Journal: Molecular Therapy Oncology

Article Title: Antigen-binding affinity is a key determinant of the durable antitumor activity of CD5 CAR-T cells

doi: 10.1016/j.omton.2026.201158

Figure Lengend Snippet: Low-affinity CD5 CAR-T cells exhibit reduced fratricide and exhaustion phenotypes (A) Viability of CAR-T cells measured on day 3 after cell seeding. Data are presented as mean ± SD from multiple donors ( n = 3). (B) Culture supernatants were collected on day 3 after cell seeding. IFN-γ and TNF-α levels secreted by CAR -T cells were measured by cytometric bead array. Data are presented as mean ± SD from multiple donors ( n = 3). (C) CD69 expression level of CAR-T cells on day 10 after seeding. Fluorescence intensity was quantified by flow cytometry. Data are presented as mean ± SD from multiple donors ( n = 3). (D) Total T cell number harvested on day 10 after cell seeding. Data are presented as mean ± SD from multiple donors ( n = 3). (E) Expression levels of PD-1 and LAG-3 (surface markers), and TOX (intracellular marker) in CAR-T cells on day 10 after seeding. Surface markers were analyzed by flow cytometry, and TOX expression was assessed via intracellular staining following fixation and permeabilization. Data are presented as mean ± SD from multiple donors ( n = 3). All statistical significance was assessed using a linear mixed-effects model with donor as a random effect and Tukey’s test.

Article Snippet: The following antibodies were used: BioLegend, CD5-PE (clone UCHT2), CD69-PE (clone FN50), LAG3-FITC (Clone 7H2C65), mIgG2a-AF647 (clone RMG2a-62), and streptavidin-AF647; BD Biosciences, CD4-V500 (clone RPA-T4), CD8-V450 (clone RPA-T8), αβ TCR-PE (clone IP26), and γδ TCR-BV421 (clone 11F2); Thermo Fisher Scientific, PD-1-PE (clone J105) and CD27-PE (clone O323); Miltenyi Biotec, TOX-APC (clone REA473), Vδ1-PE-Cy7 (clone REA173), and Vδ2-V500 (clone 123R3); Cell Signaling Technology, Myc-AF647 or PE (clone 9B11).

Techniques: Expressing, Fluorescence, Flow Cytometry, Marker, Staining

Journal: Molecular Therapy Oncology

Article Title: Antigen-binding affinity is a key determinant of the durable antitumor activity of CD5 CAR-T cells

doi: 10.1016/j.omton.2026.201158

Figure Lengend Snippet: Affinity-tuned A2 variants provide functional evidence that antigen-binding affinity plays a key role in regulating fratricide and T cell exhaustion (A) Binding level of recombinant anti-CD5 scFvs to CD5-positive and CD5-negative cell lines, as measured by flow cytometry. Data are presented as mean ± SD from technical replicates ( n = 3). (B) Affinity properties of anti-CD5 scFvs as determined by biolayer interferometry using Octet system. A2 scFv was used as controls. (For definitions of K D , K a , K dis , and curve fit parameters, refer to legend.) (C) Viability of CAR-T cells measured on day 3 after cell seeding. Data are presented as mean ± SD from multiple donors ( n = 3). (D) Culture supernatants were collected on day 3 to assess IFN-γ and TNF-α secretion, measured using cytometric bead array. Data are presented as mean ± SD from technical replicates ( n = 3). (E) Total T cell number harvested on day 10 after cell seeding. Data are presented as mean ± SD from multiple donors ( n = 3). (F) CD69 expression levels in CAR-T cells on day 10 after cell seeding, measured by surface staining and flow cytometry. Data are presented as mean ± SD from multiple donors ( n = 3). (G) Expression levels of PD-1 and LAG-3 (surface markers) and TOX (intracellular) in CAR-T cells on day 10 after seeding. Surface markers were analyzed by flow cytometry, and TOX expression was assessed via intracellular staining. Data are presented as mean ± SD from multiple donors ( n = 3). (H) 3 × 10 5 Myc + CAR-T cells were co-cultured with 1 × 10 5 Jurkat cells every 3–4 days for repeated antigen stimulation. Expansion of Myc + CAR-T cells was measured weekly by flow cytometry. Data are presented as mean ± SD from technical replicates ( n = 3). Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test for (A, D, and H) and a linear mixed-effects model with donor as a random effect followed by Tukey’s test for (C–G).

Article Snippet: The following antibodies were used: BioLegend, CD5-PE (clone UCHT2), CD69-PE (clone FN50), LAG3-FITC (Clone 7H2C65), mIgG2a-AF647 (clone RMG2a-62), and streptavidin-AF647; BD Biosciences, CD4-V500 (clone RPA-T4), CD8-V450 (clone RPA-T8), αβ TCR-PE (clone IP26), and γδ TCR-BV421 (clone 11F2); Thermo Fisher Scientific, PD-1-PE (clone J105) and CD27-PE (clone O323); Miltenyi Biotec, TOX-APC (clone REA473), Vδ1-PE-Cy7 (clone REA173), and Vδ2-V500 (clone 123R3); Cell Signaling Technology, Myc-AF647 or PE (clone 9B11).

Techniques: Functional Assay, Binding Assay, Recombinant, Flow Cytometry, Expressing, Staining, Cell Culture

Journal: Molecular Therapy Oncology

Article Title: Antigen-binding affinity is a key determinant of the durable antitumor activity of CD5 CAR-T cells

doi: 10.1016/j.omton.2026.201158

Figure Lengend Snippet: High-affinity C7 variant-based CAR5 exhibited increased levels of both fratricide and T cell exhaustion compared to the C7 wild-type CAR5 (A) Binding level recombinant anti-CD5 scFvs to CD5-positive and CD5-negative cell lines, as measured by flow cytometry. Data are presented as pooled mean ± SD from technical replicates ( n = 3). (B) Affinity properties of anti-CD5 scFvs as determined by biolayer interferometry using Octet system. C7 scFv was used as controls. (For definitions of K D , K a , K dis , and curve fit parameters, refer to legend.) (C) Viability of CAR-T cells measured on day 3 after cell seeding. Data are presented as mean ± SD from multiple donors ( n = 3). (D) Culture supernatants were collected on day 3 to assess IFN-γ and TNF-α secretion, measured using cytometric bead array. Data are presented as mean ± SD from technical replicates ( n = 3). (E) Total T cell number harvested on day 10 after cell seeding. Data are presented as mean ± SD from multiple donors ( n = 3). (F) CD69 expression levels in CAR-T cells on day 10 after cell seeding, measured by surface staining and flow cytometry. Data are presented as mean ± SD from multiple donors ( n = 3). (G) Expression levels of PD-1 and LAG-3 (surface markers) and TOX (intracellular) in CAR-T cells on day 10 after seeding. Surface markers were analyzed by flow cytometry, and TOX expression was assessed via intracellular staining. Data are presented as mean ± SD from multiple donors ( n = 3). (H) 3 × 10 5 Myc + CAR-T cells were co-cultured with 1 × 10 5 Jurkat cells every 3–4 days for repeated antigen stimulation. Expansion of Myc + CAR-T cells was measured weekly by flow cytometry. Data are presented as mean ± SD from technical replicates ( n = 3). Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test for (A, D, and H) and a linear mixed-effects model with donor as a random effect followed by Tukey’s test for (C–G).

Article Snippet: The following antibodies were used: BioLegend, CD5-PE (clone UCHT2), CD69-PE (clone FN50), LAG3-FITC (Clone 7H2C65), mIgG2a-AF647 (clone RMG2a-62), and streptavidin-AF647; BD Biosciences, CD4-V500 (clone RPA-T4), CD8-V450 (clone RPA-T8), αβ TCR-PE (clone IP26), and γδ TCR-BV421 (clone 11F2); Thermo Fisher Scientific, PD-1-PE (clone J105) and CD27-PE (clone O323); Miltenyi Biotec, TOX-APC (clone REA473), Vδ1-PE-Cy7 (clone REA173), and Vδ2-V500 (clone 123R3); Cell Signaling Technology, Myc-AF647 or PE (clone 9B11).

Techniques: Variant Assay, Binding Assay, Recombinant, Flow Cytometry, Expressing, Staining, Cell Culture