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CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells

SIMPLE SUMMARY: Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of chemotherapy-resistant lymphomas, but CAR T cell therapy for solid tumors has been disappointing. This study examined the molecular structure, membrane organization, and mobility of HER2-specific CA...

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Autores principales: Mezősi-Csaplár, Marianna, Szöőr, Árpád, Vereb, György
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10295952/
https://www.ncbi.nlm.nih.gov/pubmed/37370693
http://dx.doi.org/10.3390/cancers15123081
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author Mezősi-Csaplár, Marianna
Szöőr, Árpád
Vereb, György
author_facet Mezősi-Csaplár, Marianna
Szöőr, Árpád
Vereb, György
author_sort Mezősi-Csaplár, Marianna
collection PubMed
description SIMPLE SUMMARY: Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of chemotherapy-resistant lymphomas, but CAR T cell therapy for solid tumors has been disappointing. This study examined the molecular structure, membrane organization, and mobility of HER2-specific CARs containing different costimulatory domains. Clustering behavior and diffusion kinetics of CARs were influenced by the costimulatory domains, proving to be significant predictors of immune synapse formation and early activation. Our results suggest that CAR T cell therapy for solid tumors needs to consider the molecular structure, membrane organization, and mobility of the chimeric antigen receptors alongside the long-term costimulation-based expansion capacity and anti-tumor activity for optimal therapeutic effect. ABSTRACT: Chimeric antigen receptor (CAR)-modified T cells brought a paradigm shift in the treatment of chemotherapy-resistant lymphomas. Conversely, clinical experience with CAR T cells targeting solid tumors has been disheartening, indicating the necessity of their molecular-level optimization. While incorporating CD28 or 41BB costimulatory domains into CARs in addition to the CD3z signaling domain improved the long-term efficacy of T cell products, their influence on early tumor engagement has yet to be elucidated. We studied the antigen-independent self-association and membrane diffusion kinetics of first- (.z), second- (CD28.z, 41BB.z), and third- (CD28.41BB.z) generation HER2-specific CARs in the resting T cell membrane using super-resolution AiryScan microscopy and fluorescence correlation spectroscopy, in correlation with RoseTTAFold-based structure prediction and assessment of oligomerization in native Western blot. While .z and CD28.z CARs formed large, high-density submicron clusters of dimers, 41BB-containing CARs formed higher oligomers that assembled into smaller but more numerous membrane clusters. The first-, second-, and third-generation CARs showed progressively increasing lateral diffusion as the distance of their CD3z domain from the membrane plane increased. Confocal microscopy analysis of immunological synapses showed that both small clusters of highly mobile CD28.41BB.z and large clusters of less mobile .z CAR induced more efficient CD3ζ and pLck phosphorylation than CD28.z or 41BB.z CARs of intermediate mobility. However, electric cell-substrate impedance sensing revealed that the CD28.41BB.z CAR performs worst in sequential short-term elimination of adherent tumor cells, while the .z CAR is superior to all others. We conclude that the molecular structure, membrane organization, and mobility of CARs are critical design parameters that can predict the development of an effective immune synapse. Therefore, they need to be taken into account alongside the long-term biological effects of costimulatory domains to achieve an optimal therapeutic effect.
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spelling pubmed-102959522023-06-28 CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells Mezősi-Csaplár, Marianna Szöőr, Árpád Vereb, György Cancers (Basel) Article SIMPLE SUMMARY: Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of chemotherapy-resistant lymphomas, but CAR T cell therapy for solid tumors has been disappointing. This study examined the molecular structure, membrane organization, and mobility of HER2-specific CARs containing different costimulatory domains. Clustering behavior and diffusion kinetics of CARs were influenced by the costimulatory domains, proving to be significant predictors of immune synapse formation and early activation. Our results suggest that CAR T cell therapy for solid tumors needs to consider the molecular structure, membrane organization, and mobility of the chimeric antigen receptors alongside the long-term costimulation-based expansion capacity and anti-tumor activity for optimal therapeutic effect. ABSTRACT: Chimeric antigen receptor (CAR)-modified T cells brought a paradigm shift in the treatment of chemotherapy-resistant lymphomas. Conversely, clinical experience with CAR T cells targeting solid tumors has been disheartening, indicating the necessity of their molecular-level optimization. While incorporating CD28 or 41BB costimulatory domains into CARs in addition to the CD3z signaling domain improved the long-term efficacy of T cell products, their influence on early tumor engagement has yet to be elucidated. We studied the antigen-independent self-association and membrane diffusion kinetics of first- (.z), second- (CD28.z, 41BB.z), and third- (CD28.41BB.z) generation HER2-specific CARs in the resting T cell membrane using super-resolution AiryScan microscopy and fluorescence correlation spectroscopy, in correlation with RoseTTAFold-based structure prediction and assessment of oligomerization in native Western blot. While .z and CD28.z CARs formed large, high-density submicron clusters of dimers, 41BB-containing CARs formed higher oligomers that assembled into smaller but more numerous membrane clusters. The first-, second-, and third-generation CARs showed progressively increasing lateral diffusion as the distance of their CD3z domain from the membrane plane increased. Confocal microscopy analysis of immunological synapses showed that both small clusters of highly mobile CD28.41BB.z and large clusters of less mobile .z CAR induced more efficient CD3ζ and pLck phosphorylation than CD28.z or 41BB.z CARs of intermediate mobility. However, electric cell-substrate impedance sensing revealed that the CD28.41BB.z CAR performs worst in sequential short-term elimination of adherent tumor cells, while the .z CAR is superior to all others. We conclude that the molecular structure, membrane organization, and mobility of CARs are critical design parameters that can predict the development of an effective immune synapse. Therefore, they need to be taken into account alongside the long-term biological effects of costimulatory domains to achieve an optimal therapeutic effect. MDPI 2023-06-07 /pmc/articles/PMC10295952/ /pubmed/37370693 http://dx.doi.org/10.3390/cancers15123081 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Mezősi-Csaplár, Marianna
Szöőr, Árpád
Vereb, György
CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells
title CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells
title_full CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells
title_fullStr CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells
title_full_unstemmed CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells
title_short CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells
title_sort cd28 and 41bb costimulatory domains alone or in combination differentially influence cell surface dynamics and organization of chimeric antigen receptors and early activation of car t cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10295952/
https://www.ncbi.nlm.nih.gov/pubmed/37370693
http://dx.doi.org/10.3390/cancers15123081
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