Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain
A robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-s...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-07-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/24/13/11035 |
| _version_ | 1797591522075475968 |
|---|---|
| author | Adinarayana Kunamneni Marena A. Montera Ravi Durvasula Sascha R. A. Alles Sachin Goyal Karin N. Westlund |
| author_facet | Adinarayana Kunamneni Marena A. Montera Ravi Durvasula Sascha R. A. Alles Sachin Goyal Karin N. Westlund |
| author_sort | Adinarayana Kunamneni |
| collection | DOAJ |
| description | A robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-specific scFvs were generated through ribosomal display technology. Soluble expression and ELISA analysis showed that one antibody, scFv77-2 had the highest binding and could be purified from bacterial cells in large quantities. Octet measurements further revealed that the CCK-B scFv77-2 antibody had binding kinetics of <i>K</i><sub>D</sub> = 1.794 × 10<sup>–8</sup> M. Molecular modeling and docking analyses suggested that the scFv77-2 antibody shaped a proper cavity to embed the whole CCK-B peptide molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force, and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic intermolecular interactions and functional in vivo studies of CCK-BR. The high affinity scFv77-2 antibody showed good efficacy with binding to CCK-BR tested in a chronic pain model. In vivo studies validated the efficacy of the CCK-B receptor (CCK-BR) scFv77-2 antibody as a potential therapy for chronic trigeminal nerve injury-induced pain. Mice were given a single dose of the CCK-B receptor (CCK-BR) scFv antibody 3 weeks after induction of a chronic trigeminal neuropathic pain model, during the transition from acute to chronic pain. The long-term effectiveness for the reduction of mechanical hypersensitivity was evident, persisting for months. The anxiety- and depression-related behaviors typically accompanying persisting hypersensitivity subsequently never developed in the mice given CCK-BR scFv. The effectiveness of the antibody is the basis for further development of the lead CCK-BR scFv as a promising non-opioid therapeutic for chronic pain and the long-term reduction of chronic pain- and anxiety-related behaviors. |
| first_indexed | 2024-03-11T01:38:40Z |
| format | Article |
| id | doaj.art-fedc6289864e44fbb4c79cd07ba34b61 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T01:38:40Z |
| publishDate | 2023-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-fedc6289864e44fbb4c79cd07ba34b612023-11-18T16:47:11ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-07-0124131103510.3390/ijms241311035Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic PainAdinarayana Kunamneni0Marena A. Montera1Ravi Durvasula2Sascha R. A. Alles3Sachin Goyal4Karin N. Westlund5Department of Internal Medicine, Mayo Clinic, Jacksonville, FL 32224-1865, USADepartment of Anesthesiology & Critical Care Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM 87131-0001, USADepartment of Internal Medicine, Mayo Clinic, Jacksonville, FL 32224-1865, USADepartment of Anesthesiology & Critical Care Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM 87131-0001, USADepartment of Anesthesiology & Critical Care Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM 87131-0001, USADepartment of Anesthesiology & Critical Care Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM 87131-0001, USAA robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-specific scFvs were generated through ribosomal display technology. Soluble expression and ELISA analysis showed that one antibody, scFv77-2 had the highest binding and could be purified from bacterial cells in large quantities. Octet measurements further revealed that the CCK-B scFv77-2 antibody had binding kinetics of <i>K</i><sub>D</sub> = 1.794 × 10<sup>–8</sup> M. Molecular modeling and docking analyses suggested that the scFv77-2 antibody shaped a proper cavity to embed the whole CCK-B peptide molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force, and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic intermolecular interactions and functional in vivo studies of CCK-BR. The high affinity scFv77-2 antibody showed good efficacy with binding to CCK-BR tested in a chronic pain model. In vivo studies validated the efficacy of the CCK-B receptor (CCK-BR) scFv77-2 antibody as a potential therapy for chronic trigeminal nerve injury-induced pain. Mice were given a single dose of the CCK-B receptor (CCK-BR) scFv antibody 3 weeks after induction of a chronic trigeminal neuropathic pain model, during the transition from acute to chronic pain. The long-term effectiveness for the reduction of mechanical hypersensitivity was evident, persisting for months. The anxiety- and depression-related behaviors typically accompanying persisting hypersensitivity subsequently never developed in the mice given CCK-BR scFv. The effectiveness of the antibody is the basis for further development of the lead CCK-BR scFv as a promising non-opioid therapeutic for chronic pain and the long-term reduction of chronic pain- and anxiety-related behaviors.https://www.mdpi.com/1422-0067/24/13/11035scFvantibody libraryribosome displaymolecular dockingchronic painnerve injury |
| spellingShingle | Adinarayana Kunamneni Marena A. Montera Ravi Durvasula Sascha R. A. Alles Sachin Goyal Karin N. Westlund Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain International Journal of Molecular Sciences scFv antibody library ribosome display molecular docking chronic pain nerve injury |
| title | Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain |
| title_full | Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain |
| title_fullStr | Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain |
| title_full_unstemmed | Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain |
| title_short | Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain |
| title_sort | rapid generation and molecular docking analysis of single chain fragment variable scfv antibody selected by ribosome display targeting cholecystokinin b receptor cck br for reduction of chronic neuropathic pain |
| topic | scFv antibody library ribosome display molecular docking chronic pain nerve injury |
| url | https://www.mdpi.com/1422-0067/24/13/11035 |
| work_keys_str_mv | AT adinarayanakunamneni rapidgenerationandmoleculardockinganalysisofsinglechainfragmentvariablescfvantibodyselectedbyribosomedisplaytargetingcholecystokininbreceptorcckbrforreductionofchronicneuropathicpain AT marenaamontera rapidgenerationandmoleculardockinganalysisofsinglechainfragmentvariablescfvantibodyselectedbyribosomedisplaytargetingcholecystokininbreceptorcckbrforreductionofchronicneuropathicpain AT ravidurvasula rapidgenerationandmoleculardockinganalysisofsinglechainfragmentvariablescfvantibodyselectedbyribosomedisplaytargetingcholecystokininbreceptorcckbrforreductionofchronicneuropathicpain AT sascharaalles rapidgenerationandmoleculardockinganalysisofsinglechainfragmentvariablescfvantibodyselectedbyribosomedisplaytargetingcholecystokininbreceptorcckbrforreductionofchronicneuropathicpain AT sachingoyal rapidgenerationandmoleculardockinganalysisofsinglechainfragmentvariablescfvantibodyselectedbyribosomedisplaytargetingcholecystokininbreceptorcckbrforreductionofchronicneuropathicpain AT karinnwestlund rapidgenerationandmoleculardockinganalysisofsinglechainfragmentvariablescfvantibodyselectedbyribosomedisplaytargetingcholecystokininbreceptorcckbrforreductionofchronicneuropathicpain |