Conventional treatments for solid tumors involve removing the malignant tissues by surgery followed by chemotherapy and/or radiotherapy. Despite the effectiveness of these therapies, the side effects of chemotherapy and radiotherapy are very high because of lack of specificity to target malignant cells. Another problem is the development of resistance to these treatments by cancer cells Targeted drug delivery strategies have been developed to improve therapeutic agents efficacy and to reduce non-specific side effects by delivering the therapeutic agents specifically to target cells. Several approaches have been investigated to achieve this aim. The differences in cell surface receptors expression have widely been used to deliver therapeutic agents by conjugating them with specific ligands such as hormones, vitamins, folic acid, peptide, transferrin, aptamers, monoclonal antibodies and their fragments. In order to deliver therapeutic agents specifically to cancer cells we have conjugated therapeutic agents to a single chain antibody or to an aptamer that bind specifically to cancer cell receptors. Two types of therapeutic agents were conjugated to the scFv-425 single chain recognizing EGFR, the photosensitizer chlorin e6 and the chemotherapy payload polyglycerol-doxorubicin dendrimer. Standard methods for protein conjugation yield heterogeneous products containing a mixture of conjugated antibodies with the effector molecules conjugated at different sites and a variable number of effector molecules conjugated to each antibody resulting in a range of molar ratios and different pharmacokinetic, efficacy and safety profiles. To avoid the protein conjugation limitations, we investigated the use of SNAP-tag technology to provide a unique conjugation site on the antibody allowing the production of homogeneous conjugate preparations. We engineered a construct in which the coding sequence of a scFv that binds specifically to EGFR (scFv-425) was genetically fused to the SNAP tag cassette, which endows the antibody with a SNAP-tag and therefore allows site-specific conjugation to BG-modified substrates. This conjugation method can be applied to any antibody–therapeutic agent combination as long as the antibody carries the SNAP-tag and the substrate is modified with a BG group. We found that scFv-425-CAT-AD-SNAP conjugated to BG-modified Ce6 can selectively kill EGFR+ cells in four human tumor-derived cell lines representing epidermal, breast and cervical carcinomas (A431, MDA-MB-231, MDA-MB468 and SiHa) after exposure to light. Also scFv-425-CAT-AD-SNAP conjugated to BG-modified dendritic polymer that loaded with doxorubicin molecules kill three human tumor-derived cell lines representing epidermal, breast and pancreatic carcinomas (A431, MDA-MB-468 and Panc-1). In conclusion, we have demonstrated for the first time the conjugation of therapeutic agents to a scFv using SNAP-tag technology. This rapid and efficient method produces a homogeneous conjugate with defined pharmacokinetic and therapeutic profiles, and provides proof of principle that SNAP-tag technology can be used to conjugate therapeutic agents for cancer therapy, avoiding the off-target effects that have thus far limited the development of targeted drug delivery. In another approach RNA aptamer that binds specifically to cancer cell receptor has been used to deliver siRNA against cancer cells. siRNAs silence gene expression by triggering the sequence-specific degradation of mRNAs, but the targeted delivery of such reagents remains challenging and a significant obstacle to therapeutic applications. One promising approach is the use of RNA aptamers that bind to tumor-associated antigens as targeting moieties to achieve the delivery of siRNAs to tumor cells bearing specific antigens. RNA-based constructs are advantageous because they are inexpensive to synthesize and they have a low immunogenicity. We therefore joined an aptamer recognizing avB3 integrin to a siRNA that targets eukaryotic elongation factor 2 gene, and achieved for the first time the targeted delivery of a siRNA to tumor cells expressing avB3 integrin causing inhibition of cell proliferation and induction of apoptosis in tumor cells. In conclusion, we have demonstrated for the first time that an aptamer specifically binding to avB3 integrin can be used to deliver a cytotoxic siRNA to tumor cells expressing avB3 integrin. This allows the development of therapeutic modalities composed solely of RNA, reducing production costs and potential immunogenicity compared to protein therapeutics. The low cost, low immunogenicity and minimal off-target effects achieved by the specific targeting of tumor cells suggest that aptamer-siRNA chimeras could represent an exciting and efficacious new paradigm for cancer therapy with a significant potential impact in the clinic
