Cargando…

Developing and characterizing a single-domain antibody (nanobody) against human cytotoxic T-lymphocyte-associated protein 4 (hCTLA-4)

OBJECTIVE(S): Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is the most important human immune checkpoint that modulates T cells activity and brings about immune-homeostasis. Accordingly, checkpoint inhibitor cancer therapy has been approved as a growing method to block over-expressed immune...

Descripción completa

Detalles Bibliográficos
Autores principales: Sotoudeh, Nazli, Noormohammadi, Zahra, Habibi-Anbouhi, Mahdi, Kazemi-Lomedasht, Fatemeh, Behdani, Mahdi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Mashhad University of Medical Sciences 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8751747/
https://www.ncbi.nlm.nih.gov/pubmed/35083014
http://dx.doi.org/10.22038/IJBMS.2021.57774.12851
_version_ 1784631743191449600
author Sotoudeh, Nazli
Noormohammadi, Zahra
Habibi-Anbouhi, Mahdi
Kazemi-Lomedasht, Fatemeh
Behdani, Mahdi
author_facet Sotoudeh, Nazli
Noormohammadi, Zahra
Habibi-Anbouhi, Mahdi
Kazemi-Lomedasht, Fatemeh
Behdani, Mahdi
author_sort Sotoudeh, Nazli
collection PubMed
description OBJECTIVE(S): Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is the most important human immune checkpoint that modulates T cells activity and brings about immune-homeostasis. Accordingly, checkpoint inhibitor cancer therapy has been approved as a growing method to block over-expressed immune checkpoints, such as CTLA-4 receptors. Considering the competitive characteristics of single-domain antibodies with monoclonal antibodies, we tried to develop a camelid Nanobody against human CTLA-4. MATERIALS AND METHODS: We have constructed the VHH gene library by using immunized-camel peripheral blood mononuclear cells and carrying out the Nested-PCR technique. VHH-library was screened by phage display technique and specific nanobodies against CTLA-4 protein were selected and amplified with bio-panning steps. Stronger binders were screened by Periplasmic Extract-ELISA, followed by estimating the complexity of the library. Specific anti-CTLA-4 Nanobody and 3hCTL55, with longer CDR3 and a higher binding rate, were selected for more assays. RESULTS: Results revealed the existence of two different clones in the library with 10(8) binders. In comparison with seven different antigens, using the ELISA technique confirmed the specificity of Nanobody 3hCTL55 against human CTLA-4 antigen. We calculated Nanobody 3hCTL55 affinity for human CTLA-4 antigen at 50×10(-9) M, approximately. Performing western blot and Flow-cytometry techniques showed that Nanobody 3hCTL55 was able to specifically detect and attach both commercial human CTLA-4 protein and human CTLA-4 antigen on the cell surface and in the cell lysate. CONCLUSION: Taken together, this developed camelid-specific anti-CTLA-4 Nanobody 3hCTL55, selected from a high-quality immune library by phage display technique, may be effective for further study about cancer diagnosis and cancer-therapy purposes.
format Online
Article
Text
id pubmed-8751747
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Mashhad University of Medical Sciences
record_format MEDLINE/PubMed
spelling pubmed-87517472022-01-25 Developing and characterizing a single-domain antibody (nanobody) against human cytotoxic T-lymphocyte-associated protein 4 (hCTLA-4) Sotoudeh, Nazli Noormohammadi, Zahra Habibi-Anbouhi, Mahdi Kazemi-Lomedasht, Fatemeh Behdani, Mahdi Iran J Basic Med Sci Original Article OBJECTIVE(S): Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is the most important human immune checkpoint that modulates T cells activity and brings about immune-homeostasis. Accordingly, checkpoint inhibitor cancer therapy has been approved as a growing method to block over-expressed immune checkpoints, such as CTLA-4 receptors. Considering the competitive characteristics of single-domain antibodies with monoclonal antibodies, we tried to develop a camelid Nanobody against human CTLA-4. MATERIALS AND METHODS: We have constructed the VHH gene library by using immunized-camel peripheral blood mononuclear cells and carrying out the Nested-PCR technique. VHH-library was screened by phage display technique and specific nanobodies against CTLA-4 protein were selected and amplified with bio-panning steps. Stronger binders were screened by Periplasmic Extract-ELISA, followed by estimating the complexity of the library. Specific anti-CTLA-4 Nanobody and 3hCTL55, with longer CDR3 and a higher binding rate, were selected for more assays. RESULTS: Results revealed the existence of two different clones in the library with 10(8) binders. In comparison with seven different antigens, using the ELISA technique confirmed the specificity of Nanobody 3hCTL55 against human CTLA-4 antigen. We calculated Nanobody 3hCTL55 affinity for human CTLA-4 antigen at 50×10(-9) M, approximately. Performing western blot and Flow-cytometry techniques showed that Nanobody 3hCTL55 was able to specifically detect and attach both commercial human CTLA-4 protein and human CTLA-4 antigen on the cell surface and in the cell lysate. CONCLUSION: Taken together, this developed camelid-specific anti-CTLA-4 Nanobody 3hCTL55, selected from a high-quality immune library by phage display technique, may be effective for further study about cancer diagnosis and cancer-therapy purposes. Mashhad University of Medical Sciences 2021-09 /pmc/articles/PMC8751747/ /pubmed/35083014 http://dx.doi.org/10.22038/IJBMS.2021.57774.12851 Text en https://creativecommons.org/licenses/by/3.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License, (http://creativecommons.org/licenses/by/3.0/ (https://creativecommons.org/licenses/by/3.0/) ) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Article
Sotoudeh, Nazli
Noormohammadi, Zahra
Habibi-Anbouhi, Mahdi
Kazemi-Lomedasht, Fatemeh
Behdani, Mahdi
Developing and characterizing a single-domain antibody (nanobody) against human cytotoxic T-lymphocyte-associated protein 4 (hCTLA-4)
title Developing and characterizing a single-domain antibody (nanobody) against human cytotoxic T-lymphocyte-associated protein 4 (hCTLA-4)
title_full Developing and characterizing a single-domain antibody (nanobody) against human cytotoxic T-lymphocyte-associated protein 4 (hCTLA-4)
title_fullStr Developing and characterizing a single-domain antibody (nanobody) against human cytotoxic T-lymphocyte-associated protein 4 (hCTLA-4)
title_full_unstemmed Developing and characterizing a single-domain antibody (nanobody) against human cytotoxic T-lymphocyte-associated protein 4 (hCTLA-4)
title_short Developing and characterizing a single-domain antibody (nanobody) against human cytotoxic T-lymphocyte-associated protein 4 (hCTLA-4)
title_sort developing and characterizing a single-domain antibody (nanobody) against human cytotoxic t-lymphocyte-associated protein 4 (hctla-4)
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8751747/
https://www.ncbi.nlm.nih.gov/pubmed/35083014
http://dx.doi.org/10.22038/IJBMS.2021.57774.12851
work_keys_str_mv AT sotoudehnazli developingandcharacterizingasingledomainantibodynanobodyagainsthumancytotoxictlymphocyteassociatedprotein4hctla4
AT noormohammadizahra developingandcharacterizingasingledomainantibodynanobodyagainsthumancytotoxictlymphocyteassociatedprotein4hctla4
AT habibianbouhimahdi developingandcharacterizingasingledomainantibodynanobodyagainsthumancytotoxictlymphocyteassociatedprotein4hctla4
AT kazemilomedashtfatemeh developingandcharacterizingasingledomainantibodynanobodyagainsthumancytotoxictlymphocyteassociatedprotein4hctla4
AT behdanimahdi developingandcharacterizingasingledomainantibodynanobodyagainsthumancytotoxictlymphocyteassociatedprotein4hctla4