Patent Application
20130136764
The present invention falls within the field of vaccine preparation for specific immunotherapy.
At present several products for specific immunotherapy (SIT) are available on the market for the treatment of allergic diseases. However, SIT exhibits several limitations related to its profile of efficacy and safety. First of all, SIT is scarcely useful unless used in properly selected patients (selection based on the age, sensitizing allergen, entity of sensitization, etc). Secondly, SIT can determine an exaggerate release of mediators due to the stimulation of FceRI+ cells because of the administration of allergenic extracts to the patients with the possible consequence of severe adverse effects including anaphylactic shock. This implies that SIT cannot be used in those allergic diseases with more severe symptoms (bronchial asthma, atopic dermatitis, food allergy) in which the interference with allergen-specific response would be highly appreciable.
Antigen-specific immune response is mediated by two functionally distinct effector arms. While the T lymphocytes able to produce Interferon (IFN-)-gamma (TH1 lymphocytes) are responsible of protective responses against pathogens (intracellular bacteria, viruses), TH2 lymphocytes, able to produce IL-4, IL-5 and IL-13, are involved in the defence mechanisms towards parasites. A third subpopulation of lymphocytes, recently described as able to produce IL-17A (and , possibly, IL-22 and/or IFN-gamma) (TH17 lymphocytes) is responsible for the protective response against particular extracellular pathogens.
These three different cellular subpopulations are also responsible for several human diseases. In particular, allergic diseases (rhinitis, bronchial asthma, atopic dermatitis, food allergy, etc) are related to the expansion of TH2 cells specific for otherwise innocuous antigens (allergens) which are accumulated at the site of allergic inflammation (respiratory and/or gastro-intestinal tract, skin, etc). Even if several therapeutic options are available at the moment for the control of atopic diseases (steroids, anti-histamines, immunosuppressors), these drugs are only able to inhibit the activities of allergic mediators or, more generically and unspecifically, to regulate cellular activation.
Specific immunotherapy (SIT), used since the beginning of the last century, is intended to modify the allergy march and to prevent further sensitizations in atopic subjects but in the current practice it only partially meets these requirements. Actually, SIT should be based on the decrease of the activity of pathogenic TH2 cells together with a redirection of allergen-specific cells towards a more protective phenotype with production of IFN-gamma (TH1 cells). These activities might be possible by the use of novel and more powerful vaccine adjuvants which can induce the production of modulatory cytokines such as IFN-alfa and IL-12 through the interaction with particular Toll-like receptors (TLRs). Actually, some classes of low molecular weight compounds act as immunomodulatory agents both in vivo experimental animal models as well as on in vitro cultured human cells.
In particular, several evidences show that synthetic heterocycles chemically derived from adenine indeed exhibit immunomodulatory activities both in vivo and in vitro because of TLR7 activation with the induction of modulatory (IFN-alfa and others) and regulatory (IL-10) cytokines. At the same time these compounds reduce the production of TH2-related cytokines, inducing limited effects on B lymphocytes and low production of inflammatory cytokines. Finally, adenine derivatives have notable structural variability. The structural variability is strictly related to variable potencies in immunomodulation that is carried out at concentration usually ranging between 1 and 10 μM. These data suggest that these compounds may represent efficient and safe adjuvants to be used in vaccination protocols for the treatment of TH2-mediated diseases or, at least, in those pathologies where a protective IFN-gamma mediated immune response is hopeful.
However, even if the molecules that increase the vaccine potency through the stimulation of specific immune response and, possibly, redirect the immune response towards the wanted functional phenotype are the best adjuvants, it is also true that a simple combination (i.e. mixture) between an immunomodulator and proteins (from pathogens or exhibiting allergenic potential) affect several different cellular targets and, in particular, several antigen presenting cells. Thus, it would be necessary to use larger amounts of adjuvant to obtain the desired stimulatory effect with the possible amplification of adverse effects and reduction in the immune response specificity.
Thus, till now it is not yet solved how to direct an active adjuvant and an antigenic (or allergenic) protein at the same time to an unique antigen presenting cell in order to a) use lower amounts of adjuvant (with less toxic effects), b) act directly and exclusively on the antigen-specific response (reducing the release of inflammatory mediators), c) modulate the amount of adjuvant to the minimal active dose able to redirect the immune response. EP1035123 describes 4-(-amino-9-benzyl-8-hydroxy-2-purinil)tiobutirric acid and its related methyl-ester. These two compounds are described together with other similar compounds and are claimed as useful as therapeutic agents in the immunologic diseases.
The present invention solves the above mentioned problems through stable conjugated compounds of formula (II) derived from the conjugation of a protein antigen and modified adenines via a covalent bond in which the formula (II) is the following
where X═S; n is a integer number between 0 and 18; P represents a protein antigen (highly purified or recombinant protein or semipurified extract) covalently bound to the modified adenines as an example (but non exclusively) through its lysine residues, where m is a integer number which can vary from m=1 to m=number of lysine residues present on the P protein. It has been found that novel compounds of formula (II) derived from the conjugation of a protein antigen together with modified adenines are able to modify the allergen-specific response with re-direction towards a less pathogenic phenotype and, at the same time, exhibiting lower ability to stimulate the release of mediators of the allergic inflammation. This kind of redirecting response is determined by the synergistic activity of the antigen and the adenine derivative. This latter, as conjugated through a covalent bond, is active at significantly lower amounts than when used alone or simply mixed together with the antigen. This surprising synergy is possible because of the covalent conjugation between the protein of interest and the modified adenine. This result was also unexpected because of the great variability of chemical structures of adenine derivatives which exhibit different immunomodulatory activity as a) not all the structures (especially those with higher immunostimulatory activity) can be linked to protein antigens through covalent bonds, and b) it is not not trivial to covalently bind a protein to a modified adenine through a stable covalent bond.
Thus, subject-matter of the present invention are compounds with a chemical formula (II) described above for use as drugs (or as active ingredients in vaccine protocols and, in particular, in specific immunotherapy) for the treatment or, possibly, the prevention, of allergic diseases.
A further subject of the present invention are pharmaceutical compositions comprising at least one compound of formula (II) and at least another pharmaceutically acceptable ingredient. As pharmaceutical compositions vaccines are preferably considered.
A further object of the invention is a process for preparing compounds of formula (II), comprising the coupling of a protein P with an acid of formula (V),
or a corresponding activated ester, where n is as defined above.
The conjugation is advantageously obtained by the use of activated esters from modified adenines of general structure (I)
in which X═S and n represents the number of methylenic CH2 units between 0 and 18. The molecular substructure —CH2—(CH2)n—CO will be defined as linker. The compounds with general structure (I), as described above, are also subjects of the present invention. They have been demonstrated as able to efficiently react in mild conditions with allergenic proteins to produce stable conjugates with general structure (II).
Finally it is also a subject of the present invention a process for preparing compounds of formula (II) described above starting from compound of formula (III).
In the structure (II) the antigen P can be represented by any of the protein antigens including, as examples, bacterial antigens, inhaled allergens as pollen allergens or animal dander derivatives or food allergens.
In some of the embodiments the antigen P has been selected among the following allergens: semipurified extract of the house dust mite Dermotophogoides pteronyssinus (DP), purified natural allergen Der p2 (major allergen from the house dust mite Dermotophogoides pteronyssinus) (nDer), /recombinant allergen Der p2 (rDer) or ovalbumin (OVA grade V). In the compounds with formula (II) n is preferably included between 2 and 10, better n=2,3,4.
The novel compounds (II) derived from the conjugation of the above mentioned allergenic proteins together with modified adenines have been shown to exhibit immunomodulatory activity both in a in vitro system using human cells from atopic patients as well as an in vivo mouse models of allergy. The results from the biological assays in which conjugates with structure (II) with n=2 and P one of the above mentioned proteins, obtained by the combination of allergenic proteins (both highly purified or recombinant proteins or semipurified extracts) and the active ester SA-26E (compound exhibiting formula (I) with n=2) are of absolute relevance and particular significance due to their immunomodulatory activities.
In particular, it has been found that:
The above specified data obtained with specific embodiments of the invention suggest that, on the basis of the general applicability of the conjugation procedure, similar results can be also obtained with other protein antigens. According to the present invention, compounds of formula (II) can be obtained by reacting a compound having formula (V), as defined above, under conditions suitable to promote the amide bond formation between the linker carboxylic functionality and one or more lysine residues of the antigen P. To this end, it is possible to employ known conditions and catalysts for the formation of amide bonds; also, it is possible to transform the compound having formula (V) into a corresponding active ester. Among such active esters the compounds having formula (I) are to be preferred because they allow an efficient conjugation under mild conditions. Preferably, the conjugation reaction between a compound having formula (I) and an antigen P is carried out in a solution buffered at a pH between 7 and 8; more preferably at a pH between 7.2 and 7.6. Preferably, the solution is buffered with a phosphate buffer. Preferably, an excess of the active ester (I) is used compared to the moles of lysine evaluated to be present on the amount of the protein used; such an excess is preferably in the range of 1.5-8.0 equivalents. In the case of the conjugate in which n=2, P=nber P2, the best biological results have been obtained with m=1, 2; higher substitutions (that is m values greater than 2) have produced an excessive cytolytic effect on the cells. Compound having formula (I) according to the invention in which X═S can be prepared from the adenine derivative (III) known from the literature. The derivative (III) can initially be (step a) functionalized with a linker by treatment with ROC(═O)—(CH2)n-y, where Y is a leaving group such as Br, I, OTs, or OMs, R is a C1-C4 alkyl, n ranges from 0 to 18 (preferably between 2 and 10). To obtain a compound having formula (IV)
in which n and R are as above described.
Preferably Y is bromine and n=2,3,4.
Preferably in the step a about 1 equivalent of ROC(═O)—(CH2)n-y is employed and preferably the reaction is carried in anhydrous DMF and in the presence of K2CO3 as a base.
The ester (IV) so obtained is hydrolyzed, preferably in alkaline medium with KOH in a 3:1 MeOH—H2O mixture, generating acid (V). The active ester of formula (I) is prepared by treatment of acid (V) with a carbodiimide, preferably dicyclohexylcarbodiimide (in a slight excess), followed by N-hydroxysuccinimide under known conditions.
The present invention can be better understood through the following examples.
The synthesis of the active ester of formula (II) has been realized starting from modified adenines already known from the literature. As an example of the preparation of an active ester with formula (I) the procedure for the active ester (SA-26E) in which X═S and n=2 is reported. All the other active esters with general formula (I) can be prepared using a similar methodology.
Adenine derivative (III) known from the literature has initially been functionalized with a linker by treatment with ethyl 4-bromobutanoate (1 equiv.) in anhydrous DMF and in the presence of K2CO3 as a base. The product (IVa) is thus obtained in 54% yield after chromatography. The ethyl ester (IVa) so obtained is hydrolyzed in alkaline medium with KOH in a 3:1 methanol-water mixture. After 48 h, the methanol is evaporated and the pH of the resulting solution is adjusted to pH 3 by 10% NaHSO4, thus leading to the precipitation of acid (Va) (70% yield). The active ester SA26-E is prepared by dissolving the acid (Va) in anhydrous DMF and adding dicyclohexylcarbodiimide in a slight excess followed by N-hydroxysuccinimide according to standard conditions. After chromatography of the crude reaction mixture the active ester SA26-E is obtained in a 79% yield.
In a 100 mL two-necked flask, compound (III) (1.4 g, 5.12 mmol) is dissolved in 40 mL of anhydrous DMF under a nitrogen atmosphere. Molecular sieves (4 Å) are added in order to remove possible traces of water present in compound (III) and the solution is left under stirring at 25° C. for 35 min. K2CO3 (0.708 g, 5.12 mmol) is then added and the solution is left under stirring for 1 h. Ethyl 4-bromobutanoate (0.703 mL, 5.12 mmol) is then added and the reaction is left stirring at room temperature for 20 h. The solvent is then removed by distillation under vacuum heating at 36° C. and sterile water (5 mL) is added to the residue. A yellow precipitate is formed and the suspension is brought to pH 7 by a 10% KHSO4 solution in sterile water. The formed precipitated is filtered thus obtained a yellow solid (1.6 g) which is purified by chromatography on silica gel (eluant CH2Cl2/MeOH, 40:1, Rf 0.16) obtaining 1.073 g (2.77 mmol) of compound (IVa) as a pale yellow powder (54% yield).
1H NMR (400 MHz, DMSO, 25° C.) δ: 1.17 (t, J=7.0 Hz, 3H), 1.84-1.91 (m, 2H), 2.38 (t, J=7.4 Hz, 2H), 3.07 (t, J=7.3 Hz, 2H), 4.04 (q, J =7.0 Hz, 2H), 4.89 (s, 2H), 6.63 (br s, 2H), 7.24-7.32 (m, 5H), 10.3 (br s, 1H).
13C NMR (100.4 MHz, DMSO, 25° C.) δ: 15.0 (q), 25.8 (t), 30.3 (t), 32.5 (t), 43.4 (t), 60.7 (t), 101.3 (s), 128.3 (d), 128.5 (d, 2 C), 129.4 (d, 2 C), 138.1 (s), 147.9 (s), 148.9 (s), 152.7 (s), 162.0 (s), 173.3 (s). MS (ESI-MS) m/z (%): C18H21N5O35, 388 (M++1, 24), 342 (100).
Mp=123-127° C.
In a 50 mL flask, compound (IVa) (0.421 g, 1.09 mmol) is partially dissolved in 23 mL of a 3:1 mixture of MeOH and sterile water. KOH (0.184 g, 3.77 mmol) is then added which results in the complete solubilization of the starting material. The reaction is left under stirring at 25° C. for 48 h. After that time, the solution appears clear and dark yellow. After removing the MeOH under vacuum, the residual aqueous solution is acidified to pH 3 with a 10% solution of NaHSO4 in sterile water thus obtaining a pale yellow precipitate. This is filtered and washed with a slightly acid aqueous solution, then with water and finally with dichloromethane. The product is then dried under vacuum thus obtaining 274 mg (0.76 mmol) of product (Va) (70% yield).
1H NMR (400 MHz, DMSO, 25° C.) δ: 1.85-1.92 (m, 2H), 2.36 (t, J =7.4 Hz, 2H), 3.08 (t, J=6.6 Hz, 2H), 4.92 (s, 2H), 6.54 (br s, 2H), 7.28-7.36 (m, 5H), 10.1 (br s, 1H), 12.1 (br s, 1H).
13C NMR (100.4 MHz, DMSO, 25° C.) δ: 25.7 (t), 30.4 (t), 33.6 (t), 43.5 (t), 101.3 (s), 128.4 (d), 128.6 (d, 2 C), 129.4 (d, 2 C), 138.0 (s), 147.8 (s), 148.9 (s), 152.7 (s), 162.1 (s), 174.9 (s).
MS (ESI-MS) m/z (%): C16H17N5O3S 360 (M++1, 6), 342 (100), 274 (17).
Mp=230° C. (dec)
In a two-necked flask, the acid (Va) (0.249 g, 0.69 mmol) is dissolved in 5.5 mL of anhydrous DMF under stirring and a nitrogen atmosphere, in the presence of molecular sieves (4 Å). DCC (dicyclohexylcarbodiimide) (157 mg, 0.76 mmol) is then added followed by N-hydroxysuccinimide (80 mg, 0.69 mol).
The resulting solution is left under stirring at room temperature for 23 h. After that time, the solution appears cloudy. The solution is filtered and the solvent removed under high vacuum by heating at 33° C. The residue (490 mg) is purified by chromatography on silica gel (eluant CH2Cl2/MeOH, 40:1, Rf0.11) to give 247 mg (0.54 mmol) of SA26-E as bright yellow powder (78%).
1H (DMSO, 25° C.) δ: 1.94-2.01 (m, 2H), 2.77 (t, J=7.4 Hz, 2H), 2.80 (s, 4H), 3.10 (t, J=6.6 Hz, 2H), 4.88 (s, 2H), 6.53 (s, 2H), 7.24-7.34 (m, 5H), 10.1 (s, 1H).
13C NMR (100.4 MHz, DMSO, 25° C.) δ: 24.4 (t), 25.4 (t, 2 C), 29.3 (t), 33.3 (t), 42.5 (t), 100.4 (s), 127.4 (d), 127.6 (d, 2 C), 128.5 (d, 2 C), 137.0 (s), 146.9 (s), 148.0 (s), 151.8 (s), 160.8 (s), 170.2 (s).
MS (ESI-MS) m/z (%): C20H20N6O5S, 457 (M++1, 6), 342 (100).
Mp=161° C. (dec)
In a glass vial, the allergen nDer p2 (750 μg) is dissolved in 0.5 mL of a 0.1 M phosphate buffer at pH 7.4. The active ester (see Table), dissolved in DMSO (130 μL), is diluted to 1 mL with the same phosphate buffer and transferred into the vial containing the allergen (for a final volume of 1.5 mL). The solution is left under mechanical stirring at 4° C. for 18 h, and then is dialyzed. The conjugation with the protein nber p2 is carried out by employing an excess of active ester compared to the moles of lysine evaluated to be present on the amount of the protein used, as reported in the Table. The actual conjugation has been demonstrated by a MALDI-TOF analysis of the conjugates by which it is possible to observe the presence of the unconjugated protein (14.098 Kba) and the formation of conjugates having molecular masses of 14.439 and 14.781 Kba. These are present in a variable relative amount depending on the excess of active ester employed and correspond to the protein linked to one and two modified adenine fragments, respectively, having molecular mass of 341 (see
As an example of conjugation with an allergenic protein the coupling procedure between detoxified purified natural Der p2 (nber) is shown. The conjugation with the other allergenic proteins such as Ovalbumin (OVA), DP extract or rber has been realized following the same procedure but varying the amount of active ester on the basis of the numbers of lysine residues present in the protein molecule which can be potentially functionalized. In the case of nDer p2 (MW 14 KDa) six different lysines are present on the molecule and the amount of active ester suitable to obtain a ratio 1.7 or 7.5 between the moles of active ester and those of lysine residues on the protein has been used. In the following tables nber p2-conj is referred to a conjugate obtained by the use of a ratio between the moles of active ester and lysines as 1.7, Conj-5. The ratio 1.7 has been also used in the conjugation of the other allergenic proteins OVA, DP and rber to obtain the conjugates DP-conj, OVA-conj and rber-conj. When the ratio between the moles of active ester and those of lysine is 1.7 and the allergenic protein used is nber p2 (such as in the conjugates Conj-5, Con j-6, Con j-7 and Con j-8), it has been calculated that, every 10 μg conjugate, the amount of active ester actually present is 0.085 μg. Thus 0.085 μg/ml SA-26E has been thus used alone or mixed with 10 μg/ml nber p2 as control in some of the experimental models.
Circulating mononuclear cells (MNC) are isolated by density gradient (Ficoll-Hypaque) using buffy coats from 14 normal donors (Servizio Immunotrasfusionale e terapie cellulari, Azienda Ospedaliera Universitaria A. Meyer, Firenze). 200×106 MNC are separated by the use of a commercial kit (CD14 isolation kit, Miltenyi) by the addition of 400 μl anti-CD14 monoclonal antibody bound to iron beads followed by isolation on magnetic columns (LS column). 50×106 monocytes are recovered by positive selection and are extensively washed with calcium and magnesium-free sterile PBS and then seeded in complete medium plus 10% foetal calf serum in 24 flat bottomed plates at the concentration of 1×106/ml. As stimulants, the following compounds have been used: unconjugated allergens (DP or nber) (10 μg/ml), their respective conjugates (DP-conj or nber p2-conj) (10, 2.5 and 0.6 μg/ml), R-848 (2 μg/ml, 6 μM), SA-26, SA-26A or SA-26E (10, 2.5 and 0.6 μg/ml), LPS (100 ng/ml) and CpG-ODN 2006 (10 μg/ml, 1.3 μM). The plates are incubated for 36 hrs at 37° C. in 5% CO2 humidified atmosphere and the supernatants are collected after centrifugation at 1200 rpm, aliquoted and then stored at −20° C. up to the assay.
The preparation of purified BDCA4+ cells has been obtained in a similar way starting from MNC of buffy coats of the same 14 healthy donors. After density gradient separation, 500×106 MNC have been isolated by the use of a commercial kit (BDCA4 isolation kit, Miltenyi) by addition of 500 μl anti-BDCA4 monoclonal antibody bound to iron beads followed by separation on magnetic columns (LS column). By positive selection 5×106 plasmacytoid dendritic cells (PDCs) are recovered, extensively washed with calcium and magnesium-free sterile PBS and seeded in complete medium plus 10% foetal calf serum in 24-flat bottomed plates at the concentration 1×106/ml. As stimulants, the following compounds have been used: unconjugated allergens (DP or nber) (10 μg/ml), their respective conjugates (DP-conj or nber p2-conj) (10, 2.5 and 0.6 μg/ml), R-848 (2 μg/ml, 6 μM), SA-26, SA-26A or SA-26E (10, 2.5 and 0.6 μg/ml), LPS (100 ng/ml) and CpG-ODN 2006 (10 μg/ml, 1.3 μM). The plates are incubated for 36 hrs at 37° C. in 5% CO2 humidified atmosphere and the supernatants are collected after centrifugation at 1200 rpm, aliquoted and then stored at −20° C. up to the assay.
1×105 cells have been devoted to check the purity of the preparation and have been incubated in ice with 20 μl fluorochrome-conjugated anti-CD14 or anti-BDCA4 monoclonal antibodies in the dark. Cells are then washed with PBS, centrifuged, resuspended with 500 μl PBS and then analyzed at FACS. Purity was always above 98%.
For the determination of cytokines and chemokines of the innate immunity, commercial ELISAs have been used: IL-12p40 (Cytoscreen, Biosource Int, Camarillo, Calif.), IFN-alfa (Cytoscreen), TNF-alfa (Cytoscreen), IL-10 (Pharmingen), IL-6 and CXCL10 (Mb System). 100 μl/well of single supernatants are incubated in flat bottomed plates as specified by the productors and the amounts of the single cytokines are expressed as pg/ml on the basis of a reference curve by using recombinant cytokines.
The addition of LPS on purified CD14+ cells [Example 3 (step a)] stimulates, as expected, the production of relevant amounts of modulatory (IL-12) and inflammatory (TNF-alfa and IL-6) cytokines at levels similar to those obtained by the use of SA-26. On the contrary, the adenine SA-26A is unable to activate the production of any cytokine independently of the dose used and the soluble modified adenine SA-26E has an intermediate effect. The addition of 0.085 μg/ml soluble SA-26E (that is, as already specified, the corresponding amount of active ester present in Conj-5, -6, -7 and -8) is not able to stimulate the production of any cytokine. The unconjugated allergens (DP extract and nber p2) are essentially unable to induce the production of valuable cytokines from purified CD14+ cells. However, after conjugation with SA-26E, both the allergenic molecules (DP-conj and nber p2-conj) induce significant amounts of modulatory and regulatory cytokines at similar levels than those induced by other stimulants, even when used at low concentrations. The production of other cytokines such as IL-27 or the regulatory molecule MO is never observed (data not shown).
Analogously, BDCA4+ cells from the same donors are able to produce high levels of IFN-alfa, IL-10 and CXCL10 in response to R-848 whereas the adenine SA-26A is not able to induce any cytokine production. On the other hand, SA-26E exerts an intermediate effect on plasmacytoid dendritic cells depending on the dose used. However, SA-26E is completely inactive when used at 0.085 μg/ml concentration. As expected, unconjugated allergens do not stimulate cytokine production, whereas conjugated allergens DP-conj and nber p2-conj induce the production of high levels of both IFN-alfa and IL-10. Neither IL-29 or CXCL10 production are significantly modified by the addition of any of the stimulants with the only exception of R-848 (data not shown).
The following tables show the results (mean±5E) obtained from stimulated CD14+ (Tables A and B) and BDCA4+ (Tables C and D) cells with the corresponding significance.
To verify that the allergenic protein conjugated with the active ester SA-26E is indeed able to exhibit a modulatory activity higher than the simple mixture of the allergenic protein itself plus soluble SA-26E, purified CB14+ and BDCA4+ cells from two healthy donors have been cultured for 36 hrs in the presence of the native allergenic protein nber p2 (10 μg/ml), the conjugated compound nber p2-conj (Conj-5), LP5 or R-848 (100 ng/ml and 2 μg/ml, respectively) or with the mixture nber p2 and soluble SA-26E (10 μg/ml and 0.085 μg/ml respectively) in order to stimulate the production of modulatory, regulatory and pro-inflammatory cytokines. Both the unconjugated allergen, as expected, but also the mixture between the allergen and soluble active ester are unable to stimulate the production of significant amounts of cytokines from monocytes or plasmacytoid dendritic cells. Only when the allergen was conjugated together the modified adenine, relevant amounts of cytokines similar to those obtained by the use of known immunomodulatory agents such as LP5 and R-848 were obtained.
The following tables (E and F) show the data obtained in the two donors.
5×106 purified CD14+ cells were cultured in polypropylene 15 ml tubes in complete medium plus 10% foetal serum in the presence of the following products as stimulants: unconjugated allergens (DP or nber) (10 μg/ml), their respective conjugates (DP-conj or nber p2-conj) (10 μg/ml), R-848 (2 μg/ml) or nber p2 mixed together with soluble SA-26E (10 μg/ml and 0.085 μg/ml respectively). The tubes are then incubated for 2 hrs in 5% CO2 humidified atmosphere at 37° C. and then washed with 10 ml PBS, centrifuged at 1500 rpm repeating the treatment thrice and the cellular pellet finally dried by the use of a micropipette and stored at −80° C. up to the assay. The nuclear extract of the cultured cells was prepared by the addition of a mixture of reactive compounds to single tubes following the instruction (Active Motif) and the protein content of the single preparations was evaluated by Bradford method. Two different protein amounts (0.5 and 2 μg) were incubated in 96w plates coated by the immobilized oligonucleotide containing the activated NF-κB consensus site (5′-GGGACTTTCC-3′), followed by the incubation with an antibody recognizing an epitope of the NF-κB p50 subunit. An HRP-conjugated secondary antibody was added to single wells and the colorimetric read-out was quantified by spectrophotometry. Values are reported as Optical Density. As positive control, Jurkat nuclear extract was used as suggested by the manufacturer. For the specificity of the assay, a wild-type consensus oligonucleotide was used as a competitor for NF-κB binding.
HEK293 cells are detached by trypsin from the plastic surface of the culture bottle, extensively washed with calcium and magnesium-free PBS, counted and adjusted at the concentration 1×106 cells/tube in polypropylene V-bottomed tubes. After the final wash, the cells are pelleted and 100 μl transfection buffer (Amaxa) is added together with 5 μTLR3, TLR7, TLR8 or TLR9-encoding and ELAM-1 promoter NF-kB luciferase reporter plasmids. The mixture thus obtained is transferred in special cuvettes and transient transfection is obtained in a special apparatus which is able to directly introduce genetic material into the nucleus (Nucleofection®, Amaxa GmbH). The cells are then recovered and cultured at 1×105/ml in 48-flat bottomed plates in E-MEM medium plus 5% foetal calf serum for 18 hrs. After this first incubation, medium (as negative control) or the following compounds as stimulants are added for additional 18 hrs: DP, nber p2 or their respective conjugates DP-conj or nber p2-conj (10 μl/ml), nber p2 mixed with soluble SA-26E (10 μg/ml and 0.085 μg/ml respectively) or already known TLR-ligands such as Poly I:C (50 μg/ml), R-848 (2 μg/ml) or CpG-ODN 2006 (1.3 μM). Finally the luciferase activity is measured in the cellular lysates by a commercial kit (Promega).
he conjugated allergens DP-conj and nber p2-conj but not the unconjugated allergens DP and nber p2 which are actually inactive, are able to induce a significant increase in the p50 nuclear expression in purified CB14+ cells [Example 4 (step a)] exhibiting a similar effect as R-848 that exerts, as expected, a rapid and high activation of p50 even higher than the positive control itself (Jurkat cells). The effects exerted by conjugated compounds suggest that the conjugates are able to activate the NF-κB signalling pathway.
In
On transfected HEK293 cells along with the method used as indicated in [Example 4 (step b)], we have also found, as expected, that R-848 and CpG-ODN 2006 are able to induce NF-κB expression in TLR7/8 and TLR9-transfected cells, respectively, whereas Poly I:C activates TLR3-expressing cells (data not shown). The unconjugated allergenic proteins DP and nber p2 lack any activity. In addition, the mixture nber p2 and soluble SA-26E (10 μg/ml and 0.085 μg/ml respectively) is completely inactive (data not shown). Similar effects are obtained when HEK293 cells are transfected by the use of a murine TLR7-encoding plasmid together with ELAM-1 promoter NF-kB luciferase reporter.
Mononuclear cells from 10 atopic donors with sensitization towards Dermotopahgoides pteronyssinus have been isolated by density gradient and 1×106 cells cultured in 2 ml complete medium plus 5% autologous serum in the presence of DP or nber p2 allergens or their respective conjugates (DP-conj or nDer p2-conj) (all at 10 μg/ml) in 24 flat-bottomed plates (Costar). As positive control, cells are also cultured in the presence of DP or nber p2 and soluble R-848 (2 μg/ml). After 6 d incubation and addition of rIL-2 (Proleukin) (25 UI/ml), T cell blasts are further expanded by the addition of medium, fresh 5% autologous serum and 25 UI/ml rIL-2 every three days. After 15 d culture, T cell blasts from single cultures are collected, washed with sterile PBS, counted and used to assess allergen specificity, phenotype and function. To assess allergen-specificity, 1×105 T cell blasts are cultured in 96 U-bottomed plates in 0.2 ml final volume in medium plus 5% autologous serum, 1×105 autologous irradiated (9000 R) mononuclear cells as APCs, DP or nber p2 (10 μg/ml) and, after 3 d incubation, tritiated thymidine (3HTdR) (Amersham) is added. After further 16 hr incubation, single cultures are transferred on paper filters through the use of an Harvester apparatus (Tomtec) and radioactivity measured in a β-counter. T cell lines are considered as specific when stimulation index (SI) (cpm in cultures stimulated in the presence of autologous MNC plus allergen/cpm in cultures in the presence of autologous MNC alone) is above 3 as already published (Brugnolo F et al J Allergy Clin Immunol 2003 February; 111(2): 380-9; Fill L et al, J Allergy Clin Immunol 2006 August; 118(2):511-7).
For phenotypic analysis, 2×105 T cell blasts are resuspended in PBS 0.5% BSA and 0.02% sodium azide and treated with rabbit IgG to saturate unspecific sites. Cells are then incubated for 20 min with anti-CD3, anti-TCRαβ, anti-TCRγδ, anti-CD4, anti-CD8, anti-CD16, anti-CD20 and anti-CD56 monoclonal antibodies (Becton-Dickinson) or their respective isotype control antibodies (Southern Biotechnologies). At the end of the incubation, cells are washed and analysed at FACSCalibur (Becton-Dickinson) acquiring at least 104 events for each sample. The T cell lines obtained in the presence of DP-conj and nDer p2-conj exhibit higher percentages of CD3+γδ+ and CD16+CD56+ than unconjugated-specific T cell lines but similar to TCL derived in the presence of R-848.
To functionally analyze the T cell blasts, the following parameters have been considered: i) intracellular production of cytokines by cytofluorimetric analysis after polyclonal stimulation with PMA and ionomicin; ii) expression of transcription factors related to the TH1, TH2, TH17 and Treg phenotypes; iii) cytokine production in the supernatants after polyclonal stimulation with PMA and anti-CD3 monoclonal antibody; iv) mRNA expression for TH2- or TH1-related cytokines by the use of quantitative RT-PCR.
For the analysis of the intracellular cytokine production at single cell level, 1×106 T cell blasts from T cell lines specific for DP, nDer p2 or their respective conjugates are stimulated for 4 hrs (the last two in the presence of the Golgi venom Brefeldin A 5 μg/ml) with PMA (10 ng/ml) and ionomicin (1 μM) as already described (Brugnolo F et al J Allergy Clin Immunol 2003 February; 111(2): 380-9; Fill L et al, J Allergy Clin Immunol 2006 August; 118(2):511-7). After the incubation, the cells are washed with PBS pH 7.2 and then fixed for 15 min with formaladheyde 2% in PBS, washed again with 0.5% BSA in pH 7.2 PBS and finally permeabilized with pH 7.2 PBS containing 0.5% BSA and 0.5% saponin. At the end, the cells are incubated with the specific monoclonal antibody analyzing the following cytokines: IL-4, IFN-gamma, IL-5, IL-13, IL-9 and IL-17. The analysis is done by the use of FACSCalibur and the software CellQuest (Becton-Dickinson) acquiring at least 104 events in CD3+CD4+TCRαβ+ or CD3-CD16+-gated cells for each sample. For the determination of the cytokine (IL-4, IFN-gamma, IL-5, IL-13, IL-10 and IL-17) content in the supernatants, 1×106/ml T cell blasts from allergen-specific lines are stimulated with PMA (20 ng/ml) and anti-CD3 monoclonal antibody (UCHT1, Pharmingen, 50 ng/ml) in 1 ml volume for 36 hrs. The cytokine determination is based on ELISA assays with the use of commercial pairs of antibodies as already described (Brugnolo F et al J Allergy Clin Immunol 2003 February; 111(2): 380-9; Fill L et al, J Allergy Clin Immunol 2006 August; 118(2):511-7): 11-4, IL-5 and IL-10 (BD Pharmingen, Franklin Lakes, N.J.), IL-17 and IL-13 (R&D System), IFN-gamma (Endogen, Woburn, Mass.). The expression of transcription factors related to TH1, TH2, TH17 and Treg (T-bet, GATA-3, ROR C and Foxp3, respectively) phenotype has been determined by the use of quantitative real-time RT-PCR.
mRNA expression of cytokines (IFN-gamma, IL-2, IL-4, IL-5, IL-9, IL-13, IL-17, TGF-betal, IL-22, IL-27, CXCL10 and MMIF) has been determined by the use of quantitative real-time RT-PCR. In brief, total RNA is extracted from 1×106/ml T cell blasts by using the RNeasy kit and treated with nose I to eliminate any genomic DNA contamination (Qiagen) and quantified by Nanobrop (Celbio) in each mRNA sample. cDNA is then synthesized from the same template quantity (TaqMan Reverse Transcription Reagents, Applied Biosystems, Foster City, Calif.). Real-time polymerase chain reaction (PCR) is performed with an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) and all PCR amplifications are performed by MicroAmp optical 96well reaction plate with TaqMan Universal Master Mix and with Assay-on-Demand (Applied Biosystems). Each assay is carried out in duplicate and included a no-template sample as negative control comparing experimental levels with a standard curve generated with serial dilution of cDNA obtained from human MNCs. β-Actin is used as a housekeeping gene for normalization. Inducing allergen-specific T cell lines [Example 5 (step a)] we observed that the percentage of the cells able to produce IFN-gamma is significantly higher in the T cell lines obtained by the use of conjugated allergens DP-conj and nber p2-conj (Conj-5) than in those derived in the presence of unconjugated native allergens DP and nDer p2 with a parallel decrease in the percentage of IL-4 producing cells. The effect of redirection of allergen-specific cells from a prevalent TH2 phenotype towards a prevalent TH1/TH0 observed when conjugated allergens are used, is similar to the effect obtained when soluble R-848 together with unconjugated allergens DP and nber p2 is added in the culture system, whereas the mixture nber p2 and soluble SA-26E (10 μg/ml and 0.085 μg/ml, respectively) is not able to modify the cellular phenotype. No difference in the percentage of IL-9 or IL-17 producing cells is observed between allergen- or conjugated allergen-specific T cell lines. In particular, the percentage of IL-17-producing cells is very low in both conditions. Along with the reduced production of IL-4, lower percentages of IL5 and IL-13 producing cells is observed in DP-conj- and nber p2-conj specific T cell lines in comparison with unconjugated DP- or nber p2-specific TCLs (data not shown).
These results are even confirmed by the determination of the cytokine content into the supernatants. T cell blasts from T cell lines specific for DP-conj or nber p2-conj (Conj-5) produce significantly lower amounts of the TH2-related cytokines (IL-4, IL-5, IL-13) and significantly higher levels of IFN-gamma than TCLs specific for unconjugated DP and nber p2. We have never observed the production of significant amounts of IL-17 (data not shown). Analogously, quantitative real-time RT-PCR shows that T cell lines obtained in the presence of the conjugated allergens DP and nber p2-conj are able to express significantly higher levels of IFN-gamma (and significantly lower of IL-4, IL-5 and IL-13) than TCLs specific for the unconjugated allergens DP and nber p2. No difference in the production of IL-10, IL-29, IL-17, IL-22, TGF-betal, CXCL10 or MMIF was observed.
In the
The redirection of allergen-specific T cell lines from the TH2 towards the TH1/TH0 phenotype is finally confirmed also at transcriptional level. Actually T cell lines derived in the presence of DP-conj and nber p2-conj express higher levels of the TH1-related transcription factor T-bet and lower levels of GATA-3 than T cell lines specific for the unconjugated allergens DP or nber p2. We have never observed differences in the expression of TH17-related RoRC transcription factor. Analogously, no modification in the expression of the Treg-related transcription factor Foxp3 is found.
In all the experiments, the mixture between nber p2 and soluble SA-26E (10 μg/ml and 0.085 μg/ml, respectively) is completely inactive (data not shown).
The results are shown in
For the functional analysis of T cell clones specific for nber p2 and nDer p2-con j, T cell lines are initially derived from MNC of two atopic donors (G.E. and M.A.) affected by bronchial asthma and allergic rhinitis and sensitized to Dermotophogoides pteronyssinus, in the presence of nber p2- or its respective conjugate nber p2-conj. As control, from donor G.E. T cell lines specific for nber p2 in the presence of R-848 (which has been already described as able to induce a redirection of TH2 cells towards a TH1/TH0 oriented phenotype) have been also obtained. 28 allergen-specific T cell clones are obtained from T cell lines specific for nber p2 from donor G.E. and 37 from donor M.A. whereas 28 clones are obtained from T cell lines derived in the presence of nber p2-conj from both donors. Twelve clones specific for nber p2 were also obtained from donor G.E. starting from T cell lines cultured in the presence of nber p2 and soluble R-848. The clonal efficiency in the different culture conditions is shown in the table below:
The allergen-specific clones obtained in the different experimental conditions have been then assessed for their ability to produce IL-4 (μg/ml) and IFN-gamma (pg/ml) in the culture supernatants after polyclonal stimulation with PMA and anti-CD3 monoclonal antibody.
The results obtained in step (a) have been confirmed at clonal level [Example 5 (step b)] as shown in the
Mononuclear cells from peripheral blood (MNC) are isolated by density gradient (Ficoll-Hypaque) using buffy coats from Dermotophogoides pteronyssinus-sensitized donors (Servizio Immunotrasfusionale e terapie cellulari, Azienda Ospedaliero Universitaria Pediatrica A. Meyer, Firenze). 500×106 MNC are separated by the addition of 500 μ1 anti-CRTH2 monoclonal antibody for 20 min at room temperature, then extensively washed and incubated again with a secondary polyclonal antibody bound to iron microbeads and finally positively selected on a magnetic column (LS column). 10×106 cells are recovered by positive selection, extensively washed with sterile calcium and magnesium-free PBS buffer and cultured at the concentration 1×106/ml/well in complete medium plus 5% human AB serum in 24-flat bottomed plates. As stimulants, unconjugated allergens (DP or nDer p2) or conjugates (DP -conj or nDer p2-conj) (all at 10 μg/ml) have been used. Cultures have been incubated for 6 days at 37° C. in 5% CO2 humidified atmosphere and further expanded with rIL-2 25 UI/ml until 14 days when T cell blasts are collected, extensively washed, counted, adjusted at 1×106/ml concentration and polyclonally stimulated with PMA and ionomicin as already described to assess the intracellular production of IL-4 and IFN-gamma. The study of CRTH2 cells [Example 5 (step c)] has definitely confirmed the modulatory activity of conjugated compounds. In fact, CRTH2 cells, when in vitro stimulated with the unconjugated allergen, express IL-4, whereas, when cultured with conjugated allergen, the expression of IL-4 is dramatically reduced together with increased ability to produce IFN-gamma alone (TH1 cells) or together with IL-4 (TH0 cells).
The results obtained are shown in the following table:
In the
In the T cell lines no significant production of IL-17 that is claimed to be related to the induction of autoimmune diseases following TLR triggering, has been observed whereas a statistically significant reduction in the production of other TH2-related cytokines (IL-5, IL-13) is found (data not shown).
T cell lines derived from two atopic donors sensitized to Dermatophagoides pteronyssinus have been obtained as already described in step (a) by the use of unconjugated nber p2 and three different preparations of conjugated allergens (as indicated in table Example 2: nber p2-conj2, nber p2-conj5, nber p2-conj6), the three conjugated allergens being obtained by the same procedure of conjugation as described in Example 2 but mixing fixed amounts of nber p2 protein with different amounts of the active ester SA-26E (step b) (Conj-2>Conj-5) or mixing fixed amounts of nber p2 protein and similar amounts of the active ester SA-26E in different times (Conj-5 and Conj-6).
The conjugated compounds obtained with different ratios between the allergenic protein and the modified adenine (see table Example 2) tested in two atopic donors [Example 5 /step d)] are able to significantly reduce the expression of IL-4, IL-5 and IL-13 (together with the TH2-related transcription factor GATA-3) and to increase the expression of IFN-gamma in comparison with the T cell line obtained by the use of the unconjugated nber p2 (data not shown). The TH1-indicing effect is higher when the conjugated product nber p2-conj 5 (lower amount of conjugated adenine) is used in comparison with Conj-2 (higher amount of conjugated adenine) whereas similar effects as obtained when Conj-5 or Conj-6 are used. This indicates that a) the amount of active ester conjugated to the allergenic molecule is critical for the functional modulation of the allergen-specific cells; b) the amount of active ester can be easily modified in order to obtain effects of different entity; c) the better modulatory effects are obtained with lower amounts of active ester conjugated to the antigenic protein.
As expected, R-848 is indeed able to decrease the percentage of the cells able to produce IL-4 together with a parallel increase of IFN-gamma production. Conjugated compounds produced in identical experimental conditions (i.e. nber p2-conj 5 and nber p2-conj 6) but at different times exert analogous effects thus indicating the optimal repeatability of the experiments.
To demonstrate that the cytokines produced by the antigen presenting cells after the stimulation with the conjugated product are indeed responsible of the functional modification of the phenotype of allergen-specific T lymphocytes, allergen-specific T cell lines have been obtained from two atopic donors by the use of DP and DP-conj in the absence or presence of neutralizing anti-IL12 (R&D System), anti-IL-29 (R&D System), anti-IFN-alfa and anti-IFN-alfaR (both from PBL, Piscataway, N.J., USA) monoclonal antibodies, each at 5 μg/ml, using the same procedure described in step (a). As negative control, mouse IgG were used.
The redirection effect exerted by conjugated products on T cells are dependent of their ability to stimulate the production of modulatory cytokines from cells of the innate immunity. Actually, in the two donors of Example 5 (step e), the addition of anti-IL-12 or anti-IFN-alfa antibodies to the cells cultured in the presence of DP-conj is able to restore the production of IL-4 (and to reduce the production of IFN-gamma) induced by the conjugated compound, whereas the neutralization of IL-29 does not exert any inhibitory effect. The mixture of anti-IL-12 and anti-IFN is indeed able to completely inhibit the modulatory activity of the conjugated allergen thus suggesting that these two cytokines are both involved in the TH1 switching effect whereas the activity of IL-29 is not significant.
The results of the experiments are shown in the table reported below:
In order to evaluate whether the allergenic conjugate is correctly recognized by Dermatophogoides-specific T lymphocytes, T cell lines are induced from peripheral blood mononuclear cells of three atopic donors by the use of the procedure already described in step (a) with uncojugated DP allergen. At the end of the 14 d culture period, T cell blasts are collected, washed and counted and finally cultured at the concentration 1×106/ml together with 1×106 autologous irradiated (9000 R) mononuclear cells as APCs in the presence of DP or DP-conj (10 μg/ml). After three days, tritiated thymidine is added for further 16 hrs. The incorporated radioactivity in the single cultures, measured as counts per minute (cpm) and directly proportional to the amount of tritiated thymidine incorporated in the cellular DNA, is evaluated after cell transfer on cellulose filters by the use of a cell Harvester (Tomtec, Turku) and read in a beta-counter. The cell proliferation is expressed as Stimulation Index (I) which is calculated as follows:
Mean cpms in stimulated cultures×100
Mean cpms in Unstimulated Cultures
The modulatory effect is not correlated to the loss of recognition of the conjugated allergen by T cells, as DP-specific T cell lines [Example 5 (step f)] equally proliferate in response to DP or conjugated DP as shown in
100 μl whole blood from four allergic patients sensitized to Dermotophogoides pteronyssinus were incubated together with 20 μl stimulation buffer (negative control) provided by the manufacturer (Becton Dickinson) or 20 μl serial dilutions of DP or nber p2 or their respective conjugates DP-conj or nDer p2-conj (0.5-0.05-0.005 μg/ml) or 20 μl fMLP or 20 μg anti-IgE monoclonal antibody as positive control for 15 min at 37° C. in water. At the end of the incubation, the samples are rapidly put in ice for 5 min in order to arrest basophil degranulation and coloured by the addition of 20 μl mixture of three different monoclonal antibodies (FITC anti-CD63, PE anti-CD123 and PerCP anti-HLA-DR) for 20 min in ice. The lysis and fixation of the cells is obtained by the addition of 2 ml of a pre-warmed solution for 10 min at room temperature to single tubes which are finally centrifuged, washed and analysed at the cytofluorimeter acquiring at least 500 CD123+ cells for each sample.
The circulating basophils (CD123+ HLA-DR-) obtained as specified [Example 6 (step a)] easily increase the expression of the CD63 molecule when placed in the presence of anti-IgE as shown in the example (mean of the expression 41.3±6.5%) as well as when fMLP (data not shown) or unconjugated allergens at serial concentrations are added. On the contrary, the conjugated DP looses the ability to activated FCεRI+ cells in a dose-dependent manner. The results obtained by the use of the DP and DP-conj allergens are reported in the following table:
Circulating mononuclear cells (MNC) are isolated by Ficoll-Hypaque density gradient from buffy coats of healthy donors (Servizio Immunotrasfusionale e terapie cellulari, Azienda Ospedaliero Universitaria Pediatrica A. Meyer, Firenze). 200×106 MNC are then separated using the commercial CD19 isolation kit (Miltenyi) by the addition of 400 μl iron microbead-bound anti-CD19 monoclonal antibody followed by magnetic separation on LS column. 20×106 B cells are isolated by positive selection and are extensively washed by the use of calcium- and magnesium-free PBS and cultured in triplicates at the concentration of 1×106 /ml/well in complete medium plus 10% foetal calf serum in 96-U plates. As stimulants, the following compounds are used: unconjugated allergens (DP or nber p2) (10 μg/ml), their respective conjugates DP-conj or nber p2-conj (10 μg/ml), R-848 (2 μg/ml) and CpG-ODN 2006 (10 μg/ml). Plates are incubated in humidified atmosphere at 37° C. for 72 hrs and at the end 0.5 μCi tritiated thymidine is added to each well for further 16 hrs. The incorporated radioactivity is measured as already described in step (f) of Example 4.
Soluble TLR-ligands (R-848 and CpG) are able to induce high levels of incorporation of radioactive thymidine in purified B cells [Example 6 (step b)] as expressed by stimulation indexes whereas the conjugated allergens exhibit a low or absent ability to induce proliferation thus showing a better safety profile. As expected, the unconjugated allergens are unable to determine any proliferative response in B lymphocytes.
The results regarding the experiments with the purified allergen nber p2 and its respective conjugate are reported in the table below:
2 μg/ml
Ten BALB/C and ten C57BL/6 6-8 wk-old female mice (Charles River) have been intraperitoneally sensitized at day 0 and d 7 with 10 μg OVA (5 animals per strain) or OVA-conj (5 animals per strain) in the presence of alum. At d 14 and d 18 animals are stimulated intratracheally with 10 μg OVA and after 24 hrs unspecific bronchial hyperreactivity is measured administering first an aerosolized PBS solution to obtain the standard value and then an aerosolized solution containing increasing concentrations of methacolin for 3 min (6.25-12.5-25-50 mg/ml). The pletismograph in which the animal is inserted is able to monitor the values of airflow resistance.
Serum levels of total and OVA-specific IgE have been determined at d 14 and d 18 (before the intratracheal allergic stimulation) in sensitized animals by the use of commercial kits.
The sensitization with OVA conjugated with modified adenine [OVA-conj] in the experimental animals following the procedure already described [Example 7 (step b)] induce reduced levels of IgE (both total IgE as shown in the table and OVA-specific IgE as shown in the following table and
Moreover, the group of animals sensitized by the use of the conjugated compound OVA-conj exhibit a significant decrease in the methacolin-induced bronchial hyperreactivity [Example 7 (step a)] in comparison with unconjugated OVA-sensitized mice, thus suggesting that the conjugation of OVA together with the modified adenine is indeed able to significantly reduce the bronchial hyperreactivity following the inhalation of allergenic proteins such as OVA (see
| Number | Date | Country | Kind |
|---|---|---|---|
| FI2010A000171 | Aug 2010 | IT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2011/053482 | 8/4/2011 | WO | 00 | 2/5/2013 |
