A cathepsin-cleavage site between the adenovirus capsid protein IX and a tumor-targeting ligand improves targeted transduction.

Human adenoviruses have a great potential as anticancer agents. One strategy to improve their tumor-cell specificity and anti-tumor efficacy is to include tumor-specific targeting ligands in the viral capsid. This can be achieved by fusion of polypeptide-targeting ligands with the minor capsid protein IX. Previous research suggested that protein IX-mediated targeting is limited by inefficient release of protein IX-fused ligands from their cognate receptors in the endosome. This thwarts endosomal escape of the virus particles. Here we describe that the targeted transduction of tumor cells is augmented by a cathepsin-cleavage site between the protein IX anchor and the HER2/neu-binding ZH Affibody molecule as ligand. The cathepsin-cleavage site did not interfere with virus production and incorporation of the Affibody molecules in the virus capsid. Virus particles harboring the cleavable protein IX-ligand fusion in their capsid transduced the HER2/neu-positive SKOV-3 ovarian carcinoma cells with increased efficiency in monolayer cultures, three-dimensional spheroid cultures and in SKOV-3 tumors grown on the chorioallantoic membrane of embryonated chicken eggs. These data show that inclusion of a cathepsin-cleavage sequence between protein IX and a high-affinity targeting ligand enhances targeted transduction. This modification further augments the applicability of protein IX as an anchor for coupling tumor-targeting ligands.

A Kunjin replicon vector encoding granulocyte macrophage colony-stimulating factor for intra-tumoral gene therapy.

We have recently developed a non-cytopathic RNA replicon-based viral vector system based on the flavivirus Kunjin. Here, we illustrate the utility of the Kunjin replicon system for gene therapy. Intra-tumoral injections of Kunjin replicon virus-like particles encoding granulocyte colony-stimulating factor were able to cure >50% of established subcutaneous CT26 colon carcinoma and B16-OVA melanomas. Regression of CT26 tumours correlated with the induction of anti-cancer CD8 T cells, and treatment of subcutaneous CT26 tumours also resulted in the regression of CT26 lung metastases. Only a few immune-based strategies are able to cure these aggressive tumours once they are of a reasonable size, illustrating the potential of this vector system for intra-tumoral gene therapy applications.

Adenovirus targeting to HLA-A1/MAGE-A1-positive tumor cells by fusing a single-chain T-cell receptor with minor capsid protein IX.

Adenovirus vectors have great potential in cancer gene therapy. Targeting of cancer-testis (CT) antigens, which are specifically presented at the surface of tumor cells by human leukocyte antigen (HLA) class I molecules, is an attractive option. In this study, a single-chain T-cell receptor (scTCR) directed against the CT antigen melanoma-associated antigen (MAGE)-A1 in complex with the HLA class I molecule of haplotype HLA-A1 is fused with the C terminus of the adenovirus minor capsid protein IX. Propagation of a protein-IX (pIX)-gene-deleted human adenovirus 5 (HAdV-5) vector on cells that constitutively express the pIXscTCR fusion protein yielded viral particles with the pIXscTCR fusion protein incorporated in their capsid. Generated particles specifically transduced melanoma cell lines expressing the HLA-A1/MAGE-A1 target complex with at least 10-fold higher efficiency than control viruses. Whereas loading of HLA-A1-positive cells with MAGE-A1 peptides leads to enhanced transduction of the cells, the efficiency of virus transduction is strongly reduced if the HLA-A1 molecules are not accessible at the target cell. Taken together, these data provide proof of principle that pIXscTCR fusions can be used to target HAdV-5 vectors to tumor cells expressing intracellular CT antigens.

Efficient incorporation of a functional hyper-stable single-chain antibody fragment protein-IX fusion in the adenovirus capsid.

Recombinant adenoviruses are frequently used as gene transfer vehicles for therapeutic gene delivery. Strategies to amend their tropism include the incorporation of polypeptides with high affinity for cellular receptors. Single-chain antibodies have a great potential to achieve such cell type specificity. In this study, we evaluated the efficiency of incorporation of a single-chain antibody fused with the adenovirus minor capsid protein IX in the capsid of adenovirus type 5 vectors. To this end, the codons for the single-chain antibody fragments (scFv) 13R4 were fused with those encoding of pIX via a 75-Angstrom spacer sequence. The 13R4 is a hyper-stable single-chain antibody directed against beta-galactosidase, which was selected for its capacity to fold correctly in a reducing environment such as the cytoplasm. A lentiviral vector was used to stably express the pIX.flag.75.13R4.MYC.HIS fusion gene in 911 helper cells. Upon propagation of pIX-gene deleted human adenovirus-5 vectors on these cells, the pIX-fusion protein was efficiently incorporated in the capsid. Here, the 13R4 scFv was functional as was evident from its capacity to bind its ligand beta-galactosidase. These data demonstrate that the minor capsid protein IX can be used as an anchor for incorporation of single-chain antibodies in the capsids of adenovirus vectors.

Self-reported morbidity and disability among Trappist and Benedictine monks.

Studies of religious groups that impose prudent lifestyles on their members show low mortality rates in these groups, but it is unclear whether their morbidity rates are also low. The authors studied the prevalence of self-reported morbidity and disability among Trappist and Benedictine monks in the Netherlands. A health interview survey was administered in seven monasteries; the response rate was 67% (n = 134). Rates of morbidity and disability among monks were compared with those among all Dutch males by calculation of standardized morbidity ratios, adjusting for age and (in the case of morbidity) education. Self-reported morbidity among monks was similar to that in the general population (e.g., for one or more chronic conditions: standardized morbidity ratio = 1.07, 95% confidence interval 0.89-1.26), but rates of disability related to activities of daily living were much higher (e.g., for any trouble sitting down and getting up from a chair: standardized morbidity ratio = 2.21, 95% confidence interval 1.44-3.32). The authors hypothesize that a prudent lifestyle may prolong life, but at the expense of a higher prevalence of disability.

[Differences in risk factors for disease and health problems between monks and the general population in The Netherlands]. / Verschillen in risicofactoren voor ziekte en in gezondheidsproblemen tussen kloosterlingen en de algemene bevolking in Nederland.

The aim of this study was to determine whether the austerely living Trappist and Benedictine monks have a lower prevalence of a number of risk factors and health problems than the general Dutch population. A written questionnaire was submitted to monks of 7 monasteries. The response was 67 per cent (134 monks). The data were compared with data from the national Health Interview Survey of 1989, which used an almost identical questionnaire. Adjustment was made for differences in age and education. Monks consume less alcohol and tobacco and have a more austere diet. Their average Quetelet index is lower. The prevalence of cardiovascular disease is lower. On the other hand, monks more often report a number of other chronic diseases, physical complaints, and problems with activities of daily life. They more often have contact with general practitioners and with consultants. The lower prevalence of a number of risk factors among monks reflects their austere way of life. It is not certain whether the lower prevalence of cardiovascular diseases can be attributed to this way of life. The fact that, in general, health problems are more prevalent among monks suggests that changes in lifestyle do not necessarily lead to compression of morbidity.