Library-based selection of retroviruses selectively spreading through matrix metalloprotease-positive cells.

Viruses conditionally replicating in cancer cells form an attractive novel class of antitumoral agents. To engineer such viruses infectivity can be coupled with proteolytic activity of the target cell by modifying the envelope (Env) protein of murine leukaemia virus (MLV) with blocking domains that prevent cell entry unless they are cleaved off by tumour-associated proteases like the matrix metalloproteases (MMP). Here we show that MLV variants selectively spreading through MMP-positive cells can be evolved from virus libraries, in which a standard MMP-2 substrate peptide connecting the blocking domain CD40L with the Env protein was diversified. Passaging the virus library on human fibrosarcoma or glioma cell lines resulted in the selection of about 10 virus clones, of which the three most frequent ones were shown to become activated by MMPs and to be replication competent on MMP-positive cells only. On these cells, the selected linker peptides improved the spreading by several orders of magnitude in vitro, as well as in tumour xenografts in vivo, approaching the kinetic of the unmodified wild-type virus. The data suggest that retroviral protease substrate libraries form a potent tool for the engineering of viruses conditionally replicating in a given cancer cell type of interest.

Directed evolution of retroviruses activatable by tumour-associated matrix metalloproteases.

Protease-activatable retroviral vectors offer the possibility of targeted gene transfer into cancer cells expressing a unique set of proteases as, for example, the matrix metalloproteases (MMPs). However, it is difficult to predict which substrate sequence will be optimally cleaved by a given tumour cell type. Therefore, we developed a novel approach that allows the selection of MMP-activatable retroviruses from libraries of viruses displaying combinatorially diversified protease substrates. Starting from a virus harbouring a standard MMP-2 substrate motif, after only two consecutive cycles of diversification and in vivo selection, MMP-activatable viruses were recovered. Biochemical characterization of the selected viruses revealed that their linker peptides showed a considerably increased sensitivity for MMP-2 cleavage, and interestingly also improved the particle incorporation rate of the Env protein. Owing to the optimized linker peptide, the selected viruses exhibited a greatly enhanced spreading efficiency through human fibrosarcoma cells, while having retained the dependency on MMP activation. Moreover, cell entry efficiency and virus titres were considerably improved as compared to the parental virus displaying the standard MMP-2 substrate. The results presented imply that retroviral protease substrate libraries allow the definition of MMP substrate specificities under in vivo conditions as well as the generation of optimally adapted tumour-specific viruses.

A hyperfusogenic gibbon ape leukemia envelope glycoprotein: targeting of a cytotoxic gene by ligand display.

An important goal in cancer gene therapy is the development of novel targeted cytotoxic genes. The observation that transfection of a GaLV envelope glycoprotein lacking an R peptide into human cells results in considerable cell-cell fusion and subsequent cell death prompted us to explore the potential for using this fusogenic membrane glycoprotein (FMG) as a targeted cytotoxic gene. As proof of principle, we therefore displayed epidermal growth factor (EGF) on the N terminus of GaLV envelope glycoproteins both with and without an R peptide (GaLV R+ and GaLV R-). Transfection of the GaLVR+ envelope expression plasmids did not cause cell-cell fusion. The GaLV R+ envelopes were incorporated into retroviral vectors whose infectivity was investigated on EGF receptor-positive and -negative cells. The vector incorporating an N-terminally unmodified envelope was able to infect all human cell lines tested. Infectivity of the vector incorporating an envelope on which EGF was displayed was restricted on EGF receptor-positive cells (but not on EGF receptor-negative cells) and could be restored by protease cleavage of the displayed domain or competition with exogenous ligand. The cell-cell fusion capacity of the GaLV R- envelope glycoproteins (N-terminally unmodified and with N-terminal display of both EGF and insulin-like growth factor I [IGF-I]) was investigated by plasmid DNA transfection. While the N-terminally unmodified GaLV R- fused all human cell types tested, fusogenicity of GaLV R- on which EGF or IGF-I was displayed was considerably restricted on receptor-positive cells. "Reciprocal" competition experiments showed that fusogenicity could be restored by competition only with the relevant exogenous ligand. Thus the specificity of cell-cell fusion by a hyperfusogenic GaLV envelope glycoprotein can be regulated by N-terminal display of growth factor ligands. There is therefore significant potential for further development of the targeting of the cell-killing capability of this fusogenic viral glycoprotein by using strategies similar to those we have developed for the targeting of retroviral vectors.

Modification of retroviral tropism by display of IGF-I.

We have displayed insulin-like growth factor I (IGF-I) as an N-terminal extension of 4070A (amphotropic) retroviral envelope protein. Western blot demonstrated that chimaeric envelope proteins were incorporated into retroviral particles. Interaction between the displayed IGF-I and cell-surface receptors impaired gene delivery. The magnitude of this inhibitory effect was smallest on NIH 3T3 cells, greater on NIH 3T3 cells over-expressing insulin receptor, and greatest on NIH 3T3 cells over-expressing human type-I IGF receptor. Hence, both the number of ligand receptors and their affinity for the displayed ligand influenced the level of gene delivery. The inhibitory effect was abrogated by cleaving the displayed domain from the underlying envelope protein with factor Xa protease, and by the addition of free ligand to the infection. Addition of IGF-I or insulin caused a dose-dependent increase in titre. Possible mechanisms for receptor-mediated inhibition of gene delivery by IGF-displaying vectors are discussed, together with the implications of these results for practical applications of retroviral display and for understanding the mechanism of virus entry.

Homodimerization and hetero-oligomerization of the single-domain trefoil protein pNR-2/pS2 through cysteine 58.

The single-domain human trefoil proteins [pNR-2/pS2 and human intestinal trefoil factor (hITF)] have seven cysteine residues, of which six are involved in maintaining the structure of the trefoil domain. The seventh does not form part of the trefoil domain and is located three residues from the C-terminus. The ability of the pNR-2/pS2 single trefoil domain protein to dimerize was examined by using recombinant protein with either a cysteine or a serine residue at this position by equilibrium ultracentrifugation, laser-assisted desorption MS, gel filtration and PAGE. pNR-2/pS2 Cys58 formed dimers, whereas pNR-2/pS2 Ser58 did not. Experiments in which the dimer was treated with thiol agents demonstrated that the dimer was linked via a disulphide bond and that the intermolecular disulphide bond was more susceptible to reduction than the intramolecular disulphide bonds. To examine whether dimeric pNR-2/pS2 was secreted by oestrogen-responsive breast cancer cells, which are known to express pNR-2/pS2 mRNA, conditioned medium was separated on non-denaturing polyacrylamide gels, transferred to PVDF membrane and reacted with antiserum against pNR-2/pS2. Monomeric and dimeric pNR-2/pS2 were detected but the majority of the protein reactivity was associated with a larger protein. Treatment of this protein with thiol agents suggested that it is an oligomer containing pNR-2/pS2 linked to another protein by a disulphide bond. These studies suggest that the biological action of pNR-2/pS2 single-domain trefoil protein might involve the formation of homodimers or oligomers with other proteins.

High-resolution solution structure of human pNR-2/pS2: a single trefoil motif protein.

pNR-2/pS2 is a 60 residue extracellular protein, which was originally discovered in human breast cancer cells, and subsequently found in other tumours and normal gastric epithelial cells. We have determined the three-dimensional solution structure of a C58S mutant of human pNR-2/pS2 using 639 distance and 137 torsion angle constraints obtained from analysis of multidimensional NMR spectra. A series of simulated annealing calculations resulted in the unambiguous determination of the protein's disulphide bonding pattern and produced a family of 19 structures consistent with the constraints. The peptide contains a single "trefoil" sequence motif, a region of about 40 residues with a characteristic sequence pattern, which has been found, either singly or as a repeat, in about a dozen extracellular proteins. The trefoil domain contains three disulphide bonds, whose 1-5, 2-4 and 3-6 cysteine pairings form the structure into three closely packed loops with only a small amount of secondary structure, which consists of a short alpha-helix packed against a two-stranded antiparallel beta-sheet. The structure of the domain is very similar to those of the two trefoil domains that occur in porcine spasmolytic polypeptide (PSP), the only member of the trefoil family whose three-dimensional structure has been previously determined. Outside the trefoil domain, which forms the compact "head" of the molecule, the N and C-terminal strands are closely associated, forming an extended "tail", which has some beta-sheet character for part of its length and which becomes more disordered towards the termini as indicated by (15)N{(1)H} NOEs. We have considered the structural implications of the possible formation of a native C58-C58 disulphide-bonded homodimer. Comparison of the surface features of pNR-2/pS2 and PSP, and consideration of the sequences of the other human trefoil domains in the light of these structures, illuminates the possible role of specific residues in ligand/receptor binding.

Production and comparison of mature single-domain 'trefoil' peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58.

The preparation and purification of recombinant mature pNR-2/pS2, a single-domain member of the 'trefoil' family of cysteine-rich secreted proteins, is described. Analysis of recombinant pNR-2/pS2 by ion-exchange chromatography showed that it was heterogeneous. The heterogeneity was reduced by treatment with thiol-group-containing reagents, suggesting that it is caused by the odd number of cysteine residues in mature pNR-2/pS2, and this view was reinforced by mutation of the extra-trefoil domain cysteine residue, Cys58, to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58 proteins under non-denaturing conditions confirmed that the Ser58 mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser58 co-migrates as a single band with pNR-2/pS2 secreted from breast-cancer cells in culture. Treatment of recombinant pNR-2/pS2 proteins with various thiol-group-reactive reagents indicated that cysteine is the most effective at producing recombinant pNR-2/pS2 that co-migrates with pNR-2/pS2 secreted by breast-cancer cells. Dithiothreitol appeared to denature the proteins, and GSH was relatively ineffective. pNR-2/pS2 Cys58 treated with cysteine and untreated pNR-2/pS2 Ser58 had the same apparent molecular mass, measured by gel filtration, as pNR-2/pS2 secreted from breast-cancer cells. This is the first report of the production of a recombinant mature single-domain trefoil peptide and should greatly facilitate elucidation of the structure and function of pNR-2/pS2.

A biotype of Enterobacteriaceae intermediate between Citrobacter and Enterobacter.

The authors isolated two strains of an unnamed bacterial biotype with characteristics intermediate between those of Enterobacter and Citrobacter. The organisms did not produce acetyl-methyl carbinol, but decarboxylated lysine. Apart from the latter trait, they most closely resemble H2S-negative Citrobacter freundii. They differ biochemically from all other currently accepted species of enterobacteriaceae. Their pathogenic significance appears similar to that of the two genera they most closely resemble. Only by recognition and study of additional strains can their identity be more definitively delineated and their significance more fully assessed.