ES2353070T3 - VIRAL / LIGANDO SYSTEMIC GENE SUPPLY SYSTEM AND GENE THERAPY. - Google Patents

Abstract

Virus-ligand complex that is directed to a cell for the delivery of a virus to a target cell inside a host animal, wherein said virus-ligand complex consists of a ligand and a virus, and in which the ligand is non-covalently bound to the virus, and is transferrin, a transferrin receptor antibody, a fragment of a transferrin receptor antibody, EGF or VEGF.

Description

Systemic Gene Supply System viral / ligand and gene therapy.

Background of the invention 1. Technical field

The present invention relates to improvements in gene transfer and in gene therapy technology. More specifically, the invention provides compositions and methods for the in vivo and in vitro viral delivery of nucleic acids in man and other animals to a specific organ, tissue, or tumor. The use of this invention to deliver a therapeutic gene, for example, wtp53, can lead to increased sensitivity to conventional radiation and chemotherapy.

2. Description of the prior art background

WO98 / 40508 describes vectors adenovirals with modified tropism.

Miller and Vile (1998) unveil vectors targeted for gene therapy.

Snitkovsky and Young unveil vectors cell-specific viral mediated by a protein soluble retroviral (fusion) receptor-ligand.

Gene supply and gene therapy using viral vectors have been the subject of considerable research A lasting goal in therapy gene for cancer is a systemic delivery system that selectively target tumor cells, including metastasis. Nucleic acids can enter cells by viral vectors to produce a therapeutic effect desired on these cells. For example, a gene can be introduced to replace a defective gene that interferes with the function cellular, for example, p53. Nucleic acids can also enter the cells to produce a therapeutic effect desired in the host animal, for example, for vaccination, immunotherapy, or antisense expression.

Viral vectors have been developed to take advantage of the cellular input mechanisms that viruses use to transfer their nucleic acids to host cells. Recombinant retroviral and adenoviral vectors have been constructed using this strategy, to obtain in vitro and in vivo transfer. Approximately 80% of the gene therapy protocols that have been approved for clinical trials use viral vectors (Wivel and Wilson, 1998). Adenoviral vectors offer advantages for some forms of gene therapy, because they can penetrate non-dividing cells and carry out a relatively bulky (> 8 kb) payload of foreign DNA (Berkner, 1988). In addition, adenoviral particles can be purified and produced in titres greater than 10 11 PFU / ml (Wivel and Wilson, 1998). A disadvantage of these systems, however, is the limited cellular tropism of the viruses and the significant problem of directing the viral particles, (which) has yet to be resolved for any of the therapeutic viruses commonly used in clinical trials. for cancer In accordance with this, procedures have been developed to alter cell tropism and continue to be investigated.

It has been described (Roux et al ., 1989), a system that changes the tropism of retroviruses through a bifunctional conjugate. The bifunctional conjugate contains an antibody directed against the viral envelope and, on the other hand, an antibody directed to a specific cell membrane marker for the target cell.

Goud et al . (1988) described the bifunctional conjugates consisting of two monoclonal antibodies (MAbs). The MAbs were directed against the gp70 protein of the Moloney retrovirus envelope and the human transferrin receptor. These conjugates allowed the retrovirus to penetrate otherwise non-tolerant target cells.

WO 92/06180 (Wu et al ., 1992), discloses a method for changing the tropism of a virus, providing the surface of the virus with a molecule that binds to a surface receptor of the target cell, which results in to a virus with specificity for cells (which possess) the cell surface receptor. In exposure, a retrovirus or a hepatitis B virus is chemically modified with carbohydrate molecules that bind to the asialoglycoprotein receptor. Wu et al . they disclose only in vitro procedures for the introduction of foreign genes into cells .

Adenoviral vectors offer advantages for some forms of gene therapy, because they can penetrate the cells that do not divide and can carry a DNA sequence strange about 8 kb. Adenoviral particles can purify and produce degrees higher than 10 11 PFU / ml.

An impediment to the use of recombinant adenoviruses is their limited ability to impact specific cell types. Several studies have reported gene transfer using non-recombinant adenoviruses with DNA complexes, through receptor-mediated endocytosis, providing adenovirus the ability to release the content of endosomes (Cotten et al ., 1992; Wagner et al ., 1992). These procedures use transferrin-polylysine / DNA complexes to internalize adenoviruses bound or not. Wagner et al . (1992) modified an adenoviral vector by conjugation to the polylysine and causing it to complex with transferrin-polylysine / DNA conjugates, which led to the production of ternary transferrin-polylysine / adenovirus-polylysine / DNA complexes. A similar approach to target cells is the binding of transferrin (Tf) to adenoviral particles, in order to take advantage of the fact that the transferrinic receptor (TfR) is elevated in many tumor types (Miyamoto et al., 1994; Baselga and Mendelsohn, 1994). Schwarzenberger et al . (1997) describes molecular conjugation vectors (MCV). MCVs are constructed by condensing with polylysine (PL) a plasmid containing the gene of interest, and binding this PL to an adenovirus that is not competent for replication (endosomolytic agent), and binding PL to streptoavidin to impact biotinylated ligands. However, it has been reported (Cotten et al ., 1992; Wagner et al ., 1992; Schwarzenberger et al ., 1997) that the usual procedures for covalent binding of Tf to adenovirus, or the generation of Tf-polylysine-adenovirus conjugates , often result in a decrease in infectivity, possibly due to the severe conditions that are required to produce the modified Tf viruses.

Another approach links the cross-linking of the Fab fragment of an antifibrous neutralizing antibody or a "hump" monoclonal antibody to a ligand, such as folate or FGF2 (Rogers et al ., 1997; Douglas et al ., 1996). Adenoviral vectors that complex with the chimeric ligand-Fab have shown some promise in tumor localization and show reduced hepatic toxicity in vivo , compared to the original adenoviruses.

Curiel et al ., (US Patent No. 5,521,291 and No. 5,547,932) disclose multiple adenoviral-polycationic conjugates for internalizing nucleic acids in eukaryotic cells. Some of these conjugates use transferrin that binds to a substance that has an affinity for nucleic acid as an internalizing factor.

Cotten et al . (US Patent No. 5,693,509) discloses adenovirus with the ability (which has already been reported) to efficiently penetrate cells in which they cannot normally do so, while retaining their ability to gene expression and / or their endosomolytic properties Transferrin is covalently bound to adenoviral particles, so that they can undergo receptor-mediated endocytosis. The process oxidizes the transferrin to a form that contains aldehyde groups in the carbohydrate part and binds the oxidized transferrin to the adenovirus, under reduction conditions. It is also known that it cannot be predicted if the infectivity is maintained after the modification.

Low et al . (US Patents 5,108,921; No. 5,416,016; and No. 5,635,382) disclose methods for enhancing transport across the membranes of exogenous molecules, using ligands such as folate, biotin, thiamine, and the like. The methods can also use anti-idiotypic antibodies or other molecules that can bind to ligand receptors. Other ligands that are disclosed include niacin, pantothenic acid, riboflavin, pyridoxal, and ascorbic acid.

Xu et al . (1997) and Xu et al . (1999) found that the addition of Tf to a cationic liposome could significantly increase the ability of liposomes to deliver exogenous genes, including the normal p53 gene, both in vitro and in vivo . Very significantly, they achieved a highly selective impact with respect to a wide variety of human tumor cells that developed as xenografts in nude mice.

Publications and other materials that are used herein to illustrate the background of the invention or provide additional details regarding the practice, incorporated as a reference, and for convenience, grouped respectively in the attached list of References.

Summary of the invention

The present invention provides improvements for administration of viral vectors for, for example, the gene delivery, virally mediated, to target cells.

In one aspect, the invention provides a virus-ligand complex that targets a cell, for the supply of a virus to a target cell inside a host animal, where said complex virus-ligand is formed by a ligand and a virus, said ligand being non-covalently bound to the virus, (the which ligand) is transferrin, a receptor antibody of transferrin, a fragment of a receptor antibody from transferrin, EGF or VEGF.

In addition, the present invention provides a vector for systemic delivery of a virus to a target cell, said vector comprising a ligand directed to the cells, which does not is covalently linked to said virus, said vector being able to be prepared by mixing a composition formed by a directed ligand to the cells with a virus, in an aqueous medium, where said ligand does not covalently bind to said virus, and where said ligand is formed by transferrin, a transferrin receptor antibody, a fragment of a transferrin receptor antibody, EGF or VEGF.

The present invention still further provides a procedure for the preparation of a vector for the systemic supply of a virus to a target cell, comprising said vector a ligand that targets a cell bound so not covalent to said virus, which comprises mixing a composition constituted by said ligand that targets a cell with said virus in an aqueous medium, said ligand binding in a non- covalent to said virus, in which said ligand is constituted by transferrin, a transferrin receptor antibody, a fragment of a transferrin receptor antibody, EGF or VEGF

The procedure that is disclosed for the Mix production prevents particle inactivation virals that might otherwise be caused by severe chemical processing The mixture is prepared in a simple way and is appropriate for systemic administration (eg parenteral) To the human patient.

In another aspect, the invention provides a virus-ligand complex directed to cells, or a vector, as described above to use it as a medicine.

In addition, the present invention provides a vector for use in delivering a virus to a cell target inside an animal, in which the vector comprises a virus-ligand complex directed to cells, wherein said virus-ligand complex comprises a ligand and a virus, and in which the ligand is not covalently bound to the virus, being (this ligand) transferrin, an antibody transferrin receptor, a fragment of a receptor antibody transferrin, EGF or VEGF.

These are suitable for systemically administered in vivo to man or another animal, so as to carry out the targeted supply of the viral particle content.

The present invention further provides the use of a virus for the preparation of a therapeutic agent  comprising a vector comprising a complex virus-directed ligand to cells, for the supply of said virus to a target cell inside a animal, in which said virus-ligand complex is formed by a ligand and a virus, where the ligand is not covalently bound to the virus, being (this ligand) transferrin, a transferrin receptor antibody, a fragment of a Transferrin receptor antibody, EGF or VGEF.

In a preferred aspect, said vector encodes wild type p53 and said therapeutic agent is to be administered to an animal that receives radiation treatment or chemotherapy in addition of said therapeutic agent. In this regard, the use of the invention to deliver a therapeutic gene, wild type p53, will lead to radiation sensitization and agents Chemotherapeutic

Brief description of the figures

Figure 1. Expression of the reporter gene of the β-galactosidase in cells JSQ-3 after infection with the targeted vector by Tf that carries the lacZ gene. 5x104 cells were seeded JSQ-3 / well in a plate with 24 wells. 24 hours then the cells were washed once with EMEM without serum, adding 0.3 ml of EMEM without serum or antibiotics to each well. The As5LacZ or Tf-Ad5LacZ complexes with different proportions of transferrin / virus in 200 µL of EMEM, it added to duplicate wells. The proportions of (between) 5 x 10 2 to 5 x 10 5 Tf / virion molecules. The virus / cell ratios were 500 and 1,000 particles viral / cell (pt / cell). After 4 hours of incubation at 37 ° C, 5% CO2, by rotating mixing the test tube a once every 2 minutes, 0.5 ml of EMEM was added to the wells with 20% serum. After 2 days in culture, the cells were washed once with PBS, and lysed in lX reporter lysis buffer (Promise) Cell lysates were treated with 100 µL of 150 \ muM O-nitrophenyl-? -Galactopyranoside in 20 mM Tris (pH 7.5) containing 1 mM MgCl2, and 450 µM β-mercaptoethanol at 37 ° C for 30 minutes. The reaction was stopped by adding 150 µl / well of 1M Na 2 CO 3, the absorbance being measured at 405 nm. The purified β-galactosidase was used to make a standard curve. The results were expressed as milliUnities (mU) of equivalents of β-galactosidase per mg protein totals

Figures 2A-F. Analysis histochemical systemic delivery (adenoviral directed by Tf) of the β-galactosidase reporter gene in a mouse xenograft model. Nude nude mice DU145 tumor carriers were injected intravenously once with Tf-AdLacZ. Three days after the injection, the animals were sacrificed, and the tumor and normal tissues were They extracted and stained with X-gal. Figure 2A shows the tumor from an animal treated systemically with Tf-Adp53 with a ratio of 1.5 x 10 5 Tf / virion molecules. Figure 2B shows the liver that corresponds to figure 2A. Figure 2C shows tumors from of an animal treated systemically with Tf-Adp53 with a ratio of 2.9 x 10 5 Tf / virion molecules. 2D figure shows the liver corresponding to figure 2C. Figure 2E shows tumors from an animal treated systemically with Tf-Adp53 with a ratio of 5.8 x 10 5 Tf / virion molecules. Figure 2F shows the corresponding liver to figure 2E. Bar = 50 µm.

Figure 3. Expression of exogenous wtp53 in xenografted DU145 tumors after intravenous injection of Tf-Adp53. Nude nude mice bearing DU145 xenograft tumors were injected intravenously with Tf-Adp53 or non-directed Adp53. 48 hours later, the animals were sacrificed, normal and tumor tissues were removed and proteins were isolated to perform a Western blot analysis. Proteins isolated from the tumor and organs of an untreated mouse, as well as parental DU145 cells, were included as controls. 100 µg of the total proteins of each of these samples were loaded on the rails. 2.5 µg of total proteins of DU145 cells infected in vitro with Adp53 were also included. The p53 protein bands were detected using the Ab-2 antip53 monoclonal antibody and the Western blot ECL kit. Band 1 = human exogenous wtp53; Band 2 = p53 DU145 exogenous human; Band 3 = endogenous mouse p53.

Figure 4. Effect of the combination of adenoviral p53 targeting systemically delivered tumors and radiation treatment in xenografted JSQ-3 tumors in vivo . Tf-Adp53 was produced in a ratio of 1 x 10 5 Tf / virion molecules. In the tail vein of nude nude mice bearing JSQ-3 100-200 mm 3 xenograft tumors, 1 x 10 10 viral particles / mouse / injection (equivalent to 3 x 10 ^) were injected 8} pfu) of Tf-Adp53, or of non-directed Adp53. At the start of the day after the first intravenous injection, 30 Gy of ionizing radiation were administered to the animals at the tumor site, in daily divided doses of 2 Gy. 8 months after discontinuation of treatment, no further growth was observed in animals that received the combination treatment. Since the error was too small to be displayed, no "traces" of errors are found in the Tf-Adp53 (+) Radiation group. The "stroke" represents the duration of treatment (approximately 3 weeks). All animal experiments were carried out in accordance with the Georgetown University Institutional Guidelines for the care and use of laboratory animals.

Figures 5A-H. Chemosensitization of lung metastases of B16 mice to cisplatin (CDDP) by p53-adenoviral directed to the tumor, administered systemically. Metastases are induced in normal synergic C57 / Bl / 6 mice by intravenous injection of 1 x 10 5 cells. Four days later, The treatment was started. Tf-Adp53 and Tf-AdLacZ were produced in the proportion of 1.5 x 10 5 Tf / virion molecules. They were administered intravenously 1 x 10 10 viral particles / mouse / injection (equivalent to 3 x 10 8 pfu) of Adp53, Tf-Adp53 or Tf-AdLacZ, 3 days / week, up to a total of 12-13 doses. CDDP was injected intraperitoneally at a dose of 3-5 mg / kg every 2-4 days with a total of 8-13 doses. The Lungs of animals after a course of treatment. Lungs of untreated animals (Figures 5A and 5E); Lungs of animals treated with CDDP alone (Figure 5B); Animal lungs treated with non-directed Ad-p53 + CDDP (Figure 5C); Lungs of animals treated with Tf-LacZ plus CDDP (figure 5F); Lungs of animals treated with Tf-Adp53 alone (Figure 5G); Animal lungs treated with Tf-Adp53 plus CDDP (figures 5D and 5H).

Figure 6. Effect of the p53-adenoviral combination targeting the tumor, systemically delivered, and chemotherapy of the MDA-MB-435 xenograft tumors, in vivo .

Figure 7. Expression of the β-galactosidase in injected DU145 cells intratumorally, they were injected with adenovirus with a LacZ, not directed, or with adenovirus with LacZ, transferrin-directed.

Detailed description of the invention

The normal development of mice that are missing the wtp53 and the G1 blocking observations after the irradiation in cells expressing p53, suggests that wtp53 exerts regulatory functions, rather than during development and cell proliferation, after alteration of DNA or Stress from it. Since it seems that many anticancer therapies Conventional (chemotherapies and radiation) induce DNA damage and seem to exert their action inducing apoptosis, the alterations in the p53 pathway could lead convincingly to the failure of therapeutic regimens.

The lack of function of the wtp53 has been associated also with an increase in radiation resistance. It has been also discovered that the presence of mtp53 and the absence consequently of a block G1 correlate with an increase in radiation resistance in some human tumors and progenies cell phones. These include human tumor cell lines representative of head and neck cancer, lymphoma, bladder, breast, Thyroid, ovary and brain.

Based on these considerations, gene therapy to restore the function of wtp53 in tumor cells, could re-establish the control ("check") points of the cell cycle that depend on p53 and the apoptotic pathway, thereby leading to the reversal of the chemo / radiation resistant phenotypes. According to this model, chemosensitivity, together with apoptosis, was restored by expressing wtp53 in non-small cell lung carcinomas of mouse xenografts carrying mtp3. The chemoplansitivity to cisplatin of xenografts involving the p53 null pulmonary tumor cell H1299 and T98G glioblastoma cells has been demonstrated, as well as that of WiDr colon cancer xenografts. It was also shown in breast SK-Br-3 tumor cells, by means of adenoviral transduction of wtp53, the increase in cell death due to doxorubicin or mitomycin C. However, some conflicting reports indicated that the relationship between p53 expression and chemoresistance may have a specific cellular or tissue component. Transfection of wtp53 by an adenoviral vector has also been shown to sensitize ovarian and colorectal tumor cells to radiation. Likewise, it has been reported that the supply of adenovirus-mediated wtp53 restored functional apoptosis in a radiation-resistant head and neck tumor progeny (SCCHN) squamous cell carcinoma, leading to radiosensitization of these cells in vitro. . More significantly, the combination of adeno-wtp53 injected intratumorally and radiation, led to a complete long-term tumor regression of established SCCHN xenograft tumors.

The usual invention starts from the conventional use of intratumoral injection of non-directed viral vectors or even from the systemic supply of non-directed vectors, for the delivery of therapeutic molecules for gene therapy, as disclosed, for example. , by Roth et al . (US Patent No. 5,747,469).

The data presented herein demonstrate the superior ability of such complexes to specifically impact and sensitize tumor cells (due to the expression of the wtp53 gene), both primarily and in metastatic tumors, to radiation and / or chemotherapy both in vitro as in vivo .

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The present invention relates to the need of delivering therapeutic molecules systemically with a high degree of specificity of cellular impact and high efficiency. When is administered systemically, this supply system can achieve, and specifically impact both the primary disease as in metastatic, when the target cells are cells human cancerous As a result of the version delivery normal wild-type tumor suppressor gene p53 using this system, the inventors have shown that tumors are Sensitize to radiation therapy and / or chemotherapy.

The high transfection efficiency of this system it gives rise to such a high degree of awareness that not only causes cancer growth inhibition, but the preexisting tumors and metastases are completely eliminated for a long period of time.

The specific embodiments of the description provide pharmaceutically acceptable compositions comprising a mixture of transferrin ligand and particles vector virals capable of delivering a nucleic acid to impact on host cells. Simply, the mixture of viral vectors (for example, retroviral vectors or adenovirals) with a ligand directed to cells, in a vehicle appropriate such as sterile water for injection, increases the efficiency of transfection in a way superior to that obtained With only the viral vector. A simple mixture of the viral vector and Transferrin, for example, increases cell transfection, by example, of human cancer cells, which express the receptor transferrinic

The use of transferrin is especially advantageous in relation to gene transfer in, or in the gene therapy for a wide variety of human cancers. A wide variety of human cancer cells contains receptors for transferrin. The presence of transferrin in complexes and vectors of the present invention, allows vectors virals impact (target) efficiently and specifically in the cancer cells

Other ligands, such as proteins, peptides, hormones, antibodies and fragments thereof, will be useful for specifically direct viral vectors to cells that contain receptors for said ligands, or that may internalize these by receptor-mediated endocytosis. The ligand, for example, can be an original protein or recombinant that works to enhance viral vector binding to the target cell. Examples of ligands include insulin, toxins, EGF, VEGF, FGF, IGF, heregulin, other proteins bacterial or viral, estrogen and progesterone.

Ligands that impact cellularly according to present invention are transferrin, a receptor antibody of this, a fragment of a transferrin receptor antibody, EGF or VEGF

Although the description conceives the use of a cell-directed ligand that is naturally found in a type of virus when this ligand is mixed with a second type of virus, the description does not conceive the use of a ligand cell-directed in its natural association with the virus that encoded The description conceives the mixture of a ligand directed to cells encoded by a virus associated with the type of virus which encodes said ligand, when the ligand is in a amount greater than what is normally associated with the virus that is found naturally.

The procedure by which a complex between the ligand and the viral particle is such that a large number of ligand molecules line the surface of it and increase its stability, because it travels through the current blood In addition, the large number of ligand molecules on the surface can also serve to decrease immunogenicity of the virus, blocking viral antigens.

The invention includes the use of viruses expression recombinants. Viral vectors, such as adenovirus recombinants, AAV vectors (US Patent No. 5,139,941), vectors retrovirals, herpes simplex virus (US Patent No. 5,288,641), cytomegalovirus (CMV), vaccine virus, avian smallpox virus (FPV), Canarian Smallpox Virus (CPV) (US Patent No. 5,833,975; No. 5,762,938; and No. 5,378,457), Sindbis virus, virus chimeric or hybrid and the like, can be used in accordance with the invention.

The invention also provides procedures. to prepare a viral vector-transferrin mixture which advantageously avoids the complicated processing steps and the nasty chemicals that have been described, related to previously used procedures, which they used MAbs, linkers, polylysine, etc., to join the Transferrin to viral vectors.

According to the invention, virus-ligand mixtures can be prepared in any carrier or vehicle (typically an aqueous carrier), so as to provide a composition that is pharmaceutically appropriate for in vitro or in vivo administration . Typically, the composition will be buffered at an appropriate pH, and may contain appropriate auxiliary components, such as osmolar adjusting agents, antibiotics, etc.

The amounts of viral particles used in the mixtures and which are administered terminally to the host animal (or administered to the cells in vitro will be determined) by those skilled in the art, based on well-known principles of gene transfer and therapy described in the scientific literature. The mixtures of the invention are administered by methods analogous to those described above for the administration of viral vectors, so that gene transfer or gene therapy is carried out in vitro or in vivo . The invention improves with respect to existing technology, providing compositions and methods for the systemic administration of viral vectors. Parenteral administration (especially intravenous or intraarterial administration) of the mixtures is preferred. It is envisioned that, in some cases, substantially smaller doses (for example, 30 times) of the viral particles may be administered, due to the improvement in efficiency caused by the invention. Alternatively, in other cases, known doses may be used, which lead to an increase in genetic transfer. Likewise, concentrations of viral particles, ligands and auxiliary agents will be appropriately selected within the compositions of the invention.

The specific embodiments of the invention provide the use of the compositions of the invention, together with radiation treatment and / or chemotherapy.

The invention is not limited to any vector particular viral, or to any routes or modes of administration of the compositions of the vector mixture viral-ligand. The total desired dose may determined experimentally, and can be provided uniquely or multiple a patient who needs gene therapy.

The data presented in the examples indicate that Tf-adenovirus complexes can produce remarkably high levels of gene expression in tumors, (compared) with those seen with adenoviral vectors not directed. The gene delivery procedure described in This Report is based on the procedure relatively simple to get the viruses marked with Tf. The union is not covalent and does not imply chemical reactions that could produce unwanted products, and perhaps toxic, avoiding (in this way) the severe chemical conjugation and the complicated stages of processing that have been described, related to previously used procedures that used MAbs, linkers, polylysine, etc., to bind Tf to viral vectors. These chemical modifications of viruses decrease inevitably the infectivity of the virus and can produce aggregates possibly too large to penetrate the tumor capillaries Although clinical trials are conducted with viral vectors that are injected intratumorally for therapy gene including oncolytic viruses, are not carried out usually clinical studies with targeted viruses (which do not present) covalent modifications.

Although evidence is appearing that the single gene therapy with the p53 agent is not enough to completely eliminate long-term tumors, this combination described herein of adenovirus Tf-directed and radiation / chemotherapy conventional has been able to achieve, not only the inhibition of growth, but tumor regression, demonstrating an effect synergistic

The in vivo studies described herein demonstrate that the combination of Tf-Adp53 gene systemic therapy and conventional radiotherapy and / or chemotherapy is remarkably more effective than any single treatment. In the clinical setting, radiation doses of 65 to 75 Gy for bulky tumors and 45 to 50 Gy for microscopic tumors are commonly used in the treatment of head and neck cancer. From what is known, (side effects that are associated with high doses of radiation or chemotherapy), tumor sensitization will be of immense clinical benefit, so as to allow lower effective doses than in conventional treatment. In addition, in the case of radiation, the systemic restoration of the function of wtp53, which leads to a decrease in the dose of treatment with said radiation, whose decrease has been found effective, will allow further therapeutic intervention so that tumors do not reappear .

The sensitization of tumors to chemotherapy and radiation will lead to an increase in the efficacy of the usual treatment modalities. In addition, for this specific combinatorial tumor treatment, there is also the potential to decrease the necessary dose of both types of conventional modalities (of anticancer treatment), thus decreasing the severe side effects that are often associated with such treatments. In the in vivo studies described in the Examples, systemic administration of Tf-Adp53, combined with radiation, resulted in total and long-term tumor regression when an amount as small as 3 x 10 8 was used. pfu of the virus that impacts the tumor. This dose is approximately equivalent to that used by Kataoka et al . (1998) using Adp53 and 2-Me. In this study, a partial inhibition of tumor growth was observed (two thirds in the lung colonies count).

Although this system may well eliminate the need for intratumoral injection of gene therapy vectors, in the case of very aggressive cancers it may also be beneficial to use both systemic and intratumoral treatments. This system can also be adapted to assist in the supply of other viral cancer treatments. For example, the usual Onyx oncolytic virus assays do not support a systemic supply. ONYX-015 is a genetically modified adenovirus that efficiently replicates in and eliminates tumor cells (which are) deficient in the tumor suppressor activity of wtp53 (cells "deficient in p53") and does not do so in normal cells. The specific modification of the virus prevents it from replicating efficiently in normal cells. Clinical studies with ONYX-015 are usually carried out for head and neck cancer, pancreatic cancer and ovarian carcinoma (Heise et al , 1997; Hall et al ., 1998; Linke, 1998; Kirn et al ., 1998) . Our ability to direct viruses to tumors can significantly improve the effectiveness of other cancer treatments that rely on viruses, such as ONYX-015.

Very significantly, the administration systemic means that both the primary tumor and the metastases distant can be achieved with therapeutic genes. This shows a absolute contrast with what can be obtained with the injection intratumoral The procedure described herein memory can adapt to any recombinant viruses existing ones that are targeted (to target cells). It is also independent of the gene that will be supplied. Also this system could, as mentioned above, be used also with oncolytic viruses. The use of Tf for directing (viruses to target cells) is especially attractive in relation to p53 gene therapy for human cancers, because a broad spectrum of cancers express high levels of TfR and in more than 50% of (these) cancers, the p53 gene. Therefore, these findings demonstrate that the potential clinical of this adenoviral delivery system Tf-directed, constitutes a more effective and Efficient gene therapy for cancer, a way that will help fulfill the initial promise of gene therapy in war against this disease

In the following examples, the Illustrative embodiments of the present invention.

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Example 1 Transferrin enhances transduction efficiency adenoviral A. Preparation of the mixture adenovirus-transferrin

The holo-transferrin (Tf, saturated iron, Sigma), was dissolved in sterile water (in a proportion) of 5 mg / ml. In this study, the insufficient replication adenovirus, serotype 5, called Ad5LacZ (Ad5CMV ntbeta-gal, Gene Transfer Vector Core, University of Iowa) was used, which contained the E. coli LacZ gene under the control of the CMV promoter, at a concentration 1.1 x 10 12 particles (pt) / ml (containing 5.5 x 10 9 plaque forming units, pfu / ml) in PBS plus 3% sucrose. Tf was first diluted to 0.5 mg / ml in 10 mM HEPES buffer, pH 7.4, then Tf was added to 50 µl of a HEPES buffer in a dilution multiplied by 10, in series. Ad5LacZ was then added to the test tubes, so that the Tf / virus ratios were comprised between 1 x 10 2 and 1 x 10 6 Tf / virion molecules. The test tubes were then incubated at room temperature for 10-15 minutes, rotating the test tubes once every two minutes, and then adding 150 µl of EMEM without serum to each tube.

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B. In vitro transduction using adenovirus / Tf mixture

We use an insufficient serotype 5 replication adenovirus called AdLacZ (which contains the E. coli LacZ gene under the control of the CMV promoter) and the Tf-modified form of this virus (Tf-AdLacZ), to optimize the capacity of Tf- AdLacZ to deliver the reporter gene, cultures of the JSQ-3 cell line (Weichselbaum et al , 1988), derived from a human squamous cell carcinoma of the head and neck (SCCHN), were infected (Bischoff et al ., 1996) with AdLacZ or with Tf-AdLacZ produced using different proportions of Tf with respect to the virus and viral particles, with respect to the cells.

5 x 10 4 cells were seeded JSQ-3 / well in a plate with 24 wells. 24 hours then, the cells were washed once with EMEM without serum, adding 0.3 ml of EMEM without serum or antibiotics to each well. Ad5LacZ or Tf-Ad5LacZ complexes were added to duplicate wells, with different proportions of transferrin / virus, in 200 µl of EMEM. The proportion virus / cells was of the order of between 20 and 2,000 particles viral / cell (pt / cell). After 4 hours incubation at 37 ° C, 5% CO2, with occasional rotation of the test tubes once every 2 minutes, 0.5 ml of EMEM was added to the wells with 20% serum. After 2 days in culture, the cells are washed once with PBS, lysing in 1X reporter lysis buffer (Promise) Cell lysates were treated with 100 µL of 150 \ muM O-nitrophenyl-? -Galactopyranoside in 20 mM Tris (pH7.5) containing 1 mM MgCl2, and 450 µM β-mercaptoethanol at 37 ° C for 30 minutes. The reaction was stopped by adding 150 µl / well of 1 M Na 2 CO 3. The absorbance was measured at 405 nm. To build a standard curve was used? -galactosidase (Boehringer) purified. The results were expressed as milliUnities (mU) of equivalents of β-galactosidase per mg of proteins totals

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C. Histochemical staining

For histochemical studies of Tf-Ad5LacZ transduction, confluent cells were transfected in 60% in 24-well plates for 5 hours with transfecting solutions, as described above. After another 2 days in culture, the cells were fixed and stained with X-gal (Xu et al ., 1997). Transfection efficiency was calculated as the percentage of blue-stained cells.

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D. Results and exposure

In certain proportions of Tf / virus or virus / cells, expression with Tf-AdLacZ was order of 3 to 4 times higher than the one that was appreciated with Unaddressed AdLacZ (see Figure 1). With a viral dose of 500 pt / cell or 2.5 MOI (multiplicity of infection, or pfu / cell), 10 mU / mg product protein β-galactosidase of the reporter gene was expressed using Ad5LacZ only. The transduction with the mixture transferrin-virus Tf-Ad5LacZ (500 Tf / pt molecules) produced 25 mU / mg of expression protein from the reporter gene Tf-Ad5LacZ (5,000 molecules of Tf / pt) produced 30 mU / mg of expression, and Tf-Ad5LacZ (50,000 Tf / pt molecules) produced 38.8 mU / mg of expression, which represents 2.5, 3 and 3.8 times, respectively, of more gene transduction than that achieved with only Ad5LacZ. With a dose of 1000 pt / cell or 5 MOI, Tf-Ad5LacZ (500 Tf / pt molecules) produced 2.4 times more expression of the reporter gene and Tf-Ad5LacZ (5,000 Tf / pt molecules) produced 3.3 times more expression gene that only Ad5LacZ. Tf-Ad5LacZ (50,000 Tf / pt) molecules resulted in 2.6 times more expression gene, it seems that saturation was reached. Therefore, the optimal ratio of the Tf-Ad5LacZ complex seemed be around 500-50000 molecules of Tf / pt, preferably about 5000 molecules of Tf / pt. It is believed that the large number of transferrin molecules that are used to coating the surface of viral particles increased the stability, because it moved through the bloodstream. The large number of transferrin molecules on the surface of the virion, can also serve to reduce immunogenicity of the virus, blocking the exposure of its antigens.

Histochemical staining showed that Ad5LacZ alone resulted in an efficiency in transduction of 20-30%, while the adenovirus forming complex with transferrin, Tf-Ad5LacZ, (5000 molecules of Tf / pt), produced an efficiency of 70-90%.

The previous results showed that adenoviral-transferrin mixtures may enhance substantially adenoviral gene transduction.

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Example 2 Systemic adenoviral gene supply directed by the transferrin in a JSQ-3 xenograft model in nude mice A. Preparation of the complex adenovirus-transferrin

The transferrina-Ad5LacZ complex it was prepared by means similar to those used for preparation described in Example 1. 1 x 10 9 -1 x 10 10 pt Ad5LacZ (1 x 10 12 pt / ml in PBS, plus 3% sucrose) mixed with different amounts of TF (4 to 5 mg / ml in water) and proportions of the order from 1 µg to 1 mg Tf / 1 x 10 10 pt, or 7.5 x 10 2 -7.5 x 10 5 Tf / virion molecules. The mixtures were incubated at room temperature for 5-10 minutes (with rotation of test tubes once every two minutes), to allow the complex to form TfAd5LacZ. PBS (pH 7.4) was added to each tube to dilute up to 1 x 10 9 -1 x 10 10 pt / 0.2-0.3 ml / injection in the mouse.

Two types of human tumors were established as xenografts in nude mice by subcutaneous injection of the SCCHN cell line used in previous culture experiments (JSQ-3) or of the human prostate cancer cell line DU145 (Isaacs et al , 1991; Asgari et al , 1997). The tumor model of nude mice was established by subcutaneous injection of JSQ-3 cells or DU145 cells in the flank of nude female mice 4-6 weeks old (Xu et al , 1997). The tumors were allowed to grow to a size of 1-2 cm 3. 1 x 10 9 - 1 x 10 10 pt Ad5LacZ complexing with different amounts of Tf at 200-300 µl, each mouse was injected through the tail vein with a 1 cc syringe and a 30G needle In the control group, Ad5LacZ was injected. Three days after injection, the tumors were removed, as well as the organs of the mice, cut into 1 mm sections, washed once with PBS, and fixed with 2% formaldehyde / 0.2% glutaraldehyde for 4 hours at room temperature. The fixed tumor sections were washed 4 times, each for 1 hour, and stained with X-Gal solution plus 0.1% NP-40 (pH 8.5) at 37 ° C at night. Tumor sections subjected to staining were submerged and sectioned using normal histological procedures, undergoing contrast staining with nuclear red. Four sections per tumor were examined to evaluate the expression of the β-galactosidase gene, indicated by the blue-stained cells.

For a quantitative trial of β-galactosidase, another section was used tumor. The tissues were homogenized and lysed in a buffer of 1X reporter lysis (Promega). The lysates were added to a plate with 96 wells and a quantitative test of β-galactosidase as described in the Example 1.

In some experiments, the quantitative trial of the β-galactosidase was carried out using a Luminescent Detection Equipment II β-galactosidase (Clontech).

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B. Results and Exposure

To test the systemic directed viral gene delivery, viral vectors were injected intravenously into well established solid tumor models. Two solid tumor xenograft models were used to test the targeted viral gene delivery system. 1 x 10 10 pt Ad5LacZ alone or forming complexes with different amounts of Tf, were injected intravenously into nude mice bearing human tumor xenografts of 1-2 cm 3 size of JSQ-3 and DU145 cells. Three days later, the tumors in which Tf-Ad5LacZ had been injected showed an increase in blue-stained X-Gal cells, when compared to Ad5LacZ (which had been injected) alone (<1%). Efficiency increased as a result of an increase in Tf / pt ratios, from 5% to> (more than) 35% (0.01 mg-0.6 mg / 10 10 pt). Efficiency began to decrease with Tf / pt> ratios (of more than) 0.6-1 mg / 10 10 pt (4.5 to 6 x 10 5 Tf / virion molecules approximately). This is shown in Figures 2A, 2C and 2E, in which the percentage of cells expressing β-galactosidase (indicated by X-gal staining) actually decreases, since the ratio of TF / virion molecules) increases from 2.9 x 10 5 Tf / virion up to 5.8 x 10 5 Tf / virion (Figures 2C and 2E). In addition, in the ratio 1.5 x 10 5 Tf / virion, expression of β-galactosidase was not evident in the liver (Figure 2B), or in other organs including the spleen and lung, while the Expression of said hepatic β-galactosidase was minimal but clearly detectable in a ratio of 2.9 x 10 5 Tf / virion molecules (Figure 2D). On the contrary, the injection of Tf-AdLacZ obtained with higher ratios of Tf / virion, resulted in a notable liver staining (Figure 2F). Therefore, based on these findings, it was determined that the ratio of 1.5 x 10 5 Tf / virion molecules, which demonstrated significant tumor transfecting efficiency, while maintaining the highest degree of tumor specificity, was optimal for the studies. The optimal conditions appeared to be 0.01-0.2 mg / 10 10 pt (7.5 x 10 3 -1.5 x 10 5 Tf molecules per virion) for JSQ-3 and about 0.03-0.5 mg / 10 10 pt for DU145. Therefore, the Tf / pt ratios can be optimized in vivo in different tumor models. It should be noted that in vitro , the optimal Tf / pt ratios are much smaller than (necessary) in vivo , so that, in the latter condition, more transferrin may be required to stabilize the virus, in order to improve the impact (of this one) on the cells. The ratio of 0.2 mg Tf / 10 10 pt (1.5 x 10 5 Tf molecules per virion) has been used in subsequent in vivo gene therapy experiments for JSQ-3 tumors.

The quantitative trials of β-galactosidase also confirmed the increase substantial gene expression in murine tumors that are injected intravenously with adenoviruses directed by transferrin, when compared with those performed with adenovirus only.

The viral supply to the organs to which directed, it was also observed in murine livers, with the complex Tf-Ad5LacZ injected intravenously, but the liver supply has a different preferred Tf / pt ratio, for example, 0.5 mg-1.3 mg Tf / 10 10 pt (3.75 x 10 5 -9.75 x 10 5 Tf / virion molecules). In mice that were injected intravenously only with Ad5LacZ, only a limited number of hepatocytes were stained blue, (<1-5%), while the animals that were injected intravenously with Tf-Ad5LacZ, showed an increase in the number of blue-stained hepatocytes (10-40%). The difference of preferred proportions Tf / pt between tumor (viral) and hepatic supply, illustrates that systemic viral delivery systems, according to the present invention, can be optimized so that they are selective for different targets.

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Example 3 Adenovirus-mediated gene delivery directed by transferrin and protein expression of p53 in vivo in a xenograft model of nude mice DU145

The ability of the adenoviral vector directed by transferrin to selectively deliver the p53 gene to tumors was examined. The defective replication adenovirus serotype 5, which carries the normal human p53 gene, was used in these studies. This virus, called Adp53, was used to produce Tf-Adp53. Tf-Adp53 was obtained by mixing holo-transferrin with Adp53 in 10 mM HEPES, pH 7.4, with a ratio of 1.5 x 10 5 Tf / virion molecules. After incubation for 10 minutes at 4 ° C, phosphate buffered solution (PBS), pH 7.4, was added to bring the final volume to 300 µl / mouse and constitute a final concentration of 5% dextrose. Three days after intravenous injection of these viruses into nude mice bearing DU145 subcutaneous tumors, the mice were sacrificed, the tumors and organs were removed, Western blot analysis was performed for p53 protein expression (see Figure 3). The antibody used in these studies reacts with both normal and mutated forms of human p53, and cross-reacts with mouse p53. The p53 band representing the virally transduced wild-type p53 migrates in the gel above the mutated form of p53 found in DU145 cells. This can be seen in the two left lanes of Figure 3 if DU145 cells are compared with DU145 cells infected in the Adp53 culture. An upper band, which represents virally encoded wtp53, was evident in DU145 cells infected in vitro with Adp53. It should be taken into consideration that the first lane (DU145 + Adp53) contains only 2.5 µg of protein, while all other lanes of Figure 3 contain 100 µg of total proteins.

Western blot analysis of tumors from mice that received targeted Adp53 (i.e., Tf-Adp53), revealed an upper band (exogenous p53) and a lower band (endogenous DU145 p53) that arose in what looked like a large single band . It is clear that there is significantly more exogenous p53 in tumors from mice that received Tf-Adp53 than in tumors from mice that were treated with non-directed Adp53. The liver and other vital organs of mice treated with targeted Tf-Adp53 showed little or no exogenous wtp53. Contrary to this, treatment with non-directed Adp53 resulted in a higher level of exogenous hepatic p53. As expected, the tumors of the untreated mice contained only endogenous DU145 p53 and the organs of these animals contained only endogenous murine p53. These results further confirm that Tf-Adp53, and not unaddressed Adp53, can selectively impact tumors in vivo , and that p53 is efficiently expressed in tumor tissue after intravenous administration.

Example 4 Systemic adenovirus-mediated gene delivery directed by transferrin in vivo in a SCCHN xenograft model of nude mice, in conjunction with radiation treatment

The definitive trial regarding the usefulness of an adenoviral delivery system aimed at gene therapy is its effectiveness in treating tumors when administered systemically. It has been established that the loss of functional p53 may contribute to the radiation-resistant phenotype (Bristow et al ., 1996). We previously demonstrated that the combination of wtp53 and conventional radiation treatment could eliminate established long-term xenograft tumors (Xu et al ., 1999; Pirollo et al ., 1997). The results in this example show the ability of Tf-Adp53 to sensitize the xenografts of human tumor cells with respect to radiation therapy.

Xenografts were induced in female mice athymic nudes (NCr-nu-nu) of between 4 and 6 weeks of age, by subcutaneous injection of 4 x 10 6 JSQ-3 cells (in Matrigel ™, a collagen matrix) in the lower loin above the tail of each animal. The progeny of JSQ-3 cells was derived from a SCCHN recurring and is known to be highly radioresistant. Be let the tumors develop to a size of 100-200 mm3. The targeted adenovirus, called Targeted Ad-p53, was prepared by mixing transferrin with adenovirus carrying DNA encoding wtp53, as described in Example 1. For this experiment, the adenovirus particles (pt) per plaque forming unit (pfu). The animals were divided into four groups: (i) Untreated, (-) radiation; (ii) Ad-p53 not directed (+) Radiation; (iii) Ad-p53 directed (-radiation); (iv) Ad-p53 directed (+ radiation). The mice (in the groups ii, iii and iv) were injected intravenously, through the tail vein, every three to four days with 1 x 10 10 pt / mouse / injection (equivalent to 3 x 10 8 pfu) of Targeted Adp-p53 (a ligand mixture was injected Tf and virus) or non-directed (without ligand). They were administered in total 6 injections The next day after intravenous injection Initially, the animals (in groups II and IV) were secured in a lead bra, which allowed only the area to radiate tumor, and that the first fractional dose of 2.0 Gy of radiation 137 Cs ionizer will be administered using an irradiator J.L. Shepard and Associates Mark I. Therefore, animals are administered 2.0 Gy / day for 5 consecutive days, followed by 2 days without radiation treatment. The cycle was repeated until a Total 30 Gy had been administered. Tumor sizes are measured weekly by a blind procedure.

Untreated animals and those they received Ad-p53 directed without radiation, were sacrificed due to tumor weight on day 52 (see figure 4). He treatment with Ad-p53 not directed more radiation, delayed tumor growth during the course of treatment. However, once it ceased, the tumors in these animals they began to grow in size, so that on day 121 they had also to be sacrificed due to tumor weight. So on the contrary, the tumors in the animals that received Adp-53 directed by Tf, combined with radiation, they experienced a complete regression, during the treatment and even after it ceased, so that after 8 months after such treatment, the tumors did not reproduce in These animals.

These results, together with those in Figure 3, demonstrate that systemically administered Tf-directed adenoviruses can selectively deliver wt-p53 to tumors, leading to their sensitization to conventional radiotherapy. Very importantly, the combined treatment of Tf-Adp53 plus radiation resulted in the eradication of long-term tumors. Recently, Kataoka et al (1998), reported that the combination of 2-methoxystradiol (2-Me) and systemic delivery of non-targeted Adp53 in a murine model, using A549 cells, partially inhibits the metastatic growth of lung tumors. This combinatorial treatment resulted in a two-thirds reduction in the pulmonary colonial count. Although certainly promising, the tumor cells that remained after this treatment, would have developed very certainly and, eventually, killed the animal. Contrary to this, our results with Adp53 Tf-directed, apparently produced a total long-term regression (usually 8 months) of SCCHN subcutaneous tumors.

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Example 5 Systemic adenovirus-mediated gene delivery directed by transferrin in vivo in a SCCHN xenograft model of nude mice, in conjunction with radiation treatment

JSQ-3 xenografts were induced in NCr nu-nu mice as in Example 4. It was left that the tumors reach the size of 50-60 mm3. Targeted adenovirus, with (Ad-p53 directed) or without (Ad directed) DNA encoding the human p53 wt), it was prepared by mixing transferrin with adenovirus as it was described in Example 1. The particles of the adenovirus (pt) per plaque forming unit (pfu). The animals are They divided into five groups: (i) Untreated, (-) radiation; (ii) Ad-p53 not directed, (+) Radiation; (iii) Ad directed, (+ radiation); (iv) Ad-p53 directed, (-) radiation), (v) directed Ad-p53, (+) radiation. The mice were injected intravenously, through the vein of the tail, every three or four days with 3 x 10 9 pt / mouse / injection. A total of 5 injections were administered. Three days after initial intravenous injection, the animals were secured in a lead bra, which allowed only the area to radiate tumor, administering the first fractional dose of 2.0 Gy of ionizing radiation of 137 Cs using a J.L. Shepard and Associates Mark I. So, animals are given administered 2.0 Gy / day for 5 consecutive days, followed by 2 days without radiation treatment. The cycle was repeated until a Total 26 Gy had been administered. Tumor sizes are measured weekly by a blind procedure. As in the  Example 4, tumors in untreated animals and those they received Ad-p53 directed without radiation, they showed a continuous development, so that on day 50 the animals will They sacrificed due to tumor weight. Tumors in the group treated with radiation and Ad-directed without wt p53, demonstrated some minimal inhibition of radiation from growth during treatment, weight tumors increased by volume once treatment ended. A similar recurrence (in tumor development), but even more importantly, it took place after treatment, in the group of animals that received Adp-p53 directed, but no radiation. This contrasts importantly with the mice they received Adp-53 directed combined with radiation. As it look at Example 4, the tumor regression continued in these animals more than eight months after the completion of the entire treatment. Eight months in the life of a mouse equals 30 years In the life of a man.

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Example 6 Retroviral Gene Transduction transferrín-directed

Retroviral vectors constitute one of the gene therapy vectors most widely used in trials clinical. As with adenoviral vectors, vectors retrovirics show poor specificity and immunogenicity significant.

The insufficient replication retroviruses containing the E. coli LacZ gene, RvLacZ (A LacZ, Gene Transfer Vector Core, (Gene Transfer Vector Core, University of Iowa), with 1 x 10 0 particles (pt) / ml, containing 3 x 10 7 transformation units (TU) / ml, were used in this study.Transferrin and the Tf-RvLacZ complex were prepared similarly to that described in Example 1. Briefly, Tf were first diluted to 0.5 mg / ml in 10 mM HEPES buffer, pH 7.4, then adding different amounts of Tf to 50 µl of HEPES buffer in a serial dilution, then RvLacZ was added to the test tubes , so that the Tf / virus ratios were of the order of 1 x 10 2 to 1 x 10 6 Tf / virion molecules The test tubes were incubated at room temperature for 10-15 minutes with occasional rotation of the test tubes once every two minutes, then adding 150 µl of EMEM without serum to each tube. performed retroviral transduction in vitro as described in Example 1. The virus / cell ratio was of the order of 100 to 2000 viral pt / cell.

With a viral dose of 500 pt / cell, or 1.5 MOI (or TU / cell), 3.4 mU / mg proteins β-galactosidase were expressed only by the RvLacZ retrovirus. With the virus complexing with the transferrin, the administration of Tf-RvLacZ (500 Tf / pt molecules) resulted in 6.8 mU / mg of expression proteins β-galactosidatic, while the administration of Tf-RvLacZ (5000 molecules of Tf / pt) resulted in an expression of 9 mU / mg, which represents a gene transduction 2 and 3 times more intense than that obtained by the administration of only RvLacZ. The increase in transduction gene reached a plateau with a proportion of 50,000 molecules Tf / pt. With a dose of 1000 pt / cell, or 3 MOI, Tf-RvLacZ (5000 Tf / pt molecules) produced a reporter gene expression more than 2.1 times and Tf-RvLacZ (50,000 molecules of Tf / pt) produced a 3 times more expression than RvLacZ alone. Histochemical staining showed that RvLacZ only showed a transductive efficiency of 20-30%, while the Tf-RvLacZ forming complex with transferrin (5000 molecules of Tf / pt), showed a transduction efficiency of 60-80%. The results showed that the administration of a mixture of transferrin and retrovirus can substantially enhance the retroviral gene transduction.

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Example 7 Systemic adenovirus-mediated gene delivery directed by transferrin in vivo in a murine syngene model, in conjunction with chemotherapy

It was examined, in an animal model immunocompetent, the viral delivery system's ability to p53, directed by the ligand, to sensitize the cells tumor with respect to chemotherapy. The selected model for these studies it was the metastatic pulmonary model of melanoma murine B16. In multiple experiments, cells were injected B 16, through the tail vein, in C57 / BL / 6 mice immunocompetent In this model, the tumor colonies lungs are easily visible at two-three weeks, due to melanin expression by cells Tumor Four days after cell injection B 16, started the treatment with the combination of Adp53, Tf-Adp53 and / cisplatin (CDDP). 1 x 10 10 pt viral / mouse / injection (equivalent to 3 x 10 8 pfu), with a 1.5 x 10 5 Tf / virion molecules ratio, was administered systemically and intravenously through a tail vein three times per week for a total of 12-13 treatments viral CDDP was administered intraperitoneally (3-5 mg / kg) every 2-4 days with a Total 8-13 doses of CDDP administered. One day after all treatments (five weeks after initial injection of B16 cells), the animals' lungs and underwent perfusion with 10% formaldehyde.

As shown in the figures 5A-H, there is a marked difference in the lungs obtained from the animals that received the treatment combinatorial and those of other groups, in two separate experiments. While the CDDP alone and Tf-Adp53 only demonstrated some effect when compared to animal lungs untreated, a significant number of tumor colonies Similarly, the lungs of animals treated with the Tf-AdLacZ control virus plus CDDP, evidence that it is only a drug effect. Plus significantly, the animals that received non-targeted Adp53, together with CDDP, they also present multiple and voluminous colonies tumors, which indicate a minimal effect of non-directed Adp53 when supplied systemically. However, contrary to this, the lungs of animals that received adenoviral p53 directed transferrine (Tf-Adp53) combined with CDDP, they are virtually free of obvious tumor metastases. These findings demonstrate that the Adp53 Tf-directed, supplied systemically, it can sensitize cells Tumors to conventional chemotherapy, in addition to the conventional radiotherapy In addition, Tf-Adp53 can also function effectively in a murine synergetic model, in addition to the nude mouse models that have been used previously.

The Tf molecule that was used in all our experiments, is the human Tf. It is known that the TfR of a given species can bind to Tf from various other species (Aisen, 1998). However, reference was initially made to the fact that the selectivity that was appreciated with human tumors in the naked mice could be attributed to the human Tf that we were using, was, in the normal tissues of the host, being preferably recognized by tumor TfR with respect to TfR murine The system of synergic model that involves cells of melanoma of B16 mice that develop in the mouse C57 / BL / 6, reassures in this regard. In this model, the TfR in the TfR tumor and in normal tissues are both murine TfR. Without However, we were able to demonstrate that the adenoviruses that form complex with human Tf they lodge in tumors that have levels elevated TfR murine. This finding suggests that it is the level of TfR expression, more than the species-related phenomenon, the one that counts to target xenografted tumors humans in the nude mouse models that have been described previously and the one that feeds the possibility that you can achieve the ultimate goal of treating human tumors that Develop in man.

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Example 8 Herpes simplex virus transduction directed by the transferrin

Herpes simplex virus (HSV) is a vector competent replication viral, widely used in the gene therapy, especially in the central nervous system (Walker, 1999). As with adenoviral or retroviral vectors, HSV shows poor specificity and immunogenicity significant.

To explore the possibility of using the transferrin management strategy to direct (and do target with) the competent replication viral vectors G207, is formed an HSV complex provided with a LacZ reporter gene (Walker, 1999) with the human tranferrina. G207 (1.1 x 10 9 pfu / ml, Neuro Vir, Inc., in PBS) was mixed with Human holo-transferrin (Sigma, 5 mg / ml, in water) with different proportions, in the same way as for Tf-AdLacZ, as described in Example 1. In a group of experiments, HSV was heat treated by incubating it at 37 ° C for 10 minutes before mixing with Tf. The treatment Thermal mentioned can inactivate the HSV.

For transduction experiments, 8 x 10 4 JSQ-3 cells / well were seeded in a plate with 24 wells. 24 hours later, the cells were washed a time with serum-free EMEM, then adding 0.3 ml of EMEM without serum or antibiotics to each well. HSV complexes or Tf-HSV with different proportions of tranferrin with respect to the virus, in 200 µM EMEM, they were added to the wells, with an MOI = 1. After 4 hours incubation at 37 ° C and 5% CO2, by rotating mixing the test tube a once every 2 minutes, 0.5 ml of EMEM with 20% serum was added, to the wells. After 2 days in culture, the cells were washed once with PBS, and lysed in 1X reporter lysis buffer (Promise) Cell lysates were treated with 100 µL of 150 \ muM O-nitrophenyl-? -Galactopyranoside in 20 mM Tris (pH 7.5) containing 1 mM MgCl2, and 450 µM β-mercaptoethanol at 37 ° C for 30 minutes. The reaction was stopped by adding 150 µl / well of 1M Na 2 CO 3, the absorbance being measured at 405 nm. The purified β-galactosidase (Boehringer) is used to make a standard curve. The results are expressed as milliUnities (mU) of equivalents of β-galactosidase per mg of total proteins. The results are shown in Table 1.

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TABLE 1 Transferrin enhances transducer efficiency of HSV

one

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Transferrin enhances the efficiency of HSV transduction, a competent replication viral vector. When HSV was thermally inactivated, the formation of the complex with Tf still showed an enhancement in transducer efficiency. With a Tf / virion ratio of 50,000 and an MOI = 1, TF-HSV resulted in an expression of the reporter gene more than double that produced by the HSV devoid of address of Tf. The results show that the strategy of address with transferrin can be used for viral vectors competent replication such as HSV.

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Example 9 Systemic adenovirus-mediated gene delivery directed by transferrin in vivo in an MDA-MB-435 xenograft model of nude mice, in conjunction with the chemotherapeutic agent Docetaxel (Taxotere)

To further demonstrate the usefulness of this directed adenoviral delivery system, a second was used tumor model, the cell line MDA-MB-435, derived from cancer human breast In addition, since chemotherapy often constitutes Selective treatment for breast cancer, this experiment tested the capacity of the delivery system of this invention to sensitize the chemotherapeutic agent Docetaxel (Taxotere) commonly used, xenograft tumors of breast cancer human. Xenografts were induced in nude nude mice females (NCr nu-nu), with an age of 4-6 weeks, by subcutaneous injection of 2.5 x 10 6 MDA-MB-435 cells the breast fat pad of each animal. The tumors were allowed  will develop to a size of 40-50 mm3. Targeted adenovirus, called Tf Adp53, was prepared by mixing transferrin with adenovirus carrying DNA encoding wt p53, as described in Example 1. The animals were divided into five groups: (i) Not treated, (-) Taxotere; (ii) Taxotere alone; (iii) Adp53 not addressed (+) Taxotere; (iv) Ad-p53 directed (-) Taxotere; (v) Adp53 directed (+) Taxotere. The rats they were injected intravenously, through the tail vein, each three or four days with 5 x 10 10 pt / mouse / injection of Adp53 directed (a mixture of ligand Tf and virus was injected) or non-directed  (without ligand). A total of 12 injections were administered. Up to date next after the initial viral injection, the drug treatment The animals were provided Taxotere intravenously, at a dose of 7.5 mg / kg every three or four days up to a total of 11 injections. They were measured weekly tumor sizes blindly over a total of 6-8 tumors per group. The average of the tumor volumes per group (mm3) ± standard error, with with respect to time (days), (figure 6). While treating with only Tf-Adp53 had no effect, treatment with Taxotere alone or with the non-directed Adp53 + Taxotere, induced some growth inhibition, indicating a drug effect. Without However, a level of growth inhibition was observed, even more importantly, in tumors from animals who received Ad-p53 Tf-directed, combined with Taxotere. These findings show the synergistic effect. of the combined treatment and demonstrate not only that the complex Adp53 Tf-directed of the invention, is effective in Multiple human tumor models, but can be used also to sensitize tumors with respect to agents Chemotherapeutic

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Example 10 Adenovirus-mediated gene expression directed by transferrin, in vivo , in a DU145 xenograft model of nude mice

A DU145 cell line, derived from a prostate cancer, showed an improvement in expression in intratumoral injections The serotype was used in this example 5 adenovirus with defective replication, which carried the LacZ gene. Nude nude mice (nu / nu) were injected subcutaneously using Matrigel ™ to produce tumors. Tf-Ad-LacZ was prepared as in the Example 1. The Tf / pt ratio was 0.1-0.2 mg / 10 10 pt, as described in Example 2. Mice They had 2 tumors each, but only one was injected.

After 24 hours of intratumoral injection of 3 x 10 10 particles / tumor, tumors were removed, were they cut into pieces, quickly frozen in liquid nitrogen, and were sprayed in a Bessman tissue spray. The β-galactosidatic enzyme activity using the β-galactosidase test system Chemiluminescent Inc, Glacto-Star ™, from Tropix, Inc according to the manufacturer's protocol. The results are shown in figure 7. Tf-Ad-LacZ resulted in an increase in the expression of the β-galactosidase greater than 3.4 times, compared (the expression observed) with Ad-LacZ. The results show that the directed transferrin strategy can be used to increase expression in injections intratumoral

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US Patent No. 5,833,975

WO 92/06180

WO 98/40508

Claims (46)

1. Virus-ligand complex that is directs a cell to supply a virus to a cell target inside a host animal, wherein said complex virus-ligand is constituted by a ligand and a virus, and in which the ligand is non-covalently bound to the virus, and is transferrin, a receptor antibody of the transferrin, a fragment of a receptor antibody from the transferrin, EGF or VEGF. 2. Virus-ligand complex that is directs to a cell according to claim 1, wherein said virus and said ligand are not associated with each other naturally. 3. Virus-ligand complex that is directs to a cell according to claim 1 or 2, wherein said Virus comprises a therapeutic nucleic acid. 4. Virus-ligand complex that is directs to a cell according to claim 3 or 2, wherein said therapeutic nucleic acid encodes a peptide or protein therapeutic 5. Virus-ligand complex that is directs to a cell according to claim 4 wherein said acid Therapeutic nucleic encodes the wild-type p53. 6. Virus-ligand complex that is directs a cell according to any of the claims above, in which said virus is a retrovirus or a adenovirus 7. Virus-ligand complex that is directs a cell according to any one of claims 1 to 5, wherein said virus is selected from the group constituted by adeno-associated virus, herpes simplex virus, cytomegalovirus, vaccine virus, avian smallpox virus, canarypox virus, and Sindbis virus. 8. Virus-ligand complex that is directs a cell according to any of the claims above, in which said virus is a chimeric virus, a virus hybrid, or a recombinant virus. 9. Virus-ligand complex that is directs a cell according to any of the claims above, in which said ligand that targets a cell is an original protein or a recombinant protein. 10. Virus-ligand complex that is directed to a cell, according to any of the claims above, in which said ligand that targets a cell and said virus, are present in a proportion comprised in the 100 to 1,000,000 range of ligand molecules per virion. 11. Virus-ligand complex that is directed to a cell according to claim 10, wherein said ligand that targets a cell and said virus are present in a proportion in the range of 6,700 to 400,000 ligand molecules per virion. 12. Virus-ligand complex that is directed to a cell according to any of the claims above, in which said ligand that targets a cell and said virus, are present in a proportion comprised in the range of 1 µg to 10 mg of said ligand per 10 10 virions 13. Virus-ligand complex that is directed to a cell according to claim 12, wherein said ligand that targets the cell and said virus are present in a proportion in the range of 10 \ mug to 600 \ mug of said ligand for 10 10 virions. 14. Vector for the systemic supply of a virus to a target cell, said vector comprising a ligand that it is directed to a cell non-covalently bound to said virus, wherein said vector can be prepared by mixing a composition consisting of a ligand that targets a cell with a virus in an aqueous medium and in which said ligand is constituted by transferrin, a transferrin receptor antibody, a fragment of a transferrin receptor antibody, EGF or VEGF 15. Vector according to claim 14, wherein said virus and said ligand are not naturally associated between yes. 16. Vector according to claim 14 or 15, in which said virus comprises a therapeutic nucleic acid. 17. Vector according to claim 16, wherein said therapeutic nucleic acid encodes a peptide or a protein therapeutic 18. Vector according to claim 17, in the that said therapeutic nucleic acid encodes the type p53 wild. 19. Vector according to any of the claims 14 to 18, wherein said virus is a retrovirus or an adenovirus

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20. Vector according to any of the claims 14 to 18, wherein said virus is selected from among the group consisting of adeno-associated virus, herpes simplex virus, cytomegalovirus, vaccine virus, virus of avian smallpox, canarypox virus and viruses Sindbis 21. Vector according to any of the claims 14 to 20, wherein said virus is a virus chimeric, a hybrid virus, or a recombinant virus. 22. Vector according to any of the claims 14 to 21, wherein said ligand that is directed to A cell is an original protein or a recombinant protein. 23. Vector according to any of the claims 14 to 22, wherein said ligand that is directed to a cell and said virus are present in a proportion in the range of 100 to 1,000,000 molecules of ligating by virion. 24. Vector according to claim 23, wherein said ligand that targets a cell and said virus are present in a proportion in the range of 6,700 to 400,000 ligand molecules per virion. 25. Vector according to any of the claims 14 to 24, wherein said ligand that is directed to a cell and said virus are present in a proportion in the range of 1 µg to 10 mg of said ligand by 10 10 virions. 26. Vector according to claim 25, wherein said ligand that targets a cell and said virus are present in a proportion in the range of 10 µg at 600 µg of said ligand per 10 10 virions. 27. Procedure for preparing a vector for the systemic supply of a virus to a target cell, said vector comprising a ligand that is directed to a cell, not covalently bound to said virus, which comprises mixing a composition constituted by said ligand that targets a cell with said virus in an aqueous medium, said ligand joining non-covalent way to said virus, in which said ligand is constituted by transferrin, a receptor antibody of the transferrin, a fragment of a receptor antibody from the transferrin, EGF or VEGF. 28. Method according to claim 27, in wherein said aqueous solution includes one or more buffering agents, a agent that adjusts osmolarity or an antibiotic. 29. Virus-ligand complex that is directed to a cell according to any of claims 1 to 13, for use as a medicine. 30. Vector according to any of the claims 14 to 28, for use as a medicine. 31. Use of a virus for preparation of a therapeutic agent comprising a vector comprising a virus-ligand complex that targets a cell, for supplying said virus to a target cell inside of an animal, in which said virus-ligand complex it consists of a ligand and a virus, and in which the ligand it is non-covalently bound to the virus, and is transferrin, a transferrin receptor antibody, a fragment of a Transferrin receptor antibody, EGF or VEGF. 32. Use according to claim 31, in which said animal is a human. 33. Use according to claim 31 or 32, wherein said therapeutic agent is administered systemically. 34. Use according to claim 31 or 32, wherein said therapeutic agent is administered parenterally. 35. Use according to claim 31 or 32, wherein said therapeutic agent is administered intravenously or intraarterially 36. Use according to claim 31 or 32, wherein said therapeutic agent is administered intratumorally. 37. Use according to any of the claims 31 to 36, wherein said vector encodes p53 of wild guy 38. Use according to any of the claims 31 to 37, wherein said therapeutic agent is administers an animal that receives chemotherapy in addition to that therapeutic agent 39. Use according to any of the claims 31 to 37, wherein said therapeutic agent is administer to an animal that receives radiation treatment in addition of said therapeutic agent. 40. Use according to any of the claims 31 to 39, wherein said animal has cancer of head and neck, bladder, breast, thyroid, ovaries, brain, prostate, a melanoma or a lymphoma. 41. Use according to any of the claims 31 to 33 and 37 to 40, wherein said virus comprises a nucleic acid encoding wild-type p53, in which also said ligand that targets a cell, is transferrin, and said therapeutic agent being also administered systemically 42. Use according to claim 33 or 36, in which the target cell is a cell cancer selected from a head and neck cancer, cancer Bladder, breast cancer, thyroid cancer, ovarian cancer, prostate cancer, melanoma and lymphoma; the vector essentially consisting of the virus and directly joining a receptor that is overexpressed in the cancer cell.

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43. Use according to claim 42, in the virus sensitizes the cancer cell to radiation or to Chemotherapy 44. Use according to claim 43, in which the virus comprises a nucleic acid of interest, whose expression in the cancer cell sensitizes the cancer cell to radiation or chemotherapy. 45. Use according to claim 40, in which the virus comprises a nucleic acid that, when expressed in cancer cells, sensitizes the cell to radiation or to chemotherapy; binding the ligand directly to a receptor in the cancer cell; Y the vector essentially consisting of The virus and the ligand.

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46. Vector for use in supply from a virus to a target cell inside an animal, in the that the vector comprises a virus-ligand complex which is directed to a cell, in which said complex virus-ligand is constituted by a ligand and a virus, and in which the ligand is non-covalently bound to the virus, and is transferrin, a receptor antibody of the transferrin, a fragment of a receptor antibody from the transferrin, EGF or VEGF.