Human FAP Stable Cell Line - HT1080

A major limitation facing systemic gene therapy is the lack of novel, tissue-specific AAV (adeno-associated virus) vectors. Bispecific antibodies can be employed to redirect AAVs to specific target receptors. Here, researchers demonstrate that inserting the short linear epitope "2E3"-derived from human proprotein convertase subtilisin/kexin type 9 (PCSK9)-into various surface loop regions of the VP capsid protein enables the dissociation of AAV from its natural receptors (i.e., "detargeting"), thereby facilitating a bispecific antibody-mediated retargeting strategy. They selected several disease-associated membrane proteins as targets: fibroblast activation protein (FAP; highly expressed on activated fibroblasts in tumor stroma and fibrotic tissues) and programmed death-ligand 1 (PD-L1; significantly overexpressed in various cancers). Upon co-incubation with bispecific antibodies recognizing both the 2E3 epitope and either FAP or PD-L1, the resulting bispecific antibody/rAAV complexes were able to selectively transduce cells expressing the corresponding receptors. This versatile platform holds promise as a valuable tool for investigating the roles of disease-associated cell types and lays the groundwork for the development of novel gene therapies.

Purified rAAV-2E3 variants were pre-incubated with the bispecific KiH-2E3-FAP antibody to form FAP-targeting complexes. Provided the antibody complex did not interfere with rAAV-2E3 uncoating or cytoplasmic release, viral uptake should occur via FAP-binding-mediated AAV endocytosis, ultimately leading to viral transgene expression in FAP-expressing cells. Researchers investigated the ability to retarget the novel rAAV-2E3 capsid to huFAP-expressing HT1080 cells by combining various titers of rAAV-2E3 variants (50,000 to 5,000 VG/cell) with different dilutions of the bispecific KiH-2E3-MO36 antibody. Control experiments using AAV2 on huFAP-expressing HT1080 cells confirmed successful transduction, showing that up to 97% of cells expressed GFP, with mean fluorescence intensity (MFI) decreasing as the number of viral genomes (VG) per cell was reduced (Figure 1A, B). Variant rAAV-2E3.v2 showed no transduction activity at any antibody concentration (Figure 1C). Variants rAAV-2E3.v4 and rAAV-2E3.v5 exhibited high levels of transgene expression even without bispecific antibody binding, and GFP expression trended downward at the highest bispecific antibody concentrations; consequently, no additional retargeting effect could be detected against this high background expression (Figure 1D, E). Variant rAAV-2E3.v6 showed no GFP expression without pre-incubation with the bispecific antibody, but GFP expression increased significantly at specific antibody-to-viral-genome ratios (Figure 1F). Pre-incubation of parental AAV2 with high concentrations of the bispecific antibody did not affect AAV2 transduction of the HT1080 cell line (Figure 1G).

Figure 1. Retargeting novel rAAV-2E3 to FAP expressing cells using KiH bispecific antibodies. (Kuklik J, et al., 2021)