Figure 5.

In vivo evaluation of CARs from novel binders in murine model of chronic HBV infection. Persistent HBV replication was established in Bl6.Rag1^-/^- mice using AAV-HBV. 1x10⁶ C8-, 4D06- or 4D08-CAR CD45.1^+/+ syngeneic T cells per mouse were transferred at week 5 after AAV-HBV infection (n=3). Two animals served as mock control. End-point analyses were performed at day 21 or 35 as indicated. Kinetics of (A) HBsAg, (B) HBeAg and (C) ALT levels in serum are given. (D) HBV pregenomic RNA (pgRNA) was quantified in liver tissue lysates obtained at day 35 through RT-PCR. (E) Immunohistochemistry of HBcAg expression in the liver of one representative animal per treatment group. Visible dark brown precipitates in the nucleus indicate HBV-positive hepatocytes. Scale bar represents 300 μm. (F) Number of HBcAg⁺ hepatocytes per mm² as determined by quantitative analysis of immunohistochemistry stainings. (G) Frequencies of IFNγ-secreting CAR⁺ CD8⁺ T cells from blood, liver or spleen detected by intracellular cytokine staining after 16h ex vivo stimulation with plate-bound HBsAg. Symbols and lines represent individual mice.