Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein

An antibody-cytokine fusion protein, composed of the murine single-chain cytokine interleukin-12 (IL-12) genetically fused to a human IgG3 specific for the human tumor-associated antigen HER2/neu maintains antigen binding, cytokine bioactivity, and IL-12 heparin-binding activity. This latter propert...

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Detalles Bibliográficos
Autores: Luria Pérez, Rosendo, Candelaria, Pierre V., Daniels Wells, Tracy R., Rodríguez, José A., Helguera, Gustavo Fernando, Penichet, Manuel L.
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2019
País:Argentina
Institución:Consejo Nacional de Investigaciones Científicas y Técnicas
Repositorio:CONICET Digital (CONICET)
Idioma:inglés
OAI Identifier:oai:ri.conicet.gov.ar:11336/106007
Acceso en línea:http://hdl.handle.net/11336/106007
Access Level:acceso abierto
Palabra clave:MONOCLONAL
ANTIBODY
INTERLEUKIN 12
CYTOKINE
https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
id AR_e0d103be08a03f61b1e24c19bca2114f
oai_identifier_str oai:ri.conicet.gov.ar:11336/106007
network_acronym_str AR
network_name_str Argentina
repository_id_str
dc.title.none.fl_str_mv Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein
title Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein
spellingShingle Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein
Luria Pérez, Rosendo
MONOCLONAL
ANTIBODY
INTERLEUKIN 12
CYTOKINE
https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
title_short Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein
title_full Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein
title_fullStr Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein
title_full_unstemmed Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein
title_sort Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein
dc.creator.none.fl_str_mv Luria Pérez, Rosendo
Candelaria, Pierre V.
Daniels Wells, Tracy R.
Rodríguez, José A.
Helguera, Gustavo Fernando
Penichet, Manuel L.
author Luria Pérez, Rosendo
author_facet Luria Pérez, Rosendo
Candelaria, Pierre V.
Daniels Wells, Tracy R.
Rodríguez, José A.
Helguera, Gustavo Fernando
Penichet, Manuel L.
author_role author
author2 Candelaria, Pierre V.
Daniels Wells, Tracy R.
Rodríguez, José A.
Helguera, Gustavo Fernando
Penichet, Manuel L.
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv MONOCLONAL
ANTIBODY
INTERLEUKIN 12
CYTOKINE
https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
topic MONOCLONAL
ANTIBODY
INTERLEUKIN 12
CYTOKINE
https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
description An antibody-cytokine fusion protein, composed of the murine single-chain cytokine interleukin-12 (IL-12) genetically fused to a human IgG3 specific for the human tumor-associated antigen HER2/neu maintains antigen binding, cytokine bioactivity, and IL-12 heparin-binding activity. This latter property is responsible for the binding of the cytokine to glycosaminoglycans (GAGs) on the cell surface and the extracellular matrix and has been implicated in modulating IL-12 bioactivity. Previous studies indicate that the p40 subunit of human and murine IL-12 is responsible for the heparin-binding activity of this heterodimeric cytokine. In the present study we used bioinformatic analysis and site-directed mutagenesis to develop a version of the antibody-(IL-12) fusion protein without heparin-binding activity. This was accomplished by replacing the basic arginine (R) and lysine (K) residues in the cluster of amino acids 254–260 (RKKEKMK) of the murine IL-12 p40 subunit by the neutral non-polar amino acid alanine (A), generating an AAAEAMA mutant fusion protein. ELISA and flow cytometry demonstrated that the antibody fusion protein lacks heparin-binding activity but retains antigen binding. A Tcell proliferation assay showed IL-12 bioactivity in this construct. However, the IL-12 bioactivity is decreased compared to its non-mutated counterpart, which is consistent with an ancillary role of the heparin-binding site of IL-12 in modulating its activity. Thus, we have defined a cluster of amino acid residues with a crucial role in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein.
publishDate 2019
dc.date.none.fl_str_mv 2019-08
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/106007
Luria Pérez, Rosendo; Candelaria, Pierre V.; Daniels Wells, Tracy R.; Rodríguez, José A.; Helguera, Gustavo Fernando; et al.; Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein; Academic Press Ltd - Elsevier Science Ltd; Cytokine; 120; 8-2019; 220-226
1043-4666
CONICET Digital
CONICET
url http://hdl.handle.net/11336/106007
identifier_str_mv Luria Pérez, Rosendo; Candelaria, Pierre V.; Daniels Wells, Tracy R.; Rodríguez, José A.; Helguera, Gustavo Fernando; et al.; Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein; Academic Press Ltd - Elsevier Science Ltd; Cytokine; 120; 8-2019; 220-226
1043-4666
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://linkinghub.elsevier.com/retrieve/pii/S1043466619301012
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.cyto.2019.04.004
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Academic Press Ltd - Elsevier Science Ltd
publisher.none.fl_str_mv Academic Press Ltd - Elsevier Science Ltd
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1799195388977086464
spelling Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion proteinLuria Pérez, RosendoCandelaria, Pierre V.Daniels Wells, Tracy R.Rodríguez, José A.Helguera, Gustavo FernandoPenichet, Manuel L.MONOCLONALANTIBODYINTERLEUKIN 12CYTOKINEhttps://purl.org/becyt/ford/3.4https://purl.org/becyt/ford/3An antibody-cytokine fusion protein, composed of the murine single-chain cytokine interleukin-12 (IL-12) genetically fused to a human IgG3 specific for the human tumor-associated antigen HER2/neu maintains antigen binding, cytokine bioactivity, and IL-12 heparin-binding activity. This latter property is responsible for the binding of the cytokine to glycosaminoglycans (GAGs) on the cell surface and the extracellular matrix and has been implicated in modulating IL-12 bioactivity. Previous studies indicate that the p40 subunit of human and murine IL-12 is responsible for the heparin-binding activity of this heterodimeric cytokine. In the present study we used bioinformatic analysis and site-directed mutagenesis to develop a version of the antibody-(IL-12) fusion protein without heparin-binding activity. This was accomplished by replacing the basic arginine (R) and lysine (K) residues in the cluster of amino acids 254–260 (RKKEKMK) of the murine IL-12 p40 subunit by the neutral non-polar amino acid alanine (A), generating an AAAEAMA mutant fusion protein. ELISA and flow cytometry demonstrated that the antibody fusion protein lacks heparin-binding activity but retains antigen binding. A Tcell proliferation assay showed IL-12 bioactivity in this construct. However, the IL-12 bioactivity is decreased compared to its non-mutated counterpart, which is consistent with an ancillary role of the heparin-binding site of IL-12 in modulating its activity. Thus, we have defined a cluster of amino acid residues with a crucial role in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein.Fil: Luria Pérez, Rosendo. University of California at Los Angeles; Estados Unidos. Hospital de Niños de México "Federico Gómez"; MéxicoFil: Candelaria, Pierre V.. University of California at Los Angeles; Estados UnidosFil: Daniels Wells, Tracy R.. University of California at Los Angeles; Estados UnidosFil: Rodríguez, José A.. University of California at Los Angeles; Estados UnidosFil: Helguera, Gustavo Fernando. University of California at Los Angeles; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Penichet, Manuel L.. University of California at Los Angeles; Estados UnidosAcademic Press Ltd - Elsevier Science Ltd2019-08info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/106007Luria Pérez, Rosendo; Candelaria, Pierre V.; Daniels Wells, Tracy R.; Rodríguez, José A.; Helguera, Gustavo Fernando; et al.; Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein; Academic Press Ltd - Elsevier Science Ltd; Cytokine; 120; 8-2019; 220-2261043-4666CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://linkinghub.elsevier.com/retrieve/pii/S1043466619301012info:eu-repo/semantics/altIdentifier/doi/10.1016/j.cyto.2019.04.004info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2024-05-08T13:51:01Zoai:ri.conicet.gov.ar:11336/106007instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982024-05-08 13:51:01.411CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
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