Prostate Cancer-Specific and Potent Antitumor Effect of a DD3-Controlled Oncolytic Virus Harboring the PTEN Gene

Prostate cancer is a major health problem for men in Western societies. Here we report a Prostate Cancer-Specific Targeting Gene-Viro-Therapy (CTGVT-PCa), in which PTEN was inserted into a DD3-controlled oncolytic viral vector (OV) to form Ad.DD3.E1A.E1B(Δ55)-(PTEN) or, briefly, Ad.DD3.D55-PTEN. The woodchuck post-transcriptional element (WPRE) was also introduced at the downstream of the E1A coding sequence, resulting in much higher expression of the E1A gene. DD3 is one of the most prostate cancer-specific genes and has been used as a clinical bio-diagnostic marker. PTEN is frequently inactivated in primary prostate cancers, which is crucial for prostate cancer progression. Therefore, the Ad.DD3.D55-PTEN has prostate cancer specific and potent antitumor effect. The tumor growth rate was almost completely inhibited with the final tumor volume after Ad.DD3.D55-PTEN treatment less than the initial volume at the beginning of Ad.DD3.D55-PTEN treatment, which shows the powerful antitumor effect of Ad.DD3.D55-PTEN on prostate cancer tumor growth. The CTGVT-PCa construct reported here killed all of the prostate cancer cell lines tested, such as DU145, 22RV1 and CL1, but had a reduced or no killing effect on all the non-prostate cancer cell lines tested. The mechanism of action of Ad.DD3.D55-PTEN was due to the induction of apoptosis, as detected by TUNEL assays and flow cytometry. The apoptosis was mediated by mitochondria-dependent and -independent pathways, as determined by caspase assays and mitochondrial membrane potential

Association of warfarin dose with genes involved in its action and metabolism

We report an extensive study of variability in genes encoding proteins that are believed to be involved in the action and biotransformation of warfarin. Warfarin is a commonly prescribed anticoagulant that is difficult to use because of the wide interindividual variation in dose requirements, the narrow therapeutic range and the risk of serious bleeding. We genotyped 201 patients for polymorphisms in 29 genes in the warfarin interactive pathways and tested them for association with dose requirement. In our study, polymorphisms in or flanking the genes VKORC1, CYP2C9, CYP2C18, CYP2C19, PROC, APOE, EPHX1, CALU, GGCX and ORM1-ORM2 and haplotypes of VKORC1, CYP2C9, CYP2C8, CYP2C19, PROC, F7, GGCX, PROZ, F9, NR1I2 and ORM1-ORM2 were associated with dose (P < 0.05). VKORC1, CYP2C9, CYP2C18 and CYP2C19 were significant after experiment-wise correction for multiple testing (P < 0.000175), however, the association of CYP2C18 and CYP2C19 was fully explained by linkage disequilibrium with CYP2C9*2 and/or *3. PROC and APOE were both significantly associated with dose after correction within each gene. A multiple regression model with VKORC1, CYP2C9, PROC and the non-genetic predictors age, bodyweight, drug interactions and indication for treatment jointly accounted for 62% of variance in warfarin dose. Weaker associations observed for other genes could explain up to ∼10% additional dose variance, but require testing and validation in an independent and larger data set. Translation of this knowledge into clinical guidelines for warfarin prescription will be likely to have a major impact on the safety and efficacy of warfarin. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00439-006-0260-8 and is accessible for authorized users

Targeting tissue factor on tumour cells and angiogenic vascular endothelial cells by factor VII-targeted verteporfin photodynamic therapy for breast cancer in vitro and in vivo in mice

<p>Abstract</p> <p>Background</p> <p>The objective of this study was to develop a ligand-targeted photodynamic therapy (tPDT) by conjugating factor VII (fVII) protein with photosensitiser verteporfin in order to overcome the poor selectivity and enhance the effect of non-targeted PDT (ntPDT) for cancer. fVII is a natural ligand for receptor tissue factor (TF) with high affinity and specificity. The reason for targeting receptor TF for the development of tPDT is that TF is a common but specific target on angiogenic tumour vascular endothelial cells (VEC) and many types of tumour cells, including solid tumours and leukaemia.</p> <p>Methods</p> <p>Murine factor VII protein (mfVII) containing a mutation (Lys341Ala) was covalently conjugated via a cross linker EDC with Veterporfin (VP) that was extracted from liposomal Visudyne, and then free VP was separated by Sephadex G50 spin columns. fVII-tPDT using mfVII-VP conjugate, compared to ntPDT, was tested <it>in vitro </it>for the killing of breast cancer cells and VEGF-stimulated VEC and <it>in vivo </it>for inhibiting the tumour growth of breast tumours in a mouse xenograft model.</p> <p>Results</p> <p>We showed that: (i) fVII protein could be conjugated with VP without affecting its binding activity; (ii) fVII-tPDT could selectively kill TF-expressing breast cancer cells and VEGF-stimulated angiogenic HUVECs but had no side effects on non-TF expressing unstimulated HUVEC, CHO-K1 and 293 cells; (iii) fVII targeting enhanced the effect of VP PDT by three to four fold; (iii) fVII-tPDT induced significantly stronger levels of apoptosis and necrosis than ntPDT; and (iv) fVII-tPDT had a significantly stronger effect on inhibiting breast tumour growth in mice than ntPDT.</p> <p>Conclusions</p> <p>We conclude that the fVII-targeted VP PDT that we report here is a novel and effective therapeutic with improved selectivity for the treatment of breast cancer. Since TF is expressed on many types of cancer cells including leukaemic cells and selectively on angiogenic tumour VECs, fVII-tPDT could have broad therapeutic applications for other solid cancers and leukaemia.</p

Natural killer cells are crucial for the efficacy of Icon (factor VII/human IgG1 Fc) immunotherapy in human tongue cancer

<p>Abstract</p> <p>Background</p> <p>Icon is a novel, dual neovascular- and cancer cell-targeting immunotherapeutic agent and has shown efficacy in the treatment of cancer, wet form macular degeneration and endometriosis. However, its underlying mechanism remains to be investigated. The objective of this study is to elucidate the mechanism of Icon immunotherapy in cancer using a squamous carcinoma human tongue cancer line TCA8113 <it>in vitro </it>and <it>in vivo </it>in severe combined immunodeficiency (SCID) mice.</p> <p>Results</p> <p>We showed that Icon, as a chimeric factor VII and human IgG1 Fc immunoconjugate, could separately induce murine natural killer (NK) cells and activate complement to kill TCA8113 cancer cells <it>in vitro </it>via antibody dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, Icon-NK ADCC had a significantly stronger effect than that of Icon-CDC. Moreover, Icon could completely eradicate established human tongue tumour xenografts <it>in vivo </it>in the CB-17 strain of SCID mice that have functional NK cells at a normal level, whereas it was less effective in SCID/Beige mice that do not have functional NK cells.</p> <p>Conclusions</p> <p>We conclude that NK cells are crucial for the efficacy of Icon immunotherapy in the treatment of cancer. The results also suggest that impaired NK level/activity could contribute to the resistance to therapeutic antibodies that are currently under investigation in preclinical and clinical studies.</p

Replicating Single-Cycle Adenovirus Vectors Generate Amplified Influenza Vaccine Responses

Head-to-head comparisons of conventional influenza vaccines with adenovirus (Ad) gene-based vaccines demonstrated that these viral vectors can mediate more potent protection against influenza virus infection in animal models. In most cases, Ad vaccines are engineered to be replication-defective (RD-Ad) vectors. In contrast, replication-competent Ad (RC-Ad) vaccines are markedly more potent but risk causing adenovirus diseases in vaccine recipients and health care workers. To harness antigen gene replication but avoid production of infectious virions, we developed “single-cycle” adenovirus (SC-Ad) vectors. Previous work demonstrated that SC-Ads amplify transgene expression 100-fold and produce markedly stronger and more persistent immune responses than RD-Ad vectors in Syrian hamsters and rhesus macaques. To test them as potential vaccines, we engineered RD and SC versions of adenovirus serotype 6 (Ad6) to express the hemagglutinin (HA) gene from influenza A/PR/8/34 virus. We show here that it takes approximately 33 times less SC-Ad6 than RD-Ad6 to produce equal amounts of HA antigen in vitro. SC-Ad produced markedly higher HA binding and hemagglutination inhibition (HAI) titers than RD-Ad in Syrian hamsters. SC-Ad-vaccinated cotton rats had markedly lower influenza titers than RD-Ad-vaccinated animals after challenge with influenza A/PR/8/34 virus. These data suggest that SC-Ads may be more potent vaccine platforms than conventional RD-Ad vectors and may have utility as “needle-free” mucosal vaccines. IMPORTANCE Most adenovirus vaccines that are being tested are replication-defective adenoviruses (RD-Ads). This work describes testing newer single-cycle adenovirus (SC-Ad) vectors that replicate transgenes to amplify protein production and immune responses. We show that SC-Ads generate markedly more influenza virus hemagglutinin protein and require substantially less vector to generate the same immune responses as RD-Ad vectors. SC-Ads therefore hold promise to be more potent vectors and vaccines than current RD-Ad vectors