HER2 and HLA-A*02 dual CAR-T cells utilize LOH in a NOT logic gate to address on-target off-tumor toxicity
Background One of the major challenges in chimeric antigen receptor (CAR)-T cell therapy for solid tumors is the potential for on-target off-tumor toxicity due to the expression of CAR tumor antigens in essential tissues and organs. Here, we describe a dual CAR NOT gate incorporating an inhibitory C...
| Main Authors: | , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMJ Publishing Group
2023-12-01
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| Series: | Journal for ImmunoTherapy of Cancer |
| Online Access: | https://jitc.bmj.com/content/11/12/e007426.full |
| _version_ | 1827149978306347008 |
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| author | Sarit Tabak David Bassan Leehee Weinberger Jason Yi Tanya Kim Caitlin Schnair Kristina Vucci Neta Chaim Nir Bujanover Yael Lopesco Orit Foord Adi Sharbi-Yunger Michael R Weist Gregor B Adams Limor Levy-Knafo Richard L Kendall Frank J Calzone |
| author_facet | Sarit Tabak David Bassan Leehee Weinberger Jason Yi Tanya Kim Caitlin Schnair Kristina Vucci Neta Chaim Nir Bujanover Yael Lopesco Orit Foord Adi Sharbi-Yunger Michael R Weist Gregor B Adams Limor Levy-Knafo Richard L Kendall Frank J Calzone |
| author_sort | Sarit Tabak |
| collection | DOAJ |
| description | Background One of the major challenges in chimeric antigen receptor (CAR)-T cell therapy for solid tumors is the potential for on-target off-tumor toxicity due to the expression of CAR tumor antigens in essential tissues and organs. Here, we describe a dual CAR NOT gate incorporating an inhibitory CAR (iCAR) recognizing HLA-A*02 (“A2”) that enables effective treatment with a potent HER2 activating CAR (aCAR) in the context of A2 loss of heterozygosity (LOH).Methods A CAR-T cell screen was conducted to identify inhibitory domains derived from natural immune receptors (iDomains) to be used in a NOT gate, to kill A2− HER2+ lung cancer cell lines but spare A2+ HER2+ lung cancer cell-lines with high specificity. The extensive analysis of lead candidates included T-cell activation and killing, assays of reversibility and durability in sequential challenges, target cell specificity in mixed 3D spheroids and 2D cultures, and the characterization of CAR expression level and cell-trafficking.Results A leukocyte immunoglobulin-like receptor B1 (LIR1) iDomain iCAR was identified as most effective in regulating the cytotoxicity of a second generation HER2 aCAR. Target transfer experiments demonstrated that the ‘on’ and ‘off’ cell state of the LIR1 NOT gate CAR-T cell is both durable and reversible. Protection required iCAR signaling and was associated with reduced aCAR and iCAR surface expression. iCAR regulation was sufficient to generate high target specificity in a 3D adjacent spheroid assay designed to model the interface between clonal A2 LOH foci and normal tissue. However, we observed significant bystander killing of A2+ cells in admix culture through aCAR dependent and independent mechanisms. LIR1 NOT gate CAR-T cells conferred protection against H1703-A2+ tumors and high efficacy against H1703-A2− tumors in-vivo. We observed that the iCAR is inactive in A2+ donors due to cis-binding, but Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout of HLA-A fully restored iCAR activity.Conclusions We have preclinically validated an iCAR NOT gate technology broadly applicable for targeting HER2 expression in the context of A2 LOH. This approach is designed to prevent off tumor toxicity while allowing highly potent antitumor activity. |
| first_indexed | 2024-03-08T16:57:48Z |
| format | Article |
| id | doaj.art-5bdd2400c00d4244be00e6c3a41bb971 |
| institution | Directory Open Access Journal |
| issn | 2051-1426 |
| language | English |
| last_indexed | 2025-03-20T21:26:46Z |
| publishDate | 2023-12-01 |
| publisher | BMJ Publishing Group |
| record_format | Article |
| series | Journal for ImmunoTherapy of Cancer |
| spelling | doaj.art-5bdd2400c00d4244be00e6c3a41bb9712024-08-12T02:10:10ZengBMJ Publishing GroupJournal for ImmunoTherapy of Cancer2051-14262023-12-01111210.1136/jitc-2023-007426HER2 and HLA-A*02 dual CAR-T cells utilize LOH in a NOT logic gate to address on-target off-tumor toxicitySarit Tabak0David Bassan1Leehee Weinberger2Jason Yi3Tanya Kim4Caitlin Schnair5Kristina Vucci6Neta Chaim7Nir Bujanover8Yael Lopesco9Orit Foord10Adi Sharbi-Yunger11Michael R Weist12Gregor B Adams13Limor Levy-Knafo14Richard L Kendall15Frank J Calzone16Research, ImmPACT-Bio, Rehovot, IsraelResearch, ImmPACT-Bio, Rehovot, IsraelResearch, ImmPACT-Bio, Rehovot, IsraelResearch, ImmPACT-Bio US, West Hills, California, USAResearch, ImmPACT-Bio US, West Hills, California, USAResearch, ImmPACT-Bio US, West Hills, California, USAResearch, ImmPACT-Bio US, West Hills, California, USAResearch, ImmPACT-Bio, Rehovot, IsraelResearch, ImmPACT-Bio, Rehovot, IsraelResearch, ImmPACT-Bio, Rehovot, IsraelResearch, ImmPACT-Bio US, West Hills, California, USAResearch, ImmPACT-Bio, Rehovot, IsraelResearch, ImmPACT-Bio US, West Hills, California, USAResearch, ImmPACT-Bio US, West Hills, California, USAResearch, ImmPACT-Bio, Rehovot, IsraelResearch, ImmPACT-Bio US, West Hills, California, USAResearch, ImmPACT-Bio US, West Hills, California, USABackground One of the major challenges in chimeric antigen receptor (CAR)-T cell therapy for solid tumors is the potential for on-target off-tumor toxicity due to the expression of CAR tumor antigens in essential tissues and organs. Here, we describe a dual CAR NOT gate incorporating an inhibitory CAR (iCAR) recognizing HLA-A*02 (“A2”) that enables effective treatment with a potent HER2 activating CAR (aCAR) in the context of A2 loss of heterozygosity (LOH).Methods A CAR-T cell screen was conducted to identify inhibitory domains derived from natural immune receptors (iDomains) to be used in a NOT gate, to kill A2− HER2+ lung cancer cell lines but spare A2+ HER2+ lung cancer cell-lines with high specificity. The extensive analysis of lead candidates included T-cell activation and killing, assays of reversibility and durability in sequential challenges, target cell specificity in mixed 3D spheroids and 2D cultures, and the characterization of CAR expression level and cell-trafficking.Results A leukocyte immunoglobulin-like receptor B1 (LIR1) iDomain iCAR was identified as most effective in regulating the cytotoxicity of a second generation HER2 aCAR. Target transfer experiments demonstrated that the ‘on’ and ‘off’ cell state of the LIR1 NOT gate CAR-T cell is both durable and reversible. Protection required iCAR signaling and was associated with reduced aCAR and iCAR surface expression. iCAR regulation was sufficient to generate high target specificity in a 3D adjacent spheroid assay designed to model the interface between clonal A2 LOH foci and normal tissue. However, we observed significant bystander killing of A2+ cells in admix culture through aCAR dependent and independent mechanisms. LIR1 NOT gate CAR-T cells conferred protection against H1703-A2+ tumors and high efficacy against H1703-A2− tumors in-vivo. We observed that the iCAR is inactive in A2+ donors due to cis-binding, but Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout of HLA-A fully restored iCAR activity.Conclusions We have preclinically validated an iCAR NOT gate technology broadly applicable for targeting HER2 expression in the context of A2 LOH. This approach is designed to prevent off tumor toxicity while allowing highly potent antitumor activity.https://jitc.bmj.com/content/11/12/e007426.full |
| spellingShingle | Sarit Tabak David Bassan Leehee Weinberger Jason Yi Tanya Kim Caitlin Schnair Kristina Vucci Neta Chaim Nir Bujanover Yael Lopesco Orit Foord Adi Sharbi-Yunger Michael R Weist Gregor B Adams Limor Levy-Knafo Richard L Kendall Frank J Calzone HER2 and HLA-A*02 dual CAR-T cells utilize LOH in a NOT logic gate to address on-target off-tumor toxicity Journal for ImmunoTherapy of Cancer |
| title | HER2 and HLA-A*02 dual CAR-T cells utilize LOH in a NOT logic gate to address on-target off-tumor toxicity |
| title_full | HER2 and HLA-A*02 dual CAR-T cells utilize LOH in a NOT logic gate to address on-target off-tumor toxicity |
| title_fullStr | HER2 and HLA-A*02 dual CAR-T cells utilize LOH in a NOT logic gate to address on-target off-tumor toxicity |
| title_full_unstemmed | HER2 and HLA-A*02 dual CAR-T cells utilize LOH in a NOT logic gate to address on-target off-tumor toxicity |
| title_short | HER2 and HLA-A*02 dual CAR-T cells utilize LOH in a NOT logic gate to address on-target off-tumor toxicity |
| title_sort | her2 and hla a 02 dual car t cells utilize loh in a not logic gate to address on target off tumor toxicity |
| url | https://jitc.bmj.com/content/11/12/e007426.full |
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