. J., Smith, L. A., Ho, M., Pires-Alves, M., Wilson, B. A., & Marks, J. D. (2015). Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B. Toxins, 7(9), 3405-3423. https://doi.org/10.3390/toxins7093405