EP1425303A2

EP1425303A2 - Use of proteins for the production of a medicament for stimulating the innate non specific immune system

Info

Publication number: EP1425303A2
Application number: EP02762464A
Authority: EP
Inventors: Martin Herrmann; Wolf Bertling; Joachim R. Kalden; Reinhard E. Voll; Angelo Manfredi; Wasilis Kolowos; Christian Reiser; Walter Woith
Original assignee: responsif GmbH
Current assignee: responsif GmbH
Priority date: 2001-09-13
Filing date: 2002-08-24
Publication date: 2004-06-09
Also published as: JP2005505567A; CA2460331A1; WO2003022883A3; HU0104335D0; WO2003022883A2; US20050059587A1; DE10145254A1; HUP0104335A2

Abstract

The invention relates to the use of a protein for producing a medicament for stimulating an innate non specific immune system, wherein the protein contains a) an annexine V or a largely similar molecule thereto or b) an active fragment of annexine V or a largely similar molecule thereto.

Description

Use of a protein in the manufacture of a medicament for stimulation. _'of the innate unspecific immune system

The invention relates to the use of a protein for the manufacture of a medicament and a medicament for stimulating the innate unspecific immune system. It also relates to a method for producing the medicament.

Pathological cell death, necrosis, is associated with swelling of the cell organelles and the cell itself. The cytoplasmic membrane becomes permeable and the cell contents are released. In contrast, the programmed cell death, apoptosis, initially preserves the cell membrane. The cells are removed before their potentially dangerous contents are released into the environment. In an early apoptosis stage, the cells are subject to surface changes, for example a modification of the carbohydrates and the exposure of anionic phospholipids, in particular phosphatidylserine (PS) on the outside of the cytoplasmic membrane. The latter is caused by the downregulation of the ATP-dependent aminophospholipid translocase, which specifically transports aminophospholipids from the outside to the inside of the membrane. In addition, a non-specific lipid flip site called scramblase is activated, which leads to an acceleration of the phosphatidylserine flip-flop mechanism. Phosphatidylserine located on the outside of the cytoplasmic membrane serves as a recognition signal for the removal of apoptotic cells.

It is known that preincubation of apoptotic lymphocytes with Annexin V (AxV) effectively blocks the uptake of apoptotic cells by murine peritoneal macrophages, macrophages of the J774 cell line of the mouse and bone marrow macrophages. ciert (Krahling, S., et al., Cell Death Differ. 6 (1999), 183-189). In addition, Annexin V generally leads to strong inhibition of apoptotic cell uptake by both activated and non-activated macrophages. Due to its Ca ²⁺ channel activity, Annexin V also slows down apoptosis in CEM cells.

Eliminating apoptotic cells normally does not induce inflammation or an immune response. In direct contact with plasma, apoptotic cells can produce procoagulatory and, under certain conditions, pro-inflammatory effects. On monocytes / macrophages, however, normally immunomodulating effects of apoptotic cells dominate through the interaction of the apoptotic cells via thrombospondin with CD36 on phagocytes.

Lectin-like molecules, such as the vitronectin receptor (CD51 / CD61), thrombospondin, CD36 and CD14, are receptors that recognize surface changes on apoptotic cells. CD14 appears to be necessary for phagocytosis of lymphocytes and lipid-symmetric erythrocytes in activated and non-activated macrophages.

The phagocytosis of apoptotic lymphocytes by macrophages is stereospecifically inhibited by phosphatidyl-L-serine liposomes. A receptor responsible for the recognition of phosphatidyl-L-serine is defined by antibodies which have been caused by immunization with TGF-β and β-glucan-stimulated macrophages (Fadok, VA, et al., Nature 405 (6782) (2000 ), 85-90). Other receptors important for the detection of apoptotic cells include the scavenger receptors, the LPS receptor CD14, the thrombospondin receptor CD36, the vitronectin receptor CD51 / CD61, the complement receptors and the macrosialin CD68. In addition, pentraxins, collectines, different complement factors, ß2-glycoprotein, the annexins and gas-6 serve as adapter proteins.

It is also known that mice injected with apoptotic human T cells showed significantly reduced humoral responses to T cells compared to control mice injected with viable cells (Ponner, BB, et al., Scand. J. Immunol. 47: 343-347 (1998). It was also observed that immunization with apoptotic cancer cells induced drastically reduced cytotoxic T cell responses compared to living growth-blocked cells (Ronchetti, A., et al., J. Immunol. 163 (1999), 130-136 ). This shows that the engulfment phagocytosis of apoptotic cells does not lead to an efficient antigen presentation and activation of T and B lymphocytes. It is possible that rapid engulfment phagocytosis of apoptotic cells by macrophages and their anti-inflammatory response prevents the uptake and efficient presentation of antigens originating from apoptotic cells by dendritic cells. Together with monlect / macrophage production of interleukin-10 (IL-10) after contact with apoptotic cells, these findings can explain the poor efficiency of cancer vaccines containing apoptotic cells.

From Stach C. M. et al. , Gell Death and Differentiation (2000) 7, 911-915 it is known that Annexin V stimulates the humoral immune response. This article contains no information on stimulation of the cellular immune response or the innate unspecific immune system.

The object of the invention is to eliminate the disadvantages of the prior art. In particular, it is intended to be used and a medicament against tumors, viruses, bacteria and parasites are given. A method for producing the medicament is also to be specified.

This object is solved by the features of claims 1, 14 and 22. Expedient embodiments of the inventions result from the features of claims 2 to 13, 15 to 21 and 23 to 33.

According to the invention, the use of a protein for the manufacture of a medicament for stimulating the innate unspecific immune system is provided, the protein

a) Annexin V or a largely similar molecule

or

b) comprises an effective fragment of Annexin V or the molecule which is largely similar to it.

Surprisingly, it has been shown that the use of the protein according to the invention leads to a pro-inflammatory response of the monocytes or macrophages and thus to an increase in cellular immunity. The claimed protein is ideal for combating tumors, viruses, bacteria and parasites.

The effect of the protein according to the invention is obviously based on the fact that apoptotic cells are changed by loading with Annexin V in such a way that they are able to activate the innate unspecific immune system, in particular natural killer cells (NK cells) and macrophages. Furthermore, the effective concentration of the monokine TNF-α is increased by the administration of Annexin V. The monokine TNF-α is particularly important for the recruitment of NK cells in the Peritoneum (Smyth, MJ et al., 1998, J. Exp. Med. 9: 1611-1619). Furthermore, the effect of the protein according to the invention is in particular also attributed to the fact that it binds to phosphatidylserine and inhibits its anti-inflammatory potency. This function of the protein is not only fulfilled when it is annexin V. It is also met if the protein only comprises a molecule similar to Annexin V or an effective fragment of Annexin V or a molecule largely similar to the fragment. - A fragment is "effective" in particular when it binds to phosphatidylserine and contributes to the stimulation of a pro-inflammatory cellular immune response. The term “similar molecules” is understood to mean those molecules which bind in particular to phosphatidylserine and to a certain extent have an identity with Annexin V.

According to one embodiment, an amino acid sequence of the protein can correspond to the amino acid sequence of SEQ ID No. 1 or No. 2 or at least 50%, preferably at least 60%, particularly preferably at least 70%, very particularly preferably at least 80%, identical to it his. Identity can be determined e.g. according to the method of Altschul, S.F. et al. (1997), Nucleic Acids Res. 25: 3389-3402.

The term “identity” is understood here to mean the extent to which two nucleotide or amino acid sequences are invariant.

The amino acid sequence of SEQ ID No. 2 is an N-terminal deletion mutant of the amino acid sequence of SEQ ID No. 1, which lacks the eight amino acids 3 to 10, that is, the amino acids Lys Tyr Thr Arg Gly Thr Val Thr. Appropriately, the Annexin V is non-human Annexin V, preferably the Annexin V of the chicken. A comparison of the amino acid sequence of chicken annexin V with human annexin V shows that both proteins are 78.2% identical. Chicken annexin V has a theoretical isoelectric point (p1) of 5.60, while human annexin V has a theoretical isoelectric point of 4.94. The sequence of the human annexin can be called up under the accession number P08756 in the protein database "SWISS-PROT". Both annexins are immunogenic in the animal model.

The immune response can be stimulated by blocking, covering, masking and / or removal of extracellular membrane-located phosphatidylserine. The protein can thus be used expediently to combat bacteria, viruses, parasites or tumors. The protein is preferably used as an adjuvant. In this context, it can be used preferably in tumor therapy, for tumor vaccines, in virus therapy, in particular for the treatment of retrovirus infections, lentivirus infections, infections with Treponema pallidum, the Sindbis virus, Trypanosoma brucei and HIV infections and for the treatment of malaria and in malaria immunization.

In addition to the active substance, the medicament can further comprise human tumor cells, wherein the tumor cells can be apoptotic and / or necrotic tumor cells. The apoptosis and / or necrosis of the tumor cells can occur spontaneously or have been induced. Possible inducers for apoptosis and / or necrosis are radiation of the tumor cells ex vivo or in vivo or contact of the tumor cells with cytostatics. In this context, chemicals such as H ₂ 0 ₂ or staurosporine, medications such as corticosteroids, chemotherapeutic agents such as doxies are particularly suitable. orubicin, cis-platinum or hydroxy-urea, UVB and UVC radiation, as well as ß, γ or X-rays. The apoptotic and / or necrotic tumor cells are preferably brought into contact with the active substance.

According to a further provision of the invention, a medicament is provided for stimulating the innate unspecific immune system with a protein which

al) Annexin V or a largely similar molecule

or

a2) an effective fragment of Annexin V or the largely similar molecule and

bb) includes apoptotic and / or necrotic tumor cells.

Because of the advantageous configurations, reference is made to the preceding description. The features mentioned therein can also be configurations of the medication.

According to a further aspect of the invention, a method for producing the medicament according to the invention is provided, whereby apoptotic and / or necrotic tumor cells are brought into contact with a protein which

a) Annexin V or a largely similar molecule

or

b) comprises an effective fragment of Annexin V or the molecule which is largely similar to it. Because of the advantageous embodiments, reference is again made to the previous statements. The features specified there can also be used to design the method.

The protein used in this method is preferably produced by expression using transformed strains of Escherichia coli and subsequent purification. The Escherichia coli BL21 (DE3) strain is preferably used. The strain of Escherichia coli used can be transformed with an expression plasmid which is capable of expressing chicken annexin V or an active fragment thereof. For example, the expression plasmid pDJ2-AnXV, which in addition to the gene expressing chicken annexin V, contains an IPTG-inducible tac promoter and an antibiotic resistance cassette. Kanamycin is preferably used as the antibiotic for separating the non-transformed bacteria from the transformed bacteria.

The cells obtained using the transformed strains of Escheri chia coli, which after culture contain Annexin V of the chicken in a nutrient medium, are digested in a buffer. The solution obtained, which contains the Annexin V of the chicken, is purified by means of column chromatography, using a salt gradient, advantageously a sodium chloride gradient. The active ingredient is obtained with a purity that exceeds 95%.

The suitability of annexin V of the chicken or an active fragment thereof as an active ingredient is based on the finding that the masking of phosphatidylserine with this active ingredient leads to a pro-inflammatory effect of apoptotic and / or necrotic cells and thus promotes the cellular immune response against apoptotic cells. Chicken Annexin V increases the immunogenicity of autologous or syngeneic apoptotic cells clearly. It is therefore suitable to increase the efficiency of vaccines that contain apoptotic cells. This is particularly important if the cells have been irradiated before the vaccine is injected. Without the use of the active ingredient Annexin V of the chicken, the immunogenicity of these cells is low, since the uptake of apoptotic cells causes an anti-inflammatory reaction of human monocytes / macrophages. By treating the cells with chicken annexin V, this anti-inflammatory effect of apoptotic and / or necrotic cells is reduced, thus significantly increasing their immunogenicity.

The anti-inflammatory effect of phosphatidylserine-exposing cells can be blocked with the protein according to the invention. Furthermore, the protein can effect an immunostimulation by blocking, covering, masking and / or removing extracellular, preferably membrane-located phosphatidylserine. The protein can therefore be used as a modulator of a cellular immune response.

An important area of application for the blockade of the anti-inflammatory effects of phosphatidylserine-exposing cells results from the resulting specific "adjuvant effect". After blocking the anti-inflammatory principle of action, cells carrying phosphatidylserine are processed via a pro-inflammatory, immunostimulatory alternative route which leads to a massively increased immune response. For this "adjuvant effect" there are different fields of application, e.g. in human medicine.

On the one hand, the immunogenicity of tumor vaccines can be increased if they consist of irradiated and thus largely apoptotic tumor cells. Furthermore, it is possible to achieve a cellular immune response against those tumor cells that are cytostatically affected for therapeutic reasons. deals or have been radioactively irradiated in situ. In this case, a tumor-specific cellular immune response would increase the success of the therapy when eliminating the residual tumor mass. Also in the treatment of viral infections, for example enveloped viruses that expose phosphatidylserine, the blocking of the phosphatidylserine-dependent anti-inflammatory effect leads to a specific immune stimulation. The treatment of infections with lentiviruses and HIV must be seen as a particularly important example in this context. A penetration of the viruses, which is "unnoticed" by the cell, depending on the phosphatidylserine, leads to virus persistence in the long-lived monocyte / macrophage pool. Virus persistence in the vast majority of infected people results in death after a more or less long latency period. Chicken annexin V is suitable for the treatment of people infected with HIV, since inflammatory phagocytosed apoptotic material triggers a "respiratory burst" in the phagocytes and thus leads to the destruction of the virus genomes.

The same applies to the treatment of infections with bacteria exposing to phosphatidylserine (e.g. Treponema pallidum) or infections and parasitic diseases that lead to exposure of phosphatidylserine to infected host cells (e.g. Sindbis virus, Plasmodium fälciparum, Trypanosoma brucei).

The invention is explained in more detail below with reference to the drawings. Show it:

1 the expression kinetics of Annexin V of the chicken in transformed Escherichia Coli BL21 (DE3) on the basis of a polyacrylamide gel electrophoresis,

Fig. 2 is a polyacrylamide gel electrophoresis of samples of the individual purification steps of Annexin V des Chicken from transformed Escherichia Coli BL21 (DE3),

3 Ratios of the secreted cytokines as a function of the incubation of murine peritoneal macrophages with radioactively irradiated tumor cells which had previously been treated with chicken annexin V or not with annexin V,

4a shows the proportion of tumor-free animals as a function of the time and the injection of different amounts of irradiated tumor cells which had been treated after irradiation with or without chicken annexin V, the animals having been treated before the injection of living tumor cells,

4b shows the proportion of tumor-free animals as a function of the time and the injection of irradiated tumor cells which were treated after irradiation with or without chicken annexin V, the animals being injected with living tumor cells before the treatment,

Fig. 5a tumor latency of the mice that did not reject the tumor, depending on the dose of irradiated tumor cells and the administration of Annexin V and

5b survival of mice which have not rejected the tumor, depending on the dose of irradiated tumor cells and the administration of Annexin V,

6 shows the proportion of tumor-bearing animals as a function of the duration after tumor inoculation, 7 shows the survival rate as a function of the duration after tumor inoculation,

8 shows the [ ³ H] thymidine uptake of INA6 TUl cells above the AxV concentration and

Fig. 9 tabular summary of the results of the animal groups examined.

A. Expression and Purification of Chicken Annexin V

AI. Expression of Chicken Annexin V

For expression of chicken annexin V (cAnxV), expression plasmid pDJ2-AnxV was used, which in addition to the gene coding for cAnxV contains an IPTG-inducible tac promoter and a kanamycin resistance cassette. E.coli BL21 (DE3) was transformed with the expression plasmid and expression kinetics were carried out. 2xYT with 50 mg / 1 kanamycin was used as the nutrient medium.

For the preparation of cAnxV, a 100 ml preculture was inoculated with freshly transformed E. coli BL21 (DE3) and shaken at 37 ° C. for 8 h. 5 l of the main culture were mixed with 5 ml of the preculture and shaken at 37 ° C. for 16 to 20 h. The addition of IPTG was dispensed with, since in this case the same expression yields were achieved with and without induction. The cells were then harvested by centrifugation. The cell wet weight was 16 to 21 g. The expression kinetics is shown in FIG. 1. The expression kinetics showed that E. coli BL21 (DE3) is suitable for expression of cAnxV in the medium used even without induction with IPTG. A2. Purification of Annexin V of the chicken

The according to para. 1 cells obtained were resuspended in an Al buffer (20 mM Tris / HCl pH 7.5, 2 raM EDTA) and disrupted by high pressure (Gaulin). Insoluble constituents of the digestion suspension were removed by high-speed centrifugation. The soluble supernatant containing cAnxV was applied to a Q-Sepharose ff column (column 1) (25 ml, amersham pharmacia, Freiburg) equilibrated in buffer AI. The target protein was eluted by a linear one

NaCl gradient. The fractions containing cAnxV were pooled and dialyzed against a buffer A2 (50 mM Na acetate pH 5.6). The dialysate was applied to a Resource S column (column 2) equilibrated in A2 (6 ml, amersham pharmacia, Freiburg) and cAnxV eluted with a linear NaCl gradient. The combined fractions containing cAnxV were concentrated by means of ultrafiltration (Pall Filtron, USA) and applied to a Superdex 200 pg column (column 3) (amersham pharmacia, Freiburg), equilibrated in 10 mM Na phosphate pH 7.2, 140 mM NaCl , Homogeneous cAnxV was eluted from the column. 30 mg cAnxV with a purity exceeding 95% were isolated from 20 g cells (wet mass). 2 shows the results of the cleaning using columns 1 to 3 on the basis of a polyacrylamide gel electrophoresis.

Alternatively, instead of the specified columns for column 1 Q-Sepharose XL (amersham pharmacia, Freiburg), for column 2 SP-Sepharose HP (amersham pharmacia, Freiburg) and / or for column 3 Sephacryl S200 HR (amersham pharmacia, Freiburg) be applied.

Alternatively, a hydrophobic column can be used instead of the size exclusion chromatography (SEC) performed using column 3. In this case the fractions containing cAnxV that elute from column 2 are combined and Increase to 1.5 M with solid ammonium sulfate. The protein solution is applied to a Phenylsepharose ff column (15 ml, amersham pharmacia, Freiburg) and cAnxV eluted with a linear gradient from 1.5 to 0 M ammonium sulfate.

B. Immunization of mice with annexin V-treated, apoptotic tumor cells

Bl. Mice and tumor cells

Six to eight week old female C57BL / 6 mice were kept in a pathogen-free animal testing facility in accordance with the guidelines of the European Community. The syngeneic H-2b lymphoma line RMA was provided by Angelo A. Manfredi (Hospitale San Raffaele, 20132 Milan, Italy). The H-2b melanoma cell line B16F1 was obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cell lines were regularly tested for mycoplasma infection

B2. cell death

RMA cells were as described in Bellone, M., et al. , J. Immunol. 159 (1997), 5391-5399 described, irradiated with ultraviolet light, then treated for 1 h at 37 ° C. with 50 μg / ml mitomycin C and washed. The externalization of phosphatidylserine was followed by staining with FITC-labeled Annexin V (Bender MedSystems, Valter Occhiena, Torino, Italy). The ion tightness of the plasma membrane was determined using propidium iodide. The cells were analyzed 0, 3, 9 and 18 h after irradiation. The specificity of Annexin V binding was determined by treating irradiated cells with increasing concentrations (1500 mg / ml) of unlabeled Annexin V or bovine serum albumin (BSA) and subsequent determination of the remaining Annexin V - fluorescein isothio- cyanate binding determined by flow cytometry (FACScan, Becton-Dickinson, San Jose, USA).

B3. macrophages

Macrophages were isolated by adhering to plastic from inflammatory peritoneal lavages which had been induced by injection of thioglycolate (Fadok, V.A., et al., Nature 405 (2000), 85-90).

B4. Determination of cytokine release from macrophages after co-culture with irradiated tumor cells

Macrophages were co-cultivated in different ratios with irradiated tumor cells. The cytokines TNF-alpha, IL-lbeta, IL-10 and TGF-beta were measured in the culture supernatant using specific commercial ELISAs (DuoSet ELISA, R&D Systems, Minneapolis, MN).

B5. Immunization and healing

The mice were immunized by subcutaneous (sc) injection in the balls of 1, 5 or 10 × 10 ⁶ irradiated RMA cells. The irradiated cells had been preincubated with or without 100 μg / ml chicken Annexin V for 20 min at room temperature in an isotonic buffer containing Ca ²⁺ . On day 15, the mice received a boost injection on the right flank. On day 30, the effectiveness of the immunization was tested by sc injection of 2.5 x 10 ⁴ living RMA cells in the opposite flank. Twice a week, buttons were then used to check whether the animals had grown ore. One animal was considered tumor positive if the mean of the two diameters perpendicular to each other was> 2 mm. If a tumor grew> 10 mm or ulcerated, the infected animals were kills. Animals in which no tumor was palpable within 10 weeks after the injection of live RMA cells were rated as tumor negative. These tumor-free mice were again injected with 2.5 x 10 ⁴ living RMA cells in the opposite flank in order to check the duration of the protection. The specificity of the antitumor activity was checked in the protected animals by sc administration of 2.5 × 10 ⁴ syngeneic B16F1 melanoma cells (Bellone, M., et al., J. Immunol. 158 (1997), 783-789). To determine the effect of differences in the treatment of established RMA lymphomas, 2.5 x 10 ⁴ living RMA cells were injected sc into the left flank. After the tumor had grown for three days, the animals on the opposite flank were treated with 1 or 5 × 10 ⁶ irradiated RMA cells with or without Annexin V of the chicken (one vaccination per week for three weeks). Tumor growth was determined as described above.

B6. Result

Murine peritoneal macrophages have been incubated with different amounts of radioactively irradiated tumor cells (bTZ) (syngeneic RMA tumor cells) which had previously been treated with either Annexin V of the chicken or not with Annexin V of the chicken. After 24 hours, the culture supernatant was harvested and the release of the cytokines TNF-alpha, IL-1-beta,

IL-10 and TGF-beta measured with ELISAs. FIG. 3 shows the results of the tests with a bTZ / Mph ratio of 5 to 1. The black bars show the results of irradiated tumor cells which have been treated with chicken annexin V. The white bars show the results of irradiated tumor cells that have not been treated with chicken annexin V. The individual values of the measurements are summarized again in Table 1 below. Table 1

* P <0.05 ** P <0.01

Treatment of apoptotic cells with chicken annexin V shifts the cytokine pattern of phagocytes towards inflammation.

Irradiated syngeneic tumor cells treated with chicken annexin V protect and heal mice injected with a lethal tumor dose. The treatment leads to a long-lasting specific anti-tumor immunity.

4a and b each show the proportion of tumor-free animals as a function of the time and the injection of different amounts of irradiated tumor cells (bTZ), which had been incubated with or without Annexin V of the chicken after the irradiation. The results shown in Figures 4a and b with filled symbols relate to irradiated cells that have been treated with chicken's Annexin V. The white symbols relate to results with irradiated cells that were treated without chicken's Annexin V.

FIG. 4a shows results of experiments in which live tumor cells were injected into mice after injection with irradiated tumor cells, which had been incubated after the irradiation with or without chicken annexin V (vaccination approach). Live tumor cells were injected on day 0 (1st arrow) and in the surviving animals on day 72 (2nd arrow).

C57B1 / 6 mice were injected subcutaneously into the right flank twice according to the following scheme:

with PBS with 1 x 10 ⁶ irradiated tumor cells (bTZ) with 5 x 10 ^s irradiated tumor cells (bTZ) with 1 x 10 ⁷ irradiated tumor cells (bTZ)

On day 0, two weeks after the last booster injection, 2.5 x 10 ⁴ living RMA tumor cells were injected into the opposite left flank of the mice. On day 72, the injection was repeated in the surviving animals (arrow). 4a shows that the incubation with Annexin V of the chicken (black triangles) significantly increases the tumor rejection compared to the injection with untreated irradiated tumor cells (white triangles) in all groups.

In order to investigate the effect on pre-established tumors, 2.5 × 10 ⁴ living RMA tumor cells were injected subcutaneously into the left flank of C57B1 / 6 mice. Each cohort contained 10 mice. Four days later, either 1 x 10 ⁶ or 5 x 10 ^s irradiated RMA tumor cells were injected into the right flank. This injection was carried out after treatment of the irradiated cells with (filled symbols) or without (open symbols) Annexin V of the chicken. 4b shows the time course of the tumor-free animals. When 5 x 10 ⁶ tumor cells are injected, treatment of the irradiated tumor cells with chicken annexin V significantly increases their immunogenicity and the resulting tumor rejection. As shown in Figure 5, in the mice that were not fully protected, both tumor latency (Figure 5a) and survival were increased when the irradiated tumor cells were treated with chicken annexin V prior to injection ,

C. Data from SCID mice Cl. aim

Determination of the influence of AxV treatment of apoptotic cells on the innate unspecific immune system.

C2. material and methods

C2.1. cell

The human plasma cell line INA-6 was established from a pleural effusion of an eighty-year-old patient with plasma cell leukemia. It grows as a single cell suspension and shows the morphology of plasma blasts. The INA-6 Tul subcell line originates from a passage in SCID mice. In contrast to the original cell line, INA-6 Tul proliferates independently of interleukin-6 and, after intraperitoneal (ip) injection in SCID mice, forms palpable tumors (Burger, R. et al. (2001) Hematol. J 2: 42-53).

C2.2. Induction of apoptosis in INA-6 Tul cells

Apoptotic INA-6 Tul cells were generated by UV irradiation for 40 seconds and subsequent cultivation for 12 hours. At this dose, the cells show an apoptosis rate of approx. 90%, with an increase in the radiation dose changing the rate only insignificantly. C2.3. SCID mice

30 female, six-week-old SCID mice (Charles River Wiga GmbH) were used for the experiment. Due to a congenital defect, the SCID mice do not have a functional specific immune system in the form of mature T and B lymphocytes. The non-specific immune system of the animals is unaffected. In order to switch off an existing activity of natural killer cells (NK cells), the animals were exposed to whole-body gamma radiation with an individual dose of 2 Gy.

C2.4. Inoculation with INA-6 Tul

24 hours after the irradiation, the animals were injected with 2 × 10 ⁷ vitals (vitality rate approx. 90%) INA-6 Tul cells in 1 ml culture medium (RPMI 1640 with 20% fetal bovine serum) ip.

C2.5. Therapeutic immunization

24 hours after tumor inoculation, the mice were divided into three groups of equal size. The animals in the control group were injected 1 ml each with Hanks' Balanced Salt Solution (HBSS). The animals in the second group received 1 ml HBSS with 2 x 10 ⁷ apoptotic INA-6 Tul cells ip administered. Finally, the animals of the third group were injected with 1 ml HBSS each with 2 × 10 ⁷ apoptotic INA-6 Tul cells, the cells having been incubated with 100 μg chicken annexin V at room temperature 30 minutes before the ip administration. 2-6. Statistical evaluation of the data

The statistical analysis of the data was carried out by the log rank test using the software package SPSS 10.0.7 from SPSS Ine.

C2.7. Complementary experiment

In order to rule out a direct toxic effect of Annexin on INA-6 Tul cells, a proliferation test was carried out. For this purpose, 10 ⁴ INA-6 Tul cells per well were placed in flat-bottom microtiter plates with 0.2 ml medium (RPMI-1640 with 10% fetal bovine serum and 2.5 ng / ml human recombinant interleukin-6) and different amounts of annexin V (6 pg / ml to 50 μg / ml) incubated for 72 hours, the cells being pulsed in the last 6 hours with 1 μCi [ ³ H] -thymidine (Amersham) per well. The cells were then harvested and analyzed in a β-scintillation counter. The values in FIG. 8 show the mean [ ³ H] -thymidine incorporation in cpm (counts per minute) from triplicates with the simple standard deviation as an error bar. The curve clearly shows that Annexin V has no influence on the proliferation of INA-6 Tul cells.

C3. Observations during the animal study

48 hours after tumor inoculation, the animals in the second group, but especially the animals in the third group, showed strong general symptoms such as shaggy fur and weight loss, while the animals in the control group looked healthy on the outside. One animal from the third group was received on the third and fifth day after the tumor inoculation and one animal from the second group was received on the 18th day after the tumor inoculation. The respective cause of death could not be determined. However, it can almost certainly be will conclude that the animals died of the induced myeloma at this early stage. Therefore, when evaluating the experiment, only the remaining nine and eight animals from the second and third groups were taken into account. Animals in the final stage of the disease were killed before further investigation.

C4. Results

On day 29 after the inoculation, seven of the ten animals in the control group showed pea-sized tumors at the site of the inoculation. Comparable observations were also made in the second group, where seven out of nine animals showed significant tumor formation. Surprisingly, none of the eight animals treated with Annexin V-loaded apoptotic INA-6 Tul cells (group 3) were found to have a tumor at this time. The differences of the data regarding Tumor growth between group 3 and group 2 (p = 0.0001) or the control group (p = 0.004) are statistically significant (Fig. 9, table).

The mean survival after tumor inoculation was 54.8 days in the control group, while the mice in the second group even had a slightly shortened survival of 50.4 days on average. In contrast, the mean survival of six of the eight mice in the third group was significantly higher at 71.3 days. In addition, the remaining two animals in the third group survived the predetermined end of the observation period (day 95) by nine days. Animals with delayed tumors primarily showed liver infiltrates and ascites, while the ulcers grew locally rather early. The differences in survival data between group 3 and group 2 (p = 0.0004) or the control group (p = 0.0095) are statistically significant (Fig. 6, 7, table).

C5. Conclusions

The involvement of non-specific NK cells in addition to cytotoxic T cells directed to specific antigens in successful tumor defense is generally known.

Due to a genetic defect, SCID mice do not have a specific immune system, which means that they do not have any functional cytotoxic T cells. However, they are able to prevent tumor growth with the help of their macrophages and NK cells through nitrogen monoxide metabolites and / or perforin-mediated cytotoxicity (Cifone, MG, Ulisse, S., and Santoin, A. ( 2001) Int Immunopharmacol 1 (8): 1513-1524).

The available data show that the therapeutic administration of annexin-loaded apoptotic cells significantly delay the establishment of a human tumor in SCID mice and thus extend the survival time.

Apparently apoptotic cells are changed by the annexin loading in such a way that they are able to activate the functioning unspecific immune system - in particular NK cells and macrophages - of the SCID mice.

Above all, the regulation of the monokine TNF-alpha by annexin-loaded apoptotic cells (Fig. 6, 7) could be responsible for the activation of the NK cells. TNF-alpha is particularly important for the recruitment of NK cells into the peritoneum, since TNF-alpha-deficient mice are deficient here (Smyth, M.J. et al. (1998) J Exp Med 9: 1611-1619).

Claims

claims

1. Use of a protein for the manufacture of a medicament for stimulating the innate nonspecific Iraraun system, said protein

a) Annexin V or a molecule largely similar to it,

or

2. Use according to claim 1, wherein the protein binds to phosphatidylserine.

3. Use according to claim 1 or 2, wherein an amino acid sequence of the protein of the amino acid sequence of SEQ ID No. 1 or No. 2 corresponds or is at least 50%, preferably at least 60%, particularly preferably at least 70%, very particularly preferably at least 80%, identical with it.

4. Use according to one of the preceding claims, wherein the Annexin V is non-human Annexin V.

5. Use according to claim 4, wherein the non-human Annexin V is the Annexin V of the chicken.

6. Use according to one of the preceding claims, wherein the stimulation of the immune response by a blockade,

Veiling, masking and / or removal of extracellular membrane-located phosphatidylserine is effected.

7. Use according to any one of the preceding claims, wherein the protein is used to combat bacteria, viruses, parasites or tumors.

8. Use according to one of the preceding claims, wherein the protein as an adjuvant, preferably in tumor therapy, for tumor vaccines, in virus therapy, in particular for the treatment of retrovirus infections, lentivirus infections, infections with Treponema pallidum, Sindbis virus, Trypanosoma brucei and HIV infections and for Treatment of malaria as well as in malaria immunization.

9. Use according to any one of the preceding claims, wherein the medicament further comprises human tumor cells.

10. Use according to claim 9, wherein the tumor cells are apoptotic and / or necrotic tumor cells.

11. Use according to claim 10, wherein the apoptosis and / or necrosis is triggered by radiation of the tumor cells ex vivo or in vivo.

12. Use according to claim 10 or 11, wherein the apoptosis and / or necrosis is triggered by bringing the tumor cells into contact with cytostatics.

13. Use according to any one of claims 9 to 12, wherein the tumor cells are brought into contact with the protein.

14. Medicament for stimulating the innate non-specific immune system with a protein which

al) Annexin V or a molecule largely similar to it, or a2) an effective fragment of Annexin V or the largely similar molecule

and

bb) includes apoptotic and / or necrotic tumor cells.

15. Medicament according to claim 14, wherein the protein binds to phosphatidylserine.

16. Medicament according to claim 14 or 15, wherein an amino acid sequence of the protein corresponds to the amino acid sequence of SEQ ID No. 1 or No. 2 or at least 50%, preferably at least 60%, particularly preferably at least 70%, very particularly preferably at least 80%, is identical to it.

17. Medicament according to claim 14 to 16, wherein the Annexin V is non-human Annexin V.

18. Medicament according to claim 17, wherein the non-human Annexin V is the Annexin V of the chicken.

19. Medicament according to one of claims 14 to 18, wherein it further comprises human tumor cells.

20. Medicament according to claim 19, wherein the tumor cells are apoptotic and / or necrotic tumor cells.

21. Medicament according to one of claims 19 or 20, wherein the tumor cells are in contact with the protein.

22. The method for producing the medicament according to any one of claims 14 to 21, wherein apoptotic and / or necrotic Tumor cells are brought into contact with a protein which

a) Annexin V or a molecule largely similar to it,

or

23. The method of claim 22, wherein the protein binds to phosphatidylserine.

24. The method according to claim 22 or 23, wherein an amino acid sequence of the protein corresponds to the amino acid sequence of SEQ ID No. 1 or No. 2 or at least 50%, preferably at least 60%, particularly preferably at least 70%, very particularly preferably at least 80%, is identical to it.

25. The method according to any one of claims 22 to 24, wherein the Annexin V is non-human Annexin V.

26. The method of claim 25, wherein the non-human annexin V is the annexin V of the chicken.

27. The method according to any one of claims 22 to 25, wherein the apoptosis and / or necrosis is triggered by irradiation of the tumor cells ex vivo or in vivo.

28. The method according to any one of claims 22 to 27, wherein the apoptosis and / or necrosis is triggered by contacting the tumor cells with cytostatics.

29. The method according to any one of claims 22 to 28, wherein the protein by expression by means of transformed strains of Escherichia coli and subsequent cleaning is produced.

30. The method of claim 29, wherein the transformed strain of Escherichia coli is Escherichia coli BL21 (DE3).

31. The method according to claim 29 or 30, wherein pDJ2-AnXV is used as the expression plasmid.

32. The method according to any one of claims 29 to 31, wherein the cleaning of the expressed Annexin V is carried out by means of column chromatography.

33. The method of claim 32, wherein the column chromatography is carried out using a salt gradient.