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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Mashhad University of Medical Sciences
2021
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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 |
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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 |
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