Recombinant Mouse Antibody (5a19) is capable of binding to HBV Pres1 Region , expressed in Chinese Hamster Ovary cells (CHO).
Figure 1 Lymphocytes obtained from a healthy donor were retrovirally grafted with cTCR containing scFv recognizing HBV S protein (S-C8), HBV L protein (L-5a19), or carcinoembryonic antigen (CEA) or used as control (PBL). Flow cytometric analysis using a PE-conjugated anti-CD3 and an FITC-conjugated anti-human Ig-Fc antibody, which detects the extracellular IgG1 CH2CH3 spacer domain of the receptors, were performed to identify cTCR grafted T cells.
Figure 2 Cytotoxic T-cell response and cytokine secretion.
T cells grafted with the cTCR directed either against HBV S (S-C8, open diamond) or L protein (L-5a19, open circle) or against CEA (CEA, solid square) or unmodified cells (PBL, solid rectangle) were cultured together with HBV (upper panel) HepG2.2.15 cells or HBV (lower panel) HepG2 cells. T cells were added in different dilutions to obtain indicated effector to target cell (E:T) ratios and cocultured for 72 hours. (A) Specific lysis of target cells in 3 parallel assays is shown. Secretion of IFN- (B) and IL-2 (C) into cell culture media was measured by ELISA. Mean SD is given. (D) Antigen-specific proliferation was determined by flow cytometry of CFSE stained, cTCR grafted T cells after 48 hours. One representative staining (E:T 0.8:10) out of 4 stainings is shown.
Figure 3 HBV-infected primary human hepatocytes are eliminated by antigen-specific, engineered T cells.
Primary human hepatocytes were infected with HBV and cultured for 3 days prior to the addition of redirected T cells (HBV target cells: black columns; HBV control cells: grey columns). T cells grafted with cTCR S-C8, L-5a19, or CEA, respectively, or unmodified cells (PBL) were added as an effector to target cell ratio of 2:1 and cocultured for 96 hours. (A) Liver transaminase (ALT) levels were determined in cell culture media as a marker for hepatocyte lysis. Mean values and standard deviations obtained from 3 independent infection experiments are given. For comparison, ALT levels of CD95-treated cells undergoing apoptosis are shown. (B) IFN- and (C) IL-2 were measured in the culture supernatant by ELISA. (D) HBeAg and (E) HBsAg were determined in cell culture supernatants by ELISA. (F) Western blot analysis of HBV-infected PHH cells for intracellular albumin and HBV core proteins. (G) HBV rcDNA and (H) HBV cccDNA were quantified by real-time PCR in cellular DNA preparations from infected hepatocyte cultures upon coculture with redirected T cells.
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For Research Use Only. Not For Clinical Use.