WO2003061684A2 - Tumour treatment compositions comprising hsp70 and tumour necrosis factor - Google Patents

Abstract

The invention relates to a novel pharmaceutical composition comprising HSP70 and tumour necrosis factor and its use in systemic tumour treatment.

Description

Novel pharmaceutical compositions for tumour treatment

Field of the invention

The invention relates to a novel pharmaceutical composition comprising HSP70 and tumour necrosis factor and its use in systemic tumour treatment.

Background to the invention

Tumour necrosis factor (TNF) is a cytokine with very potent anti-tumour activity, especially when used in combination with interferon-γ. Unfortunately, systemic administration of TNF at relatively high doses induces a systemic inflammatory response syndrome, characterized by bowel necrosis, liver damage and severe hypotension, leading to death (Tracey and Cerami, 1993), such that its therapeutic potential is limited mainly to clinical protocols where TNF can be administered locally, e.g. using isolated limb perfusion (Eggermont, 1998). TNF was indeed found to be a mediator in endotoxin-induced shock, but a role for TNF has also been demonstrated in inflammatory bowel disease, since application of a monoclonal antibody directed against TNF led to improvement of the disease, both in an animal model (Watkins et al., 1997) and in patients with Crohn's disease (D'Haens et al., 1999). TNF inhibitors and anti-TNF antibodies have also proven their efficacy in treating arthritis (Lorenz, 2000; Emery, 2001). In diabetes it is still doubtful whether TNF plays a deleterious role (Hunger et al., 1997) or a protective role (Cope et al., 1997). To broaden the therapeutic margin of TNF, it is necessary to understand the process by which TNF causes induction of lethality and to identify all molecular species involved. We previously reported that TNF-induced lethal shock is preceded by the release of hypotension-causing nitric oxide, hypothermia, and release in the serum of secondary cytokines such as IL-6. The kinetics and amplitude of IL-6 induction by TNF were found to correlate very well with the lethal outcome, although IL-6 is not a causative mediator. Both hypotension and liver toxicity have been proven to be dose-limiting. We are studying several ways to inhibit the toxicity induced by TNF and we have for example demonstrated in the past that acute phase proteins confer protection against TNF-induced toxicity and lethality (Libert et al., 1994; Libert et al., 1996; Van Molle et al., 1997; Van Molle et al., 1999). In this invention we have surprisingly found that the heat shock protein HSP70 significantly leads to attenuation of TNF-induced lethality and furthermore we show that a pharmaceutical composition comprising HSP70 and TNF can be used systemically to eliminate tumours. It has been suggested in the art that whole body hyperthermia at 40 degrees C° with immune therapy using TNF could eliminate cancer cells (Rosen P. (1989) Med. Hypotheses 29(1 ):45). Hyperthermia induces a lot of effects in the body including the induction of a heat shock response. Although a potentially, protective role for HSP70 has been documented over the years and the use of HSP70 for intracellular targeted delivery of compounds for the treatment of cancer has been described (WO 00/31113) it could not be predicted that HSP70 is a crucial molecule that can be used as a protectant for the plethora of side-effects that occur when TNF is used systemically as an anti-tumour agent.

Figures

Figure 1: Generation of hsp70.1 -deficient mice

(A) Targeting strategy. (B) Southern blot analysis of representative mouse tail DNA digested with Apal and hybridized with probe A. (C) Northern blot analysis of representative HS-induced mouse embryonic fibroblast mRNA hybridized with probe B. The hsp70.1-NeoR fusion mRNA was calculated to be 2.4 kb based on the sequence of the targeting vector.

Figure 2: Increase in body temperature and HSP70 induction in tissue homogenates of mice after whole-body HS

(A) Rectal body temperature was monitored and recorded (•, n = 12) during HS until 10 min after HS (black line, HS induction period). (B) Mice were subjected to HS and killed at different timepoints. 50 μg total protein of homogenized tissue was separated on 7.5% acrylamide gel and transferred to a nitrocellulose membrane. Blots were developed with an anti-HSP70 antibody.

Figure 3: HS treatment 12 h before TNF challenge prevents lethality Mice were subjected to whole-body HS and challenged i.v. with an LD₁₀₀ of 15 μg TNF 2 h (•, n = 6), 6 h (o, n = 8), 12 h (■, n = 45), 24 h (Δ, n = 6) or 48 h (A, n = 6) after HS. Controls were challenged with TNF without prior HS (o, n = 44). Figure 4. Effect of HS on TNF-induced hypothermia as well as TNF-induced NO and IL-6 production

(A) Mice were subjected to whole-body HS and challenged i.v. after 12 h with 15 μg TNF (•, n = 12). Control mice were challenged i.v. with 15 μg TNF without prior HS (o, n = 12). Rectal body temperature was measured at the indicated timepoints after TNF treatment. (B-C) Mice were subjected to whole-body HS and challenged i.v. after 12 h with 15 μg TNF (•, n = 30). Control mice were challenged i.v. with 15 μg TNF without prior HS (o, n = 30). 1 , 3, 6, 9, 12 or 24 h after TNF administration, blood was withdrawn and analyzed for NOχ^" and IL-6 concentrations (n = 6 per timepoint).

Figure 5. HS pretreatment prevents TNF-induced bowel damage Mice were kept at room temperature or subjected to whole-body HS and challenged i.v. after 12 h with 15 μg TNF. Parts of the jejunum were homogenized for assessment of DNA fragmentation and DEVDase activity (A). Other parts of the jejunum were fixed and embedded in paraffin, followed by hematoxylin/eosin staining (B-D), TUNEL assay (E-G) or immunohistochemistry (H-J). (B, E and H) PBS-treated mice; (C, F and I) TNF-treated mice; (D, G and J) TNF-treated mice 12 h after HS (scale bars, 10 μm).

Figure 6. HS-induced protection is absent in hsp70.1 -deficient mice Wt mice (•, n = 13) and hsp70.1 -deficient mice (■, n = 12) were HS treated and challenged i.v. after 12 h with 15 μg TNF. Control wt mice (o, n = 13) and hsp70.1- deficient mice (D, n = 12) were injected with 15 μg TNF without prior HS. Liver, jejunum and colon were removed from parallelly HS-treated animals 6 h and 12 h after HS. (insert) Western blot for HSP70 6 h and 12 h after HS.

Figure 7.

B16BI6 melanoma model (panel A and B)

Mice were inoculated with 6 x 10⁵ B16BI6 cells at day 0 and were randomized and stratified into 4 different experimental groups at day 10. The 1^st group (O, n=8) received PBS from day 11 until day 20. The 2^nd group (D; n=8) was subjected to daily heat shock for 20 min at 42°C starting from day 10 until day 19 and received PBS injections from day 11 until day 20, 12 h after ending the heat shock treatment. The 3^rd group (•, n=10) was treated with 10 μg TNF and 5.000 IU IFN-gamma on day 11-15 and on day 17-20 and with 5 μg mTNF and 2.500 IU IFN-gamma on day 16. The 4^th group (■, n=10) was subjected to heat shock daily from day 10 until day 19 and treated with TNF and IFN- gamma (same dose scheme as 3^rd group) from day 11 until day 20, at 12 h after ending the heat shock exposure. TSI was determined daily from day 10 until day 21 The TSI is demonstrated in fig 7A and survival curves in fig 7B. Period of treatment with TNF/IFN- gamma is indicated with ♦.

PG19 melanoma model (panel C and D).

Mice were inoculated with 5 x 10⁶ PG19 cells at day 0 and were randomized and stratified into 4 different experimental groups at day 10. The 1^st group (O, n=8) received PBS from day 11 until day 20. The 2^nd group (D; n=8) was subjected to daily heat shock for 20 min at 42°C starting from day 10 until day 19 and received PBS injections from day 11 until day 20, 12 h after ending the heat shock treatment. The 3^rd group (•, n=10) was treated with 10 μg TNF and 5.000 IU IFN-gamma on day 11-15 and on day 18-20 and with 5 μg mTNF and 2.500 IU IFN-gamma on day 16 and 17. The 4^th group (■, n=10) was subjected to heat shock daily from day 10 until day 19 and treated with TNF and IFN-gamma (same dose scheme as 3^rd group) from day 11 until day 20, at 12 h after ending the heat shock exposure. TSI was determined daily from day 10 until day 21 The TSI is demonstrated in fig 7C and survival curves in fig 7D. Period of treatment with TNF/IFN-gamma is indicated with ♦. LLC model (panel E and F) Mice were inoculated with 5 x 10⁵ LLC cells at day 0 and were randomized and stratified into 4 different experimental groups at day 10. The 1^st group (O, n=8) received PBS from day 11 until day 20. The 2^nd group (D; n=8) was subjected to daily heat shock for 20 min at 42°C starting from day 10 until day 19 and received PBS injections from day 11 until day 20, 12 h after ending the heat shock treatment. The 3^rd group (•, n=10) was treated with 10 μg TNF and 5.000 IU IFN-gamma on day 11-15 and on day 18-20 and with 5 μg mTNF and 2.500 IU IFN-gamma on day 16 and 17. The 4^th group (■, n=10) was subjected to heat shock daily from day 10 until day 19 and treated with TNF and IFN- gamma (same dose scheme as 3^rd group) from day 11 until day 20, at 12 h after ending the heat shock exposure. TSI was determined daily from day 10 until day 21 The TSI is demonstrated in fig 7E and survival curves in fig 7F. Period of treatment with TNF/IFN- gamma is indicated with ♦. Detailed description of the invention

In the present invention we demonstrate that the endogenous HSP70 induction in several organs confers significant protection against lethality induced by a systemic administration of high doses of tumor necrosis factor (TNF). It is further shown that the production of HSP70 prevents high production of interleukin-6 and nitric oxide, and reduces severe damage and apoptosis of the enterocytes in the bowel. We find that mice deficient in the major inducible hsp70.1 gene are no longer protected by high doses of TNF. It is surprisingly shown that HSP70 induction can be applied successfully in an anti- tumour protocol based on TNF and interferon-γ, leading to a significant inhibition of lethality but not to a reduction of anti-tumour capacity.

Heat shock (HS) proteins (HSPs) are a class of conserved molecules present in all prokaryotes and eukaryotes studied so far. Under normal physiological conditions, the expression of these proteins is very low (Craig and Gross, 1991). In stress situations, a very strong synthesis of these proteins has been observed (Lindquist and Craig, 1988). The main function of HSPs is to operate as an intracellular chaperone for aberrantly folded or mutated proteins, and to provide cytoprotection against stress conditions. For this reason, the presence of a cellular stress response in cancer cells reduces their sensitivity to chemical stress caused by insufficient tumor perfusion or chemotherapy (Arrigo, 2000; Jolly and Morimoto, 2000; Sarto et al., 2000). One of the major HSPs is HSP70, named after its molecular mass of approximately 70 kDa. The subfamily, consisting of at least seven members in the mouse, contains both constitutive and inducible forms (Lindquist and Craig, 1988). As constitutive members in the mouse, the 70-kDa HS cognate HSC70 (Giebel et al., 1988) as well as the 75-kDa and 78-kDa glucose-regulated proteins GRP75 (Domanico et al., 1993) and GRP78 (Kozutsumi et al., 1989) have been described. HSP70.1 and HSP70.3 are both inducible (Hunt et al., 1993), while the spermatocyte-specific HSP70.2 is expressed during the meiotic phase of spermatogenesis (Allen et al., 1988; Zakeri et al., 1988). The testis-specific HSC70 (HSC70t) is expressed in postmeiotic spermatids (Maekawa et al., 1989; Matsumoto and Fujimoto, 1990). Recently, several reports have dealt with the protecting capacities of HSP70 against various toxic stimuli. HS was demonstrated to prevent lethality induced by lipopolysaccharide (LPS) in rats and mice (Ryan et al., 1992; Hotchkiss et al., 1993). This protection also correlated with HSP70 upregulation in several organs (Hotchkiss et al., 1993). Ischemic preconditioning of the liver also leads to strong HSP70 induction, which results in resistance to subsequent ischemia-reperfusion injury of the liver in the rat (Kume et al., 1996). Injection into mice of amphetamine increases the body temperature, resulting in a strong induction of HSP70 (Nowak, 1988). Treatment of rats with amphetamine reduces the hepatotoxicity induced by acetaminophen and bromobenzene (Salminen et al., 1997).

In one embodiment the invention provides a pharmaceutical composition comprising

HSP70 and TNF.

With the term ΗSP70' it is meant in this invention the gene and gene product designated as HSP70.1 - also called HSPA1A (designated as SEQ ID NO: 6 and its corresponding nucleotide sequence designated as SEQ ID NO: 5). When HSP70 is exogenously added to a patient in need it is preferred that the wild type HSP70 is used to avoid for example an undesired immune response. However, it can also be foreseen that variants of HSP70 with for example a homology of 90% or more or fragments of HSP70 containing similar protective and functional characteristics as HSP70 can be used in a pharmaceutical composition comprising TNF.

The wording "a pharmaceutical composition comprising HSP70" can either mean that the levels of HSP70 are raised endogenously or that the HSP70 is added exogenously. Endogenously raised levels of HSP70 can be obtained by inducing hyperthermia in patients. Several methods and apparatuses for inducing a controlled hyperthermia are known in the art and examples are described in US 6,149,674 and US 6,245,094. Alternatively hyperthermia can be induced as an (undesired) side-effect of certain drugs but preferably said hyperthermia is induced under medically controlled conditions. For example it is known that amphetamine increases the body temperature, resulting in a strong induction of HSP70 (Nowak, 1988). It is also known that endogenous levels of HSP70 can be induced by stress stimuli which can either be physiological (growth factors and hormonal stimulation), environmental (e.g. heavy metals (Liu J. et al (2001) Toxicol. Sci. 61(2):314; Waelput W. et al (2001) J Exp Med 194(11):1617) and ultraviolet radiation) or pathological (inflammation, autoimmune reactions, and viral, bacteriological or parasitic infections).

Thus in a particular embodiment the invention provides a pharmaceutical composition comprising HSP70 and TNF and wherein HSP70 is endogenously induced by heat. In yet another embodiment HSP70 can be induced by the usage of non-toxic HSP70 inducers such as for example geranylgeranylacetone (Hirakawa T. et al (1996) Gastroenterology 11(2): 345 and JP2001097853).

According to the invention it is also possible to add HSP70 exogenously to a patient in need. HSP70 can either be purified from for example heat induced organs or preferably it can be made in a recombinant manner. Methods for the recombinant expression of proteins are well known in the art. One way for the manufacture of recombinant HSP70 is described in WO 00/31113. Yet another way of exogenous administration of HSP70 to a patient is by means of gene therapy. In vivo and in vitro gene therapeutic methods are herein further explained. The pharmaceutical composition comprising HSP70 and TNF can be applied to the patient at the same time but it is preferred that the levels of HSP70 are raised before TNF is administered. Thus according to the invention the levels of HSP70 are raised 6 hours, 12 hours, 18 hours or 24 hours or 30 hours before TNF is administered. Accordingly a hyperthermia is induced before a pharmaceutical composition comprising TNF is administered or HSP70 protein is administered preceding the administration of a pharmaceutical composition comprising TNF.

In another embodiment the invention provides a pharmaceutical composition comprising HSP70, TNF and interferon-gamma.

In yet another embodiment the invention provides a pharmaceutical composition comprising HSP70, TNF, interferon-gamma and a chemotherapeutic agent.

Chemotherapeutic agents are well known in the art and are those medications that are used to treat various forms of cancer. These medications are given in a particular regimen over a period of weeks. Most chemotherapeutic agents have the ability to directly kill cancer cells. Examples of chemotherapeutic agents comprise busulfan, cisplatin, cyclophosphamide, methotrexate, daunorubicin, doxorubicin, melphalan, vincristine, vinblastine, and chlorambucil.

In a particular embodiment said chemotherapeutic compound is melphalan.

In another embodiment the invention provides pharmaceutical compositions as described herein above for use as a medicament. In yet another embodiment the invention provides the use of pharmaceutical compositions as described herein above for the manufacture of a medicament for systemic tumor treatment.

The term 'medicament to treat' relates to a composition comprising molecules as described above and a pharmaceutically acceptable carrier or excipient (both terms can be used interchangeably) to treat cancer as indicated above. Suitable carriers or excipients known to the skilled man are saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives. Other suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers. The 'medicament' may be administered by any suitable method within the knowledge of the skilled man. The preferred route of administration is parenterally. In parental administration, the medicament of this invention will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with the pharmaceutically acceptable excipients as defined above. However, the dosage and mode of administration will depend on the individual. Generally, the medicament is administered so that the proteins or agents from the pharmaceutical composition of the present invention are given at a dose between 1 μg/kg and 10 mg/kg, more preferably between 10 μg/kg and 5 mg/kg, most preferably between 0.1 and 2 mg/kg. Preferably, it is given as a bolus dose. Continuous infusion may also be used and includes continuous subcutaneous delivery via an osmotic minipump. If so, the medicament may be infused at a dose between 5 and 20 μg/kg/minute, more preferably between 7 and 15 μg/kg/minute.

Another aspect of administration of HSP70 involves the use of gene therapy to deliver a polynucleotide encoding HSP70. Thus the present invention can use the nucleic acid of HSP70 for the transfection of cells in vitro and in vivo. This nucleic acid can be inserted into any of a number of well-known vectors for the transfection of target cells and organisms as described below. The nucleic acid is transfected into cells, ex vivo or in vivo, through the interaction of the vector and the target cell. Said nucleic acid, under the control of a promoter, then expresses HSP70, thereby mitigating the effects of absence or shortage of HSP70. Such gene therapy procedures have been used in the art to correct acquired and inherited genetic defects, cancer, and viral infection in a number of contexts. The ability to express artificial genes in humans facilitates the prevention and/or cure of many important human diseases, including many diseases which are not amenable to treatment by other therapies (for a review of gene therapy procedures, Nabel & Feigner, TIBTECH 11 :211-217 (1993); Mintani & Caskey, TIBTECH 11 :162-166 (1993); Mulligan, Science 926-932 (1993); Dillon, TIBTECH 11 :167-175 (1993); Van Brunt, Biotechnology 6(10):1149-1154 (1998); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1); 31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology (Doerfler & Bόhm eds., 1995); and Yu et al., Gene Therapy 1 :13-26 (1994)). Delivery of the gene or genetic material into the cell is the first critical step in gene therapy treatment of disease. A large number of delivery methods are well known to those of skill in the art. Preferably, the nucleic acids are administered for in vivo or ex vivo gene therapy uses. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in, e.g., US Pat. No. 5,049,386, US Pat No. 4,946,787; and US Pat. No. 4,897,355 and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Flegner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration). The preparation of lipid: nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710- 722 (1995); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871 , 4,261 ,975, 4,485,054, 4,501 ,728, 4,774,085, 4,837,028, and 4,946,787). The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo). Conventional viral based systems for the delivery of nucleic acids could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues. The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vector that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised on c/s-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum c/s-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); PCT/US94/05700.

In applications where transient expression of the nucleic acid is preferred, adenoviral based systems are typically used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., U.S. Patent No. 4,797,368; WO 93/24641 ; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka. Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Hermonat & Muzyczka, Proc. Natl. Acad. Sci. U.S.A. 81 :6466-6470 (1984); and Samulski et al., J.Virol. 63:03822-3828 (1989). In particular, at least six viral vector approaches are currently available for gene transfer in clinical trials, with retroviral vectors by far the most frequently used system. All of these viral vectors utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent. pLASN and MFG-S are examples are retroviral vectors that have been used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn et al., Nat. Med. 1 :1017-102 (1995); Malech et al., Proc. Natl. Acad. Sci. U.S.A. 94/22 12133-12138 (1997)); Pa317/pl_ASN was the first therapeutic vector used in a gene therapy trials. (Blaese ef al., Science 270:475-480 (1995)). Transduction efficiencies of 50% greater have been observed for MFG-S packaged vectors (Ellem ef al. Immunol Immunother. 44(1): 10-20 (1997); Dranoff et al., Hum. Gene Ther. 1 :111-2 (1997)). Recombinant adeno-associated virus vectors (rAAV) are a promising alternative gene delivery systems based on the defective and non-pathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system (Wagner et al., Lancet 351 :9117 1702-3 (1998). Replication-deficient recombinant adenoviral vectors (Ad) are predominantly used in transient expression gene therapy, because they can be produced at high titer and they readily infect a number of different cell types. Most adenovirus vectors are engineered such that a transgene replaced the Ad E1a, E1b, and E3 genes; subsequently the replication deficient vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in the liver, kidney and muscle system tissues. Conventional Ad vectors have a large carrying capacity. An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Sterman et al., Hum. Gene Ther. 9:7 1083-1089 (1998); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513 (1998)). Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. In many gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type. A viral vector is typically modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the viruses outer surface. The ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al., Proc. Natl. Acad. Sci. U.S.A. 92/9747-9751 (1995), reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other pairs of virus expressing a ligand fusion protein and target cell expressing a receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to non-viral vectors. Such vectors can be engineered to contain specific uptake sequences thought to favour uptake by specific target cells. Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intra-peritoneal, intra-muscular, sub-dermal, or intra- cranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector. Ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., via re-infusion of the transfected cells into the host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from the subject organism, transfected with a nucleic acid (gene or cDNA), and re-infused back into the subject organism (e.g., patient). Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique (3^rd ed. 1994)) and the references cited therein for a discussion of how to isolate and culture cells from patients).

In one embodiment, stem cells are used in ex vivo procedures for cell transfection and gene therapy. The advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow. Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM- CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med. 176: 1693-1702 (1992)). Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), and lad (differentiated antigen presenting cells) (see Inaba et al., J. Exp. Med. 176:1693-1702 (1992)). Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing therapeutic nucleic acids can be also administered directly to the organism for transduction of cells in vivo. Alternatively, naked DNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. The nucleic acids are administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. Materials and Methods Animals

Female C57BL/6 mice were obtained from Iffa-Credo (Saint Germain-sur-l'Arbresle, France) and were used at the age of 8-10 weeks. Hsp70.1 -deficient mice were bred as homozygotes in our facilities. Mice were kept in a temperature-controlled, air-conditioned animal house with 14-10 h light/dark cycles; they received food and water ad libitum.

Generation of hsp70.1 -deficient Mice

The mouse hsp70.1 genomic clone was isolated from a 129 strain mouse genomic library (Stratagene, La Jolla, CA) by a standard plaque hybridization procedure using a 3'- noncoding sequence of the hsp70.1 gene as specific probe. The targeting vector was constructed as follows. The hsp70.1 -coding sequence was replaced with the PMC1- NeoR gene (Stratagene) fragment from which the promoter region was removed. When homologous recombination occurs in a correct way, the NeoR fusion gene is expressed in-frame starting from the hsp70.1 promoter. E14 ES cells were transfected with 30 μg of the linear targeting vector DNA per 5 x 10⁷ cells by electroporation (800 V, 300 mF; Bio- Rad Laboratories, Richmond, CA). G418 selection (150 mg/ml) was initiated 24 h after electroporation; G418-resistant colonies were obtained after 7-10 days of selection. The targeted disruption was confirmed by Southern blot analysis. Chimeric mice were produced by injection of ES cells into F1 (C57BL/6J x CBA/CaLac) blastocysts. Heterozygotes for the targeted allele were obtained by breeding of chimeras with C57BL/6J mice (CleaJapan, Tokyo, Japan). Finally, homozygote knockout mice were produced by interbreeding of heterozygotes.

Reagents

Recombinant murine TNF and murine IFN-γ were produced in Escherichia coli and were purified to homogeneity in the Ghent laboratory. Two different batches of TNF were used. For antitumor experiments, TNF had a specific activity of 9.1 x 10⁷ lU/mg, the endotoxin contamination being <100 EU/mg. In all other experiments, TNF with a specific activity of 1.2 x 10⁹ lU/mg and an endotoxin contamination of ~6 UE/mg was used. IFN-γ had a specific activity of 1.1 x 10⁸ lU/mg. The endotoxin contamination was assessed in a chromogenic Limulus amebocvte lysate assay (Coatest; Chromogenix, Stockholm, Sweden). N-(1-naphthyl)ethylenediamine dihydrochloride, paraffin and sulfanilamide were purchased from Sigma Chemical Co. (St. Louis, MO). Histo-clear was obtained from National Diagnostics (Atlanta, GA).

HS Induction

An empty mouse cage and a tray filled with water were placed in a hybridization oven (Amersham Pharmacia Biotech, Rainham, UK) for at least 2 h at 42°C. These conditions should provide a relative humidity of 75% (Dietrich et al., 2000), which favors efficient HS (Fujio et al., 1987; Nowak et al., 1990). Mice were placed in the cage for 20 min at 42°C, after which they were transferred to a clean cage at room temperature.

Injections and Blood Collections

I.v. injections were performed in 0.2 ml and s.c. injections in 0.1 ml; i.p. injections had a volume of 0.5 ml. Prior to injection, cytokines and reagents were diluted in an LPS-free isotonic solution. Blood was taken by cardial puncture during avertin anesthesia. Blood was allowed to clot for 30 min at 37°C and for at least 1 h at 4°C, followed by two centrifugations at 20,000x g for 3 min. Serum was stored at -20°C until use.

Tissue Embedding, Tissue Sectioning and Staining

Tissues were removed after cervical dislocation and immediately fixed in a 4% paraformaldehyde solution at room temperature. After passing through baths of 50, 70, 95 and 100% ethanol and 100% Histo-clear, tissues were embedded in paraffin. 4 μm sections were prepared with a microtome, followed by hematoxylin/eosin staining. TUNEL staining was performed with the Deadend colorimetric apoptosis detection system (Promega Biotec, Madison, Wl). Tissue sections were treated according to the manufacturer's instructions. Briefly, samples were treated for 10 min with a proteinase K solution and then incubated with TdT enzyme and biotinylated dUTPs for 60 min at 37°C. After blocking the endogenous peroxidase activity, sections were incubated with horseradish peroxidase conjugated to streptavidin and developed with diaminobenzidine, yielding dark brown staining of nuclei. For immunohistochemistry, paraffin sections were incubated overnight at room temperature with a goat anti-mouse HSP70 antibody (1/50; Santa Cruz Biotechnology, Santa Cruz, CA). A rabbit anti-goat biotin-coupled antibody was used for 45 min at room temperature (1/400) as secondary antibody. The signal was amplified by tyramide signal amplification (Du Pont, Wilmington, DE); visualization was obtained with an AEC+ chromogen substrate (Dako, Carpinteria, CA).

Tumor Cell Culture and Inoculation B16BI6 melanoma cells, PG19 melanoma cells and Lewis Lung Carcinoma (LLC) cells were kept in culture in DMEM. Cells were harvested by treatment for 5 min with cell dissociation buffer (Life Technologies, Paisley, UK). After three washes in an LPS-free isotonic solution, cells were counted and brought at a concentration of 6 x 10⁶/ml for the B16BL6 cells and 5 x 10⁷/ml for PG19 and LLC cells. They were injected s.c. in 100 μl into the back right limb.

TSI, Bodyweight and Body Temperature

TSI (tumor size index) was obtained by multiplying the smaller and larger diameters of the tumor. Bodyweight and rectal body temperature were recorded with an electronic balance (Mettler Toledo, Prague, Czech Republic) and an electronic thermometer (model 2001 ; Comark Electronics, Littlehampton, UK), respectively.

Tissue Isolation and Homogenization

Tissues were homogenized with a homogenizer (model RZR 2020 from Heidolph- Instruments, Kelheim, Germany) in 2 ml ice-cold glycerol buffer (10% glycerol, 5 mM EDTA, 10 mM Tris/HCI pH 7.4, 200 mM NaCI), supplemented with 1 mM PMSF. The homogenates were centrifuged at 13,000x g for 20 min at 4°C; the supernatant was stored at -20°C until further analysis.

Assays

Serum IL-6 was determined with a 7TD1 bioassay (Van Snick et al., 1986). Nitrate and nitrite levels (NO_x ^") in the serum were assessed as previously described (Granger et al., 1991). This procedure, slightly adapted by us, is based on the detection of nitrite by a complex reaction resulting in a dark purple color (Griess, 1879). 30 μl of each sample or a nitrate standard was transferred to a V-bottom 96-well plate. As blanks, serum from untreated animals was used. To reduce nitrate to nitrite (nitrate as such cannot be determined with this method), 30 μl of a bacterial suspension of nitrate reductase containing Pseudomonas oleovorans was added at 5.0 x 10⁹ CFU/ml, followed by incubation at 37°C for 4 h. Plates were centrifuged at 1 ,300x g for 5 min; 40 μl of supernatant was transferred to a second V-bottom plate. Detection of nitrite was achieved by adding 80 μl of Griess reagent, consisting of 1 vol of 1% sulfanilamide in 5% phosphoric acid and 1 vol of 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in bidistilled water. After thorough mixing, 10% TCA was added for protein precipitation and the plate was centrifuged at 1,300x g for 15 min. Finally, 120 μl supernatant was removed and added to a flat bottom plate, which was read at 540 nm (test) and 655 nm (background).

Western Blot 50 μg of total protein of the different tissue homogenates was loaded on a 7.5% polyacrylamide gel and run at 150 V. After electrophoretic separation, proteins were blotted on a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) for 2 h at 120 mA. Blots were blocked with 1% BSA and 0.1% Triton X-100 overnight at 4°C. They were incubated for 2 h at room temperature with a biotinylated mouse anti-HSP70 antibody (SPA-810B; Stressgen Biotechnologies Corporation, Victoria, Canada), diluted 1/3,000 in BSA/Triton X-100. After three washes, blots were incubated with streptavidin- AP (1/1500; BioSource International, Camarillo, CA) and developed with NBT/BCIP (Roche Molecular Biochemicals, Basel, Switzerland).

DNA Fragmentation ELISA

20%-homogenates of jejunum samples were made in glycerol buffer supplemented with 1 mM PMSF, 0.3 mM aprotenin, 1 mM leupeptin and 1 mM oxidized glutathione, and centrifuged for 30 min at 13,000x g. The supernatant was stored at -20°C. DNA fragmentation was quantified by immunochemical determination of histone-complexed DNA fragments in a microtiter plate (Salgame et al., 1997). Briefly, plates were coated with an antibody against histone H2B. After blocking, homogenates were added and a biotinylated detection antibody specific for the nucleosome subparticle of histones H2A, H2B and DNA (Losman et al., 1992) was administered. Detection was performed with alkaline phosphatase-conjugated streptavidin (Sanvertech, Boechout, Belgium) and substrate (Sigma). Jejunum from PBS-treated animals was regarded as 100%.

Fluorogenic Substrate Assay for Caspase Activity

Caspase-like activities were determined by incubation of jejunum homogenate (containing 50 μg of total protein) with 50 μM of the fluorogenic substrate Ac-DEVD-amc (Peptide Institute, Osaka, Japan) in 150 μl cell-free system buffer containing 10 mM HEPES, pH 7.4, 220 mM mannitol, 68 mM sucrose, 2 mM NaCI, 2.5 mM KH₂P0₄, 0.5 mM EGTA, 2 mM MgCI₂, 5 mM pyruvate, 0.1 mM PMSF and 1 mM DTT. Release of fluorescent amc was measured for 1 h at 2-min intervals by fluorometry (Cytofluor; PerSeptive Biosystems, Cambridge, MA). Data are expressed as units of cleavable ac- DEVD-amc activity after application of a serial dilution of free amc as a standard, where one unit is the activity releasing 1 pmol/amc in 1 min at 30°C.

Statistical Analysis Survival curves (Kaplan-Meyer plots) were compared using a log-rank test. Final lethality was compared with a chi² test. Means ± SD were compared with a student's t-test. ^*, ** and *** represent p = 0.01-0.05, p = 0.001-0.01 and p <0.001 , respectively.

Examples Generation of hsp70.1 -deficient Mice

The hsp70.1 genomic structure, the targeting vector and the targeted allele are shown in Figure 1A. After restriction digest of genomic DNA with Apal and hybridization with probe A, a 6.7-kb fragment was obtained for wild type (wt) mice. The targeted allele provided a fragment of 9.3 kb due to loss of the unique Apal site in the hsp70.1 gene by homologous recombination with the targeting vector (Figure 1A). The latter was constructed with the neomycin-resistant (NeoR) gene without promoter as positive selection marker. Thus the NeoR gene can be expressed from the hsp70.1 gene promoter, provided accurate homologous recombination had occurred. The frequency of the homologous recombination was 2/78 of G418-resistant clones. Two cloned embryonic stem (ES) cells were injected into blastocysts. The resulting chimera mice bred, so that hsp70.1 heterozygous mice (+/-) were obtained. After 8 generations of backcross with C57BL/6J mice, hsp70.1 homozygous mice (-/-) were generated from crossbreeding between heterozygotes. The hsp70.1 (+/+), (+/-), (-/-) genotypes were born according to expected Mendelian ratios of 1 :2:1. Screening of ES cells and mice was performed by Southern blot, yielding a single fragment of 6.7 kb for wt, a 9.3 kb fragment for knockout and both fragments for heterozygote mice (Figure 1B). To further confirm the targeted disruption of the hsp70.1 gene, we examined hsp70.1 mRNA transcription by Northern blot analysis (Figure 1C). Cultures of wt, homozygous and heterozygous embryonic fibroblasts were cultured at 42°C for 1 h, followed by 12-h incubation at 37°C. Probe B, specific for the hsp70.1 gene, hybridized with the 3.0 kb mRNA for the wt allele and the 2.4 kb mRNA for the targeted allele. These results confirm that correct homologous recombination had occurred in ES cells. Hsp70.1 gene knockout mice developed normally and were fertile. Gross and histopathological observations revealed no apparent differences between knockout and wt mice under specific pathogen-free conditions.

HS Treatment of Mice Induces HSP70 in Several Organs

A number of different methods to induce HS have been described previously. Some of these methods demonstrate HSP70 induction in several organs in rats and mice (Hotchkiss et al., 1993; Kume et al., 1996). First, we evaluated the increase in core body temperature during the HS induction method used. Mice (n = 12) were HS treated for 20 min. The rectal body temperature was monitored from HS start until 10 min after HS end and was recorded every 2 min. The body temperature increased in a linear way up to 10 min after HS start and then displayed a plateau level of about 41.5°C until HS end. 6 min after HS end, all mice had regained their normal body temperature of approximately 37°C (Figure 2A). To test whether the HS conditions used had led to a strong HSP70 induction, mice were HS treated for 20 min; 2, 6, 12, 24, 48, 72 or 96 h thereafter mice were killed. Seven organs (liver, lung, duodenum, jejunum, colon, heart and spleen) were removed and homogenized in glycerol buffer. The homogenates were analyzed by Western blot for HSP70 presence (Figure 2B). HSP70 induction in the liver was clear at the earliest timepoint. The expression was maximal between 6 and 24 h following HS and diminished after 48 h. In control mice (not subjected to HS), no detectable HSP70 was present. A similar HSP70 induction profile was also observed in lung samples, high levels being reached from 6 to 24 h after HS. In the duodenum, HSP70 was detected only between 6 and 12 h after HS. A very high expression was also observed in the jejunum 6 and 12 h after HS. In the colon, the expression was low at 2 h, but remained strong 6 to 48 h after HS. In the heart, there was only induction 24 h after HS, while no HSP70 could be detected in the kidney (results not shown). These data demonstrate that the method used for HS application results in a high increase in core body temperature and in a strong HSP70 induction in several organs.

HS Induction Prevents TNF-induced Lethality

Based on our data on HSP70 induction, we evaluated the effect of HSP70 upregulation on TNF-induced lethality. In most of the organs tested, the induction is maximal from 6 to 24 h after HS. Therefore, we subjected mice to HS and challenged them i.v. with 15 μg TNF, i.e. a 100% lethal dose, 6 h (n = 8), 12 h (n = 45) or 24 h (n = 6) later. Mice were also challenged with TNF 2 h (n = 6) or 48 h (n = 6) after HS (low or no expression of HSP70). As controls, mice (n = 44) were challenged with TNF without prior HS. We show that mice are very significantly protected from TNF-induced lethality when treated with TNF 12 h after HS (Figure 3): a statistically significant difference was observed, both in survival time (p <0.0001) and in final lethality (12/45 dead HS-pretreated mice vs 44/44 dead control mice; p <0.0001). When mice were challenged with TNF 6 h after HS, a highly significant delay in lethality (p = 0.0066) was found. However, eventually all mice died as a result of TNF toxicity. No significant protection was observed when mice were challenged 2, 24 or 48 h after HS. From these data we conclude that mice are maximally protected 12 h after HS treatment.

Effect of HS on TNF-induced Hypothermia, NO and IL-6 Injecting mice with a high (lethal) dose of TNF results in massive production of NO (leading to hypotension) and IL-6. High levels of nitrate and nitrite (stable end products of NO) and IL-6 were observed in the serum after TNF challenge (Van Molle et al., 1997). It is believed that sustained levels of IL-6 (Libert et al., 1990) and NO are representative of a lethal outcome. First we studied the effect of HS treatment on the drop in temperature induced by TNF injection. Mice were HS pretreated (n = 12) and challenged i.v. 12 h later with 15 μg TNF. Control mice (n = 12) received TNF without prior HS. HS-pretreated mice have significantly less TNF-induced hypothermia compared to mice challenged with TNF without prior HS treatment (p <0.001 from 6 to 28 h after the challenge) (Figure 4A). In both groups the body temperature dropped, but HS-pretreated mice recovered between 12 and 24 h after the challenge. By 48 h after the TNF challenge, all HS- pretreated surviving mice had regained normal body temperatures. In order to study the effect of HS on TNF-induced NO and IL-6, mice were pretreated with HS (n = 30) and challenged 12 h later i.v. with 15 μg TNF. Control mice (n = 30) were challenged i.v. with TNF without HS pretreatment. 1 , 3, 6, 9, 12 or 24 h after the challenge blood was taken by cardial puncture (6 mice per timepoint) and serum was prepared. HS pretreatment significantly reduced the NO production (Figure 4B). A highly significant difference was found 9 h (p = 0.0019), 12 h (p <0.0001) and 24 h (p <0.0001) after the challenge for HS- pretreated vs control mice. We also observed a strong inhibiting effect of HS on TNF- induced IL-6 production. IL-6 induction (Figure 4C) was significantly lower in HS- pretreated mice than in control mice at 3 h (p = 0.0233), 6 h (p = 0.0025), 9 h (p = 0.0042), 12 h (p = 0.0002) and 24 h (p = 0.0001) after TNF challenge. These data demonstrate that HS pretreatment significantly prevents TNF-induced NO and IL-6 production.

HS Prevents TNF-induced Bowel Damage

Challenging mice with TNF results in severe bowel swelling and damage (Tracey et al., 1986; Piguet et al., 1998). To evaluate the HS effect on TNF-induced bowel damage, mice were subjected to HS (n = 12), followed after 12 h by an i.v. challenge with 15 μg TNF. Control mice (n = 12) were challenged i.v. with 15 μg TNF without prior HS and killed after 1 h. The small intestine (duodenum, jejunum and ileum) was removed and weighed with its content (n = 12), which was significantly high (p <0.0001 compared to mice treated with PBS). HS pretreatment greatly prevented exudation (p <0.0001 compared to mice treated with TNF without prior HS) (Figure 5A). The small intestine is indeed known to increase dramatically in weight at early timepoints (0.5-1.5 h) after TNF challenge due to excessive fluid exudation (Piguet et al., 1998).

In order to investigate in more detail the TNF-induced tissue damage and HS- induced protection, parts of the jejunum were removed and 20%-homogenates (n = 6) were made. The extent of apoptosis was determined using a DNA fragmentation ELISA. The degree of background apoptosis in PBS-treated mice was regarded as 100%, being a measure for normal physiologically occurring apoptosis. TNF significantly increased the extent of apoptosis in the jejunum (p = 0.0029 as compared to PBS treatment), while HS pretreatment significantly inhibited this tissue damage (p = 0.0232 as compared to TNF treatment without prior HS) (Figure 5A). Since activation of caspases is a typical hallmark for apoptosis, we also determined the caspase activity in the homogenates. Cleavage of the chromogenic substrate acetyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)- aminomethylcoumarin (ac-DEVD-amc) is a measure of caspase-3 and caspase-7 activity, both known as executioner caspases. In homogenates of mice treated with PBS (n = 6), there was some background caspase-like activity, but that activity was significantly increased after TNF challenge (p = 0.0053 as compared to PBS treatment) (Figure 5A). HS pretreatment prevents apoptosis, since DEVDase activity was significantly lower as compared to a TNF challenge without prior HS (p = 0.0030).

To investigate the TNF-induced tissue damage at the cellular level, parts of the jejunum were removed and stained with hematoxylin/eosin, analyzed by immunohistochemistry for HSP70 induction or analyzed for apoptosis by TUNEL assay. Treatment with TNF (Figure 5C) resulted in severe damage of the jejunum compared to untreated animals (Figure 5B). The villi are flattened due to loss of their upper part with denudation at the top. The crypts are distended, and contain mucus and debris. We also observed loss of goblet cells. When mice were HS-pretreated 12 h before the TNF challenge, the damage was markedly reduced (Figure 5D). There is less shortening of the villi and the crypts retain their normal architecture. Tissue sections were also analyzed by TUNEL assay to stain apoptotic cells. In control mice, no TUNEL staining was found (Figure 5E); in contrast, the top of the villi of TNF-treated mice showed positive TUNEL staining, as visualized by brown nucleus staining (Figure 5F, arrows). In HS- pretreated mice, TUNEL staining was completely absent (Figure 5G). With immunohistochemistry we only observed detectable HSP70 in the jejunum from HS- treated mice (Figure 5J), while only background staining was detected in PBS-injected mice (Figure 5H) and TNF-injected mice (Figure 51) without prior HS. The expression was maximal at the top of the villi, demonstrating that HSP70-expressing cells were protected against TNF-induced apoptosis. These data demonstrate that HS pretreatment completely prevents the TNF-induced tissue damage (apoptosis) at the site of the jejunum.

HS-induced Protection Is Abrogated in hsp70.1 -deficient Mice

In order to investigate whether induction of HSP70 is responsible for HS-conferred protection, we used hsp70.1 -deficient mice. First, we analyzed the induction of HSP70 in hsp70.1 -deficient mice generated after HS. Wt and hsp70.1 -deficient mice were HS treated and 12 h later sacrificed to remove liver, jejunum and colon (organs where we showed strong HSP70 induction after HS). While induction of HSP70 is very obvious in liver, jejunum and colon of wt mice, no induction whatsoever was found 12 h after HS (Figure 6A). These data illustrate that induction of HSP70 is completely absent in hsp70.1 -deficient mice 12 h after HS treatment.

Wt (n = 13) and hsp70.1 -deficient mice (n = 12) were subjected to HS and 12 h later challenged with 15 μg TNF. As control groups we also treated wt (n = 13) and hsp70.1- deficient (n = 12) mice without prior HS with 15 μg TNF (we observed no difference in sensitivity to TNF between wt and hsp70.1 -deficient mice; results not shown). HS treatment significantly prevented TNF-induced lethality in wt mice (p <0.0001), while protection was completely absent in hsp70.1 -deficient mice (p >0.05) (Figure 6B). We also observed that the drop in body temperature induced by TNF in deficient mice was not inhibited by HS treatment (results not shown). These data demonstrate that induction of HSP70 is very crucial for protection conferred by HS treatment.

HS Application in an Antitumor Protocol

Inoculation of C57BL/6 mice with B16BI6 melanoma cells is a syngeneic tumor model. Application of TNF in combination with IFN-γ induces regression of these tumors, but is accompanied with high mortality (Brouckaert et al., 1986). In order to reduce or inhibit this toxicity, we evaluated the application of HS induction in an antitumor protocol. Since this experiment involves a 10-day HS treatment, we investigated whether ten consecutive HS treatments led to a feedback on HSP70 induction, resulting in lack of protection. To that end, mice (n = 11) were HS treated for 10 consecutive days and were injected i.v. with 15 μg TNF 12 h after the tenth HS treatment. As controls, mice were treated with 15 μg TNF 12 h after a single HS treatment (n = 12) or without HS treatment (n = 12). To analyze hsp70 induction, liver, jejunum and colon were removed from parallelly HS-treated mice at the time of TNF injection (12 h after HS). A single HS treatment induced high HSP70 levels; after 10 consecutive HS treatments, the expression of HSP70 was still high, even higher as compared to a single HS treatment. Furthermore, the increased number of HS treatments led to a significant protection against TNF lethality, although statistically not better than a single HS treatment. We also observed a better protection against TNF- induced hypothermia. 9 and 12 h after TNF challenge, a significant difference in body temperature was found between mice treated 10 times or only once with HS (p = 0.0041 and p <0.0001 for 9 and 12 h, respectively). These data indicate that repeated HS treatments do not lead to downmodulation of hsp70 expression or tolerization against HSP70 induction, and that increased HSP70 levels provide a better protection (dose response between HSP70 levels and degree of protection).

In a further experiment 3 different tumor models were used: B16BI6 melanoma model, PG19 melanoma model and Lewis Lung Carcinoma tumor model (LLC). The results are shown in figure 7 and the legend of figure 7 describes the experimental set-up in detail. The combination of TNF and IFN-γ can be administered sub-cutaneously in the vicinity of the tumor or intravenously however at least in our models here we used the former application since it allowed the best tumor regression. Mice treated with PBS displayed drastic tumor growth, while TNF/IFN-γ treatment led to complete regression of the tumor as compared to PBS-treated mice. Importantly, HS pretreatment did not inhibit this anti- tumor activity. HS induction had a significantly protective effect against lethality induced by a TNF/IFN-γ treatment. A significant difference, both in survival time and final lethality, was found compared to TNF/IFN-γ-treated mice without prior HS. These data demonstrate that HS induction not only prevents TNF-induced lethality, but also allows application of TNF in combination with IFN-γ as a safe anti-tumor strategy.

Recombinant HSP70 and adenoviral vectors comprising HSP70

We will make use of the B16BI6 melanoma, PG19 melanoma and Lewis lung carcinoma (LLC) mouse models. We have already seen that application of heat shock allows a safer ant-tumor therapy with TNF in combination with IFN-gamma. We will now also apply recombinant HSP70 and adenoviral vectors comprising HSP70 as adjuvant during therapy with TNF and IFN-gamma.

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Claims

Claims

1. A pharmaceutical composition comprising HSP70 and TNF wherein said HSP70 is endogenously induced by heat.

2. A pharmaceutical composition according to claim 1 further comprising interferon-gamma.

3. A pharmaceutical composition according to claim 1 or 2 further comprising a chemotherapeutic compound.

4. A pharmaceutical composition according to claims 1-3 for use as a medicament.

5. Use of a pharmaceutical composition according to claims 1-3 for the manufacture of a medicament for systemic tumour treatment.