Single-chain variable fragment affinity tuning can optimize anti-AML CAR-NK cell functionality
Background Natural Killer (NK) cells have intrinsic anticancer activity that can be redirected toward acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) engineering. Here, we study the functional consequences of CAR binding affinity and targeted epitope on CAR-NK cell activation, cyto...
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| Main Authors: | , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMJ Publishing Group
2025-02-01
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| Series: | Journal for ImmunoTherapy of Cancer |
| Online Access: | https://jitc.bmj.com/content/13/2/e010763.full |
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| author | Marianna Zahurak Ravi Varadhan Ilias Christodoulou Ruyan Rahnama Jun Choe Huilin Yang Monika Kizerwetter Christian Guaraca Natalie Jordan Holl Stamatia C Vorri Megan Zinsky Danielle G Jones Nikol Garcia Espinoza Yun-Huai Kuo Jamie B. Spangler Challice L. Bonifant |
| author_facet | Marianna Zahurak Ravi Varadhan Ilias Christodoulou Ruyan Rahnama Jun Choe Huilin Yang Monika Kizerwetter Christian Guaraca Natalie Jordan Holl Stamatia C Vorri Megan Zinsky Danielle G Jones Nikol Garcia Espinoza Yun-Huai Kuo Jamie B. Spangler Challice L. Bonifant |
| author_sort | Marianna Zahurak |
| collection | DOAJ |
| description | Background Natural Killer (NK) cells have intrinsic anticancer activity that can be redirected toward acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) engineering. Here, we study the functional consequences of CAR binding affinity and targeted epitope on CAR-NK cell activation, cytolytic synapse formation, and antitumor activity.Methods We characterized NK-92 and primary NK cell populations expressing variant affinity AML-specific CARs containing single-chain variable fragments (scFvs, 26292 or 7G3) targeting two epitopes on CD123. 26292 affinity variants were discovered through directed evolution of an error-prone mutagenic library, while 7G3 affinity variants were previously reported. The resulting CAR-NK cell panel was studied with in vitro binding, activation, and cytotoxicity studies and in mouse xenograft models.Results 26292 and 7G3 CARs of variable CD123 binding affinities were highly expressed in NK cells and conferred antigen-specific activation in vitro. High-resolution imaging demonstrated greater clustering of high-affinity 7G3 CAR-NK cells and consequent AML target cell death in a short-term time lapse. Low-affinity 7G3 CAR-NK cells exhibited enhanced antigen density discrimination with greater membrane-proximal signaling, cytokine production, and cytotoxicity. In longer-term assays, low-affinity 7G3 CAR-NK cells demonstrated more sustained killing of AML cells. In vivo testing highlighted greater expansion of low-affinity 7G3 CAR-NK cells in two xenograft models.Conclusions Expression of 26292 and 7G3 CARs with a range of CD123 binding affinities in NK cells leads to antigen-specific activation and cytotoxicity against AML. Affinity-based differences in functional activation and antitumor activity are dependent on time course and are scFv/epitope specific. |
| format | Article |
| id | doaj-art-52c2834ebe0845d88c186dc6ec6f2846 |
| institution | Kabale University |
| issn | 2051-1426 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | BMJ Publishing Group |
| record_format | Article |
| series | Journal for ImmunoTherapy of Cancer |
| spelling | doaj-art-52c2834ebe0845d88c186dc6ec6f28462025-02-07T07:35:15ZengBMJ Publishing GroupJournal for ImmunoTherapy of Cancer2051-14262025-02-0113210.1136/jitc-2024-010763Single-chain variable fragment affinity tuning can optimize anti-AML CAR-NK cell functionalityMarianna Zahurak0Ravi Varadhan1Ilias Christodoulou2Ruyan Rahnama3Jun Choe4Huilin Yang5Monika Kizerwetter6Christian Guaraca7Natalie Jordan Holl8Stamatia C Vorri9Megan Zinsky10Danielle G Jones11Nikol Garcia Espinoza12Yun-Huai Kuo13Jamie B. Spangler14Challice L. Bonifant15Oncology, Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USAOncology, Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USAOncology, Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USAOncology, Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USAOncology, Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USADepartment of Chemical and Biomolecular Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USADepartment of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USADepartment of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USAOncology, Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USAOncology, Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USAOncology, Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USAOncology, Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USADepartment of Chemical and Biomolecular Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USADepartment of Chemical and Biomolecular Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USAOncology, Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USAOncology, Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USABackground Natural Killer (NK) cells have intrinsic anticancer activity that can be redirected toward acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) engineering. Here, we study the functional consequences of CAR binding affinity and targeted epitope on CAR-NK cell activation, cytolytic synapse formation, and antitumor activity.Methods We characterized NK-92 and primary NK cell populations expressing variant affinity AML-specific CARs containing single-chain variable fragments (scFvs, 26292 or 7G3) targeting two epitopes on CD123. 26292 affinity variants were discovered through directed evolution of an error-prone mutagenic library, while 7G3 affinity variants were previously reported. The resulting CAR-NK cell panel was studied with in vitro binding, activation, and cytotoxicity studies and in mouse xenograft models.Results 26292 and 7G3 CARs of variable CD123 binding affinities were highly expressed in NK cells and conferred antigen-specific activation in vitro. High-resolution imaging demonstrated greater clustering of high-affinity 7G3 CAR-NK cells and consequent AML target cell death in a short-term time lapse. Low-affinity 7G3 CAR-NK cells exhibited enhanced antigen density discrimination with greater membrane-proximal signaling, cytokine production, and cytotoxicity. In longer-term assays, low-affinity 7G3 CAR-NK cells demonstrated more sustained killing of AML cells. In vivo testing highlighted greater expansion of low-affinity 7G3 CAR-NK cells in two xenograft models.Conclusions Expression of 26292 and 7G3 CARs with a range of CD123 binding affinities in NK cells leads to antigen-specific activation and cytotoxicity against AML. Affinity-based differences in functional activation and antitumor activity are dependent on time course and are scFv/epitope specific.https://jitc.bmj.com/content/13/2/e010763.full |
| spellingShingle | Marianna Zahurak Ravi Varadhan Ilias Christodoulou Ruyan Rahnama Jun Choe Huilin Yang Monika Kizerwetter Christian Guaraca Natalie Jordan Holl Stamatia C Vorri Megan Zinsky Danielle G Jones Nikol Garcia Espinoza Yun-Huai Kuo Jamie B. Spangler Challice L. Bonifant Single-chain variable fragment affinity tuning can optimize anti-AML CAR-NK cell functionality Journal for ImmunoTherapy of Cancer |
| title | Single-chain variable fragment affinity tuning can optimize anti-AML CAR-NK cell functionality |
| title_full | Single-chain variable fragment affinity tuning can optimize anti-AML CAR-NK cell functionality |
| title_fullStr | Single-chain variable fragment affinity tuning can optimize anti-AML CAR-NK cell functionality |
| title_full_unstemmed | Single-chain variable fragment affinity tuning can optimize anti-AML CAR-NK cell functionality |
| title_short | Single-chain variable fragment affinity tuning can optimize anti-AML CAR-NK cell functionality |
| title_sort | single chain variable fragment affinity tuning can optimize anti aml car nk cell functionality |
| url | https://jitc.bmj.com/content/13/2/e010763.full |
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