somatostatin r5/sstr5 antibody  (Bio-Techne corporation)

Journal: Journal for immunotherapy of cancer

Article Title: Development of anti-somatostatin receptors CAR T cells for treatment of neuroendocrine tumors.

doi: 10.1136/jitc-2022-004854

Figure Lengend Snippet: Figure 1 Expression of SSTR2 and SSTR5 by NET cell lines. (A) WB of membrane extracts from CM, BON1, QGP1, H727 and CNDT2.5 cell lines. Na+/K+ATPase membrane expression was used as loading control. (B) Representative flow cytometry analysis of SSTR2 and SSTR5 expression in CM and BON1 cells as well as in HAP1 cells as negative control. (C) Representative confocal microscopy evaluation of SSTR2 and SSTR5 expression in CM and BON1 cells. For both flow cytometry and confocal microscopy experiments permeabilization procedures were omitted to allow detection of membrane SSTRs only. SSTRs, somatostatin receptors; WB, Western blot.

Article Snippet: NET cells were fixed in paraformaldehyde and incubated overnight at 4°C with a mouse mAb against SSTR2 (R&D Systems, cat#MAB4224) or a rabbit polyclonal Ab targeting SSTR5 (Novus Biologicals, cat#NB100- 74540).

Techniques: Expressing, Membrane, Control, Flow Cytometry, Negative Control, Confocal Microscopy, Western Blot

Journal: Journal for immunotherapy of cancer

Article Title: Development of anti-somatostatin receptors CAR T cells for treatment of neuroendocrine tumors.

doi: 10.1136/jitc-2022-004854

Figure Lengend Snippet: Figure 3 Anti-SSTR CAR T cells exhibit antigen-specific tumoricidal activity. (A) Anti-SSTR CARs endow human T lymphocytes with reactivity against SSTR-expressing targets. By in vitro BLI assay, CAR T cells induced cell death in up to 58% of Luc+ NET cell lines as compared with UT T cells at an E:T ratio of 1:1. The percentage of specific tumor cell lysis was calculated as the ratio between CAR T cell and UT T cell antitumor activity using the formula: % lysis=1-(mean BLI signal in the presence of anti-SSTR CAR T cells/mean BLI signal in the presence of UT cells) x 100%. (B) In vitro BLI assay to evaluate the cytolytic activity of CAR T cells as compared with UT T cells according to increasing E:T ratios after 24 hours of coculture with NET cells. The degree of cytotoxicity induced by CAR T cells increased when the number of effector cells increased. (C) IFN-γ and (D) TNF-α release on 24 hours coculture of NET cells with CAR T cells or UT T cells at an E:T ratio of 1:1. Cytokine production was measured in culture supernatants by ELISA. CD3/CD28 T cell stimulation through TransAct was used for positive control experiments. (E) In vitro BLI assay to investigate the cytolytic activity of CAR T cells as compared with UT T cells against mutant CM cells and parental cells at an E:T ratio of 1:1. (F) Evaluation of the cytotoxic potential of CAR T cells against CM cells harboring wild-type or mutated SSTR2 and/or SSTR5 according to increasing E:T ratios after 24 hours of coculture. (G) IFN-γ and (H) TNF-α release on 24 hours coculture of CAR T cells or UT T cells with mutated or parental CM cells at an E:T ratio of 1:1. All experiments were carried out in technical triplicate using lymphocytes from three healthy donors. Mean values and standard errors are represented in figure. *P<0.05, **p<0.01. CAR, chimeric antigen receptor; E:T, effector:target; NET, neuroendocrine tumor; SSTR, somatostatin receptor; UT, untransduced.

Article Snippet: NET cells were fixed in paraformaldehyde and incubated overnight at 4°C with a mouse mAb against SSTR2 (R&D Systems, cat#MAB4224) or a rabbit polyclonal Ab targeting SSTR5 (Novus Biologicals, cat#NB100- 74540).

Techniques: Activity Assay, Expressing, In Vitro, Lysis, Enzyme-linked Immunosorbent Assay, Cell Stimulation, Positive Control, Mutagenesis

Journal: Journal for immunotherapy of cancer

Article Title: Development of anti-somatostatin receptors CAR T cells for treatment of neuroendocrine tumors.

doi: 10.1136/jitc-2022-004854

Figure Lengend Snippet: Figure 5 Anti-SSTR CAR T cells infiltrate human xenografts and murine SSTR-expressing organs. (A) Visualization of SSTR2 (UMB1 mAb) and SSTR5 (UMB5 mAb) within tumor xenografts by IHC. Magnification: ×20. Scale bar: 100 µm. (B) Explanted CM tumor xenografts as well as SSTR-expressing organs such as murine spleen, pancreas and brain were lysed and subjected to DNA extraction. The infiltration of CAR T cells was demonstrated by PCR using primers specific for the anti-SSTR CAR sequence. The purified CAR construct DNA was used for positive control experiments. Distilled water was used in negative control experiments. (C) FAM-labeled and HEX-labeled probes against the CAR sequence and the MKL2 reference gene, respectively, were employed in ddPCR experiments to quantify the infiltration of CM (blue dots) and BON1 xenografts (red dots) by anti-SSTR CAR T cells. An inverse correlation can be observed between CAR T cell infiltration and tumor bioluminescence increase relative to baseline. (D) Anti-SSTR CAR T cells recognize and kill the MIN6 cells, a murine NET cell line expressing SSTR2/5. By in vitro BLI, CAR T cells induced cell death in the 48% of Luc+ MIN6 cells as compared with UT T cells at an E:T ratio of 1:1 after 72 hours of coculture. (E) IFN-γ release on 24 hours coculture of NET cells with CAR T cells or UT T cells at an E:T ratio of 1:1. (F) Histopathological analysis of human NET xenografts and murine pancreas and brain by H&E staining. Extensive necrosis foci could be identified within tumors (arrowhead). Representative microphotographs show the absence of necrosis or other tissue damages in the context of the murine pancreas and brain. Magnification: x20. Scale bar: 100 µm. **P<0.01. CAR, chimeric antigen receptor; ddPCR, droplet digital PCR; E:T, effector:target; IHC, immunohistochemistry; NET, neuroendocrine tumor; SSTR, somatostatin receptor; UT, untransduced.

Article Snippet: NET cells were fixed in paraformaldehyde and incubated overnight at 4°C with a mouse mAb against SSTR2 (R&D Systems, cat#MAB4224) or a rabbit polyclonal Ab targeting SSTR5 (Novus Biologicals, cat#NB100- 74540).

Techniques: Expressing, DNA Extraction, Sequencing, Purification, Construct, Positive Control, Negative Control, Labeling, In Vitro, Staining, Digital PCR, Immunohistochemistry