WO2004009130A1 - Tumor treating composition and methods - Google Patents

Abstract

The invention relates to the treatment of tumors an in particular to methods and compositions for the treatment of solid vascular tumors. In one aspect, the method comprises the over-expression of VHL in the tumor.

Description

TUMOR TREATING COMPOSITION AND METHODS

Technical Field

The invention relates to the treatment of tumors and in particular to a composition for the treatment of solid vascular tumors. The invention also relates to compositions and methods of use in such treatments.

Background

Non Hippel-Lindau (NHL) disease is an autosomal dominant familial cancer syndrome that predisposes affected individuals to a variety of highly vascular tumors (1, 2). The most common tumors are hemangioblastomas of the central nervous system, renal cell carcinoma (RCC), and pheochromocytoma. NHL kindreds have germline mutations in the NHL gene, and somatic inactivation or loss of the remaining wild-type NHL allele is linked to tumor formation.

NHL is a tumor suppressor, whose functional inactivation stimulates tumor formation in a variety of ways, in particular by increasing the stability of Hypoxia Inducible Factor - 1 (HIF-1) (2, 3). HIF-1 regulates cellular adaption to changes in the oxygen availability by regulating genes involved in angiogenesis, erythropoiesis, energy and iron metabolism, tissue matrix metabolism, and cell survival decisions; which are key factors for tumor growth and survival (4-6). HIF-1 is an β heterodimer of which the β subunit is expressed constitutively and is not significantly affected by hypoxia, whereas levels of the α subunit rise markedly with hypoxia, and fall rapidly under normoxic conditions. A 35 amino acid subdomain of the α domain of the 30 kDa von Hippel-Lindau protein (pNHL) binds elongin C, which recruits additional proteins including elongin B, cullin-2, the RJΝG-H2 protein Rbxl/Rocl, and ubiquitin conjugating enzyme E2, to form a ubiquinating complex. The β domain of pNHL binds hypoxia-inducible factor (HIF) subunits HIF-1 α and HIF- 2α, targeting them for ubiquitination and proteasomal destruction in a NHL α-domain- dependent manner (7). The binding of HIF-lα subunits to NHL, and their rapid degradation by the NHL ubiquitinating complex under normoxic conditions, is regulated by oxygen and iron-dependent hydroxylation of Pro-564 within HIF-1 (8). Mutation of the ct and β domains of NHL either prevents formation of a NHL ubiquitinating complex, and or binding to HIF-1, respectively, leading to stabilization of HIF-1 (3, 7). A hypoxic phenotype results in which increased levels of HIF-1 induce the synthesis of hypoxia- inducible genes such as vascular endothelial growth factor (NEGF), platelet derived growth factor, and glucose transporter- 1 (Glut-1), which assist tumor growth by stimulating tumor angiogenesis, and metabolism (9-12).

Reintroduction of wild-type NHL into the NHL-negative tumor RCC in which both NHL alleles are either inactivated or lost, restores NHL-mediated functions, and leads to a loss of tumorigenicity in nude mice (13).

The development and growth of tumors is complex. Despite the positive results in tumor treatment described to date, there would be distinct advantages in providing alternative options which contribute to the available treatments.

Summary of the Invention

In one aspect, the invention provides a method of treating tumors in an animal, the method comprising at least the over-expression of NHL in a tumor.

In a further aspect, the invention provides a method of treating small tumors in an animal by engineered over-expression of NHL in the tumor.

In another aspect, the invention provides a method of inhibiting tumor angiogenesis in an animal, the method comprising at least the over-expression of NHL in a tumor.

In another aspect, the invention provides a method of enhancing tumor cell apoptosis in an animal, the method comprising at least the over-expression of NHL in a tumor.

A method of any one of the above mentioned aspects preferably includes the step of administering to the animal an agent adapted to effect over-expression of NHL in a tumor. Preferably the agent is a vector adapted to express NHL. Preferably, the vector is a nucleic acid vector. Alternatively, the vector is a viral vector comprising nucleic acid in a viral capsid.

Preferably, the agent is one which allows for over-expression of native NHL within the tumor.

Preferably, the agents adapted to effect over-expression of NHL in a tumor are administered intratumorally. Alternatively, the agents are administered systemically.

In another aspect, the invention provides a composition for use in tumor treatment in an animal, the composition comprising an effective amount of an agent adapted to over- express NHL in a tumor, together with one or more suitable carriers.

In another aspect, the invention provides a composition for inhibiting tumor angiogenesis in an animal, the composition comprising an effective amount of an agent adapted to over- express NHL in a tumor, together with one or more suitable carriers.

In another aspect, the invention provides a composition for enhancing tumor cell apoptosis in an animal, the composition comprising an effective amount of an agent adapted to over- express NHL in a tumor, together with one or more suitable carriers.

In a further aspect, the invention provides a method of treating tumors, enhancing tumor cell apoptosis, or inhibiting tumor angiogenesis in an animal, the method comprising at least the administration of an agent which mimics the function of NHL in a tumor (a NHL function mimicking agent).

In another aspect, the invention provides a composition for use in tumor treatment, enhancing tumor cell apoptosis, or inhibiting tumor angiogenesis in animals, the composition comprising an effective amount of an agent adapted to mimic NHL in a tumor, together with one or more suitable carriers. In another aspect, the invention provides a method of assessing efficacy of over-expressed NHL in animal tumor treatment, the method comprising the steps of engineering over- expression of NHL in tumors of varying sizes in an animal followed by determining the effect this engineered over-expression has on tumor growth.

Preferably a method of the invention involves the administration of a vector adapted to express NHL which is administered in an amount between about 5 μg and 2 mg.

In another aspect, the invention provides a method of treating tumors, enhancing tumor cell apoptosis or inhibiting tumor angiogenesis in an animal, the method comprising at least the step of administering to the animal an effective amount of NHL.

Drawings

These and other aspects of the present invention, which should be considered in all its novel aspects, will become apparent from the following description, which is given by way of example only, with reference to the accompanying figures, in which:

Figure 1 Illustrates that intratumoral injection of an expression plasmid encoding

NHL downregulates HIF-1 α and NEGF in tumors. (A) Immunohistochemistry to analyze the expression of plasmids injected into tumors. Tumors of 0.4 cm diameter were injected with empty pcDΝA3 vector (pCDNA3), or an expression plasmid encoding NHL. Tumor sections prepared two days after plasmid injection were stained (brown) for NHL with the rabbit polyclonal anti-NHL antibody FL-181. Magnification, xlOO. (B) Overexpression of NHL by intratumoral injection of a NHL expression plasmid downregulates HIF-1 α. EL-4 tumors as in (A) were stained with the mouse anti-mouse HIF-1 α mAb Hlα67. Magnification, xlOO. (C) Overexpression of NHL by intratumoral injection of a NHL expression plasmid downregulates NEGF expression. EL-4 tumor sections as in (A), but prepared 4 days after plasmid injection, were stained with the

Ab-1 rabbit polyclonal antibody against NEGF. Magnification, xlOO. (D) Western blot analysis of homogenates of tumor cells extracted from tumors. Tumor cell homogenates prepared from tumors as in (A) were injected with empty plasmid (lane 1), or NHL (lane 2). They were resolved by SDS- PAGE, and Western blotted with antibodies against NHL and HIF-1 α, and NEGF as indicated. (E) Decrease in the percentage of HIF-1 α positive- staining cells after injection of a NHL plasmid. The numbers of HIF-1 α positive cells in sections (x40 magnification) illustrated in (B) were counted in 10 blindly chosen random fields, n, number of tumors assessed. There was a significant (P<0.01) difference in the numbers of HIF-l positive cells in sections of tumors injected with empty pCDΝA3 plasmid versus tumors injected with NHL plasmid.

Figure 2 Illustrates that intratumoral injection of an expression plasmid encoding

NHL eradicates small tumors. (A) Small EL-4 tumors, approximately 0.1 cm in diameter, were injected at day 0 with expression plasmids encoding

NHL, or empty plasmid (Control), and tumor size was recorded for 12 days. Complete tumor regression is denoted by vertical arrows. Mice were euthanased when tumors reached 1 cm in diameter (denoted by stars).

Figure 3 Illustrates that intratumoral injection of an expression plasmid encoding

NHL inhibits tumor angiogenesis. (A) Illustrated are sections prepared from 0.4 cm tumors injected 4 days earlier with empty pcDΝA3 vector

(pCDNA3), or an expression plasmid encoding NHL. Sections were stained with anti-CD31 antibody MEC13.3 to visualize blood vessels. (B) Measurement of tumor vascularity. Tumor blood vessels stained with the anti-CD31 mAb were counted in 5 blindly chosen random fields to record mean blood vessel counts per section (40^χ magnification field), n, number of tumors assessed. A significant (P < 0.01) difference in mean vessel counts between tumors injected with therapeutic plasmid vectors versus tumors injected with empty pCDΝA3 plasmid is donated by stars.

Figure 4 Illustrates that intratumoral injection of an expression plasmid encoding

NHL enhances tumor cell apoptosis. (A) Tumor sections were prepared from 0.4 cm diameter tumors injected 4 days earlier with either empty pCDΝA3 vector, or a plasmid encoding NHL. Tumor sections were stained by TUNEL analysis for apoptotic cells (here colored grey). Magnification xlOO. (B) TUNEL positive cells were counted to record the apoptosis index (Al) (40 x magnification field), n, number of tumors assessed.

Detailed Description

The von Hippel-Landau (NHL) tumor suppressor is lost or mutated in patients with NHL cancer syndrome, and in the majority of patients with sporadic renal cell carcinomas (RCCs). NHL binds the α subunits of hypoxia-inducible factor (HIF)-Iα, which stimulates tumor angiogenesis and glycolysis, targeting them for ubiquitination and proteasomal destruction. Reintroduction of the NHL gene product (pNHL) inhibits the growth, tumorigenicity, and invasiveness of RCC cells.

The present inventors have now found that intratumoral injection of small (0.1 cm diameter) subcutaneous tumors derived from mouse EL-4 thymic lymphoma cells with a plasmid encoding NHL to over-express NHL in the tumor, resulted in the down-regulation of HIF-1 α and vascular endothelial growth factor (NEGF), and inhibited tumor angiogenesis. There was increased tumor cell apoptosis, accompanied by complete eradication of tumors.

In accordance with these findings the invention relates to methods for inhibiting tumor angiogenesis, enhancing tumor cell apoptosis, and ultimately treating tumors in an animal. The methods of the invention are particularly applicable to the treatment of solid vascular tumors, particularly small tumors. The methods may find application in the treatment of hemangioblastomas of the central nervous system, renal cell carcinomas, and pheochromocytoma.

As used herein, the term "vascular tumor" should not be taken to imply that such tumors are highly vascular.

As used in relation to the invention, the term "treating" or "treatment" and the like should be taken broadly. They should not be taken to imply that an animal is treated to total recovery. Accordingly, these terms include amelioration of the symptoms or severity of a particular condition or preventing or otherwise reducing the risk of further development of a particular condition.

It should be appreciated that methods of the invention may be applicable to various species of animal, preferably mammals, more preferably humans.

Methods of the invention involve the over-expression of NHL in a tumor. The term "over- expression" should be taken to refer to an increase in NHL expression above the baseline expression level for a particular tumor. "Over-expression" may occur by increasing expression from an endogenous NHL gene (ie that native to the tumor, or to surrounding or adjacent tissue) or via introduction of a NHL-expressing transgene (as will be elucidated further herein).

Accordingly, a method of the invention includes the administration to an animal of an effective amount of an agent adapted to effect over-expression of NHL in a tumor. In addition, the inventors contemplate methods involving the administration of agents adapted to mimic the function of NHL (ie NHL mimetics), or to up-regulate such agents within the tumor.

In the context of the invention an "effective amount" of an agent to be administered to an animal is an amount necessary to at least partly attain a desired response.

In an alternative embodiment, NHL may be administered to increase its levels in a tumor.

In a preferred embodiment of the invention an agent adapted to effect over-expression of

NHL in a tumor is a nucleic acid expression vector. Alternatively, the agent is a viral vector comprising a nucleic acid vector contained within a viral capsid.

Persons of general skill in the art to which the invention relates will readily appreciate nucleic acid expression vectors of use in the invention. However, by way of example expression vectors that contain the CMN promoter are highly active in tumors. One explicit example, in the form of pCDΝA3 (Invitrogen), is provided herein after under the heading "Methods". Such expression vectors may be constructed according to standard techniques and/or manufacturers instructions, having regard to the published nucleic acid sequence of NHL and/or the published amino acid sequence thereof. The nucleic acid and protein sequences for both human and murine NHL are available on publicly accessible databases. For example, human NHL is available on GenBank under the accession number AF010238. The murine NHL sequence information is available on GenBank under the accession number AF513984. A specific example of how such a vector may be constructed is provided herein after under the heading "Methods".

It should be appreciated that expression vectors of the invention may include various regulatory sequences. For example, they may include tissue specific promoters, inducible or constitutive promoters. Further, they may include enhancers and the like which may aid in increasing expression in certain circumstances. Persons of general skill in the art to which the invention relates will appreciate various regulatory regions which may provide benefit having regard to the tumor to be treated.

In respect of viral vectors, the inventors contemplate the use of such vectors as adeno- associated virus, lentivirus, adenovirus, retroviruses. Viral vectors may be constructed according to standard procedures in the art. The paper Xu, R., Sun, X., Chan, D., Li, H.,

Tse, L-Y., Xu, S., Xiao, W., Kung, H., Krissansen, G.W., and Fan, S-T. Long-term expression of angiostatin suppresses metastatic liver cancer in mice. Hepatol. 37:1451-60, 2003 provides details of appropriate viral vectors. It will be appreciated that viral vectors will generally be attenuated such that they do not posses their original virulence.

As noted herein before, the effect of NHL on a tumor can also be achieved via the use of agents or factors that stimulate endogenous NHL expression including those that stimulate NHL gene transcription, translation, or protein stability. Examples of such agents include "nonselective" (indomethacin) and COX-2-selective (ΝS-398) non steroidal anti- inflammatory drugs (NSAIDs)" (14). Skilled persons may appreciate other appropriate agents. Reagents that mimic the effects of NHL include drugs that interact with NHL effectors, and stimulate a response similar to that of NHL. Peptides and pharmaceutical type reagents based on the NHL protein sequence or structure could be used as NHL mimetics.

It should be appreciated that agents or compounds of use in the invention may be modified to assist their function in vivo for example by reducing their immunogenicity or increasing their lifetime in vivo. Agents may be modified (for example by addition of a carrier peptide or membrane translocating motif (for example Chariot™ peptide; Active Motif, Carlsbad, CA, USA) as will be known in the art) or formulated with additional agents to allow for their cell permeability and the like. Persons of ordinary skill in the art to which the invention relates will readily appreciate appropriate modifications. However, by way of example, the agents may be PEGylated to increase their lifetime in vivo, based on, e.g., the conjugate technology described in WO 95/32003.

Administration of agents of use in methods of the invention may occur by any means capable of increasing expression of NHL, or a mimetic thereof, in a tumor. Such methods include intratumoral admimstration and systemic administration. Intratumoral administration may occur via injection (as exemplified herein after) or alternatively direct injection into blood vessels supplying the tumor could occur. Systemic administration may occur by any standard means readily known to the skilled person in the art to which the invention relates having regard to the information herein and to the agent to be administered.

By way of general example, modes of administration may include oral, topical, systemic (eg. transdermal, intranasal, or by suppository), parenteral (eg. intramuscular, subcutaneous, or intravenous injection), intratumoral (eg. by injection, using bollistics); by implantation, and by infusion through such devices as osmotic pumps, transdermal patches, and the like.

Persons of general skill in the art to which the invention relates will be able to readily appreciate the most suitable mode of administration having regard to the therapeutic agent to be used and the tumor to be treated. While compounds or agents of use in the invention may be administered alone, in general, they will be administered as pharmaceutical compositions in association with at least one or more carriers and/or excipients. Accordingly, compounds may be administered as naked DNAs, or using virus technologies, or as recombinant proteins, peptides, or pharmaceutical compositions, or by other means that any person of ordinary skill in the art would be able to devise.

Compositions may take the form of any standard known dosage form including tablets, pills, capsules, semisolids, powders, sustained release formulation, solutions, suspensions, elixirs, aerosols, liquids for injection, or any other appropriate compositions. Persons of ordinary skill in the art to which the invention relates will readily appreciate the most appropriate dosage form having regard to the nature of the tumor to be treated and the active agents to be used without any undue experimentation. It should be appreciated that one or more active agents described herein may be formulated into a single composition.

As previously mentioned, compounds or agents compatible with this invention might suitably be administered by a sustained-release system. Suitable examples of sustained- release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L- glutamate, poly (2 -hydroxy ethyl methacrylate), ethylene vinyl acetate, or poly-D-(-)-3- hydroxybutyric acid (EP 133,988). Sustained-release compositions also include a liposomally entrapped compound. Liposomes containing the compound are prepared by methods known per se: DE 3,218,121 ; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP

102,324. Ordinarily, the liposomes are of the small (from or about 200 to 800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mole percent cholesterol, the selected proportion being adjusted for the most efficacious therapy.

Suitable carriers and/or excipients will be readily appreciated by persons of ordinary skill in the art, having regard to the nature of the agent to be formulated. However, by way of example, suitable liquid carriers, especially for injectable solutions, include water, aqueous saline solution, aqueous dextrose solution, and the like, with isotonic solutions being preferred for intravenous, intraspinal, and intracisternal administration and vehicles such as liposomes being also especially suitable for admimstration of agents, such as naked nucleic acid vectors to tumors.

In addition to standard diluents, carriers and/or excipients, compositions of the invention may be formulated with additional constituents, or in such a manner, so as to decrease the immunogenicity of an agent to be administered, or help protect its integrity and prevent in vivo degradation, for example. Persons of ordinary skill in the art to which the invention relates will readily appreciate constituents and techniques to this end.

The compositions may be formulated in accordance with standard techniques as may be found in such standard references as Gennaro AR: Remington: The Science and Practice of th

Pharmacy, 20 ed., Lippincott, Williams & Wilkins, 2000, for example.

As will be appreciated, the dose of an agent or composition administered, the period of administration, and the general administration regime may differ between subjects depending on such variables as the severity of symptoms, the type of tumor to be treated, the mode of administration chosen, type of composition, size of a unit dosage, kind of excipients, the age and/or general health of a subject, and other factors well known to those of ordinary skill in the art.

The issue of what is, or is not, a small tumor would be readily discernible by trial and error, following the surprising findings disclosed herein.

In relation to intratumoral administration of a nucleic acid vector adapted to express NHL, dosages of between about 5 μg and about 2 mg, may be appropriate, however, this is not definitive.

Admimstration may include a single daily dose or administration of a number of discrete divided doses as may be appropriate. An administration regime may also include admimstration of one or more of the active agents, or compositions comprising same, as described herein. The invention will now be further described with reference to the following non-limiting examples.

Examples

Methods

Mice and cell lines. Male C57BL/6 mice, 6-8 weeks old, were obtained from the Animal Resource Unit, Faculty of Medicine and Health Science, University of Auckland, Auckland, New Zealand. The EL-4 thymic lymphoma, which is of C57BL/6(H-2^b) origin, was purchased from the American Type Culture Collection (Rockville, MD, USA). It was cultured at 37°C in DMEM medium (Gibco BRL, Grand Island, NY, USA), supplemented with 10% foetal calf serum, 50 U/ml penicillin/streptomycin, 2 mM L-glutamine, ImM pyruvate.

Expression plasmids. A cDNA fragment encoding full-length (546bp) mouse NHL was

PCR amplified using IMAGE clone 63956 as a template, and the primers 5'-AGG CGG CGG GGG AGC CCG GTC CTG AGG AGA TGG AGG CTG GGC GGC CGC GGC CGG TGC TGC GCT CG-3' and 5 '-ACT CTC AAG GTG CTC TTG GCT CAG TCG CTG TAT GTC CTT CCG CAC ACT TGG GTA G -3'. The resulting PCR product was used as a template for further amplication with the primers 5' -GGG AAT TCC AAT AAT

GCC CCG GAA GGC AGC CAG TCC AGA GGA GGC GGC GGG GGA GCC CGG TCC TG-3' and 5'-GGT CTA GAT CAA GGC TCC TCT TCC AGG TGC TGA CTC TCA AGG TGC TCT TGG CTC A-3'. The PCR product was subcloned into pCDΝA3 (Invitrogen). All constructs were verified by DNA sequence analysis.

Gene transfer of expression plasmids in situ and measurement of anti-tumor activity. Purified plasmids were diluted to 1 mg/ml in a solution of 5% glucose in 0.01% Triton X- 100, and mixed in a ratio of 1 :3 (wt:wt) with DOTAP cationic liposomes (Boehringer Mannheim, Mannheim, Germany), as described previously (15). Tumors were established by injection of 2 x 10⁵ EL-4 tumor cells into the right flank of mice, and growth determined by measuring two perpendicular diameters. Animals were killed when tumors reached more than 1 cm in diameter, in accord with Animal Ethics Approval (University of Auckland). Once tumors reached either 0.1 cm in diameter, they were injected with 100 μl expression plasmid (100 μg). Empty pCDNA3 vector served as a control reagent. All experiments included 6 mice per group, and each experiment was repeated at least once.

Immunohistochemistry. Tumor cryosections (10 μm) prepared 2 days following injection of plasmids were incubated overnight with either a rabbit polyclonal antibody against a peptide corresponding to N-terminal amino acids 1-181 of NHL (FL-181, Santa Cruz Biotechnology, Inc), a mouse anti-mouse HIF-1 α mAb (Hlα67, Νovus Biologicals, Inc., Littleton, CO, USA), or a rabbit polyclonal antibody against NEGF (Ab-1, Lab Vision Corporation; CA, USA). Rabbit antibody-stained sections were subsequently incubated for

30 min with appropriate secondary antibodies (NECTASTAIΝ Universal Quick kit, Vector Laboratories, Burlϊngame, CA), and developed with Sigma FAST DAB (3,3'- diaminobenzidine tetrahydrochloride) and CoCl enhancer tablets (Sigma). Sections were counterstained with Mayer's hematoxylin. The Vector M.O.M. Immunodetection Kit (Vector Laboratories, Inc.Burlingame, CA) was used to detect the mouse anti-HIF- lα mAb. The total number of HIF-1 positive cells in 10 randomly selected fields was counted, and the percentage of positive staining cells was calculated (percentage of positive cells = number of positive cells x 100/ total number of cells).

Assessment ofvascularity. Methodology to determine tumor vascularity has been described previously (16, 17, 18). Briefly, 10 μm frozen tumor sections, prepared 4 days after injection of 0.4 cm diameter tumors with plasmid, were immunostained with the anti-CD31 antibody MEC13.3 (Pharmingen, CA). Stained blood vessels were counted in five blindly chosen random fields (0.155 mm²) at 40^χ magnification, and the mean of the highest three counts was calculated. The concentric circles method (19, 20) was used to assess vascularity, where 5 to 6 tumor sections were analysed for each plasmid-injected tumor.

In situ detection of apoptotic cells. Serial sections of 6 μm thickness were prepared from excised tumors that had been frozen in liquid nitrogen, and stored at -70°C. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labelling (TUΝEL) staining of sections was performed using an in situ apoptosis detection kit from Boehringer Mannheim, Germany. Briefly, frozen sections were fixed with 4% paraformaldehyde solution, permeabilized with a solution of 0.1% Triton-XlOO and 0.1% sodium citrate, incubated with TUNEL reagent for 60 min at 37°C, and examined by fluorescence microscopy. Adjacent sections were counterstained with haematoxylin and eosin. The total number of apoptotic cells in 10 randomly selected fields was counted. The apoptotic index was calculated as the percentage of positive staining cells, namely Al

= number of apoptotic cells x 100/total number of nucleated cells.

Western blot analysis. Tumors previously injected with either empty plasmid, or VHL expression plasmids were excised, minced with scissors and homogenized in protein lysate buffer (50mmol/L Tris pH 7.4, 100 μmol/L EDTA, 0.25mol/L sucrose, 1% SDS, 1% NP40, 1 μg/ml leupeptin, 1 μg/ml pepstatin A and 100 μmol/L phenylmethylsulfonyl fluoride) at 4°C using a motor-driven Virtus homogenizer (Nirtus, Gardiner, ΝY). Tumor lysates from each treatment group were pooled, and debris removed by centrifugation at 10,000 x g for 10 min at 4°C. Protein samples (100 μg) were resolved on 10% polyacrylamide SDS gels under reducing conditions, and electrophoretically transferred to nitrocellulose Hybond C extra membranes (Amersham Life Science, Buckingham,

England). After blocking the membranes with 5% bovine serum albumin in Tween 20/Tris-buffered saline (TTBS; 20 mmol/L Tris, 137 mmol/L ΝaCl pH 7.6, containing 0.1% Tween-20), blots were incubated with primary antibodies, and subsequently with horseradish peroxidase-conjugated secondary antibodies. They were developed by enhanced chemiluminescence (Amersham International, Buckingham, England), and exposure to x-ray film. Band density was quantified using Scion Image software (Scion Corporation, Frederick, MD).

Statistical analysis. Results were expressed as mean values + standard deviation (SD). A student's t test was used for evaluating statistical significance, where a value less than 0.05 (P< 0.05) denotes statistical significance.

Results

Engineered over-expression of VHL eradicates small tumors. Whether over-expressed

NHL might effectively down-regulate HIF-1 pathways, and angiogenesis in tumors already expressing functional NHL has not been able to be predicted with certainty. In tumors, NHL not only has to contend with HIF-1 induced by hypoxia, but potentially also HIF-1 induced in response to tumor-derived factors such as v-src, and insulin-like growth factor- 1 receptor (IGF-1R) ligands (IGF-I, IGF-II, and insulin) (21, 22).

To test the latter notion, small EL-4 tumors, 0.1 cm in diameter, were established in the right flank of C57BL/6 mice, and injected with a DNA/liposome transfection vehicle containing either 100 μg of empty pCDNA3, or VHL-pCDNA3 plasmids.

Immunohistochemical analysis of tumor sections, prepared 2d following plasmid injection, revealed that VHL was over-expressed in tumors injected with NHL plasmid, compared to tumors injected with empty plasmid which displayed low levels of endogenous NHL (Figure 1A). This result was confirmed by Western blot analysis of lysates of tumor cells extracted from tumors, which revealed exogenous NHL was over-expressed 2-fold and was of the expected size of 20 kDa (Figure ID). Tumors grew rapidly following injection of empty pCDΝA3 plasmid, reaching 1 cm in size within 12d. In contrast, tumors injected with NHL plasmid rapidly regressed within one week of injection, and completely disappeared (Figure 2 A). Thus, like antisense HIF-1 α monotherapy previously reported (16), engineered over-expression of NHL monotherapy is effective against small tumors.

Intratumoral injection of a VHL plasmid down-regulates the expression of HIF-1 a and its effector molecule VEGF. In order to understand the mechanisms responsible, in part, for the anti-tumor activity exhibited by exogenous NHL, we examined tumors that had been injected with a NHL plasmid for the levels of HIF-lα, and its effector NEGF. Gene transfer of NHL led to complete downregulation of HIF-lα expression in a proportion (20%) of tumor cells, as revealed by immunohistochemistry (Figures IB and E), and supported by Western blot analysis (Figure ID). However, a major proportion of tumor cells appeared to retain some HIF-1 α expression (Figures IB and E). Similarly, NHL therapy led to down-regulation of tumoral NEGF expression (Figures 1C and D).

VHL therapy reduces tumor blood vessel density, and increases apoptosis. Injection of a NHL plasmid into tumors inhibited tumor angiogenesis, as evidenced by a statistically significant (p<0.05) reduction in tumor blood vessel density (Figures 3 A and B), in accord with reductions in the angiogenic factors HIF-1 α, and NEGF. The median and 90th centile distances to the nearest CD31 -labelled venules from an array of points within tumors treated with NHL plasmid were significantly (both p<0.05) longer than those for tumors treated with empty vector (Table 1). The median and 90th centile distances to the nearest CD31 -labelled venules from an array of points within tumors treated with NHL were significantly longer than those for tumors treated with either empty pCDΝA3 (P<0.025) plasmid (Table 1).

Table 1. Vessel density measured by the concentric circle method

The median and 90th centile distances (± SD) to the nearest CD31 -labelled venules from an array of points within tumors injected with either empty pcDNA3 or NHL plasmids were determined. P values refer to distances to labelled venules in tumors treated with NHL plasmid versus empty pcDΝA3 plasmid.

Since tumors were deprived of tumor blood vessels, and survival factors, we examined whether they underwent programmed death as measured by in situ labelling of fragmented DNA using the TUNEL method. A small number of apoptotic cells were detected in tumors injected with empty plasmid (Figure 4A), whereas tumor apoptosis was almost doubled following injection of NHL plasmid (Figure 4A, and refer to Apoptosis Index in Figure 4B). The apoptotic index (Al) for tumors injected with NHL was significantly

(P<0.001) different from that of tumors treated with empty pCDΝA3 vector.

Discussion

It has been demonstrated for the first time that intratumoral injection of VHL is able to cause complete rejection of small established EL-4 tumors that are not known to carry germline mutations in the NHL gene. NHL therapy caused reductions in the levels of HIF- lα and NEGF in tumors, with a consequent reduction in tumor angiogenesis, and increased tumor cell apoptosis. While tumors did not reappear for three weeks after complete rejection, we cannot discount the possibility that they had regressed to microscopic dormant nodules, rather than being completely eradicated, as occurs with anti-angiogenic therapy employing angiostatin (23) and endostatin (24). The complete and permanent regression of tumors in response to a single injection of NHL gene is similar to that achieved with antisense HIF-lα therapy (16), which is unusual for anti-angiogenic agents where transient suppression of tumor growth is the norm. For antisense HIF-lα therapy, the mechanism involved the ΝK cell-dependent rejection of tumors (16).

The fact that NHL therapy was very effective at inducing tumor cell apoptosis suggests NHL may have a more predominant role in regulating cell survival, in comparison to its role in regulating HIF-1 α expression. NHL appears to exhibit both pro-apoptotic and anti- apoptotic effects, depending on the cellular context. Reintroduction of NHL into NHL- negative RCC cells in vitro provides protection against the cytotoxic effects of serum withdrawal (25), glucose deprivation (26), and UN irradiation (27). It appears to protect renal cells from chemically-induced apoptosis and UN irradiation by inducing Bcl-2 and

Bcl-xL expression, and accumulation of cyclin-dependent kinase inhibitors p27 and p21 (28). Since an anti-apoptotic role is counterintuitive to NHL's role as a tumor suppressor, it was argued that loss of NHL may provide selective pressure for tumor cells to override apoptosis (28). The latter results were obtained in in vitro assays, and may not reflect the conditions required for the survival of tumor cells in vivo. Our results suggest that the pro- apoptotic effects of NHL in vivo outweigh its anti-apoptotic effects. It has previously been reported that EL-4 tumors already express the anti-apoptotic factors Bcl-2 (29), and survivin (30), and yet NHL therapy leads to enhanced EL-4 cell apoptosis in vivo. A number of potentially tumor suppressive, and pro-apoptotic properties of NHL have been reported. Thus, NHL interacts with fibronectin, and assists in the assembly of a fibronectin matrix, which can suppress cellular properties associated with malignancy (31). RCC cells engineered to express NHL differentiate and undergo growth arrest when grown to high density on collagen I, whereas NHL negative cells continue to proliferate (32), suggesting that NHL induces differentiation and growth arrest via the integration of cell to cell and cell to matrix signals. Similarly, reintroduction of the NHL gene into mutant RCC cells resulted in growth suppression in vitro, but only when the cells were grown as spheroid cultures, suggesting the effects of NHL are highly dependent on multicellular growth conditions that mimic the basic nature of solid tumors (33). NHL stabilizes actin organization, increases cell adhesion, and inhibits cell motility and invasiveness of tumor cells through focal adhesion formation, and by increasing the expression of tissue inhibitors of metalloproteinases (TIMPS), and decreasing the expression of matrix metalloproteinases 2 and 9 (34). Thus, engineered over-expression of VHL may increase the sensitivity of EL-4 tumors to inhibitory signals from the extracellular matrix.

There are various other mechanisms by which NHL may inhibit tumor growth. NHL interacts in vivo with heteronuclear ribonucleoprotein (hnRΝP) A2, an RΝA-binding protein that binds a cis-acting instability element in the Glut-1 3'-UTR and protects Glut-1 mRΝA from degradation (35). NHL downregulates the expression of hnRΝP A2, which leads to a decrease in Glut-1 mRΝA. NHL therapy would be expected to inhibit Glut-1 gene transcription, as well as destabilizing already synthesized Glut-1 mRΝA, leading to increased inhibition of tumor glycolysis, and decreased tumor metabolism. Reintroduction of the wild-type NHL gene product into RCC cells results in the accumulation of p27, and causes RCC cells to exit the cell cycle and enter GO/quiescence (36). Some tumors such as

RCC are dependent on insulin-like growth factor- 1 (IGF-1) for tumor growth and invasion. Reintroduction of NHL into RCC cells inhibits IGF-1 receptor signalling via an interaction with protein kinase Cδ, leading to an inhibition of tumor growth and invasion (37).

In both the present report, and a previous publication, we demonstrated that challenging mice with increasing numbers of parental EL-4 cells, causes increased reductions in the generation of anti-tumor CTL, indicating that EL-4 cells are immunosuppressive. One mechanism used by EL-4 cells to evade the immune response involves secretion of TGF-β (38). Thus, the tumorigenicity of EL-4 cells is suppressed by therapy with soluble type II TGF-β receptor (39). Although not tested here, the success of NHL therapy may be due in part to the fact that NHL represses TGF-β 1 mRΝA and protein levels by decreasing the half-life of TGF-β 1 mRΝA (40). Reintroduction of NHL into RCC cells was found to neutralize TGF-β activity, causing the regression of established RCC tumors without the development of drug resistance (40). TGF-β 1 has proangiogenic effects (41), as evidenced by the finding that targeted disruption of either the TGF-β 1 gene or its type II receptor results in defective placental vasculogenesis (42). It appears to synergize with NEGF and bFGF in mediating an angiogenic response (40). NHL also suppresses tumor cell invasion and angiogenesis by upregulating the expression of urokinase-type plasminogen activator mRNA and protein, and conversely downregulating the expression of plasminogen activator 1 mRNA and protein (43). It post-transcriptionally down-regulates the expression platelet derived growth factor (PDGF), and NEGF (44, 45).

Unlike conventional anti-angiogenic agents, NHL therapy appears to inhibit an array of pathways required for tumor growth, and survival. The fact that NHL therapy can cause complete tumor regression is unexpected, and suggests that NHL may, like antisense HIF- 1 α therapy, expose tumors to the innate immune system which senses danger signals from damaged cells.

While in the foregoing description there has been made reference to specific components or integers of the invention having known equivalents then such equivalents are herein incorporated as if individually set forth.

Although this invention has been described by way of example only and with reference to possible embodiments thereof it is to be understood that modifications or improvements may be made without departing from the scope or spirit of the invention as defined in the appended claims.

The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in any country.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprising" and the like, are^'to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of "including, but not limited to". References

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Claims

CLAIMS:

1. A method of treating tumors in an animal, the method comprising at least the over- expression of VHL in a tumor.

2. A method of treating small tumors in an animal by engineered over-expression of VHL in the tumor.

3. A method of inhibiting tumor angiogenesis in an animal, the method comprising at least the over-expression of VHL in a tumor.

4. A method of enhancing tumor cell apoptosis in an animal, the method comprising at least the over-expression of VHL in a tumor.

5. A method of any one of claims 1 to 4 wherein it includes the step of administering to the animal an agent adapted to effect over-expression of VHL in a tumor.

6. A method as claimed in claim 5 wherein the agent adapted to effect over- expression of VHL in a tumor is a vector adapted to express VHL.

7. A method as claimed in claim 6 wherein the vector is a nucleic acid vector.

8. A method as claimed in claim 6 wherein the vector is a viral vector comprising nucleic acid in a viral capsid.

9. A method as claimed in any one of claims 1 to 5 wherein the agent allows for over- expression of native VHL within the tumor.

10. A method as claimed in any one of claims 1 to 9 wherein the agents adapted to effect over-expression of VHL in a tumor are administered intratumorally.

11. A method as claimed in any one of claims 1 to 9 wherein, the agents are administered systemically.

12. A composition for use in tumor treatment in an animal, the composition comprising an effective amount of an agent adapted to over-express VHL in a tumor, together with one or more suitable carriers and/or excipients.

13. A composition for inhibiting tumor angiogenesis in an animal, the composition comprising an effective amount of an agent adapted to over-express VHL in a tumor, together with one or more suitable carriers and/or excipients.

14. A composition for enhancing tumor cell apoptosis in an animal, the composition comprising an effective amount of an agent adapted to over-express VHL in a tumor, together with one or more suitable carriers and/or excipients.

15. A method of treating tumors, enhancing tumor cell apoptosis, or inhibiting tumor angiogenesis in an animal, the method comprising the admimstration of an agent which mimics the function of VHL in a tumor.

16. A composition for use in tumor treatment, enhancing tumor cell apoptosis, or inhibiting tumor angiogenesis in animals, the composition comprising an effective amount of an agent adapted to mimic VHL in a tumor, together with one or more suitable carriers and/or excipients.

17. A method as claimed in any one of claims 1 to 11 and 15, wherein the tumor is a small tumor.

18. A method of assessing efficacy of over-expressed VHL in animal tumor treatment, the method comprising the steps of engineering over-expression of VHL in tumors of varying sizes in an animal followed by determining the effect this engineered over-expression has on tumor growth.

19. The use of an agent adapted to effect over-expression of VHL in a tumor in the manufacture of a medicament for use in tumor treatment, enhancing tumor cell apoptosis, or inhibiting tumor angiogenesis in animals.

20. The use of an agent which mimics the function of VHL in a tumor in the manufacture of a medicament for use in tumor treatment, enhancing tumor cell apoptosis, or inhibiting tumor angiogenesis in animals.

21. A method of treating tumors, enhancing tumor cell apoptosis or inhibiting tumor angiogenesis in an animal, the method comprising at least the step of administering to the animal an effective amount of VHL.