The investigation of the death-inducing potency of a recombinant Adenovector expressing Mda-7-tlyp-1 on different cancer cell lines.

Aim: The potency of Adenovector expressing Mda7-tLyp1 (Ad-Mda7-tLyp1) for death induction was evaluated on the breast (MCF7), liver (HepG2), and gastric (MKN45) cancer cell lines. Background: Mda-7 could be a possible complementary to traditional cancer therapy, and tethering to tumor-homing peptides (THPs) might improve its therapeutic efficacy. Methods: After the preparation of recombinant Ad-Mda7-tLyp1 and Ad-Mda7, the expression of recombinant proteins was analyzed by ELISA. Adenovectors were transduced (MOI=2-5) into Hep-G2, MCF7, MKN45, and normal skin fibroblast, then tumor-killing effect was measured by cytopathic effect (CPE) monitoring, MTT viability test, BAX gene expression analysis, and Caspase3/7 assay. Results: ELISA assay revealed a sustained level of recombinant protein secretion following Adenovector transduction. In CPE microscopy, all cancer cell lines showed a significant reduction (≥50%) in their normal phenotype after receiving Ad-Mda7-tLyp1 and Ad-Mda7. The viability was significantly lower compared to the control, indicating an anti-proliferating effect. In parallel, the viability test showed that Ad-Mda7 and Ad-Mda7-tLyp1 have a significant killing effect (≥50%) on MCF-7, Hep-G2, and MKN45 compared to normal fibroblast (P≤0.05). BAX gene expression analysis showed that both Ad-Mda7-tLyp1 and Ad-Mda7 vectors induced >2-fold increase of apoptosis (P<0.05), particularly in MCF7. Similarly, caspase3/7 activity showed a significant increase (P<0.05) following Ad-Mda7, and Ad-Mda7-tLyp1 transduction into cancer cell lines, but not in normal fibroblasts. Conclusion: The newly constructed Ad-Mda-tlyp1 showed a suitable tumor cell killing activity and enough specificity on studied cell lines.

Fiber manipulation and post-assembly nanobody conjugation for adenoviral vector retargeting through SpyTag-SpyCatcher protein ligation.

For adenoviruses (Ads) to be optimally effective in cancer theranostics, they need to be retargeted toward target cells and lose their natural tropism. Typically, this is accomplished by either engineering fiber proteins and/or employing bispecific adapters, capable of bonding Ad fibers and tumor antigen receptors. This study aimed to present a simple and versatile method for generating Ad-based bionanoparticles specific to target cells, using the SpyTag-SpyCatcher system. The SpyTag peptide was inserted into the HI loop of fiber-knob protein, which could act as a covalent anchoring site for a targeting moiety fused to a truncated SpyCatcher (SpyCatcherΔ) pair. After confirming the presence and functionality of SpyTag on the Ad type-5 (Ad5) fiber knob, an adapter molecule, comprising of SpyCatcherΔ fused to an anti-vascular endothelial growth factor receptor 2 (VEGFR2) nanobody, was recombinantly expressed in Escherichia coli and purified before conjugation to fiber-modified Ad5 (fmAd5). After evaluating fmAd5 detargeting from its primary coxsackie and adenovirus receptor (CAR), the nanobody-decorated fmAd5 could be efficiently retargeted to VEGFR2-expressing 293/KDR and human umbilical vein endothelial (HUVEC) cell lines. In conclusion, a plug-and-play platform was described in this study for detargeting and retargeting Ad5 through the SpyTag-SpyCatcher system, which could be potentially applied to generate tailored bionanoparticles for a broad range of specific targets; therefore, it can be introduced as a promising approach in cancer nanotheranostics.

Immunovirotherapy: The role of antibody based therapeutics combination with oncolytic viruses.

Despite the fact that the new drugs and targeted therapies have been approved for cancer therapy during the past 30 years, the majority of cancer types are still remain challenging to be treated. Due to the tumor heterogeneity, immune system evasion and the complex interaction between the tumor microenvironment and immune cells, the great majority of malignancies need multimodal therapy. Unfortunately, tumors frequently develop treatment resistance, so it is important to have a variety of therapeutic choices available for the treatment of neoplastic diseases. Immunotherapy has lately shown clinical responses in malignancies with unfavorable outcomes. Oncolytic virus (OV) immunotherapy is a cancer treatment strategy that employs naturally occurring or genetically-modified viruses that multiply preferentially within cancer cells. OVs have the ability to not only induce oncolysis but also activate cells of the immune system, which in turn activates innate and adaptive anticancer responses. Despite the fact that OVs were translated into clinical trials, with T-VECs receiving FDA approval for melanoma, their use in fighting cancer faced some challenges, including off-target side effects, immune system clearance, non-specific uptake, and intratumoral spread of OVs in solid tumors. Although various strategies have been used to overcome the challenges, these strategies have not provided promising outcomes in monotherapy with OVs. In this situation, it is increasingly common to use rational combinations of immunotherapies to improve patient benefit. With the development of other aspects of cancer immunotherapy strategies, combinational therapy has been proposed to improve the anti-tumor activities of OVs. In this regard, OVs were combined with other biotherapeutic platforms, including various forms of antibodies, nanobodies, chimeric antigen receptor (CAR) T cells, and dendritic cells, to reduce the side effects of OVs and enhance their efficacy. This article reviews the promising outcomes of OVs in cancer therapy, the challenges OVs face and solutions, and their combination with other biotherapeutic agents.

Vitamin D receptor gene polymorphisms and risk of breast cancer in Iranian women.

OBJECTIVES: Vitamin D deficiency is a driving force of common cancers like breast cancer. Vitamin D receptor (VDR) can play a tumor suppressor role by helping the precise function of vitamin D in cells such as modulation TGF-ß signaling pathway. This study aimed to investigate the association of VDR gene variants and susceptibility to breast cancer in Iranian women. METHODS: Genomic DNAs were isolated from blood samples of 161 women with breast cancer and 150 healthy women. After amplification of five positions of VDR gene, the prepared amplicons were digested with TaqI, ApaI, BsmI, Cdx2, and FokI restriction enzymes. RESULTS: Subsequently, the digested products were electrophoresed on the 1.5% agarose gel. Odds ratios (ORs) for breast cancer were calculated for genotypes and estimated haplotypes. Binary logistic regression analysis showed FokI (rs2228570), BsmI (rs1544410), and ApaI (rs7975232) polymorphisms had the significant distribution in patients than to the normal group. Analysis of linkage disequilibrium for all pairs of SNPs showed that D'-value between SNP TaqI and SNP BsmI was significantly (p ≤ 0.05). We observed that four major haplotypes of ApaI, BsmI, FokI, Cdx2, and TaqI SNPs significantly were in high frequency than predicted frequency. Among these four haplotypes, CGTAT haplotype was in a higher significant association than others with breast cancer risk (p-value = 0.0001). CONCLUSION: Our results showed that FokI, BsmI, and ApaI of VDR polymorphisms associated with the risk of breast cancer in Iranian population.

Isolation of Polyclonal Single-Chain Fragment Variable (scFv) Antibodies Against Venomous Snakes of Iran and Evaluation of Their Capability in Neutralizing the Venom.

Several species of dangerous snakes are found in Iran and, according to the Emergency Response Center of Iran from 2002 to 2011, 53,787 Iranians have suffered from snakebite. Although the mortalities caused by snakebite are very low, snakebite-related amputations are still a major concern. Currently, anti-venom polyclonal antibodies derived from animals, such as horses are used to treat snakebites; however, in some cases they can cause anaphylactic shock and serum sickness. In line with this premise, generation of recombinant anti-venom antibodies can be considered as an alternative strategy. Single-chain fragment variable (scFv) antibodies offer several advantages compared to the whole antibodies, including ease of production, high affinity and specificity. In the present study, scFv antibodies were selected against the venom of the most poisonous snakes in Iran using phage display technology. Phage particles harboring anti-venom specific scFv were separated and scFv antibodies were produced in bacteria. In-vitro assay showed that polyclonal scFvs specifically bind to the venom. Furthermore, in-vivo experiment in mice BALB/c indicated effective toxin neutralization using 20 µg of polyclonal scFv. Our study indicates the neutralizing capacity of anti-venom polyclonal scFvs, although further neutralization assays are needed to confirm their effectiveness.

CEA Plasmid as Therapeutic DNA Vaccination against Colorectal Cancer.

BACKGROUND: Human colorectal cancer cells overexpress carcinoembryonic antigen (CEA). CEA is a glycoprotein which has shown to be a promising vaccine target for immunotherapy against colorectal cancer. OBJECTIVES: To design a DNA vaccine harboring CEA antigen and evaluate its effect on inducing immunity against colorectal cancer cells in tumor bearing mice. METHODS: In the first step the coding sequence of the CEA was cloned into the pcDNA3.1 vector. The mice were injected with the vaccine construct and the immune responses were monitored during the experiment period. The specific IgG anti-CEA, IFN-γ, IL-2 and IL-4 were measured by ELISA and levels of IFN-γ was detected by ELISpot assay. The lymphocyte proliferation was assessed using a 5-bromo-2-deoxyuridine (BrdU) cell proliferation assay kit. RESULTS: Immunization of the mice with the CEA plasmid resulted in stimulation of CEA-specific T cell and antibody responses. The serum level of specific IgG antibodies against CEA was increased in immunized mice. Moreover, the injection of CEA plasmid led to the stimulation of T-helper-1 by increase in the secretion of IFN-γ, IL-2 and lymphocyte proliferation response. CONCLUSION: As the CEA DNA vaccine displayed encouraging antitumor effects, therefore, we suggest that it can be a potential therapeutic modality for colorectal cancer and is worthy of further investigation.