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. 2022 Jun 22;30(11):3341–3357. doi: 10.1016/j.ymthe.2022.06.011

Figure 6.

Figure 6

The function of adaptor and protector

(A) Construction of adaptor (CFS) and protector (HF) proteins. The CFS was composed of a humanized anti-EpCAM scFv, the ectodomain of CXADR, and a phage T4 fibritin polypeptide. The HF was composed of a humanized scFv against the hexon protein of ADV and a phage T4 fibritin polypeptide.

(B–E) A diagram (B) for intravenous administration of ADV with or without CFS and HF proteins on NOD-SCID mice with H520 xenograft. ADV was incubated with CFS, HF, and CFS + HF for 2 h, and this ADV/protein complex was injected into the mice via the tail vein. The amount of ADV was 1.0 × 1010 VP and concentration of the protein was 1.0 × 10−7 pmol/VP. After administration of ADV, ADV + CFS, ADV + HF, and ADV + CFS + HF for 48 h, the tumor, liver, lung, and kidney isolated from the mice were analyzed for distribution of ADV expressing GFP and SaCas9. The representative images (C) and relative abundance (D) of GFP bioluminescence and quantitative real-time-PCR analyses of SaCas9 (E) were demonstrated. n = 3.

(F) ADV expressing GFP was incubated with CFS and HF proteins for 2 h, followed by adding serum of C57BL/6N mice for 1 h. The amounts of proteins and serum were 1.0 × 10−7 pmol/VP and 5 μL, respectively. The mixture was added in the LEWIS cells (mouse lung cancer cell line) for 48 h. The multiplicity of infection (MOI) of ADV was 1:500. The relative infection efficiencies were detected by comparing the ratios of GFP by flow cytometry.

(G–I) A diagram (G) for intratumoral and intravenous administration of ADV with or without CFS and HF on LEWIS xenograft (C57BL/6N mice). ADV was incubated with CFS, HF, and CFS + HF for 2 h, and this ADV/protein complex was injected into the mice via intratumor and tail vein. The amount of ADV was 1.0 × 1010 VP and concentration of the protein was 1.0 × 10−7 pmol/VP. The ADV distribution in the isolated tumor, liver, lung, and kidney of C57BL/6N mice was detected after 2 days by quantitative real-time-PCR analyses of SaCas9 (H). The abundance of anti-ADV antibody in serum of C57BL/6N mice was administrated with ADV/protein complex as indicated by ELISA analyses after 7 days (I). The control mice were untreated. i.t., intratumor; i.v., intravenous, via tail vein. n = 3. ∗p ＜ 0.05, unpaired 2-tailed t test in (D–(F), (H), and (I).