WO2006128679A2 - Phosphatidylinositol composition and 3'-phosphorylated lysophosphatidylinositols in the treatment of cancer and autoimmune diseases - Google Patents

Abstract

The invention relates to a combination or a combination compound comprising a 31-phosphorylated form of phosphatidylinositol and a secretory (s) phospholipase A2. Also provided are pharmaceutical compositions comprising 3'-phosphorylated lyso-phosphatidylinositols. Furthermore, the invention relates to a pharmaceutical composition comprising the combination or combination compound of the invention. Moreover, the present invention provides for the use of a combination or a combination compound of the invention, for a preparation of a pharmaceutical composition for the treatment of a proliferative disorder and/or tumorous disease. Moreover, the present invention provides for a method for the generation of a cellular lysate of normal or tumorous/tumor cells comprising the steps of: (a) contacting non-tumorous cells or tumorous/tumor cells with the combination or combination compound of the invention; and (b) obtaining the resulting cellular lysate.

Description

Phosphatidylinositol combination composition and 3'-phosphorylated lyso- phosphatidylinositols in the treatment of cancer and autoimmune diseases

The invention relates to a combination or a combination compound comprising a 3¹- phosphorylated form of phosphatidylinositol and a secretory (s) phospholipase A2. Also provided are pharmaceutical compositions comprising 3'-phosphorylated lyso- phosphatidylinositols. Furthermore, the invention relates to a pharmaceutical composition comprising the combination or combination compound of the invention. Moreover, the present invention provides for the use of a combination or a combination compound of the invention, for a preparation of a pharmaceutical composition for the treatment of a proliferative disorder and/or tumorous disease. Moreover, the present invention provides for a method for the generation of a cellular lysate of normal or tumorous/tumor cells comprising the steps of: (a) contacting non- tumorous cells or tumorous/tumor cells with the combination or combination compound of the invention; and (b) obtaining the resulting cellular lysate.

Cancer is among the leading causes of death in industrialized nations. As treatment options for and prevention of infectious diseases and cardiovascular disease continue to improve, and the average life expectancy increases, cancer is likely to become the most common fatal disease. In developed countries, about one person in three receives a diagnosis of cancer during his or her lifetime and almost one in four dies from it. Malignant tumors are divided into carcinomas (which arise from epithelial precursor cells), sarcomas (which arise largely from mesenchymal tissues) and lymphomas (which arise from precursors of red and white blood cells). Therefore, curing cancer requires that all the malignant cells be removed or destroyed without killing the patient. The overt manifestation and initial clinical presentation of cancer usually occur at a late stage in the disease process when the capacity for invasion has already been unleashed. By the time of diagnosis, a high proportion of patients have occult or even clinically detectable metastases. The capacity of conventional cytotoxic approaches to succeed in the face of this advanced, accelerating disease has been limited (Astrow, Lancet, 343: 494-495, 1994; Bailar, N Engl J Med, 314: 1226-1232, 1986).

Frequent failure of conventional approaches such as chemotherapy or hormone therapy have led to the development of immunotherapeutic regimen for the treatment of metastatic cancers. In strongly varying protocols, cytokines, lymphocytes and modified tumor cells have been applied to mobilize the immune system against the growing tumor. Nonspecific immunotherapy has administered cytokines like interferon-alpha (Coppin, Cochrane Database Syst Rev CD001425, 2000.) or interleukin-2 alone (von der Maase, Eur J Cancer, 27: 1583-1589, 1991; Margolin, Semin Oncol, 27: 194-203, 2000) or in combination (McDermott, J Clin Oncol, 23: 133-141 , 2005). Cytokine-based immunotherapy is limited by sometimes severe side effects, barely tolerable by the mostly elderly patients. The passive transfer of tumor- infiltrating lymphocytes (TIL) or lymphokine-activated killer (LAK) cells activated and expanded in vitro turned out to be ineffective (Margolin, 2000, loc. cit.). In the active, specific immunotherapy the immune system is prompted to specifically target and attack distinct tumor-associated antigens. This approach is a vaccination by nature although therapeutic (as opposed to prophylactic vaccination such as the known vaccinations which serve to prevent infectious diseases). The advantage of the active, specific immunotherapy is in its specificity and in the development of an immunologic memory that should be able to prevent recurrence of the disease. Within this category, gene-modified tumor cells producing GM-CSF, have been used to vaccinate cancer patients against their tumors (Simons, JCancer Res, 57: 1537- 1546, 1997). In this trial, biopsies of intradermal sites of injection with GM-CSF gene- modified tumor cells contained distinctive dendritic cell infiltrates pointing toward an important role of these cells in the induction of tumor immunity. Dendritic cells are indeed highly attractive for the active specific immunotherapy since their natural function is to induce antigen-specific immunity (Banchereau, Nature, 392: 245-252, 1998).

Also proteosome modulating compounds have been described in anticancer therapy. For example, WO 2004/004711 describes two anticancer compounds modulating proteasome activity for preparing a medicament for cancer treatment. The proteasome modulating compounds may be, inter alia, an intercolant.

DCs affect both innate and adaptive immune responses (Bancherau, 1998 loc. cit.). DCs elicit and tune antigen-specific T and B cell responses. According to a well accepted concept that has emerged during recent years the maturation state of DCs swings the decision between tolerance and immunity ( Steinman, Annu Rev Immunol, 21: 685-711 , 2003). While immature DCs maintain tolerance by silencing antigen- specific T lymphocytes or by actively converting them into regulatory T cells, mature DCs promote immunity. Maturation is induced by a multitude of signals frequently referred to as "danger" signals (Matzinger, Science, 296: 301-305, 2002) and DCs have learned to respond to these signals using various receptors of the TNF or toll- like receptor families (Steinman, 2003, loc. cit.). A hallmark of "danger" is inflammation. Infectious agents usually provoke inflammatory responses characterized by the production of many cytokines including TNF-alpha and IL-1β which are both known to trigger DC maturation (Sallusto, J Exp Med, 179: 1109- 1118, 1994).

During the last decade the enhanced interest in DCs was accompanied by a boost of the research in basic and clinical immunology and by a re-emergence of interest in the tumor immunosurveillance concept. After periods of considerable scepticism, today the issue is not if - but, rather, how - tumor immunosurveillance works to prevent the development of cancer in the immunocompetent host (Dunn, Immunity, 21: 137-148, 2004). Lymphocytes of the innate (NK, NKT cells and gamma-delta T cells) and adaptive immune system (gamma-delta T cells) are required to prevent the development of both chemically-induced and spontaneous tumors. The two critical functions of these lymphocytes in tumor cell eradication are the ability to produce IFN-gamma and the ability to kill.

The fact that DCs activate and/or modulate all these lymphocyte subsets has stimulated great interest in DC-based immunotherapeutic approaches to control and/or eliminate human tumors. The cumbersome attempt to validate the concept of DC-based immunotherapy of human tumors in clinical trials has recently been initiated (Schuler, Curr Opin Immunol, 15: 138-147, 2003). The discovery that DCs can be cultured in sufficient numbers from abundant circulating precursor cells paved the way for clinical research. A frequently used protocol is based on a two step culture system (Sallusto 1994, loc. cit., Romani, J Exp Med, 180: 83-93, 1994): in the first step monocytes are differentiated into immature DCs (= monocyte-derived DCs, moDCs) in the presence of GM-CSF and IL-4. Then, inflammatory stimuli induce a maturation step, which is characterized by a re-programming that enhances the immunostimulatory potential of the DCs, in healthy individuals and in cancer patients (Thurnher, Exp Hematol, 25: 232-237, 1997; Radmayr, lnt J Cancer, 63: 627-632, 1995). In addition to the monocyte pathway, DCs can be cultured from early hematopoietic progenitor cells (Banchereau 1998, loc. cit.).

The immunity-initiating potential of DCs is currently exploited in clinical settings. In such studies DCs are generated from patient peripheral blood precursor cells in vitro, loaded with antigens, matured and injected back into the patient to treat diseases like cancer (Schuler, 2003, loc. cit., HoItI, Lancet, 352: 1358, 1998; HoItI, J Urol, 161: 777 '-782, 1999; HoItI, Clin Cancer Res, 8: 3369-3376, 2002; HoItI, Cancer Immunol Immunother, 2004).

Ramoner (2005) Blood 105:3583-3587 suggests a vaccine based on dendritic cells to be administered to the treatment of cancer-like diseases.

Perrin-Cocon (2004) Eur. J. Immunol. 34:2293-2302 describes a stimulatory effect of sPLA2 on DCs.

In de Vries (2003) Clin. Cancer Res. 9:5091-100, the maturation of dendritic cells is described to be a prerequisite for inducing immune responses, at least in advanced melanoma patients.

Schnurr (2001) Cancer Res. 61 :6445-50 shows that T cells specific for pancreatic carcinoma cells can be generated in vitro by lysate-pulsed dendritic cells. The use of unfractionated tumor-derived antigens in the form of tumor cell lysates (or whole tumor cells) is proposed. Moreover, the provision of a tumor vaccine against pancreatic carcinoma based on DCs is suggested.

In WO 03/006634 the use of oxidized lipoproteins for differentiation of precursor cells into mature dendritic cells (DC) is described, whereby oxidized lipoproteins are selected among VLDL oxidized and/or IDL oxidized and/or LDL oxidized lipoproteins. The precursor cells are preferably monocytes.

Phospholipases A2 (PLA2; phosphatidylcholine-2-acylhydrolase, EC 3.1.1.4) represent a growing family of enzymes that catalyze the hydrolysis of the sn-2 fatty acyl ester bond of membrane glycero-3'-phospholipids to release free fatty acids and lysophospholipids such as arachidonic acid (AA) and lysophosphatidylcholine (lyso- PC) (Balsinde, FEBS Lett, 531: 2-6, 2002; Murakami, Crit Rev Immunol, 17: 225-283, 1997; Lambeau, Trends Pharmacol Sci, 20: 162-170, 1999). PLA2 have been assigned to several groups and classified according to cellular localization, amino acid sequence, molecular mass, and calcium (Ca²⁺) requirement for enzymatic activity (Six, Biochim Biophys Acta, 1488: 1-19, 2000). The extracellular or secreted PLA2 (sPLA2) are characterized by high disulfide bridge content, low molecular mass (as 14 kD), the requirement of millimolar concentration of Ca²⁺ for catalysis, and wide fatty acid selectivity in vitro. sPLA2 products such as AA and lyso-PC are themselves potent mediators that have been implicated in the regulation of cellular functions (Serhan, Faseb J, 10: 1147-1158, 1996). In addition AA and lyso-PC are subject to further metabolism. While AA can be converted into eicosanoids including the prostaglandins, leukotrienes, thromboxanes and lipoxins, lyso-PC can be metabolized to generate lysophosphatidic acid (LPA) or platelet-activating factor (PAF) (Serhan, 1996, loc. cit.).

Several mammalian and venom sPLA2s have been identified (Valentin, Biochimie, 82: 815-831 , 2000). The different venom sPLA2 have been classified into four groups based on their primary structures. The bee venom (bv) sPLA2 was the founding member of group III. The overall three-dimensional structure of bv-group III sPLA2 reveals the striking features of the PLA2 fold including a well-defined Ca²⁺-loop, three large α-helices, and a ^β wing-like structure. Group III sPLA2 from bee venom is made up of about 135 amino acids and has 5 disulfide bridges. Consistent with these catalytic and structural similarities between venom and mammalian sPLA2, evidence has been obtained that venom sPLA2 can indeed behave like mammalian sPLA2 and induce physiologic effects such as migration of endothelial cells and neurite outgrowth (Rizzo, Blood, 96: 3809-3815, 2000; Nakashima, Biochem J, 376: 655- 666, 2003; Nakashima, Brain Res, 1015: 207-211 , 2004).

WO 01/59129 describes a mammalian secreted group III phospholipase A2 (PLA2) and the use thereof in methods for screening various chemical compounds and mentions a method for identifying a biologically active compound capable of inhibiting the catalytic activity of sPLA2. Also described is a pharmaceutical composition containing the identified compound for treating diseases, like cancer.

Thorne (1996) J. Virol. 70:8502-7 discusses the lysis of virus-infected cells induced by the activity of cytosolic PLA2 (cPLA2).

It is discussed that the apoptotic death of (virus-) infected cells generates signals that can be perceived by immune cells. Furthermore these signals might, next to the indication of the location of a virus, target infected tissue for removal by phagocytes.

In Voelkel-Johnson (1996) J. Immunol. 156:201-7 it is suggested that the activity of cPLA2 is both necessary and rate-limiting for programmed cell death.

Masamune (2001) Pancreas 22:75-83 describes the induction of apoptosis in rat pancreatic AR42J cells by lysophosphatidylcholine (lyso-PC). Lyso-PC is therein generated by the activity of PLA2 on phospholipids. PLA2 catalyzes hydrolysis of phosphatidyl-choline (PC) and produces lyso-PC. However, lyso-PC-induced apoptosis described by Masamune (2001) loc. cit. only occurs at elevated concentrations.

Shaposhnikova (2001) Ivz. Akad. Nauk. Ser. Biol. 2:249-52 relates, according to Masamune (2001) loc. cit. also to the induction of tumor cell death. While Masamune (2001) loc. cit. attributes the cell death to high concentrations of lyso-PC generated by the activity of PLA2 on phospholipids, Shaposhnikova (2001) describe high concentrations of PLA2 inducing apoptotic cellular death.

You (2004) Zhonghua Zhong Liu Za Zhi 26:333-6 summarizes the generation of T cell-mediated antitumor response in vitro by autologous dendritic cells pulsed with tumor lysates in patients with non-small cell lung cancer.

Watala (1990) Comp. Biochem. Physiol. C. 97:187-94 investigated the hemolytic potency and phospholipase activity of some crude venoms derived from bee and wasps. It is said that the incubation of red blood cells with purified bee venom PLA2 was not accompanied by lysis and, when supplemented with purified melittin, the increase of red blood cells was approximately 30%.

Melittin is said to be the principal protein component of bee venom and is thought to function as a lytic agent (Terwilliger (1982), J. Biol. Chem. 257:6010-6015). In particular, melittin apparently activately PLA2 (Shaposhnikova (1998), Ivz. Akad. Ser. Biol.; Saini (1999), Toxicon. 37:1605-19). In Saini (1999) it is reported that transient activation of phospholipase D (PLD) by melittin at the point of initiation of cytolysis, suggested a role for PLD in melittin-mediated membrane disruption/cytolysis by an uncharacterized signal transduction mechanism. However, Shaposhnikova (1998) loc. cit. and Shaposhnikova (1997); FEBS Lett. 410:285-8 teach that melittin can induce necrosis but does not induce apoptosis, even at high concentrations it inhibits apoptosis.

Phosphatidylinositol (Ptdlns) and its phosphorylated derivatives are collectively referred to as phosphoinositides (PIs) (Vanhaesebroeck, Annu Rev Biochem, 70: 535-602, 2001). Phosphoinositides are quantitatively minor phospholipids of cell membranes but their metabolism is highly active and accurately controlled. They exert their role either as precursors of second messengers such as inositol 1 ,4,5- trisphosphate (lns(1 ,4,5)P3) and diacylglycerol (DAG) or directly by interacting with proteins and orchestrating the spatio-temporal organization of key intracellular signal transduction pathways (Payrastre, Cell Signal, 13: 377-387, 2001 ; Toker, Cell MoI Life Sci, 59: 761-779, 2002). Ptdlns, the basic building block for the intracellular inositol lipids in eukaryotic cells, consists of D-myo-inositol-1 -phosphate (lns(1)P) linked via its phosphate group to diacylglycerol. The inositol head group of Ptdlns has five free hydroxyl groups, as many as three of which have been found to be phosphorylated in cells, in different combinations. The 2 and 6 positions in these lipids have not been documented to become esterified with phosphate. PIs all reside in membranes and are substrates for kinases, phosphatases, and lipases resident in or recruited to these membranes (Vanhaesebroeck, 2001 , loc. cit.).

Ptdlns is the most abundant Pl in mammalian cells (approximately 10% of total cell glycerophospholipids) and can undergo sequential and reversible phosphorylations at position 3, 4 and 5 by specific kinases. Ptdlns(4)P, Ptdlns(4,5)P2 (each representing about 10% of total PIs) and Ptdlns are kept in a steady state in cell membranes because of continuous phosphorylation/dephosphorylation reactions by specific 4 and 5 -kinases and -phosphatases which are not yet fully identifed. The pool of Ptdlns(4,5)P2 can serve as a substrate for phospholipases C (PLCs) which, upon agonist-dependent activation, make the second messengers lns(1 ,4,5)P3 and DAG (Payraste, 2001 , loc. cit.; Toker, 2002, loc. cit. Berridge, Annu Rev Biochem, 56: 159-193, 1987). Ptdlns, Ptdlns(4)P and Ptdlns(4,5)P2 can also be phosphorylated by a family of Pl 3-kinases displaying different substrate specificity (Katso, Annu Rev Cell Dev Biol, 17: 615-675, 2001). As clearly shown in the last years, Pl 3-kinases play a critical role in agonist-stimulated signaling pathways (Rameh, J Biol Chem, 274: 8347-8350, 1999). The best-known Pl 3-kinase products, Ptdlns(3,4)P2 and Ptdlns(3,4,5)P3, are rapidly and transiently produced in response to agonist-mediated cell activation (classically, their level never exceed 10 % of Ptdlns-(4,5)P2). Ptdlns(3)P is constitutively present in small quantities and its level is generally rather stable in mammalian cells, although minor changes are noted in some conditions. Ptdlns(5)P and Ptdlns(3,5)P2 are also quantitatively minor but their level can change upon cell activation. The 3'-phosphorylated PIs, and also Ptdlns(4,5)P2, can specifically interact with protein-targeting modules. Through high- affnity lipid-protein interactions, newly synthesized PIs can locally recruit specific signaling proteins in response to extracellular stimuli. Thus, Pl-metabolizing enzymes, which allow rapid and reversible formation of microdomains enriched in specific Pl species, provide a powerful system to spatially restrict and regulate membrane signals (Pendaries, FEBS Lett, 546: 25-31 , 2003).

In many instances, the link between Ptdlns(3)P biology and the function of these proteins remain obscure. Pl 3-kinase products often are associated with cell survival and stimulation of proliferation (Vanhaesebroeck, 2001 , loc. cit.). Pl 3-kinase activation as a prerequistite of generation of 3'-phosphorylated PIs is required for growth factor induced DNA synthesis and regulation of the cell cycle. Pl 3-kinase products are critical in S phase entry and in operating the action of cyclins (GiIIe, J Biol Chem, 274: 22033-22040, 1999). The mainstream view concerning the role of Pl 3-kinase in apoptosis is that activation is responsible for cell survival (Yao, Science, 267: 2003-2006, 1995; Vemuri, Development, 122: 2529-2537, 1996; Kennedy, Genes Dev, 11: 701-713, 1997; Dudek, Science, 275: 661-665, 1997; Kulik, MoI Cell Biol, 17: 1595-1606, 1997). Pl 3-kinase is generally considered to protect cells from apoptosis via its downstream target Akt/PKB. Indeed, this protein kinase delays cell death in a variety of cell types upon different apoptotic stimuli (Dudek, 1997, loc. cit.; Downward, J. Curr Opin Cell Biol, 10: 262-267, 1998). However, the properties and the modes of action of PIs still are unclear and uncertainty exists.

The technical problem underlying the present invention was the provision of means and methods for treatment of malignancies, in particular immunological disorders (like auto-immune diseases) and cancer.

The solution to the technical problem is provided herein and is characterized in the appended claims and the corresponding embodiments provided herein below.

Accordingly, the present invention relates to a combination or a combination compound comprising a 3'-phosphorylated form of phosphatidylinositol and a secretory (s) phospholipase A2 (sPLA2), whereby preferably said phospholipase A2 is a phospholipase A2 of group III, more preferably said phospholipase A2 is a venom sPLA2 and most preferably said venom sPLA2 is bee venom sPLA2 (bv sPLA2). Furthermore, the present invention also relates to the pharmaceutical use of lyso-phosphatidylinositols in their 3'-phosphorylated form, in particular lyso- Ptdlns(3,4)P2.

As documented in the appended examples, it was surprisingly found that the combination and/or the combinatorial use of 3'-phosphorylated phosphatidylinositol and a secretory phospholipase A2 (sPLA2), in particular bee venom sPLA2 (bv sPLA2) provides for means and methods useful not only in the efficient lysis of cells, like tumor/cancer cells, but also useful in the generation of a cellular lysate(s) to be employed in pharmaceutical approaches, in particular in vaccination approaches and in form of an immunological adjuvant. As documented in the appended examples, also in cell-free systems the 3'-phosphorylated phosphatidylinositols are substrates of sPLA2 and the resulting product(s) is/are lyso-phosphatidylinositols (lyso-Ptdlns). In particular, lyso-Ptdlns(3,4)P2 can be obtained when Ptdlns(3,4)P2 is employed as a corresponding substrate for sPLA2 (for example, from bee venom) and said resulting lyso-Ptdlns can also be employed in the medical indications provided herein and may also be employed to generate the herein described cellular lysates, e.g., lysates form normal or tumor cells.

The work illustrated in the appended examples, tables and figures documents that surprisingly phosphatidylinositols (Ptdlns) with a 3'-phosphorylation, i.e. a phosphate group esterified to position 3 of the inositol head group, synergize efficiently with bv- sPLA2 in inducing the lysis of non-transformed normal cells and of cancer cells. This specific synergistic effect was unexpected and could not be observed with Ptdlns with a 4- or a 5-phosphorylation. Ptdlns or its derivatives have fatty acids attached to positions sn-1 and sn-2. As documented herein, the term "lyso" relates in context of Ptdlns ("lyso-Ptdlns") to Ptdlns or its derivates wherein (a) fatty acid(s) is/are missing from either the sn-1 or the sn-2 position. In the presented studies, 3'-phosphorylated Ptdlns with the unsaturated fatty acid palmitic acid (C16) were most potent, however, also 3'-phosphorylated Ptdlns with other saturated fatty acids, such as stearic acid (C18) are envisaged in context of this invention. If one acyl group was a polyunsaturated fatty acid such as arachidonic acid (C20), the ability to synergize with bv-sPLA was only slightly reduced, yet still very useful. Accordingly, also other unsaturated fatty acids such as the monounsaturated oleic acid (C 18) are envisaged on the 3'-phosphorylaetd Ptdlns to be employed in accordance with this invention. A preferred hierarchy of the structural requirements of Ptdlns derivatives with regard to the ability to synergize with bv-sPLA2 is shown and documented in the appended examples and figures. The combination provided herein as well as the herein described lyso-Ptdlns are particularly useful in the generation of cellular lysates. These lysates (as obtained by methods disclosed herein) are immunogenic since they enhance survival of dendritic cells (DC) and induce the maturation of CD83+ DCs with increased immunostimulatory capacity.

In general, the lysates may be obtained by the following, non-limiting protocol: normal or tumorous/tumor cells are incubated at appropriate cell densities (for instance 10⁵cells/ml) in cell culture vessels (microtiter plates or flasks) in a CO₂ incubator in aqueous culture media such as RPMI 1640 or lactated Ringer's solution which must be devoid of proteins. The cellular lysate can be obtained by adding 10 μg/ml bv- sPLA2 (range: 1 to 100 μg/ml) and 10 μM 3'-phosphorylated Ptd Ins-derivatives (range: from 1 to 100 μM). The addition of this combination results in complete lysis of the cells within several hours (2-16 hours). The cell culture as a whole now represents the cellular lysate consisting of lysed cells (cell extract or cell debris) dissolved or suspended in the culture media. The cellular lysate also represents a copious mixture of cellular antigens. The cellular lysate can be harvested from the culture vessel by suction pipetting. In principle, the cellular lysate can be utilized in its crude form without any modification. Alternatively, the crude cellular lysate can be further processed by homogenisation and/or by the addition of, inter alia, proteins, serum components or protease inhibitors to stabilize the lysate. In another embodiment, the lysate can also be fractionated by centrifugation, filtration or chromatography. The lysate may also be adsorbed to a carrier material (substrate) such as salts (aluminium or calcium salts) or (micro) particles. The crude or processed lysate may also be packaged, enclosed or encapsulated within (complex) structures, such as liposomes. The crude or processed lysate may also be subjected to lyophilisation in order to concentrate the antigenic material contained in the lysate and to facilitate storage of the lysate.

The cell lysates as generated by the combined treatment with 3'-phosphorylated inositol derivatives and (bv) sPLA2 or by treatment with 3' phosphorylated lyso-Ptdlns as characterized in this invention display all features of a vaccine with adjuvant properties. In case of a cancer cell, the cell lysate itself contains the complete spectrum of potentially relevant target tumor antigens. The generation of a useful cell lysate/cellular lysate is detailed herein below and illustrated in the appended examples. Lysates may also be obtained by direct exposure of the cells to lyso- Ptdlns in their 3'-phosphorylated form, like lyso-Ptdlns(3,4)P2. For vaccination purposes antigens are admixed with commercial adjuvants which serve to enhance immunogenicity. Such adjuvants usually contain hydrophobic substances which are known to augment both antibody production and cell-mediated immunity. In addition, adjuvants frequently contain bacterial products which induce local inflammation at the site of administration. Today its known that inflammation serves to recruit immune cells to the site of vaccine administration and to promote DC maturation which favors the induction of the desired immune responses (Banchereau, 1998, loc. cit.; Matzinger, 2002, loc. cit.). Also be employed in context of this invention may be lysate comprising lyso-Ptdlns, like lyso-Ptdlns(3,4)P2 or lysates generated by the use of lyso-Ptdlns, like lyso-Ptdlns(3,4)P2. Said lyso-Ptdlns are, in context of this invention, in their 3'-phosphorylated form. Corresponding lyso-Ptdlns may also comprise lyso-Ptdlns (3,4,5) D3 or lyso Ptdlns (3,4,5)R3-AA.

The lysates as obtained by the methods provided herein are particularly useful in medical, pharmaceutical as well as diagnostic settings.

Accordingly, said lysates may, inter alia, be used as therapeutic agent for the treatment or prevention of malignancies in humans and/or animals, be administered as an agent for the treatment or prevention of infectious diseases in humans and/or animals, be administered in humans and/or animals in order to prevent transplant rejection, be administered as an immunomodulating agent, be administered in aqueous solutions, be administered in aqueous solutions containing viable or inactivated cells, be administered as a cell lysate or as a combination of cell lysate with viable cells resuspended in buffers, like lactated Ringer's solution containing 1- 20% heat-inactivated human serum, proteins or protease inhibitors, be administered, inter alia, intradermal^, subcutaneously, intravenously, intranodally, intratumourally, orally, or directly into lymphoid organs, be applied by single or repetitive administration, be administered in various concentrations (lysates derived from cell numbers ranging e.g. from about 1x10⁵ to 1x10⁸ per administration, or lysates derived from cell numbers ranging e.g. from about 1x10⁵ to 1x10⁸ combined with e.g. about 1x10⁵ to 1x10⁸ DCs per administration), be administered by employing technical injection devices (e.g. syringes or similar), be administered in combination with adjuvants, be applied as a reconstituted lyophilisate, be used as an enhancer of, inter alia, vitality and/or viability, or enhancer of cell-survival for the in vitro and in vivo generation of DCs, and be used in diagnostic procedures (e.g. comparison of lysates derived from normal cells versus lysates derived from malignant cells).

Without being bound by theory, the rapid cell lysis induced by the combined administration of 3'-phosphorylated Ptdlns and bv-sPLA2 indicates the presence of detergent activity previously attributed to /yso-phospholipids generated by sPLA2 enzyme activity (Murakami, 1997, loc. cit.). The present invention demonstrates that 3'-phosphorylated Ptdlns derivatives most potently interact with bv-sPLA2 in generating such cell-lysing activity. In the cell lysates generated with Ptdlns derivatives as disclosed herein and bv-sPLA2, the bee venom enzyme may serve as a helper antigen (in analogy to the role of keyhole limpet hemocyanin KLH). Furthermore, adjuvants may contain factors that promote DC maturation such as bacterial products. The purpose of DC maturation is to increase the immunostimulatory capacity of the DCs and thus to promote the initiation of an immune response. The illustrative cellular lysates of the invention, like the Ptdlns(3,4)P2-bv-sPLA2 lysates meet this criterion since they induce the survival and maturation of CD83+ DCs with increased immunostimulatory capacity, as clearly documented in the appended examples. Accordingly (and as detailed below) the present invention provides for cellular extracts particularly useful in promoting cellular survival of dendritic cells and their maturation.

Furthermore, it was surprisingly found that during a cooperative interaction between sPLA2 and 3' phosphorylated Ptdlns, 3'-phosphorylated lyso-Ptdlns is already formed at concentrations between 1 and 2 μM; see also appended table 5. This is in contrast to the data presented by Masamune (2001, loc. cit.) who employed phosphatidyl-choline in elevated concentrations of 50 μ M and more. Accordingly, for some of the medical/pharmaceutical uses provided herein, it is envisaged that the herein described phosphatidylinositol combination(s) or the lyso-phosphatidylinositols in their 3'-phosphorylated form are preferably administered locally rather than systemically; routes of administration include but are not restricted to intradermal, subcutaneous, intranodal, introtumoral application or topical administration onto the skin. It is envisaged that, in accordance with the data provided herein (and in particular in appended table 5), lyso-phosphatidylinositol may be administered to an individual in need of such treatment in a range of 1 to 25 μM, more preferably in a range of 1 to 10 μM. The person skilled in the art is readily in a position to understand that in certain circumstances also higher concentrations may be comprised in the corresponding dosage form. For example, in case the provided dosage form comprises additional constituents, like e.g. proteins, the comprised dosage/per dosage form of lyso-phosphatidylinositol as described herein may be higher than 10 μM or even 25 μM.

In addition, the methods of the invention allow the effcient generation of (tumor) cell lysates which can be loaded onto DCs in vitro and in vivo. The DCs become activated and mature as a consequence of the inherent stimulatory activity of the lysate. Alternatively, such lysates can themselves be considered vaccines which can be injected into cancer patients, preferably into tissues enriched with DCs (skin, lymphoid organs, etc.). Under such conditions, the lysate are useful in activating and maturing resident DCs in situ. Lysates derived from normal cells (alone or in combination with DCs) can, for example, be applied as immunogens to induce immune reactions against normal self-antigens. Usually, immune responses against normal self-antigens result in autoimmunity. Hence, lysates derived from normal cells can be applied to induce autoimmunity in experimental systems (e.g. animals). This approach enables investigation of autoimmunity and facilitates identification of antigens involved in autoimmune reactions.

As detailed below, it is also envisaged to use the combination of the invention for a medical regimen, whereby tumor cell lysis in vivo induced with susequent recruitment and activation of immune cells, including DCs. The combination(s) of the present invention may be administered in various formulations that may also serve to mediate retarded drug release. As shown in the appended examples, also lyso- phosphatidylinositols in their 3'-phosphorylated form are of use in the herein described pharmaceutical applications and applications for the generation of cellular lysates.

In context of this invention the term "3'-phosphorylated" and "3-phosphorylated" are equivalent term and are used interchangeably.

In context of this invention, the inventive combination may be used in vitro or may be administered in vivo. Also envisaged is the in vivo and in vitro use of 3'- phosphorylated lyso-phosphatidylinositols, like lyso-Ptdlns(3,4)P2, as described herein. In the in vivo as well as in the in vitro uses and methods employing the inventive combination the sequential, separates as well as the simultaneous use of the individual compounds of the inventive combination is envisaged. Accordingly, the present invention relates to a combination/combination product/combination compound as defined herein as well as a combined preparation for simultaneous, separate or sequential use. The compounds comprised in said inventive combination may, accordingly, be administered to cells (e.g. in order to prepare a cellular lysate as described herein and as being part of this invention) or to a patient (preferably a human patient) in a sequential and separate manner (i.e. one after the other) or simultaneously (i.e. together in one preparation, for example in form of a mixture). As is illustrated herein below and in the appended examples, also lyso- phosphatidylinositols in their 3'-phosphorylated form may be employed in the medical and pharmaceutical uses described herein.

The methods provided herein, e.g. the administration of the combination provided herein is also to be used to lyse DCs to generate a non-viable DC-derived vaccine with either immunogenic or tolerogenic properties. A major advantage of such a vaccine relates to the simplified logistics of a vaccine consisting of a cell lysate as compared to a vaccine consisting of viable cells.

In general, DC-based vaccines consist of viable DCs (range e.g. about 1x10⁵ - 1x10⁸ cells) treated with proteins, peptides or nucleic acids with inherent immunogenic properties. In addition to this concept, the method provided herein allows the generation of a vaccine consisting of lysates derived in the range e.g. from about 1x10⁵ - 1x10⁸ normal cells, tumor cells or DCs or a combination thereof. These lysates can be resuspended in buffers like lactated Ringer's solution containing 1- 20% heat-inactivated serum. The resulting vaccine preparations represent "nonviable lysates" which can be further processed, for example cryopreserved or lyophilized. It is evident for the skilled artisan that these lysates have distinct advantages over the use of known DC-based vaccines. Inter alia, storage and logistics of the medical tools provided herein are more convenient compared to conventional viable DC-based vaccines.

The terms "combination" or "combination compound" as employed herein comprise also a product which comprises the individual compounds disclosed herein, namely a 3'-phosphorylated form of phosphatidylinositol and a secretory phospholipase A2 (sPLA2). Accordingly, said term also comprises a kit comprising these two individual compounds. Preferably, said kit is to be employed in medical settings and/or pharmaceutical settings. Also use in basic scientific research is envisaged. In context of this invention the term "combination" or "combination compound" also comprises a mixture of the individual two constituents of the inventive combination product, i.e. a mixture of 3'-phosphorylated form of phosphatidylinositol and a secretory phospholipase A2 (sPLA2). The kit as described herein is useful in medical settings (as pharmaceutical) but also as research product.

The 3'-phosphorylated phosphatidylinositol to be employed and used in accordance with this invention is a compound of the formula I

wherein

R¹ and R² are independently selected from C4-24 alkyl and C_4-24 alkenyl; and R⁴ and

R⁵ are independently selected from OH and OPO₃ ^2".

Accordingly, preferred 3'-phosphorylated phosphatidylinositol (3'-phosphorylated forms of phosphatidylinositol) are compounds of the formula I above, whereby R³, R⁴, R⁵ are selected as documented in the following table:

Said 3'-phosphorylated phosphatidylinositol is preferably phosphatidylinositol-3,4- diphosphate (Ptdlns(3,4)P2) or phosphatidylinositol-3,4,5-triphosphate (Ptdlns(3,4,5)P3) and most preferred are the 3'-phosphorylated phosphatidylinositols: 1 -(1 ,2-dihexadecanoylphosphatidyl)inositol-3,4,5-triphosphate), 1 -(1 ,2- dihexadecanoylphosphatidyl)inositol-3,4-diphosphate), 1 -(1 -octadecanoyl-2-

(5Z,8Z,1 1Z,14Z)-eicosatetraenoylphosphatidyl)inositol-3,4,5-triphosphate), 1 -(1 ,2- dihexadecanoylphosphatidyl)inositol-3-phosphate), or 1-(1 ,2- dihexadecanoylphosphatidyl)inositol-3,5-diphosphate).

It is envisaged that the 3'-phosphorylated form of phosphatidylinositol is a salt, most preferably a sodium salt or an ammonium salt, in a preferred embodiment, the 3¹- phosphorylated phosphatidylinositol is 1-(1 ,2-dihexadecanoylphosphatidyl)inositol- 3,4,5-triphosphate hexasodium salt), 1-(1 ,2-dihexadecanoylphosphatidyl)inositol-3,4- diphosphate tetrasodium salt), 1-(1-octadecanoyl-2-(5Z,8Z,11Z,14Z)- eicosatetraenoylphosphatidyl)inositol-3,4,5-triphosphate heptasodium salt), 1-(1 ,2- dihexadecanoylphosphatidyl)inositol-3-phosphate ammonium salt), and/or 1-(1 ,2- dihexadecanoylphosphatidyl)inositol-3,5-diphosphate tetrasodium salt).

Corresponding examples are given in the experimental part.

The term "lyso-Ptdlns" as employed herein relates to 3'-phosphorylated lyso- phosphatidylinositols, like lyso-Ptdlns(3,4)P2. As illustrated in the appended examples, such lyso-Ptdlns may easily be obtained, also in cell-free systems.

As pointed out above, the combination of the invention is particularly useful in treatment regimes of tumorous disease or autoimmune disorders. Also useful in this context are cellular lysates obtained by being the inventive combination. Accordingly, the present invention also relates to a pharmaceutical composition comprising the combination or combination compound as defined herein. Also useful in the treatment of tumorous disease or autoimmune disorders are the herein described lyso-phosphatidylinositols (lyso-Ptdlns), in their 3'-phosphorylated form. As documented in the appended examples, 3'-phosphorylated lyso-phosphatidylinositols can be obtained, inter alia, in cell-free systems, since phosphatidylinositols in their 3¹- phosphorylated form (see also "preferred 3'-phosphorylated phosphatidylinositols as described herein above under formula I) can also be a substrate of sPLA2 in cell-free systems. For example, as documented in the experimental part, sPLA2 can hydrolyze Ptdlns (in particular Ptdlns(3,4)P2) to their lyso-form (for example, lyso- Ptdlns(3,4)P2). Accordingly, in context of the present invention, also pharmaceutical compositions are described which comprise 3'-phosphorylated phosphatidylinositols in their lyso-form, i.e. lyso-Ptdlns. Such 3'-phosphorylated phosphatidylinositols in their lyso-form comprise in particular 1-O-acyl-sn-glycero-3-phosphoinositol-3,4- bisphosphate, i.e. lyso-Ptdlns(3,4)P2 or i-O-acyl-sn-glycero-S-phosphoinositol-SAδ- trisphosphate, i.e. lyso-Ptdlns(3,4,5)P3; The remaining fatty acid (acyl) at the sn-1 position may be palmitic acid (C16:0) but may also be replaced by other fatty acids that vary in their number of carbon atoms as well as in the number of double bonds, for instance, arachidonic acid (C20:4). Alternatively, such 3'-phosphorylated phosphatidylinositols in their lyso-form comprise 2-O-acyl-sn-glycero-3- phosphoinositol-3,4-bisphosphate, i.e. lyso-Ptdlns(3,4)P2 or 2-O-acyl-sn-glycero-3- phosphoinositol-3,4,5-trisphosphate, i.e. lyso-Ptdlns(3,4,5)P3; The remaining fatty acid at the sn-2 position may be palmitic acid (C16:0) but may also be replaced by other fatty acids that vary in their number of carbon atoms as well as in the number of unsaturated double bonds, e.g. arachidonic acid (C20:4).

Therefore, the present invention also relates to the use of lyso-phosphatidylinositols in their 3'-phosphorylated form in the preparation of a pharmaceutical composition, like for the treatment and/or amelioration of (a) tumorous disease(s), like cancer or the treatment or amelioration of an autoimmune disorder.

The stereochemistry of triacylglycerols and other glycerolipids is described by the "stereospecific numbering" (sn) system as recommended by a commission of the International Union of Pure and Applied Chemistry (IUPAC) - International Union of Biochemistry (IUB) (= Joint Commission on Biochemical Nomenclature (JCBN)). http://www.chem.qmul.ac.uk/iupac/lipid/

Stereospecific Numbering. In order to designate the configuration of glycerol derivatives, the carbon atoms of glycerol are numbered stereospecifically. The carbon atom that appears on top in a Fischer projection that shows a vertical carbon chain with the hydroxyl group at carbon-2 to the left is designated as C-1. To differentiate such numbering from conventional numbering conveying no steric information, the prefix 'sn' (for stereospecifically numbered) is used. This term is printed in lower-case italics, even at the beginning of a sentence, immediately preceding the glycerol term, from which it is separated by hyphen. Derivatives of glycerolipids acids resulting from hydrolytic removal of one of the two acyl groups at sn-1 and sn-2 may be designated by the prefix 'lyso', e.g. lysophosphatidylcholine (IUPAC-IUB Commission on Biochemical Nomenclature (CBN). The nomenclature of lipids. Eur J Biochem. 1967 2(2):127-31).

As pointed out above, the pharmaceutical composition of the invention comprises a 3-phophorylated form of phosphatidylinositol and a secretory phospholipase A2 (sPLA2) whereby said individual compounds may be administered to a patient in need of treatment simultaneously or sequentially. Also provided in this invention is the use of a combination or a combination compound as defined herein, for a preparation of a pharmaceutical composition for the treatment of a proliferative disorder and/or tumorous disease. Said tumorous disease is in particular cancer, like an urogenitcal cancer, a breast cancer, a pleural cancer, a bronchial cancer, a lung cancer, an oral cavity cancer, a pharynx cancer, an oesophagus cancer, a stomach cancer, a colon cancer, a rectum cancer, a liver cancer, a pancreas cancer, a larynx cancer, a melanoma of skin, a brain cancer, a nervous system cancer, a thyroid cancer, a Non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, leukemia, cancer of the cervix uteri, cancer of the corpus uteri, ovary cancer. In a preferred embodiment of this use said urogenital cancer is cancer of kidney, bladder, prostate and/or testis.

The combination or a combination compound as defined herein may also be employed for the in vitro generation of a cellular lysate, for example, a cellular lysate of non-tumorous cells (normal cells) or of tumorous/tumor cells. Said cells may be cultured cells or cells derived from biopsies. Said cells may also be primary cultured cells. The appended examples document how a corresponding cellular lysate may be obtained and generated. Accordingly, the present invention also provides for a method for the generation of a cellular lysate of normal or tumorous/tumor cells comprising the steps of:

(a) contacting non-tumorous cells or tumorous/tumor cells with the combination or combination compound as defined above and comprising a 3-phophorylated form of phosphatidylinositol and a secretory phospholipase A2 (sPLA2); and

(b) obtaining the resulting cellular lysate. Step (b) of the above recited method may comprise the addition of stabilizing agents like serum components, proteins, protease inhibitors. All lysates may, inter alia, be obtained by fractionation, centrifugation, filtration chromatography, adsorption or lyophilisation.

The method described above may also comprise the direct contact of non-tumorous or tumor cells with lyso-Ptdlns, like lyso-Ptdlns(3,4)P2 in step (a), i.e. said cells are directly contacted with the herein defined 3'-phosphorylated lyso-Ptdlns.

In a preferred embodiment of this invention, the above recited method is carried out with tumorous/tumor cells preferably selected from urogenital cancer cells, breast cancer cells, kidney cancer cells, prostate cancer cells, lung cancer cells, pleural cancer cells, bronchial cancer cells, oral cavity cancer cells, pharynx cancer cells, oesophagus cancer cells, stomach cancer cells, colon cancer cells, rectum cancer cells, liver cancer cells, pancreas cancer cells, larynx cancer cells, cells of melanoma of skin, brain cancer cells, nervous system cancer cells, thyroid cancer cells, cells of Non-Hodgkin lymphoma, cells of Hodgkin's disease, multiple myeloma cells, leukemia cells, cells of cancer of the cervix uteri, cells of cancer of the corpus uteri, or cells of ovary cancer. The corresponding obtained lysates are particularly useful in the preparation of "cellular vaccines". Lysates derived from normal or tumor cells are also useful in the maturation of dendritic cells, as illustrated in the appended examples. Also useful in context of the medical interventions described herein are lysates comprising 3'-phosphorylated forms of lyso-Ptdlns, in particular lyso- Ptdlns(3,4)P2. Such lysate may be produced by the methods described herein and also in cell-free systems as documented in the appended examples. Accordingly, the present invention also provides for pharmaceutical compositions comprising 3'- phosphorylated lyso-Ptdlns, in particular lyso-Ptdlns(3,4)P2. Therefore, the present invention also provides for the use of 3'-phosphorylated lyso-Ptdlns in the preparation of a pharmaceutical for the in vitro or the in vivo immunomodulation of blood cells, like the maturation of immature dendritic cells (DC) to mature dendritic cells (DC).

The method provided herein above may also comprise a step

(c) of eliminating and/or inactivating inherent nucleic acid molecules, bacterial contamination or viral contamination.

Again, corresponding embodiments are provided in the appended examples, wherein said elimination and/or inactivation comprises the treatment of the obtained lysate with radioactive radiation, sterile-filtration, or sterilization by heating. As discussed herein, the obtained cellular lysates are useful in medical settings. Accordingly, the present invention also relates to a pharmaceutical composition comprising a cell lysate as generated by the use provided above or as obtained by the method as disclosed and illustrated herein, namely employing 3-phophorylated form of phosphatidylinositol and a secretory phospholipase A2 (sPLA2). The cell lysate may also by a lysate comprising lyso-Ptdlns, like lyso-Ptdlns(3,4)P2, as described herein.

This pharmaceutical composition comprising the cellular lysates is particularly useful in the in vitro and/or in vivo immunomodulation of blood cells. Accordingly, the invention also provides for the use of the cell lysate as generated by the use described above or as obtained by the method disclosed herein for the preparation of a pharmaceutical composition for the in vitro and/or in vivo immunomodulation of blood cells. Most preferably said immunomodulation of blood cells is the maturation of immature dendritic cells (DC) to mature dendritic cells (DC).

It is also envisaged that the pharmaceutical composition comprising the cellular lysate obtained by the inventive method is to be administered in combination with cytokines, like TNF-α or an interleukin. Preferably, said interleukin is interleukin-1β.

The same embodiment is also envisaged for the pharmaceutical use of the herein described pharmaceutical compositions comprising lyso-phosphatidylinositols in their 3'-phosphorylated form.

The cell lysate/cellular lysate as obtained by the method described herein or the lyso- Ptdlns as described herein are particularly useful in the treatment, amelioration and/or prevention of proliferative disorder and/or a tumorous disease or of an autoimmune disease. Corresponding proliferation disorders comprise cancers, like urogenitcal cancer, breast cancer, pleural cancer, bronchial cancer, lung cancer, oral cavity cancer, pharynx cancer, oesophagus cancer, stomach cancer, colon cancer, rectum cancer, liver cancer, pancreas cancer, larynx cancer, melanoma of skin, brain cancer, nervous system cancer, thyroid cancer, Non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, leukemia, cancer of the cervix uteri, cancer of the corpus uteri, ovary cancer.

Said autoimmune disease to be treated with said cell lysate/cellular lysate or with the lyso-Ptdlns as described herein may be selected from the group consisting of allergy, multiple sclerosis, diabetes mellitus, in particular Type I diabetes, rheumatic arthritis, Lupus erythematosus, and Morbus Crohn. Also envisaged is the use in graft-versus- host reactions or in transplantation medicine (also in xenotransplantation, heterotransplantation or homotransplantations.)

In the following an illustrative clinical use of the invention in cancer therapy is exemplified by the treatment of human renal cell carcinoma.

Renal cell carcinoma (RCC) collectively refers to a group of neoplasms originating from proximal tubular epithelial cells. RCC accounts for 2-3% of all malignancies and is the most common cancer of the kidney. Patients with metastatic RCC have a poor prognosis and a median survival time of less than a year reflecting the lack of effective treatment. RCC affects more than 30.000 individuals each year in Europe and has a peak incidence in the 5th and 6th decade of life. About one third of the patients has metastatic disease at diagnosis and 30 - 50 % of initially localized RCCs eventually metastasize. Several lines of evidence indicate that the immune system can control the development of RCCs. Past and currents efforts therefore aim at mobilizing the immune system against the growing tumor (immunotherapy). Two forms of immunotherapy can be distinguished. In the therapeutic approach, immunotherapy is used to treat already existing metastases of RCC. In the adjuvant approach, immunotherapy is performed subsequent to surgical removal of the primary tumor in an attempt to prevent or delay the formation of metastases which is known to occur in 30-50% of the patients. According to the invention disclosed herein, the tumor cell lysate is, for example, obtained by treating RCC cells with PLA2, preferably bv-sPLA2 and 3'- phosphorylated Ptd Ins-derivatives. RCC cells can either be derived from the patient's own tumor (autologous, primary) or from one or more permanent, well-characterized RCC cell lines such as the A-498 cell line. By introducing large numbers of RCC cells (e.g., 10⁶, preferably 10⁷, 10⁸ or 10⁹ or even more than 10⁹) ample supply of the tumor lysate can be generated. The obtained tumor lysate may be concentrated by lyophilisation and divided into 100 aliquots each corresponding to 10⁷ cells (when, for example, starting with 10⁹ cells) and stored chilled or frozen until use. Immediately before treatment an aliquot may be thawn and reconstituted with sterile, injectable water. The reconstituted tumor lysate is taken up in a hypodermic syringe and injected into patient. Routes of administration include intracutaneous, intradermal, intranodal and intratumoral injection (the latter are performed under ultrasound or CT control). Preferred routes may be intradermal and intranodal injection, since dendritic cells are abundant at these sites. Different routes of administration can be combined in a single treatment. Corresponding routes are described herein.

Also be used as a pharmaceutical may be the herein described lyso-Ptdlns, which may be obtained by the methods disclosed herein.

Dendritic cells will pick up and process the antigens contained in the tumor lysate present at the injection site. In addition, dendritic cells will be activated and matured by the tumor lysate due to the inherent stimulatory properties of the lysate provided by the invention. The antigen-loaded and activated dendritic cells will migrate to regional lymph nodes (except injection has been performed intranodally) and will there activate T-lymphocytes which are specific for antigens of the tumor lysate. The induced immune response will therefore be directed against RCC and will cause the stop of tumor growth or induce tumor rejection. Since the injection of the tumor lysate induces an immune response it can be considered a "vaccination". In the first cycle, two additonal vaccinations are performed in monthly intervals to fresh up and boost the primary immune response against the cancer, in particular RCC. Additional treatment cycles can be performed depending on the clinical course of the disease. The clinical course of the disease is routinely assessed by means of computed tomography (CT) scans of the patient.

As an alternative to the direct injection of the obtained and inventive tumor lysate into tissues rich in dendritic cells (epidermis, lymph nodes). The tumor lysate can be also loaded onto cultured dendritic cells in vitro. For this purpose, dendritic cells are cultured from the peripheral blood of a cancer patient, for example, a RCC patient using published protocols. A protocol frequently used by persons skilled in the art is based on a two-step culture system in which monocytes are differentiated into mature dendritic cells. After step 1 , the immature dendritic cells are loaded with the RCC tumor lysate which is prepared, stored and reconstituted as described above. The dendritic cells will pick up, process and present the antigens contained in the tumor lysate. In step 2 of the protocol, the antigen-loaded dendritic cells are usually activated by the addition of one or more pro-inflammatory factors to induce the full maturation of the cells. In the present protocol, however, the tumor lysate itself will induce the maturation of the dendritic cells due to the inherent stimulatory properties of the lysate provided by the invention. It is of note that the herein described embodiments for the obtained tumor lysate apply, mutatis mutandis, to the pharmaceutical use of the herein described lyso-Ptdlns, like lyso-Ptdlns(3,4)P2 or to lysates comprising said lyso-Ptdlns, like lyso-Ptdlns(3,4)P2.

The mature, antigen-loaded dendritic cells are harvested, washed and injected into the patient using a hypodermic syringe. Possible routes of administration include intracutaneous, intradermal, intranodal injection as well as intravenous infusion. The persons skilled in the art may divide the total dose of 10⁷ cells and administer, for instance, 3 x 10⁶ cells intranodally (ultrasound-guided) and 7 x 10⁶ cells intravenously.

After injection the dendritic cells will induce immune responses in vivo against the antigens of the tumor lysate and thus also directed against the patient's cancer cells, for example RCC. Vaccination with tumor lysate-loaded dendritic cells may also repeated. For example, a monthly interval may be employed to boost the primary immune response in the patient. Yet, another clinical application of the invention is the direct intratumoral administration of a combination of a 3'-phosphorylated form of phosphatidylinositol (Ptdlns) and PLA2, preferably bv-sPLA2. Also envisaged is the medical use of the herein described lyso-Ptdlns, in particular lyso-Ptdlns(3,4)P2. For these purposes, the individual substances are dissolved in appropriate aqueous solutions such as lactated Ringer's solution and subsequently admixed to obtain a final solution (5 ml) which contains the 3'-phosphorylated forms of phosphatidylinositol (for example, 20 to 500 μM, preferably 50 to 200 μM, more preferably 100 μM) and bv-sPLA2 (for example, 20 to 500 μg/ml, preferably 50 to 200 μg/ml, more preferably 100 μg/ml). In this setting, either substance may be used at a rather high concentration (as compared to the lysate approach) since substantial dilution may occur within the tumor tissue. The final solution containing both, 3'-phosphorylated Ptdlns and PLA2, preferably bv-sPLA2, may be injected directly into tumor tissue using a hypodermic needle under ultrasound or CT control. This treatment form should induce tumor cell lysis in situ. As a consequence, immune cells including dendritic cells will be recruited into the tumor tissue, where they pick up and process tumor antigens for subsequent presentation to T-lymphocytes in the lymph nodes draining the tumor tissue. The activated T-lymphocytes will leave the lymph node, enter the tumor tissue and kill tumor cells. Depending on the clinical course, intratumoral injections may be repeated ad libitum.

As documented herein, also the herein described lyso-Ptdlns in their 3¹- phosphorylated form may be employed in the herein described medical indications and setting The medical indications and settings described herein are intended to be employed in patients in need of a corresponding treatment, in particular human patients but also animals, in particular mammals.

Accordingly, the present invention also provides for methods of treating individuals in need of such a treatment, said methods comprising the administration of the combination or combination compound as described herein and/or the administration of a cellular lysate of non-tumorous cells of tumorous/tumor cells and/or the administration of lyso-Ptdlns, like lyso-Ptdlns(3,4)P2 to the patient/individual.

The Figures show: Figure 1: Combined treatment of A498 cells with Ptdlns(3,4)P2 and bv- sPLA2 induces cell lysis.

Figure 1 shows phase contrast microscopy images of treatment of A498 cells with bv-sPLA2 and Ptdlns(3,4)P2 in combination or either alone. A498 cells cultured under serum-free conditions were left untreated (C=control), or treated with Ptdlns(3,4)P2 (10 μM), with sPLA2 (10 μg/ml), or the combination of both, Ptdlns(3,4)P2 and bv-sPLA2. Cell cultures were examined under a phase contrast microscope two hours after administration of the reagents. Addition of FCS prevented cell lysis.

Figure 2: Treatment of moDCs with lysates generated by the combined administration of Ptdlns(3,4)P2 and bv-sPLA2 enhances moDCs yield.

Figure 2 shows the treatment of moDCs with lysates generated by the combined administration of Ptdlns(3,4)P2 and bv-sPLA2. Immature day- 5 moDCs were pulsed with A498 cell lysates generated either by conventional freezing and thawing (standard lysate), or by combined action of Ptdlns(3,4)P2 (10 μM) and bv-sPLA2 (10 μg/ml). After 48 h of incubation in the presence or absence of the moDC maturation stimuli TNF-alpha and IL1-β, cell numbers were determined by counting in a Neubauer chamber.

Figure 3: Treatment of moDCs with lysates generated by the combined administration of Ptdlns(3,4)P2 and bv-sPLA2 matures moDCs.

Figure 3 shows the treatment of moDCs with lysates generated by the combined administration of Ptdlns(3,4)P2 and bv-sPLA2. Immature day- 5 moDCs were treated with A498 cell lysates generated by conventional freezing and thawing (standard lysate) or by the combined actions of Ptdlns(3,4)P2 (10 μM) and bv-sPLA2 (10μg/ml). Control treatments were performed by treating moDCs with viable intact A498 cells or A498 cells treated with either Ptdlns(3,4)P2 or bv-sPLA2 alone. After two days of incubation in the presence (right panel) or absence (left panel) of the maturation stimuli TNF-alpha and IL-1β, FACS analysis of CD83⁺ expression was performed to determine the maturation status of moDCs.

Figure 4: MoDCs treated with lysates generated by the combined administration of Ptdlns(3,4)P2, and bv-sPLA2 display enhanced T- cell stimulatory capacity.

Figure 4 shows the treatment of moDCs with lysates generated by the combined administration of Ptdlns(3,4)P2 and bv-sPLA2. Lysate-treated moDCs were used as stimulators of CD14⁺-depleted PBMCs in allogenic mixed leukocyte reactions. After 5 days of co-incubation, proliferation was determined by assessing [³H] thymidine incorporation. Shown are mean values (cpm) ± SD of triplicate measurements of two independently performed experiments.

Figure 5: Precursor ion scans of 241 showing bv-sPLA2 products of the Ptdlns(3,4)P2 substrate in a cell free system.

A: Cell culture medium as a control containing B: bv-sPLA2 (10 μg/ml) C: a mixture of bv-sPLA2 (10 μg/ml) and Ptdlns(3,4)P2 (10 μM) and D: Ptdlns(3,4)P2 only. The lyso-Ptdlns(3,4)P2 product of Ptdlns(3,4)P2 is observable at m/z 571 , an in-source bis-dephosphorylated fragment, in the presence of 10 mM ammonium acetate. The precursor ion scan 241 in negative ion mode corresponds to the loss of the InsP head group. The asterix indicates the lyso- Ptdlns derived from an in source fragmentation of the C32:0 Ptdlns used as internal standard (ISTD).

Figure 6: Precursor scan 241 of bv-sPLA2 products of Ptdlns(3,4)P2 in a cell free system to detect lyso-Ptdlns(3,4)P2 by attenuated in source fragmentation using piperidine.

A: Cell culture medium containing Ptdlns(3,4)P2 only, B: Cell culture medium containing a mixture of bv-sPLA2 (10 μg/ml) and Ptdlns(3,4)P2 (10 μM). Lyso- Ptdlns(3,4)P2 observable at m/z 731, was detected by the precursor ion scans 241 with the corresponding loss of InsP head group. The lyso-Ptdlns observable at m/z 571, was produced from an in-source fragmentation of the Ptdlns(3,4)P2 and also from the C32:0 Ptdlns used as internal standard (C32:0 Ptdlns-ISTD). Other in source fragments are also observable e.g. product ions seen at m/z 651 and 673 and products thereof are from in-source fragmentation of (M-H)^" parent ion m/z 969 and at (M-2H+Na)^" m/z 991 respectively

A better understanding of the present invention and of its many advantages will be seen from the following examples, offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Example 1 : Materials and methods employed in context of this invention

Phosphatidylinositols

Phosphatidylinositol in its abbreviated version reads as Ptdlns. Ptdlns and phosphorylated Ptdlns derivatives used are well known in the art and were purchased from Cayman Chemical, Ann Arbor, Ml, USA. The corresponding Cat. Nos. are indicated in the following:

1. Ptdlns(3,4)P2: Phosphatidylinositol-3,4-bisphosphate (1 ,2-dipalmitoyl, ammonium salt), (formal name: 1-(1 ,2-dihexadecanoylphosphatidyl)inositol-3,4-diphosphate, tetrasodium salt); Cat. No 64922.

2. Ptdlns(3,4,5)P3: Phosphatidylinositol-3,4,5-trisphosphate (1 ,2-dipalmitoyl, ammonium salt), (formal name: 1-(1 ,2-dihexadecanoylphosphatidyl)inositol-3,4,5- triphosphate, hexasodium salt); Cat. No 64920.

3. Ptdlns(3,4,5)P3-AA: Phosphatidylinositol-3,4,5-trisphosphate (1-stearyl, 2- arachidonoyl, sodium salt), (formal name: 1-(1-octadecanoyl-2-(5Z,8Z,11Z,14Z)- eicosatetraenoylphosphatidyl)inositol-3,4,5-triphosphate,heptasodium salt); Cat. No 64930.

4. Ptdlns(4,5)P2: Phosphatidylinositol-4,5-bisphosphate (1 ,2-dipalmitoyl, ammonium salt), (formal name: 1-(1 ,2-dihexadecanoylphosphatidyl)inositol-4,5-diphosphate, tetraammonium salt); Cat. No 64924. D-myo-inositol1 ,3,4-triphosphat (sodium salt) (lns(1 ,3,4)P3) (formal name: D-myo- inositol-1 ,3,4-tris(dihydrogen phosphate), hexasodium salt) was also purchased from Cayman Chemical, Ann Arbor, Ml, USA (Cat. No 60972).

L-α-Phosphatidylinositol (synonym: 1 ^-Diacyl-sn-glycero-S-phospho-i-D-myo- inositol-Na) was purchased from Biomol, Hamburg, Germany (Cat. No PH-100).

Lvsophosphatidylinositols

1. Lyso-Ptdlns(3,4)P2: Lyso-phosphatidylinositol-3,4-bisphosphate (1-O-acyl-sn- glycero-3-phosphoinositol-3,4-bisphosphate);

2. Lyso-Ptdlns(3,4,5)P3: Lyso-phosphatidylinositol-3,4,5-trisphosphate (1-O-acyl-sn- glycero-3-phosphoinositol-3,4,5-triphosphate);

3. Lyso-Ptdlns(3,4,5)P3-AA: Lyso-phosphatidylinositol-3,4,5-trisphosphate (1-acyl, 2- arachidonoyl.sodium salt)

Secretory phospholipase A2 (Type III) sPLA2 from bee venom (bv-sPLA2) was purchased from Cayman Chemical, Ann Arbor, Ml, USA (Cat. No 60500). sPLA2 is known in the art and bv_sPLA2 is, inter alia, encoded by the following DNA sequence:

1 atgcaagtcg ttctcggatc cttgttcctt ctcctcctct ctacctctca cggatggcaa 61 atcagggata ggatcgggga taacgagttg gaggaacgga taatatatcc aggaacgtta 121 tggtgcgggc atggtaacaa gtcgtccggc ccgaacgagc taggtcggtt caagcacacg 181 gatgcatgct gtcgaaccca cgacatgtgc ccggacgtga tgtcagctgg tgaatcgaag 241 cacggcctga ccaacacggc ctcccacacc aggttgtcgt gcgactgcga cgacaagttc 301 tatgattgtc ttaaaaattc ggcggacacg attagctcgt atttcgtagg gaagatgtac 361 ttcaatctga tagacacgaa gtgttacaaa ctggagcatc ctgtcaccgg gtgcggtgag 421 agaaccgagg gtcgttgtct tcactacacc gtggacaaaa gcaaaccgaa agtgtaccaa 481 tggttcgatc ttcgcaagta ttga [SEQ ID NO.1]

A corresponding bv-sPLA2 protein sequence is

1 mqwlgslfl lllstshgwq irdrigdnel eeriiypgtl wcghgnkssg pnelgrfkht 61 daccrthdmc pdvmsagesk hgltntasht rlscdcddkf ydclknsadt issyfvgkmy 121 fnlidtkcyk lehpvtgcge rtegrclhyt vdkskpkvyq wfdlrky [SEQ ID NO.2]

sPLA2 activity was routinely monitored using the colorimetric sPLA2 assay kit from Cayman Chemical Company, Ann Arbor, Ml, USA, which is based on the synthetic substrate diheptanoyl thiophosphorylcholine. Corresponding other assay lists are known in the art.

Cell culturing

Cell lines

Tumor cell lines used in the present invention are indicated in the following:

1. A498 = ATCC no: HTB-44, human kidney carcinoma (as described in Fogh J. et al. 1977 and Brossart et al. 1998).

2. T-47D = ATCC no: HTB-133, ductal carcinoma, mammary gland, breast, pleural (as described in Keydar et al. 1979).

3. DU145 = ATCC no: HTB-81 , carcinoma, prostate (as described in Stone et al. 1978).

4. BEAS-2B = ECACC no: 95102433, human, bronchial epithelium, normal (as described in Lechner et al. 1985).

Cell lines were propagated at 37°C and 5% CO₂ in medium consisting of RPMI 1640 supplemented with 10 mM Hepes, non-essential aminoacids (1x), 1 mM Na-pyruvate (all from Cambrex Bio Science, Verviers, Belgium), Glutamax (1x) (Invitrogen, Paisley Scotland, UK), 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories, Linz, Austria) and 10% FCS (heat-inactivated, 30 min., 56⁰C).

Generation of cell Ivsates

Generation of cell lysates using Ptdlns-derivatives plus bv-sPLA2 20.000 cells were plated in 200 μl medium without serum in 96-well flat-bottomed tissue culture plates (Falcon, BD, Franklin Lakes, NJ, USA). Alternatively, 300.000 cells were seeded in 3 ml medium without serum in 6-well flat-bottomed tissue culture plates (Costar, Corning Inc. Corning, NY, USA). Lysis was induced by the addition of 10 μg/ml bv-sPLA2 and 10 μM Ptdlns-derivatives.

Generation of freeze-thaw ("standard") lysates

Standard lysates were generated by seeding 300.000 cells in 3 ml medium without serum in 6-well flat-bottomed tissue culture plates. Lysis was induced by three cycles of freezing (-80⁰C, 2 h) and thawing (37°C incubator, 1 h).

Measurement of tumor cell proliferation

Cells were incubated in 96-well plates in medium without serum for 48 h in the presence or absence of Ptdlns-derivatives and bv-sPLA2. During the last 16 h cells were pulsed with 50 μl fresh medium containing 10% FCS and 1 μCi/well (37 kBq/well) [³H] thymidine (ICN Biomedicals, Eschwege, Germany). Cells were harvested with a Tomtec harvester (Hamden, CT, USA ), liquid scintillation counting was performed with a Chamelon multi label reader (HVD-Life Science, Vienna, Austria). Results are mean cpm ± SD of triplicate wells. Values were normalized by setting controls to 100%. For the calculation of inhibition (Tab. 3 and 4), proliferative effects of single substances were totalized and were compared with the inhibitory effects achieved by the combined treatment with Ptdlns-derivatives plus bv-sPLA2.

Generation of monocvte-derived DCs (moDCs)

At the local Institute for Blood Transfusion, healthy individuals underwent a standard leukapheresis procedure performed with the Cobe Spectra cell separator (Gambro BCT, Lakewood, CO, USA). Using the MNC program at a continuous whole-blood inlet flow rate ranging from 50 ml to 70 ml/min, 3 L to 5 L of whole blood were processed. ACD-A solution (Baxter, Vienna, Austria) at a 1 :12 ratio was used for anticoagulation. Subsequently, apheresis products (50-100 ml) were transferred to a cell culture flask (Costar, Corning, NY, USA) and adjusted to 200 ml using CliniMACS PBS/EDTA Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 2% heat-inactivated human AB serum from the local Institute of Blood Transfusion. CD14⁺ monocytes were separated from apheresed cells by positive selection using the CD14-Reagent, the CliniMACS Tubing Set 600 (for up to 20x10⁹ cells) and the CliniMACS Instrument. All steps were carried out according to the manufacturer's instructions.

CD14⁺ cells (50 x 10⁶ in 50 ml) were cultured in 162 cm² cell culture flasks (Costar, Corning, NY, USA) in AIM-V (Invitrogen, Grand Island, NY, USA) containing 1 % heat- inactivated human AB serum, 10 mM HEPES and 50 μM 2-mercaptoethanol (Merck, Darmstadt, Germany) as well as a combination of recombinant human GM-CSF (1000 U/ml) (Leucomax, Novartis, Basel, Switzerland) and recombinant human IL-4 (1000 U/ml) (CellGenix, Freiburg, Germany). After 2 days of culture, 50 ml of fresh medium containing supplements were added. Sterility testing of day-5 moDCs was performed at the local Institute of Hygiene. All tests were negative (100% sterility). Day-5 moDCs were harvested and frozen in liquid nitrogen using a standard protocol (50% AIM-V, 40% human AB serum, 10% DMSO).

Day-5 moDCs were thawed, counted and replated in 6-well plates at 1 ,8 x 10⁶ cells per well in 3 ml of fresh medium consisting of RPMI 1640 supplemented with 10 mM Hepes, non-essential aminoacids (1x), 1 mM Na-pyruvate , Glutamax (1x), 100 U/ml penicillin and 100 μg/ml streptomycin, GM-CSF (2000 U/ml), IL-4 (500 U/ml) and alternatively recombinant human TNF-a (200 U/ml) and IL-1β (2 ng/ml)(both from R&D Systems, Minneapolis, MN, USA.).

Treatment of moDCs with cell lysates

Cell lysates were generated as described above. Control treatments with single substances were also performed. The lytic activity of lysates was quenched by the addition of 20% fetal calf serm (FCS) or, alternatively, by lipoprotein deficient FCS (Sigma-Aldrich, St.Louis, Mo, USA). Subsequently, lysate preparations were added to moDCs cultures. After 48 h at 37°C, moDCs were harvested, washed with PBS and prepared for analysis (cell counting, flow cytomtry and T-cell assay). Flow cytometry (FACS)

MoDCs treated with lysate preparations were subjected to FACS analysis using anti- CD83-PE- and anti-lgG1k-PE-antibodies (isotype control), a FACSCalibur and CellQuest software (all from BD, Mountain View, CA, USA).

T-cell assay

The immunostimulatory capacities of the differentially treated moDCs were determined in an allogeneic mixed leukocyte reaction (MLR). MoDCs were used as stimulator cells of allogeneic peripheral blood mononuclear cells (PBMCs) depleted of CD14⁺ cells (= CD14⁺ -depleted PBMCs). CD14⁺ -depleted PBMCs (10⁶AnI) were stimulated with moDCs at a ratio of 40:1 in flat-bottomed 96-well plates in AIM-V (Invitrogen, Grand Island, NY, USA). Cultures were pulsed during the last 16 h of a day-5 culture at 37⁰C and 5% CO₂ with 1 μCi/well (37 kBq/well) [3H] thymidine. Cells were harvested with a Tomtec harvester and liquid scintillation counting was performed with a Chameleon Multi label reader. Results are mean cpm ± SD of triplicate wells.

Limulus amebocvte Ivsate (LAP assay

To test for potential endotoxin (LPS) contamination, bv-sPLA2 was subjected to the Limulus Amebocyte Lysate (LAL) assay from Biowhittaker. Endotoxin concentration of bv-sPLA2 at the maximum final concentration used in the experiments (10μg/ml) corresponded to 4,1 pg LPS/ml (i.e. 2,8x10² EU/ml). Endotoxin levels of Ptdlns(3,4)P2 at 10 μM corresponded to 0,11 pg LPS/ml (7,6XiO^-4 EUZmI). Endotoxin levels of Ptdlns(4,5)P2 at 10 μM corresponded to 0,62 pg LPS/ml (4,3x10^'3 EU/ml). At these concentrations, LPS fails to induce moDC maturation as documented in corresponding titration assays. Example 2: Lysis of cancer cells and normal non-proliferating cells by combined treatment of A498 cells with Ptdlns derivatives with phosphorylation at position 3 of the inositol head group and bv-sPLA2

Ptdlns and various derivatives (10 μM) were tested in combination with bv-sPLA2 (1- 10 μg/ml) to induce the lysis of the well-characterized kidney cancer cell line A498. Only Ptdlns and its 3'-phosphorylated derivatives synergize with bv-sPLA2 to induce A498 cell lysis (data summarized in table 1). In contrast, Ptdlns(4,5)P2 or 3'- phosphorylated Inositol (Ins) lacking the phosphatidyl failed to induce cell lysis. Figure 1 depicts phase contrast microscopy images which demonstrate that the combination of bv-sPLA2 and Ptdlns(3,4)P2 synergistically induced lysis of A498 cells while either substance alone had virtually no effect. Cell lysis affected the majority of the cells and occurred within 2 h. Cell lysis only occurred under serum (protein)-free conditions. Addition of FCS prevented cell lysis.

The combination of bv-sPLA2 and Ptdlns(3,4)P2 also induced the lysis of normal non-proliferating cells such as PBMCs as well as immature and mature moDCs (table 2).

Table 1

Treatment of A498 kidney cancer cells with Ptdlns derivatives in combination with bv-sPLA2 induces cell lysis.

A498 cells were treated under serum-free conditions with bv-sPLA2 (10 μg/ml) in combination with the indicated substances (each at 10 μM). Cell lysis was evaluated by phase contrast microscopy. ("+" cell lysis; "-" no lysis).

Table 2

Treatment of non-proliferating normal cells with 3"-phosphorylated Ptdlns in combination with bv-sPLA2 induces cell lysis.

PBMCs and moDCs (immature and mature) were treated under serum-free conditions with Ptdlns(3,4)P2 (10 μM), or Ptdlns(4,5)P2 (10 μM) respectivly, in combination with bv-sPLA2 (10μg/ml). Cell lysis was evaluated by phase contrast microscopy. ("+" cell lysis; "-" no lysis).

Cell lysis

Cells bv-sPLA2 + Ptdlns(3 ,4)P2 bv-sPLA2 + Ptdlns(4,5)P2

PBMCs + — moDCs (immature) + — moDCs (mature) + —

Example 3: Lysis and growth inhibition of cancer cells derived from various tissues with Ptdlns derivatives with phosphorylation at position 3 of the inositol head group and bv-sPLA2

To quantify the synergistic effect of bv-sPLA2 and Ptdlns(3,4)P2, A498 cells were treated with bv-sPLA2 and Ptdlns(3,4)P2 either alone or in combination. After 32 h of treatment A498 cells were pulsed with [³H] thymidine and cell proliferation was measured as [³H] thymidine incorporation. Either substance alone had indeed little effect on cell proliferation (19% and 12% inhibition, respectively), while the combination dramatically inhibited cell proliferation (81 % inhibition) (data shown in table 3). To investigate whether the observed effect was cell type-specific or not, other cell lines derived from different tissues were also tested. bv-sPLA2 and Ptdlns(3,4)P2 exhibited a similar synergistic effect in the T-47D breast cancer (ductal carcinoma) cell line, in DU 145 prostate cancer cells and in immortalized BEAS-2B bronchial epithelial cells (data shown in table 3). bv-sPLA2 and Ptdlns(3,4)P2 synergistically induced the lysis of cancer cells derived from various tissues (kidney, prostate, lung, breast). Table 3

Treatment of cancer cells from various tissues with the combination of 3'- phosphorylated Ptdlns and bv-sPLA2 inhibits cell proliferation and induces cell lysis. Various transformed cell lines were cultured under serum-free conditions and either left untreated (C) or treated with Ptdlns(3,4)P2 (10 μM), with bv-sPLA2 (10 μg/ml, and 5 μg/ml for T-47D respectively) or a combination of both, Ptdlns(3,4)P2 and bv-sPLA2. After 48 h of incubation, proliferation was determined by assessing [3H] thymidine incorporation. Shown are normalized mean values, untreated controls were set to 100 %. Inhibition induced by the single substances (Ptdlns(3,4)P2 or bv- sPLA2) were totalized and compared with the inhibition induced by the simultaneous treatment with both substances. Cell lysis was evaluated by phase contrast microscopy. ("+" cell lysis; "-" no lysis).

Example 4: Structural requirements of Ptdlns derivatives for synergistic effects with bv-SPLA2.

In order to deduce the structural requirements of Ptdlns derivatives for the observed synergistic effects with bv-sPLA2, the effects of Ptdlns or its derivatives alone or in combination with bv-sPLA2 on A498 cell proliferation were examined. In combination with bv-sPLA2, the 3'-phosphorylated Ptdlns were most potent (Ptdlns(3,4,5)P3 > Ptdlns(3,4,5)P3-AA > Ptdlns(3,4)P2) in inhibiting [³H] thymidine incorporation into A498 cells (data summarized in table 4). Unphosphorylated Ptdlns also showed synergistic activity with bv-sPLA2 although at a lower level. Ptdlns(4,5)P2 lacking 3'- phosphorylation and lns(1 ,3,4)P3 lacking the phosphatidyl group failed to act synergistically with bv-sPLA2. These data reveal a structural hierarchy in which the Ptdlns triphosphate (P3) with saturated fatty acids at sn-1 and sn-2 is most potent. The 3'-phosphorylation of the Ptdlns appears to be critically since the three most potent substances are all 3'-phosphorylated. In contrast, lns(1 ,3,4)P3 alone exhibited merely weak activity. Accordingly, the phosphatidyl rest is of high relevance without being bound by theory, one function of the phosphatidyl may be that of an acceptor for fatty acids (either be saturated or unsaturated) and thus exhibit different activities in combination with bv-sPLA2 (table 4). Surprisingly, only Ptdlns derivatives with phosphorylation at position 3 of the inositol head group display potent synergy with bv-sPLA2.

Example 5: lmmunomodulating properties of cell lysates generated through the combined treatment with Ptdlns(3,4)P2 and bv-sPLA2

lmmunomodulating properties of cell lysates generated through the combined treatment with Ptdlns(3,4)P2 and bv-sPLA2 inducing maturation of human DCs were examined. For this purpose, A498 Ptdlns(3,4)P2-bv-sPLA2 lysate was added to cultures of immature moDCs. The A498 Ptdlns(3,4)P2-bv-sPLA2 lysate was pretreated with serum in order to quench its lytic activity. Serum components such as albumin serve as scavengers, which bind and inactivate lipids including lysophospholipids. Otherwise, the moDCs would also be lysed. A conventional lysate of A498 cells which was generated through three cycles of freezing at -80⁰C and rapid thawing at 37°C was used as a control. Freeze-thaw cycles represent an established procedure for the preparation of cell lysates (Sauter et al. 2000) and result in complete cell lysis as assessed by trypan blue exclusion (data not shown). The treatment with the lysates was performed in the presence or absence of TNF-α and IL-1β which are well established maturation-inducing cytokines (8). After 48 h of treatment of day-5 moDCs with the A498 cell lysate, moDCs were harvested, counted and prepared for flow cytometry. An intriguing finding at this step was the consistently higher moDC yield in the presence of the Ptdlns(3,4)P2-bv-sPLA2 lysate as compared to the freeze-thaw lysate (Fig. 2). These data indicate that the administration of Ptdlns(3,4)P2-bv-sPLA2 lysate improves survival of the moDCs and thus results in higher DC yields as compared to the conventional freeze-thaw lysate. MoDCs were then stained for the expression of CD83 which is one of the most reliable markers of moDC maturation (maturation is a late differentiation step of DC development which generates immunity-stimulating DCs) (Rieser et al. 1997). While only 12% of untreated moDCs expressed the CD83 marker (Fig. 3 a), 67% of the moDCs treated with the Ptdlns(3,4)P2-bv-sPLA2 lysate were CD83-positive (Fig. 3 d). In contrast, only 17% of the moDCs treated with the freeze-thaw lysate expressed CD83 (Fig. 3 c), and coculture of moDCs with viable, non-lysed A498 cells even appeared to reduce CD83 expression (10% positive cells, Fig. 3 b). Either substance alone induced the maturation of 28% (Ptdlns(3,4)P2) and 37% (bv-sPLA2) of the moDCs (Fig. 3 e+f). These data indicate that the Ptdlns(3,4)P2-bv-sPLA2 lysate induces moDC maturation. Similar results were obtained when moDC maturation was induced with TNF-α and IL-1β (Fig. 3 g-l). Under these conditions, 80% of the cells were CD83-positive after treatment with the Ptdlns(3,4)P2-bv-sPLA2 lysate (Fig. 3 j) as compared to 55% of CD83-positive moDCs after treatment with the conventional lysate (Fig. 3 i). Taken together, in contrast to the conventional freeze- thaw lysate the Ptdlns(3,4)P2-bv-sPLA2 lysate induces maturation on its own and enhances the maturation induced by TNF-α and IL-1 β (Fig. 3).

Example 6: Increased T-cell stimulatory capacity of moDCs treated with the Ptdlns(3,4)P2-bv-sPLA2 lysate

The purpose of moDC maturation is to increase T-cell stimulatory capacity. The T-cell stimulatory capacity of moDCs treated either with the conventional A498 lysate or the Ptdlns(3,4)P2-bv-sPLA2 lysate in the allogeneic MLR was compared. Treatment with the cell lysates was performed both in the presence and absence of TNF-α plus IL- 1β. moDCs treated with the Ptdlns(3,4)P2-bv-sPLA2 lysate were always more potent in inducing the proliferation of allogeneic T cells than moDCs treated with the conventional lysate (Figure 4). Table 4

The inhibitory potential of bv-sPLA2 combined with Ptdlns-derivatives is dominated by 3'-phosphorylated Ptdlns.

A498 cells cultured under serum-free conditions were left either untreated (control, C) or treated with the indicated Ptdlns-derivatives (each 10 μM), with bv-sPLA2 (10 μg/ml), or a combination of both, the Ptd Ins-derivative and bv-sPLA2. After 48 h of incubation, proliferation was determined by assessing [³H] thymidine incorporation. Shown are normalized mean values, untreated controls were set to 100 %. Inhibition induced by single substances (Ptdlns derivative or bv-sPLA2) were totalized and compared with the inhibition induced by the simultaneous treatment with both substances. Cell lysis was evaluated by phase contrast microscopy. ("+" cell lysis; "-" no lysis).

Example 7: bv-sPLA2 cleaves Ptdlns(3,4)P2 to generate lyso-Ptdlns(3,4)P2

Reagents

Methanol HPLC grade and Chloroform p.a were purchased from Merck (Darmstadt, Germany). 1 ,2 dipalmitoylphosphatidylinositol (C32:0 Ptdlns-ISTD), piperidine and ammonium acetate (MS grade) were purchased from Sigma-Aldrich (Vienna, Austria)

Mass spectrometry

Lipids were extracted from cell culture and media control samples using a modified procedure of Folch et al. (Folch et al. (1957), J Biol Chem. 226(1 ):497-509) omitting the salt addition. Mass spectrometric analyses were performed on a triple-quadrupole mass spectrometer, the API4000 Qtrap (Applied Biosystems/MDS Sciex Toronto, Canada) operated in negative ionisation modes with a turbo-ionspray source and Analyst v1.4.1 data system (Applied Biosystems/MDS Sciex Toronto, Canada). Samples (50 μl) were injected via an CTC-HTC-PAL autosampler (Zwingen, Switerland) or infused (100 μl) with a syringe into the electrospray source at a flow rate of 25 μl/min by a binary Agilent HP 1100 HPLC pump, (Bόblingen, Germany) or 10 μl/min with a syringe pump (Harvard, Massachusetts, USA) respectively. For HPLC a carrier solvent of 4:1 CH₃OH / CHCI₃ was used. Nitrogen was used for the collision and sheath gas. Precursor scans (PS in negative ion mode) of lipid head groups included PS 255, 241 , 321 and 401.

Sample preparation for mass spectrometric lipid analysis of A498 cells

A498 cells (10⁵/ml) were incubated in serum-free medium (6 ml) in the presence or absence of bv-sPLA2 (10 μg/ml), Ptdlns(3,4)P2 (10 μM) or a combination thereof for 2 h at 37°C and 5% CO₂. Supernatants and cells were harvested, 4.5 ml of this suspension were concentrated by lyophilisation. Samples were then reconstituted in 300 μl H₂O and transferred into a 10 ml glass tube. 1 ml of methanol was used to wash residual material followed by a further wash with 2 ml of chloroform and 3 ml of chloroform/methanol (2:1 v/v). Finally 250 pmol of 1 ,2 dipalmitoylphosphatidylinositol was added to each tube as an internal standard and the mixture was incubated for 1 h in the dark at room temperature whilst shaking. Then, 1.5 ml of destilled H₂O was added to the mixture and shaken for further 30 min. The samples were centrifuged for 10 min at 1500 x g and the lower organic phase was collected and dried under a gentle stream of nitrogen. Prior to ESI-MS/MS (electrospray mass spectrometry) analysis the samples were reconstituted in 1 ml_ of chloroform/methanol/H₂0 (2:1 :01 v:v:v). containing 10 mM ammonium acetate.

Sample preparation for mass spectrometric lipid analysis in cell free media

Serum-free medium (2 ml) supplemented with bv-sPLA2 (10 μg/ml), Ptdlns(3,4)P2 or a combination thereof was incubated for 2 h at 37°C and 5% CO₂ and was processed and analysed as previously described but with the following changes to improve recovery of the Ptdlns(3,4)P2 and its purported lyso product. After the 1 h incubation in the dark at room temperature whilst shaking, water was substituted with a 10 mM ammonium acetate solution and the upper phase including the interface was dried and reconstituted with methanol (200 μl) for desalting with a (pre-washed) reverse- phase extraction cartridge (Isolut C18 endcapped, 50 mg). The samples were desalted on-column with 2 ml of destilled water and then eluted with 300 μl methanol and 500 μL of chloroform/methanol/H₂0 (2:1 :01 v:v:v). The combined eluants were dried with a gentle stream of nitrogen gas. Prior to mass spectrometric analyses the samples were reconstituted in 400 μL of chloroform/methanol/H₂0 (2:1 :01 v:v:v) containing 30 mM piperidine.

B v-s P L A2 hydrolyzes Ptdlns(3,4)P2 to generate lyso-Ptdlns(3,4)P2

Aforementioned data outlines a series of combined effects that mediate the inhibitory actions of bv-sPLA2 and Ptdlns(3,4)P2, including abrogation of signal transduction and membrane damage. One further possibility for a combined interaction is that Ptdlns(3,4)P2 serves as a substrate of bv-sPLA2 which cleaves fatty acids from the sn-2 position to generate the corresponding lyso-Ptdlns(3,4)P2 with putative cytotoxic properties. This hypothesis was tested in a set of experiments, where bv-sPLA2 (10 μg/ml) was incubated with Ptdlns(3,4)P2 (10 μM) in the absence of A498 cells for 2 hours. Commercially available 1 ,2-dipalmitoylphosphatidylinositol (C32:0 Ptdlns- ISTD) was added to samples prior to extraction as an internal standard (ISTD) for quantification of all samples including lyso-Ptdlns(3,4)P2. The reaction products were extracted and subjected to analysis by mass spectroscopy. Lipids other than Ptdlns in general were monitored in this system requiring ammonium acetate. These are not the ideal conditions for observing highly phosphorylated Ptdlns as they undergo in- source defragmentation giving rise to product ions including losses of fatty acid and phosphate causing artefactual ions correspondingly. Hence analysis of the bv-sPLA2 digest of the Ptdlns(3,4)P2 gives rise to a prominent peak corresponding to a monoacyl-Ptdlns at m/z 571. Supporting evidence of this is shown by the addition of piperidine to the cell free lysis mixture prior to ESI-MS/MS analysis. Piperidine is reported to stabilise the phosphate groups minimising the in-source fragmentation (Wenk (2003), Nat Biotech. 21 :813-817). The mass spectrometry analysis of the Ptdlns(3,4)P2 (denoted C32:0 PtdlnsP2) by the InsP head group specific ion scan PS 241 , clearly show this (Fig. 6A and B) and to a lesser extent in PS 321 and PS 401 scans (data not shown), albeit with severely diminished ionisation responses and an ever present in-source fragmentation series of product ions. The lyso product is clearly observed in figure 6B at m/z 731.3 supporting the original premise that Ptdlns(3,4)P2 is a substrate for bv-sPLA2 in the original A498 tumor cell lysis experiments (Tab. 5). A small background of m/z 571 is also present in the ISTD+media only (Fig. 5A) due to back ground in-source fragmentation of the ITSD itself. This small amount of the m/z 571 is due to the same lipid backbone and InsP head group and therefore requires substraction from other analyses. Future ISTD choices when available will avoid this complication. In the cell lysis experiments summarised in table 5, lyso-Ptdlns(3,4)P2 was increased from 0.08 μM to 0.98 μM, a 12 fold increase, when bv-sPLA2 and Ptdlns(3,4)P2 were incubated together with cells. However, the administration of bv-sPLA2 or Ptdlns(3,4)P2 alone did not increase lyso-Ptdlns(3,4)P2 levels significantly above background (<0.2 μM). Taken together these data demonstrates that bv-sPLA2 hydrolyzes Ptdlns(3,4)P2 to generate lyso-Ptdlns(3,4)P2. This hydrolysis occurs in the presence and absence of cells but is somewhat reduced in the presence of A498 cells. The data provided herein document that lyso-Ptdlns(3,4)P2 which is generated by bv-sPLA2-mediated hydrolysis of Ptdlns(3,4)P2 (Figures. 5, 6 and table 5) is involved in the observed antitumor action. Accordingly, 3'-phosphorylated phosphatidylinositols are of particular medical use.

Table 5

Generation of lyso-Ptdlns(3,4)P2

Quantification of lyso-Ptdlns(3,4)P2 (m/z 571) was based on precursor scan 241.

Cell free A498 cells

Treatment lyso-Ptdlns(3,4)P2 [μM] lyso-Ptdlns(3,4)P2 [μM]

Control (medium only) 0.43 -

Control (cells + medium) 0.08 bv-sPLA2 0.50 0.1

Ptdlns(3,4)P2 0.11 0.2 bv-sPLA2 + Ptdlns(3,4)P2 2.06 0.98

These data demonstrate that during the herein described co-operative interaction between sPLA2 and 3'-phosphorylated Ptdlns, the 3'-phosphoarylated lyso-Ptdlns are readily formed and low concentrations of 1 to 2 μM. that is in surprising contrast to the elevated concentrations provided in Masamune (2001 , loc. cit.).

Claims

Claims

1. A combination or a combination compound comprising a 3'-phosphorylated form of phosphatidylinositol and a secretory (s) phospholipase A2.

2. The combination or combination compound of claim 1 , wherein said phospholipase A2 is a phospholipase A2 of group III.

3. The combination or combination compound of claim 1 or 2, wherein said phospholipase A2 is a venom sPLA2.

4. The combination or combination compound of claim 3, wherein said venom sPLA2 is bee venom sPLA2 (bv sPLA2).

5. The combination or combination compound of any one of claims 1 to 4, wherein said 3'-phosphorylated phosphatidylinositol is a compound of the formula I

wherein

R¹ and R² are independently selected from C_4-24 alkyl and C_4-24 alkenyl; and R⁴ and R⁵ are independently selected from OH and OPO₃ ^2'.

6. The combination or combination compound of claim 5, wherein said 3'- phosphorylated phosphatidylinositol is phosphatidylinositol-3,4-biphosphate (Ptdlns(3,4)P2) or phosphatidylinositol-3,4,5-triphosphate (Ptdlns(3,4,5)P3).

7. The combination or combination compound of claims 5 and 6, wherein said 3'- phosphorylated phosphatidylinositol is: 1-(1 ,2- dihexadecanoylphosphatidyl)inositol-3,4,5-triphosphate), 1-(1 ,2- dihexadecanoylphosphatidyl)inositol-3,4-diphosphate), 1 -(1 -octadecanoyl-2- (5Z,8Z, 11 Z, 14Z)-eicosatetraenoylphosphatidyl)inositol-3,4,5-triphosphate), 1 - (1 ,2-dihexadecanoylphosphatidyl)inositol-3-phosphate), or 1-(1 ,2- dihexadecanoylphosphatidyl)inositol-3,5-diphosphate).

8. The combination or combination compound of any one of claims 1 to 7, wherein said 3'-phosphorylated form of phosphatidylinositol is a salt.

9. The combination or combination compound of claim 8, wherein said salt is a sodium salt or an ammonium salt.

10. A pharmaceutical composition comprising the combination or combination compound of any one of claims 1 to 9.

11. A pharmaceutical composition comprising a 3'-phosphorylated lyso- phosphatidylinositol.

12. The pharmaceutical composition of claim 11, wherein said 3'-phosphorylated lyso-phosphatidylinositol is lyso-phosphatidylinositol-3,4-biphosphate (lyso- Ptdlns(3,4)P2).

13. Use of a combination or a combination compound of any one of claims 1 to 9 or of 3'-phosphorylated lyso-phosphatidylinositol for a preparation of a pharmaceutical composition for the treatment of a proliferative disorder and/or tumorous disease.

14. The use of claim 13, wherein said tumorous disease is cancer.

15. The use of claim 14, wherein said cancer is an urogenitcal cancer, a breast cancer, a pleural cancer, a bronchial cancer, a lung cancer, an oral cavity cancer, a pharynx cancer, an oesophagus cancer, a stomach cancer, a colon cancer, a rectum cancer, a liver cancer, a pancreas cancer, a larynx cancer, a melanoma of skin, a brain cancer, a nervous system cancer, a thyroid cancer, a Non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, leukemia, cancer of the cervix uteri, cancer of the corpus uteri, ovary cancer.

16. The use of claim 15, wherein said urogenital cancer is cancer of kidney, bladder, prostate and/or testis.

17. Use of a combination or a combination compound for any one of claims 1 to 9 or of 3'-phosphorylated lyso-phosphatidylinositol for the in vitro generation of a cellular lysate.

18. The use of claim 17, wherein said cellular lysate is a cellular lysate of non- tumorous cells or of tumorous/tumor cells.

19. Method for the generation of a cellular lysate of normal or tumorous/tumor cells comprising the steps of:

(a) contacting non-tumorous cells or tumorous/tumor cells with the combination or combination compound of any one of claims 1 to 9 or with 3'-phosphorylated lyso-phosphatidylinositol; and

(b) obtaining the resulting cellular lysate.

20. The method of claim 19, wherein said step (b) comprises addition of stabilizing agents selected from the group consisting of serum components, proteins, protease inhibitors, fractionation, centrifugation, filtration chromatography, adsorption or lyophilization.

21. The method of claim 19, wherein said tumorous/tumor cells are tumor cells selected from urogenital cancer cells, breast cancer cells, kidney cancer cells, prostate cancer cells, lung cancer cells, pleural cancer cells, bronchial cancer cells, oral cavity cancer cells, pharynx cancer cells, oesophagus cancer cells, stomach cancer cells, colon cancer cells, rectum cancer cells, liver cancer cells, pancreas cancer cells, larynx cancer cells, cells of melanoma of skin, brain cancer cells, nervous system cancer cells, thyroid cancer cells, cells of Non-Hodgkin lymphoma, cells of Hodgkin's disease, multiple myeloma cells, leukemia cells, cells of cancer of the cervix uteri, cells of cancer of the corpus uteri, or cells of ovary cancer.

22. The method of claims 19 to 21 , which further comprises the step

(c) of eliminating and/or inactivating inherent nucleic acid molecules, bacterial contamination or viral contamination.

23. The method of claim 22, wherein said elimination and/or inactivation comprises the treatment of the obtained lysate with radioactive radiation, sterile-filtration, or sterilization by heating.

24. A pharmaceutical composition comprising a cell lysate as generated by the use of claim 17, as obtained by the method of any one of claims 19 to 23 or comprising a cell lysate that comprises a 3'-phosphorylated lyso- phosphatidylinositol.

25. Use of the cell lysate as generated by the use of claim 17 or as obtained by the method of any one of claims 19 to 23 or a cell lysate that comprises a 3'- phosphorylated lyso-phosphatidylinositol for the preparation of a pharmaceutical composition for the in vitro and/or in vivo immunomodulation of blood cells.

26. Use of a 3'-phosphorylated lyso-phosphatidylinositol for the preparation of a pharmaceutical composition for the in vitro and/or in vivo modulation of blood cells.

27. The use of claim 25 or 26, wherein said immunomodulation of blood cells is the maturation of immature dendritic cells (DC) to mature dendritic cells (DC).

28. The use of any one of claims 25 or 27, wherein said pharmaceutical composition is to be administered in combination with cytokines.

29. The use of claim 28, wherein said cytokine is TNF-α or an interleukin.

30. The use of claim 29, wherein said interleukin is interleukin-1β.

31. Use of the cell lysate as generated by the use of claim 17 or as obtained by the method of any one of claims 19 to 23, or of a lysate comprising 3'- phosphorylated lyso-phosphatidylinositol or use of 3'-phosphorylated lyso- phosphatidylinositol for the preparation of a pharmaceutical composition for the treatment of proliferative disorder and/or a tumorous disease or of an autoimmune disease.

32. The use of claim 31 , wherein said autoimmune disease is selected from the group consisting of allergy, multiple sclerosis, diabetes mellitus, rheumatic arthritis, Lupus erythematosus, Morbus Crohn, and autoimmune disease by transplantation.