Fig. 1.

Secretion of an anti-HER2 therapeutic antibody, TZB, by transduced tumor cells. (A) Schematic of paracrine delivery by SHREAD of a therapeutic antibody, TZB. The heavy and light chains of the therapeutic antibody are expressed from the cytomegalovirus (CMV) promoter using a furin-2A site, which leads to stochiometric expression of secreted chains via a previously described ribosome “skipping” mechanism and correct trimming of the heavy chain (22, 23, 49). Transgenes encoding the therapeutic antibody, TZB, and a transcriptionally coupled eGFP reporter are packaged into the genome of the SHREAD gene therapy platform, which consists of a retargeted and shielded Ad5 that is specific for the tumor surface antigen, HER2 (1) (15, 16). Upon administration of the virus, a subset of tumor cells is transduced via HER2 (green cells; 2) and acts of a therapeutic biofactory, secreting antibodies (black) in a paracrine fashion from within the tumor microenvironment where they diffuse locally (yellow arrow; 3). The secreted antibody then exerts therapeutic effects directly on the tumor (e.g., by the formation of pores; 4). (B) Schematic of a bispecific DARPin adapter that blocks the natural tropism of Ad5 while redirecting specificity to the chosen cell-surface biomarker (Top) (15). The Ad5 knob-binding module (orange) with SHP trimerization module (yellow), a protein from phage 21, is fused to a retargeting module (green) that allows for specific transduction of tumors expressing biomarkers for which it is specific (e.g., HER2). Schematic of the engineered trimeric protein-based “shield” that is coated on the Ad5 capsid (Bottom). The shield (magenta) binds spanning the threefold symmetry axes between neighboring pentameric hexon proteins on the viral capsid as previously described (16). Together with the retargeting adapter, it comprises the SHREAD gene therapy platform, which allows for improved tumor specificity and prevention of liver- and immune-based clearance mechanisms in immunodeficient mouse models (16). (C) Transduction of cell lines in vitro via the naked virus (natural, cyan), blocked virus (by a retargeting adaptor without the targeting DARPin, gray), HER2-retargeted virus (without shield [orange] and with shield [magenta]). Transduction is measured with a reporter virus that expresses fluorescent TdTomato by flow cytometry. Error bars represent SD for n = 3 replicates. (D) ESI-MS of commercially produced Herceptin (Genentech; Top) and TZB purified from culture supernatants of transduced BT474 cells (Bottom) following treatment with PNGase F and dihtiothreitol (DTT). Both show very similar profiles. (E) XTT of BT474 cells treated with recombinant TZB (Herceptin, Genentech, black), TZB purified from culture supernatants of CHO (dark gray) or BT474 (green) cells transduced with Ad5-TZB or with Ad-D1.3 (light gray), or a recombinant control antibody (anti-human PD1 antibody, nivolumab, Opdivo, BMS, white). Error bars represent SD for n = 3 replicates.