Journal: Science advances

Article Title: Multi-armored allogeneic MUC1 CAR T cells enhance efficacy and safety in triple-negative breast cancer.

doi: 10.1126/sciadv.adn9857

Figure Lengend Snippet: Fig. 4. TGFBR2KO armored UCARTM1 shows resistance to inhibitory effects of TGFB1. (A) Schematic representation of the non-alloreactive CAR T cell with TRAC and TGFBR2 knockout with TALEN (UCARTM1∆TGFBR2). (B) Functional phenotyping of TGFBR2 knockout edited UCARTM1 compared to the unedited UCARTM1 via flow cytometry analysis of pSMAD2/3 staining in the presence of TGFB1. (C) Flow cytometry analysis of the percentage of CD25+ cells present in UCARTM1 and UCARTM1∆TGFBR2 following activation with MUC1 recombinant protein, in the presence or absence of TGFB1 (n = 2). Statistical significance was calculated using unpaired t test. (D) Proliferation assay for MUC1 recombinant protein activated UCARTM1 and UCARTM1∆TGFBR2 in the presence or absence of TGFB1 at days 0, 4, 6, 8, and 11 (n = 2 technical replicates per time point). Statistical significance was determined using a two-way ANOVA. (E) Design of in vivo experiment and individual tumor growth comparison of tumors treated intravenously with 5 million UCARTM1∆TGFBR2, UCARTM1, NTD, or PBS (n = 4 to 5 per cohort) for donor 1. Experiment repeated with donor 2 is shown in the Supplementary Materials. Two- way ANOVA was performed for comparisons over time for tumor growth. (F) Design of in vivo experiment, individual tumor growth comparison of tumors, and (G) Kaplan- Meier survival analysis of cohorts treated intratumorally with 2 million UCARTM1∆TGFBR2 (n = 4), UCARTM1 (n = 4), NTD (n = 2), or PBS (n = 3). Each point represents a biological replicate for (E) to (G). Statistical significance was calculated using mixed-effects analysis comparisons over time for tumor growth and the log-rank Mantel-Cox test. *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns (not significant) indicates P > 0.05. (H) TGFB1 ELISA analysis of HCC70-GFP tumors averaging 50 to 100, 250, 450, and 600 mm3 per mm3 of tumor (n = 2 to 6). Each point represents a biological replicate. Treatments were done using 2 × 106 or 5 × 106 CAR+ cells as indicated on each in vivo design.

Article Snippet: IL- 12, IFNG, and TGFB1 ELISAs IL- 12 levels were measured with Quantikine ELISA Human IL- 12 p70 Immunoassay (R&D Systems, no. D1200) using 30 μl of blood.

Techniques: Knock-Out, Functional Assay, Flow Cytometry, Staining, Activation Assay, Recombinant, Proliferation Assay, In Vivo, Comparison, Enzyme-linked Immunosorbent Assay

Journal: Science advances

Article Title: Multi-armored allogeneic MUC1 CAR T cells enhance efficacy and safety in triple-negative breast cancer.

doi: 10.1126/sciadv.adn9857

Figure Lengend Snippet: Fig. 5. PD1KO/IL-12KI and TGFBR2KO armored CAR T cells clear tumors within weeks without relapse with less CAR T cell expansion. (A) Schematic representation of the non- alloreactive CAR T cell with TRAC, PDCD1, and TGFBR2 knockouts and IL-12 knock-in. (B) Proliferation assay of MUC1 recombinant protein–activated UCARTM1∆PD1/IL12 and UCARTM1∆PD1/IL12; ∆TGFBR2 with or without TGFB1 (n = 2 technical replicates per time point). Two-way ANOVA was performed for statistical significance over time. (C) Cytotoxic assay of T47D cells cocultured with NTD, UCARTM1, UCARTM1∆TGFBR2, UCARTM1∆PD1/IL12, and UCARTM1∆PD1/IL12; ∆TGFBR2in the presence of TGFB1 with a 10-to-1 E:T ratio. CAR T cells were rechallenged after 24 hours (n = 3). Each point represents a technical replicate. Statistical significance was calculated using unpaired t test. (D) Design of in vivo experiment. (E) Tumor growth curve for cohorts intravenously treated with 5 million UCARTM1∆PD1/IL12; ∆TGFBR2, UCARTM1∆PD1/IL12, UCARTM1∆TGFBR2, UCARTM1, NTD, or PBS (n = 5 to 6 per cohort) and (F) flow cytometry analysis of PB collected from all cohorts, 40 days after treatment for number and percent of hCD45+ cells. Two-way ANOVA was performed for comparisons for tumor growth, and ordinary one-way ANOVA was performed to determine the statistical significance for (F). (G) Color mapping of tumor clearance and inflammation in the mam- mary glands of UCARTM1∆PD1/IL12- and UCARTM1∆PD1/IL12; ∆TGFBR2-treated cohorts (n = 5 and 6, consecutively) and representative dissection images of tumor cell injected (inj.) and no tumor cell injected mammary fat pad (MFP) (Control; Ctrl) of one animal each. (H) Kaplan-Meier survival analysis of cohorts treated with UCARTM1∆PD1/IL12; ∆TGFBR2, UCARTM1∆PD1/IL12, UCARTM1∆TGFBR2, UCARTM1, NTD, or PBS (n = 4 to 6 per cohort). Each point represents a biological replicate for (E) to (G). Statistical significance was calculated using the log-rank Mantel-Cox test in (H). *P < 0.05, **P ≤ 0.01, ****P ≤ 0.000, ns (not significant) indicates P > 0.05. All treatments were done using 5 × 106 CAR+ cells.

Article Snippet: IL- 12, IFNG, and TGFB1 ELISAs IL- 12 levels were measured with Quantikine ELISA Human IL- 12 p70 Immunoassay (R&D Systems, no. D1200) using 30 μl of blood.

Techniques: Knock-In, Proliferation Assay, Recombinant, In Vivo, Flow Cytometry, Dissection, Injection, Control

Journal: International Journal of Nanomedicine

Article Title: IL-12-Overexpressed Nanoparticles Suppress the Proliferation of Melanoma Through Inducing ICD and Activating DC, CD8 + T, and CD4 + T Cells

doi: 10.2147/IJN.S442446

Figure Lengend Snippet: CPP transferred IL-12 into B16F10 cells and CPP-IL-12 induced immunogenic cell death. ( A ) The structure of IL-12 vector. ( B ) Western blot detection of IL-12 expression. ** P < 0.01, Student’s t -test. ( C ) ELISA. CPP-IL-12 increased IL-12 levels in the supernatant of B16-F10 cells, and 2.0 W/cm 2 of laser further increased IL-12 levels. ** P < 0.01, ANOVA. ( D ) Live/dead staining assay. Scale bars=100 μm. ( E ) The number of dead cells. CPP-IL-12 increased and 2.0 W/cm 2 of laser further increased the number of dead cells. ** P < 0.01, ANOVA. ( F ) Cell apoptosis analysis. CPP-IL-12 increased and laser treatment further increased the number of apoptotic cells. ( G ) Immunofluorescence. Green color, CALR expression. Scale bars=2 μm. ( H, I ) ELISA analysis of extracellular and intracellular HMGB1 levels, respectively. * P < 0.05, ** P < 0.01, ANOVA. (J) ATP levels. The ATP levels decreased in CPP-IL-12-treated cells, and laser treatment further reduced ATP content. ** P < 0.01, ANOVA. ( K ) CPP increased CD80 + /CD86 + expression of DC.

Article Snippet: IL-12 and HMGB1 levels were estimated using ELISA kits (CSB-E04600m and CSB-E08225M, CUSABIO, Wuhan, China) in accordance with the manufacturer′s instructions.

Techniques: Plasmid Preparation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence