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Sleeping Beauty (SB) transposon, a type of nonviral integrative vectors, provides an alternative to modify primary T cells. Creative biolabs has developed SB transposon CAR vector pSBCAR1 CAIX (MSC8) h(ICOSBBζ), which is constructed for the engineering of T cells to target human CAIX. The T cells are genetically modified through transduction with a nonviral vector expressing scFv of anti-CAIX antibody linked to ICOS (CD278) signaling domain and CD137 (4-1BB), CD3-zeta signaling domains. And the vector product was designed for the treatment of Renal cell carcinoma.
CAR Construction : Fig.1 Serial dilutions of purified Fab were injected on a rhCAIX-coated CM5 sensor chip. Values were corrected for binding to the reference flow cell. The solid lines represent the theoretical curves for each Fab concentration calculated according to the 1 : 1 binding model. KA, ¾ and KD values calculated by the 1 : 1 binding model are shown in the table. |
CAR Construction : Fig.1 Selection of CAIX-specific antibodies. Antibody binding to (A) wild-type (dashed) or CAIX-transfected (shaded) SKRC 17 cells, (B) HeLa cells under normoxic (dashed) or hypoxic (shaded) conditions and (C) murine CT26 cells under normoxic (dashed) or hypoxic (shaded) conditions was studied by flow cytometry. Sample analyses are shown for SC3 and MSC8 Fab antibody fragments. Fab ESC 1 1 recognizing human fibroblast activation protein and antibodies against either human (M75) or murine CAIX (M- 100) served as controls. |
CAR Construction : Fig.3 Antibody binding to carbonic anhydrase isoforms II, IV, IX, XII and XIV was analyzed in ELISA on recombinant proteins. ESC1 1 IgG recognizing human fibroblast activation protein served as negative control. Coating was confirmed by staining with a Penta-His antibody. |
CAR Construction : Fig.2 In vitro carbonic anhydrase activity assay on HCTl 16 membrane fragments. (A) Time course for C02 hydration with membrane-free buffer (dotted), HCT l 16-EV membrane-fragments (grey), HCTl 16-CAIX membrane fragments (black) and HCTl 16-CAIX membrane fragments blocked with MSC 8 Fab (dashed). (B) Carbonic anhydrase activity, normalized to a scale where activity measured in HCTl 16-CAIX and HCTl 16-EV membrane fragments are one and zero, respectively. Asterisks denote significant reduction in activity (MSC5, MSC 10„MSC12 and MSC8 Fab antibodies). (C) Time course for CO2 hydration with HCTl 16-EV membrane-fragments (grey), HCTl 16-CAIX membrane- fragments (black) and HCTl 16-CAIX membrane fragments blocked with MSC3 IgG (dashed black), MSC8 IgG (dotted black) or acetazolamide (dashed grey). (D) Dose-response curves for MSC 8 Fab (grey) and MSC 8 IgG (black) influence on CAIX-activity (using the same normalization algorithm as in B). |
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