Several approaches have been used to improve the Ad transduction efficiency of cells normally resistant to Ad. In the best cases, genetically modified Ad vectors containing heterologous peptides, such as RGD in the HI loop of the fiber (13
) or at the C-terminal end (64
), were reported to increase the transduction efficiency up to 500-fold across a broad range of cell types. However, cells of hematopoietic origin with low levels of integrin expression (29
) are not expected to be good targets of RGD-modified Ad. An additional limitation of such a genetic approach is the small size of the targeting peptide tolerated by the virus capsid structure (8
). Further extension of productive Ad-cell interactions by selective strategies is thus important to broaden Ad tropism for medical purposes. Toward this goal, we have designed bispecific and simple-to-produce fusion proteins. We have demonstrated that a bispecific fusion protein consisting of the ectodomain of CAR fused to the immunoglobulin Fc domain improves the transduction efficiency of hematopoietic cells devoid of the primary Ad receptor CAR by up to 250-fold. This transduction increase is highly significant and fully dependent on the presence of high-affinity Fcγ receptor I (CD64). The transduction increase is significantly reduced by CTLA4-Ig protein or an anti-human CD64 monoclonal antibody, which bind to the high-affinity Fcγ receptor I. The inclusion of the CAR ectodomain in our fusion construct is useful since this domain confers high-affinity binding to the fiber protein of Ad. If all the fibers are decorated with CARex-Fc, we expect that such modified virus prefers to bind to Fcγ receptors rather than to the receptor of native Ad, CAR.
Accordingly, we have demonstrated that coincubation of virus with CARex-Fc strongly reduced Ad-mediated expression in CAR-positive, Fcγ receptor-negative A549 cells, arguing for the correct folding of at least the D1 domain of CARex-Fc (19
). In addition, the successful production of a virus-neutralizing monoclonal anti-human CAR-specific antibody using the CARex-Fc protein to boost the immune response, together with the demonstration of direct binding of the CARex-Fc protein to CD64-positive cells, adds further evidence for the functionality of our hybrid adapter protein.
Other strategies to alter Ad host range have involved conjugates containing blocking anti-Ad fiber knob Fab (15
) or an anti-FLAG epitope antibody (65
). Most closely related to our approach is the use of a bispecific protein consisting of the neutralizing scFv antibody fragment recognizing the fiber knob fused to the EGF ligand, termed the adenobody (60
). In this system, viral gene delivery to cells expressing the EGF receptor was enhanced 16-fold. Since all the cells tested in these experiments also expressed CAR, the contribution of the additional EGF domain to retargeting Ad was difficult to define. Besides the EGF receptor, additional receptors like the folate receptor (15
) and CD3 on T lymphocytes (62
) have been used as targets for bispecific conjugates to extend Ad tropism. A similar approach was described for retroviruses by using a bispecific soluble virus receptor fused to EGF (6
). In this system, improved transduction was obtained by preincubating cells with the hybrid protein and then incubating them with virus. Alternatively, virus was incubated with the fusion protein and purified and then the complex was added to target cells.
In contrast to several of the previous reports, our approach was effective for cells lacking the natural virus receptor. The presence of the high-affinity Fcγ receptor I on the target cells was sufficient for a robust Ad-mediated gene expression. It is not known at present if CARex-Fc-modified Ad entered the CAR-less cells by a clathrin-dependent pathway, which was suggested to operate for Ad infection of fibroblast-type cells (59
). Possibly, our CARex-Fc-modified Ad is taken up by a clathrin-independent route similar to Fc receptor-mediated endocytosis in phagocytic cells (2
). This pathway operates in an actin-dependent manner, typically with particles larger than 0.5 μm. Since the CARex-Fc bridging protein seems to be dimeric, multiple Ads could become cross-linked and thus might induce phagocytosis. However, the low-affinity Fcγ receptors II and III, which primarily recognize immune complexes (55
), were inefficient for CARex-Fc-Ad infection, suggesting that the CARex-Fc-Ad complexes were poorly recognized by the low-affinity Fcγ receptor uptake system. Further experiments are necessary to clarify the mechanisms of Ad-mediated transgene delivery into Fcγ receptor I-positive cells.
Nonetheless, CARex-Fc-modified Ad may have clinical potential. CD64 is expressed on monocytes, macrophages, and subtypes of AML cells but not on maturating dendritic cells (7
) (Fig. ). Since global survival rates for patients with AML are poor, the further development of immunotherapy and immune vaccine approaches is a valuable goal to pursue (4
). The concept of immune gene therapy for cancer is based on the presumption that the host immune system is capable of recognizing tumor-associated antigens. For AML, little information is available about potential immunostimulatory antigens. Recently, isolation of AML-specific T-cell clones was reported (38
), but no corresponding peptide sequences have so far been published. High levels of transiently expressed cytokines and immune modulators might be sufficient to activate the immune system in vivo and thereby achieve the therapeutic goal (34
). Cells of the hematopoietic system are particularly suitable for gene therapy, since the techniques for bone marrow and blood cell transplantations are well established and, importantly, the transductions can be performed ex vivo. Primary AML cells can be obtained from patient bone marrow aspirates or peripheral blood mononuclear cells and can be kept in culture in the presence of interleukin-3, stem cell factor, or kit ligand (for a review, see reference 4
). However, none of the currently used gene therapy vectors allow efficient gene transfer to malignant cells of hematopoietic origin. Therefore, the development of CARex-Fc for Ad-mediated gene transfer provides a promising alternative to specifically transfer therapeutic genes into CD64-positive AML malignancies. For other types of malignancies, alternative cell surface markers may serve as targets. Replacement of the Fc portion of CARex-Fc with other polypeptide sequences that bind to surface markers, such as receptor ligands or single-chain antibodies, may allow the production of a whole range of bifunctional, soluble Ad receptor-ligand fusion proteins.