cd28 mab  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: CAR T cells targeting Nectin-4 safely overcome resistance to anti-Nectin-4 antibody-drug conjugate in solid tumors

doi: 10.1101/2025.09.30.679440

Figure Lengend Snippet: A. Schematic illustration of the protocol. B. SUM190PT tumor growth control by a single injection of CARTN4 cells in vivo . SUM190PT cells were orthotopically injected into the mammary fat pad of female NSG mice. Mice received a single i.v. injection of CART19 as vehicle control or CARTN4 cells. n = 5 CD19-CAR and n = 5 Nectin-4-CAR animals with 2 tumors each were examined in one experiment. C. CD3+GFP+ absolute count per gram of tumor and CD4 rate are higher in Nectin-4 tumor. Tumors were harvested and dissociated 10 days after CAR-T cells treatment. D. Effector memory and EMRA T-cells rate in CD4 T-cells in tumor at day 10. E. Flow cytometry results illustrating the higher frequency of TEMRA in CD19-CAR-T cells and the higher frequency of effector memory T-cells in Nectin-4-CAR-T cells. F. PD1 and TIM3 double positive T-cells rate in CD4 T-cells in tumors at day 10. G. Flow cytometry results illustrating the higher frequency of PD1 and TIM3 double positive T-cells in CD19-CAR-T cells showing a more exhausted phenotype. H. ELISA assay dosing TNFα and IFNγ in tumor dissociation supernatants revealing higher concentrations of both cytokines in CARTN4 / tumors conditions. I. IHC on mouse SUM190PT tumor 10 days after CAR treatment showing a massive CARTN4 cell infiltration. Each point represents one tumor. 4 tumors were harvested from 2 mice per condition. Data in panels B, C, D, F and H are presented as Mean +/- SEM. P-values were calculated using a 2way ANOVA in panel B and using a t-test in panel C, D, F and H. Source data are provided in the Source Data file.

Article Snippet: Human PBMCs were activated with purified anti-human CD3 mAb ( clone OKT3, Miltenyi Biotec) at 50 ng/mL and purified anti-human CD28 mAb ( clone CD28.2, Biolegend) at 125 ng/mL in CTS OptimizerTM T-cell expansion media (ThermoFisher Scientific) supplemented with 5% human AB serum (Fisher Scientific), 5% CTS Immune cell serum (ThermoFisher Scientific) and 300 U/mL IL-2 (Miltenyi Biotec) at density of 2x10 cells/mL for 48 hrs.

Techniques: Control, Injection, In Vivo, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: CAR T cells targeting Nectin-4 safely overcome resistance to anti-Nectin-4 antibody-drug conjugate in solid tumors

doi: 10.1101/2025.09.30.679440

Figure Lengend Snippet: A. Schematic illustration of the protocol. B. SUM190PT tumor total regression with a double injection of CARTN4 cells in vivo . SUM190PT cells were orthotopically injected into the mammary fat pad of female NSG mice. Mice received a double i.v. injection of CART19 as vehicle control or Nectin-4-CAR T cells. n = 5 CD19-CAR and n = 5 Nectin-4-CAR animals with 2 tumors each were examined in one experiment. C. CD3+GFP+ absolute count per gram of tumor is higher in Nectin-4 tumor until the 12 th day. Tumors were harvested and dissociated at day 10, 12 and 20 after CAR-T cells treatment. D. CD4 and CD8 rate in CAR-T cells showing a increase in the CD8 population at day 20 for CARTN4 cells. E. Effector memory T-cells rate in CD4 T-cells increases over time until reaching that of CARTN4 cells at day 20 whereas EMRA T-cells rate decreases. F. Flow cytometry results illustrating the higher frequency of TEMRA in CART19 cells and the higher frequency of effector memory T-cells in Nectin-4-CAR-T cells at day 12. G. PD1 and TIM3 double positive T-cells rate in CD4 T-cells in tumors at day 10. E. Flow cytometry results illustrating the higher frequency of PD1 and TIM3 double positive T-cells in CART19 over time until reaching that of CART1N4 at day 20. H. ELISA assay dosing TNFα and IFNγ in tumor dissociation supernatants revealing higher concentrations of both cytokines in CARTN4 / tumors whatever the day. 4 tumors were harvested from 2 mice per condition. Data in panels B, C, D, E, G and I are presented as Mean +/- SEM. P-values were calculated using a 2way ANOVA. Source data are provided in the Source Data file.

Article Snippet: Human PBMCs were activated with purified anti-human CD3 mAb ( clone OKT3, Miltenyi Biotec) at 50 ng/mL and purified anti-human CD28 mAb ( clone CD28.2, Biolegend) at 125 ng/mL in CTS OptimizerTM T-cell expansion media (ThermoFisher Scientific) supplemented with 5% human AB serum (Fisher Scientific), 5% CTS Immune cell serum (ThermoFisher Scientific) and 300 U/mL IL-2 (Miltenyi Biotec) at density of 2x10 cells/mL for 48 hrs.

Techniques: Injection, In Vivo, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay