Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling

Landoni, Elisa; Fucà, Giovanni; Wang, Jian; Chirasani, Venkat R.; Yao, Zhiyuan; Dukhovlinova, Elena; Ferrone, Soldano; Savoldo, Barbara; Hong, Lee K.; Shou, Peishun; Musio, Silvia; Padelli, Francesco; Finocchiaro, Gaetano; Droste, Miriam; Kuhlman, Brian; Shamshiev, Abdijapar; Pellegatta, Serena; Dokholyan, Nikolay V.; Dotti, Gianpietro

doi:10.1158/2326-6066.cir-20-0451

Cited by 29 publications

(37 citation statements)

References 48 publications

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“…Similarly, a recent study showed how the biophysical balance of the CAR ectodomain is an important determinant of CAR T cell efficacy (30). Using the 763.74 scFv, Dotti and colleagues developed a CAR against chondroitin sulphate proteoglycan 4 (CSPG4), which is a glycosylated transmembrane protein overexpressed in malignant melanoma.…”

Section: Advanced Car Design: Understanding How the Physiochemical Properties Of The Ectodomain Influence Car Functionmentioning

confidence: 99%

“…Using the 763.74 scFv, Dotti and colleagues developed a CAR against chondroitin sulphate proteoglycan 4 (CSPG4), which is a glycosylated transmembrane protein overexpressed in malignant melanoma. Regardless of the costimulatory endodomain (4-1BB or CD28), the inherent instability of the scFv culminated in spontaneous cytokine release, tonic signaling, and progressive dysfunction over time (30). Using computational modeling strategies, the authors identified critical residues within the framework regions of the CAR targeting domain (isoleucine 123 as well as glutamine 127) that regulated the scFv stability (30).…”

Section: Advanced Car Design: Understanding How the Physiochemical Properties Of The Ectodomain Influence Car Functionmentioning

confidence: 99%

“…Regardless of the costimulatory endodomain (4-1BB or CD28), the inherent instability of the scFv culminated in spontaneous cytokine release, tonic signaling, and progressive dysfunction over time (30). Using computational modeling strategies, the authors identified critical residues within the framework regions of the CAR targeting domain (isoleucine 123 as well as glutamine 127) that regulated the scFv stability (30). Advanced structural analyses using Eris software, revealed a number of amino acid substitutions that would further stabilize the underlying framework of the scFv; attenuate tonic signaling, and enhance the overall anti-tumor function of CSPG4-specific CAR T cells (30).…”

Section: Advanced Car Design: Understanding How the Physiochemical Properties Of The Ectodomain Influence Car Functionmentioning

confidence: 99%

“…Using computational modeling strategies, the authors identified critical residues within the framework regions of the CAR targeting domain (isoleucine 123 as well as glutamine 127) that regulated the scFv stability (30). Advanced structural analyses using Eris software, revealed a number of amino acid substitutions that would further stabilize the underlying framework of the scFv; attenuate tonic signaling, and enhance the overall anti-tumor function of CSPG4-specific CAR T cells (30). As the authors found no connection between CD28 vs 4-1BB endodomains and the degree of tonic signaling, future studies are necessary to understand how CAR costimulation impacts CAR desensitization in the absence of antigen engagement (30).…”

Section: Advanced Car Design: Understanding How the Physiochemical Properties Of The Ectodomain Influence Car Functionmentioning

confidence: 99%

“…Advanced structural analyses using Eris software, revealed a number of amino acid substitutions that would further stabilize the underlying framework of the scFv; attenuate tonic signaling, and enhance the overall anti-tumor function of CSPG4-specific CAR T cells (30). As the authors found no connection between CD28 vs 4-1BB endodomains and the degree of tonic signaling, future studies are necessary to understand how CAR costimulation impacts CAR desensitization in the absence of antigen engagement (30). These findings highlight the benefit of structural modeling to design CARs that can overcome the inherent instability of scFvs in the CAR format, and enhance persistence following adoptive transfer (30).…”

Section: Advanced Car Design: Understanding How the Physiochemical Properties Of The Ectodomain Influence Car Functionmentioning

confidence: 99%

See 4 more Smart Citations

In Like a Lamb; Out Like a Lion: Marching CAR T Cells Toward Enhanced Efficacy in B-ALL

Kozani

O’Connor

2021

Molecular Cancer Therapeutics

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Combining synthetic biology with adoptive T-cell transfer has led to promising advances in the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL). Chimeric antigen receptors (CARs) are synthetic receptors that redirect T-cell specificity against cancer. CARs include “built-in” signaling domains that reprogram T-cell metabolism, enhance effector function, and support long-term persistence. Despite their success in blood-based malignancies, relapse can occur in CD19-redirected CAR T-cell therapies for several reasons, including poor engraftment, impaired in vivo proliferation, and T-cell senescence. Herein, we explain how subtle alterations in CAR design may overcome barriers to effective adoptive immunotherapy. We also discuss how the physiochemical properties of the single-chain variable fragment (scFv) affect differentiation and persistence. Moreover, we describe innovative advances in CAR engineering and provide insight into the development of humanized scFvs whose proposed benefits include increased persistence and improved clinical outcomes. Tumor cells can evade CAR T-cell–mediated detection and elimination due to the emergence or presence of CD19-negative leukemic cell subpopulations. We also discuss the opportunities and challenges in targeting other B-ALL–associated antigens. Identifying alternate targets is fundamentally necessary to restore the success of CAR T-cell therapies in CD19-negative patients with B-ALL.

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Section: Advanced Car Design: Understanding How the Physiochemical Properties Of The Ectodomain Influence Car Functionmentioning

confidence: 99%

Section: Advanced Car Design: Understanding How the Physiochemical Properties Of The Ectodomain Influence Car Functionmentioning

confidence: 99%

Section: Advanced Car Design: Understanding How the Physiochemical Properties Of The Ectodomain Influence Car Functionmentioning

confidence: 99%

Section: Advanced Car Design: Understanding How the Physiochemical Properties Of The Ectodomain Influence Car Functionmentioning

confidence: 99%

Section: Advanced Car Design: Understanding How the Physiochemical Properties Of The Ectodomain Influence Car Functionmentioning

confidence: 99%

See 3 more Smart Citations

In Like a Lamb; Out Like a Lion: Marching CAR T Cells Toward Enhanced Efficacy in B-ALL

Kozani

O’Connor

2021

Molecular Cancer Therapeutics

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Utilizing induced neural stem cell‐based delivery of a cytokine cocktail to enhance chimeric antigen receptor‐modified T‐cell therapy for brain cancer

Woodell

Landoni

Valdivia

et al. 2023

Bioengineering & Transla Med

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Chimeric antigen receptor (CAR)‐modified T‐cell therapy has shown enormous clinical promise against blood cancers, yet efficacy against solid tumors remains a challenge. Here, we investigated the potential of a new combination cell therapy, where tumor‐homing induced neural stem cells (iNSCs) are used to enhance CAR‐T‐cell therapy and achieve efficacious suppression of brain tumors. Using in vitro and in vivo migration assays, we found iNSC‐secreted RANTES/IL‐15 increased CAR‐T‐cell migration sixfold and expansion threefold, resulting in greater antitumor activity in a glioblastoma (GBM) tumor model. Furthermore, multimodal imaging showed iNSC delivery of RANTES/IL‐15 in combination with intravenous administration of CAR‐T cells reduced established orthotopic GBM xenografts 2538‐fold within the first week, followed by durable tumor remission through 60 days post‐treatment. By contrast, CAR‐T‐cell therapy alone only partially controlled tumor growth, with a median survival of only 19 days. Together, these studies demonstrate the potential of combined cell therapy platforms to improve the efficacy of CAR‐T‐cell therapy for brain tumors.

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Novel chimeric antigen receptor‐expressing T cells targeting the malignant mesothelioma‐specific antigen sialylated HEG1

Kouro,

Higashijima,

Horaguchi

et al. 2024

Intl Journal of Cancer

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The selection of highly specific target antigens is critical for the development of clinically efficient and safe chimeric antigen receptors (CARs). In search of diagnostic marker for malignant mesothelioma (MM), we have established SKM9‐2 monoclonal antibody (mAb) which recognizes a MM‐specific molecule, sialylated Protein HEG homolog 1 (HEG1), with high specificity and sensitivity. In this study, to develop a novel therapeutic approach against MM, we generated SKM9‐2 mAb‐derived CARs that included the CD28 (SKM‐28z) or 4‐1BB (SKM‐BBz) costimulatory domain. SKM‐28z CAR‐T cells showed continuous growth and enhanced Tim‐3, LAG‐3, and PD‐1 expression in vitro, which might be induced by tonic signaling caused by self‐activation; however, these phenotypes were not observed in SKM‐BBz CAR‐T cells. In addition, SKM‐BBz CAR‐T cells exhibited slightly stronger in vitro killing activity against MM cell lines than SKM‐28z CAR‐T cells. More importantly, only SKM‐BBz CAR‐T cells, but not SKM‐28z CAR‐T cells, significantly inhibited tumor growth in vivo in a MM cell line xenograft mouse model. Gene expression profiling and reporter assays revealed differential signaling pathway activation; in particular, SKM‐BBz CAR‐T cells exhibited enhanced NF‐kB signaling and reduced NFAT activation. In addition, SKM‐BBz CAR‐T cells showed upregulation of early memory markers, such as TCF7 and CCR7, as well as downregulation of pro‐apoptotic proteins, such as BAK1 and BID, which may be associated with phenotypical and functional differences between SKM‐BBz and SKM‐28z CAR‐T cells. In conclusion, we developed novel SKM9‐2‐derived CAR‐T cells with the 4‐1BB costimulatory domain, which could provide a promising therapeutic approach against refractory MM.

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Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling

Cited by 29 publications

References 48 publications

In Like a Lamb; Out Like a Lion: Marching CAR T Cells Toward Enhanced Efficacy in B-ALL

In Like a Lamb; Out Like a Lion: Marching CAR T Cells Toward Enhanced Efficacy in B-ALL

Utilizing induced neural stem cell‐based delivery of a cytokine cocktail to enhance chimeric antigen receptor‐modified T‐cell therapy for brain cancer

Novel chimeric antigen receptor‐expressing T cells targeting the malignant mesothelioma‐specific antigen sialylated HEG1

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