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

Journal: bioRxiv

Article Title: Ex vivo drug testing in metastatic biopsies reveals patient-specific vulnerabilities to cancer targeting and immune activating drugs

doi: 10.64898/2026.02.06.704037

Figure Lengend Snippet: ( A ) Schematic illustration of the drug response assay. Created with BioRender.com. TME, tumor microenvironment; IF, immunofluorescence. ( B ) Cellular composition of malignant pleural effusions (MPEs) from five NSCLC patients as determined by flow cytometry. ( C ) ATP-based viability of a MPE in different culture media. Each dot represents the mean ± standard deviation (shaded envelope) of n = 5 technical replicates from one representative donor (P179). ( D ) Flow cytometric analysis of MPE viability in HPLM medium. Each dot represents an individual donor (n = 5). P-values were calculated using Wilcoxon matched-pairs signed rank test. ( E ) Log2-fold change (Log2FC) in cellular composition of MPEs after 5 days in HPLM medium compared to baseline, analyzed by flow cytometry. Each dot represents an individual donor. Samples from n = 5 donors were measured. To illustrate fold changes, cell fractions below the 2%-detection limit were excluded from analysis. P-values were determined using a one-sample Wilcoxon test against zero. ( F ). As for (E), but for marker expression of cancer cells (PD-L1, Nectin-4, TROP2) and T cells (PD-1). ( G ) Cytokine secretion and immune checkpoint expression of MPEs (n = 5) after 5 days of ex vivo culture. Color scale represents Z-scores normalized across patients for each cytokine or marker. nMFI, mean fluorescence intensity normalized to fluorescence minus one (FMO) control. ( H ) Relative cell loss of two non-adherent cell lines following the optimized IHC liquid handling protocol. Dots represent outliers among technical replicate wells across n = 3 biological replicates (384-well plates). P-values were calculated using a one-sample Wilcoxon test. ( I ) Pseudocolor plot depicting MFIs of CD45 and EpCAM signal for all segmented masks of a spike-in of MCF-7 cells in lymph node cells. Cell populations were classified based on manual gating. ( J ) Number of cancer (left) and immune cells (right) detected using our image analysis pipeline. Box plots represent data of n = 10 technical replicate wells. ( K ) Cancer cell fractions in malignant pleural effusions (MPEs) from NSCLC patients (n = 5), determined by EpCAM-based immunocytochemistry. The dashed line indicates the detection limit (10 cells per well) of the drug response assay.

Article Snippet: Cells were resuspended in 100 μL of FACS buffer (1% (w/v) BSA, 2 mM EDTA in DPBS) per 1 x 10 6 cells and stained using the following antibodies: Alexa Fluor® 488 anti-human CD3, clone HIT3a (BioLegend, #300319, RRID:AB_493690), PerCP/Cyanine5.5 anti-human CD14, clone HCD14 (BioLegend, # 325621, RRID:AB_893252), PE anti-human CD279 (PD-1), clone EH12.2H7 (BioLegend, # 329905, RRID:AB_940481), PE/Cyanine7anti-human CD11c, clone Bu15 (BioLegend, # 337215, RRID:AB_2129791), APC-Vio770 anti-human CD19, clone LT19 (1:50; Miltenyi Biotec, # 130-098-073, RRID:AB_2661296), Brilliant VioletTM 421 anti-human CD45, clone 2D1 (BioLegend, # 368522, RRID:AB_2687375), Brilliant VioletTM 605 anti-human HLA-DR, clone L243 (BioLegend, # 307639, RRID:AB_11219187), Brilliant VioletTM 785 anti-human CD56 (NCAM), clone 167 (BioLegend, # 362549, RRID:AB_2566058), Alexa Fluor® 488 anti-human CD326 (EpCAM), clone Co17-1A (BioLegend, # 369808, RRID:AB_2650905), PE anti-human TROP2, REAfinityTM (1:50; Miltenyi Biotec, # 130-115-097, RRID:AB_2726914), APC anti-human Nectin-4, REAfinityTM (1:50; Miltenyi Biotec, # 130-116-103, RRID:AB_2727350), Brilliant VioletTM 421 anti-human CD274 (B7-H1, PD-L1), clone 29E.2A3 (BioLegend, # 329714, RRID:AB_2563852), Brilliant VioletTM 785 anti-human CD45, clone HI30 (BioLegend, # 304048, RRID:AB_2563129), Zombie AquaTM (1:1,000; BioLegend, # 423102), Zombie NIRTM (1:500; BioLegend, # 423106).

Techniques: Immunofluorescence, Flow Cytometry, Standard Deviation, Marker, Expressing, Ex Vivo, Fluorescence, Control, Immunocytochemistry