Journal: Frontiers in Immunology

Article Title: Engineered antibody cytokine chimera synergizes with DNA-launched nanoparticle vaccines to potentiate melanoma suppression in vivo

doi: 10.3389/fimmu.2023.1072810

Figure Lengend Snippet: Design, pharmacokinetic and pharmacodynamic profiles of novel ACC TA99-HL2-KOA1. (A) Model depiction of engineered cytokine mimic HL2-KOA1 binding to IL-2Rα. IL-2 shown in blue, IL-2Rα shown in grey. Positions in contact with F42 shown as grey spheres. F42 and F42V shown as pink and red spheres respectively. Clash scores provided in total Rosetta energy units (REU). (B) Reducing SDS-PAGE analysis comparing the migration patterns of TA99-WT, TA99-Neo2/15, TA99-HL2-KOA1, pVAX backbone control transfection supernatants. (C) Binding of recombinant TA99-WT, TA99-Neo2/15, and TA99-HL2-KOA1 to recombinant human Tyrp1 compared to a murine isotype IgG2a control. (D) Binding of recombinant TA99-WT, TA99-Neo2/15, and TA99-HL2-KOA1 to recombinant human IL2-Rα compared to human IL2-Fc control. (E) Binding of recombinant TA99-WT, TA99-Neo2/15, and TA99-HL2-KOA1 to recombinant human IL2-Rβ compared to human IL2-Fc control. (F) Geometric mean fluorescence intensity of phosphorylated STAT5 in CD25+ CD4+ CD19- T cells from naïve mice (n=6) treated with indicated ACC. (G) Pharmacokinetic profile of recombinant TA99-WT and TA99-Neo-2/15 antibodies; C57BL/6 mice were injected intraperitoneally with 100µg of ACC constructs (n=5 mice per group). Serum at the indicated timepoints was assessed for TYRP1 binding by ELISA. (H) Total serum cytokine level over 14-day period in terms of area under the curve (AUC) in mice treated with ACC. (I) Timecourse of serum TNFα levels in sera of mice administered 100µg ACC. Two sera pools were used for (H, I) . (J) Survival curves of mice (n=10 mice per group) post tumor challenge following treatment with anti-PD1 alone, or anti-PD1 (200µg) + TA99-Neo2/15 (50µg) + TriVax (10µg each of Trp2, Gp100 and Tyrp1), or anti-PD1 (200µg) + TA99-HL2-KOA1 (50µg) + TriVax. 1x10 5 B16F10 cells subcutaneously into the right flank of C57BL/6 mice on Day 0. Green arrows indicate administration of treatment. (K) Survival curves of mice (n=10 mice per group) post tumor challenge following treatment with anti-PD1 alone, or anti-PD1 (200µg) + TA99-HL2-KOA1 (50µg) + TriVax, or anti-PD1 + TA99 (50µg) + Human IL2 (5µg) + TriVax. Challenge and treatment scheme was performed as in (J) Error bars represent standard deviation; non-parametric Mann Whitney T test compared with TA99-WT used in (F) ; non-parametric Kruskal-Wallis ANOVA was used in (I) ; log-rank test was used to compare between differences in all survival curves; *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: In addition, serially diluted recombinant protein variants of antibody-cytokine chimera or recombinant mouse Fc-tagged IL2 (IL2-Fc) (Molecular Innovations, Cat# MIL2-FC-0.05MG) were used in place of serially diluted mouse sera.

Techniques: Binding Assay, SDS Page, Migration, Control, Transfection, Recombinant, Fluorescence, Injection, Construct, Enzyme-linked Immunosorbent Assay, Standard Deviation, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: Characterization of the Therapeutic Effects of Novel Chimeric Antigen Receptor T Cells Targeting CD38 on Multiple Myeloma

doi: 10.3389/fonc.2021.703087

Figure Lengend Snippet: Cytotoxicity and proliferation of CD38 CAR-T cells in vitro . (A) The ability of CAR-T cells to kill tumor cells was measured by luciferase assay. CD38 CAR-T cells or pan-T cells were incubated with RPMI-gfp-luc cells or Raji-luc cells, and the luciferase signal was determined after 12 hours. (B) The ability of CAR-T cells to kill tumor cells was measured by an IncuCyte S3 system. RPMI-gfp-luc cells were incubated with CD38 CAR-T cells or pan-T cells at an E:T ratio of 1:1. Total integrated GFP intensity per well was assessed as a quantitative measure of live, GFP + tumor cells. The values were normalized to the starting values. (C) The ability of CD38 CAR-T cells to kill tumor cells was determined by flow cytometry. CD38 CAR-T cells or pan-T cells were incubated with RPMI-gfp-luc cells, Raji-luc cells, Daudi cells or K562-hBCMA cells for 12 hours. (D) CD38 CAR-T cells were incubated with RPMI-gfp-luc cells for 12 hours, and the secretion of cytokines was measured with a flow cytometry-based CBA kit. The graph shows the secretion of TNFα, IFN-γ, IL-2 and granzyme B. (E) The proliferation of CD38 CAR-T cells. The proliferation ability was measured by the CFSE-based assay. Data were obtained from three replicates and are presented as the mean ± SEM (n=3). *indicates p value < 0.05; **< 0.01; ***< 0.005 and ****< 0.001, comparison of two groups was performed by Student’s t-test. TNF-α, tumor necrosis factor α; IFN-γ, interferon γ; IL-2, interleukin 2.

Article Snippet: PBMCs were activated with 100 ng/mL soluble anti-CD3, clone OKT3 (Sino Biological, GMP-10977-H001) and 100 U/mL IL-2 (Sino Biological, GMP-11848-HNAE) and then cultured at 37°C in a 5% CO 2 incubator for 48 hours.

Techniques: In Vitro, Luciferase, Incubation, Flow Cytometry, CFSE Assay

Journal: Cell reports. Medicine

Article Title: Targeting neoadjuvant chemotherapy-induced metabolic reprogramming in pancreatic cancer promotes anti-tumor immunity and chemo-response.

doi: 10.1016/j.xcrm.2023.101234

Figure Lengend Snippet: Figure 6. Targeting CD36 synergistically promoted AG-mediated killing of PDAC in preclinical models (A) Visual presentation of subcutaneous xenograft murine PDAC tumor models (C57 mice) for each group. (B) Measurement of tumor volumes showed CD36 blockage synergistically promoted AG-mediated killing of PDAC in subcutaneous xenograft murine PDAC tumor models. (C) Measurement of tumor weights showed CD36 blockage synergistically promoted AG-mediated killing of PDAC in subcutaneous xenograft murine PDAC tumor models (n = 5). (D) Representative IHC staining showed Ki67 expression in subcutaneous xenografts treated with different regimens. (E) t-Distributed stochastic neighbor embedding (TSNE) analyses showed the clustering for CD36+ CD8+ T cells and GZMB+ CD8+ T cells. (F) Flow cytometry revealed that more CD8+ T cells infiltrated PDAC with NAC, while the percentage of CD36+ CD8+ T cells also increased (n = 5) (mean with standard deviation). (G) ELISA results showed the combination of AG and CD36 blockade significantly improved IFN-g and tumor necrosis factor a (TNF-a) levels intratumorally (n = 5). (H) Representative image of orthotopic murine models of PDAC. (I) Kaplan-Meier curve revealed the combination of CD36 blockade and AG significantly prolonged the survival interval of mice that received orthotopic PDAC cell transplantation (n = 10). Circle or square referred to a happened event (death or censored). Censored event means the mice is still alive at the time point that we ended follow-up. (J) CD36 blockade synergistically with AG regimens optimally narrowed the PDAC tumor size in a humanized PDX model (n = 10). (K) Representative IHC staining image of CD36-high and -low PDAC. (L) Kaplan-Meier curve showed increased CD36 expression predicted worse prognosis of PDAC patients with adjuvant AG chemotherapy. The statistical sig- nificance shown in this figure was detected using t test.

Article Snippet: The ELISA kits used in the present study were as follows: Human IFN-gamma ELISA Kit (absin, abs510007); Human IL-2 ELISA Kit (Boster, EK0397); Mouse TNF alpha ELISA Kit (absin, abs520010) and Mouse IFN- gamma ELISA Kit (absin, abs520007).

Techniques: Immunohistochemistry, Expressing, Flow Cytometry, Standard Deviation, Enzyme-linked Immunosorbent Assay, Transplantation Assay, Adjuvant