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[Cancer Research 66, 7694-7700, August 1, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Oncolytic Efficacy and Enhanced Safety of Measles Virus Activated by Tumor-Secreted Matrix Metalloproteinases

Christoph Springfeld1, Veronika von Messling1, Marie Frenzke1, Guy Ungerechts1, Christian J. Buchholz2 and Roberto Cattaneo1

1 Molecular Medicine Program and Virology and Gene Therapy Track, Mayo Clinic College of Medicine, Rochester, Minnesota and 2 Medizinische Biotechnologie, Paul-Ehrlich-Institut, Langen, Germany

Requests for reprints: Roberto Cattaneo, Molecular Medicine Program, Mayo Clinic Rochester, Guggenheim 1838, 200 First Street Southwest, Rochester, MN 55902. Phone: 507-284-0171; Fax: 507-266-2122; E-mail: Cattaneo.Roberto{at}mayo.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cancer cells secrete matrix metalloproteinases (MMP) that degrade the extracellular matrix and are responsible for some hallmarks of malignant cancer. Many viruses, including a few currently used in oncolytic virotherapy clinical trials, depend on intracellular proteases to process their proteins and activate their particles. We show here for measles virus (MV) that particle activation can be made dependent of proteases secreted by cancer cells. The MV depends on the intracellular protease furin to process and activate its envelope fusion (F) protein. To make F protein activation cancer cell specific, we introduced hexameric sequences recognized by an MMP and identified the mutant proteins most effective in fusing MMP-expressing human fibrosarcoma cells (HT1080). We showed that an MMP inhibitor interferes with syncytia formation elicited by mutant F proteins and confirmed MMP-dependent cleavage by Edman degradation sequence analysis. We generated recombinant MVs expressing the modified F proteins in place of furin-activated F. These viruses spread only in cells secreting MMP. In nude mice, an MMP-activated MV retarded HT1080 xenograft growth as efficiently as the furin-activated MV vaccine strain. In MV-susceptible mice, the furin-activated virus caused lethal encephalitis upon intracerebral inoculation, whereas the MMP-activated did not. Thus, MV particle activation can be made dependent of proteases secreted by cancer cells, enhancing safety. This study opens the perspective of combining targeting at the particle activation, receptor recognition, and selective replication levels to improve the therapeutic index of MV and other viruses in ongoing clinical trials of oncolysis. (Cancer Res 2006; 66(15): 7694-700)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Oncolytic virotherapy, the selective killing of tumor cells by viruses, is a promising experimental treatment for cancer. Virotherapy clinical trials started early in the 20th century, but despite encouraging case reports, overall results were not convincing enough for inclusion into standard treatment regimens (1). Recently, a better understanding of the determinants of viral tropism allowed to develop new strategies for enhancing the specificity of viral replication in cancer cells (2). Current virotherapy clinical trials are based on engineered adenoviruses, herpes simplex viruses, vaccinia viruses, measles viruses (MV), attenuated strains of Newcastle disease virus (NDV), and reovirus (2, 3). Most of these viruses replicate preferentially in tumor cells because mutations that favor the transformation process often weaken innate immunity (4–7). Additional levels of specificity for replication in tumor cells are being engineered.

The MV is a nonintegrating negative-strand RNA virus. Wild-type MV infection occasionally causes regression of hematologic malignancies (8). Additionally, the live attenuated Edmonston vaccine strain can reduce or eliminate human lymphoma, myeloma, ovarian cancer, and glioma xenografts (8). Patients have been treated in clinical trials of cutaneous lymphoma (9) and ovarian cancer.3 MV tumor selectivity can be improved by engineering the recognition of designated receptors: targeting domains displayed on the attachment protein (hemagglutinin) sustain cell entry through tumor antigens (10, 11). When these specificity determinants are displayed on a hemagglutinin protein unable to recognize the natural receptors CD46 and CD150 (12), targeting at the receptor recognition level is complete (13).

To add another layer of cancer cell specificity, we aimed at making also MV particle activation tumor specific. The viral fusion (F) protein requires proteolytic activation to function. MV F does not restrict tropism because it is activated by the ubiquitous trans-Golgi protease furin (14). However, activation of the murine paramyxovirus Sendai (SeV) F protein depends on tryptase Clara and mini-plasmin. Because these proteases are expressed only in the lungs, SeV replication is restricted to this organ (15). Similarly, F protein cleavage is the main virulence determinant of the poultry pathogen NDV (16, 17), of which nonpathogenic strains are currently in phase I/II clinical trials (18, 19). Maisner et al. previously engineered recombinant MV requiring an exogenous protease for activation of infectivity (20). To confine MV spread strictly to tumor tissue, we aimed at making proteolytic activation of the MV F protein dependent on tumor-specific proteases.

Nearly all types of human cancer cells express matrix metalloproteinases (MMP; ref. 21). These enzymes degrade the extracellular matrix and are responsible for some hallmarks of malignant cancer, including invasiveness and metastasis (22). MMP inhibitors are currently tested in phase III clinical trials (23). Genetically modified toxins activated by MMPs have been generated as potential cancer therapeutics (24), and retroviruses with a blocking domain fused to the viral glycoprotein via an MMP-cleavable linker were shown to infect selectively cancer cells (25).

We have generated recombinant MV with MMP-activated F proteins. These viruses fuse MMP-expressing cells but not other cell types, are oncolytic in a mouse xenograft model with MMP-expressing tumor cells, and are apathogenic when given intracerebrally into susceptible mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and viruses. Vero, HT1080, and 293T cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in DMEM containing 10% FCS with penicillin/streptomycin. Recombinant viruses were rescued using a vaccinia-based system (26) on a 293T cell line stably expressing the MV F protein.4 To grow viral stocks, subconfluent cell monolayers in 15-cm dishes were infected at a multiplicity of infection of 0.03. When most cells formed syncytia, they were scraped into 1 to 2 mL Opti-MEM (Invitrogen, Carlsbad, CA) and subjected to one cycle of freeze-thawing. After low-speed centrifugation to remove cell debris, the supernatant was aliquoted. The TCID50 was determined by the method of Kärber (27) on Vero cells. Because certain viruses do not cause a cytopathic effect on Vero cells, infected cells were identified by monitoring green fluorescent protein (GFP) expression. The titers of MVgreen, MVgreen-MMP-A, and MVgreen-MMP-A1 produced in HT1080 cells, and thus activated, were measured in Vero cells. All three viruses reached titers higher than 106 TCID50, and MVgreen grew to slightly higher titers than the other two viruses. Only MVgreen grew in Vero cells, to a 107 titer.

Mutagenesis and cloning. Modifications of the F protein cleavage site were done in the eukaryotic expression plasmid pCG-F (28) either by overlap-extension PCR or with the Quick-Change mutagenesis kit (Stratagene, La Jolla, CA). Plasmids with full-length MV genomes and expressing GFP were generated by exchanging the NarI-PacI fragment in pMeGFPNV (29) with the corresponding fragments from pCG-F mutants. Viruses derived from these plasmids were named MVgreen, MVgreen-MMP-A, and MVgreen-MMP-A1. For the pathogenicity assays, viruses that do not express GFP were generated based on the MV-NSe genome without a reporter GFP gene (30).

Fusion assays and immunoblots. HT1080 or Vero cells were transfected in 12-well plates at about 85% density with 2 µg of each pCG-F and pCG-H (28) using Lipofectamine 2000 (Invitrogen), and syncytia formation was monitored. To quantify the fusion efficiency of the recombinant viruses, 50% confluent HT1080 cells were inoculated at a multiplicity of infection of 0.01. Thirty-eight hours after infection, the number of nuclei per syncytium in 20 fields was counted in triplicate experiments. Immunoblot analysis (31) was done using a rabbit peptide antiserum against the F cytoplasmic tail ({alpha}Fcyt; ref. 32; dilution, 1:10,000), an anti-rabbit-horseradish peroxidase conjugate, and the enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ).

Protein sequence analysis. HT1080 cells were infected with the recombinant virus MVgreen-MMP-A1 and lysed in radioimmunoprecipitation assay buffer after 48 hours, and the F protein was immunoprecipitated with the {alpha}Fcyt antibody and protein A agarose (Affi-Gel, Bio-Rad, Hercules, CA). The immunoprecipitated proteins were separated on a 10% polyacrylamide gel, blotted to a polyvinylidene difluoride membrane (Sequi-Blot, Bio-Rad) and visualized by Coomassie blue staining. A protein band with a size corresponding to F1 was excised and sequenced by Edman degradation (Mayo Clinic protein core facility) with an Applied Biosystems 492 Procise cLC sequencer (Applied Biosystems, Inc., Foster City, CA).

Animal experiments. All animal experiments were approved by and done according to the guidelines of the Mayo Clinic Rochester Institutional Animal Care and Use Committee. To determine pathogenicity, 6- to 7-week-old Ifnarko-CD46Ge mice (33) were injected i.c. with 2 x 104 TCID50 of the different viruses in 20 µL Opti-MEM (Invitrogen) or Opti-MEM only as control. Mice were observed daily and euthanized when showing unequivocal signs of encephalitis.

For the xenograft model, 4- to 6-week-old athymic nude mice (Harlan, Indianapolis, IN) were injected s.c. with 2 x 105 HT1080 cells (34). After 9 days, 30 mice were distributed into three groups with similar average tumor sizes. The tumors were injected with a daily dose of 1.4 x 106 TCID50 of the different viruses or Opti-MEM for 5 days, measured every 2 to 3 days, and the tumor volumes were calculated according to the formula: V = a2b / 2, where a is the shortest diameter, and b is the longest diameter. Because large tumors tended to ulcerate, mice were euthanized when the tumors reached 1,000 mm3 volume.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MV F1 NH2 terminus tolerates additional residues. The uncleaved F protein precursor (F0) is processed into a larger COOH-terminal fragment (F1) and a smaller NH2-terminal fragment (F2; Fig. 1A ). F1 contains a conserved hydrophobic fusion peptide at its NH2 terminus. In all but two paramyxoviruses, the first F1 amino acid is phenylalanine. Although furin cleaves directly downstream of a basic amino acid stretch, typically RX(K/R)R (one-letter amino acid code; Fig. 1A), one of the MMPs (MMP-2) cleaves in the center of a six-amino-acid motif (35). Replacement of the furin cleavage site with an MMP-2 site would, therefore, add amino acids upstream of the fusion peptide or alter the conserved plenylalanine, or both. However, in the Tupaia paramyxovirus, an isoleucine precedes the phenylalanine (31). This suggested that additional residues upstream of the fusion peptide may not interfere with protein function.


Figure 1
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  		Figure 1. Schematic drawing of the MV F protein, sequences at the F2{downarrow}F1 junction, and functional analysis. A, top, schematic drawing. Signal peptide (SP), F2{downarrow}F1 cleavage site ({downarrow}), fusion peptide (FP), transmembrane region (TM), and cytoplasmic tail (CT). Bottom, amino acid sequence of the F2{downarrow}F1 junction. Unmodified (F) sequence and the sequence with the three-amino-acid (MLS, underlined) insertion (F+3). B, Syncytia formation after transfection of Vero cells with the plasmids encoding the unmodified F protein and the modified protein (F+3).

 
To test this hypothesis, we constructed a plasmid encoding an F protein with the three amino acids MLS inserted between the furin cleavage site and the fusion peptide (Fig. 1A, F + 3). When this plasmid was cotransfected with another one encoding the MV hemagglutinin protein into Vero cells, large syncytia formed (Fig. 1B, right). Thus, even if syncytia formation induced by the standard F protein was slightly more efficient (Fig. 1B, left), three amino acids upstream of the fusion peptide seemed to be compatible with function.

Modified MV F proteins that selectively fuse MMP-expressing cells. Next, we constructed three plasmids encoding modified F proteins with the six amino acids defining the MMP-2 cleavage site PLG{downarrow}MLS inserted in the F2{downarrow}F1 junction region (Fig. 2, top ). This MMP-2 cleavage site was previously used to generate a recombinant anthrax toxin selectively activated in MMP-2-expressing cells (24). In protein F-MMP-A, the six amino acids were inserted downstream of a furin cleavage site, leaving it intact (Fig. 2, top, second line). In proteins F-MMP-B and F-MMP-C, two or four of the five basic amino acids defining the furin cleavage sequence were deleted, respectively (Fig. 2, top, asterisks in third and fourth lines). When either mutated F plasmid was cotransfected with a plasmid encoding hemagglutinin into Vero cells, fusion was below background (Fig. 2, left, third, fourth, and fifth rows; background was one syncytium with 4-5 nuclei per about 2,000 cells). In contrast, large syncytia formed after transfection with a plasmid encoding the unmodified F protein (Fig. 2, left, second row). Thus, the inserted residues interfere with F-MMP-A fusion function.


Figure 2
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  		Figure 2. Modified F proteins selectively fuse MMP-expressing cells. Top, amino acid sequence of the F2{downarrow}F1 junction of unmodified fusion and the modified proteins (F-MMP-A, F-MMP-B, and F-MMP-C). The six-amino-acid sequence recognized by MMP is underlined. Deleted amino acids (*). Bottom, Vero cells (left) or MMP-expressing HT1080 cells (right) were mock transfected or transfected with plasmids encoding the unmodified F protein or the three mutants as indicated. In all experiments, a plasmid encoding MV H was cotransfected.

 
Transfection of the same set of plasmids into MMP-2-positive human fibrosarcoma cells (HT1080) resulted in syncytia formation, with F-MMP-A being the most and F-MMP-C the least efficient mutated F protein (Fig. 2, right, compare third, fourth, and fifth rows); fusion of HT1080 cells by F-MMP-A was reduced compared with the unmodified protein (Fig. 2, right, compare third row with second row). Therefore, insertion of an MMP-cleavage site without deletion of the furin cleavage site results in more efficient fusion.

Analysis of the modified F proteins. F proteins expressed in Vero and HT1080 cells were analyzed with an antibody detecting uncleaved F0 precursor and cleaved F1 protein. As expected, in Vero cells, the unmodified F protein was cleaved efficiently, whereas cleavage of all three mutated proteins was minimal (Fig. 3, top , compare the intensity of the F0 and F1 bands in lanes F, A, B, and C). In addition, expression levels of the mutant proteins were reduced compared with the unmodified protein (Fig. 3, top, compare lane F with lanes A, B, and C). Because very low amounts of the mutated F1 fragments were produced, we were not in the position of sequencing their NH2 termini (see below).


Figure 3
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  		Figure 3. Immunoblot analysis of F protein processing in cells that do or do not express MMP. Top, extracts of Vero cells; bottom, extracts of HT1080 cells. Lane F, extracts of cells transfected with a plasmid coding for unmodified F protein; lanes A, B, and C, extracts from cells expressing the corresponding mutated proteins. Right, position of the F0 precursor and the F1 subunit.

 
In protein extracts of HT1080 cells, about half of the F-MMP-A, F-MMP-B, and F-MMP-C proteins were in the F1 form, below the 80% to 90% efficiency monitored with the unmodified protein (Fig. 3, bottom, compare the intensity of the F0 and F1 bands in lanes F, A, B, and C). Cleavage of all mutated F proteins occurred at similar levels; however, F-MMP-A fused more efficiently HT1080 cells than the other two proteins (Fig. 2, right; data not shown).

Generation of MMP-activated recombinant MV. Because F-MMP-A was most fusogenic, we focused on this protein when attempting to rescue recombinant MVs. Towards this aim, the F coding region of an infectious MV genome expressing GFP (29) was replaced with the corresponding F-MMP-A coding segment. Using a modified virus rescue method based on the overlay of the helper cells with HT1080 cells, we obtained a recombinant virus and named it MVgreen-MMP-A. When Vero cells were infected with this virus, no cytopathic effect was monitored (Fig. 4, middle, first ). Under activation of GFP fluorescence by UV light, single infected cells were identified (Fig. 4, middle, second), as expected, because viral stocks produced on MMP-expressing HT1080 cells are activated and can infect Vero cells but cannot fuse them. Often, pairs of neighboring infected cells were observed, suggesting that infected cells divided. In contrast, in MMP-expressing HT1080 cells syncytia formed (Fig. 4, middle, third and fourth) and reached similar sizes as those forming after infection of HT1080 or Vero cells with MVgreen (Fig. 4, top, third and fourth, and first and second, respectively).


Figure 4
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  		Figure 4. Growth of unmodified and MMP-activatable MV. The growth of MVgreen, MVgreen-MMP-A, and MVgreen-MMP-A1 was compared on Vero cells (six panels on the left), or HT1080 cells (all others). The MMP-inhibitor GM6001 was added to the medium of the HT1080 cells in the six panels on the right. Cells were infected with a TCID50 of 0.01 and, 42 hours later, were photographed under visible light (first, third, and fifth) or UV light (second, fourth, and sixth) to activate GFP fluorescence.

 
In addition to MVgreen-MMP-A with the F protein containing the cleavage site PLG{downarrow}MLS, we generated a virus with the cleavage site PQG{downarrow}LYA (MVgreen-MMP-A1). This cleavage site, differing in four positions from the one above, was selected for preferential recognition by the proteases expressed in HT1080 cells in an evolutive protocol based on a retroviral library expressing combinatorially diversified recognition hexamers (36). As predicted, this virus was more efficient than MVgreen-MMP-A in fusing HT1080 cells (Fig. 4, bottom, third and fourth; compare with the corresponding panels in the middle). Fusion efficiency was quantified by counting the nuclei per syncytium in HT1080 cells. Compared with MVgreen set as 100%, the number of nuclei per syncytium was 61 ± 2% for MVgreen-MMP-A and 75 ± 3% for MVgreen-MMP-A1. Comparative analysis of the levels of F protein cleavage by immunoblot revealed similar levels of cleavage for the A1 as for the A protein (data not shown; see Discussion).

MMP specificity: accuracy of cleavage and sensitivity to a broad-spectrum MMP inhibitor. To verify whether the modified F proteins are cleaved as predicted in HT1080 cells, we collected protein extracts of MVgreen-MMP-A1–infected cells, immunoprecipitated the F protein, blotted it to a membrane, and excised the Coomassie blue–stained band corresponding to the F1 fragment. Edman degradation of this material yielded the NH2-terminal sequence LYAFAG, with three amino acids upstream of the phenylalanine defining position 1 of the fusion peptide. We did not observe alternative sequence products. This sequence proves formally that three additional residues at the F1 NH2 terminus do not abolish protein fusogenicity.

To confirm that fusion depends on MMP activity, we used an MMP inhibitor. We infected HT1080 cells with the viruses MVgreen, MVgreen-MMP-A, and MVgreen-MMP-A1 and added the broad spectrum MMP inhibitor GM 6001 (N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide; ref. 37) or an appropriate control (N-t-butoxycarbonyl-L-leucyl-L-tryptophan methylamide) to the medium. GM 6001 completely inhibited fusion by MVgreen-MMP-A and MVgreen-MMP-A1 (Fig. 4, middle and bottom, fifth and sixth, respectively) but not of the furin-activated parental virus MVgreen (Fig. 4, top, fifth and sixth).

MMP-activated MV retains oncolytic activity. To test whether the MMP-activated MV retains oncolytic activity, we established s.c. tumors by injection of 2 x 105 MMP-expressing HT1080 cells in the flank of athymic nude mice. After 9 days, when the tumors were palpable, the mice were distributed into three groups (mean tumor volume: 119, 116, and 118 mm3, respectively). In this xenograft model, MV infection retards tumor growth rather than abolishing it (38). Mice were then treated with five daily i.t. injections of 1.4 x 106 tissue culture infectious dose 50% (TCID50) of either MVgreen-MMP-A1, MVgreen as a positive control, or culture medium as negative control. Tumor growth in the MVgreen-MMP-A1–treated group (Fig. 5A, {blacksquare} ) was retarded to a level equivalent to that of the MVgreen-treated control group (Fig. 5A, {blacktriangleup}). This was reflected in the survival curves of the three groups (Fig. 5B). Survival of mice treated with either MVgreen or the MVgreen-MMP-A1 was similarly prolonged to about double that of the mock-treated animals. Thus, MVgreen-MMP-A1 retained oncolytic activity.


Figure 5
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  		Figure 5. Comparative analysis of the oncolytic efficacy and pathogenicity of an MMP-activatable and the parental virus. A and B, nude mice bearing s.c. HT1080 cells xenografts were treated with MVgreen-MMP-A1 ({blacksquare}), MVgreen ({blacktriangleup}), or mock infected with tissue culture medium (bullet). A, average tumor volumes measured in the three groups of mice, starting at the beginning of treatment (day 9 after implantation) and ending at day 24, when more than half the animals in the control group had to be euthanized. B, Kaplan-Meyer survival plot representation of the same data. C, genetically modified mice were infected i.c. with MV-MMP-A1, MV, or control media as above. The Kaplan-Meyer survival plot uses the same symbols for each group as above.

 
MMP-activated MV has reduced pathogenicity. We then sought to determine whether the pathogenicity of the virus expressing F-MMP-A1 was reduced. MV does not cause disease in normal mice but causes lethal encephalitis when injected intracerebrally in adult mice expressing the MV receptor human CD46 and defective for the {alpha}/ß IFN receptor (33). These hosts were used to compare the virulence of different MV strains (39), including recombinant viruses with a F protein cleavage activation signal modified to become trypsin dependent (20).

Since it was recently found that GFP expression from an additional transcription unit situated in the same position as in MVgreen attenuates virulence of a morbillivirus (40), we recloned F-MMP-A1 into an MV genome without a reporter gene, generating MV-MMP-A1. To assess its pathogenicity, nine animals were injected intracerebrally with 2 x 104 TCID50 (titer determined on HT1080 cells). Control groups were injected with equivalent amounts of MV (n = 9) or culture medium (n = 3). Although all but one mouse injected with MV died 4 to 8 days after injection, none of the control mice or those injected with MV-MMP-A1 died, indicating that this virus is not pathogenic. The pathobiology of the MMP-activatable MV was not characterized in the lungs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The success of oncolytic virotherapy depends on strict restriction of viral replication to cancer cells. To augment selectivity, we took advantage of the tumor microenvironment. We generated an MMP-activated MV and showed that it retains full oncolytic activity while completely losing the capacity to induce lethal encephalitis. Thus, activation of a virus particle can be made dependent of proteases secreted by cancer cells. Conditional activation by tumor-specific proteases can be engineered in other viruses currently used in cancer clinical trials.

The principle of transforming a noxious agent into a therapeutic by altering its activation was recently applied to a toxin: an anthrax toxin, which activity was made dependent on intermolecular complementation of subunits cleaved either by MMP or by the urokinase plasminogen activator, is safe, and effective in the treatment of tumor xenografts in mice (41). An MMP-activatable defective virus was also generated and shown to retard human xenograft growth, but this recombinant mouse paramyxovirus (SeV) was assembly deficient and propagated only through cell fusion (42). The MMP-activated MV generated here is fully assembly competent. If necessary, its fusogenicity can be enhanced without compromising particle formation by modifying the glycoprotein cytoplasmic tails (32, 43).

In the proteins F-MMP-A and F-MMP-A1, the furin recognition sequence was maintained but moved six residues upstream of the fusion peptide. This had three consequences in Vero cells: cleavage of these proteins was minimal; fusion function was completely compromised; and expression was reduced. On the other hand, in MV-MMP-A1–infected HT1080 cells, the F protein was cleaved efficiently. Sequence analysis of the F1 protein NH2 terminus revealed the three expected amino acids upstream of the fusion peptide, excluding subsequent trimming. Our study also indicated that the upstream sequences MLS and LYA are compatible with efficient membrane fusion. Work with the SeV F protein revealed that the sequence MTS, but not LGL or LWA, upstream of the fusion peptide is compatible with efficient membrane fusion (42), but Edman degradation analysis of the SeV F1 NH2 terminus was not presented.

In HT1080 cells, the F protein retaining the complete furin cleavage sequence was more fusogenic than the two proteins retaining only part of it, even if immunoblot analysis revealed similar ratios of precursor (F0) to product (F1) proteins. Thus, levels of F0 cleavage monitored by immunoblot do not directly correlate with biological activity monitored by fusion assays. Because in F-MMP-C no potential cleavage site for furin or related enzymes exists, these data suggest that proteases other than MMP cleave F-MMP-C in HT1080 cells. These proteases may leave a number of residues upstream of the fusion peptide, which may interfere with the membrane fusion process.

Even if the mechanisms of proteolytic cleavage/activation of the mutant MV F proteins are not fully understood, this study shows in animal models that restriction of F protein activation to cancer cells can add a layer of specificity to oncolytic MV. Provided that the appropriate amino acids are tolerated upstream of the F1 protein fusion peptide, it should be possible to engineer recombinant MV activated by specific MMP, as done for the "A1" sequence (36). Analogously, it should be possible to identify hexamers preferentially cleaved by proteases secreted by tumors resected from patients (44). These sequences will then be transferred to the MV F gene as done here to generate a panel of recombinant MV with specific recognition sequences for individual treatment of patients with tumors expressing specific MMP. The cancer cell–dependent activation principle established here with MV particles can be applied to other enveloped viruses that rely on intracellular proteases for activation, including those currently in cancer clinical trials, as NDV or reovirus.

Suppressing the patient's immune system is becoming an important option in gene therapy clinical trials (45, 46), and immunosuppression has been considered also in virotherapy preclinical studies (47). To achieve tight tumor specificity, and thus efficacy and safety, the combination of different targeting layers seems ideal: particle activation in the tumor microenvironment can be combined with cell entry through a designated receptor, and with restriction of viral replication to cells defective in innate immunity by modification of the IFN antagonist protein V (48). Recombinant MV with combinations of targeting modalities may flank and then replace simpler derivatives of the MV vaccine strain in ongoing clinical trials.


   Acknowledgments
 
Grant support: Mayo and Siebens Foundation, NIH grant R01 CA90636, Deutsche Forschungsgemeinschaft research scholarship grants SP 694/1-1 (C. Springfeld) and UN254/1-1 (G. Ungerechts), and Bundesministerium für Bildung und Forschung grant 01GU0504 (C.J. Buchholz).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Sompong Vongpunsawad for excellent technical support, Ben Madden and the Mayo Protein Core Facility for excellent service, Joshua Burgess for experimental help, and Patricia Devaux for helpful discussions.


   Footnotes
 
Note: C. Springfeld is currently at the Department of Gastroenterology, Infectious Diseases, and Intoxications, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany.

V. von Messling is currently at Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Québec, Laval H7V 1B7, Canada.

3 E. Galanis, personal communication. Back

4 293-FMVvac, Springfeld and Cattaneo, unpublished. Back

Received 2/21/06. Revised 4/ 7/06. Accepted 5/25/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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