PL227489B1 - Active substance with immunostimulating and anti-metastatic properties, originating from bacteriophage lysate - Google Patents

Description

The subject of the invention is an active substance of bacterial origin (LOS-OH E. coli B) with an immunostimulatory and antimetastatic effect derived from loss-of-disease lysates.

Cancer, the rapid unregulated pathological proliferation of abnormal cells, is the second most common cause of human death after cardiovascular disease. It is estimated that neoplastic diseases cause about 20% of all deaths (from any cause) in Poland (OECD, 2011). Currently, it is estimated that one in three women and one in 2 men in the United States will develop cancer (Bloom, 2011). The most common cancers in the world are, in the order of mortality: in men - lung, stomach, liver, colon and rectal cancers, esophagus, prostate and in women - cancers of the breast, lung, stomach, colon, rectum and cervix.

Although there are many types of cancer therapy that can influence tumor growth, in many cases the therapeutic effect is not sustained. Drug resistance and irremovable metastatic lesions are responsible for the vast majority of cancer-related deaths (up to 90%). As the knowledge about the mechanisms of the complex metastasis process is still very limited, the possibilities of preventing the development of metastasis are also limited. Even the time of metastasis to occur is difficult to predict as it can take a long time, sometimes decades, for metastasis to develop (Bragado, 2012). Breast cancer is an example: approximately 30% of women diagnosed with early stage disease progress to metastatic breast cancer for whom treatment options are limited. Continuing this example, treatment regimens based on taxanes and anthracycline antibiotics are now recommended for first-line chemotherapy for breast cancer. They typically give a 30-70% response rate, however, the response is often unstable with a time to progression of 6 to 10 months. Patients who develop disease progression or resistance to chemotherapy may be treated with other chemotherapy drugs, but response rates are generally low (20-30%); the median duration of response is less than 6 months (Rivera, 2010).

Current anti-cancer treatment strategies rely heavily on chemotherapy and radiotherapy, which are often ineffective and cause severe side effects. The promising breakthrough with biological therapies has been limited to only certain types of cancer and the use of targeted specific antibodies.

Accordingly, there is a significant need to develop new agents and new therapeutic strategies that circumvent common drug resistance mechanisms. It is very difficult to directly affect the cells of metastasis. A chance to limit the possibility of metastasis may be modulation of the host's immune mechanisms, which are used by metastatic cells to spread and penetrate into distant organs or tissues (Mina, 2011).

The present inventors have shown in previous studies that bacteriophage preparations can be anti-tumor and anti-metastatic in mice with LLC mouse lung cancer or B16 melanoma. In the course of the research, they confirmed the binding of the T4 bacteriophage to eukaryotic cancer cells and normal blood cells. The antimetastatic effect of T4 bacteriophage was observed after intraperitoneal administration of both whole lysates and purified bacteriophage preparations (Dabrowska, 2004). Moreover, the purified preparations slowed down the growth of BI6 melanoma primary tumors, and the effect depended on the dose and method of administration (Dabrowska, 2005). The influence of the combined use of T4 bacteriophage preparations and cytostatics on the development of experimental tumors was also investigated. Compounds with different mechanisms of action on neoplastic cells, such as cyclophosphamide, cisplatin and 5-fluorouracil, were analyzed. Synergy in the interaction of BPT4 and cyclophosphamide has been observed. However, no influence of BPT4 on the antitumor and antimetastatic activity of other cytostatics was observed. The diversified effect of the interaction of various compounds with antitumor activity is probably due to differences in the mechanism of their antitumor activity. However, this is undoubtedly an important cycle of experiments that allows to state that bacteriophage preparations can be used in cancer patients without adversely affecting the effect of anticancer treatment (Szczaurska-Nowak, 2009). The effect of bacteriophage preparations on the migration of neoplastic cells in vitro was also investigated. T4 and HAP bacteriophage preparations showed an activity that inhibited the migration of B16 murine melanoma and Hs294T human melanoma cells (Dabrowska, 2008).

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For years, the present inventors have been conducting research on the identification of active substances / active substances among the components of mixtures formed as a result of the lysis of E. coli B bacteria by the bacteriophage T4, responsible for the observed antitumor effect (Szczaurska-Nowak, 2008). Research on the explanation of the observed effects suggests that both phage and bacterial components, formed during lysis caused by bacteriophages (components of unpurified bacteriophage preparations), may be responsible for the antitumor activity. There are indications that the role of bacteriophages themselves is likely to induce the observed anti-cancer and anti-metastatic effects. Preliminary studies on the interaction of bacteriophage preparations with eukaryotic cells indicate the process of bacteriophage binding to β3 integrins as one of the possible mechanisms of T4 bacteriophage antimetastatic activity (Dabrowska, 2004). We have shown that the capsid protein of bacteriophage T4 (gp24) contains in its sequence a KGD motif that is homologous to the RGD motif and capable of binding β3 receptor integrins. It is likely that the bacteriophage's ability to interact with eukaryotic cells may be due to its ability to interact with the β3 integrin. It has previously been shown that bacteriophages have the ability to bind to tumor cells capable of expressing avβ3 integrin. Moreover, the use of β3 integrin blockers (anti-^ 3 antibodies and a synthetic peptide containing the KGD sequence, which is a ligand for integrins from this group) decreased the ability of bacteriophages to bind to neoplastic cells (Dabrowska, 2004). The involvement of integrin receptors in the binding of bacteriophages was investigated in tests on blood cells isolated from the blood of a patient suffering from Glanzmampa thrombasthenia (mutation causing blocking of allbj © and / or αvβ3 integrin function). It has been shown that both the platelets and lymphocytes of a patient with thrombasthenia are unable to interact with bacterial virus particles at a level comparable to the cells of a healthy person. In order to confirm the role of this mechanism in the anti-metastatic activity of bacteriophages, transfection of B16 siRNA melanoma cells (short interfering RNA) was performed to block the expression of the β3 receptor. It was shown that T4 bacteriophage did not affect the number of lung metastases in mice with B16 melanoma cells, characterized by a 55% reduced surface expression of the β3 integrin subunit. At the same time, a significant reduction in the number of metastases was observed in mice loaded with untransfected B16 cells. A strain of T4-derived bacteriophages was selected, capable of stronger binding to tumor cells (named HAP1). Both the parental T4 strains and the HAP1 lineage significantly inhibited the number of experimental B16 melanoma metastases (administered intravenously) in the lungs of mice. However, the HAP1 bacteriophage preparations showed a stronger antitumor activity than the T4 preparations, both in the case of tumors and metastases. A mutation absent from the T4 genome has been detected in the HAP1 genome: there is a non-sense mutation in the hoc gene of the HAP1 phage, which prevents the formation of a functional Hoc protein, and gpHoc is absent in the HAP1 capsid (Dabrowska et al. 2007).

The T4 bacteriophage is extremely widespread in the environment and in living organisms. It belongs to the microorganisms inhabiting the intestines of many animals and humans. The most important host of this bacteriophage is Escherichia coli, symbiotically found in most mammals, which is why this bacteriophage is so common. Its importance relates both to the regulatory effect it has on the gut flora in humans and animals and to its potential utility in the field of biotechnology. T4 is one of the best-described bacteriophages. The complete sequence of its genome is known. The complex structure of its capsid was also characterized quite well. All these features made it a model for studying the infectious mechanisms of bacteriophages, providing an extensive and detailed description of this complicated process (Figure, 2010).

The composition of the crude bacteriophage preparations suggests that components of bacterial origin, formed during lysis caused by bacteriophages, may also be responsible for the anti-tumor activity. The components of the active substance with antitumor activity may be bacterial amphiphilic glycolipids such as e.g. modified lipopolysaccharides (LPS, endotoxin) in Gram-negative bacteria or other building blocks of bacteria (e.g. peptidoglycan, lipoproteins, lipoteichoic acids, bacterial porins, nucleic acids and their fragments - CpG DNA). All these molecules, which are products of bacterial cell breakdown, are referred to as pathogen molecular patterns (PAMP) and belong to the key activators of the mechanisms of innate immunity (Toll-like receptors, TLR, Toll-likereceptors), as well as acquired immunity. (Aderem, 2000).

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A characteristic feature of most Gram-negative bacteria is the presence of thermostable, amphiphilic macromolecules anchored in their outer membrane, called lipopolysaccharides (LPS) because of their structure. LPS determines the stability and proper functioning of the bacterial cell, protects against the defensive mechanisms of the infected macroorganism and is an important factor in bacterial virulence in the case of sepsis and septic shock. A single LPS molecule consists of three distinct regions: lipid A, core oligosaccharide, and an O-specific chain (O-antigen). From a medical point of view, LPS is called an endotoxin because its appearance in the blood circulation is associated with diseases such as sepsis and septic shock. Lipid A is the region recognized by receptors on cells of the immune system. E. coli B bacteria have a rough type of LPS (R-LPS), called lipooligosaccharide (LOS) due to the lack of the O antigen in its structure. The available information on the interaction of bacteriophage proteins with endotoxins on the surface of bacteria is still incomplete (Prehm, 1976, Andres, 2010, 2012, 2013).

It cannot be ruled out that the observed antitumor effect of bacteriophage preparations is the result of the interaction of several substances of bacterial origin or their complexes with molecules of bacteriophage origin, independently influencing tumor development or mutually modulating their activity. Initial studies were conducted comparing the effects of unpurified bacteriophage preparations with the anti-tumor effects induced by the use of bacteriophage preparations of various purities, as well as purified components of the cell wall of Gram-negative bacteria, such as LPS, modified LPS and MPL E. coli particles. Both molecules, LPS and MPL, belong to the group of potential elements of the sought active substance. Initial studies were carried out in which it was shown that LPS fragments with lipid A in their structure enhance the antimetastatic activity of both the T4 bacteriophage preparation and the LPS. A strong antimetastatic effect was also observed when LPS was used in combination with polymyxin B or anti-LPS serum (both serum and antibiotic complex LPS). LPS, polymyxin and serum alone had no effect on the number of B16 melanoma metastases or on the antimetastatic activity of T4 bacteriophage.

Lipopolysaccharides and lipids A have been used in anti-cancer therapy since the 9th century. In 1891, the doctor William Coley observed that bacterial infection prolonged the survival time of patients with bone sarcoma (sarcoma). He administered supernatants from the cultures of Streptococcus pyogenes and Serratia mercescens bacteria (the so-called Coley toxins) to his patients, improving the condition of patients. In the light of current knowledge, it seems that the main factor responsible for the therapeutic effect of Coley's toxins was the induction of increased production of tumor necrosis factor (TNF) in patients' organisms (McCarthy, 2006), as well as some interleukins such as: IL-1, IL- 12, IL-15 and IL18.0d during Cooley's studies (1891-1936) various biological preparations, mainly based on modified substances of bacterial origin, were tried to enhance the immune response of the organism. The mechanism of action of these preparations was associated with the activation of macrophages, vascular endothelial cells, increased cell infiltration in the tumor and peritumor environment, induction of cytokine secretion or stimulation of non-specific cytotoxic cells on tumor cells.

Despite the fact that bacterial lipopolysaccharides (LPSs, endotoxins) have been studied as potential anti-cancer agents with immunomodulatory effects for many years, the concept of using LPS in treating people with cancer has failed due to its general toxicity and weak anti-cancer effect (Goto, 1996). ). The basics of LPS toxicity are well understood, and the main cause of the poor anti-cancer effects is LPS tolerance resulting from repeated doses of LPS or lipid A (Rockwell, 2010). The existence of this phenomenon has raised some doubts as to the usefulness of LPS / lipid A and their analogues in cancer therapy. Most clinical trials in cancer patients with LPS failed to maintain the anti-cancer effect that was initially present (Rockwell, 2010). The conducted research clearly confirmed that lipid A LPS is responsible for the observed effect of LPS / lipid A tolerance. In order to transform LPS into a less toxic form, various lipid A analogs have been designed and synthesized for years (Reisser, 2010). Studies on appropriately modified lipids A showed that as a result of the modification they lost partially or completely their toxic properties, while acting as LPS antagonists (an example may be monophosphorylated lipid A (MPL) (Persing, 2002). MPL is obtained from lipid A as a result of removal of the phosphate group from the Cl position of the proximal GlcN By analysis

Properties of the lipid A derivative MPL (monophosphoryl lipid A) described by Cluff C. et al. 2005, it was found that it was possible to separate the beneficial effects of this molecule on the immune system from its toxicity (Cluff, 2005). MPL has been shown to interact with cells through the Toll like receptor (TLR4) (Vasselon T., 2002). LPS and MPL have similar immunostimulatory properties, however the latter molecule is at least 100 times less toxic (Cluff, 2005).

MPL has been shown to be safe and an adjuvant in vaccines because it has the ability to activate antigen presenting cells (APCs) and induce the cytokine cascade (Baldridge, 1999). Other examples of lipid A derivatives are also known, such as Tolerin, ONO-4007, DT-5461 and SDZ MRL 953 (Rockwell, 2010). They are synthetic derivatives of lipid A. SDZ MRL 953 and ONO-4007 are triacylated analogs of lipid A. DT-5461 is tetraacylated lipid A (Reisser, 2010). The results of a few Phase I clinical trials in cancer patients (Bardou, 2010) treated with MPL or with the lipid analogs A mentioned above are known. MPL and other lipid analogues

And they were safe and well tolerated by patients. Trials with MPL with S. minnesota ₂ or S. typhimurium (iv doses of 10,250 μg / m body surface area) did not show an antitumor effect in the group of treated patients. Similar negative results were obtained in phase I clinical trials using the previously described SDZ analogues MRL 953, ONO-4007, ONO-174. Despite the good tolerance of patients to these compounds and the observed increased concentration of IL-6 and TNFa, no anti-tumor effect was observed. In contrast, the analogues showed positive effects in the prevention of septic shock and bacterial infections in Phase I clinical trials. Several documents related to the use of LPS molecules as anti-cancer preparations have also been identified. An example is the de-O-acylated lipooligosaccharide of the deep rough mutant Salmonella minnesota 1167-R595 (LOS type Re, lipid A substituted with two Kdo molecules) (CAI 171786A1). The LOS of the Neisseria meningitides LgtB mutant containing a triple-sugar outer core connected with a fragment of lipid A with reduced toxicity (de-O-acetylation, e.g. as a result of hydrazine) was proposed as an adjuvant for vaccines characterized by increased immunostimulatory activity, increased binding to the receptor dendritic cells (W02005107798A1). Document CZ290331B1 describes a glucosamine disaccharide obtained from Echerichia coli 011LB4 lipid A which was O-deacylated at the 3 and 3 'positions by alkaline hydrolysis (0.2 M NaOH, 37 ° C, 1.5 h). This disaccharide exhibits lower endocytotoxicity, but retains biological activity such as immunomodulatory and anti-tumor activity. It should be noted that the E. coli 0: 111 LPS is a smooth LPS composed of lipid A, a core oligosaccharide and an O-specific chain. DLPS has been shown to be less toxic than monophosphorylated lipid A (MPL) and has immunostimulatory effects. E. coli J5 de-O-acylated LOS has been claimed as a vaccine against infections caused by Gram-negative bacteria (WO 199529662). EP2664345 (A2) describes a vaccine consisting of a pathogen-derived antigen and a deacylated, non-toxic lipooligosaccharide (LOS) rough Escherichia coli CIA05 strain (KCCM 10374). LOS was inactivated by alkaline hydrolysis (2 N NaOH, 140 min, 60 ° C). In the described technology, the LOS derivative acted as an adjuvant that induces the maturation of dendritic cells. Document US20120100169 (A1) describes a cervical cancer vaccine which contains the L1 virus-like (VLP) antigens of the human HPV virus (papillomavirus) and a deacylated, non-toxic LOS of Escherichia coli, preferably KCCM 10374 strain, used as an adjuvant. LOS was deacylated by alkaline hydrolysis (0.2 N NaOH, 140 min, 60 ° C). This vaccine induces a TH1 and Th2 type immune response against HPV. Examples using de-O-acylated LOS as adjuvants in antibacterial or antiviral vaccines are also described in US4929604A, EP306496B1, US8506971 B2, US6531131 B1. The use of O-deacylated LPS derivatives as vaccine antigens is described in WO 1993013797 A2 (Detoxified LPS Vibrio cholerae conjugates with proteins such as cholera toxin), EP2481419A2 (Neisseria vaccine), US20120164145A1 (Helicobacter pylon vaccine) and others. The fatty acids in the derivatives of LOS, LPS and lipids A are usually removed using NaOH, hydrazine or enzymatically using acyloxyacyl hydrolase. In summary, O-deacylated LOSs have been used as components of the glycoconjugate antigen or as adjuvants in antibacterial vaccines and anti-cancer formulations.

The subject matter of the invention is defined in the appended claims.

List of figures:

Fig. 1. Structures of LOS (A) and LOS-OH (B) of E. coli B. * LOS-OH molecule is devoid of phosphoethanolamine (PEtn).

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Fig. 2. Average number of experimental B16 melanoma metastases in the lungs of mice treated with preparations resulting from the lysis of E. coli B bacterial cells observed on the 21st day of the experiment. * p = 0.035765 versus control, Mann-Whitney U test.

Figure 3. MALDI-TOF spectra of the FB fraction used in the in vivo studies showing anti-metastatic (A), synthetic LOS-OH (B) and LOS (C) activity in E. coli B.

Fig. 4. ESI-MS spectrum of the de-O-acylated derivative - LOS-OH of A. coli B.

Fig. 5. Number of experimental B16 melanoma metastases in the lungs of C57BL / 6 mice treated with either LOS or LOS-OHsynt B. The analysis was performed in the lungs collected on days 14 and 21 of the experiment. The graph shows the mean number of lung metastases from 5 mice of each group (days 14 and 21 of the experiment). * p <0.05 compared to control, Mann-Whitney U test.

Fig. 6 The number of experimental B16 melanoma metastases in the lungs of C57BL / 6 mice treated with LOS or LOS-OH E. coli B were assessed on day 21 of the experiment. One hour prior to intravenous administration _{of 5} B16 tumor cells (2 x 10 ⁵ kom./mysz), mice were intraperitoneally administered formulations LOS and LOS-OH E. coli B, at doses of 250 and 2500 .mu.g / kg body weight. The administration of preparations was repeated 4 times - one hour before the inoculation of the cells and on the 1st, 7th and 14th day of the experiment. The graph shows the mean number of lung metastases from 9-10 mice for each group on day 21 of the experiment. * p <0.05 compared to control, ** p <0.05 compared to E. coli B LOS 2500 μg / kg, *** p <0.05 compared to E. coli B LOS 250 μg / kg.

Fig. 7. Effect of E. coli B LOS and LOS-OH preparations on the ability of J774A.1 cells to produce IL-6 and TNF-α. Cells with a density of 1 x 10 ⁶ / ml were stimulated with LOS and LOS-OH A. coli B at concentrations of 10, 1 and 0.1 μg / ml for 4 (for TNF-α) or 24 (for IL-6) hours. The control (K) was unstimulated cells. After this time, culture supernatants were collected in which the concentration of cytokines was determined by ELISA.

Fig. 8. Effect of E. coli B LOS and LOS-OH preparations on the stimulation of BM-DC cells to produce cytokines. Cells with a density of 1x10 ⁶ / ml were incubated in the presence of LOS and LOS-OH A. coli B at concentrations of 10 and 1 µg / ml for 24 hours. The control was unstimulated cells. Supernatants were harvested from the cultures and the concentration of IL-6, IL-10 and IL-12 was assessed by ELISA.

Fig. 9. A, B - Evaluation of the ex vivo biological activity of the E. coli B LOS-OH preparation compared to LOS on splenocytes obtained from healthy mice. Cells (2 million / ml) were stimulated for 24 and 48 hours with E. coli B LOS and LOS-OH at a concentration of 1 µg / ml. The level of IL-6 and IFN-γ production was assessed by ELISA in the collected supernatants. Unstimulated cells were used as controls. C - Assessment of the percentage of IL-10 + cells among CD19 + lymphocytes after stimulation with LOS and LOS-OH A. coli B. Spleen cells collected from healthy mice were incubated with the test preparations for 24 and 48 hours. Brefeldin A (10 g / ml) and monensin (2 μΜ / ml) were added for the last 6 hours of stimulation. Controls were E. coli B LOS and LOS-OH unstimulated cells. Cells were stained with APC-labeled anti-CD19 antibodies, permeabilized, and incubated with PerCP-Cy5.5 conjugated anti-IL-10 antibodies.

Fig. 10. Percentage of CD4 + (A), CD8 + (B) T cells, CD49b + cells (NK cells) (C), CD19 + cells (B cells) (D) among splenocytes from B16 melanoma-bearing mice treated with LOS or LOS- OH E. coli B. Spleen cells were harvested on day 21 of the experiment (Example 6). The splenocytes were labeled with APC (allophycocyanin) -conjugated anti-CD4, PE-Cy7-conjugated anti-CD8 (phycoerythrin and cyanin 7 conjugate of the fluorochromes), PE (phycoerythrin) anti-CD19b (phycoerythrin) FITC-conjugated (fluorescein) anti-CD19 antibodies. The control was spleen cells taken from untreated, tumor-bearing mice. The graphs show the mean values obtained by analyzing cells from 3 mice in each group.

Fig. 11. Production level of IL-6 (A), IFN-γ (B), IL-10 (C) in supernatants from 48 hours of ConA stimulated splenocyte culture. Spleen cells collected on day 21 of the experiment were grown at a density of 2x10 ⁶ / ml and in the presence of ConA (0.5 µg / ml). The enzyme immunoassay ELISA was used to assess the concentration of this cytokine. The graph shows the mean values obtained from 3 mice of each group.

Fig. 12. Production level of IL-6 (A), IFN-γ (B), IL-10 (C) In supernatants from a 48-hour culture of splenocytes stimulated with E. coli B LOS. 2 x 10 ⁶ / ml and in the presence of E. coli B LOS (1 μg / ml). The enzyme immunoassay ELISA was used to assess the concentration of this cytokine. The graph shows the mean values obtained from 3 mice of each group.

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DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an active ingredient of bacterial origin showing immunostimulatory and anti-metastatic activities, its identification, isolation, structural analysis and method of preparation. The indicated active substance was isolated from a bacteriophage preparation (bacteriophage lysate), which is a mixture of the decay products of the bacteriophage T4, bacteria

Gram-negative - Escherichia coli B and the culture medium. The identified active substance is E. coli B de-O-acylated lipooligosaccharide (LOS-OH) with the structure shown in Fig. 1. LOS-OH E. coli B is composed of a core oligosaccharide having a new structure, covalently bonded with a disaccharide> 6) -pD-Glcp, N4P- (16) -aD-GlcpN1P of lipid A, which is substituted at the N2 and N2 'position with R-3-hydroxytetradecanoic acids [14: 0 (3-OH)].

The new active ingredient, E. coli B LOS-OH, object of the present invention, was identified in the organic phase (butanol phase, FB) obtained during the extraction of bacteriophage lysate (Examples 1,2, 3). Identification of the LOS-OHis derivative. E. coli B in the bacteriophage lysate indicates an active participation in this modification of the factor present at one of the stages of the bacteriophage lytic cycle. In addition, the nature of the E. coli B LOS modification indicates a factor with de-O-acylase activity, as the structure of LOS-OH differs from the native LOS by the lack of all primary, esterically bound fatty acids (Fig. 1). The active substance was isolated directly from the bacteriophage lysate (Examples 1 and 3), the significant disadvantage of which is the contamination in the form of complete LOS particles, which are pyrogens. After carrying out the structural analysis of LOS and LOS-OH, it turned out that LOS-OH in pure form could be obtained by hydrazinolysis of E. coli B LOS (Example 4). Both the LOS-OH containing FB lysate fraction and the synthetic LOS-OH obtained from purified E. coli B LOS preparations showed antimetastatic properties in in vitro, ex vivo and in vivo experiments (Examples 2 and 5-8). Both molecules have a unique structure so far not identified in natural preparations of Gram-negative bacteria, which differs from the structure postulated in earlier studies by one glucose residue (Prehem, 1976) (examples 3 and 4).

The biological activities of the isolated LOS-OH lysate and the synthetic LOS-OH were determined by means of routine in vitro and ex vivo tests designed to detect anti-metastatic properties and the ability to stimulate immune system cells involved in tumorigenesis. As described in the experimental section below, the in vivo antimetastatic and pre-proliferative activity of LOS-OH was tested using a B16 human melanoma mouse model and compared, in some experiments, to chemical anti-tumor agents such as cisplatin. The butanol fraction (FB) of the bacteriophage T4 lysate containing, inter alia, The identified E. coli B LOS-OH showed a pre-metastatic effect in the in vivo experiment. In lungs collected from mice with B16 melanoma, after administration of selected fractions of bacteriophage lysate, it showed statistically significant (p = 0.035765) reduction in the number of metastases in the group of mice that received FB at a dose of 100EU. The active substance identified in the FB phase after the extraction of the bacteriophage T4 lysate turned out to be de-O-acylated, in positions 03 and 03 ', of the LOS E. coli B derivative (Fig. 1), in which it was possible to obtain a synthetic form directly from purified LOS of E. coli B (without interference with the bacteriophage T4). Both the LOS-OH containing FB lysate fraction and the synthetic LOS-OH obtained from purified E. coli B LOS preparations showed antimetastatic properties in in vitro, ex vivo and in vivo experiments (Examples 2 and 5-8). Both molecules have a unique structure so far not identified in natural preparations of Gram-negative bacteria, which differs from the structure postulated in earlier studies by one glucose residue (Prehm, 1976) (examples 3 and 4). The obtained derivative, LOS-OH E. coli B (LOS-OHsynt), was used in in vitro, ex vivo (examples 8, 9) and in vivo (examples 5, 6, 7) studies. The obtained results indicate the effect of LOS-OH _synt on the reduction of the metastatic potential of neoplastic cells.

In vivo experiments carried out in a mouse model showed that the use of both LOS-OH and LOS E. coli B as antimetastatic therapeutics inhibits the increase in the number of metastases in the lungs of mice with BI6 melanoma. The toxicity of LOS molecules excludes them as potential antimetastatic therapeutics. On the other hand, the use of a modified LOS derivative, ie LOS-OH E. coli B, seem to be an excellent therapeutic agent in the conducted research. The metastasis increment for the 21st day of the experiment is given in Table 1. The metastasis increment for the 21st day of the experiment was calculated using the following formula:

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No. 21 day - day - the number of metastases in a given group on the 21st day of the experiment,

No. 14. day - number of metastases in a given group on the 14th day of the experiment.

T a b e l a 1. Lung metastasis increment in B16 melanoma-bearing mice treated with LOS or LOS-OH E. coli B. Calculated based on the data of Example 5.

Preparations The rate of increase in the number of metastases control 2.74 LOS 25 pg / kg 1.73 LOS 250 pg / kg 1.63 7 ,,. 70: - .: 7,: 77: --- 77 '-7-7 -: - · 777.:7 - ^:: -. . 7:77: 7 -7-7-7: - 7-7 '77' ^: 77 -'- 7 7 ' LOS-OH 25 pg / kg 2.40 LOS-OH 250 pg / kg 1.33

A summary of the mean results obtained after the analysis of the effect of administration of the preparations on the activation of immunocompetent cells in mice with B16 melanoma collected on the 21st day of the experiment is presented in Table 2. The differences in the percentage of the CD4 ⁺ , CD8 ⁺ , CD49b ⁺ and CD19 ⁺ populations among splenocytes are presented. The profile of cytokines produced by splenic cells after stimulation with concanavalin A and LOS E. coli B was also compared.

T a b e l a 2. Comparison of the activity of immunocompetent cells from B16 melanoma-bearing and LOS or LOS-OH E. coli B treated mice on day 21 of the experiment.

Percentage of spleen cells Production of cvtokines after stimulation of mitogenania CD4 + [% | CD8 + [% | CD49b + [%] CD 19+ [% l IL-6 [ng / ml] LOS ConA IFNs [ng / ml] LOS ConA IL-10 [ng / ml] LOS ConA Healthy 17.71 13.20 5.32 61.42 0.10 || 2.47 0.02 0.33 3.86 control 14.36 10.55 4.60 67.28 0.12 0.33 2.84 0.12 0.24 2, + 5 MOOSE 25 pg / kg 14.4+ 11.58 4.36 66.51 :: 3? $ 0.04 0.25 B 7'3 ' 1.68 0.03 0-20 ¹ '1.52 MOOSE 250 pg / kg 15.44 12.27 4.22 65.50 0.21 0.29 7.02 0.18 0.50 2.61 LOS-OH 25 pg / kg 13.38 and '. ,,! m. ' r '> T r _f t · ,; . ·· -. 9.97 3.91 69.67. 0.20 0.46 and 7: / 7:: - 0.12 7 L: -: · ... · .. · 0.73 3.19 LOS-OH 250 pg / kg 15.74 12.55 5.17 62.76 0.25 0.25 7.67 0.29 0.6+ 2.15

The presence of a developing tumor in the body changes the size of the individual populations of spleen cells and changes in the profile of cytokines produced by these cells. Among splenocytes collected from mice with B16 melanoma, a decrease in the percentage of CD4 ⁺ , CD8 ⁺ T lymphocytes and NK cells (CD49b ⁺ ) and an increase in the percentage of B (CD19 ⁺ ) lymphocytes in relation to

Normal mice. Analyzing the profile of cytokines produced by cells obtained from normal and control mice, a slight increase in the production of IL-6 and IFN-γ and a decrease in the production of IL-10 can be found both after E. coli B ConA and LOS stimulation by splenic cells taken from mice with melanoma.

Four times administration of LOS and LOS-OH E. coli B preparations in the dose of 250 μg / kg body weight inhibited the development of colonies of tumor cells in the lungs of mice with B16 melanoma. The lowest metastatic growth rate in these groups correlates with an increase in the percentage of CD4 ⁺ and CD8 ⁺ T cells and a reduction in the percentage of CD19 ⁺ cells (B lymphocytes), as well as a higher production of IL-6, IL-10 after LOS and IFN-γ stimulation after stimulation ConA and LOS E. coli B compared to untreated mice. The effect of LOS-OH compared to LOS E. coli B (at doses of 250 μg / kg) is slightly stronger. Additionally, in the group of mice treated with LOS-OH E. coli B at a dose of 250 µg / ml, an increase in the percentage of NK cells was demonstrated.

The results presented show that E. coli B LOS-OH exhibits immunostimulatory and anti-metastatic activities, making it a candidate for use as an anti-metastatic drug or adjuvant in anti-cancer vaccines.

Examples

Example 1. Identification of the antimetastatic T4 bacteriophage lysate fraction (LOS-OH E. coli B)

Cultures of the E. coli B strain, which was deposited in accordance with the Budapest Treaty under accession number B / 00085 on February 23, 2015 at the Polish Collection of Microorganisms at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław, were carried out in a fermentor in vol. 9.5 L (BioFlo 415) on liquid LB medium, enriched with glucose, yeast extract and casein hydrolyzate. The following fermentation parameters were used: 24 h culture, 37 ° C, agitation 200, aeration 5, pH ~ 7.0. After 24 h, phage (titer 6.x 109 - 2.2x1011 pfu - plaque forming unit) were added to the culture. To isolate the active substance from a bacteriophage lysate with an antimetastatic effect, elements of the technology described in the national patent application P 382800) (Boratyński, patent application, 2007) and the international patent application PCT PL 2008000045 (Boratyński, PCT 2008000045) were used. A two-phase extraction of a bacteriophage lysate (called the water fraction, FW) was applied using butanol as an organic substance (called the butanol phase, FB) into which hydrophobic molecules such as LOS E. coli B and its derivatives were extracted. 400 ml of bacteriophage lysate and 300 ml of butanol (ratio 4: 3) were used for the extraction. After extraction, both phases were separated. The FW phase was freeze-dried while the FB was dried on an evaporator and then freeze-dried. Endotoxin levels were checked in all fractions obtained (Table 3) using a commercially available LAL test according to the manufacturer's instructions (Pierce LAL Chromogenie Endotoxin Quantitition Kit, Thermo Scientific, USA). The antimetastatic activity of the preparations obtained after extraction was verified in in vivo studies using the B16 mouse melanoma metastasis model, and all experiments were performed in C57B1 / 6 mice (example 2). The fractions showing the highest antimetastatic and antproliferative activities were subjected to composition analysis by MALDI-TOF mass spectrometry (Example 3). Based on the results of the MALDI-TOF MS analyzes, E. coli B LOS-OH was identified in the FB phase (Fig. 3A). The mass spectrum of LOS-OH isolated from bacteriophage lysate (Fig. 3A) and its interpretations are described in Example 3.

Example 2: Evaluation of the antimetastatic activity in the aqueous and butanol fractions of bacteriophage lysate.

The studies were performed using the B16 murine melanoma metastasis model. All experiments were performed in C57B1 / 6 mice (5-6 mice / group). Bacteriophage lysate fractions (according to Table 1) were administered to animals once, intraperitoneally (i.p.) one hour before intravenous (i.v.) implantation of B16 melanoma tumor cells, cisplatin at a dose of 10 mg / kg body weight was administered on day 4 after tumor cell inoculation. The LAL test was used to determine the level of endotoxins present in the tested fractions. The endotoxin level administered to the animals was 2.5 or 100 EU / mouse. In the control group, PBS was used instead of bacteriophage or cisplatin preparations. On the 21st day of the experiment, the mice were sacrificed, the lungs removed from the animals were weighed and then fixed in a 4% buffered formalin solution, then the visible metastatic foci were counted. The results of the experiment in the form of the mean number of B16 melanoma metastases in the lungs of the tested mice are presented in Fig. 2. The assessment of the number of lung metastases from mice with B16 melanoma after administration of selected lysate fractions was performed.

Bacteriophage bacteria showed a statistically significant (p = 0.035765) reduction in the number of metastases in the group of mice that received FB at a dose of 100EU. Lower LOS content in FB as well as in FW showed only a tendency to reduce the number of metastases.

T a b e l a 3. The level of endotoxins present in the tested preparations determined by the LAL test. The presence of A. coli B LOS and LOS-OH was confirmed by mass spectrometry (Fig. 3) and immunoblotting with anti-OSJ5 serum.

Phase Titre in the LAL test Presence of LOS and LOS-OH dotblot / mass spectra FW 135 EU / ml NO FB 3.4xl () ⁷ EU / ml YES

Example 3. Identification of the active substance (LOS-OH) in the bacteriophage T4 lysate fractions.

DotBloting and MALDI-TOF mass spectrometry were used to identify LOS and LOS derivatives in the FW and FB fractions that reduced the number of metastases. During the dotblot analysis, rabbit polyclonal sera obtained by immunizing rabbits with the LOS core oligosugar conjugate E. coli J5 (OSJ5) with tetanus toxoid (anti-OSJ5-TT) were used (Lukasiewicz, 2002). The antibodies contained in the serum allow the detection of the core oligosaccharide of the J5 type and, as it turned out, cross-react with a similar structure characteristic for LOS E. coli B. Therefore, they were used to identify fragments and complete LOS E. coli B molecules in individual fractions of the bacteriophage lysate T4. The results of the immunoblotting showed that during the extraction of the bacteriophage lysate, whole and modified LOS E. coli B particles are extracted into the FB phase, which correlated with the simultaneous analysis of individual fractions using the commercial LAL test (Table 3).

The presence of LOS and LOS-OH E. coli B molecules was checked in the selected FB fraction by MALDI-TOF mass spectrometry. A matrix of 2,4,6-trihydroxyacetophenone (THAP) was used at a concentration of 200 mg / ml, dissolved in methanol and mixed with a nitrocellulose solution (30 mg / ml in 1: 1 acetone / propanol). The prepared matrix was mixed with a 4: 1 nitrocellulose solution. A sample of the E. coli B FB (1 mg / ml), LOS and LOS-OH phases was dissolved in 50% methanol with 5 mM EDTA and then desalted with the ammonium DOWEX 50WX8 ion exchange resin. Then the samples were mixed with the matrix (1: 1) and 100 mM of basic ammonium citrate (1: 1) was added to the mixture. The spectra were analyzed in the negative mode using the AutoflexIII apparatus (Bruker, Germany). Figure 3 shows the MALDI spectrum of the FB fraction, which in vivo showed antimetastatic properties and the spectrum of the synthetic LOS-OHSYNT and LOS E. coli B. The mass spectrum of the FB fraction (Fig. 3A) showed ions at m / z 2211.31 Da [M _los - _oh -H] ^- , m / z 2233.71 Da [M _LOS - _OH -H + Na] ^- corresponding to the LOS-OH molecule (LOS E. coli B stripped of four primary and secondary ester-linked fatty acids and phosphoethanolamine residues). Characteristic ions from m / z 1716.95 and 1976.07 Da were also observed on the spectrum, which were derived from the lipid A molecule, indicating the presence of native LOS molecules in the tested fraction. The ions at m / z 1213.57, m / z 953, 13, m / z 923.71 correspond to sugar fragments of LOS and LOS-OH molecules.

Example 4. Preparation of the synthetic form of LOS-OH E. coli B

The active substance identified in the FB phase after the extraction of the bacteriophage T4 lysate turned out to be de-O-acylated at positions 03 and 03 ', a derivative of the E. coli B LOS, in which it was possible to obtain a synthetic form directly from purified E. coli LOS B (without the interference of the T4 bacteriophage). The E. coli B was cultivated as described in Example 1, and the resulting bacterial mass after centrifugation, washing and lyophilization was extracted according to the method described for rough LOS (Galanos, 1969) to obtain the E. coli B LOS used as control in in vitro and in vivo. Briefly, killed and freeze-dried E. coli B bacteria (20 g) were suspended in 100 ml of phenol / chloroform / petroleum ether (bp 40-60 ° C) (2/5/8, v / v / v) and then broken for 3 minutes with an ultrasonic probe until a homogeneous mixture was obtained. The mixture was then separated by centrifugation (15 min. At 3500 x g / 4 ° C). The supernatant was collected, and 80 ml of phenol / chloroform / petroleum ether (b.p. 40-60 ° C) was added to the pellet and homogenized with an ultrasonic probe (3 minutes). Again the mixture was separated by centrifugation (15 min. At 3500 x g / 4 ° C). The supernatants after centrifugation were combined and dried. The dried supernatant was warmed to room temperature and mQ water was added dropwise. Precipitation of LOS was observed.

The mixture was left for 10 minutes for complete precipitation of LOS. The mixture was centrifuged (15 min / 3500 x g / 4 ° C). The supernatant was decanted to which mQ water was added dropwise to check that all of the LOS had precipitated. The tube with the sediment was turned upside down to dry completely. 10 ml of 80% phenol was added to the pellet, stirred and centrifuged (15 min. At 3500 x g / 4 ° C). The supernatant was poured off and again added to the pellet 10 ml of 80% phenol and centrifuged (15 min. 3500 x g / 4 ° C). Then, after drying, 10 ml of diethyl ether were added to the precipitate, the mixture was broken using an ultrasonic probe, and centrifuged (15 min / 3500 x g / 4 ° C). The washing with diethyl ether was repeated twice. MQ water was added to the sediment and it was freeze-dried. The resulting crude LOS preparation was purified from proteins and nucleic acids by ultracentrifugation (105,000 x g, 4 hours / 4 ° C). The ultracentrifugation supernatant was decanted, leaving a pellet containing aggregated LOS. Purified LOS was suspended in water and lyophilized. The LOS extraction efficiency in relation to bacterial dry matter was approx. 2%. After the performed extractions, the LOS of E. coli B was checked by MALDI-TOF spectrometry in order to confirm the correctness of the structure. The structure of the isolated E. coli B LOS is shown in figure 1. The unique structure of E. coli B LOS was identified by NMR spectroscopy and MALDI-TOF mass spectrometry. The presented LOS structure of E. coli B differs from that previously published by Prehm et al. (Prehm, 1976), and the difference results from the lack of one glucose residue in the core oligosaccharide, which is consistent with the results obtained by mass spectrometry and NMR (Table 4). ).

TABLE 4. Chemical shift values for the atoms of ¹ H and ¹³ C the components of the core oligosaccharide LOS E. coli B.

Rest Hl / Cl H2 / C2 H3 (H3 _a "H3 _e ,) / C3 H4 / C4 H5 / C5 H6.6 '/ C6 H7.7' / C7 hs, sycą ppm -> 5-a-Kdo n / a 95.5 1.84, 2.19 33.4 4.07 65.5 4.10 73.1 3.77 71.7 3.63 69.0 3.46, 3.83 53.8 -> 3-L-a-D-Hepp 5.11 99.8 3.96 70.9 4.04 77.3 4.55 ¹ 71.6 4.14 71.3 4.04 68.6 3.65 62.9 > 3.7-L-a-u-Hep 5.06 102.7 4.31 69.4 3.96 73.6 3.95 65.4 3.59 72.7 4.11 68.1 3.54, 3.70 69.4 L-a-D-Hepp 4.89 100.0 3.88 70.0 3.79 70.5 3.79 66.1 3.53 71.3 3.97 68.7 3.59 63.1 a-D-GIcp 5.21 100.3 3.48 71.9 3.73 73.0 3.34 69.7 3.80 72.2 3.68, 3.88 60.4 Gly 168.0 3.93 40.2 PEtn 3.23 40.0 4.14 62.4

The oligosaccharide of the LOS core of E. coli B, composed of two α-Kdo molecules, three LD-Hepp, α-D-Glcp and pyrophosphoethanolamine (PPEtn), covalently substitutes lipid A composed of the disaccharide 3-D-GlcpN4P- (1 ^ 6) -aD-GlcpN1P substituted with primary: amide-linked 14: 0 (3-OH) acid residues and 14: 0 (3-OH) ester-linked acid residues and secondary acid residues 12: 0 and 14: 0. The mass spectrum of the whole LOS E. coli B molecule was obtained as described in example 3. The LOS spectrum of E. coli B had the characteristic om / z 1796.17 Da ion, which corresponds to the mass of the six-residue fragment of lipid A created during mass analysis (ion source). fatty acids, two glucosamine residues, and two phosphate groups (Figure 1 for LOS). The ion with m / z 3178.87 Da corresponds to the mass of the complete LOS molecule and the ion with m / z 3055.02 Da to the LOS molecule devoid of any phosphoethanolamine (PEtn) residue.

From the obtained LOS E. coli B, the de-O-acylated derivative LOS-OH _{synt was obtained} using a hydrazinolysis reaction with anhydrous hydrazine (Patel, 1993). Anhydrous hydrazine was obtained according to the published method (Fukada, 1993). To 50 mg of LOS E. coli B was added 4 ml of anhydrous hydrazine, pipetted with a pipette. Reactions were carried out in a sealed tube at 37 ° C for 30 min, and then the reaction mixture was added dropwise to acetone (cooled at -20 ° C) leading to

E. coli B LOS-OH was precipitated. The mixture was centrifuged (-10 ° C, 15,000 x g, 20 min), and then the obtained pellet was lyophilized. The structures of the derivative obtained were analyzed by spectrometry

ESI-MS ⁿ (Fig. 4, Table 3). The LOS molecule of E. coli B was depleted by chemical methods of all ester bound fatty acid residues and of the phosphate group (Fig. 1). The obtained derivative, LOS-OH E. coli B (LOS-OH _synt ), was used in in vitro, ex vivo (examples 8, 9) and in vivo (examples 5, 6, 7) studies.

Table 3. Populations of molecules observed in the ESI MS spectrum of the prepared LOS-OH derivative of E. coli B.

Observed ion [M-4H] ⁴ Calculated mass (monoisotope) Interpretation 534.5 ⁴ ' 2142.0 M- (2xl4: 0 (3-OH) +14: 0 + 12: 0) -Hep 540.2 ' 2164.8 M- (2xl4: 0 (3-OH) +14: 0 + 12: 0) -Hep + Na 551.7 ⁴ ' 2210.8 M- (2xl4: 0 (3-OH) +14: 0 + 12: 0) -PEtn 557.2 ⁴ ' 2232.8 M- (2xl4: 0 (3-OH) +14: 0 + 12: 0) -PEtn + Na 571.7 ⁴ ' 2290.8 M- (2xl4: 0 (3-OH) +14: 0 + 12: 0) -Etn 577.2 ⁴ 2312.8 M- (2xl 4: 0 (3 -OH) +14: 0 + 12: 0) -Etn + Na 582.5 ^1- 2334.0 M- (2xl4: 0 (3-OH) +14: 0 + 12: 0) 588.2 ^4- 2356.8 M- (2xl4: 0 (3OH) +14: 0 + 12: 0) + Na 592.2 ⁴ ' 2435.6 M- (14: 0 (3-OH) +14: 0 + 12: 0) -PEtn

M = the complete E. coli B LOS structure shown in Fig. 1 (M = 3178.5 Da).

Example 5: Study of the anti- _{metastatic activity of LOS-OH synt} in a B16 murine melanoma metastasis model.

In an experimental metastasis model of mouse melanoma B16, it was verified antimetastatic activity LOS and LOS-OH _Synth E. coli B. For this purpose, the mice of the strain C57BL / 6 are given intravenously ₅ ^SYNT B16 melanoma cells (3 x 10 ⁵ kom./mysz) hour after first administration of preparations. The preparations were administered intraperitoneally in four replications: one hour before the administration of the tumor cells, and then on the 1st, 7th and 14th day of the experiment (Scheme 1). On days 14 and 21 of the experiment, biological material (lungs, spleens) was collected from the mice for further ex vivo analyzes (Example 6). The lungs were weighed and then fixed in a 4% buffered formalin solution, then the visible metastatic lesions were counted. Figure 5 shows the mean number of B16 melanoma cell metastases in the lungs of C56BL / 6 mice (5 mice per group) after 3 or 4 administrations of the preparations (LOS or LOS-OH E. coli B). On the 14th day of the experiment, the highest number of metastases was recorded in the control group and the group receiving LOS 250 μg / kg, while the lowest number of metastases was observed in the group treated with 250 μg / kg of LOS-OH E. coli B. On day 21 of the experiment, the greatest effect was also the LOS-OH dose of E. coli B of 250 μg / kg (p = 0.007291 compared to control, Mann-Whitney U test) had the effect of reducing the number of metastases. The obtained results indicate the effect of LOS-OH _Synt on reducing the metastatic potential of neoplastic cells. The effect of the administration of the preparations on the systemic response was assessed on the basis of the functional characteristics of the splenocytes collected (on days 14 and 21) from mice participating in the experiment (groups treated with LOS and LOS-OH _Synt E. coli B) (Example 8).

Scheme 1. Scheme of the experiment described in example 5

PL 227 489 B1

Example 6: Study of the LOS-OH activity of E. coli B

In example 7, the influence of LOS E. coli B and LOS-OH _synt on the dose-dependent antimetastatic activity was investigated in an experimental model of melanoma metastasis ₅

B16 in mice. Mice of the strain C57BL / 6 mice (7-8 weeks old) were intravenously given B16 melanoma cells (2 x 10 ⁵ kom./mysz) and preparations of LOS and LOS-OH (in two doses of 250 and 2500 .mu.g / kg body weight) by. the dosing regimen shown in Scheme 2. The number of experimental lung metastases in the mice with B16 melanoma and treated with the preparations was assessed and the results are presented in Fig. 6. The obtained results were statistically analyzed using the Mann-Whitney test, which showed statistically significant differences in the number of metastases. between: (I) the control and the groups receiving LOS E. coli B 2500 µg / kg (P = 0.001086) and LOS-OH E. coli B 250 µg / kg (P = 0.027284); (II) E. coli B LOS 2500 μg / kg group and E. coli B LOS 250 μg / kg groups (P = 0.000444), E. coli B LOS-OH 2500 μg / kg (P = 0, 000280) and 250 μg / kg (P = 0.000276); (III) E. coli B LOS 250 µg / kg group and E. coli B LOS-OH 250 µg / kg group (P = 0.012412).

Scheme 2 The experimental procedure described in Example 7. The mice of the strain C57BL / 6 mice, d ożylnie B16 melanoma cells (2 x 10 ⁵ kom./mysz). LOS and LOS-OH E. coli B preparations in doses of 250 and 2500 μg / ml were administered four times: one hour before the administration of tumor cells and on the 1st, 7th and 14th day of the experiment. On the 14th and 21st days

Example 7: Evaluation of the effect of LOS-OH E. coli B on the stimulation of biological activity of immune cells in in vitro and ex vivo culture conditions (healthy animals).

J774A cells were used to test the biological activity of LOS-OH E. coli B against cells of the immune system. 1 macrophage derived (TIB-67 ™, ATCC®, Manassas, VA), myeloid derived dendritic cells (BM-DC) and splenocytes from healthy C57BL / 6 mice. The purpose of functional tests using both myeloid and lymphoid cells was to determine differences in cytokine production after stimulation with LOS-OH E. coli B preparation. LOS E. coli B preparation was used as control. All cells were grown under standard conditions (37 ° C, 5% CO ₂ and 95% humidity), in plastic bottles or on Costar 6-well plates (NY, USA). J774A.1 cells were cultured in DMEM ATCC culture medium supplemented with 10% FBS at low density for not less than 7 days. The culture was kept for 3-5 weeks. The culture of BM-DC cells, with an initial density of 1 x 10 ⁶ cells / ml, was grown for 8 days in RPMI 10% FBS (MH) culture medium supplemented with GM-CSF (40 ng / ml) and IL-4 (10 ng / ml). ml). Cells were fed twice. On the sixth day, cells were removed from the culture flasks using trypsin EDTA solution, resuspended in MH supplemented with 10% FBS and GM-CSF (40 ng / ml) and IL-4 (5 ng / ml) and adjusted to a density of 1 x 10 ⁶ cells / ml in 12-well plates. Spleens were collected from healthy C57BL / 6 mice and frozen. Prior to mounting the assay, the splenic cell suspension was thawed and resuspended in RPMI with 10% FBS adjusting to a density of 2 x ¹⁰⁶ / ml.

Activation of the J774A.1 lineage. Cells were removed from the plates by repeated pipetting, rinsed, resuspended in appropriate culture medium to bring them to a density of 1x10 ⁶ / ml, and applied to 24-wells (Costar, NY, USA). 50 μl of the appropriate dilution of stimulators were added to the cultures so that their final concentrations in the well were successively 10, 1; 0.1 and 0.01 pg / ml. After 4 and 24 hours incubation supernatants were collected from the culture. The level of the concentration of cytokines TNF-α (4 hours) and IL-6 (24 hours) in supernatants from the culture of cells stimulated with LOS was assessed.

PL 227 489 B1 and LOS-OH E. coli B (Figure 7). The control was unstimulated cells. Dose-dependent differences in the production of both cytokines were observed depending on the preparation used and the stimulator concentration used. It was shown that LOS E. coli B has a greater ability to stimulate cells to produce IL-6 and TNF-α compared to LOS-OH E. coli B. The highest production of IL-6 and TNF-α was observed after LOS stimulation (10 μg / ml) and it was more than twice as high as after adding LOS-OH E. coli B at the same concentration. Only cells incubated with 10 µg / ml LOS-OH were able to produce the tested cytokines. Therefore, the two highest concentrations of E. coli B LOS and LOS-OH, ie 10 and 1 μg / ml, were used for subsequent ex vivo tests carried out on BM-DC cells.

Activation of the BM-DC lineage. The LOS and LOS-OH E. coli B preparations were added to the cell cultures prepared the day before (1 x 10 ⁶ / ml), bringing the final concentrations to 10 and 1 µg / ml. Incubation of BM-DC with the tested preparations at concentrations of 10 and 1 μl was carried out for 24 hours, after which supernatants from the culture were collected, in which the levels of IL-6, IL-10 and IL-12 were assessed by means of an ELISA assay (Fig. 8) (Becton Dickinson, USA). Unstimulated cells were used as a control.

Activation of splenocytes. The splenocytes were grown for 24 and 48 hours. in the presence of LOS and LOS-OH E. coli B (1 µg / ml). In all collected supernatants, the level of cytokine production (IL-6, IFN-γ) was analyzed by ELISA (Becton Dickinson, USA) (Fig. 9). The differentiation of the production level was shown, depending both on the cultivation time and the type of preparation used. There was no IFN-y after the 24 hour incubation. However, the presence of IFN-γ was detected in the 48-hour supernatants, and the highest cytokine concentration was recorded after incubation with E. coli B LOS-OH (60 pg / ml). In the case of IL-6, the increase in cytokine production was found only after stimulation with LOS E. coli B preparation. Its size ranged from 100 pg / ml (after 24 hours) to 250 pg / ml (after 48 hours of culture). Application of LOS-OHE. coli B did not induce the production of this cytokine.

Additionally, the percentage of cells capable of producing IL-10 among CD19 ⁺ lymphocytes was analyzed (Fig. 10C). Cells were stimulated with LOS and LOS-OH for 24 and 48 hours. and subjected to the procedure of intracytoplasmic protein staining. Intracytoplasmic protein staining method. 6 hours before the end of the culture, brefeldin A (10 μg / ml) and monensin (2 μ) were added to retain the proteins produced inside the cells. The splenocytes were removed from the wells, centrifuged and stained with APC-conjugated anti-CD19 antibodies (allophycocyanin) (eBioscience, USA) for 30 min. The cells were then permeabilized by overnight incubation with Fixation / Permabilization reagent (eBioscience, USA). Samples were washed in Flow Cytometry Staining Buffer (eBioscience, USA) and an anti-CD16 / CD32 antibody (Fc Block, (eBioscience, USA)) was added to the pellet to block the receptor for Fc fragments of antibodies. Incubation for 15 min was followed by the addition of PerCP-Cy5.5 conjugated anti-IL-10 antibodies (tandem of fluorochromes) (eBioscience, USA). The stained cells were washed twice and analyzed on a FACSCalibur flow cytometer (Becton Dickinson, USA). After a shorter incubation time (24 hours), the percentage of IL-10 ⁺ cells among CD19 ⁺ lymphocytes did not show significant differences and was for LOS (10.0%), for LOS-OH E. coli B (5.2%). Extended to 48 hours stimulation caused a significant variation in the percentage of positive cells. Cultivation in the presence of LOS induced 28.1% of IL-10 ⁺ cells among CD19 +, while the use of LOS-OH E. coli B slightly increased the percentage of these cells (6.3%). The control was unstimulated splenocytes, among which IL-10 ⁺ cells accounted for 6.7% and 9.8% of the CD19 + cell population after 24 and 48 hours of stimulation, respectively. Conclusions: The LOS-OH E. coli B preparation turned out to be a much weaker stimulator of immune cells compared to LOS. Although at high doses (e.g. 10 μg / ml) its activity was comparable to that of the LOS E. coli B preparation, it lost the ability to activate even at a concentration of 1 μg / ml.

Example 8: Effect of E. coli B LOS-OH administration on the activation of immunocompetent cells in mice with B16 melanoma.

The experiment was an extension of the ex vivo studies for the spleen samples obtained in Example 6. To test the effect of E. coli B LOS-OH on splenic cell activity, an analysis of the surface phenotype of cells taken from mice with B16 melanoma and treated (Example 6) was performed. The effect of the tested preparations on the degree of stimulation of the anti-tumor response was determined on the basis of changes in the activity of splenic cells obtained from mice on the 21st day of the experiment. The assessment was performed on the basis of phenotypic changes in 4 major splenocyte populations (CD4, CD8, CD19 and CD49b) and their ability to produce cytokines.

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Analysis of phenotypic changes in the population of CD4, CD8, CD19 and CD49b splenocytes. Spleen cells were thawed and resuspended in PBS with 2.5% FBS to bring the suspension to a density of 2 x ¹⁰⁶ cells / ml. Tubes were filled with 50 μl of cell suspension and 50 μl of appropriately diluted antibody solution was added.

The antibodies used were anti-CD4 (APC) (Becton Dickinson, USA), anti-CD8 (PE-Cy7) (Becton Dickinson, USA), anti-CD49b (FITC) (Becton Dickinson, USA) and anti-CD19 (PE) ( Becton Dickinson, USA). Incubation was carried out for 45 min. at 4 ° C. The reaction was stopped by adding 2 ml of PBS with 2.5% FBS, centrifuged and discarded supernatants. Cells were resuspended in 200 µl PBS with 2.5% FBS and analyzed on a flow cytometer.

The mean values for the percentages of CD4 + cells for the three mice in each group are shown in Figure 10A. The presented data show that the percentage of CD4 ⁺ cells decreased in all groups of tumor-bearing mice compared to the group of healthy mice (17.71 ± 4.95%). The percentage of cells in the control mice (tumor-bearing, untreated) was 14.36 ± 1.34%. The use of both LOS and LOS-OH E. coli B at a dose of 250 μg / kg body weight slightly increased the percentage of CD4 ⁺ cells, which was 15.44 ± 1.12% or 15.74 ± 2.02%, respectively. A similar percentage was obtained for the group receiving LOS E. coli B at the dose of 25 µg / kg body weight. Only after the application of LOS-OH E. coli B at the dose of 25 μg / kg body weight, a slight decrease in the percentage of CD4 ⁺ T cells (13.38 ± 0.55%) was observed. However, in none of the groups of mice with melanoma CD4 ⁺ cell abundance reached the value found in normal mice, and administration of LOS-OH A. coli B at a dose of 250 µg / kg body weight did not correlate with the reduced number of metastases. However, lowering the percentage of CD4 ⁺ splenocytes may affect the ability of the overall population to support the emerging anti-metastatic response.

The mean percentages of CD8 + T cells in the treatment groups are shown in Figure 10B. The percentage of CD8 + cells among splenocytes from mice in the control group is 10.50 ± 0.66%. However, the lowest percentage of CD8 ⁺ cells in the groups of mice bearing tumors and / or treated with the preparations as compared to healthy mice (13.20 ± 3.07%) was demonstrated after administration of LOS-OH E. coli B at the dose of 25 μg / kg ( 9.97 ± 0.76%). The use of both LOS and LOS-OH E. coli B at a dose of 250 μg / kg resulted in the smallest decrease in the percentage of positive cells (12.27 ± 1.26% and 12.55 ± 0.38%, respectively). Thus, despite similar trends in changes in the size of the CD8 + cell population, only the use of a high dose of E. coli B LOS-OH was associated with a reduction in the number of metastases.

A comparison of changes in the percentage of CD49b + cells among splenocytes obtained from melanoma-bearing mice treated with LOS or LOS-OH E. coli B is shown in Figure 10C. There was a slight reduction in the percentage of NK cells among splenocytes from tumor-bearing mice as compared to healthy mice. However, all changes ranged from 3.91 ± 0.37% (after administration of LOS-OH, 25 μg / kg body weight) to 5.17 ± 0.07% (after administration of LOSOH, 250 μg / kg body weight) ). As in the previous subpopulation, the highest percentage of CD49b ⁺ cells was also observed after high doses of the preparation.

B lymphocytes are the major cell population in the spleen under normal conditions. Therefore, it was interesting to determine whether administration of E. coli B LOS-OH to mice with B16 melanoma will vary the percentage of CD19 + cells. Figure 10D shows the results of the analysis of the changes in the percentage of CD19 + cells as a function of the type and dose of the administered preparations. In healthy mice, this percentage was 61.42 ± 8.77%. An increase in the percentage of B lymphocytes among splenic cells taken from mice with B16 melanoma and / or treated with the preparations was observed after the administration of 25 μg / kg body weight LOS-OH E. coli B (69.69 ± 1.03%), showed the opposite trend in relation to to the percentage of T and NK lymphocytes. As in the case of the application of 250 μg / kg of the LOS-OH E. coli B preparation, which was accompanied by the lowest percentage of B lymphocytes (62.8 ± 1.03%).

Summing up, although only trends in the size of the assessed splenocyte populations were observed, it can be suggested that the administration of a high dose of E. coli B LOS-OH induced an increase in the percentage of CD4 ⁺ , CD8 ⁺ and CD49b ⁺ cells compared to the control and a decrease in the percentage of CD19 cells. ⁺ . The comparison of the results obtained with the use of both preparations emphasized that the changes in the percentage of the population of spleen cells depend on the type of the preparation used.

Analysis of the ability to produce cytokines by ConA-activated splenocytes. A suspension of splenocytes at a density of 2 million / ml of 10% RPMI was stimulated with concanavalin A (Con A, T cell mitogen) or with E. coli B LOS, (equivalent to a B cell stimulator). E. coli B LOS was used at a concentration of 1 µg / ml, and ConA at a concentration of 0.5 µg / ml. Incubation was carried out for 48

PL 227 489 B1 h. After this time, supernatants were collected from above the cultures, in which the concentration of IL-6, IFN-γ and IL-10 was assessed by means of ELISA tests (Becton Dickinson, USA; eBioscience, USA). The level of IL-6 and IFN-γ production (produced mainly by Th 1 lymphocytes) and IL-10 usually associated with immunosuppressive activity was assessed.

Splenocytes collected from normal, ConA stimulated mice showed IL-6 production (Fig. 11A) at 0.1 ng / ml. Cells obtained from tumor bearing mice did not show the ability to secrete this cytokine in greater amounts. Moreover, the administration of LOS E. coli B at a dose of 25 μg / kg reduced the level of production of this cytokine. Only the use of LOS E. coli B in a dose of 250 μg / kg or LOS-OH E. coli B in a dose of 25 μg / kg resulted in an increase in the production of IL-6 to approx. 0.2 ng / ml. The highest level of IL-6 was demonstrated after administration of 250 µg / kg LOS-OH E. coli B. The obtained results suggest the influence of a high dose of LOS-OH E. coli B on the increase in the production of pro-inflammatory cytokine in the systemic immune response, visible even in the activation of splenic cells.

The production level of IFN-γ (Fig. 11B) by splenocytes obtained from normal and ConA stimulated mice was 2.47 ng / ml. On the other hand, a significant increase in the production of IFN-γ was observed after the use of both 250 pg / kg LOS E. coli B (7.00 ng / ml) and both doses of LOS-OH A. coli B: 6.13 ng / ml and 7, 67 ng / ml (for 25 and 250 μg / kg, respectively). The use of a low dose of LOS E. coli B did not increase the production of this cytokine. It seems extremely important at this point to emphasize the relationship between a slight increase in the percentage of splenocytes capable of producing IFN-γ and the primary (in vivo) stimulation of these cells to produce this cytokine after ex vivo stimulation. The data shown in Figure 12B show that a high dose of A. coli B LOS-OH appears to be sufficient to (indirectly or even directly) induce a cellular response that is presumably responsible for the reduction in lung metastases in treated mice.

Con A-stimulated cells obtained from mice bearing B16 melanoma and treated with LOS 250 μg / kg, LOS-OH E. coli B 25 μg / kg, or LOS-OH 250 μg / kg were found to be able to produce small amounts of IL-10 (respectively 0.5 ng / ml, 0.73 ng / ml or 0.64 ng / ml). As with the production of IFN-γ and IL-6, only the splenocytes in these groups showed a greater ability to produce IL-10. This increased reactivity of splenocytes obtained from mice treated with both doses of E. coli B LOS-OH indicates the involvement of T cells in stimulating the anti-melanoma cell response and the in vivo phase of the response at the time of collection of the material. It should be noted that there is a slight but apparent inverse trend in cytokine production depending on the dose of E. coli B LOS used (Fig. 11).

The obtained results suggest that the use of a high dose of E. coli B LOS-OH increases the level of IL-6 (pro-inflammatory cytokine) production. This amount of IL-6 appears to be sufficient to induce a cellular response responsible for reducing the number of metastases in the lungs of the treated mice. The increased reactivity of splenocytes obtained from mice treated with two different doses of E. coli B LOS-OH is manifested by the ability of these cells to produce IL-10. This indicates the involvement of T lymphocytes in stimulating the response against melanoma cells in vivo at the time of collection of the material.

The profile of cytokines produced by splenocytes after E. coli B LOS stimulation. Analysis of the splenocyte phenotype showed changes in the percentage of both T lymphocytes and CD19 ⁺ cells depending on the type and dose of the preparation used. Analysis of the cytokine profile produced after Con A stimulation of splenic cells showed significant production of IFN-γ and only slight production of IL-10. The next stage of the research was therefore to determine the reactivity of the tested splenocytes against LOS E. coli B, which can be considered a B lymphocyte stimulator. The concentration of cytokines was assessed by means of enzyme immunoassays ELISA. Figure 12A summarizes the results obtained after analyzing the IL-6 concentration.

The level of IL-6 production by spleen cells taken from normal mice was about 0.2 ng / ml and was the lowest among all groups tested. In the groups of mice bearing tumors and treated with the preparations, significant differences in production were observed after administration of LOS-OH E. coli B in vivo. The lowest concentration of this cytokine was found in supernatants of cells obtained from mice receiving 250 mg / kg bw. (0.25 ng / ml), while the production of IL-6 increased to 0.45 ng / ml after using a lower dose. The decrease in the ability of splenocytes to produce a pro-inflammatory cytokine by the action of E. coli B LOS assessed in supernatants from cultures containing an increased percentage of B lymphocytes may suggest a dose-dependent stimulation of a specific population of splenic cells in mice treated with E. coli B LOS-OH.

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IFN-γ production by spleen cells stimulated in vitro with E. coli B LOS ranged from 0 to 0.29 ng / ml (Fig. 12B). Cells of normal mice did not show the production of this cytokine at the sensitivity level of the test, while the use of LOS-OH E. coli B at a dose of 250 µg / kg body weight caused a slight increase in the production of this cytokine to 0.29 ng / ml. Splenocytes from mice in the remaining groups produced this cytokine in trace amounts. The low concentration of IFN-γ in splenocyte culture seems to be a secondary phenomenon, but emphasizing the significant contribution of the administration of LOS-OH E. coli B at a dose of 250 μg / kg to the activation of the cellular response.

The production of IL-10 by E. coli B LOS stimulated spleen cells taken from tumor-bearing mice and treated with the preparations is significantly lower than that of the production of this cytokine by splenocytes obtained from healthy mice (3.8 ng / ml). The variation in the level of IL-10 production appears to be largely related to the individual response of the treated mice. However, in this case, splenocytes from mice receiving LOS-OH E. coli B at a dose of 250 mg / kg belonged to the group significantly responsive to stimulation. Another group showing significant sensitivity to LOS E. coli B ex vivo stimulation was the group receiving 25 µg / kg of this preparation in vivo. There is an increasing tendency to IL-10 production in the group of mice receiving a lower dose of E. coli B LOS-OH (both after in vitro stimulation with ConA and LOS) and a significant reduction in the ability of splenocytes to produce IL-10 ex vivo after administration high dose LOS-OH A. coli B (Fig. 12C).

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Claims (9)

1. Compound of formula 1: where: R1 is 14: 0 (3-O-14: 0) or H, R2 is 14: 0 (3-OH) or 14: 0 (3-O-12: 0), R3 is H or 14: 0 (3-OH), R4 is 14: 0 (3-OH). 2. A compound according to claim 1, wherein R1 is H, R2 is 14: 0 (3-OH), R3 is H and R4 is 14: 0 (3-OH). 3. The compound according to p. 1, where R1 is 14: 0 (3-0-14: 0), R2 is 14: 0 (3-O-12: 0), R3 is 14: 0 (3-OH) and R4 is 14: 0 (3-OH). 4. A method for the preparation of a LOS-OH compound as defined in claim 1 The method of claim 2, characterized in that a) cells of the E. coli B bacterial strain are multiplied, PL 227 489 B1 b) the obtained bacterial cells are separated and lyophilized, c) extracting a compound according to claim 1; 3, d) the compound obtained in step c is treated with anhydrous hydrazine and precipitated from the reaction mixture, especially in excess of acetone, to obtain the compound according to claim 2. 5. The method according to p. A process as claimed in claim 3, characterized in that in step c) extraction with phenol / chloroform / petroleum ether mixture and precipitation with water are performed, followed by washing successively with phenol and diethyl ether and precipitation with water. 6. A compound as defined in claim 2 for use as a medicine. 7. A compound as defined in claim 2 for use in the prevention or treatment of cancer. 8. The compound for use according to claim 1 5 in the prevention of metastasis. 9. The compound for use according to claim 1 As a component of anti-cancer vaccines, especially as an adjuvant.