Journal: ACS nano

Article Title: Eradication of Established Tumors by Chemically Self-Assembled Nanoring (CSAN) Labelled T Cells

doi: 10.1021/acsnano.8b01308

Figure Lengend Snippet: Bispecific CSANs are constructed from αCD3 DHFR2, αEpCAM DHFR2, and BisMTX to form multivalent nanorings. Effector T cells are engaged with bispecific CSANs which tightly bind, due to their high avidity, and generate PAR T cells that facilitate the cell lysis of target cells. Additionally, the use of clinically relevant concentrations of Trimethoprim disassembles CSANs on the cell surface.

Article Snippet: Cell Lines, Culture Conditions and T cell isolation MCF-7 breast cancer cells and U87 MG human glioblastoma cells were obtained from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Construct, Lysis

Journal: ACS nano

Article Title: Eradication of Established Tumors by Chemically Self-Assembled Nanoring (CSAN) Labelled T Cells

doi: 10.1021/acsnano.8b01308

Figure Lengend Snippet: (A) Target cell lysis was evaluated at a set 10:1 E:T ratio with increasing concentrations of CSANs as well as (B) at a set concentration of CSANs (50 nM) with increasing E:T ratio. Unactivated PBMCs were co-cultured with media, monospecific CSANs or bispecific CSANs in the presence or absence of target MCF-7 cells. CD69 expression levels was measured on (C) CD4+ T cells and (D) CD8+ T cells following a 12-hour incubation while CD25 expression was measured on (E) CD4+ Tells and (F) CD8+ T cells following a 24-hour incubation. CD4+ and CD8+ T cells were gated on the lymphocyte population as determined by the forward and side scatter as well as live/dead cell dye. Following incubation, the media was analyzed for (G) IL-2 and (H) IFN-γ by ELISA. Data shown was obtained from one donor (n=3), but representative of three donors (Figure S16 & S17). *P<0.05 with respect to αEpCAM CSAN and no treatment controls, by 2-tailed Student’s t test.

Article Snippet: Cell Lines, Culture Conditions and T cell isolation MCF-7 breast cancer cells and U87 MG human glioblastoma cells were obtained from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Lysis, Concentration Assay, Cell Culture, Expressing, Incubation, Enzyme-linked Immunosorbent Assay

Journal: ACS nano

Article Title: Eradication of Established Tumors by Chemically Self-Assembled Nanoring (CSAN) Labelled T Cells

doi: 10.1021/acsnano.8b01308

Figure Lengend Snippet: (A) CD8+ and CD4+ T cells were isolated separately by negative selection and used for targeting cell killing with 50 nM Bispecific CSANs and a 10:1 E:T ratio at various CD4:CD8 ratios. Cytokine production produced from CD4+ and CD8+ T cells isolated by negative selection. Unactivated PBMCs were co-cultured with media, monospecific CSANs or bispecific CSANs in the presence or absence of target MCF-7 cells for 24 hours at various CD4:CD8 ratios. Following incubation, the media was analyzed for (B) IFN-γ and (C) IL-2 by ELISA. Data shown was obtained from one donor (n=3), but representative of three donors (Figure S18 & S19). *P<0.05 with respect to antiEpCAM CSAN and no treatment controls, by 2-tailed Student’s t test.

Article Snippet: Cell Lines, Culture Conditions and T cell isolation MCF-7 breast cancer cells and U87 MG human glioblastoma cells were obtained from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Isolation, Selection, Produced, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay

Journal: ACS nano

Article Title: Eradication of Established Tumors by Chemically Self-Assembled Nanoring (CSAN) Labelled T Cells

doi: 10.1021/acsnano.8b01308

Figure Lengend Snippet: The phenotype of T cells in the peripheral blood from mice treated with PBMCs and PARs formed by modifying PBMCs with αEpCAM/αCD3 bispecific CSANs was analyzed on day 0, 4 and 30. They are displayed as the proportion of (A) CD8+ cells and (B) CD4+ cells by flow cytometry with staining for CCR7and CD45RO (n = 5). All SD < 1.9%.

Article Snippet: Cell Lines, Culture Conditions and T cell isolation MCF-7 breast cancer cells and U87 MG human glioblastoma cells were obtained from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Flow Cytometry, Staining

Journal: ACS nano

Article Title: Eradication of Established Tumors by Chemically Self-Assembled Nanoring (CSAN) Labelled T Cells

doi: 10.1021/acsnano.8b01308

Figure Lengend Snippet: NSG mice were inoculated in the mammary fat pad with 1.0×106 MCF-7 cells. Tumors were allowed to grow to 300 mm3 prior to infusing mice with 20×106 PBMCs to allow for uman T cell engraftment. Four days following PBMC infusion mice were injected with 64Cu-radiolabeled αEpCAM/αCD3 bispecific CSANs, 64Cu-radiolabeled αEpCAM/αCD3 bispecific CSANs ten minutes following the infusion of 10 mg/kg αEpCAM mAb (moc31), and 64Cu-radiolabeled non-targeted αCD3 CSANs. Mice were sacrificed and organs were harvested to determine a (A) full biodistribution analysis at 24 hours, (B) the tumor uptake over time, and (C) ratio of CSANs that are bound to T cells or in free plasma circulation. Results are reported as (A & B) percentage injected dose per gram of tissue and (C) ratio of T cell:Plasma (n = 4), representing approximately 87.5 ± 2.4 % percent of radioactivity in the isolatiod blood samples. Statistical significance, *p < 0.05 with respect to block and αCD3 CSAN controls (two tailed T-test).

Article Snippet: Cell Lines, Culture Conditions and T cell isolation MCF-7 breast cancer cells and U87 MG human glioblastoma cells were obtained from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Injection, Clinical Proteomics, Radioactivity, Blocking Assay, Two Tailed Test