Half-Life Extension and Biodistribution Modulation of Biotherapeutics via Red Blood Cell Hitch-Hiking with Novel Anti-Band 3 Single-Domain Antibodies
Small therapeutic proteins are receiving increased interest as therapeutic drugs; however, their clinical success has been limited due to their rapid elimination. Here, we report a half-life extension strategy via strategy via red blood cell red blood cell (RBC) hitch-hiking. This manuscript details...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/1/475 |
| _version_ | 1797625593397772288 |
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| author | Toan D. Nguyen Brandon M. Bordeau Yu Zhang Anna G. Mattle Joseph P. Balthasar |
| author_facet | Toan D. Nguyen Brandon M. Bordeau Yu Zhang Anna G. Mattle Joseph P. Balthasar |
| author_sort | Toan D. Nguyen |
| collection | DOAJ |
| description | Small therapeutic proteins are receiving increased interest as therapeutic drugs; however, their clinical success has been limited due to their rapid elimination. Here, we report a half-life extension strategy via strategy via red blood cell red blood cell (RBC) hitch-hiking. This manuscript details the development and characterization of novel anti-RBC single-domain antibodies (sdAbs), their genetic fusion to therapeutic antibody fragments (TAF) as bispecific fusion constructs, and their influence on TAF pharmacokinetics and biodistribution. Several sdAbs specific to the band 3 antigen were generated via phage-display technology. Binding affinity to RBCs was assessed via flow cytometry. Affinity maturation via random mutagenesis was carried out to improve the binding affinity of the sdAbs. Bi-specific constructs were generated by fusing the anti-RBC sdAbs with anti-tissue necrosis factor alpha (TNF-α) TAF via the use of a glycine-serine flexible linker, and assessments for binding were performed via enzyme-linked immunosorbent assay and flow cytometry. Pharmacokinetics of anti-RBC sdAbs and fusion constructs were evaluated following intravenous bolus dosing in mice at a 1 mg/kg dose. Two RBC-binding sdAbs, RB12 and RE8, were developed. These two clones showed high binding affinity to human RBC with an estimated K<sub>D</sub> of 17.7 nM and 23.6 nM and low binding affinity to mouse RBC with an estimated K<sub>D</sub> of 335 nM and 528 nM for RB12 and RE8, respectively. Two derivative sdAbs, RMA1, and RMC1, with higher affinities against mouse RBC, were generated via affinity maturation (K<sub>D</sub> of 66.9 nM and 30.3 nM, respectively). Pharmacokinetic investigations in mice demonstrated prolonged circulation half-life of an anti-RBC-TNF-α bispecific construct (75 h) compared to a non-RBC binding control (1.3 h). In summary, the developed anti-RBC sdAbs and fusion constructs have demonstrated high affinity in vitro, and sufficient half-life extension in vivo. |
| first_indexed | 2024-03-11T09:58:34Z |
| format | Article |
| id | doaj.art-19f2fea4a573472090640d6b81da03cd |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T09:58:34Z |
| publishDate | 2022-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-19f2fea4a573472090640d6b81da03cd2023-11-16T15:33:55ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-12-0124147510.3390/ijms24010475Half-Life Extension and Biodistribution Modulation of Biotherapeutics via Red Blood Cell Hitch-Hiking with Novel Anti-Band 3 Single-Domain AntibodiesToan D. Nguyen0Brandon M. Bordeau1Yu Zhang2Anna G. Mattle3Joseph P. Balthasar4Department of Pharmaceutical Sciences, University at Buffalo, Buffalo, NY 14214, USADepartment of Pharmaceutical Sciences, University at Buffalo, Buffalo, NY 14214, USADepartment of Pharmaceutical Sciences, University at Buffalo, Buffalo, NY 14214, USADepartment of Pharmaceutical Sciences, University at Buffalo, Buffalo, NY 14214, USADepartment of Pharmaceutical Sciences, University at Buffalo, Buffalo, NY 14214, USASmall therapeutic proteins are receiving increased interest as therapeutic drugs; however, their clinical success has been limited due to their rapid elimination. Here, we report a half-life extension strategy via strategy via red blood cell red blood cell (RBC) hitch-hiking. This manuscript details the development and characterization of novel anti-RBC single-domain antibodies (sdAbs), their genetic fusion to therapeutic antibody fragments (TAF) as bispecific fusion constructs, and their influence on TAF pharmacokinetics and biodistribution. Several sdAbs specific to the band 3 antigen were generated via phage-display technology. Binding affinity to RBCs was assessed via flow cytometry. Affinity maturation via random mutagenesis was carried out to improve the binding affinity of the sdAbs. Bi-specific constructs were generated by fusing the anti-RBC sdAbs with anti-tissue necrosis factor alpha (TNF-α) TAF via the use of a glycine-serine flexible linker, and assessments for binding were performed via enzyme-linked immunosorbent assay and flow cytometry. Pharmacokinetics of anti-RBC sdAbs and fusion constructs were evaluated following intravenous bolus dosing in mice at a 1 mg/kg dose. Two RBC-binding sdAbs, RB12 and RE8, were developed. These two clones showed high binding affinity to human RBC with an estimated K<sub>D</sub> of 17.7 nM and 23.6 nM and low binding affinity to mouse RBC with an estimated K<sub>D</sub> of 335 nM and 528 nM for RB12 and RE8, respectively. Two derivative sdAbs, RMA1, and RMC1, with higher affinities against mouse RBC, were generated via affinity maturation (K<sub>D</sub> of 66.9 nM and 30.3 nM, respectively). Pharmacokinetic investigations in mice demonstrated prolonged circulation half-life of an anti-RBC-TNF-α bispecific construct (75 h) compared to a non-RBC binding control (1.3 h). In summary, the developed anti-RBC sdAbs and fusion constructs have demonstrated high affinity in vitro, and sufficient half-life extension in vivo.https://www.mdpi.com/1422-0067/24/1/475single-domain antibodyred blood cell hitch-hikinghalf-life extensionpharmacokineticsphage displaybispecific antibody |
| spellingShingle | Toan D. Nguyen Brandon M. Bordeau Yu Zhang Anna G. Mattle Joseph P. Balthasar Half-Life Extension and Biodistribution Modulation of Biotherapeutics via Red Blood Cell Hitch-Hiking with Novel Anti-Band 3 Single-Domain Antibodies International Journal of Molecular Sciences single-domain antibody red blood cell hitch-hiking half-life extension pharmacokinetics phage display bispecific antibody |
| title | Half-Life Extension and Biodistribution Modulation of Biotherapeutics via Red Blood Cell Hitch-Hiking with Novel Anti-Band 3 Single-Domain Antibodies |
| title_full | Half-Life Extension and Biodistribution Modulation of Biotherapeutics via Red Blood Cell Hitch-Hiking with Novel Anti-Band 3 Single-Domain Antibodies |
| title_fullStr | Half-Life Extension and Biodistribution Modulation of Biotherapeutics via Red Blood Cell Hitch-Hiking with Novel Anti-Band 3 Single-Domain Antibodies |
| title_full_unstemmed | Half-Life Extension and Biodistribution Modulation of Biotherapeutics via Red Blood Cell Hitch-Hiking with Novel Anti-Band 3 Single-Domain Antibodies |
| title_short | Half-Life Extension and Biodistribution Modulation of Biotherapeutics via Red Blood Cell Hitch-Hiking with Novel Anti-Band 3 Single-Domain Antibodies |
| title_sort | half life extension and biodistribution modulation of biotherapeutics via red blood cell hitch hiking with novel anti band 3 single domain antibodies |
| topic | single-domain antibody red blood cell hitch-hiking half-life extension pharmacokinetics phage display bispecific antibody |
| url | https://www.mdpi.com/1422-0067/24/1/475 |
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