Use of a ribonuclease of the T2 family having actin-binding activity for inhibiting tumor angiogenesis

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DESCRIPTION

Use of a T2 Family Ribonuclease Having Actin Binding Activity to Inhibit Tumor Angiogenesis

Field and background of the invention

The present invention relates to the use of a T2 family ribonuclease or a polynucleotide encoding it to inhibit tumor angiogenesis in a subject. The present disclosure also relates to pharmaceutical compositions containing, as active principle, a ribonuclease of the T2 family or a polynucleotide that encodes it for treating proliferative diseases or disorders in general and cancer in particular.

There is continuing interest, both within the medical community and among the general population, in the development of new therapeutic agents for the treatment of cell proliferative diseases and disorders such as cancer.

Agents exhibiting anti-proliferative, anti-colonization, anti-differentiation and / or anti-development properties against mammalian cells can potentially be used as anti-cancer drugs. As such, these agents are widely sought from both natural and synthetic sources.

RIBASES are ribonucleases (RNases) that exhibit biological activity that is distinct from their ability to degrade RNA. RIBASAs and their structural homologues are known to effect a large number of cellular reactions (Rybak, M. et al., 1991, J. Biol. Chem. 266: 21202-21207; Schein, CH 1997 Nature Biotechnol. 15: 529- 536). EDN and ECP, two major proteins found in the secretory granules of cytotoxic eosinophils (members of the RNase A family) are believed to be involved in the immune response. The self-incompatible plant style S-RNases (members of the RNase T2 family) arrest the growth of the pollen tube and thus prevent fertilization. RC-RNase, produced by bullfrog oocytes, inhibits, in vitro, the growth of tumor cells such as the P388 and L1210 leukemia cell lines and is effective for the in vivo killing of sarcoma 180, Erlich, and ascitic fluid cells. Mep II (Chang, CF. et al 1988, J. Mol Biol 283: 231-244). Some RNases have limited ribonuclease activity, an example of which includes angiogenins that stimulate blood vessel formation (Fett, JW 1985, Biochemistry 24: 5480-5486).

Living organisms use extracellular RNases for defense against pathogens and tumor cells. For example, ECP is secreted in response to attack by parasites (Newton, DL. 1992, J. Biol. Chem. 267: 19572-19578) and exhibits antibacterial and antiviral activity. This activity is also exhibited by Zinc-a2-glycoprotein (Zna2gp), an RNase present in most human body fluids including blood, seminal plasma, breast milk, synovial fluid, saliva, urine, and sweat (Lei G, et al. , 1998, Arch Biochem Biophys. 15 Jul; 355 (2): 160-4).

The specific mechanism by which extracellular RNases act in cellular reactions is unknown.

The main barrier to the cytotoxic activity of some RNases is the cell membrane. ECP has been found to form channels in both artificial and cellular membranes. Supposedly, ECP released from the granular membrane together with EDN (eosinophilic RNase, which is responsible for the destruction of cerebellar Purkinjie cells) transfers EDN to the intercellular space. Entry of the fungal toxin a-sarcin (a member of the RNase A family) into target cells depends on viral infection that permeates the cell membrane (Rybak, M. et al., 1991, J. Biol. Chem. 266: 21202-21207). It is also possible that RNases enter the cell through endocytosis. When the Golgi-altering drugs retinoic acid or monensin were used to artificially deliver BS-RNase to cells, cytotoxicity increased dramatically (Wu Y, et al., 1995, J Biol Chem. 21; 270 (29): 17476-81).

The cytotoxicity of RNases can be used for therapeutic purposes. Human RNase L is activated by interferon and inhibits viral growth. Expression of the gene for human RNase L together with that of a 2'5'-A synthetase in tobacco plants is sufficient to protect plants from cucumber mosaic virus and to prevent replication of potato virus Y. Human immunodeficiency virus 1 (HIV-1) induces blockage in RNase L antiviral pathways (Schein, CH 1997 Nature Biotechnol. 15: 529-536.). RNases can be fused with specific membrane protein antibodies to create immunotoxins. For example, the fusion of RNase with antibodies to the transferrin receptor or to the CD5 T lymphocyte antigen leads to the inhibition of protein synthesis in tumor cells that carry a specific receptor for each of the above toxins (Rybak, M. et al., 1991, J. Biol. Chem. 266: 21202-21207; Newton DL, et al., 1998, Biochemistry 14; 37 (15): 517383). Since RNases are less toxic to animals, they may have fewer undesirable side effects than currently used immunotoxins.

The cytotoxicity of cytotoxic ribonucleases appears to be inversely related to the strength of the interaction between a ribonuclease inhibitor (RI) and RNase. The ribonuclease inhibitor (RI) is a naturally occurring molecule found within cells of the vertebrae that serves to protect these cells from the potentially lethal effects of ribonucleases. The ribonuclease inhibitor is a 50 kDa cytosolic protein that binds to RNases with varying affinity. For example, RI binds to members of the bovine pancreatic ribonuclease A (RNase A) superfamily of ribonucleases with inhibition constants spanning ten orders of magnitude, with Ki ranging from 10^-6 to 10^-16 M.

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A-RNases

ONCONASE, like RNase A and BS-RNase, is a member of the RNase A superfamily. Members of the RNase A superfamily share approximately 30% identity in amino acid sequences. Most of the non-conserved residues are located in surface loops and appear to play a significant role in the dedicated biological activity of each RNase. ONCONASE was isolated from Leopard frog (Rana pipiens) oocytes and early embryos. It has antitumor effect on a variety of solid tumors, both in situ and in vivo (Mikulski SM, et al., 1990 J. Natl. Cancer 17; 82 (2): 151-3). ONCONASE has also been found to specifically inhibit HIV-1 replication in infected H9 leukemia cells at non-cytotoxic concentrations (Youle RJ, et al., 1994, Proc. Natl. Acad. Sci. 21; 91 (13): 6012-6).

Although the RNase activity of ONCONASE is relatively low, it is accepted that the enzymatic and cytotoxic activities thereof are associated to some degree. The tertiary structure of A-RNases is believed to differentiate between cytotoxic and non-cytotoxic types. For example, differences between the tertiary structure of onconase and RNase A are believed to be responsible for the increased cytotoxicity observed for ONCONASE. ONCONASE, unlike RNase A, contains a blocked Glu 1 N terminal residue (pyroglutamate) that is essential for both enzymatic and cytotoxic activities. This unique structure allows ONCONASE to permeate into target cells (Boix E., et al., 1996, J. Mol. Biol. 19: 257 (5): 992-1007). Furthermore, in ONCONASE the Lys9 residue replaces the Gln11 residue of RNase A, which is believed to effect the structure of the active site. Furthermore, the differences in the amino acid sequence of the primary structure between ONCONASE and RNase A cause topological changes in the periphery of the active site that effect its specificity (Mosimann SC, et al., 1992, Proteins 14 (3): 392 -400).

Cytotoxicity differences between A-RNases are also attributed to their ability to bind to RI. Bovine seminal ribonuclease (BS-RNase) is 80% identical in amino acid sequence to RNase A, but unlike other members of the RNase A superfamily, BS-RNase exists in a dimeric form. The quaternary structure of BS-RNase has been shown to prevent binding to RI, thus allowing the enzyme to retain its ribonucleolytic activity in the presence of RI (Kim et al., 1995, J. Biol. Chem. 270 No. 52 : 31097-31102). ONCONASE, which shares a high degree of homology with RNase A, is resistant to RI binding. The RIONCONASA complex has a K_d at least one hundred million times lower than that of the RI-RNase A complex. The lower binding affinity of ONCONASE for RI prevents effective inhibition of ribonucleolytic activity and could explain why ONCONASE is cytotoxic at low concentrations whereas RNase A is not .

Binding to the cell surface receptor has been suggested to be the first stage in ONCONASE cytotoxicity. Nothing is known about the nature of ONCONASE receptors on mammalian cell surfaces. ONCONASE can bind to cell surface carbohydrates as in the case of ricin, or it can bind to receptors originally developed for physiologically imported molecules such as polypeptide hormones (Wu Y, et al., 1993, J. Biol. Chem. fifteen; 268 (14): 10686-93). In mice, ONCONASE was eliminated from the kidneys 50-100 times slower than RNase A. The lower elimination rate of ONCONASE is explained as a result of its greater ability to bind to tubular cells and / or by its resistance. to proteolytic degradation. The strong retention of ONCONASE in the kidneys could have clinical implications (Vasandani VM, et al., 1996, Cancer Res. 15; 56 (18): 4180-6). ONCONASE can also bind to Purkinjie cell EDN receptors (Mosimann SC, et al., 1996, J. Mol. Biol. 26; 260 (4): 540-52). The specificity of ONCONASE is also expressed in its tRNA preference. In rabbit reticulocyte lysate and Xenopus oocytes, ONCONASE was found to inhibit protein synthesis by degradation of tRNA, rather than rRNA or mRNA. In contrast, RNase A primarily degrades rRNA and mRNA (Lin JJ, et al., 1994, Biochem. Biophys. Res. Commun. 14; 204 (1): 156-62).

Treatment of susceptible tissue cultures with ONCONASE results in the accumulation of cells arrested in the G 1 phase of the cell cycle, which have a very low level of RNA contents (Mosimann SC, et al., 1992, Proteins 14 (3): 392-400). In glioma cells ONCONASE inhibited protein synthesis without a significant reduction in cell density, showing that ONCONASE is also cytotoxic to cells as well as being cytostatic (Wu Y., et al., 1993, J. Biol. Chem. fifteen; 2 68 (14): 10686-93). ONCONASE, combined with chemotherapeutic agents, can overcome multidrug resistance. Treatment with vincristine and ONCONASE increased the median survival time (MST) of mice bearing vincristine-resistant tumors up to 66 days, compared to 44 days in mice treated with vincristine alone (Schein, CH, 1997, Nature Biotechnol. 15 : 529-536). In addition, some chemotherapeutic agents can act in synergy with ONCONASE. In human pancreatic adenocarcinoma and human lung carcinoma tumor cell lines treated with a combination of ONCONASE and tamoxifen (anti-estrogen), trifluoroperazine (Stelazine, calmodulin inhibitor) or lovastatin (3-hydroxyl-3-methylglutatil coenzyme inhibitor (A HMG-CoA) reductase) stronger growth inhibition was observed than in cells treated with ONCONASE only (Mikulski SM, et al., 1990, Cell Tissue Kinet. 23 (3): 237-46). Therefore, a possibility of developing combination therapy regimens with greater efficacy and / or less toxicity is evident.

Bovine seminal RNase is a unique member of the RNase A family, since it is the only RNase that contains a dimer of RNase A-like subunits linked by two disulfide bonds. In addition, it maintains allosteric regulation both by substrate and by reaction products. Regulation occurs in the phase of

ES 2 397 052 ​​T3 hydrolysis of cyclic nucleotides. It has the ability to cleave both single and double stranded RNA. BS-RNase is highly cytotoxic. It has an antitumor effect in vitro on mouse leukemic cells, human embryonic lung and HeLa cells, mouse neuroblastoma cells and human fibroblasts and mouse plasmacytoma cell lines. When administered in vivo to rats bearing solid carcinomas (thyroid follicular carcinoma and its lung metastases), BS-RNase induces a drastic reduction in tumor weight, with no detectable toxic effects in treated animals (Laccetti, P. et al., 1992 , Cancer Research 52: 4582-4586). Artificially monomerized BS-RNase has higher ribonuclease activity but lower cytotoxicity than native dimeric BS-RNase (D'Allessio G., et al., 1991, TIBS: 104-106). This, again, indicates the importance of molecular structure for biological activity. It appears that like ONCONASE, BS-RNase binds to recognition site (s) on the surface of target cells, prior to penetration into target cells.

Besides being cytotoxic, BS-RNase is also immunosuppressive. BS-RNase can block the proliferation of activated T lymphocytes, and prolong the survival of transplanted skin grafts in allogeneic mice. The immunosuppressive activity of SB-RNase is explained by the need to protect sperm from the female immune system.

T2-RNases

In plants, self-compatibility is abundant and it is effective in preventing self-fertilization. Pollen that carries a particular allele at the S locus, which controls self-incompatibility, is unable to fertilize plants that carry the same S allele. In many self-incompatible plants, especially members of Solanaceae and Rosaceae, SRNase, a member of the T2 family -RNase, is secreted by female organs. S-RNase specifically recognizes self pollen and stops its growth in the stigma or style before fertilization occurs (Clarke, AE and Newbigin, E., 1993, Ann. Rev. Genet. 27: 257-279). The growth arrest of the pollen tube is believed to be a direct consequence of RNA degradation, however the mode of entry of S-RNase into the tube cell is not yet clear.

Members of the RNase T2 family were identified for the first time in fungi (Egami, F. and Nakamura, K. 1969, Microbial ribonucleases. Springer-Verlag, Berlin). Since then, they have been discovered in a wide variety of organisms, ranging from viruses to mammals. In particular, T2-RNases show a much broader distribution than the exhaustively described RNase A family. However, the in vivo role of T2-RNases in mammalian cells is not yet known.

In microorganisms, it is generally accepted that extracellular T2-RNases contribute to the digestion of polyribonucleotides present in the growth medium, thus giving rise to nutrients that can be diffused. They can also act as defense agents (Egami, F. and Nakamura, K., 1969, Microbial ribonucleases. Springer-Verlag, Berlin).

In plants, T2-RNases play a role in the pollination process, significantly limiting the elongation of the polionic tubes that are directed towards the ovules (Roiz, L. and Shoseyov, O., 1995, Int. J. Plant Sci. 156: 37-41, Roiz L. et al., 1995, Physiol. Plant. 94: 585-590). To date, the mechanism by which these RNases affect pollen tubes is unclear.

Therefore, there are few examples of cytotoxic ribonucleases that can be used effectively as cancer treatment agents. New ribonucleases with anti-proliferation, anti-colonization, anti-differentiation and / or anti-development activities are needed for mammalian cells to enhance the spectrum of therapeutic agents available for treatment of human cancers, thus opening new horizons in the field of cancer treatment.

There is therefore a widely recognized need and it would be highly advantageous to have a new ribonuclease that has potential utility in the treatment of tumor angiogenesis.

Summary of the invention

The scope of the present invention is defined by the claims and any information that is not within the claims is provided for information only.

In accordance with one aspect of the present disclosure there is provided a method of preventing, inhibiting and / or reversing the proliferation, colonization, differentiation and / or development of abnormally proliferating cells in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a T2 family ribonuclease.

In accordance with another aspect of the present disclosure there is provided a method of preventing, inhibiting and / or reversing the proliferation, colonization, differentiation and / or development of abnormally proliferating cells in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a polynucleotide that encodes and is capable of expressing in vivo a recombinant ribonuclease of the T2 family.

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According to yet another aspect of the present disclosure methods are provided for (i) treating a tumor in a subject; (ii) prevent, inhibit and / or reverse the development of a tumor in a subject; (iii) prevent, inhibit and / or reverse the transformation of a benign tumor into a malignant tumor in a subject; (iv) prevent, inhibit and / or reverse tumor angiogenesis in a subject; (v) reduce the number of individual tumors; (vi) reduce tumor transformation in a subject; (vii) reducing various malignant tumors in a subject; and (viii) preventing, inhibiting, and / or reversing the transformation of a tissue into a tumor in a subject, each of the procedures being performed by administering to the subject a therapeutically effective amount of a T2 family ribonuclease or a therapeutically effective amount of a polynucleotide that encodes and is capable of expressing in vivo a recombinant ribonuclease of the T2 family.

According to yet another aspect of the present disclosure there is provided a pharmaceutical composition comprising, as an active principle, a ribonuclease of the T2 family, and a pharmaceutically acceptable carrier.

According to a further aspect of the present disclosure there is provided a pharmaceutical composition comprising, as an active principle, a polynucleotide that encodes and is capable of expressing in vivo a recombinant ribonuclease of the T2 family, and a pharmaceutically acceptable carrier.

According to yet another aspect of the present disclosure there is provided a method for preparing a medicament useful for preventing, inhibiting and / or reversing the proliferation, colonization, differentiation and / or development of abnormally proliferating cells comprising the step of combining a T2 family ribonuclease with a pharmaceutically acceptable carrier.

According to a still further aspect of the present disclosure there is provided a method for preparing a medicament useful for preventing, inhibiting and / or reversing the proliferation, colonization, differentiation and / or development of abnormally proliferating cells comprising the step of combining a polynucleotide that encodes and is capable of expressing in vivo a recombinant ribonuclease of the T2 family with a pharmaceutically acceptable carrier.

In accordance with additional features in preferred embodiments of the invention described below, the T2 family ribonuclease is substantially devoid of ribonucleolytic activity. As used herein the phrase "substantially lacks ribonucleolytic activity" refers to (i) an inactivated ribonuclease (natural or recombinant) of the T2 family that has 0-10% ribonucleolytic activity compared to a similar ribonuclease , not inactivated; and / or (ii) a recombinant mutant (natural or human-induced) ribonuclease of the T2 family that has 0-10% ribonucleolytic activity compared to a similar, non-mutant ribonuclease. Inactivation of the ribonucleolytic activity of the T2 family ribonuclease can be effected by a procedure selected from the group consisting of boiling, autoclaving, and chemical denaturation.

According to further additional features in the described preferred aspects the abnormally proliferating cells are cancer cells.

According to further additional features in the described preferred embodiments, the step of administering to the subject the therapeutically effective amount of the RNase of the T2 family is carried out by a mode of administration selected from the group consisting of oral administration, topical administration, transmucosal administration, parenteral administration, rectal administration and by inhalation.

According to further additional features in the described preferred embodiments the T2 family ribonuclease is RNase B1.

According to further additional features in the described preferred embodiments the ribonuclease of the family of ribonuclease T2 is selected from the group consisting of RNase T2, RNase Rh, RNase M, RNase Trv, RNase Irp, RNase Le2, RNase Phyb, RNase LE, RNase MC, RNase CL1, RNase Bsp1, RNase RCL2, RNase Dm, RNase Oy and RNase Tp.

According to further additional features in the preferred aspects described the abnormally proliferating cells are cells associated with a proliferative disorder or disease selected from the group consisting of papilloma, blastoglioma, Kaposi's sarcoma, melanoma, lung cancer, cancer of ovarian, prostate cancer, squamous cell carcinoma, astrocytoma, head cancer, neck cancer, bladder cancer, breast cancer, colorectal cancer, thyroid cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, leukemia, lymphoma, Hodgkin's disease, Burkitt's disease, arthritis, rheumatoid arthritis, diabetic retinopathy, angiogenesis, restenosis, stent restenosis, and vascular graft restenosis.

The present invention successfully addresses the shortcomings of presently known configurations by characterizing novel activities of ribonucleases of the T2 family useful in inhibiting tumor angiogenesis.

Brief description of the drawings

The invention is described herein, by way of example only, with reference to the accompanying drawings.

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With specific reference now to the detailed drawings, it is emphasized that the details shown are by way of example and for illustrative analysis purposes of the preferred embodiments of the present invention only, and are presented to provide what is believed to be the most useful description. and easily understandable of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings revealing to those skilled in the art how they may be carried out in practice. the many forms of the invention.

In the drawings:

FIGURE 1 is a graphical representation of the absorbance and RNase activity of Aspergillus niger RNASE B1 isolated according to the teachings of Roiz, L. and Shoseyov, O., 1995, Int. J. Plant Sci. 156: 37-41 . Graph A represents fractions obtained by EMD-TMAE column chromatography of a crude filtrate, while graph B represents the fraction obtained from MONO-Q column chromatography of the active fractions that resulted from EMD-TMAE chromatography of the filtrate in stupid. The solid line represents absorbance at 280 nm and the dashed line represents RNase activity.

FIGURE 2 is an SDS-PAGE zymogram illustrating the increase in RNase B1 protein concentration throughout the purification steps used. Lane 1 represents crude filtrate; Lane 2 represents the eluate from the EMD-TMAE column; Lane 3 represents the eluate from the MONO-Q column; Lane 4 represents lane 3 eluate tested in situ for RNase activity and stained with toluidine blue; Lane 5 represents purified RNase after deglycosylation by PNGase F. Lanes 1-3 and 5 are stained with coomassie blue.

FIGURE 3 is a graph illustrating the in vitro effect of different concentrations of B1 RNase on peach pollen germination (solid line with black squares) and pollen tube length (dashed line with boxes).

FIGURES 4a and 4b illustrate the effect of RNase B1 on the growth of the peach pollen tube in the stigma and upper part of the style. Figure 4a represents the control flower, while Figure 4b is a flower treated with RNase B1 before pollination. Bar = 0.2 mm.

FIGURES 5a and 5b illustrate the effect of RNase B1 on pollen tube growth in the stigma of a mandarin flower. Figure 5a represents a control flower that was exposed to open pollination for 48 hours. Figure 5b represents a flower that was treated with RNase B1 prior to pollination. Bar = 0.1 mm.

FIGURES 6a and 6b illustrate viability assay performed on nectarine seeds. Figure 6a represents a control seed produced by an untreated flower, while Figure 6b represents a seed produced by a flower treated with RNase B1. Bar = 0.3mm.

FIGURE 7 illustrates the effect of RNase B1, untreated, boiled or autoclaved, on the length of the pollen tube of lily cv. Osnat.

FIGURES 8a and 8b illustrate the effect of RNase B1 in pollen tubes of lilies growing in vitro and stained with IKI.

FIGURES 9a and 9b illustrate still images captured from integrated video images showing organelle movement and localization in pollen tubes untreated (Figure 9a) and treated (Figure 9b) with RNase B1.

FIGURES 10a and 10b illustrate the effect of RNase B1 on actin filaments from a growing lily pollen tube. Figure 10a represents the control pollen tube while Figure 10b represents the pollen tube treated with RNase B1. Both pollen tubes were excised and stained with TRITC phalloidin for visualization after experimentation.

FIGURE 11 is a representation of Scatchard depicting the binding of RNase B1 with actin. At actin concentration (gM), Rf - concentration of free RNase B1 (gM), Rb - concentration of bound RNase B1 (gM).

FIGURES 12a-c illustrate immunooro silver stained lily pollen tubes grown for 1 hour. Figure 12a represents a control, while Figures 12b and 12c are both pollen tubes treated with RNase B1. The pollen tube of Figure 12b was incubated with rabbit preimmune serum, while the pollen tube of Figure 12c was incubated with polyclonal rabbit anti-RNase B1 antibody.

FIGURES 13a and 13b illustrate the effect of different concentrations of RNase B1 on HT29 colon cancer cell viability. Repeated cell samples were cultured for 48 hours or 72 hours at 37 ° C, visualized using trypan blue differential staining and counted. Figure 13a represents the total cell numbers while Figure 13b represents the percentage of dead cells.

FIGURE 14 illustrates the effect of RNase B1 on HT29 cell clonogenicity. Repeated cell samples were preincubated with growth medium in the absence or presence of RNase B1 10^-6 M for 48 hours, trypsinized, washed, resuspended in growth medium without RNase B1 in serial dilutions, and seeded in 96-well microtiter plates to colonize for 14 days. Colonies were counted after fixation and methylene blue staining.

FIGURE 15 illustrates the effect of the period of exposure to RNase B1 on the clonogenicity of HT29 cells. Repeat cell samples were preincubated with growth medium containing RNase B1 10^-6 M for 48 hours and then allowed to colonize in growth medium containing the same concentration of RNase B1, or in medium without RNase B1. Colonization was carried out in microtiter plates

ES 2 397 052 ​​T3 of 96 wells for 7 days. Each treatment contained different starting numbers of cells per well. Colonies were counted after fixation and visualization in methylene blue. Cells preincubated and colonized in growth medium without RNase B1 acted as a control.

FIGURES 16a-c illustrate the effect of RNase B1 on the colonization capacity of HT29 cells. Control cells (Figure 16a) were pre-incubated for 48 hours in growth medium without RNase B1 and then trypsinized and incubated with the same growth medium in 96-well microtiter plates for colonization. Figure 16b represents cells that were pre-incubated for 48 hours in growth medium containing RNase B1.^-6 M and then allowed to colonize in growth medium without RNase B1. Figure 16c represents cells that were pre-incubated and then colonized in growth medium containing RNase B1.^-6 M. Cell colonies were visualized using methylene blue staining.

FIGURE 17 is a scheme of in vivo experiments performed in rats, describing the treatment for each group of 6 rats.

FIGURE 18 demonstrates the effect of two different pHs on the release rate of RNase B1 from CAP microcapsules. The microcapsules containing 10 mg of RNase B1 were suspended in 10 ml of 0.1 M HCl (pH 1) or 0.1 M Tris buffer (pH 8) and incubated at 37 ° C with shaking. Top solution samples were taken every 30 minutes for RNase activity assays.

FIGURES 19a-d demonstrate the effect of RNase B1 and / or DMH on the growth rate of rats, as shown by body weight at the end of each experiment. The initial weight of the rats was approximately 200 grams. n = 6.19a - PBS, RNase B1 or I-RNase B1 was provided by osmotic pumps at weeks 1-9 after the first DMH injection (preventative treatment). Rats treated as described above, but in the absence of DMH, were used as control. 19b - PBS, RNase B1 or I-RNase B1 was provided by osmotic pumps at weeks 12-17 after the first DMH injection (therapeutic treatments). 19c - Rats were fed microcapsules containing RNase B1 or glucose as a preventive treatment. Rats that had been treated with RNase B1 in the absence of DMH were used as controls. 19d - Rats were fed microcapsules containing RNase B1 or glucose.

FIGURES 20a-c demonstrate RNase activity in feces of rats implanted with osmotic pumps containing RNase B1 (20a), I-RNase B1 (20b) or PBS (20c), as a preventive treatment. As a control, rats were treated with RNase B1 or PBS in the absence of DMH. RNase activity was determined as described in the Examples section below.

FIGURE 21 demonstrates RNase activity in feces of rats fed microcapsules containing RNase B1 or glucose as a preventive treatment. As a control, rats were fed RNase B1 or glucose in the absence of DMH. RNase activity was determined as described in the Examples section below.

FIGURE 22 shows the number of aberrant crypt foci (ACF) in the distal colon (5 cm) of rats that had been implanted with osmotic pumps as a preventive treatment (n = 6).

FIGURES 23a-c demonstrate the effect of RNase B1 on different parameters examined in the distal colon (5 cm) of rats fed with microencapsulated RNase B1 or glucose, as preventive treatment (n = 6). 23a - number of tumors per colon; 23b - tumor size; 23c - ACF per colon.

FIGURES 24a-d demonstrate different types of tumors, as photographed on the inner mucosa surface 1 hour after excision. 24a - red tumors; 24b - white tumors. 24c - a pink tumor and a red tumor; 24d - distribution of three types of tumors in rats fed with microcapsules containing glucose or RNase B1, as preventive treatment.

FIGURES 25a-d show histopathological examination of tumors stained with Mayer's hematoxylin and martius yellow. 25a - an adenoma or adenopapilloma - a benign tumor; 25b - adenocarcinoma, in which mucosal cells have penetrated below the submucosa; 25c - a well-developed adenocarcinoma, in which the tissue arrangement is completely disturbed; 25d - tumor adenoma and adenocarcinoma type distribution pattern in rat colons treated with encapsulated glucose or RNase B1, as preventive treatment.

FIGURES 26a-c demonstrate the effect of RNase B1 on different parameters examined in the distal colon (5 cm) of rats treated with osmotic pumps containing PBS, RNase B1 or I-RNase B1 as therapeutic treatment. 26a - number of tumors per colon; 26b - distribution of tumors according to size; 26c distribution of tumors according to color, indicating angiogenesis.

FIGURES 27a-c demonstrate the effect of RNase B1 on different parameters examined in the distal colon (5 cm) of rats fed microencapsulated RNase B1 or glucose as therapeutic treatments. 27th number of tumors per colon; 27b - distribution of tumors according to size; 27c - distribution of tumors according to color, indicating angiogenesis.

FIGURES 28a-b show cultured HT-29 4-d human colon carcinoma cells stained with TRIRC for actin. 28a - control cells; 28b - cells that were grown in the presence of 10-6 M RNase B1.

FIGURES 29a-b show cultured HT-29 4-d human colon carcinoma cells immunostained for membrane actin. 29a - control cells; 29b - cells grown in the presence of 10-6 M RNase B1.

FIGURES 30a-c show cultured HT-29 4-d human colon carcinoma cells immunostained with FITC. Anti-RNase B1 was used as the primary antibody. 30a - control cells; 30b - cells that were grown in the presence of RNase B1, showing RNase B1 bound on the cell surface; 30c - preimmunized serum (PIS) was used as the primary antibody.

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FIGURE 31 demonstrates the effect of different protein treatments on lily pollen tube length. The pollen tubes were cultured in vitro for 1 hour at 25 ° C as described in the section on

Examples below.

Description of the preferred embodiments

The scope of the present invention is defined by the claims and any information that does not fall within the claims is provided for information only.

The present disclosure teaches the use of a T2 family ribonuclease or a polynucleotide encoding it, which inhibits tumor angiogenesis. The present disclosure further teaches pharmaceutical compositions that contain, as active principle, a ribonuclease of the T2 family or a polynucleotide that encodes it to treat proliferative diseases or disorders in general and cancer in particular.

The use of ribonucleases with cytotoxic activity to inhibit tumor cell proliferation is not new and has been previously demonstrated in the art. A family A ribonuclease known commercially as ONCONASE has been shown to inhibit cell proliferation in tumor tissue in clinical trials. Several other RNases of the RNase A superfamily have also been shown to have cytotoxic activity in addition to their ribonucleolytic activity.

Although the cytotoxicity of some ribonucleases depends to some degree on ribonucleolytic activity, the level of ribonucleolytic activity does not always correlate with the level of cytotoxicity observed for ribonucleases. In addition, there are several examples of ribonucleases that do not exhibit cytotoxic activity at all, but perform well as ribonucleases. The best known example is RNase A. In other cases, the reaction rate is scarified for more specific binding or improved function in some other capacity. For example, the active site of angigenin is blocked by side chains that are not present in RNase A, making it 10,000 times less active on general substrates, but more specific in cleavage of ribosomal RNA. BS-RNase is a faster nuclease when it is monomeric. However, its cytotoxicity is higher, and inhibition by ribonuclease inhibitor is considerably reduced in the dimeric form. Glycosylated RNase B is less active than RNase A on most substrates, whereas a frequently observed deamidation in BS-RNase (asparagine 67 to isoaspartate) reduces the activity of RNase A mutants by cleaving an entire chain of binding structures. H in protein (reviewed in Shein, CH 1997. Nature Biotechnol 15: 529-536).

Ribonucleases of the T2 family are characterized by their unique molecular characteristics. A comparison between RNase members of families A and T2 is summarized below in Table 1 (amino acid location is after RNase A and RNase T2 in families A and T2, respectively).

TABLE 1

<td>Characteristic</td><td>RNase A</td><td>RNase T2</td>

<td>Molecular mass</td><td>11-14 kDa (with the exception of BSRNase)</td><td>24-36 kDa.</td>

<td>Optimal temperature for RNase activity:</td><td>37 ° C</td><td>50-60 ° C</td>

<td>Optimal pH for RNase activity:</td><td> 6,5-8</td><td> 3,5-5</td>

<td></td><td>Not glycosylated</td><td>12-25% of the total molecular mass</td>

<td>Base specificity:</td><td>Specific to pyrimidine base</td><td>Non-specific with preference for adenylic acid.</td>

ES 2 397 052 ​​T3 (continued)

<td>Characteristic</td><td>RNase A</td><td>RNase T2</td>

<td>Disulfide bonds:</td><td>Four: Common: Cys28-84, Cys40-96, Cys58110. In pancreatic RNases the fourth SS bond is located between Cys6572, forming a loop containing Glu69 and Asn71, which are part of the nucleotide binding site. In ONCONASE and bullfrog lectin Cys87-Cys104 they form a COOH-terminal loop, which is located near the active site. Angiogenins have only 3 disulfide bonds.</td><td>Five: Cys3-20, Cys10-53, Cys19-120, Cys63-112 and Cys182-213.</td>

<td>Mechanism of RNase activity:</td><td>Active site Two stages in RNA extension (i) His12 acts as a general base and removes a proton from the 2'-hydroxyl group of RNA. His 119 acts as a general acid, donating a proton from the O 5 'of the outgoing nucleotide. (ii) The resulting 2'3'cyclic nucleotides are hydrolyzed, with the roles of His12 and His119 reversed. Lys41 stabilizes the pentavalent transition state. Substrate binding sites: GLn11 and Phe120 form hydrogen bonds with the substrate. In ONCONASE and glu11 bullfrog lectin forms H bond with the substrate phosphate. Gln96, Asn71, Glu111, of which Asn71 is the most conserved, could catalyze RNA cleavage.</td><td>Active site RNA catalysis is similar to RNase A. His46 and His109 act as general acid and base catalysts. Glu 105 and Lys108 could play a role in the polarization of the P = O bond of the substrate or in the stabilization of the transition state pentacovalent. Substrate binding sites: His104 (in plants it is Tyr or Asp) could act as the phosphate receptor of the substrate. There are two recognition sites: The main site (B1) contains Tyr57, Trp49 and Asp51. Asp51 is responsible for the recognition of the adenine base. A minor site (B2) contains Phe101, Gln95, Asn94, Ser93, Pro92, and Gln32.</td>

Ribonucleases of the T2 family have been identified in numerous microorganisms, as well as in plants, where they play an active role in the pollination process, selectively limiting the elongation of pollen tubes that are directed towards the ovules.

As discovered by the inventors of the present invention and as further detailed hereinbelow in Examples 1, 2, and 6, RNase B1, a ribonucleolytically active or non-active ribonucleolytically T2 ribonuclease, specifically binds actin. in elongation pollen tubes to thereby inhibit elongation of pollen tubes and also mammalian cell actin.

Actin is known to form filaments that are essential cytoskeletal components of cells, active both in maintaining cell structure and in supporting intracellular transport of organelles. As a result, actin filaments participate in many cellular processes during the life cycle of normal and abnormal cells, including proliferation, colonization, differentiation, transformation, and other aspects of development including tissue formation. Numerous studies have shown that actin also participates in various cellular processes that control the generation of cancer cells (Jordan, MA &amp; Wilson, L. 1998. Curr. 15 Opin. Cell Biol. 10: 123-130; Jammy, PA &amp; Chaponnier, C. 1995. Curr. Opin. Cell Biol. 7: 111-117: Sigmond, SH

nineteen ninety six. Curr. Opin. Cell Biol. 8: 66-73; Tapon, N. et al. 1997. Curr. Opin. Cell Biol. 9: 86-92). Thus, for example, actin filaments participate in abnormal cell proliferation (Assoian, RK &amp; Zhu, X. 1997. Curr. Opin.

Cell Biol. 9: 93-98). Malignant cells more sensitive to cytochalasin B than normal cells were found

ES 2 397 052 ​​T3 (Hemstreet GP et al. 1996. J. Cell Biochem. 25S: 197-204).

Since actin is a highly conserved protein, which maintains a high level of homology between evolutionarily distant organisms, it has been hypothesized that the actin-binding activity of RNase B1, which inhibits elongation of the pollen tube, can be used, without be limited by this theory, to specifically bind to mammalian cell actin, to thereby inhibit proliferation, colonization, differentiation and / or development thereof.

While reducing the present disclosure to practice and as further described in Example 2 and 5 of the Examples section, exogenous RNase B1 specifically binds to membrane actin and causes a disorder of the cellular actin network. As shown in Examples 3-5, the effect of RNase B1 on mammalian cancer cells was further investigated in vitro and in vivo. As clearly demonstrated there, RNase B1 (i) substantially reduces the proliferation and / or colonization of adenocarcinoma cells grown in culture; and (ii) reduces the number of aberrant crypt foci (ACF), reduces the number and size of tumors, interferes with tumor angiogenesis, reduces the malignancy of tumors and the transition from adenoma to adenocarcinoma in a rat model of carcinoma. colon, in a preventive and / or therapeutic manner, while having no obvious side effects on healthy tissue in the colon or elsewhere.

One or more ribonucleases of the T2 family are collectively referred to herein as T2-RNase. Similarly, one or more polynucleotides encoding one or more ribonucleases of the T2 family are collectively referred to herein as a polynucleotide encoding a T2-RNase (or the same).

Therefore, in accordance with one aspect of the present invention there is provided a method of inhibiting tumor angiogenesis. The method according to this aspect of the present invention is carried out by administering to the subject a therapeutically effective amount of a ribonuclease of the T2 family or of a polynucleotide that encodes and is capable of expressing in vivo a recombinant ribonuclease of the T2 family, per se itself or as an active principle of a pharmaceutical composition.

Therefore, according to another aspect of the present invention there is provided a pharmaceutical composition that comprises, as an active principle, a ribonuclease of the T2 family, a polynucleotide that encodes and is capable of expressing in vivo a recombinant ribonuclease of the T2 family and a pharmaceutically acceptable carrier.

According to yet another aspect of the present invention there is provided a method for preparing a medicament useful for preventing, inhibiting tumor angiogenesis comprising the step of combining a ribonuclease of the T2 family or a polynucleotide that encodes and is capable of expressing in vivo a Recombinant ribonuclease of the T2 family, with a pharmaceutically acceptable carrier.

The drug is preferably identified as providing a treatment for a specific proliferative disorder or disease, such as a specific cancer. Such printed identification can be made on, for example, a container containing the drug or on a brochure, as is well known in the art.

The method and pharmaceutical composition of the present disclosure can be used to, for example, (i) treat a tumor in a subject; (ii) prevent, inhibit and / or reverse the development of a tumor in a subject; (iii) prevent, inhibit and / or reverse the transformation of a benign tumor into a malignant tumor in a subject; (iv) prevent, inhibit and / or reverse tumor angiogenesis in a subject; (v) reduce the number of individual tumors in a subject; (vi) reduce tumor size in a subject; (vii) reducing a number of malignant tumors in a subject; and / or (viii) prevent, inhibit and / or reverse the transformation of a tissue into a tumor in a subject.

T2-RNase can be derived from a native source, as further exemplified in Example 1 below, or, alternatively, can be produced as a recombinant protein using a polynucleotide (see Table 2 below and the following descriptions) and expression system appropriate. The expression and purification of recombinant proteins are well known in the art and can be accomplished by any one of a plurality of alternative techniques described in detail in any one of several textbooks and laboratory protocol books, including, for example, "Molecular Cloning: A laboratory Manual ”Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley &amp; Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Volumes 1-4, Cold Spring Harbor Laboratory Press, New York (1998).

ES 2 397 052 ​​T3

TABLE 2

<td colspan="2">Source</td><td>Name (Prot)</td><td>Name (Gen)</td><td>Reference or references</td><td>GeneBank Access Number</td>

<td colspan="6"></td>

<td rowspan="4">Bacteria</td><td>Aeromonas hydrophila</td><td></td><td>RNI locus</td><td>Favre, D. et al. 1993. J. Bacteriol. 175: 3710-3722.</td><td>Q07465</td>

<td>Haemophilus influenzae</td><td>RNase HI0526</td><td>Locus RN26</td><td>Fleischmann, RD, et al. 1995. Science 269: 496-512.</td><td>P44012</td>

<td>Escherichia coli</td><td>RNase I</td><td>RNI locus</td><td>Meador, J. III. &amp; Kennell, D. 1990. Gene 95: 1-7. Oshima, T., et al. 1996. DNA Res. 3: 137-155. Henikoff, S. &amp; Henikoff, JG 1994. Genomics 19: 97-107.</td><td>P21338</td>

<td>Aspergillus oryzae</td><td>RNase T2</td><td>rnt B</td><td>Kawata Y. et al. 1988. Eur J. Biochem 176 (3): 683-97. Kawata Y. et al. 1990. Eur J. Biochem 187: 255-62. Ozeki K, et al. 1991. Curr Genet. 19: 367-73.</td><td>P10281</td>

<td rowspan="3">Mushrooms</td><td>Rhisopus niveus</td><td>RNase Rh</td><td></td><td>Horiuchi, H. et al. 1988. J. Biochem. 103: 408-418. Kurihara, H. et al. 1992. FEBS Lett. 306: 189-192. Kurihara, H. et al. 1996. J. Mol. Biol. 255: 310-320. Ohgi, K. et al. 1991. J. Biochem. 109: 776785.</td><td>P08056</td>

<td>Trichoderma viride</td><td>RNase Trv</td><td></td><td>Inada, Y. et al. 1991. J. Biochem. 110 (6), 896-904.</td><td>P24657</td>

<td>Lentinula edodes (shiitake mushroom)</td><td>RNase Irp</td><td></td><td>Kobayashi, H. et al. 1992. Biosci. Biotechnol. Biochem. 56: 2003-2010.</td><td>AAB24971</td>

<td rowspan="3"></td><td>L. edodes</td><td>RNase Le2</td><td></td><td>Kobayashi, H. et al. 1992. Biosci. Biotechnol. Biochem. 56: 2003-2010. Shimada, H. et al. 1991. Agric. Biol. Chem. 55: 1167- 1169.</td><td>P81296</td>

<td>Irpex lacteus</td><td>RNase Irp1</td><td></td><td>Watanabe, H., et al. 1995. Biosci. Biotechnol. Biochem. 59: 2097-2103.</td><td>AAB35880</td>

<td>Physarum polycephlum</td><td>RNase Phyb</td><td></td><td>Inokuchi, N. et al. 1993. J. Biochem. 113: 425-432.</td><td>P81477</td>

ES 2 397 052 ​​T3 (continued)

<td colspan="2">Source</td><td>Name (Prot)</td><td>Name (Gen)</td><td>Reference or references</td><td>GeneBank Access Number</td>

<td rowspan="4">Floors</td><td>Arabidopsis thaliana</td><td>RNS2</td><td>RNS2 locus</td><td>Green, PJ 1993. Proc. Natl. Acad. Sci. USA 90: 5118- 5122.</td><td>P42814</td>

<td>A. thaliana</td><td>RNase 3</td><td>RNS3 locus</td><td>Bariola, PA, et al. 1994. Plant J. 6: 673685.</td><td>P42815</td>

<td>A. thaliana</td><td>RNase 1</td><td>RNS1 locus</td><td>Bariola, PA, et al. 1994. Plant J. 6: 673685.</td><td>P42813</td>

<td>Lycopersicon esculentum (cultivated tomato)</td><td>RNase LE</td><td>RNALE</td><td>Kock, M. et al. 1995. Plant Mol. Biol. 27: 477-485. Jost, W. et al. 1991. Eur. J. Biochem. 198: 1-6.</td><td>P80022</td>

<td rowspan="2"></td><td>L. esculentum</td><td>RNase LX</td><td>RNLX</td><td>Kock, M., et al. 1995. Plant Mol. Biol. 27: 477-485. Loffler, A., et al. 1993. Eur. J. Biochem. 214: 627633.</td><td>P80196</td>

<td>Nicotiana alata (tobacco,)</td><td>S-RNase</td><td>S</td><td>Anderson, MA, et al. 1986. Nature 321: 38-44. Matton, DP et al. 1995. Plant Mol. Biol. 28: 847-858. McClure, BA et al. 1989. Nature 342: 95-97.</td><td>P04002</td>

<td rowspan="3"></td><td>Malus domestica (Apple tree)</td><td>S-RNases</td><td>S</td><td>Sassa, H., et al. 1996. Mol. Gen. Genet. 250: 547-557.</td><td></td>

<td>Pyrus pyrifolia (Japan Pear)</td><td>S-RNases</td><td>S</td><td>Norioka, N., et al. 1996. J. Biochem. 120; 335-345.</td><td></td>

<td>Momordica charantia (melon bitter)</td><td>RNase MC</td><td>RNMC locus</td><td>Blaxter, ML, et al. 1996. Mol. Biochem. Parasitol. 77: 77-93. Ide, H. et al. 1991. FEBS Lett. 284: 161164. Ide, H. et al. 1991. FEBS Lett. 289: 126.</td><td>P23540</td>

<td rowspan="2">Animals</td><td>Gallus gallus (chicken)</td><td>RNase CL1</td><td></td><td>Uchida, T. et al. 1996. Biosci. Biotechnol. Biochem. 60: 1982-1988.</td><td>JC5126</td>

<td>Rana catesbeiana (bullfrog)</td><td>RNase RCL2</td><td></td><td>Yagi, H. et al. 1995. Biol. Pharm. Bull. 18: 219-222. Liao, YD et al. 1996. Protein Expr Purif. 7: 194202. Liao YD, et al. 1994. Eur J Biochem. 222: 215-20. Liao, YD et al. 1998. J. Biol. Chem. 273: 6395-401</td><td>PC2347</td>

ES 2 397 052 ​​T3 (continued)

<td colspan="2">Source</td><td>Name (Prot)</td><td>Name (Gen)</td><td>Reference or references</td><td>GeneBank Access Number</td>

<td rowspan="4"></td><td>Drosophyla melanogaster</td><td>RNase DM</td><td>DmRNase</td><td>Lankenau, DH et al. 1990. Chromosome 99: 111-117. Hime, G., et al. 1995. Gene 158: 203-207.</td><td>X15066</td>

<td>Crassostera gigus (Pacific Oyster)</td><td>RNasa Oy</td><td>Locus JX0295</td><td>Watanabe, H. et al. 1993. J. Biochem. 114: 800-807.</td><td>JX029</td>

<td>Todarodes pasificus (Japanese squid)</td><td>RNase Tp</td><td></td><td>Kusano, A. et al. 1998. Biosci. Biotechnol. Biochem. 62: 87-94.</td><td>PMID 9501521</td>

<td>Homo sapiens</td><td>Forerunner of RNase 6</td><td>RNase 6PL</td><td>Trubia, M. et al. 1997. Genomics 42: 342-344.</td><td>NP003721</td>

For some applications it may be beneficial to use a ribonuclease that is substantially devoid of ribonucleolytic activity, which may have or cause unwanted side effects. As used herein the phrase "substantially devoid of ribonucleolytic activity" refers to (i) an inactivated ribonuclease (natural or recombinant) of the T2 family that has 0-10% ribonucleolytic activity compared to a similar ribonuclease, not inactivated; and / or (ii) a recombinant mutant (isolated natural or man-induced) ribonuclease of the T2 family that has 0-10% ribonucleolytic activity compared to a similar, non-mutant ribonuclease. Inactivation of the ribonucleolytic activity of the T2 family ribonuclease can be accomplished by a process selected from the group consisting of boiling, autoclaving, and denaturation or chemical inactivation.

As further detailed in Examples 2 and 6 below, it has been shown by the inventors that the anti-proliferation, anti-colonization, anti-differentiation and / or anti-development activities of RNase B1 are not dependent on its ribonucleolytic activity, since boiled, autoclaved RNase B1 and chemically inactivated (acetylated), having little (10%) or substantially no (0-10%) ribonucleolytic activity retained substantially all of its antiproliferation activities, anti-colonization, anti-differentiation and / or anti-development.

Therefore, a T2-RNase protein according to the present invention can be used either in a native, active ribonucleolytic form, or, alternatively, in a silent, or repressed ribonucleolytic form, which has none (0%) or has little (up to 10%) ribonucleolytic activity but it maintains its other activities. As such, the term "T2-RNases" is intended to encompass all anti-proliferative, anti-colonization, anti-differentiation, and / or anti-developmental forms of the protein, regardless of other activities of the protein.

It will be appreciated that the use of a T2-RNase, directly or expressed from a polynucleotide, that exhibits a desired activity and is still devoid of, or suppressed in, ribonucleolytic activity is particularly advantageous since ribonucleolytic activity can produce side effects. unwanted in a subject.

A polypeptide representing the amino acid sequence of a T2-RNase as defined herein can be produced by any of a number of procedures well known in the art. For example, the polypeptide can be produced synthetically by standard peptide synthesis techniques, for example using standard 9-fluorenylmethoxycarbonyl (F-Moc) chemistry (see, for example, Atherton, E. and Sheppard, RC 1985, J. Chem Soc. Chem. Comm. 165) or conventional butyloxycarbonate (T-Boc) chemistry, although it is observed that, more recently, the fluorenylmethoxycarbonyl (Fmoc) / tert-butyl system, developed by Sheppard has found an increasingly wide application (Sheppard, RC1986 Science Tools, The LKB Journal 33, 9).

Alternatively, a T2-RNase protein can be isolated or purified by procedures well known in the art from organisms known to express this protein. Such organisms include, for example, Aeromonas hydrophila, Haemophilus influenzae, Escherichia coli, Aspergillus oryzae, Aspergillus phoenicis, Rhisopus niveus, Trichoderma viride, Lentinula edodes, Irpex lacteus; Physarum polycephlum, Arabidopsis thaliana, Lycopersicon esculentum, Nicotiana alata, Malus domestica, Pyrus pyrifolia, Momordica charantia, Gallus gallus, Rana catesbeiana, Drosophyla melanogaster, Crassostera gigus, Todarodes pasificus and Homo sapiens. It is anticipated, however, that other organisms not yet known to produce T2-RNase, once discovered as such, could also be used as a source of T2-RNase in accordance with the present invention.

Alternatively and preferably a T2-RNase protein can be produced recombinantly by expressing a polynucleotide encoding it, using an appropriate expression vector system. Preferably,

ES 2 397 052 ​​T3 selects an expression system that provides suitable post-translational modifications. Suitable expression vector systems include, but are not limited to, mammalian cells infected with a virus (eg, adenovirus, retrovirus, herpes simplex virus, fowl pox virus); insect cells infected with a virus (eg, baculovirus); genetically modified plants or plant cells transformed with a plasmid, a plant virus or an Agrobacterium; transformed microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA. Vector expression control elements vary in their strengths and specifications depending on the host-vector system used, any one of several suitable transcription and translation elements can be used. A recombinantly produced T2-RNase can be purified from host cells by affinity chromatography, electrophoresis, high performance liquid chromatography (HPLC), immunoprecipitation, sedimentation, or any other method known in the art.

A purified T2-RNase can be used to prepare a medicament according to the present invention by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, immobilizing or lyophilizing procedures with the addition of vehicles and / or Appropriate pharmaceutically acceptable excipients or alternatively may be linked to appropriate delivery vehicles as described hereinbefore.

A polynucleotide according to the present invention may encode a native T2-RNase protein, the term of which in this context describes a T2-RNase having both antiproliferative and ribonucleolytic activities or, alternatively, a polynucleotide according to the present invention may encode a silent or repressed T2-RNase mutant, having little or no ribonucleolytic activity, to express (e.g., transcribe and translate) in vivo into a protein that is substantially devoid of ribonucleolytic activity.

As such, the term "polynucleotide" when used herein in the context of T2-RNases in general, or in the context of any specific T2-RNase, refers to any polynucleotide sequence that encodes an active T2-RNase in the prevention, inhibition and / or reversal of proliferation, colonization, differentiation and / or development of abnormally proliferating cells, which have or are substantially devoid of ribonucleolytic activity. Polynucleotides encoding a T2-RNase devoid of ribonucleolytic activity can be obtained using known molecular biology techniques, such as random mutagenesis, site-directed mutagenesis, and enhanced evolution techniques. Site-directed mutagenesis can easily be employed because amino acid residues essential for the ribonucleolytic activity of T2-RNases have been recognized (see Kusano et al., 1998. Biosci. Biothechnol. Biochem. 62: 87-94, and Table 2 above). .

Therefore, the present invention can be used to treat conditions, syndromes or diseases characterized by abnormally proliferating cells, such as cancer cells or others, such as, but not limited to, papilloma, blastoglioma, Kaposi's sarcoma, melanoma, lung cancer. , ovarian cancer, prostate cancer, squamous cell carcinoma, astrocytoma, head cancer, neck cancer, bladder cancer, breast cancer, colorectal cancer, thyroid cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, leukemia, lymphoma, Hodgkin's disease, Burkitt's disease, arthritis, rheumatoid arthritis, diabetic retinopathy, angiogenesis, restenosis, stent restenosis, and vascular graft restenosis.

As used herein the terms "cancer" or "tumor" are clinically descriptive terms that encompass a multitude of diseases characterized by cells showing abnormal cell proliferation. The term "tumor", when applied to tissue, generally refers to any abnormal tissue growth, characterized by abnormal and excessive cell proliferation. A tumor can be "benign" and unable to spread from its original focus, or "malignant" or "metastatic" and capable of spreading beyond its anatomical site to other areas throughout the host's body. The term "cancer" is an old term that is generally used to describe a malignant tumor or the pathology that arises from it. Alternatively, the technique refers to an abnormal growth as a neoplasm, and a malignant growth as a malignancy.

Any ribonuclease of the T2 family having the anti-angiogenesis activities described herein and exemplified in the Examples section below can be used as a therapeutic agent in accordance with the teachings of the present invention. Similarly, any polynucleotide encoding a T2 family ribonuclease having the anti-angiogenesis activities described herein can be used as a therapeutic agent in accordance with the teachings of the present invention. A non-exhaustive list of T2 family ribonucleases is provided in Table 2, above. As further exemplified by the examples below, RNase B1, which is a member of the T2 family, has antiangiogenesis as determined by in vivo and in vitro assays. Furthermore, RNase B1 has been shown to bind to actin even when treated to rid it of ribonuclease activity. Therefore, the present invention provides three different assays with which a person of ordinary skill in the art could test a given ribonuclease for its anti-angiogenesis activities, these are an in vitro assay to determine the effect of the ribonuclease assayed on cancer cells, in vivo assay to determine the effect of the tested ribonuclease on tumor development, and another in vitro assay to determine the ability of the tested ribonuclease to bind to cellular and / or free actin. Without limiting the present invention by any theory, it is believed that an ability of a ribonuclease to bind actin is indicative that said ribonuclease has anti-proliferation, anti-colonization, anti-differentiation and / or anti-development activities.

ES 2 397 052 ​​T3

A ribonuclease according to the present invention can be administered to an organism, such as a human or any other mammal, by itself, or in a pharmaceutical composition in which it is mixed with suitable carriers or excipients.

As used herein a "pharmaceutical composition" or "medicament" refers to a preparation of one or more of the ribonucleases or polynucleotides that encode them as described herein with other chemical components such as physiologically carriers and excipients. suitable. The purpose of a pharmaceutical composition is to facilitate the administration of a compound to an organism.

As used herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

The pharmaceutical compositions can also include one or more additional active ingredients, such as, but not limited to, anti-inflammatory agents, antimicrobial agents, anesthetics, and the like in addition to the main active ingredient.

Pharmaceutical compositions of the present invention can be manufactured by procedures well known in the art, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, immobilizing, or lyophilizing procedures.

Pharmaceutical compositions for use according to the present invention can therefore be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and adjuvants, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. The appropriate formulation depends on the selected route of administration.

Therefore, effecting the administration of the pharmaceutical composition of the present invention includes a suitable pharmaceutical carrier and an effective amount of a T2-RNase or a polynucleotide encoding it, and is administered, for example, topically, intraocularly, parenterally, orally, intranasally, intravenously, intramuscularly, subcutaneously, or by any other effective means by procedures well known in the art.

For intravenous, intramuscular, or subcutaneous injection, a T2-RNase or a polynucleotide encoding it can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For example, a physiologically appropriate solution containing an effective amount of a T2-RNase or a polynucleotide encoding it systemically can be administered to the bloodstream to treat a cancer or tumor that cannot be directly reached or anatomically isolated. A physiologically appropriate solution containing an effective amount of a T2RNase or a polynucleotide encoding it can be injected directly into a target tumor or cancer tissue by needle in amounts effective to treat the tumor cells of the target tissue.

For transmucosal administration, appropriate penetrants for the permeation barrier are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition of the present invention can be easily formulated by combining a T2-RNase or a polynucleotide encoding it with pharmaceutically acceptable carriers well known in the art. Such carriers allow a T2-RNase or a polynucleotide encoding it to be formulated as tablets, pills, lozenges, capsules, liquids, gels, syrups, pastes, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid carrier, optionally grinding the resulting mixture, and processing the mixture into granules, after adding suitable adjuvants if desired, to obtain tablets or dragee cores. Particular excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

The dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions can be used which may optionally contain acacia, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to tablets or dragee coatings for identification or to characterize different combinations of active ingredient doses.

Additional pharmaceutical compositions, which can be used orally, include snap-fit ​​capsules composed of gelatin as well as soft, sealed capsules composed of gelatin and a plasticizer, such as glycerol or sorbitol. The snap-fit ​​capsules may contain a T2-RNase or a polynucleotide that encodes it in filler admixture such as lactose, binders, such as starches, lubricants such as talc or

ES 2 397 052 ​​T3 magnesium stearate and, optionally, stabilizers. In soft capsules, one can be dissolved or suspended.

T2-RNase or a polynucleotide that encodes it in suitable liquids, such as fatty acids, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for the selected route of administration.

Oral delivery of the pharmaceutical composition of the present invention may not be successful due to the pH and enzymatic degradation present in the gastrointestinal tract. Therefore, such pharmaceutical compositions must be formulated to avoid undesirable circumstances. For example, enteric coating can be applied to an oral solid formulation. Substances with acid resistant properties such as cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP) and acrylic resins are more commonly used to coat tablets or granules for microencapsulation. Preferably wet granulation is used to prepare enteric coated granules to avoid reactions between the active ingredient and the coating (Lin, SY and Kawashima, Y. 1987, Pharmaceutical Res. 4: 70-74). A solvent evaporation procedure can also be used. The solvent evaporation procedure was used to encapsulate insulin administered to diabetic rats to maintain blood glucose concentration (Lin, SY et al., 1986, Biomater, Medicine Device, Artificial organ 13: 187-201 and Lin, SY and col., 1988, Biochemical Artificial Cells Artificial Organ 16: 815-828). It was also used to encapsulate high molecular weight biological materials such as viral antigen and concanavalin A (Maharaj, I. et al. 1984, J. Phamac. Sci. 73: 39-42).

For buccal administration, the pharmaceutical composition of the present invention may take the form of tablets or lozenges formulated in a conventional manner.

Suppositories can be used for rectal administration as is well known in the art.

For administration by inhalation, a T2-RNase or a polynucleotide encoding it is conveniently delivered for use in accordance with the present invention in the form of an aerosol spray presentation from a pressurized container or a nebulizer with the use of a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules or cartridges of, for example, gelatin can be formulated for use in an inhaler or insufflator containing a powder mixture of a T2-RNase or a polynucleotide encoding it and a suitable powder base such as lactose or starch.

The pharmaceutical composition of the present invention can also be formulated for parenteral administration, for example, by bolus injection or continuous infusion. A composition for injection may be presented in unit dosage form, for example, in ampoules or in multiple dose containers, optionally with an added preservative. The compositions can be suspensions, solutions or emulsions in aqueous or oily vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water soluble form. Additionally, suspensions of a T2-RNase or a polynucleotide encoding it can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of a T2-RNase or a polynucleotide that encodes it to allow the preparation of highly concentrated solutions.

Alternatively, a T2-RNase or a polynucleotide encoding it may be in a powder form for constitution with a suitable vehicle, eg, sterile, pyrogen-free water, before use.

The pharmaceutical composition of the present invention can also be formulated into rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.

In addition, a cancer or tumor present in a body cavity, such as in the eye, gastrointestinal tract, genitourinary tract (eg, urinary bladder), pulmonary or bronchial system, and the like, can be given a physiologically appropriate composition (eg, a solution such as a phosphate or saline buffer, a suspension, or an emulsion, which is sterile) that contains an effective amount of a T2-RNase or a polynucleotide that encodes it by direct injection with a needle or by a catheter or other delivery tube located in the hollow organ afflicted with cancer or tumor. Any imaging device such as an X-ray, sonogram, or fiberoptic viewing system can be used to locate the target tissue and guide the needle or catheter tube into the vicinity thereof.

The pharmaceutical composition of the present invention can also be delivered by osmotic micropumps. Osmotic micropumps are implanted in one of the body cavities and the drug is constantly released

ES 2 397 052 ​​T3 to the tissue to be treated. This procedure is particularly advantageous when experiencing an immune response to the pharmaceutical composition. This procedure has been used for ONCONASA (Vasandani VM, et al.,

1996, Cancer Res. 15; 56 (18): 4180-6).

Alternatively and in accordance with another preferred embodiment of the present invention, the pharmaceutically acceptable carrier includes a delivery vehicle capable of delivering a T2-RNase or a polynucleotide encoding it to the mammalian cell of the subject.

Numerous delivery vehicles and methods are known in the art for directing proteins or nucleic acids to or into tumors or cancer cells. For example, liposomes are artificial membrane vesicles that are available to deliver proteins or nucleic acids to target cells (Newton, AC and Huestis, WH, Biochemistry, 1988, 27: 4655-4659; Tanswell, AK et al., 1990, Biochmica et Biophysica Acta, 1044: 269-274; and Ceccoll, J. et al., Journal of Investigative Dermatology, 1989, 93: 190-194). Therefore, a T2-RNase or a polynucleotide encoding it can be encapsulated with high efficiency in liposome vesicles and delivered to mammalian cells. In addition, the T2-RNase protein or nucleic acid can also be delivered to target tumor or cancer cells via micelles as described in, for example, US Patent No. 5,925,628 to Lee.

T2-RNase encapsulated in liposome or micelle or a polynucleotide that encodes it can be administered topically, intraocularly, parenterally, intranasally, intratracheally, intrabronchially, intramuscularly, subcutaneously or by any other effective means at an effective dose. to treat abnormally proliferating cells of the target tissue. The liposomes can be administered in any physiologically appropriate composition that contains an effective amount of encapsulated T2-RNase or a polynucleotide encoding it.

Alternatively and in accordance with another preferred embodiment of the present invention the delivery vehicle may be, but is not limited to, an antibody or a ligand capable of binding to a specific cell surface receptor or marker. An antibody or ligand can be directly linked to a T2-RNase protein or nucleic acid by a suitable linker, or alternatively said antibody or ligand can be provided on the surface of a liposome that encapsulates a T2-RNase or a polynucleotide that encodes it.

For example, a T2-RNase or a polynucleotide encoding it can be fused with antibodies or specific membrane protein ligands to target specific tissues or cells as previously described in the art. It will be appreciated in this regard that fusion of RNase A of the ribonuclease A superfamily with antibodies to the transferrin receptor or to the CD5 antigen of T lymphocytes leads to the inhibition of protein synthesis in tumor cells that carry a specific receptor for each. of the above toxins (Rybak, M. et al., 1991, J. Biol. Chem. 266: 21202-21207 and Newton DL, et al., 1997, Protein Eng. 10 (4): 463-70).

Pharmaceutical compositions suitable for use in the context of the present invention include compositions in which the active ingredients are contained in an effective amount to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of the active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein.

The toxicity and therapeutic efficacy of the active principles described in this document can be determined by conventional pharmaceutical procedures in cell cultures or experimental animals, for example, determining the IC50 and LD50 (lethal dose that causes death in 50% of the animals tested. ) for an active substance object. The data obtained from these cell culture assays and animal studies can be used by formulating a range of dosage for use in humans. The dosage may vary depending on the pharmaceutical form used and the route of administration used. The exact formulation, route of administration and dosage can be selected by the individual physician in light of the patient's condition. (See, for example, Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", C. 1 p.1).

Depending on the severity and sensitivity of the condition to be treated, the dosage may also be a single administration of a slow release composition, the treatment cycle lasting from several days to several weeks or until cure is effected or decrease in pathology.

The amount of a composition to be administered will, of course, depend on the subject being treated, the severity of the condition, the mode of administration, the judgment of the prescribing physician, etc.

As already mentioned hereinabove, according to one aspect of the present invention, the active principle of the pharmaceutical composition is a polynucleotide encoding a T2-RNase.

According to this aspect of the present invention, the polynucleotide is introduced into the mammalian cell together with a pharmaceutically acceptable carrier, the introduction of which results in a genetic modification of this cell, allowing the expression of a T2-RNase in it.

ES 2 397 052 ​​T3

As used herein in the specification and claims section below, the term "genetic modification" refers to a method of inserting nucleic acids into cells. Insertion can, for example, be accomplished by viral infection, injection, transfection, particle bombardment, or any other effective means of introducing nucleic acids into cells, some of which are further detailed hereinafter. After genetic modification the nucleic acid is fully or partially integrated into the genome of the cell (DNA), or remains outside the genome of the cell, thereby providing stably modified or transiently modified cells.

As such, the pharmaceutical composition according to this aspect of the present invention can be used for gene therapy.

As used herein, the phrases "gene therapy" or "gene therapy" are used interchangeably and refer to a method of therapy in which a stable or transient genetic modification of a proliferating cell or cells such as cells cancerous, leads to inhibition of the proliferation of this cell.

Any one of the polynucleotides identified in Table 2 by their Gene Bank accession number may be employed in accordance with the present invention as a polynucleotide encoding a T2-RNase. In addition, polynucleotides 40% or more homologous and / or hybridizing under mild and / or stringent hybridization conditions with the polynucleotides listed as a polynucleotide encoding a T2-RNase may also be employed, provided that the protein encoded by them is characterized as a T2-RNase and display the desired activities. Furthermore, it will be appreciated that portions, mutant chimeras, or alleles of such polynucleotides may also be employed as a polynucleotide encoding a T2-RNase in accordance with the present invention, again, provided that such mutant portions, chimeras, or alleles of such polynucleotides encode a T2-RNase showing the desired activities.

The isolation of new polynucleotides encoding T2-RNases is also envisioned. Such isolation can be accomplished using methodologies well known in the art such as, but not limited to, library screening, hybridization, PCR amplification, tagged primers, tagged degenerate primers. Both genomic and cDNA polynucleotides can therefore be employed.

A polynucleotide according to the present invention may be fused, in frame, with any other protein-encoding polypeptide to encode a fused protein using procedures well known in the art. For example, the polypeptide can be fused to a leader sequence or signal peptide for secretion. Similarly a T2-RNase protein can be fused (conjugated) to other proteins using procedures well known in the art. Many methods are known in the art to conjugate or fuse (couple) molecules of different types, including proteins. These procedures can be used in accordance with the present invention to couple a T2-RNase to other molecules such as ligands or antibodies to thereby assist in targeting and binding of T2-RNase to specific cell types. Any pair of proteins can be conjugated or fused to each other using any conjugation procedure known to one of ordinary skill in the art. Proteins can be conjugated using an N-hydroxysuccinimide ester of 3- (2-pyridyldithio) propionic acid (also called N-succinimidyl 3- (2-pyridyldithio) propionate) ("SDPD") (Sigma, Cat. No. P-3415), a glutaraldehyde conjugation procedure or a carbodiimide conjugation procedure.

According to a preferred embodiment of the present invention, the polynucleotide includes one or more segments harboring transcriptional control sequences operably linked to the T2RNase coding sequence. Such transcriptional control sequences may include, but are not limited to, promoters and enhancers as further detailed hereinbelow. These transcriptional control sequences are normally operably linked upstream of the coding region and act in the regulation of transcription and / or translation thereof.

According to another preferred embodiment of the present invention the polynucleotide encoding a T2-RNase is included within a eukaryotic expression vector. The phrase "expression vector" refers to a nucleic acid sequence that includes a sequence that encodes a T2-RNase and transcriptional control sequences and that is capable of expressing a T2-RNase within a mammalian cell.

Numerous procedures are known for inserting DNA fragments into a vector, for the purposes of mammalian gene expression, and can be used to construct an expression vector for genes encoding T2RNase that includes appropriate transcriptional / translational control sequence and the desired T2-RNase polynucleotide sequences. These methods may include in vitro DNA recombinant and synthetic techniques and in vivo genetic recombination. The expression of a polynucleotide encoding a T2-RNase can be regulated by transcriptional control sequences such that a T2-RNase is expressed in a host cell infected or transfected with the recombinant DNA molecule. For example, the expression of a T2RNase can be controlled by any promoter / enhancer element known in the art. Activation of the promoter can be tissue specific or inducible by a metabolic product or administered substance.

ES 2 397 052 ​​T3

Promoters / enhancers that can be used to control T2-RNase expression within target tissues or cells include, but are not limited to, the native RB promoter, the cytomegalovirus (CMV) promoter / enhancer (Karasuyama, H., et al. ., 1989, J. Exp. Med., 169: 13), the human β-actin promoter (Gunning, P., et al., 1987, Proc. Natl. Acad. Sci. USA, 84: 4831-4835), the glucocorticoid-inducible promoter present in the long terminal repeat of the mouse breast tumor virus (HHTV LTR) (Klessig, DF, et al., 1984, Mol. Cell Biol., 4: 1354-1362), Holoney murine leukemia virus (MULV LTR) long terminal repeat sequences (Weiss, R., et al., 1985, RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York), the SV40 early region promoter (Bernoist and Chambon, 1981, Nature 290: 304-310), the promoter contained in the 3 'long terminal repeat of Rous sarcoma virus (RSV) (Yamamoto and col., 1980, Cell 22: 787-797), the herpes simplex virus (HSV) thymidine kinase promoter / enhancer (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1441-1445), the regulatory sequences of the metallothionein (Brinster et al., 1982, Nature 296: 39-42), the adenovirus promoter (Yamada et al., 1985, Proc. Natl. Acad. Sci. USA 82 (11): 3567-71), and the herpes simplex virus lAt promoter (Wolfe, JH, et al., 1992, Nature Genetics, 1: 379-384).

Expression vectors compatible with mammalian host cells for use in tumor or cancer cell gene therapy include, but are not limited to, plasmids, retroviral vectors, adenovirus vectors, herpes viral vectors, and non-replicating fowl pox viruses, such as is disclosed, for example, in US Patent No. 5,174,993.

Various methods can be used to deliver the expression vector according to this aspect of the present invention to the target mammalian cell (s).

For example, a suitable pharmaceutically acceptable carrier such as a physiologically appropriate solution containing an effective amount of an expression vector can be administered topically, intraocularly, parenterally, orally, intranasally, intravenously, intramuscularly, intramuscularly. subcutaneously or by any other effective means.

A physiologically appropriate solution containing an effective amount of an expression vector can be administered systemically into the bloodstream to treat a cancer or tumor that cannot be reached directly or anatomically isolated.

It will be appreciated that since a "naked" expression vector can be actively taken up by mammalian cells, uptake and targeted delivery is enhanced if the expression vector is properly packaged or encapsulated.

Therefore, according to another preferred embodiment of the present invention the pharmaceutically acceptable carrier includes a delivery vehicle suitable for delivering the expression vector to mammalian cells in a targeted manner.

A viral expression vector can be introduced by a delivery vehicle to a target cell in an expressible form by infection or transduction. Such a delivery vehicle includes, but is not limited to, a retrovirus, an adenovirus, a herpes virus, and a fowlpox virus. A delivery vehicle capable of introducing the vector construct into a target cell and capable of expressing T2-RNase therein in amounts that inhibit cell proliferation can be administered by any effective method described hereinbefore.

Alternatively, such delivery vehicle may include, but is not limited to, a liposome, a micelle, an antibody, or a ligand as previously described hereinbefore.

It will be appreciated that the polynucleotides described herein can be used in the preparation of a medicament useful for inhibiting proliferation of a mammalian cell by mixing the polynucleotide with an appropriate pharmaceutically acceptable carrier.

As previously mentioned herein, polynucleotides encoding a T2-RNase can be obtained by a variety of methods, including, but not limited to, polymerase chain reaction (PCR) amplification from genomic library screening or from CDNA, using T2-RNase specific primers, use reverse transcription PCR in conjunction with T2-RNase specific primers to amplify mRNA isolated from organisms known to express T2-RNases or directly isolate DNA sequences encoding a T2-RNase from appropriate organisms. It will be appreciated in this case that the above-mentioned procedures can also be used to isolate or generate any of the active forms of a T2-RNase described hereinbefore.

The purified polynucleotide can then be inserted into appropriate expression vectors or provided with the appropriate transcriptional control sequences and prepared as described hereinbefore.

As further exemplified in the Examples section below and previously mentioned herein, an assay for determining the effects of a specific T2-RNase is also provided.

ES 2 397 052 ​​T3 or a polynucleotide encoding it according to the teachings of the present invention. Such an assay is carried out, for example, by exposing proliferating cells to a T2-RNase and following their proliferative behavior over time compared to untreated control cells. This assay can be used not only to select the most potent T2-RNase for any specific application, but also to establish the dose response, which can be translated into initial treatment dosing in in vivo experiments or during treatment of a subject, all as is further exemplified herein for T2 family RNase B1. It will be appreciated that this assay can also be used to determine the antiproliferative active part or site or a T2-RNase, or to determine the activity of generated or isolated mutants that do not exhibit ribonucleolytic activity.

Additional objects, advantages, and novel features of the present invention will become apparent to one of ordinary skill in the art upon examination of the following examples, which are not intended to be limiting.

Examples

Reference is now made to the following examples, which together with the descriptions above, illustrate the invention in a non-limiting manner.

In general, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are fully explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley &amp; Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in US Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8^to Edition), Appleton and Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980); Immunoassays available in the scientific and patent literature are extensively described, see, for example, US Patent Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, MJ, ed. (1984); "Nucleic Acid Hybridization" Hames, BD, and Higgins SJ, eds. (1985); "Transcription and Translation" Hames, BD, and Higgins SJ, eds. (1984); "Animal Cell Culture" Freshney, RI, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996). Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader.

Example 1

Characterization of B1 RNase from Aspergillus niger and its inhibitory effect on the growth of pollen tubes in fruit trees

Materials and procedures

Preparation and purification of extracellular RNase from A. niger

Aspergillus niger B1 (CMI CC 324626) was grown in liquid culture containing 1% wheat flour (w / v) and 0.05% ammonium sulfate (w / v). The mixture was adjusted to pH 3.5 with hydrochloric acid and autoclaved. An inoculum of approximately 10⁶ spores in 100 ml of medium and incubated at 30 ° C in an orbital shaker, at 200 rpm for 100 hours. The growth medium was passed through a 0.2 pm membrane and dialyzed three times against 10 volumes of 2 mM sodium acetate pH 6. Two liters of dialyzed solution were loaded onto a Fractogel EMD-TMAE 650 column (M ) 10/26 (Merck), equilibrated with 20 mM sodium acetate pH 6. Bound proteins were eluted with a linear gradient of 500 ml of 0-1.0 M sodium chloride in the same buffer, using a fast protein liquid chromatography (FPLC) system (Pharmacia) with a flow rate of 5 ml-min.^-1. The fractions showing the highest RNase activity were pooled and dialyzed against 2 mM sodium acetate pH 6, and a 50 ml aliquot was loaded onto a MONO-Q 5/5 HR column (Pharmacia), equilibrated with 20 mM sodium acetate pH 6. Elution was performed as with the EMD-TMAE column, except that only 10 ml of a 0-1.0 M saline gradient was used, at a flow rate of 1 ml-min.^-1.

Proteins were monitored at 280 nm and measured according to Bradford (Bradford, MM 1976. Anal. Biochem. 72: 248-245), using bovine serum albumin (BSA) as a standard. Different fractions were analyzed by 12.5% ​​sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmeli, U.

ES 2 397 052 ​​T3

K. 1970. Nature 227: 680-685). RNase activity was determined as previously described (Roiz and Shoseyov

1995, Int. J. Plant Sci. 156: 37-41).

The purified RNase B1 was enzymatically deglycosylated according to the procedure described by Broothaerts et al. (Broothaerts, WP et al. 1991. Sex. Pant Reprod. 4: 258-266). The enzyme was mixed with SDS 0.5% (w / v) and β-mercaptoethanol 5% (w / v) and heated at 100 ° C for 5 minutes. Once cooled, the reaction mixture was diluted

2.5 times with a buffer containing 50 mM sodium phosphate pH 7.5, 25 mM EDTA, 1% Triton X-100 (w / v) and 0.02% (w / v) sodium azide. Peptide-N-glycosidase F (PNGase F, Boehringer-Mannheim) was added to a final concentration of 20 ml-units.^-1 and incubation was carried out overnight at 37 ° C. The sample was then mixed with sample application buffer, heated at 100 ° C for 5 minutes, and analyzed by SDS-PAGE using a 12.5% ​​gel.

RNase Assays:

Optimal conditions for RNase activity were determined according to a modified Brown and Ho procedure (Brown, PH and Ho, THD 1986 Plant Physiol. 82: 801-806), using a temperature range of 20 to 100 ° C in increments. 10 ° C, and a pH range of 2.5 to 7 in 0.5 pH unit increments, established using 50 and 12 mM phosphate-citrate buffers. Samples of 10 gl each were added to 490 gl of ice cold buffer, containing 4 mg-ml yeast RNA.^-1 (Sigma). Half of each sample was used as a blank by immediately adding a stop solution containing 50 gl of 0.75% (w / v) uranyl sulfate in 25% (w / v) perchloric acid. The remaining half was incubated for 10 minutes, after which 50 gl of stop solution was added to each. After centrifugation at 15,000 X g for 5 minutes, the supernatant was diluted 20 times with distilled water and the absorbance at 260 nm was determined. One unit of RNase activity was determined as the amount of enzyme that releases soluble nucleotides at a rate of one U.A260 nm per minute.

RNase B1 was visualized by gel activity, as previously described (Roiz and Shoseyov, 1995, Int. J. Plant Scizzz. 156: 37-41). An SDS gel containing RNase B1 was renatured by washing twice for 15 minutes each with 20 mM acetate buffer at pH 3.5 containing 25% (v / v) isopropanol and then twice for 15 minutes each with tampon only. The renatured gel containing RNase B-1 was placed on a plate containing 0.1% RNA and 0.8% agarose in 20 mM acetate buffer and incubated at 37 ° C for 30 minutes. The gel was then removed and the agarose plate was stained with 0.02% (w / v) toluidine blue in water to visualize RNase activity.

The effect of RNase B1 on pollen tube growth

Peach pollen germinated cv. Almog in vitro in liquid culture, as previously described (Roiz and Shoseyov, 1995, Int. J. Plant Sci. 156: 37-41). The pollen grains were suspended in aliquots containing 100 gl of sucrose at 15% (w / v), boric acid 100 gg-ml^-1, magnesium sulfate 200 gg-ml^-1, calcium nitrate 200 gg-ml^-1 and different concentrations of RNase B1. After incubation overnight at 25 ° C in a dark chamber, the percentage of germination was recorded. The length of the pollen tube was examined with an eye micrometer.

The effect of RNase B1 treatment on pollen tube growth was also tested in vivo. Intact peach and mandarin (Citrus reticulata, Blanco cv. Murcott) flowers were sprayed in the early stages of anthesis with 100 ml-units.^-1 RNase B1 in 20 mM citrate buffer at pH 3.5. In each species additional flowers were sprayed at the same stage, on different branches, with only buffer or remained untreated as controls. After exposure to open pollination for 48 hours, the styles were fixed in acetic acid in ethanol 3: 1 (by volume) for 24 hours, washed with distilled water, and embedded overnight in 8M sodium hydroxide. After thorough washing in distilled water, the styles were cut lengthwise, each dipped in a drop of 0.1% (w / v) aniline blue in 0.1 M potassium phosphate on a slide and carefully ground with a glass coverslip. Pollen tubes were observed by epifluorescence microscopy (Olympus BX40 equipped with WIB cube).

The effect of RNase B1 on fruit formation:

Field experiments were carried out on nectarine (Prunus persica var. Nectarina Fantasia). Branches 30-40 cm long, bearing approximately 10% open flowers, were sprayed with different concentrations of RNase B1 in 20 mM citrate buffer pH 3.5 and 0.025% Triton-X 100. The untreated branches, and the branches to which only buffer and newt-X 100 were sprayed served as controls. The branches were sprayed at 2-3 day intervals during the flowering period (14 days). One month later, the number of fruits per branch was examined. For viability testing, the seeds were cut lengthwise through the embryo and immersed in 1% 2,3,5-triphenyl tetrazolium chloride in water for 4 hours at 20 ° C in a dark room. Red staining indicated viable tissues.

Experimental results

Purification and characterization of RNase B1:

A. niger grown in liquid culture produced considerable amounts of extracellular RNase B1. It was discovered that

ES 2 397 052 ​​T3 a temperature of 60 ° C and a pH of 3.5 were optimal for RNase activity, and were adopted as the standard conditions for subsequent RNase assays.

RNase B1 purification included three steps (Table 3). In a first stage a crude filtrate contained 1000 ml-units^-1 and 0.05 mg-ml were obtained^-1 protein. The crude filtrate was passed through an EMDTMAE column and the pooled active fractions (Figure 1, graph A) contained 0.1 mg-ml protein^-1, with an RNase activity of 40,000 ml-units^-1. In the final stage, the pooled fractions were passed through a MONO-Q column and the active RNase fraction was eluted (Figure 1, graph B). This fraction contained a protein concentration of 1.05 mg-ml.^-1 and RNase activity of 543,000 units-ml^-1. Two main protein bands, 40 and 32 kDa, were observed after SDS-PAGE of the purified RNase B1 fraction (Figure 2). An RNase activity gel showed active bands corresponding to the 32 and 40 kDa proteins. When subjected to PNGase F, a single protein band appeared at 29 kDa. RNase activity was preserved after PNGase digestion (not shown).

TABLE 3

<td>Purification stage</td><td>Total Units</td><td>Protein concentration (mg / ml)</td><td>Recovery (%)</td><td>Specific activity (units / mg protein)</td>

<td>Crude filtering</td><td> 0</td><td> 0,05</td><td> 100</td><td> 20.000</td>

<td>EMD-TMAE column</td><td> 0</td><td> 0,1</td><td> 56</td><td> 400.000</td>

<td>MONO-Q column</td><td> 652.200</td><td> 1,05</td><td> 32,6</td><td> 517.143</td>

The effect of RNase B1 on pollen tubes and fruit formation:

In in vitro experiments, 75% of the control pollen grains germinated and the pollen tubes reached approximately 0.5 mM in length. The addition of RNase B1 to the growth medium reduced the germination percentage and the length of the pollen tubes, in a dose-sensitive manner (Figure 3). RNase B1 had a pronounced inhibitory effect, 50 ml-units^-1, which represent 0.1 pg-ml¹ protein, were lethal, while 125 pg-ml^-1 of BSA reduced only half of pollen germinability and tube growth.

In vivo, the growth of peach control pollen tubes was observed through the stigmatic tissue directed towards the style 48 hours after pollination (Figure 4a). A similar effect was seen in tampon-only styles. In contrast, pollen grains sprouted on RNase B1-treated stigmas produced short pollen tubes, which appeared to lack any growth orientation, and did not penetrate stylar tissue (Figure 4b). In mandarins only a small part of the stigmatic tissue, the diameter of which was 2-3 mm, was captured by the field of view of the microscope. Therefore, only a few pollen tubes were observed, as shown in Figure 5. However, the difference between the normal growth of the control pollen tubes (Figure 5a) and the irregular growth of the RNase-treated pollen tubes (Figure 5b), it was clearly evident.

In nectarine cv. Fantasy, RNase B1 produced a reduction in fruit formation (Table 4). In branches that remained untreated or sprayed with X-100 newt buffer, fruit formation was 48.3% and 36.3% respectively. It seems that the low pH buffer had some inhibitory effect on fruit formation, however the branches treated with 500 and 1000 ml units^-1 of RNase B1 formed 23.3% and 18.4% of fruits, respectively, indicating a significant clearing effect of RNase, in a dose-dependent manner.

TABLE 4

<td>Treatment</td><td>Flowers (total number)</td><td>Formation of fruits (%)</td>

<td>Untreated control</td><td> 169</td><td>48.3 to *</td>

<td>Control buffer</td><td> 143</td><td>36.3 ab</td>

<td>RNase B1 500 units / ml</td><td> 148</td><td>23.3 bc</td>

<td>RNase B11000 units / ml</td><td> 106</td><td>18.4 c</td>

<td colspan="3">* Values ​​that do not share a common letter are significantly different at P = 0.05.</td>

In branches treated with RNase B1, many undeveloped fruits were observed. The viability tests showed that in the control flowers (not treated or sprayed only with buffer), the embryonic tissues were stained red, (Figure 6a), while the embryo tissues developed in the flowers treated with RNase,

ES 2 397 052 ​​T3 stained brown, which is indicative of necrosis (Figure 6b).

Extracellular RNase B1 from Aspergillus niger (RNase B1) was purified to homogeneity. It was found to contain two 32 and 40 kDa glycoprotein isoforms, sharing a 29 kDa protein core. Optimal RNase activity was observed at a temperature of 60 ° C and a pH of 3.5. In peach (Prunus persica cv. Almog) and mandarin (Citrus reticulata, Blanco cv. Murcott) the enzyme inhibited pollen germination and tube growth in vitro as well as in vivo. In field experiments, RNase caused a reduction in nectarine fruit formation (Prunus persica var. Fantasy nectarine) and inhibited normal embryonic development.

Example 2

Inhibition of pollen germination and tube growth by T2-RNase is mediated through interaction with actin

The inhibition of pollen germination and tube growth by RNase is well recognized, although the mechanism by which this enzyme interferes with the elongation process is not yet clear. As such, this study aims to decipher the role of RNase B1 in the interaction with the pollen tube elongation process.

Experimental materials and procedures:

The effect of RNase B1 on pollen tube growth:

Lily anthers (Lilium grandiflorum L. cv. Osnat) were allowed to dehisce for 24 hours at room temperature and then fresh were used or stored at -20 ° C. RNase B1 was produced and purified from Aspergillus niger growth medium filtrate as described in Example 1. Pollen was germinated in vitro in aqueous cultures of 100 µl each, containing 7% sucrose, CaNO3 1.27 mM, 0.16 mM H3BO3, 1 mM K2NO3 and KH₂PO₄ 3 mM in water (Yokota and Shimmen 1994). Some cultures were supplemented with RNase B1 having 100 units / ml of RNase activity at a final concentration of 16 pg / ml. Additional cultures were supplemented with RNase that had previously been boiled for 30 minutes resulting in loss of 50% activity, or with autoclaved RNase that lacked any catalytic activity. After 2 hours of incubation at 25 ° C in the dark, the length of the pollen tube was measured under the microscopic ocular micrometer. Pollen tubes were stained with IKI (I2 0.3% and KI 1.5% in water) to detect starch bodies.

1 hour actively spreading pollen tubes were transferred to glass cells at the microscopic stage. The pattern of pollen tube growth and organelle movement was recorded on video as modified by Heslop-Harrison and Heslop-Harrison (Heslop-Harrison, J. and Heslop-Harrison, Y. 1990. Sex Plant Reprod. 3: 187-194), using Applitec MSV-800 video presenter. Images were captured at 0.8 frames / second for 8 seconds by a Scion LG-3 frame grabber and then digitized and integrated by NIH image software. The photographs were processed using Adobe Photoshop (Adobe Systems Inc., Mountain View, CA) and Power-Point (Microsoft Co.) software.

The effect of RNase on actin filaments of pollen tubes:

Pollen was germinated in vitro in aqueous cultures with or without RNase. After overnight incubation, the pollen tubes were gently pelleted and the growth medium was replaced with tetramethylrhodamine B isothiocyanate (TRITC) labeled phalloidin.^-6 M (Sigma) in PBST buffer (150 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.02% Tween-20). For in vivo observations, lily flowers cv. Stargazer at the time of onset of anthesis and 0.5 ml of a growth medium containing 10 units / ml RNase was injected through the stigma into the style canal. Flowers injected with growth medium without RNase were used as a control. The liquids were absorbed into the style tissue for 5 hours at 25 ° C, after which the stigmas were hand pollinated by pollen of lily cv. Osnat. After 48 hours of incubation at 25 ° C, each pistil was cut lengthwise and the pollen tubes were carefully excised and removed to TRITC-TBST solution and incubated for 1 hour. The incision in the stigma did not affect the pollen tubes, since their vital protoplasts were located in the distal part, protected by callus plugs. In both in vitro and in vivo experiments, the stained pollen tubes were rinsed in TBS (TBST without Tween-20), placed on a glass slide and observed with an epifluorescent light microscope (Olympus BX40 equipped with a USH-mercury lamp). 102D).

Binding of actin with RNase B1:

The interaction between RNase B1 and actin was quantified by modifying from Simm (Simm, FC et al., 1987. Eur. J. Biochem. 166: 49-54). Globu (G-) actin from rabbit muscle (Sigma Co.) was polymerized to filamentous actin (F-) in Buffer F (10 mM Tris pH 8, 0.1 mM ATP, 0.2 mM CaCf, 0.1 KCL M and 2 mM MgCl2) for 30 minutes at room temperature. 50 µl samples each containing 30 µM F-actin were incubated overnight at 4 ° C with 1-33 µM RNase B1. As a control, each concentration of RNase was incubated with buffer F only. The samples were centrifuged at 15,000 g for 40 minutes and the RNase activity of the supernatant was determined (Roiz, L., Goren, R. and Shoseyov, O. 1995, Physiol. Plant. 94: 585-590.).

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RNase B1 immunooro silver staining in pollen tubes:

Immunoro silver staining (IGSS) was used to detect RNase binding with lily pollen tubes. Polyclonal antibodies against RNase B1 were raised in rabbit (Aminolab). 2 hour lily pollen tubes were fixed in vitro overnight in 2.5% glutaraldehyde in PBST at 4 ° C. The pollen tubes were washed for 1 hour in PBST, blocked for 1 hour in PBST containing 1% BSA and 2% skim milk, and incubated for 1 hour in anti-RNase B1, diluted 1: 500 in PBST. Rabbit preimmune serum (PIS) was used as a control. The pollen tubes were washed three times, 10 minutes each, in PBST and then incubated for 1 hour in goat anti-rabbit IgG conjugated with 5 nM gold particles, diluted 1: 100 in PBST. After two 10 minute washes in PBST and one 10 minute wash in water, a silver staining kit (BioCell Research Laboratories) was used for the final development of the reaction. The pollen tubes were soaked with the combined kit solutions for 10-15 minutes, washed in excess distilled water, and viewed under a light microscope (Olympus BX40).

Experimental results

A control sample of lily pollen tubes, germinated in vitro in RNase-free growth medium, reached approximately 300 µm in length (Figure 7). The cultures that were treated with RNase under the same conditions, reached only 160 µm in length. Boiling or autoclaving the RNase produced pollen tubes 130 and 170 µm in length, respectively. Differences between the three RNase-treated groups of pollen tubes were considered negligible.

Starch staining showed that amyloplasts from the control sample were observed propagating along the pollen tube, except in the area of ​​the tip (Figure 8a). On the other hand, IKI-stained bodies from pollen tubes treated with RNase accumulated in the tip area (Figure 8b).

Integrated video images of actively extending pollen tubes displayed cytoplasmic flow lines (Figures 9a and 9b). In the control sample, continuous longitudinal movement was more common, as was acropetal flow in the periphery of the tube and basipetal flow in the center, which formed a pattern of “reverse source” below the tip area (Figure 9a). The tip zone itself was occupied by much smaller bodies, mainly P particles, whose pattern of motion was hardly observed. In RNase-inhibited pollen tubes the tip appeared swollen, with starch and lipid particles well visible reaching the tip area (Figure 9b). Continuous motion could not be detected, but instead irregular stretched images indicated that the cytoplasmic bodies rotated randomly.

The effect of RNase on actin filament distribution was examined in pollen tubes in vitro for 1 hour and in vivo for 48 hours. In vivo pollen tubes reached approximately 3-4 cm in length, and their TRITC-phalloidin staining was more intensive than in vitro tubes. However, the mode of RNase effect was similar in both experiments. In the control, the actin microfilaments were assembled longitudinally along the axis of the tube, forming a fine network in the area of ​​the tip (Figure 10a). On the other hand, in pollen tubes treated with RNase, masses of actin accumulated in the cell wall of the tip (Figure 10b).

The interaction between RNase B1 and actin was quantified using Scatchard analysis. In the actinRNase B1 experiment, a regression line, intersecting with the abscissa at 0.45 (Figure 11) indicated that the RNase: actin molar ratio was 0.45, which implies that two actin molecules bind. to each RNase molecule.

Germinated pollen was prepared in the presence of RNase B1 for light microscopy and the localization of RNase was determined by IGSS, using anti-RNase antibodies (Figures 12a-c). In pollen tubes cultured without RNase (Figure 12a) or with RNase but treated with PIS (Figure 12b), the outer surface of the cell wall was devoid of silver staining. On the other hand, in the pollen tube treated with RNase B1, an evident immunooric silver staining appeared, which accumulated on the tip area (Figure 12c).

In this study, pollen germination and tube elongation of Lily (Lilium grandiflorum) were specifically inhibited by A. niger RNase B1. Boiled or autoclaved RNase, which lacked most of the original catalytic activity, showed a similar inhibitory effect. The results demonstrate that A. niger RNase is an actin-binding protein that has an inhibitory effect on pollen tube elongation. This binding which is not related to the catalytic activity of RNase B1 deforms the ordering of the actin filaments of the pollen tube to thereby interrupt the cytoplasmic current.

Example 3 (REFERENCE EXAMPLE)

The effect of RNase B1 on human colon cancer cells

Since the actin binding activity discovered for RNase B1 in pollen tubes pointed to a possible cytotoxic activity it was decided to examine the cytotoxic effect of RNase B1 in human colon cancer cells.

ES 2 397 052 ​​T3

Materials and procedures and experimental results

Cell culture:

All experiments were carried out in vitro. Human colon adenocarcinoma cells (HT29) were cultured in DMEM medium (Biological Industries, Bet Haemek), supplemented with 10% fetal calf serum, 1% glutamine and 10% Antibiotic-Antifungal solution (Biolab). The cells were incubated at 37 ° C in a humidified atmosphere containing 5% CO2. RNase B1 solutions were made in PBS buffer, pH 6.8.

Preliminary cell viability assay:

Cells were incubated with 50 ml flasks. Each flask contained 2 X 10⁵ cells in 7 ml of medium, in the absence or presence of different concentrations (10^-8 - 10^-6 M) of RNase B1. Cells were cultured for 48 hours or 72 hours, and then viable and non-viable cells were differentially counted using trypan blue staining.

In all treatments the total number of cells cultured for 72 hours (55-60 X 10⁵) exceeded approximately twice the number of cells obtained after 48 hours of culture (25-30 X 10⁵) (Figure 13a). The presence of RNase B1 in the growth medium did not have a significant effect on cell growth. However, a small but significant effect of RNase B1 was found on the number of dead cells at both 48 hours and 72 hours of incubation (Figure 13b).

Clonogenicity test I:

The long-term survival of tumor cells is characterized by their ability to divide and produce clones. Cells were preincubated for 48 hours in growth medium containing RNase B1 10^-6 M, then trypsinized, washed and resuspended in growth medium without RNase B1. Before seeding into 96-well microtiter plates, cells were diluted to 5-fold serial dilutions ranging from 50 to 10⁵cells in each well (200 ml). The plates were incubated for 14 days under the conditions described above, without adding new growth medium after which the colonies were fixed and stained with methylene blue. Clonogenic cells in each well were numbered after visualization of the clones. Control cells were treated as before, but preincubated for the first 48 hours in medium without RNase B1.

In both treatments a similar number of colonies was observed in wells in which 100 cells were seeded (Figure 14). The cytotoxic effect of RNase B1 appeared in wells containing higher cell densities. In wells in which 500 cells were seeded each, control cells and those treated with RNase B1 produced 180 and 100 colonies per well, respectively. Furthermore, in wells in which 1000 cells were seeded each, RNase B1-treated cells formed approximately 250 colonies per well, while control cells formed numerous colonies that were fused in a continuous layer, which therefore could not be counted and illustrated in Figure 14. Cells seeded at higher densities did not survive culture without medium change.

Clonogenicity test II:

The ability of tumor cells to proliferate and colonize in short versus continuous exposure to RNase B1 was examined. The experiment was performed as described in Clonogenicity Assay I using (i) control cells, (ii) cells preincubated with medium containing RNase B1.^-6 M and which were then allowed to colonize in growth medium without RNase B1, and (iii) cells preincubated as in (ii) and then incubated during the colonization assay in growth medium containing RNase B1 10^-6 M. In these experiments the initial densities ranged from 250-1000 cells per well and the colonization period was 7 days.

The shorter incubation period used in this experiment (7 days) compared to a 14 day preincubation resulted in non-inactivated colonies, which could be distinguished even in wells containing high cell densities. At all densities, a 48 hour preincubation in RNase B1 led to a 20-30% reduction in the cells' ability to colonize, compared to the control (Figure 15). However at each density a continuous exposure to RNase B1 led to a drastic 90% reduction in clonogenicity. Figures 16a-c show that cells treated with continuous RNase B1 (Figure 16c) were smaller and less stainable than cells that were pre-incubated for 48 hours in RNase B1 (Figure 16b) or control cells (Figure 16a). This result indicates that RNase B1 affected the growth rate of the colonies.

Therefore, as clearly shown from the results presented herein, RNase B1 from A. niger has a clear cytotoxic effect on human adenocarcinoma HT29 cancer cells. The cytotoxic effect of RNase B1 is expressed by reducing cell clonogenicity, rather than reducing cell viability. It is possible that RNase B1 has a long-term effect on tumor cells. RNase B1 causes a reduction in the growth rate of the colonies compared to the control, indicating that it may affect the ability of cells to proliferate.

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Example 4 (REFERENCE EXAMPLE)

The in vivo effect of RNase B1 on tumor development in a rat model

To further study the antineoplastic effect of RNase B1, an in vivo experiment was performed in rats.

Experimental materials and procedures

Male Charles-River-derived 4-week-old rats were divided into groups of 6. In some groups, rats were induced to develop colon cancer by five weekly injections of dimethylhydrazine (DMH). In this experiment, two modes of administration of RNase B1 were examined. RNase B1 was applied directly to the colon by osmotic micropumps, or was delivered orally using enteric coated microcapsules. The complete set of treatments that each group of rats received is described by the scheme of Figure 1. During the experiment, the rats were weighed weekly to monitor the effect of DMH and / or RNase B1 on their growth rate. In groups treated with RNase B1, feces were collected weekly from each cage and dried at 60 ° C. A 250 mg dry stool sample was ground and redissolved in phosphate buffered saline (PBS). After centrifugation, the RNase activity in the upper solution was examined as described in Roiz et al. (Roiz, L. et al., J. Amer. Soc. Hort. Sci. 125 (1): 9-14.2000).

Administration of RNase B1 to the colon by osmotic micropumps:

RNase B1 was loaded into osmotic micropumps (ALZET). The pumps were implanted subcutaneously in the abdomen of the rats. The osmotic pumps allowed a constant release of RNase B1 directly to the colon through a catheter, at a calculated concentration of 10^-6 M in the colon for at least 6 weeks, assuming the rat colon is approximately 4 ml in volume. The rats were treated as follows: Pumps containing "live" RNase B1 (RNase B1), which had full RNase activity, were implanted in the rats in one group. In rats from a second group pumps were implanted containing autoclave-inactivated RNase B1 (I-RNase B1), which lacked any RNase activity. Meanwhile, rats in a third group were implanted with pumps containing PBS, which was used as the vehicle for RNase B1 in the first two groups, and acted as controls.

To examine a possible preventive effect of RNase B1, selected DMH-treated rats received RNase B1 1-9 weeks after the first DMH injection. The rats were then sacrificed and their colons were excised and washed with PBS and then with PBS containing 0.1M dithiothreitol (DTT). The colons were then split lengthwise and fixed for at least 1 hour in 4% formaldehyde in PBS on filter paper. After staining with 0.025% methylene blue in PBS, the colon mucosa was viewed under a low magnification microscope for aberrant crypt foci (ACF). ACFs were counted in the distal colon (5 cm).

To examine a therapeutic effect of RNase B1, the rest of the rats received RNase B1 12 to 17 weeks after the first DMH injection. Colons were excised and fixed as described above and tumors were counted and measured. For histopathological examinations, each tumor was embedded in paraffin. Thin sections (10 pm) were stained and the degree of malignancy was evaluated.

Oral administration of RNase B1:

Microcapsule preparation:

Microcapsules were prepared with a modified procedure described by Lin et al. (Lin JJ, et al., 1994, Biochem. Biophys. Res. Commun. 14; 204 (1): 156-62). A mixture of 0.6 grams of lyophilized RNase B1 and 2.4 grams of glucose was ground well using a mortar. The fine powder was poured into a 1 L beaker containing 200 ml of liquid paraffin and 2 ml of Span-80 and stirred at 600 rpm for 20 minutes. Acetone-ethanol-cellulose acetate phthalate (CAP) solution was carefully added to the above stirring mixture (3.2 grams CAP in 40 ml acetone: 95% ethanol 9: 1) and allowed to stir for an additional 2 hours in a hood, to remove traces of acetone. The microcapsules were hardened by adding 30 ml of ether and dried on filter paper using a Buchner funnel and traces of liquid paraffin were removed by two additional washes with 30 ml of ether. The microcapsules were then allowed to dry overnight and passed through a fine mesh. Most of the microcapsules were between 200 and 500 pm.

In a preliminary experiment (Figure 18), CAP microcapsules were found to be insoluble at acidic pH, representing the stomach environment. However, after 1 hour in alkaline pH the maximum RNase activity was achieved, indicating that the microcapsules could easily release their contents in the intestines.

Oral administration of RNase B1 microcapsules:

The microcapsules containing RNase B1 or glucose as a placebo were mixed with ground Purina feed. Each rat treated with RNase B1 received a daily dose of 1.6 mg of RNase B1, to obtain a concentration

ES 2 397 052 ​​T3 final of 10 '⁵ M in the colon. The experimental details for oral administration were as described above for micropump administration, except for a delayed termination (11 weeks) while evaluating the preventive effect of RNase B (Figure 17).

Experimental results

The effect of different treatments on the growth rate of rats:

The initial weight of the rats was approximately 200 grams. The experiment ended at a different time for each treatment, as described above. In general, the rats reached a final body weight of approximately 400-500 grams, with no significant differences between the different treatments (Figures 19a-d). However, the DMH treated groups showed a slight reduction in body weight compared to the RNase B1 treated groups, in the presence or absence of DMH.

RNase activity in rat feces:

Figures 20a-c show changes in RNase activity in feces of rats that had been implanted with osmotic pumps containing RNase B1, I-RNase B1 or PBS in a preventive way (Figure 1) for 8 weeks. In rats treated with RNase B1 (Figure 20a) the RNase activity in the feces was 5 times higher than in rats treated with I-RNase B1 (Figure 20b) or with PBS (Figure 20c). This activity remained high for 5 weeks and then gradually decreased as the RNase B1 depot in the pumps was depleted. Basal endogenous RNase activity was detected in stool from the two subsequent groups.

A similar pattern was observed in rats fed microencapsulated RNase B1, but 8-fold higher RNase activity was detected compared to rats fed microcapsules containing only glucose (Figure 21). In this experiment, the gradual reduction of RNase activity can be explained as a result of the increase in the body weight of the rats and, as a consequence, of the colonic volume.

The effect of RNase B1 as a preventive agent

The implanted pump rats were sacrificed after 8 weeks, since infection was observed at the pump sites. At this stage only ACF were evident. ACFs are surrogate biomarkers of carcinogenic changes in the rat colon during the initiation phase of carcinogenesis. The goblet cells in the crypts become larger and intensely stained compared to normal mucosal cells. ACF counts were drastically reduced due to RNase B1 or I-RNase B1 treatments (Figure 22). No detrimental effect was observed on the colonic mucosa in rats treated with RNase B1 or I-RNase B1 in the absence of DMH.

In rats fed microencapsulated RNase B1, the experiment continued for 11 weeks after the first DMH administration. At that time, both tumors and ACF were present. RNase B1 caused a reduction in the number of tumors per colon (Figure 23a), the tumor size (Figure 23b) as well as the number of ACFs (Figure 23c) compared to the control.

In addition, a color diversity of colon tumors was observed; red tumors that had intensive blood supply (Figure 24a), white tumors almost devoid of blood vessels (Figure 24b), and “pink” tumors with only few blood vessels (Figure 24c). In the glucose-treated rats all tumors were red. On the other hand, in rats treated with RNase B1, a significant reduction in the number of reddish tumors was observed (Figure 24d); 10% and 50% of the tumors were pink and white, respectively. These results clearly indicate an antiangiogenic effect of RNase B1.

Tumors could also be distinguished by histopathological parameters, such as benign or malignant. A benign tumor called an adenoma (Figure 25a) can be defined by a spread mucous layer, and sometimes by the development of adenopapilloma, however the submucosa is intact and well defined with respect to the other layers of the colon. In a malignant tumor, termed adenocarcinoma, mucosal cells penetrate below the submucosa and eventually lead to loss of tissue ordering (Figure 25b and 25c). Examinations of the distribution of the different tumor types in rats preventatively treated with glucose or RNase B1 showed that RNase B1 clearly reduced the degree of malignancy (Figure 9d).

The effect of RNase B1 as a therapeutic agent:

In both modes of application, directly by osmotic pumps or orally, well-developed tumors were exposed to RNase B1 during weeks 12-17 of the experiment. In rats treated with osmotic pumps, RNase B1 caused a reduction in the number of tumors per colon (Figure 26a). The inhibitory effect, approximately 50% relative to the control, was more significant in rats treated with I-RNase B1.

RNase B1 also affected tumor growth, as demonstrated by the size distribution (Figure 26b). In general, most tumors were 3-5 mm in diameter, however exceptionally large tumors, in addition to 9-12 mm, appeared in PBS-treated rats. This result implies that RNase B1

ES 2 397 052 ​​T3 inhibits or stops the development of pre-existing tumors. However, no significant differences were observed between the effects of RNase B1 and I-RNase B1.

As in the RNase B1 preventive effect experiment, the angiogenesis pattern was also affected by RNase B1 applied by osmotic pumps (Figure 26c). In PBS-treated rats the majority of tumors, approximately 80%, were red in color. In contrast, in rats treated with both RNase B1 and IRNase B1 only 30% of the tumors were red, while the others were pink or white. RNase B1 appears to reduce angiogenesis also in pre-existing tumors.

In rats fed encapsulated RNase B1, the treatment effect was less significant than that obtained by osmotic pumps (Figures 27a-c). This result is explained by assuming that a very small proportion of the protein reaches the colon. As mentioned above, the microcapsules do indeed pass into the stomach, but they still have a long route through the small intestine and the cecum. An experiment was therefore carried out to test this hypothesis using CAP microcapsules loaded with a fluorescent protein and given to rats. The rats were sacrificed after six hours and the contents of their gastrointestinal tract were observed under a fluorescent microscope. It was found that in the duodenum the microcapsules began to dissolve. The solution was further processed in the ileum and jejunum. When the microcapsules reached the cecum, most of the fluorescence diffused to the contents of the cecum. Since the microcapsules were damaged, the effect of RNase B1 can be reduced due to proteases present in the intestine and cecum.

Despite the fact that orally administered RNase B1 did not reduce the number and size of pre-existing tumors, the distribution between tumor color types was slightly affected (Figure 27c). Glucose and RNase B1 treated rats had approximately 60% and 40% red tumors, respectively. It appears that orally administered RNase B1 in the present formulation also affects angiogenesis, but in a moderate way.

Example 5 (REFERENCE EXAMPLE)

The effect of RNase B1 on human HT-29 colon cancer cells in vitro

Experimental material and procedures

Cell growth conditions:

All experiments were performed on human colon adenocarcinoma cells (HT-29). The cells were cultured in 50 ml flasks containing DMEM medium (Biological Industries, Bet Haemek), supplemented with 10% fetal calf serum, 1% glutamine and 1% Antibiotic-Antifungal solution (Biolab). The cells were trypsinized, and 2 ml of medium containing 5 x 10⁴ cells in each well of a 6-well plate. Some plates were supplemented with RNase B1, at a final concentration of 10^-6 M. Cells were incubated at 37 ° C in a modified atmosphere containing 5% CO2. After 48 hours the medium in the presence or absence of RNase B1 was replaced in each well respectively, to maintain a constant supply of ingredients and RNase B1. After 4 days the medium was removed and the cell cultures were fixed in 4% formaldehyde in PBS (150 mM NaCl, 3 mM KCl, Na₂HPO₄ 10 mM, NaH₂PO₄ 2 mM) pH 7.2 for 15 minutes on ice. The cells were stained for different purposes, as follows.

Direct staining for intracellular actin:

Cells were washed in PBS and permeabilized in PBS containing 0.02% Tween-20 (PBST) for 1 hour at room temperature. After 3 washes with PBS, cells were stained for actin with tetramethylrhodamine B isothiocyanate (TRITC) labeled phalloidin.^-6 M (Sigma) for 1 hr and allowed to stand in PBS overnight at 4 ° C to remove any excess staining material. Cells were spread in water on a glass slide and visualized using Confocal Laser Scanning Microscope (LSM) 510 (Zeiss).

Immunostaining for membrane actin:

Cells were fixed with formaldehyde and washed with PBS as described above, and then incubated with rabbit anti-actin antibodies (Sigma) diluted 1: 500 in PBS for 1 hour at room temperature and washed three times with PBS. The cells were then incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (FITC) diluted 1: 100 in PBS for another hour under the same conditions, washed again and visualized as described above.

RNase immunostaining Well the cell surface:

Rabbit polyclonal antibodies (Aminolab, Israel) were raised against purified RNase B1. Anti-RNase B1 was used as the main antibody in immunostaining for HT-29, according to the procedure described above.

Experimental results

Direct staining for intracellular actin:

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In control cells growing without RNase B1, a TRITC-stained fine actin network was observed, filling the cell cytoplasm. A slight staining was observed on the membrane surface (Figure 28a). On the contrary, in cells treated with RNase B1, the membrane and the peripheral zone of the cytoplasm in each cell were intensively stained (Figure 28b), which indicates the rearrangement of the actin network in response to external addition of

RNase B1.

Immunostaining for membrane actin:

Immunostaining with FITC showed fine fluorescent spots of actin in the membrane region in control cells (Figure 29a). This result agrees with the TRITC staining shown in Figure 28a. In this experiment, no detergent was used, since the antibody barely penetrated the cytoplasm of the cells. Therefore, it appears that in these cells, membrane actin interacts with the external environment. Much weaker immunostaining was observed in cells treated with RNase B1 (Figure 29b), implying that RNase B1 previously bound to membrane actin interfered with the binding of anti-actin antibodies.

Additional cells, not treated with RNase B1, were incubated with the pre-mixed anti-rabbit actin and 1 gM actin. Similar weak fluorescence was observed, as described in Figure 29b (not shown). To eliminate the possibility of spontaneous FITC fluorescence, cells were treated as described, except that anti-actin was omitted. Fluorescent staining was not detected.

Cell surface RNase B1 immunostaining:

Very weak FITC fluorescence appeared in control cells incubated with anti-RNase B1 (Figure 30a). However, cells treated with RNase B1 showed a strong fluorescent response (Figure 30b). This result indicates a significant presence of RNase B1 on the cell surface, especially at the edges and extensions of the cells. Treatment with rabbit preimmune serum (PIS) instead of anti-RNase B1 resulted in very weak fluorescence (Figure 30c).

Example 6

The effect of IAc-RNase B1 on the growth of lily pollen tubes

Experimental materials and procedures

RNase B1 iodoacetylation:

RNase B1 iodoacetylation was performed according to Irie et al (Irie M, et al. 1986 J. Biochem. 99 (3): 627-33). RNase B1 was dissolved in 2.5 ml of 0.1 M acetate buffer, containing 0.1 M iodoacetate, to a final concentration of 10 nM. After incubation overnight at 37 ° C, the protein was desalted on a Sephadex G-column.

fifteen. The fractions containing the protein were collected and intensively dialyzed against water. After lyophilization, 10 mg of protein was obtained. The RNase activity of iodoacetylated RNase B1 (IAc) was compared to unmodified RNase B1.

The effect of IAc-RNase B1 on lily pollen tubes:

Lyophilized IAc-RNase B1 was dissolved at a final concentration of 1 or 5 gM in lily pollen tube growth medium containing 7% sucrose, 1.27 mM Ca (NO3) 2, 0.16 mM H3BO3, 1 mM KNO3 and 3 mM KH2PO4 in water (Yokota E. and Shimmen, T. 1994. Protoplasma 177: 153-162). Pollen grains of lilies (Lilium longiflorum) were germinated in vitro in tubes containing 100 gl of growth medium, in the presence or absence of IAc-RNase B1 10^-6 M. As additional controls, lily pollen was germinated at the same concentrations of RNase B1 or BSA in growth medium. After 1.5 hours of incubation at 25 ° C in the dark, the length of the pollen tubes was measured in each treatment with a light microscope.

Experimental results

RNase B1 iodoacetylation:

Iodoacetate leads to inhibition of RNase activity, by binding to histidine residues at the active site of RNase. The RNase activity of IAc-RNase B1 was 90% lower, compared to untreated RNase B1.

The effect of IAc-RNase B1 on the growth of lily pollen tubes:

The pollen tubes of control lilies reached a length of 0.26 mm (Figure 31). In this experiment BSA was used as a control, since it has no cytotoxic effect on pollen tubes. In fact, at a concentration of 10^-6 M, BSA had no significant effect on pollen tube growth. The inhibitory effect of BSA 5 x 10^-6 M can be explained as a result of the fact that pollen tubes are sensitive to changes in the osmotic potential of the growth medium. Both RNase B1 and IAc-RNase B1 showed a clear inhibitory effect on the growth of pollen tubes. At both concentrations, IAc-RNase B1 was more efficient than RNase B1, however the differences were not significant. Hence, in pollen tubes of lilies, IAc29

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RNase B1 inhibits the growth of pollen tubes in a similar way to unmodified RNase B1, showing that loss of RNase activity does not reduce its inhibitory effect.

Although the invention has been described in conjunction with specific embodiments thereof, it is clear that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to encompass all such alternatives, modifications, and variations that are within the spirit and broad scope of the appended claims. All publications, patents, patent applications and sequences identified by an accession number mentioned in the present specification are incorporated herein in their entirety by reference to the specification, to the same extent as if individually indicated that each individual publication, patent, patent application or sequence is incorporated herein by reference. Furthermore, the mention or identification of any reference to the present application should not be construed as an admission that such reference is available as prior art to the present invention.