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<t>CD19/NKG2DL</t> tandem CAR design. Schematic representation of single and tandem CAR constructs. scFv FMC63, single-chain variable fragment anti-CD19; NKG2D EC, extracellular domain of human NKG2D; (G 4 S) 3 , Glycine-Serine linker; CD8α H 55 , 55 amino acids hinge from human CD8α; CD8α H 12 , 12 amino acids hinge from human CD8α; IgG4 H 12 , 12 amino acids hinge from human IgG4; CD8α TM, transmembrane region from human CD8α; 4-1BB, intracellular domain from 4-1BB; CD3ζ, intracellular domain from human CD3ζ; 2A, 2A self-cleaving peptide; tCD34, truncated CD34; shRNA MICA/B-CD3ζ , shRNA duplex targeting MICA/B and CD3ζ.
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CD19/NKG2DL tandem CAR design. Schematic representation of single and tandem CAR constructs. scFv FMC63, single-chain variable fragment anti-CD19; NKG2D EC, extracellular domain of human NKG2D; (G 4 S) 3 , Glycine-Serine linker; CD8α H 55 , 55 amino acids hinge from human CD8α; CD8α H 12 , 12 amino acids hinge from human CD8α; IgG4 H 12 , 12 amino acids hinge from human IgG4; CD8α TM, transmembrane region from human CD8α; 4-1BB, intracellular domain from 4-1BB; CD3ζ, intracellular domain from human CD3ζ; 2A, 2A self-cleaving peptide; tCD34, truncated CD34; shRNA MICA/B-CD3ζ , shRNA duplex targeting MICA/B and CD3ζ.

Journal: Frontiers in Immunology

Article Title: Tandem CAR T-cells targeting CD19 and NKG2DL can overcome CD19 antigen escape in B-ALL

doi: 10.3389/fimmu.2025.1557405

Figure Lengend Snippet: CD19/NKG2DL tandem CAR design. Schematic representation of single and tandem CAR constructs. scFv FMC63, single-chain variable fragment anti-CD19; NKG2D EC, extracellular domain of human NKG2D; (G 4 S) 3 , Glycine-Serine linker; CD8α H 55 , 55 amino acids hinge from human CD8α; CD8α H 12 , 12 amino acids hinge from human CD8α; IgG4 H 12 , 12 amino acids hinge from human IgG4; CD8α TM, transmembrane region from human CD8α; 4-1BB, intracellular domain from 4-1BB; CD3ζ, intracellular domain from human CD3ζ; 2A, 2A self-cleaving peptide; tCD34, truncated CD34; shRNA MICA/B-CD3ζ , shRNA duplex targeting MICA/B and CD3ζ.

Article Snippet: NKG2DL were detected using antibodies directed against human MICA (FAB1300P, R&D systems), MICB (FAB1599P, R&D systems), ULBP1 (FAB1380P, R&D systems), ULBP3 (FAB1517P, R&D systems), ULBP2-5-6 (FAB1298P, R&D systems) or using a recombinant human NKG2D (rhNKG2D-Fc, 1299-NK-050, R&D systems) detected with an anti-human IgG-Fc antibody (12-4998-82, ThermoFisher).

Techniques: Construct, shRNA

CD19/NKG2DL tandem CAR expression. (A) Representative dot plots of CD19- and NKG2DL-binding domain expression detected by staining T-cells with rhCD19-Fc + anti-human IgG-Fc and CD314 antibody. (B) CD19-binding domain MFI and (C) NKG2DL-binding domain MFI. (D) Representative dot plots of CD19- and NKG2DL-binding domain expression detected by staining T-cells with rhCD19-Fc + anti-human IgG-Fc and rhMICA-Avi + streptavidin, respectively, in a subpopulation expressing CD34 marker at similar level. (E) CD19-binding domain MFI and (F) NKG2DL-binding domain MFI when staining was performed with the two ligands simultaneously (black bars) or with one ligand only (grey dashed bars). Adjusted P values (*P<0.05; ***P < 0.001; ****P < 0.0001) were determined by one-way ANOVA with Dunnet’s correction for multiple comparisons. Data presented as means (SD) of n=5 (B, D) or n=3 (E, F) . Each symbol denotes a different PBMC donor.

Journal: Frontiers in Immunology

Article Title: Tandem CAR T-cells targeting CD19 and NKG2DL can overcome CD19 antigen escape in B-ALL

doi: 10.3389/fimmu.2025.1557405

Figure Lengend Snippet: CD19/NKG2DL tandem CAR expression. (A) Representative dot plots of CD19- and NKG2DL-binding domain expression detected by staining T-cells with rhCD19-Fc + anti-human IgG-Fc and CD314 antibody. (B) CD19-binding domain MFI and (C) NKG2DL-binding domain MFI. (D) Representative dot plots of CD19- and NKG2DL-binding domain expression detected by staining T-cells with rhCD19-Fc + anti-human IgG-Fc and rhMICA-Avi + streptavidin, respectively, in a subpopulation expressing CD34 marker at similar level. (E) CD19-binding domain MFI and (F) NKG2DL-binding domain MFI when staining was performed with the two ligands simultaneously (black bars) or with one ligand only (grey dashed bars). Adjusted P values (*P<0.05; ***P < 0.001; ****P < 0.0001) were determined by one-way ANOVA with Dunnet’s correction for multiple comparisons. Data presented as means (SD) of n=5 (B, D) or n=3 (E, F) . Each symbol denotes a different PBMC donor.

Article Snippet: NKG2DL were detected using antibodies directed against human MICA (FAB1300P, R&D systems), MICB (FAB1599P, R&D systems), ULBP1 (FAB1380P, R&D systems), ULBP3 (FAB1517P, R&D systems), ULBP2-5-6 (FAB1298P, R&D systems) or using a recombinant human NKG2D (rhNKG2D-Fc, 1299-NK-050, R&D systems) detected with an anti-human IgG-Fc antibody (12-4998-82, ThermoFisher).

Techniques: Expressing, Binding Assay, Staining, Marker

Cytokine secretion, cytolytic activity and proliferation of CD19/NKG2DL tandem CAR T-cells co-cultured with CD19+ and CD19- Nalm-6 cells. (A) Expression of CD19 and NKG2DL in Nalm-6 and Nalm-6 CD19 KO cells. (B) Expression of individual NKG2DL using antibodies directed against MICA, MICB, ULBP1, ULBP3 or ULBP2-5-6 (grey histograms) vs autofluorescence (white histograms). (C) Secretion of IFN-γ, TNF-α and IL-2 cytokines after a 24-hour co-culture at 1:1 E:T ratio with Nalm-6 and Nalm-6 CD19 KO cells. (D) Cytolytic activity of CAR T-cells against Nalm-6 and Nalm-6 CD19 KO cells at 1:1 and 0.1:1 E:T ratio. Results are expressed as percentage of remaining cancer cells normalized to t0h timepoint. (E) Representative experiment showing CTV histograms of CAR T-cells after 4 days of co-culture without cancer cells or at 1:1 E:T ratio with Nalm-6 and Nalm-6 CD19 KO cells (4 individual experiments were performed on 4 different donors). Adjusted P values (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) were determined by one-way ANOVA with Dunnet’s correction for multiple comparisons. Data presented as means (SD) of n=5. Each symbol denotes a different PBMC donor.

Journal: Frontiers in Immunology

Article Title: Tandem CAR T-cells targeting CD19 and NKG2DL can overcome CD19 antigen escape in B-ALL

doi: 10.3389/fimmu.2025.1557405

Figure Lengend Snippet: Cytokine secretion, cytolytic activity and proliferation of CD19/NKG2DL tandem CAR T-cells co-cultured with CD19+ and CD19- Nalm-6 cells. (A) Expression of CD19 and NKG2DL in Nalm-6 and Nalm-6 CD19 KO cells. (B) Expression of individual NKG2DL using antibodies directed against MICA, MICB, ULBP1, ULBP3 or ULBP2-5-6 (grey histograms) vs autofluorescence (white histograms). (C) Secretion of IFN-γ, TNF-α and IL-2 cytokines after a 24-hour co-culture at 1:1 E:T ratio with Nalm-6 and Nalm-6 CD19 KO cells. (D) Cytolytic activity of CAR T-cells against Nalm-6 and Nalm-6 CD19 KO cells at 1:1 and 0.1:1 E:T ratio. Results are expressed as percentage of remaining cancer cells normalized to t0h timepoint. (E) Representative experiment showing CTV histograms of CAR T-cells after 4 days of co-culture without cancer cells or at 1:1 E:T ratio with Nalm-6 and Nalm-6 CD19 KO cells (4 individual experiments were performed on 4 different donors). Adjusted P values (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) were determined by one-way ANOVA with Dunnet’s correction for multiple comparisons. Data presented as means (SD) of n=5. Each symbol denotes a different PBMC donor.

Article Snippet: NKG2DL were detected using antibodies directed against human MICA (FAB1300P, R&D systems), MICB (FAB1599P, R&D systems), ULBP1 (FAB1380P, R&D systems), ULBP3 (FAB1517P, R&D systems), ULBP2-5-6 (FAB1298P, R&D systems) or using a recombinant human NKG2D (rhNKG2D-Fc, 1299-NK-050, R&D systems) detected with an anti-human IgG-Fc antibody (12-4998-82, ThermoFisher).

Techniques: Activity Assay, Cell Culture, Expressing, Co-Culture Assay

Cytokine secretion of CD19/NKG2DL tandem CAR T-cells upon stimulation with coated CD19 or coated MICA. IFN-γ secretion after a 24-hour exposure with increasing concentrations of (A) coated rhCD19-Avi or (B) coated rhMICA-Fc.

Journal: Frontiers in Immunology

Article Title: Tandem CAR T-cells targeting CD19 and NKG2DL can overcome CD19 antigen escape in B-ALL

doi: 10.3389/fimmu.2025.1557405

Figure Lengend Snippet: Cytokine secretion of CD19/NKG2DL tandem CAR T-cells upon stimulation with coated CD19 or coated MICA. IFN-γ secretion after a 24-hour exposure with increasing concentrations of (A) coated rhCD19-Avi or (B) coated rhMICA-Fc.

Article Snippet: NKG2DL were detected using antibodies directed against human MICA (FAB1300P, R&D systems), MICB (FAB1599P, R&D systems), ULBP1 (FAB1380P, R&D systems), ULBP3 (FAB1517P, R&D systems), ULBP2-5-6 (FAB1298P, R&D systems) or using a recombinant human NKG2D (rhNKG2D-Fc, 1299-NK-050, R&D systems) detected with an anti-human IgG-Fc antibody (12-4998-82, ThermoFisher).

Techniques:

Cytolytic activity and proliferation of CD19/NKG2DL tandem CAR T-cells upon repeated antigen stimulation. (A) Experimental design of the repeated antigen stimulation assay (B) Cytolytic activity of single and tandem CAR T-cells against HeLa CD19 cells at 1:1 E:T ratio during successive cycles of stimulation. Results are expressed as percentage of remaining cancer cells normalized to t0h timepoint. (C) Cumulative expansion of T-cells after four cycles of stimulation with HeLa CD19 cells. (D) Cytolytic activity of single and tandem CAR T-cells against HeLa WT cells at 1:1 E:T ratio during successive cycles of stimulation. Results are expressed as percentage of remaining cancer cells normalized to t0h timepoint. (E) Cumulative expansion of T-cells after four cycles of stimulation with HeLa WT cells. Adjusted P values (*P < 0.05; ***P < 0.001; ****P < 0.0001) were determined by one-way ANOVA with Dunnet’s correction for multiple comparisons. Data from (B) and (D) presented as means (SD) of n=5. Data from (C) and (E) were log-transformed to achieve a normal distribution and presented as box plots with 10 th percentile, median, and 90 th percentile (n=5). Each symbol denotes a different PBMC donor.

Journal: Frontiers in Immunology

Article Title: Tandem CAR T-cells targeting CD19 and NKG2DL can overcome CD19 antigen escape in B-ALL

doi: 10.3389/fimmu.2025.1557405

Figure Lengend Snippet: Cytolytic activity and proliferation of CD19/NKG2DL tandem CAR T-cells upon repeated antigen stimulation. (A) Experimental design of the repeated antigen stimulation assay (B) Cytolytic activity of single and tandem CAR T-cells against HeLa CD19 cells at 1:1 E:T ratio during successive cycles of stimulation. Results are expressed as percentage of remaining cancer cells normalized to t0h timepoint. (C) Cumulative expansion of T-cells after four cycles of stimulation with HeLa CD19 cells. (D) Cytolytic activity of single and tandem CAR T-cells against HeLa WT cells at 1:1 E:T ratio during successive cycles of stimulation. Results are expressed as percentage of remaining cancer cells normalized to t0h timepoint. (E) Cumulative expansion of T-cells after four cycles of stimulation with HeLa WT cells. Adjusted P values (*P < 0.05; ***P < 0.001; ****P < 0.0001) were determined by one-way ANOVA with Dunnet’s correction for multiple comparisons. Data from (B) and (D) presented as means (SD) of n=5. Data from (C) and (E) were log-transformed to achieve a normal distribution and presented as box plots with 10 th percentile, median, and 90 th percentile (n=5). Each symbol denotes a different PBMC donor.

Article Snippet: NKG2DL were detected using antibodies directed against human MICA (FAB1300P, R&D systems), MICB (FAB1599P, R&D systems), ULBP1 (FAB1380P, R&D systems), ULBP3 (FAB1517P, R&D systems), ULBP2-5-6 (FAB1298P, R&D systems) or using a recombinant human NKG2D (rhNKG2D-Fc, 1299-NK-050, R&D systems) detected with an anti-human IgG-Fc antibody (12-4998-82, ThermoFisher).

Techniques: Activity Assay, Transformation Assay

In vivo anti-tumor activity of CD19/NKG2DL tandem CD8s CAR T-cells in a B-ALL relapse model. (A) The B-ALL relapse model was established in NSG mice by injecting 0.1x10 6 Nalm-6 tumor cells (mix WT: CD19 KO 1:1) on day 0, followed by an injection of 5x10 6 CAR T-cells on day 7, and 3 re-challenges of 0.1x10 6 Nalm-6 CD19 KO cells on day 14, 21 and 28. Study was terminated on day 70. (B) Tumor burden in individual mice followed by BLI (n=5, except in mock group n=3) until D60. (C) Images depicting the tumor burden monitored by BLI at the indicated time points. (D) Kaplan–Meier curves showing survival of NSG mice injected with control, single or tandem CAR T-cells. Adjusted P value (P**<0.005) was determined by Logrank Mantel–Cox test.

Journal: Frontiers in Immunology

Article Title: Tandem CAR T-cells targeting CD19 and NKG2DL can overcome CD19 antigen escape in B-ALL

doi: 10.3389/fimmu.2025.1557405

Figure Lengend Snippet: In vivo anti-tumor activity of CD19/NKG2DL tandem CD8s CAR T-cells in a B-ALL relapse model. (A) The B-ALL relapse model was established in NSG mice by injecting 0.1x10 6 Nalm-6 tumor cells (mix WT: CD19 KO 1:1) on day 0, followed by an injection of 5x10 6 CAR T-cells on day 7, and 3 re-challenges of 0.1x10 6 Nalm-6 CD19 KO cells on day 14, 21 and 28. Study was terminated on day 70. (B) Tumor burden in individual mice followed by BLI (n=5, except in mock group n=3) until D60. (C) Images depicting the tumor burden monitored by BLI at the indicated time points. (D) Kaplan–Meier curves showing survival of NSG mice injected with control, single or tandem CAR T-cells. Adjusted P value (P**<0.005) was determined by Logrank Mantel–Cox test.

Article Snippet: NKG2DL were detected using antibodies directed against human MICA (FAB1300P, R&D systems), MICB (FAB1599P, R&D systems), ULBP1 (FAB1380P, R&D systems), ULBP3 (FAB1517P, R&D systems), ULBP2-5-6 (FAB1298P, R&D systems) or using a recombinant human NKG2D (rhNKG2D-Fc, 1299-NK-050, R&D systems) detected with an anti-human IgG-Fc antibody (12-4998-82, ThermoFisher).

Techniques: In Vivo, Activity Assay, Injection, Control

In vitro activity of CD19/NKG2DL tandem CD8s CAR T-cells against primary B-ALL bone marrow samples. Secretion of IFN-γ after 24 hours of co-culture at 1:1 E:T ratio with bone marrow specimen from two B-ALL patients. Data presented as means (SD) of n=2 technical replicates.

Journal: Frontiers in Immunology

Article Title: Tandem CAR T-cells targeting CD19 and NKG2DL can overcome CD19 antigen escape in B-ALL

doi: 10.3389/fimmu.2025.1557405

Figure Lengend Snippet: In vitro activity of CD19/NKG2DL tandem CD8s CAR T-cells against primary B-ALL bone marrow samples. Secretion of IFN-γ after 24 hours of co-culture at 1:1 E:T ratio with bone marrow specimen from two B-ALL patients. Data presented as means (SD) of n=2 technical replicates.

Article Snippet: NKG2DL were detected using antibodies directed against human MICA (FAB1300P, R&D systems), MICB (FAB1599P, R&D systems), ULBP1 (FAB1380P, R&D systems), ULBP3 (FAB1517P, R&D systems), ULBP2-5-6 (FAB1298P, R&D systems) or using a recombinant human NKG2D (rhNKG2D-Fc, 1299-NK-050, R&D systems) detected with an anti-human IgG-Fc antibody (12-4998-82, ThermoFisher).

Techniques: In Vitro, Activity Assay, Co-Culture Assay

Clinical trials of CAR NK cell agents for solid cancers

Journal: Experimental Hematology & Oncology

Article Title: The clinical landscape of CAR NK cells

doi: 10.1186/s40164-025-00633-8

Figure Lengend Snippet: Clinical trials of CAR NK cell agents for solid cancers

Article Snippet: NKG2DL , Acute Myeloid Leukemia, Myelodysplastic Syndromes , Peripheral Blood , Phase 1 , 2020-09-21 , Active, not recruiting , 61 , Nkarta Inc , NCT04623944.

Techniques: Clinical Proteomics

Clinical trials of CAR NK cell agents for hematologic cancers

Journal: Experimental Hematology & Oncology

Article Title: The clinical landscape of CAR NK cells

doi: 10.1186/s40164-025-00633-8

Figure Lengend Snippet: Clinical trials of CAR NK cell agents for hematologic cancers

Article Snippet: NKG2DL , Acute Myeloid Leukemia, Myelodysplastic Syndromes , Peripheral Blood , Phase 1 , 2020-09-21 , Active, not recruiting , 61 , Nkarta Inc , NCT04623944.

Techniques: Clinical Proteomics

Clinical trials of CAR NK cell agents for autoimmune and infectious diseases

Journal: Experimental Hematology & Oncology

Article Title: The clinical landscape of CAR NK cells

doi: 10.1186/s40164-025-00633-8

Figure Lengend Snippet: Clinical trials of CAR NK cell agents for autoimmune and infectious diseases

Article Snippet: NKG2DL , Acute Myeloid Leukemia, Myelodysplastic Syndromes , Peripheral Blood , Phase 1 , 2020-09-21 , Active, not recruiting , 61 , Nkarta Inc , NCT04623944.

Techniques: Clinical Proteomics

Clinical trials of CAR NK cell agents

Journal: Experimental Hematology & Oncology

Article Title: The clinical landscape of CAR NK cells

doi: 10.1186/s40164-025-00633-8

Figure Lengend Snippet: Clinical trials of CAR NK cell agents

Article Snippet: NKG2DL , Acute Myeloid Leukemia, Myelodysplastic Syndromes , Peripheral Blood , Phase 1 , 2020-09-21 , Active, not recruiting , 61 , Nkarta Inc , NCT04623944.

Techniques: Clinical Proteomics