The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity
To improve the efficacy and safety of chimeric antigen receptor (CAR)-expressing T cell therapeutics through enhanced CAR design, we analysed CAR structural factors that affect CAR-T cell function. We studied the effects of disulphide bonding at cysteine residues and glycosylation in the HD on CAR-T...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-04-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/23/7/4056 |
| _version_ | 1797438857094889472 |
|---|---|
| author | Sachiko Hirobe Keisuke Imaeda Masashi Tachibana Naoki Okada |
| author_facet | Sachiko Hirobe Keisuke Imaeda Masashi Tachibana Naoki Okada |
| author_sort | Sachiko Hirobe |
| collection | DOAJ |
| description | To improve the efficacy and safety of chimeric antigen receptor (CAR)-expressing T cell therapeutics through enhanced CAR design, we analysed CAR structural factors that affect CAR-T cell function. We studied the effects of disulphide bonding at cysteine residues and glycosylation in the HD on CAR-T function. We used first-generation CAR[V/28/28/3z] and CAR[V/8a/8a/3z], consisting of a mouse vascular endothelial growth factor receptor 2 (VEGFR2)-specific single-chain variable fragment tandemly linked to CD28- or CD8α-derived HD, transmembrane domain (TMD) and a CD3ζ-derived signal transduction domain (STD). We constructed structural variants by substituting cysteine with alanine and asparagine (putative N-linked glycosylation sites) with aspartate. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] formed homodimers, the former through a single HD cysteine residue and the latter through the more TMD-proximal of the two cysteine residues. The absence of disulphide bonds did not affect membrane CAR expression but reduced antigen-specific cytokine production and cytotoxic activity. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] harboured one N-linked glycosylation site, and CAR[V/8a/8a/3z] underwent considerable O-linked glycosylation at an unknown site. Thus, N-linked glycosylation of CAR[V/28/28/3z] promotes stable membrane CAR expression, while having no effect on the expression or CAR-T cell activity of CAR[V/8a/8a/3z]. Our findings demonstrate that post-translational modifications of the CAR HD influence CAR-T cell activity, establishing a basis for future CAR design. |
| first_indexed | 2024-03-09T11:44:21Z |
| format | Article |
| id | doaj.art-42f5ea30e5a340579f8c2175e2fc453d |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T11:44:21Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-42f5ea30e5a340579f8c2175e2fc453d2023-11-30T23:26:08ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-04-01237405610.3390/ijms23074056The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell ActivitySachiko Hirobe0Keisuke Imaeda1Masashi Tachibana2Naoki Okada3Laboratory of Clinical Pharmacology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita 565-0871, Osaka, JapanProject for Vaccine and Immune Regulation, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita 565-0871, Osaka, JapanProject for Vaccine and Immune Regulation, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita 565-0871, Osaka, JapanProject for Vaccine and Immune Regulation, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita 565-0871, Osaka, JapanTo improve the efficacy and safety of chimeric antigen receptor (CAR)-expressing T cell therapeutics through enhanced CAR design, we analysed CAR structural factors that affect CAR-T cell function. We studied the effects of disulphide bonding at cysteine residues and glycosylation in the HD on CAR-T function. We used first-generation CAR[V/28/28/3z] and CAR[V/8a/8a/3z], consisting of a mouse vascular endothelial growth factor receptor 2 (VEGFR2)-specific single-chain variable fragment tandemly linked to CD28- or CD8α-derived HD, transmembrane domain (TMD) and a CD3ζ-derived signal transduction domain (STD). We constructed structural variants by substituting cysteine with alanine and asparagine (putative N-linked glycosylation sites) with aspartate. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] formed homodimers, the former through a single HD cysteine residue and the latter through the more TMD-proximal of the two cysteine residues. The absence of disulphide bonds did not affect membrane CAR expression but reduced antigen-specific cytokine production and cytotoxic activity. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] harboured one N-linked glycosylation site, and CAR[V/8a/8a/3z] underwent considerable O-linked glycosylation at an unknown site. Thus, N-linked glycosylation of CAR[V/28/28/3z] promotes stable membrane CAR expression, while having no effect on the expression or CAR-T cell activity of CAR[V/8a/8a/3z]. Our findings demonstrate that post-translational modifications of the CAR HD influence CAR-T cell activity, establishing a basis for future CAR design.https://www.mdpi.com/1422-0067/23/7/4056chimeric antigen receptorCAR-T cell therapydisulphide bondsglycosylationhinge domain |
| spellingShingle | Sachiko Hirobe Keisuke Imaeda Masashi Tachibana Naoki Okada The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity International Journal of Molecular Sciences chimeric antigen receptor CAR-T cell therapy disulphide bonds glycosylation hinge domain |
| title | The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity |
| title_full | The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity |
| title_fullStr | The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity |
| title_full_unstemmed | The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity |
| title_short | The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity |
| title_sort | effects of chimeric antigen receptor car hinge domain post translational modifications on car t cell activity |
| topic | chimeric antigen receptor CAR-T cell therapy disulphide bonds glycosylation hinge domain |
| url | https://www.mdpi.com/1422-0067/23/7/4056 |
| work_keys_str_mv | AT sachikohirobe theeffectsofchimericantigenreceptorcarhingedomainposttranslationalmodificationsoncartcellactivity AT keisukeimaeda theeffectsofchimericantigenreceptorcarhingedomainposttranslationalmodificationsoncartcellactivity AT masashitachibana theeffectsofchimericantigenreceptorcarhingedomainposttranslationalmodificationsoncartcellactivity AT naokiokada theeffectsofchimericantigenreceptorcarhingedomainposttranslationalmodificationsoncartcellactivity AT sachikohirobe effectsofchimericantigenreceptorcarhingedomainposttranslationalmodificationsoncartcellactivity AT keisukeimaeda effectsofchimericantigenreceptorcarhingedomainposttranslationalmodificationsoncartcellactivity AT masashitachibana effectsofchimericantigenreceptorcarhingedomainposttranslationalmodificationsoncartcellactivity AT naokiokada effectsofchimericantigenreceptorcarhingedomainposttranslationalmodificationsoncartcellactivity |