Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells

Bispecific antibodies (BsAbs) or fusion proteins (BsAbFPs) present a promising strategy for cancer immunotherapy. Numerous BsAbs targeting coinhibitory and costimulatory pathways have been developed for retargeting T cells and antigen presenting cells (APCs). It is challenging to assess the potency...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Madhu S. Pandey, Chunlei Wang, Scott Umlauf, Shihua Lin
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	bioassay bispecific antibody potency mechanism of action qualification Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/bc79f5add5b0428696093c0f178b73f6
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:bc79f5add5b0428696093c0f178b73f6
record_format	dspace
spelling	oai:doaj.org-article:bc79f5add5b0428696093c0f178b73f62021-11-11T16:49:29ZSimultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells10.3390/ijms2221113021422-00671661-6596https://doaj.org/article/bc79f5add5b0428696093c0f178b73f62021-10-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/21/11302https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067Bispecific antibodies (BsAbs) or fusion proteins (BsAbFPs) present a promising strategy for cancer immunotherapy. Numerous BsAbs targeting coinhibitory and costimulatory pathways have been developed for retargeting T cells and antigen presenting cells (APCs). It is challenging to assess the potency of BsAb that engages two different signaling pathways simultaneously in a single assay format, especially when the two antigen targets are expressed on different cells. To explore the potency of anti-PD-L1/CD40L BsAbFP, a fusion protein that binds to human CD40 and PD-L1, we engineered CHO cells as surrogate APCs that express T cell receptor activator and PD-L1, Jurkat cells with PD-1 and NFAT-luciferase reporter as effector T cells, and Raji cell with NFkB-luciferase that endogenously expresses CD40 as accessory B cells. A novel reporter gene bioassay was developed using these cell lines that allows anti-PD-L1/CD40L BsAbFP to engages both PD-1/PD-L1 and CD40/CD40L signaling pathways in one assay. As both reporters use firefly luciferase, the effects of activating both signaling pathways is observed as an increase in luminescence, either as a higher upper asymptote, a lower EC<sub>50</sub>, or both. This dual target reporter gene bioassay system reflects potential mechanism of action and demonstrated the ability of anti-PD-L1/CD40L BsAbFP to synergistically induce biological response compared to the combination of anti-PD-L1 monovalent monoclonal antibody and agonist CD40L fusion protein, or either treatment alone. The results also showed a strong correlation between the drug dose and biological response within the tested potency range with good linearity, accuracy, precision, specificity and stability indicating properties, suggesting that this “three-cell-in-one” dual target reporter gene bioassay is suitable for assessing potency, structure-function and critical quality attributes of anti-PD-L1/CD40L BsAbFP. This approach could be used for developing dual target bioassays for other BsAbs and antibodies used for combination therapy.Madhu S. PandeyChunlei WangScott UmlaufShihua LinMDPI AGarticlebioassaybispecific antibodypotencymechanism of actionqualificationBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 11302, p 11302 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	bioassay bispecific antibody potency mechanism of action qualification Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	bioassay bispecific antibody potency mechanism of action qualification Biology (General) QH301-705.5 Chemistry QD1-999 Madhu S. Pandey Chunlei Wang Scott Umlauf Shihua Lin Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells
description	Bispecific antibodies (BsAbs) or fusion proteins (BsAbFPs) present a promising strategy for cancer immunotherapy. Numerous BsAbs targeting coinhibitory and costimulatory pathways have been developed for retargeting T cells and antigen presenting cells (APCs). It is challenging to assess the potency of BsAb that engages two different signaling pathways simultaneously in a single assay format, especially when the two antigen targets are expressed on different cells. To explore the potency of anti-PD-L1/CD40L BsAbFP, a fusion protein that binds to human CD40 and PD-L1, we engineered CHO cells as surrogate APCs that express T cell receptor activator and PD-L1, Jurkat cells with PD-1 and NFAT-luciferase reporter as effector T cells, and Raji cell with NFkB-luciferase that endogenously expresses CD40 as accessory B cells. A novel reporter gene bioassay was developed using these cell lines that allows anti-PD-L1/CD40L BsAbFP to engages both PD-1/PD-L1 and CD40/CD40L signaling pathways in one assay. As both reporters use firefly luciferase, the effects of activating both signaling pathways is observed as an increase in luminescence, either as a higher upper asymptote, a lower EC<sub>50</sub>, or both. This dual target reporter gene bioassay system reflects potential mechanism of action and demonstrated the ability of anti-PD-L1/CD40L BsAbFP to synergistically induce biological response compared to the combination of anti-PD-L1 monovalent monoclonal antibody and agonist CD40L fusion protein, or either treatment alone. The results also showed a strong correlation between the drug dose and biological response within the tested potency range with good linearity, accuracy, precision, specificity and stability indicating properties, suggesting that this “three-cell-in-one” dual target reporter gene bioassay is suitable for assessing potency, structure-function and critical quality attributes of anti-PD-L1/CD40L BsAbFP. This approach could be used for developing dual target bioassays for other BsAbs and antibodies used for combination therapy.
format	article
author	Madhu S. Pandey Chunlei Wang Scott Umlauf Shihua Lin
author_facet	Madhu S. Pandey Chunlei Wang Scott Umlauf Shihua Lin
author_sort	Madhu S. Pandey
title	Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells
title_short	Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells
title_full	Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells
title_fullStr	Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells
title_full_unstemmed	Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells
title_sort	simultaneous inhibition of pd-1 and stimulation of cd40 signaling pathways by anti-pd-l1/cd40l bispecific fusion protein synergistically activate target and effector cells
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/bc79f5add5b0428696093c0f178b73f6
work_keys_str_mv	AT madhuspandey simultaneousinhibitionofpd1andstimulationofcd40signalingpathwaysbyantipdl1cd40lbispecificfusionproteinsynergisticallyactivatetargetandeffectorcells AT chunleiwang simultaneousinhibitionofpd1andstimulationofcd40signalingpathwaysbyantipdl1cd40lbispecificfusionproteinsynergisticallyactivatetargetandeffectorcells AT scottumlauf simultaneousinhibitionofpd1andstimulationofcd40signalingpathwaysbyantipdl1cd40lbispecificfusionproteinsynergisticallyactivatetargetandeffectorcells AT shihualin simultaneousinhibitionofpd1andstimulationofcd40signalingpathwaysbyantipdl1cd40lbispecificfusionproteinsynergisticallyactivatetargetandeffectorcells
_version_	1718432262196822016

Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells

Ejemplares similares