Tumor cell targeted gene delivery by adenovirus 5 vectors carrying knobless fibers with antibody-binding domains

Abstract

Most human carcinoma cell lines lack the high-affinity receptors for adenovirus serotype 5 (Ad5) at their surface and are nonpermissive to Ad5. We therefore tested the efficiency of retargeting Ad5 to alternative cellular receptors via immunoglobulin (Ig)-binding domains inserted at the extremity of short-shafted, knobless fibers. The two recombinant Ad5's constructed, Ad5/R7-Z wt -Z wt and Ad5/R7-C2-C2, carried tandem Ig-binding domains from Staphylococcal protein A (abbreviated Z wt ) and from Streptococcal protein G (C2), respectively. Both viruses bound their specific Ig isotypes with the expected affinity. They transduced human carcinoma cells independently of the CAR pathway, via cell surface receptors targeted by specific monoclonal antibodies, that is, EGF-R on A549, HT29 and SW1116, HER-2/ neu on SK-OV-3 and SK-BR-3, CA242 (epitope recognized by the monoclonal antibody C242) antigen on HT29 and SW1116, and PSMA (prostate-specific membrane antigen) expressed on HEK-293 cells, respectively. However, Colo201 and Colo205 cells were neither transduced by targeting CA242 or EGF-R nor were LNCaP cells transduced by targeting PSMA. Our results suggested that one given surface receptor could mediate transduction of certain cells but not others, indicating that factors and steps other than cell surface expression and virus-receptor interaction are additional determinants of Ad5-mediated transduction of tumor cells. Using penton base RGD mutants, we found that one of these limiting steps was virus endocytosis. Gene Therapy (2005) 12, 211-224.

Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu

“…FibDCAR-HI-Link-ZHZH was also cloned into an Ad genome carrying a penton base with EGD instead of RGD. 40 PCR amplification and DNA sequencing verified the presence of a correct fiber and penton base insert with the expected sequence. The cosmid was also controlled by cleavage with HindIII and SpeI.…”

“…These fibers could be incorporated into viable viruses that, however, exhibit even worse growth defects than we have previously encountered in similar Affibodyliganded viruses. 12,40 These growth defects can be explained by a low-fiber content caused by the lack of the fiber knob hampering virus entry and intracellular trafficking and a loss of other growth -or viral assembly functions carried by the fiber knob. [20][21][22][23][24][25] Only in the de-knobbed virus Ad5/R7-C2C2, containing the Ig binding C2 domain from Streptococcal protein G, we found a normal fiber content indicating that this fiber somehow compensates, as far as fiber content of the virus is concerned, for the loss of the fiber knob.…”

“…10 For all recombinant fibers of this study, a pattern similar to WT was observed, showing that they were all able to form trimers (data not shown), which is in agreement with previous results with similarly constructed fibers. 10,12,40 Ligand reactivity by binding to cellular receptors. After having established the solubility and trimerization status of the fiber, the ligand reactivity was analyzed by cellbinding assay.…”

“…The FibDCAR-HILink-ZHZH was also cloned into an Ad genome with an amino-acid substitution in the penton base (RGD to EGD) to abolish binding to integrins. 40 To be able to separate the viruses, they were named Ad5/FibDCAR-HILink-ZHZH and Ad5/EGD/FibDCAR-HI-Link-ZHZH.…”

Transductional targeting of adenovirus vectors for gene therapy

“…99 This fiber modification also provided cancer cell selective infection. 100 Korokhov et al, 101 Volpers et al 102 and others 103 have developed similar targeting approaches that embody elements of both genetic fiber modification and adapterbased targeting by incorporating the immunoglobulin (Ig)-binding domain of Staphylococcus aureus protein A into the fiber C-terminus or HI-loop. As a result, these fiber-modified vectors form stable complexes with a wide variety of targeting molecules containing the Fc region of Ig.…”