EP3787670A1 - Induction of allergen-specific tregs prior to oral or sublingual immunotherapy of food allergy - Google Patents

Abstract

The present invention relates to methods for the induction of food tolerance by a combination of subcutaneous hydrogel- based immunotherapy with food allergen-derived T cell peptides in the presence of tolerance-promoting concentrations of oligodeoxynucleotides with CpG or GpC or GpG motifs (Phase A) and subsequent oral or sublingual immunotherapy with natural or recombinant food allergens (Phase B)

Description

Induction of Allergen-Specific Tregs prior to Oral or Sublingual Immunotherapy of Food Allergy

Field of the invention

The present invention relates to methods for the induction of food tolerance by a combination of subcutaneous hydrogel-based immunotherapy with food allergen-derived T cell peptides in the presence of tolerance-promoting concentrations of oligodeoxynucleotides with CpG or GpC or GpG motifs (Phase A) and subsequent oral or sublingual immunotherapy with natural or recombinant food allergens (Phase B) .

BACKGROUND OF THE INVENTION

Food allergy is a potentially life-threatening condition with no approved therapies apart from avoidance and injectable epinephrine for treatment of acute allergic reactions (for a review, see Wood, 2017) . As of today, food allergy is estimated to affect up to 8% of children and up to 2-3% of adults in the U.S. alone. The foods most often associated with food allergy in the U.S. include cow's milk, hen's egg, peanut, tree nut, wheat, soy, fish, and shellfish. Most important is the growing incidence of peanut allergy which affects 1-2% of children in the U.S. and is implicated in over half of all fatal food allergy-related deaths in the U.S. Furthermore, only 15-20% of peanut or tree nut allergic individuals "outgrow" their allergies, whereas at least 80% of milk- and egg-allergic children are expected to achieve natural tolerance to these foods by adulthood. Avoidance is currently the only approved therapy for food allergy. However, avoidance diets can be difficult and may also put children at risk of nutritional deficiencies and impaired growth. Therefore, there is a need in the field for effective therapies for peanut and other common food allergies .

Several therapeutic approaches have been evaluated in the recent past including oral, sublingual and epicutaneous immunotherapy. Oral immunotherapy (OIT) is an experimental treatment in which patients consume gradually increasing quantities of the food to which they are allergic. Most OIT protocols include an initial escalation phase, followed by dose build-up phase and maintenance phases with considerable variability depending on the study. The initial escalation phase is typically conducted over one to two days, using rapid up-dosing starting from a very small dose which is extremely unlikely to cause any adverse reaction, and progressing to a dose that is still likely safe for home administration. Generally, the initial doses are in microgram quantities of allergenic protein and increases to several milligrams by the end of this phase. If well tolerated, the dose is escalated incrementally (usually bi-weekly over a period of approx. 6 months) until a target maintenance dose is reached or the subject reaches dose-limiting symptoms. Maintenance therapy continues with daily administration in the home, and the length of maintenance therapy varies considerably, lasting from a few months to several years.

While desensitization is possible in most patients, adverse reactions are very common with OIT, with overall rates similar for each of the foods studied to date (for a review, see Wood, 2017) . Reactions are generally mild and local symptoms such as oral itching are most common. Abdominal pain is the most common symptom leading to withdrawal from treatment, and moderate reactions, such as wheezing, vomiting, and urticaria occur in a small percent of all doses. However, given that doses are given daily over an extended period of treatment, the risk for each patient is substantial. For example, during peanut OIT for 352 patients 95 reactions requiring epinephrine occurred (Wasserman et al . , 2014). Therefore, a major impediment to moving OIT treatments to clinical practice is the high percent of patients who cannot tolerate OIT. Overall, 10-20% of subjects have dropped out of OIT trials, with rates as high as 36% (for a review, see Wood, 2017) . Further studies directed at minimizing adverse reactions are therefore critically important to move these treatments forward toward clinical use. Sublingual immunotherapy (SLIT) for food allergy has also been shown to induce desensitization (for a review, see Vazquez- Ortiz and Turner, 2016) . Effector cells (mast cells, basophils) are scarce in the sublingual mucosa which reduces the risk of allergic reactions. Conversely, antigen-presenting dendritic cells (e.g. Langerhans cells) are abundant, which may promote the induction of tolerogenic T-regulatory cells. While systemic reactions are less frequent on SLIT, the increase in amount of food allergen that can be tolerated following SLIT is modest and lower than that achievable with OIT. Two studies have compared the efficacy of SLIT vs. OIT for cow's milk and peanut allergy, respectively. In both cases, OIT resulted in an increase in threshold up to 10 times that achieved with SLIT (Narisety et al . , 2015; Keet et al . , 2012) . Whether the effect of SLIT is sufficient to protect patients from accidental reactions is unclear, as the median threshold following SLIT can be relatively low. A study of peanut SLIT resulted in a median threshold for reactivity of 371 mg of peanut protein - less than 2 peanuts (Fleischer et al. , 2013) . Epicutaneous immunotherapy (EPIT) involves the application of a patch containing the food allergen to the skin (for a review, see Vazquez-Ortiz and Turner, 2016) . The epidermis is poorly vascularized, which might limit the potential for systemic reactions. In contrast, it contains high numbers of potent antigen-presenting cells, which may allow immune modulation and enhanced efficacy. Pre-clinical data in peanut- sensitized mice demonstrated that peanut-EPIT on intact skin decreased the clinical and allergen-specific Th2 responses ( ondoulet et al., 2012), and recent clinical phase I-II studies (Clinicaltrials.gov NCT01170286 and NCT01197053) confirmed this observation. No systemic reactions occurred, although local eczematous skin reactions were common. However, the immunotherapeutic efficacy of EPIT for peanut allergy is more than two-fold lower than that of SLIT and more than twenty-fold lower than that of OIT (corporate presentation of Aimmune Therapeutics, March 2017) .

In summary, there is a significant unmet need for an efficient disease-modifying treatment for food allergy and in particular for peanut allergy with a minimal risk of adverse reactions. Currently, the most plausible candidate with the most clinical data is OIT, but OIT-associated adverse reactions need to be minimized by novel treatment modalities.

The present invention solves this problem by a novel combination of subcutaneous hydrogel-based immunotherapy with food allergen-derived peptides prior to OIT or sublingual immunotherapy with intact food allergens. SUMMARY OF THE INVENTION

In one embodiment, the present invention discloses a novel combination of subcutaneous hydrogel-based immunotherapy with food allergen-derived T cell peptides (Phase A) prior to oral (OIT) or sublingual (SLIT) immunotherapy with intact food allergens (Phase B) . Subcutaneous immunotherapy with allergen- derived peptides is performed first to decrease disease- promoting effector T cells and to increase tolerance-promoting regulatory T cells (Tregs) , thereby re-directing the T cell status towards tolerance (Phase A) . OIT or sublingual immunotherapy with intact food allergens is performed thereafter to induce the generation of protective allergen- specific antibodies and to further enhance the development of a tolerogenic T and B cell status (Phase B) . This novel combination provides several important advantages in that a) Phase A modifies the allergic immune status towards tolerance without anaphylactic risks for food allergic patients since peptides are unable to cross- link IgE, b) Phase A minimizes adverse side effects of subsequent OIT or sublingual immunotherapy, and c) Phase A enhances the therapeutic efficacy of subsequent OIT or sublingual immunotherapy significantly. In another embodiment, the present invention discloses the novel application of tolerance-promoting amounts of synthetic oligodeoxynucleotides (ODN) with CpG or GpC or GpG motifs as adjuvant in hydrogel compositions for enhancement of the therapeutic efficacy of peptide immunotherapy in Phase A. In another embodiment, the present invention discloses the application of tolerance-promoting anionic PLGA spheres as an alternative adjuvant in hydrogel compositions for Phase A.

In another embodiment, the present invention discloses the application of find-me molecules in hydrogel compositions for Phase A for attracting peripheral antigen-presenting cells

(APCs) including dendritic cells (DCs) and macrophages to the site of the injected hydrogel in order to enhance phagocytosis of hydrogel-embedded components by antigen-presenting cells

(APC) . In another embodiment, the present invention discloses the application of phosphatidyl-L-serine (PS) -presenting liposomes (PS-liposomes) in hydrogel compositions for Phase A in order to enhance phagocytosis and subsequent presentation of short food allergen-derived T cell peptides by antigen-presenting cells (APCs) . PS- liposomes allow direct targeting of dendritic cells and macrophages due to the surface-exposed eat-me signal phosphatidyl-L-serine . In addition, PS-liposomes mediate tolerance-promoting effects since they mimic the tolerance- promoting effects of apoptotic cells.

In another embodiment, the present invention discloses allergen-derived T cell peptides which are suitable for Phase A immunotherapy. Preferred are T cell peptides which are derived from allergens in cow' s milk, hen ' s egg , peanut , tree nuts, wheat, soy, fish, and shellfish.

In another embodiment, the present invention discloses thermosensitive hydrogels which are suitable for subcutaneous administration and sustained local delivery of hydrogel- embedded components for peptide immunotherapy in Phase A. Preferred thermosensitive hydrogels are injectable in situ- forming gel systems which a) undergo a sol-gel-sol transition, preferably forming a free flowing sol at room temperature and a non- flowing gel at body temperature, b) can serve as depot for sufficient quantities of above listed components, c) allow the release of sufficient quantities of the embedded components over a prolonged period of at least 2 to 3 days, d) are chemically and physically compatible with all embedded components, and e) are biodegradable. Preferred biodegradable polymers approved by the FDA and used in clinical trials, include but are not limited to poly (D, L-lactic acid), poly (lactic-co-glycolic acid) (PLGA) , and copolymers of L- lactide and D,L-lactide such as PLGA-PEG-PLGA copolymers. In another embodiment, the present invention discloses therapeutically effective doses of active substances and adjuvants in hydrogel compositions for peptide immunotherapy in Phase A including CpG-ODN, plain PLGA spheres, the immune modulators vitamin D3 derivative calcipotriol and glucocorticoids including dexamethasone phosphate, allergen- derived T cell peptides, and peptide-loaded PS-liposomes .

In another embodiment, the present invention discloses pharmaceutical formulations of hydrogel compositions for peptide immunotherapy in Phase A.

In another embodiment, the present invention discloses several methods for the first subcutaneous hydrogel-based immunotherapeutic step with food allergen-derived T cell epitope-containing peptides (T cell peptides) in Phase A including a) PS- liposomal approaches using tolerance-promoting ODN, b) PS-liposomal approaches using PLGA spheres as alternative tolerance-promoting adjuvant, c) PS-liposomal approaches using tolerance-promoting immune modulators, d) PS- liposomal approaches using a combination of tolerance- promoting immune modulators and adjuvants, e) PS-liposomal approaches without CpG-ODN or other adjuvants, and f) non- liposomal approaches.

In another embodiment, the present invention discloses useful biomarkers for the induction of T cell-mediated tolerance by peptide immunotherapy in Phase A. Based on these biomarkers, reasonable induction of T cell-mediated tolerance by peptide immunotherapy in Phase A is considered to be achieved if at least 50% of CD4+ T cells express IL-10, c-Maf or LAG-3 , and the percentage of PD-1⁺ cells and TIGIT+ cells in the CD4+ T cell population has increased significantly.

In still another embodiment, the present invention discloses therapeutic protocols for a combined Phase A and Phase B immunotherapy of food allergy including an escalating dose protocol in Phase A or an identical dose protocol in Phase A, and subsequent OIT or SLIT protocols in Phase B including an escalating up-dosing phase and a subsequent maintenance phase. Specific preferred embodiments of the present invention will become evident from the following more detailed description and the claims.

DESCRIPTION OF THE DRAWINGS

Figure 1. Experimental design for the evaluation of the tolerance-inducing efficacy of hydrogel compositions containing CpG-ODN for Fel d 1-specific immunotherapy using a murine acute airway allergy model. Sensitization is performed by 3 successive intraperitoneal (IP) injections of 10 μg Fel d 1 (natural LoTox Fel d 1) with 500 μg Al(OH)₃ in 200 μΐ PBS, at days 0, 14 and 28. A control group receives 3 successive IP injections of 500 g Al(OH)3 in 200 μΐ PBS. Specific immunotherapy is performed by 3 successive subcutaneous (SC) injections (at days 42, 56 and 70) . Three groups of mice are compared. Group I: Treatment with 200 μΐ hydrogel composition of Example 16 comprising a) 15% w/v PLGA-PEG- PLGA, b) 20 μς class B CpG-ODN 1826 (approx. 3.1 nmol), c) 10 ]ig Fel d 1, and d) 0.05 nmol ATP and 0.05 nmol UTP (the concentration of both nucleotides in the composition is 250 nM) (Hydrogel + Find Me) . Group II (allergy group) : Treatment with 200 μΐ PBS via IP injections (Allergy) . Group III (control group) : Treatment with 200 μΐ PBS via IP injections (Control) . Challenge is performed by nasal instillation (NI) with 5 μg Fel d 1 in 50 μΐ PBS on days 83, 84 and 85. Control mice receive only PBS during the nasal instillations. On day 86, serum immunoglobulin profiles are determined, the airway hyperreactivity of the mice is tested by flexiVent analysis upon methacholine challenges, then BALF analyses are performed.

Figure 2. Analysis of immunoglobulin profiles in serum. Mice are bled at day 86 and analysed for Fel d 1-specific IgE, Fel d 1-specific IgA, and Fel d 1-specific IgGl by ELISA. Plates are coated with Fel d 1 in 100 μΐ 0.1 M NaHC0₃ for 6 h at 37°C, followed by blocking with 200 μΐ 3% BSA in PBS , pH 7.4, for 2 h at 37°C. After washing, 100 μΐ of 1:40 serum dilutions with PBS, pH 7.4, containing 1% BSA are incubated overnight at 4°C. The amount of bound antibody is analyzed using horseradish peroxidise-conjugated antibodies with specificity for murine heavy chain classes (IgE, IgGA, and IgGl) . Analysis is performed at 405 nm in a microplate autoreader .

Figure 3. Assessment of airway resistance to inhaled methacholine .

Three to five mice of each group are analyzed for airway resistance (opposition to flow caused by the forces of friction, defined as the ratio of driving pressure to the rate of air flow) to inhaled methacholine. A detailed description of the procedure for the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada) is provided by McGovern et al . (2013) .

Figure 4. Assessment of airway compliance to inhaled methacholine .

Three to five mice of each group are analyzed for airway compliance (a measure of the ease of expansion of the lungs, determined by pulmonary volume and elasticity) to inhaled methacholine using the forced oscillation technique.

Figure 5. TH2 cytokine pattern in the bronchoalveolar lavage fluid. The lungs from three to five mice of each group are lavaged in situ with three 3 successive washes: first with 700 μΐ PBS- BSA-protease inhibitor to collect cells and cytokines, then 2 times with 700 μΐ PBS only to collect the rest of the cells. The BAL is centrifuged and the cytokine supernatant is profiled using a panel of cytokines including IL-4, IL-5, IL- IL-13, IL-17, IFN-Y and TNF- . Figure 6. TH1 cytokine pattern in the bronchoalveolar lavage fluid.

For details see figure 5.

Figure 7. Eosinophils in the bronchoalveolar lavage fluid.

For the collection of cells from BAL samples see figure 5. The cells are analyzed by fluorescence flow cytometry. For these analyses, BAL samples are washed in phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin and 0.1% Na 3. Aliquots containing 10⁴ to 10⁵ cells are incubated with 100 μΐ of appropriately diluted antibodies for 30 min at 4°C. After staining, the cells are washed twice with the above PBS solution, and relative fluorescence intensities are determined on a -decade log scale by flow cytometric analysis using a FACScan (Beeton Dickinson) .

Figure 8. Analysis of PLGA-PEG-PLGA copolymer by gel permeation chromatography (GPC) . For details see Example 1.

Figure 9. Concentration-dependent gelling temperature of PLGA- PEG-PLGA copolymer. For details see Example 1.

Figure 10. Effect of PS-liposomes on the gelation characteristics of PLGA-PEG-PLGA copolymers. For details see Example 6.

Figure 11. Release of PS-liposomes from PLGA-PEG-PLGA hydrogels. For details see Example 7.

Figure 12: Release of ATP from PLGA-PEG-PLGA hydrogels. For details see Example 8. Figure 13. Release of liposome-complexed oligodeoxynucleotides from PLGA-PEG-PLGA hydrogels. For details see Example 9.

Figure 14. Schematic outline of the experiment in Example 13.

DETAILED DESCRIPTION OP THE INVENTION The mechanisms underlying successful oral immunotherapy (OIT) with food allergens and induction of long-term tolerance are not fully understood. Successful desensitization includes an increase in Treg cells and food-specific IgG4 antibodies, an initial increase followed by a decrease in food-specific IgE, a decrease in the number and reactivity of both mast cells and basophils (early desensitization effect) , and a reduction in skin prick test (for a review, see Cavkaytar et al . , 2014).

Long-term tolerance is thought to be induced by a major change in allergen-specific T cells, with a decrease in the proportion of IL-4-secreting Th2 cells and a concomitant increase in IL- 10-producing T regulatory cells (for a review, see Cavkaytar et al . , 2014) . As demonstrated in a recent study, OIT for peanut allergy results in the induction of Treg cells with enhanced suppressive and chemotactic functions and epigenetic changes within the FOXP3 locus of these Treg cells (Syed et al., 2014). Furthermore, expansion and affinity maturation of allergen- specific memory B cells during OIT suggest also a potential role of these cells in tolerance acquisition (Patil et al . , 2016). Sublingual food allergen immunotherapy (Food SLIT) mimics some of the immune changes seen with OIT. SLIT for food allergy can result in decreased titrated skin prick test and specific IgE levels, with associated allergen-specific increases in IgG4 (for a review, see Vazquez-Ortiz and Turner, 2016) . Based on these observations, both humoral and cellular responses as well as innate and adaptive immune responses seem to play a role in successful OIT and sublingual food allergen immunotherapy (Food SLIT) . Therefore, novel therapeutic approaches are needed to address the different arms of the immune system. In one embodiment, the present invention discloses a novel approach for the treatment of food allergy by combining subcutaneous hydrogel-based immunotherapy with food allergen- derived T cell epitope-containing peptides (Phase A) and OIT or sublingual immunotherapy with intact food allergens (Phase B) . Subcutaneous immunotherapy with allergen-derived peptides is performed first to decrease disease-promoting effector T cells and to increase tolerance-promoting regulatory T cells (Tregs) , thereby re-directing the T cell status towards tolerance (Phase A) . OIT or sublingual immunotherapy with intact food allergens is performed thereafter to induce the generation of protective allergen-specific antibodies and to further enhance the development of a tolerogenic T and B cell status (Phase B) . OIT or sublingual immunotherapy with intact food allergens in Phase B is performed as described in the literature (for reviews, see Uyenphuong and Burks, 2014; Vazquez-Ortiz and Turner, 2016) .

This novel combination provides several important advantages : a) The first immunotherapeutic step modifies the allergic immune status towards tolerance without anaphylactic risks for food allergic patients since peptides are unable to cross- link IgE and, therefore, are unable to activate mast cells. b) Due to the induction of tolerance-promoting Tregs by the first immunotherapeutic step with allergen-derived peptides, the therapeutic efficacy of subsequent OIT or sublingual immunotherapy is significantly enhanced. This concept is supported by a recent experimental animal study. Using a murine ovalbumin (OVA) -induced food allergy model, the therapeutic efficacy of oral immunotherapy with OVA could be improved significantly by concomitant application of regulatory T cell- inducer kakkonto, a traditional Japanese herbal medicine (Nagata et al., 2017). c) The increased presence of allergen-specific Tregs during the second oral or sublingual immunotherapeutic step provides another important advantage in that adverse side effects of OIT or sublingual immunotherapy are minimized. d) Furthermore, a T cell status that is already re-directed towards tolerance is likely to allow a shorter up-dosing phase which will reduce costs and provides faster relief for food allergic patients.

For the design of an efficient peptide immunotherapeutic approach in Phase A, however, there is an additional need in the field. Recent clinical studies have demonstrated that the limited success of current peptide-based immunotherapies is most likely a consequence of a) poor cellular uptake of short T cell peptides by antigen-presenting cells (APCs) and b) the lack of a suitable adjuvant capable of supporting the induction of tolerance. The present invention discloses novel technologies which provide both significantly improved cellular uptake of short T cell peptides by APCs and simultaneous efficient tolerogenic priming of such APCs by tolerance-promoting adjuvants. In one embodiment, the present invention discloses the novel application of tolerance-promoting amounts of synthetic oligodeoxynucleotides (ODN) with CpG or GpC or GpG motifs as adjuvant in hydrogel compositions for Phase A.

In another embodiment, the present invention discloses the application of tolerance-promoting anionic PLGA spheres as an alternative adjuvant in hydrogel compositions for Phase A. In still another embodiment, the present invention discloses the application of find-me molecules in hydrogel compositions for Phase A for attracting peripheral antigen-presenting cells (APCs) including dendritic cells (DCs) and macrophages to the site of the injected hydrogel in order to enhance phagocytosis of hydrogel-embedded components by antigen-presenting cells (APC) .

For the enhancement of phagocytosis and subsequent presentation of short food allergen-derived T cell peptides by APCs, the present invention discloses also the application of phosphatidyl-L-serine (PS) -presenting liposomes (PS-liposomes) in hydrogel compositions for Phase A. PS-liposomes allow direct targeting of dendritic cells and macrophages due to the surface-exposed eat-me signal phosphatidyl-L-serine . In addition, PS-liposomes mediate tolerance-promoting effects since they mimic the tolerance-promoting effects of apoptotic cells. PS-liposomes have been shown to inhibit the maturation of dendritic cells and to enhance their secretion of antiinflammatory cytokines. 1. Enhancement of peptide-based immunotherapy in Phase A by tolerogenic amounts of synthetic ODN

In one embodiment, the present invention discloses the application of tolerance-promoting amounts of synthetic oligodeoxynucleotides (ODN) with CpG or GpC or GpG motifs as adjuvant in hydrogel compositions for enhancement of the therapeutic efficacy of peptide immunotherapy in Phase A.

Promotion of tolerance by CpG-ODN. It has been demonstrated that CpG-ODN not only act as immune stimulatory agents (Hemmi et al., 2000) but can also induce strong immune suppression depending on the route of administration (Wingender et al . , 2006) and the quantity of administered CpG-ODN (Volpi et al . , 2013). The suppressive effect is mediated by indoleamine 2,3- dioxygenase 1 (IDOl) (Mellor et al . , 2005; Fallarino and Puccetti, 2006) as indicated by the observation that CpG-ODN induced T cell suppression could be abrogated by 1-methyl- tryptophan (1-MT) , an inhibitor of IDO. Studies with a set of knockout mice demonstrated that the CpG- ODN-induced immune suppression is dependent on TLR9 stimulation (Wingender et al . , 2006). Further studies revealed a previously undescribed role for TRIF and TRAF6 proteins in TLR9 signaling. At high doses of CpG-rich oligodeoxynucleotides, association of TLR9, TRIF and TRAF6 leads to activation of the alternate (noncanonical) pathway of NF- Β signaling and the induction of IRF3- and TGF- β-dependent immune-suppressive tryptophan catabolism (Volpi et al . , 2012; Volpi et al., 2013). In vivo, the TLR9-TRIF circuit and not the TLR9-MyD88 signaling is required for CpG-mediated immunosuppression.

However, application of high doses of CpG-ODN poses toxicity problems as demonstrated for CpG-ODN 7909, a synthetic 24mer single stranded ODN ( 5¹ -TCGTCGTTTTGTCGTTTTGTCGTT-3 ' ) containing 4 unmethylated CpG motifs (Jahresdorfer et al . , 2005) with a phosphorothioate backbone resistant to degradation by DNAse (class B ODN) . In a phase I study with patients suffering from chronic lymphocyte leukemia (CLL) , a single intravenous dose of CpG 7909 was well tolerated with no clinical effects and no significant toxicity up to 1.05 mg/kg. A single subcutaneous dose of CpG 7909 had a maximum tolerated dose of 0.45 mg/kg with dose limiting toxicity of myalgia and constitutional effects (Zent et al . , 2012). Subcutaneous administration of this dose level resulted in an increase in activated T cells, associated with local inflammation at the site of injection and in draining lymph nodes as has been observed in other trials of CpG 7909 (Kim et al., 2010). Both i.v. and s.c. therapy resulted in changes in natural killer ( ) cells and T cells consistent with systemic immune activation and cytokine-induced immune activation (Zent et al . , 2012) .

These clinical studies demonstrate that even maximum tolerated dose levels of CpG-ODN induce activation of the immune system via the TLR9-MyD88 pathway and not induction of tolerance via the TLR9-TRIF pathway. Based on the data of Volpi et al. (2013) , however, dendritic cells require a significant higher concentration of CpG-ODN in order to activate tolerogenic TLR9-TRIF signalling. Therefore, novel technologies are required to modulate human macrophages and DCs with CpG-ODN in a tolerogenic manner without toxicological side effects.

The present invention solves this problem by using a hydrogel- based technology for subcutaneous administration of CpG-ODN in combination with a technology for attraction of antigen- presenting cells to the subcutaneously injected hydrogel composition by hydrogel-embedded find-me signals. Thereby, the tolerance- inducing concentration of CpG-ODN can be reduced to an extent that does not exceed the maximum tolerated dose . Furthermore, encapsulation of CpG-ODN together with food allergen-derived T cell peptides in phosphatidylserine- liposomes (PS- liposomes) allows direct targeting of macrophages and dendritic cells by the tolerance-promoting eat-me signal phosphatidyl-L-serine on the surface of PS- liposomes, which further reduces the tolerance- inducing concentration of CpG-ODN significantly.

First proof of concept studies demonstrate the tolerance- promoting effects of hydrogel-embeddded CpG-ODN in a murine model of cat allergy (see figure 1 for the experimental design) . Subcutaneous treatment of Feld d 1-allergic mice with a 15% w/v PLGA-PEG-PLGA hydrogel composition containing 20 g class B CpG-ODN 1826 (approx. 3.1 nmol) , 10 g Fel d 1, and the find-me molecules ATP (0.05 nmol) and UTP (0.05 nmol), efficiently induced tolerance as indicated by analyses of serum immunoglobulins, airway responsiveness to inhaled methacholine, and cytokines in bronchoalveolar lavage (BAL) fluids . In serum, Fel d 1-specific IgE and Fel d 1-specific IgGl decreased significantly after 3 successive subcutaneous injections at days 42, 56 and 70 (figure 2) , indicating the successful induction of tolerance since IgE and IgGl are good markers of an allergic TH2 response in mice (Adel -Patient et al. , 2000) .

Analysis of airway responsiveness to inhaled methacholine showed that both airway resistance (figure 3) and airway compliance (figure 4) were back to baseline after subcutaneous immunotherapy. Last not least, BAL analyses revealed that levels of the TH2 cytokines IL-5 and IL-13 decreased significantly after immunotherapy (figure 5) which is also in accordance with a successful induction of tolerance. Furthermore, the decrease of IL-17 to baseline levels (figure 6) points to the induction of tolerance- inducing regulatory T cells, since recent studies support a hypothesis that a reciprocal relationship between regulatory T cells and TH17 differentiation pathway may exist (Bettelli et al., 2006). The effect of immunotherapy on IF -γ, the principal TH1 effector cytokine, is difficult to evaluate since significant variations in the Bal levels of this cytokines are observed in control animals, allergic animals and therapeutically treated animals (figure 6) . The increased BAL level of TNF- in therapeutically treated mice (figure 6) is due to the fact that high doses of CpG-ODN elicit TNF-a- dependent toxicity in rodents. Rodents express TLR9 in monocyte/macrophage lineage cells as well as in plasmacytoid DCs (pDCs) and B cells, whereas in humans B cells are the principal TLR9 -expressing cells (Campbell et al . , 2009) . Important is also the decreased BAL level of eosinophils after immunotherapy (figure 7) . Since the accumulation of eosinophils in BAL fluids is associated with asthma and allergy, the significant decrease of eosinophils in BAL fluids after immunotherapy points to a successful induction of tolerance .

Suitable synthetic CpG-ODN for the present invention. Currently used synthetic CpG-ODN differ from microbial DNA in that they have a partially or completely phosphorothioated backbone instead of the typical phosphodiester backbone and a poly G tail at the 3' end, 5¹ end, or both. The phosphorothioated backbone modification protects the ODN from being degraded by nucleases in the body and poly G tails enhances cellular uptake due the formation of intermolecular tetrads resulting in high molecular weight aggregates.

Based on their sequence, secondary structure and effects on human peripheral blood mononuclear cells, different classes of synthetic CpG-ODN have been defined (for a review, see Vollmer and rieg, 2009) . Class A CpG-ODN contain a central palindromic phosphodiester CpG sequence and a phosphorothioate-modified 3' poly-G tail. Class B CpG-ODN are 18-28mer linear oligodeoxynucelotides . They contain a fully nuclease-resistant phosphorothioated backbone with one or more 6mer CpG motifs. The optimal motif is GTCGTT in human and GACGTT in mouse. Class C ODN combine features of both classes A and B. They contain a complete phosphorothioate backbone and a CpG-containing palindromic motif.

For the method of the present invention, all three classes of CpG-ODN are suitable. Although class A ODNs are rapidly degraded in vivo with a half-life of nearly 5 to 10 min, they are also applicable for the method of the present invention if they are protected by encapsulation in PS-liposomes . Furthermore, for liposome-based approaches cellular uptake- enhancing poly G tails are not required. In addition, ODNs with one or more CpG motifs are suitable for the present invention which are fully nuclease-susceptible if they are protected by encapsulation in PS- liposomes . Promotion of tolerance by GpC-ODN. GpC oligodeoxynucleotides (GpC-ODN) have also the ability to modulate dendritic cells (DCs) in a tolerogenic manner via TLR7/TRIF-mediated signaling events (Volpi et al . , 2012).

Physiologically, TLR7 recognizes and responds to viral ssRNA through a signal transduction pathway leading to both induction of type I IF s, typically involved in virus elimination—and differentiation of DCs (Kawai and Akira, 2006) . It is well documented that TLR7 activation by ssRNA is mainly MyD88 dependent. However, TLR7 is also capable of mediating opposite functional effects, depending on the ligand nature and experimental setting, resulting either in Thl7-type responses in humans (Yu et al . , 2010) or in inhibition of Thl7 responses via induction of lL-10 (Vultaggio et al . , 2011) .

As demonstrated in a recent study, synthetic GpC-ODN are capable to confer highly suppressive activity on mouse and human splenic plasmacytoid dendritic cells (pDCs) via the TLR7-TRIF pathway (Volpi et al . , 2012). In this study, GpC-ODN 1826 (5' -TCCATGAGCTTCCTAAGCT -3 ' ) and 1668 (5'-

TCCATGAGCTTCCTGATGC -3 ' ) selectively conferred suppressive properties on pDCs, contingent on functional indoleamine 2,3- dioxygenase 1 (IDOl) . The induction of IDOl by these GpC-ODN was depended on autocrine TGF-β and alternate (noncanonical) NF-kB transcriptional activity. The downstream response culminating in IDOl induction, required TLR7/TIR domain- containing adapter inducing IFN- β (TRIF) -mediated signaling events (Volpi et al . , 2012). Induction of IDO by these GpC- containing ODN could also be demonstrated in human dendritic cells, allowing those cells to assist FOXP3+ T cell generation in vitro (Volpi et al . , 2012) .

For the method of the present invention, all GpC-ODN are suitable which are capable of inducing tolerance via the TLR7- TRIF pathway. Preferred GpC-ODN include but are not limited to GpC-ODN 1826 (5 ' -TCCATGAGCTTCCTAAGCTT-3 ' ) and GpC-ODN 1668 ( 5 ' - CCATGAGCTTCCTGATGC -3 ' ) both of which have been shown to confer suppressive properties on human splenic plasmacytoid dendritic cells (pDCs) , contingent on functional indoleamine 2, 3-dioxygenase 1 (Volpi et al., 2012).

Promotion of tolerance by GpG-ODN

In still another embodiment, the present invention discloses synthetic GpG oligodeoxynucleotides (GpG-ODN) capable of attenuating experimental immune diseases. Preferred GpG-ODN include but are not limited to GpG-ODN 5- TGACTGTGAAGGTTAGAGATGA-3 which has been demonstrated to suppress the severity of experimental autoimmune encephalomyelitis, to downregulate autoreactive Thl and to induce an altered isotype switching of autoreactive B cell to a protective IgGl isotype (Ho et al . , 2003). Furthermore, this GpG-ODN delayed the onset and attenuated the severity of lupus nephritis by antagonizing and blocking the activation of multiple TLRs (Graham et al . , 2010).

2. Enhancement of peptide-based immunotherapy in Phase A by tolerance-promoting anionic PLGA spheres

In another embodiment, the present invention discloses hydrogel-embedded anionic poly ( lactic-co-glycolic acid) spheres (PLGA spheres) as tolerance-promoting adjuvant for peptide immunotherapy in Phase A. PLGA belongs to a family of biodegradable polymers that are highly biocompatible. In water, PLGA biodegrades by hydrolysis of its ester linkages, which leads to metabolite monomers, lactic acid and glycolic acid. Because these two monomers are endogenous and easily metabolized by the body via the Krebs cycle, a minimal systemic toxicity is associated with the use of PLGA for drug delivery (for a review, see Makadia and Siegel, 2011) or biomaterial applications (for a review, see Gentile et al . , 2014) . Due to these properties, PLGA has been approved by the US FDA and European Medicine Agency (EMA) in various drug delivery systems for humans.

Several experimental studies have demonstrated that autoantigen- or allergen-loaded PLGA nanoparticles induce tolerogenic effects in animals ((Kim et al . , 2002; Scholl et al . , 2004; Keij zer et al . , 2011; for a review, see De Souza Reboucas et al . , 2012). OVA- loaded PLGA nanospheres (400 nm in size) were demonstrated to enhance retinaldehyde dehydrogenase enzyme activity in cervical lymph node-derived CDllc+ dendritic cells (DCs) (Keijzer et al . , 2013). The metabolic conversion of retinol to retinoic acid in DCs, which drives Treg differentiation, requires retinaldehyde dehydrogenase. Using these DCs after treatment with OVA-containing PLGA nanospheres for in vitro activation of OVA-specific T cells, an enhanced induction of tolerance-promoting CD4 (+) FoxP3 (+) T- cells via a retinoic acid and TGF-β dependent mechanism was observed (Keijzer et al . , 2013).

As demonstrated in several studies, even plain PLGA spheres have the capacity to promote tolerance (for a review, see Getts et al . , 2015) . For example, in the study of Kim et al . (2002) , oral tolerance induction in mice with collagen- induced arthritis (CIA) was investigated using PLGA nanoparticles (approx. 300 nm in size) with or without entrapped collagen II. The data show that both plain anionic PLGA spheres and collagen II-loaded anionic PLGA spheres exert tolerogenic effects, although the tolerogenic effects induced by plain PLGA spheres were less profound than those induced by collagen II-loaded PLGA nanoparticles . The observation that even plain PLGA spheres are tolerance- promoting was substantiated by the study of Jilek et al . (2004) . Using a murine phospholipase A2-induced allergy model, the authors have demonstrated that plain PLGA microspheres (size range of 1-10 μπι) can induce tolerance for as long as 6 months post-sensitization. In this study, the potential of plain cationic (polyethylenimine-coated) and anionic PLGA microspheres for modulating the allergic response triggered by bee venom phospholipase A2 (PLA2) sensitization over a 3 -week time course was analyzed. Both cationic and anionic plain PLGA microspheres were capable to downregulate allergic responses in this model and mice were even protected against an otherwise lethal challenge with phospholipase A2. For both microsphere formulations, the peak of PLA2 -specific IgG2a antibody (reflecting Thl bias) preceded that of IgGl (Th2 marker) . PLA2-specific IgGl and IgG2a production turned out to be 2 times higher using cationic microspheres compared to anionic microspheres. The initial Thl bias observed at the level of immunoglobulin was sustained transiently, followed by a mixed Thl/Th2 response supported by a similar expression of IL-4 and IF -γ. During the whole time frame a sustained production of IL-10 was observed in both groups. These data suggest a dual mechanism that does initially rely on a Th2 to Thl immune deviation and then on IL-10 -mediated suppression which can explain the protective effect against anaphylaxis. Preferred surface charge of PLGA nanoparticles. For the promotion of tolerance according to the method of the present invention, anionic PLGA nanoparticles are preferred. The preference for anionic PLGA nanospheres is supported by the observation that cationic nanoparticles can alter mitochondrial and endoplasmic reticulum function, triggering the production of reactive oxygen species and pro- inflammatory cytokines, as well as cell death (Hawang et al . , 2015; Chiu et al., 2015; Xia et al . , 2008), whereas anionic nanoparticles have been associated with little to no toxicity (Park et al . , 2011) .

Most important, however, cationic nanoparticles are likely to induce Thl-type immune responses rather than tolerance- promoting effects. Positively charged nanoparticles seem to be able to escape from lysosomes after being internalized and exhibit perinuclear localization, whereas negatively and neutrally charged nanoparticles prefer to colocalize with lysosomes (for a review, see Danhier et al., 2012) . Escape of cationic nanoparticles from lysosomes into the cytosol, however, is likely to favor presentation of allergenic or auto-immunogenic proteins adsorbed onto the surface of these cationic nanoparticles in the hydrogel via MHC class I, which leads to Thl-type immune responses. This assumption is supported by the observation that positively charged antigen- loaded nanoparticles are significantly more effective at stimulating Thl responses after either intradermal or mucosal (pulmonary) inoculation, whereas anionic particles stimulate T and B cell responses poorly under similar conditions (Fromen et al . , 2015; Bal et al . , 2011). This observation is further substantiated by a recent study in which the uptake of cationic particles by dendritic cells has been demonstrated to regulate positive costimulatory molecules in these cells (Koppolu and Zaharoff., 2013). In vitro, professional antigen- presenting cells such as DCs have been reported to engulf cationic microspheres in low acidic phagosomes, resulting in slow degradation that favors expression of the surface marker CD86, and production of TNF- Oi (Jilek et al., 2004b; Thiele et al . , 2003) . Preferred size and shape of PLGA particles. For the method of the present invention, PLGA particles with a size of more than 100 nm are suitable. Nanoparticles with a size of less than 100 nm are not suitable since they tend to interact with cellular organelles, including the mitochondria and nucleus, and these interactions can trigger cellular respiratory and gene toxicity in cells (Park et al . , 2011). This risk is reduced with increasing NP size, presumably because larger NPs tend to initiate phagocytosis, which effectively isolates particles from the more sensitive cytoplasmic environment (for a review, see Getts et al., 2015) .

Preferred for the method of the present invention are PLGA particles with a size of more than 200 nm. Small particles with a size of less than 200 nm may drain freely from subcutaneous sites of application to local lymph nodes (Xiang et al., 2013), whereas particles with a size of more than 200 nm must be phagocytosed by local phagocytes before transport to the draining lymph nodes . In order to create a local tolerogenic micro-environment at the injection site of the hydrogel composition, phagocytosis of released PLGA particles close to the injected hydrogel composition is preferred. Thereby, PLGA-particle- induced secretion of the antiinflammatory cytokines TGF-β and IL-10 by macrophages and DCs will create a tolerogenic environment in proximity of the injection site for the presentation of allergenic or auto- immunogenic proteins upon their release from the injected hydrogel composition.

Preferred for the method of the present invention are PLGA spheres. For antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs) , uptake is also impacted by shape, with spherical nanoparticles having more favorable uptake kinetics than rodshaped nanoparticles, irrespective of nanoparticle size (Champion and Mitragotri, 2006) . PLGA nanospheres with a size of 300-500 nm are rapidly taken up by local APCs as demonstrated in a recent study (Nicolete et al., 2011). The data of this study show that plain PLGA nanospheres with an average size of 389 nm are rapidly phagocytosed by murine macrophages, whereas the uptake of plain PLGA microspheres with an average size of 6.5 μπι by is slow.

Based on these observations, spherical PLGA particles with a size ranging from 200 nm to 10 μπι are applicable for the method of the present invention. Preferred are PLGA spheres with a size ranging from 300 nm to 1 μπι.

3. Enhancement of peptide-based immunotherapy in

Phase A by find-me molecules for attraction of APC

In another embodiment, the present invention discloses hydrogel-embedded find-me molecules capable of attracting APCs to the site of subcutaneously injected hydrogel compositions for peptide immunotherapy in Phase A.

Effective local uptake of hydrogel-embedded allergens, autoantigens or peptides derived thereof and empty or loaded PS- liposome by dendritic cells and macrophages in subcutaneous tissues requires the presence of released find-me signals. For example, apoptotic cells are quickly recognized and removed by phagocytes, which can be either neighboring healthy cells or professional phagocytes recruited to the site of apoptotic cell death. Phagocytes are extremely efficient in sensing and detecting the dying cells at the earliest stages of apoptosis. This is a result of find-me signals released from apoptotic and the exposure of eat-me signals on apoptotic cells.

In the recent past, several find-me signals released from apoptotic cells have been identified (for a review, see Ravichandran, 2011) . In one embodiment, the present invention utilizes these find-me signals capable of triggering effective local phagocytosis including but not limited to fractalkine (chemokine CXC3CL1) , lysophosphatidylcholine (LPC) , sphingosine-1-phosphate (SIP) and the nucleotides ATP and UTP. Both nucleotides have been described as non-redundant find-me signals released by apoptotic cells (Elliott et al . , 2009). UTP acts only on P2Y- family receptors and UDP produced via degradation of released UTP by extracellular enzymes has been shown to promote phagocytic activity via the P2Y6 nucleotide receptor. In contrast, ATP acts on P2X- and P2Y- family receptors, whereas ADP produced via degradation of released ATP by extracellular enzymes acts only on P2Y-family receptors (for a review, see Gombault et al . , 2013).

Preferred for the method of the present invention are find-me signals which can be embedded in thermosensitive hydrogels in sufficient quantities for efficient chemotaxis, which are chemically and physically compatible with such hydrogels, and which can be released from such hydrogels over a period of one to two days in a way that resembles the release of find-me from apoptotic cells. In one embodiment, only one find-me signal selected from ATP, UTP, ADP or UDP is employed. In a preferred embodiment, equimolar quantities of ATP and UTP are employed as find-me signals. Using a transwell migration assay, both nucleotides have been demonstrated to effect maximal migration of phagocytes at a concentration of about 100 nM (Elliott et al . , 2009) .

For the method of the present invention the release of ATP and/or UTP from subcutaneously injected hydrogel compositions at nanomolar concentrations is a strict requirement. At nanomolar concentrations, ATP activates receptors such as P2Y2 (EC₅₀ <1 μΜ) which mediate chemotaxis. Furthermore, it has been demonstrated that at nanomolar concentrations ATP exerts antiinflammatory effects by suppressing the secretion of pro- inflammatory cytokines and promoting the release of of antiinflammatory cytokines (for a review, see Chekeni and Ravichandran, 2011) . In contrast, at concentrations of more than 1 μΜ ATP acts as a danger signal via activation of the nucleotide receptor P2X7 (EC50 >100 μΜ) , which in turn leads to activation of the inflammasone and release of pro- inflammatory cytokines ( ono and Rock, 2008) .

4. Enhancement of peptide-based immunotherapy in Phase A by phosphatidyl^- serine liposomes In another embodiment, the present invention discloses hydrogel-embedded phosphatidyl-L-serine (PS) -presenting liposomes for peptide immunotherapy in Phase A which are a) capable of targeting antigen-presenting cells (APC) including dendritic cells (DCs) and macrophages, and b) capable of inducing tolerogenic effects in these cells after release from subcutaneously injected hydrogel compositions.

PS-mediated targeting of APC. In viable cells, PS is kept exclusively on the inner leaflet of the lipid bilayer via ATP- dependent translocases . In apoptotic cells, the concentration of PS on the outer leaflet of the lipid bilayer is estimated to increase by more than 280 -fold within only a few hours after induction of apoptosis. PS exposed on the surface of apoptotic cells represents the key signal for triggering phagocytosis by macrophages (for a review, see Hochreiter- Hufford and Ravichandran, 2013) . This is indicated by the observation that phagocytosis of apoptotic lymphocytes by macrophages was inhibited in a dose-dependent manner by PS and PS-containing liposomes, but not by liposomes containing other anionic phospholipids including phosphatidyl-D-serine (Fadok et al. , 1992) .

Clearance of apoptotic cells is initiated when phosphatidyl-L- serine-enriched membranes engage phosphatidyl-L-serine receptors. Two types of phosphatidyl-L-serine receptor have been described, those that bind the phospholipid directly and those that use bridging molecules to associate with it. Direct phosphatidyl-L-serine-binding receptors include T cell immunoglobulin and mucin receptor (TIM) proteins (TIM1, TIM3 and TIM4) ; the CD300 family members CD300a and CD300f (also known as CLM1) ; and the seven-transmembrane spanning receptors brain-specific angiogenesis inhibitor 1 (BAI1) , stabilin 1 and receptor for advanced glycosylation end products (RAGE) . The phosphatidyl-L-serine-bridging molecule MFGE8 is used for apoptotic clearance through νβ3 and νβ5 integrins, which are indirect phosphatidyl-L-serine receptors. Similarly, GAS6 and protein S (PROS) are the bridging molecules that link the indirect phosphatidylserine receptors of the tyrosine protein kinase receptor 3 (TYR03 ) -AXL-MER (TAM) family to phosphatidyl-L-serine to mediate apoptotic clearance (for a review, see Amara and Mercer, 2015) .

Liposomes containing phosphatidyl-L-serine (PS-liposomes) mimic apoptotic cells and are engulfed by phagocytes including macrophages, dendritic cells and microglia (e.g., Wu et al . , 2010) . Therefore, components encapsulated in PS-liposomes are effectively targeted to APC, resulting in optimal presentation of encapsulated T cell peptides by these APC, and optimal interaction of encapsulated ODN with endosomal/lysosomal Toll- like receptor molecules.

PS-mediated tolerogenic effects in APC. Phosphatidyl-L-serine (PS) -presenting liposomes are also capable of inducing tolerance-promoting antigen-presenting cells (APC) including dendritic cells (DCs) and macrophages. Phagocytosis of apoptotic cells inhibits the maturation of dendritic cells, their secretion of pro- inflammatory cytokines (Steinman et al . , 2000; Chen et al . , 2004), and there is evidence that PS-dependent phagocytosis of apoptotic cells transforms macrophages to an anti- inflammatory phenotype (Fadok et al._f 1998; Hoffmann et al . , 2001; Huynh et al . , 2002) .

PS-liposomes mimic the tolerogenic effects of apoptotic cells in macrophages and microglia via induction and secretion of anti-inflammatory mediators including TGF-βΙ and PGE2 (Otsuka et al., 2004; Zhang et al. , 2006b). As demonstrated in a recent study, expression of MyD88, which is essential for the signal transduction in lipopolysaccharide (LPS) stimulation, is suppressed when macrophages are treated with PS-liposomes (Tagasugi et al . , 2013).

Moreover, PS-liposomes inhibit the maturation of dendritic cells and enhance their secretion of anti-inflammatory cytokines. For example, large unilamellar PS-liposomes have been shown to inhibit the up-regulation of HLA-ABC, HLA-DR, CD80, CD86, CD40, and CD83 , as well as the production of IL- 12p70 in human DCs in response to LPS. DCs exposed to PS had diminished capacity to stimulate allogeneic T cell proliferation and to activate IF -y-producing CD4(+) T cells (Chen et al . , 2004). A corresponding effect of PS-liposomes on the maturation and immuno-stimulatory functions of murine DCs in response to l-chloro-2, 4-dinitrobenze (DNCB) was observed in the study of Shi et al . (2007) . After treatment with PS- liposomes, murine DCs displayed reduced expression of MHC II, CD80, CD86 and CD40, but increased programmed death ligand-1 (PD-L1 and PD-L2) , increased IL-10 and inhibited IL-12 cytokine production. DCs treated with PS-liposomes exhibited normal endocytic function, but their ability to stimulate allogeneic T cells was reduced, similar to immature dendritic cell. Treatment of DCs with PS-liposomes also suppressed DNCB- induced CD4(+) T cell proliferation and IFN-γ production. Furthermore, DCs treated with PS-liposomes enhanced the ratio of CD4( +) CD25 (high) Foxp3 ( +) T cells to CD4 ( +) T cells and PD- 1 expression on CD4(+) T cells.

In vivo effects of PS and PS-liposomes have also been reported For example, intravenous injection of PS in mice has been demonstrated to reduce both T cell-dependent and T cell- independent antibody production (Ponzin et al . , 1989) and intravenous injection of PS prior to the injection of bacterial LPS has been shown to reduce serum TNF-a levels (Monastra and Bruni, 1992) . Comparable effects have been observed also after injection of PS-liposomes. For example, intraperitoneal injection of PS-liposomes ameliorated the course of extrinsic allergic encephalitis induced in mice by immunization with myelin basic protein (Monastra et al . , 1993) . Also, PS-liposomes specifically inhibited responses in mice to antigens as determined by decreased draining lymph node tissue mass, reduced numbers of total leukocytes and antigen-specific CD4+ T cells and decreased levels of antigen- specific IgG in blood. TGF-β appears to play a critical role in this inhibition, as the inhibitory effects of PS-liposomes were reversed by in vivo administration of anti-TGF-β antibodies (Hoffmann et al . , 2005). A recent study demonstrated that after uptake of PS-liposomes in vitro and in vivo, macrophages secrete high levels of the anti-inflammatory cytokines TGF-β and IL-10 and upregulate the expression of CD206 (mannose receptor C type 1; MRC1) , concomitant with downregulation of pro-inflammatory markers such as TNF-a and the surface marker CD86 (Harel-Adar et al . , 2011). CD86 (also known as B7-2) is a protein expressed on antigen-presenting cells that provides costimulatory signals necessary for T cell activation and survival. In subsequent experiments, the authors demonstrated that modulation of cardiac macrophages by PS-liposomes improves infarct repair. Injection of PS- liposomes via the femoral vein in a rat model of acute myocardial infarction promoted angiogenesis and prevented ventricular dilatation and remodeling (Harel-Adar et al . , 2011) .

Preparation of loaded PS-liposomes . In another embodiment, the present invention discloses the preparation of PS-liposomes. PS-liposomes include but are not limited to PS-liposomes which contain a) one or more allergen- -derived T cell peptides, b) one or more allergen- -derived T cell peptides, and CpG-ODN or GpC-ODN or GpG-ODN, or c) one or more allergen- -derived T cell peptides, CpG-ODN or GpC-ODN or GpG-ODN, and one or more tolerance-promoting immune modulators.

PS-Liposomes are thermodynamically stable vesicles composed of one or more concentric lipid bilayers. PS-liposomes have two compartments, an aqueous central core, and a lipophilic area within the lipid bilayer. Hydrophilic molecules such as hydrophilic T cell peptides, oligodeoxynucleotides (ODN) with CpG or GpC or GpG motifs and the hydrophilic tolerance- promoting immune modulator dexamethasone phosphate can be encapsulated in the inner aqueous volume, while hydrophobic molecules such as the tolerance-promoting immune modulator calcipotriol can be incorporated into the lipid bilayer.

A variety of liposomal carrier systems have been used for encapsulating hydrophilic and incorporating hydrophobic molecules including conventional liposomes, ethosomes, niosomes, and elastic liposomes (the initial formulation approach being termed transferosomes) . Preferred for the method of the present invention are conventional PS-containing liposomes .

Conventional PS-liposomes are composed of PS and other phospholipids such as phosphatidylcholine (PC) from soybean or egg yolk, with or without cholesterol (CH) . The most common applied PS is derived from bovine brain, but other PS sources and synthetic PS preparations such as 1-palmitoyl-2-oleyl-sr.- 3 -glycerophospho-L-serine or 1, 2-distearoyl--sn-3-glycero- phospho-L-serine are also suitable. Cholesterol is used to stabilize the system. For the preparation of conventional PS- liposomes various lipid mixtures containing PS, PC and, optionally, CH are applicable including but not limited to lipid mixtures comprising molar ratios of PS: PC of 30:70 (Gilbreath et al . , 1985) or 50:50 (Fadok et al . , 2001) for PS- liposomes without cholesterol and molar ratios of PS:PC:CH of 30:30:40 (Hoffmann et al., 2005; Harel-Adar et al., 2011) for PS- liposomes with cholesterol. As demonstrated recently, however, efficient uptake by macrophages can also be achieved with liposomes containing PS as low as 6 mol% (Geelen et al . , 2012) .

Conventional PS-liposomes can be prepared in several ways. Most frequently, a film hydration method is employed, where a thin layer of lipid is deposited on the walls of a container by evaporation of a volatile solvent. An aqueous solution, optionally containing the molecule to be entrapped, is added at a temperature above the transition temperature of the lipids, resulting in the formation of multilamellar vesicles. These systems contain several lipid bilayers surrounding the aqueous core. Further processing by sonication or filter extrusion generates large unilamellar vesicles (LUV, 1-5 μιτι diameter), or small unilamellar vesicles (LUV, 0.1-0.5 μτη diameter) . PS-liposomes with 1 μτη diameter have been shown to trigger efficient uptake by macrophages (Harel-Adar et al . , 2011) .

Preparation of hydrogel formulations with PS-liposomes. For sustained local delivery of PS-liposomes at the site of allergen presentation according to the method of the present invention, different liposome-hydrogel formulations are suitable (e.g., Xing et al., 2014; Nie et al., 2011). In a preferred embodiment, thermosensitive hydrogels comprising PLGA-PEG-PLGA are employed for sustained local delivery of PS-liposomes at the site of autoantigen or allergen presentation. As demonstrated in a recent study, liposome-loaded PLGA-PEG-PLGA hydrogels exhibit still reversible thermosensitive properties (Xing et al . , 2014). However, its sol-gel and gel-precipitate transition temperatures decreased with increasing liposome concentrations. Most important, the particle size of free liposomes and those released from the PLGA-PEG-PLGA hydrogels were found to be close regardless of particle size of liposomes, indicating that the liposomes were stable in the hydrogel and intact liposomes could be released (Xing et al . , 2014) . 5. Enhancement of peptide-based immunotherapy in Phase A by tolerance-promoting immune modulators

In another embodiment, the present invention discloses tolerance-promoting immune modulators for encapsulation or incorporation in hydrogel-embedded PS-liposomes or for direct embedment into hydrogels.

Although direct embedment of tolerance-promoting immune modulators into hydrogels does not provide for maximal effective tolerizing immune modulation of APCs, it allows extension of the tolerizing effect of these hydrogel-embedded immune modulators also to other immune cells in addition to APCs. For example, dexamethasone phosphate (DexP) exerts its immunosuppressive and anti-inflammatory activity not only in dendritic cells and macrophages, but also in various other immune cells including T cells, eosinophils, mast cells, and neutrophils. In all of these cells, DexP and its receptor regulate a complex network that inhibits a variety of inflammatory pathways via several mechanisms such as expression of anti-inflammatory proteins, induction and inhibition of cytokines, inhibition of inflammatory receptors, reduced expression of adhesion molecules, and the induction of Tregs (for reviews, see Barnes, 2001; Rhen and Cidlowski, 2005; Longui, 2007; Robinson, 2010) . Suitable tolerance-promoting immune modulators for the method of the present invention are those which are capable of a) inducing tolerogenic APCs (including DCs and macrophages) and tolerance-promoting Tregs, b) suppressing effector T cell- mediated responses, and c) inhibiting pro-inflammatory cytokines and pro- inflammatory complement factors at the site of autoantigen or allergen presentation. Such immune modulators are listed in patent applications EP16001276 and EP3095440 and include but are not limited to a) vitamin D3 and selected analogs such as calcipotriol, b) glucocorticoids such as dexamethasone phosphate, c) Janus kinase inhibitors, also known as JAK inhibitors or jakinibs, such as tofacitinib, d) antagonistic cytokine molecules such as I1-4/IL-13 muteins, e) salicylate-based therapeutics for the inhibition of T FR1- mediated pathways such as acetylsalicylic acid and salicylic acid, f) peptide-based complement inhibitors such as the 13- residue cyclic peptide (H-I [CWQDWGHHRC] T-NH₂ or peptidomimetica-based complement inhibitors such as cyclic PMX53, and g) aptamer-based inhibitors of pro- inflammatory cytokines . Preferred tolerance-promoting immune modulators for direct embedment in a hydrogel include those which are soluble in the aqueous environment of the hydrogel such as the hydrophilic glucocorticoid dexamethasone phosphate and the hydrophilic citrate derivative of tofacitinib. Preferred are also those tolerance-promoting immune modulators which are characterized by a short serum half-life, since such immune modulators are removed rapidly from circulation, thereby minimizing potential systemic side effects. Glucocorticoids exhibiting a short plasma half-life (ranging between 30 min and 2 hours) and a relatively short biological half-life of 8-12 hours include cortisone and hydrocortisone, glucocorticoids exhibiting an intermediate plasma half-life (ranging between 2.5 and 5 hours) and an intermediate biological half-life of 18-36 hours include prednisone, prednisolone, methylprednisolone and triamcinolone, and glucocorticoids exhibiting a long plasma half-life (up to 5 hours) and a relatively long biological half-life of 36-54 hours include dexamethasone , betamethasone and fludrocortisone (for a review, see Longui, 2007) . However, most important for the method of the present invention is the glucocorticoid potency, which defines the capacity to elevate glycemia and which is proportional to the anti-inflammatory potency. In this respect cortisone and hydrocortisone exhibit a rather low potency, prednisone, prednisolone, methylprednisolone and triamcinolone an intermediate potency, whereas dexamethasone and betamethasone exhibit a rather high potency, which is 25- 30 -fold higher than that of cortisone or hydrocortisone (for a review, see Longui, 2007) . Therefore, glucocorticoids with a high anti-inflammatory potency are preferred for the method of the present invention despite their relatively long plasma and biological half-lives.

Hydrogel-embedded tofacitinib citrate formulations offer the possibility to use tofacitinib as supporting tolerance- promoting immune modulator at relatively low concentrations which provide therapeutic efficacy at the site of allergen or autoantigen presentation but minimize potential tofacitinib- mediated adverse effects. The hydrogel serves as sustained delivery system for tofacitinib at the site of allergen or autoantigen presentation and, thereby, eliminates peak serum levels of tofacitinib as observed after oral administrations. Furthermore, the short in vivo half-life of tofacitinib (2-3 h) minimizes systemic effects of tofacitinib upon diffusion and transport away from injected hydrogel-based compositions. Lowering the dose of tofacitinib without affecting its therapeutic efficacy is important since currently administered doses of tofacitinib for systemic treatment of arthritis is associated with potential serious adverse effects. For example, in a phase 3, randomized, double-blind, placebo- controlled clinical study with rheumatoid arthritis patients, there was an increased rate of serious infections during oral therapy with tofacitinib (at doses of 5 mg or 10 mg twice daily) as compared with placebo (Fleischmann et al . , 2012a). In months 0 to 3 of treatment, a total of 330 patients (54.1%) had 701 adverse events, with similar frequencies across the patients treated with 5 mg or 10 mg tofacitinib. The most common of these adverse events were upper respiratory tract infection, headache, and diarrhea. Twelve patients (2.0%) discontinued the study drug during this period of treatment owing to serious adverse events, with similar frequencies across the two tofacitinib-treated patient groups (Fleischmann et al . , 2012a). More serious immunologic and hematological adverse effects have also been noted resulting in lymphopenia, neutropenia, anemia, and increased risk of cancer and infection ('https://pubchem.ncbi.nlm.nih.gov) .

Preferred tolerance-promoting immune modulators for encapsulation or incorporation into PS-liposomes include but are not limited to those a) which can be encapsulated in the aqueous compartment of liposomes such as the hydrophilic glucocorticoid derivative dexamethasone phosphate and the hydrophilic tofacitinib citrate, and b) which can be incorporated into the lipid layer of liposomes such as the lipophilic vitamin D3 derivative calcipotriol.

Calcipotriol (or calcipotriene) is a synthetic derivative of calcitriol, which has similar VDR binding properties as compared to calcitriol, but has low affinity for the vitamin D binding protein (DBP) (for a review, see Tremezaygues and Reichrath; 2011) . In vivo studies in rats showed that effects of calcipotriol on calcium metabolism are 100-200 times lower as compared to calcitriol while the tolerance-promoting effects of calcipotriol are comparable to those of calcitriol (e.g., Al-Jaderi et al . , 2013) . The half-life of calcipotriol in circulation is measured in minutes (Kragballe, 1995) . The rate of clearance (serum half-life of 4 min in rats) is approximately 140 times higher for calcipotriol than for calcitriol. Furthermore, calcipotriol is rapidly metabolized and effects of the metabolites have been demonstrated to be 100 times weaker than those of the parent compound ( issmeyer and Binderup, 1991) .

6. Suitable allergen-derived T cell peptides for peptide immunotherapy in Phase A

Suitable for the method of the present invention are T cell peptides derived from various food allergens including but not limited to those derived from apple (Mai d allergens) , almond (Pru du allergens) , avocardo (Pers a allergens) , banana (Mus a allergens) , Brazil nut (Ber e allergens) , buckwheat (Fag e allergens) , carrot (Dau c allergens) , carp (Cyp c allergens) , cashew nut (Ana 0 allergens) , celery (Api g allergens) , cherry (Pru av allergens) , chestnut (Cas s allergens) , cod (Gad c allergens), cow's milk (Bos d allergens), frog (Ran e allergens), hazelnut (Cor a allergens), hen's egg (Gal d allergens) , kiwi (Act d allergens) , lobster (Pan s allergens and Horn a allergens) , melon (Cue m allergens) , maize (zea m allergens) , mussel (Pern v allergens) , mustard (Sin a allergens) , peanut (Ara h allergens) , peach (Pru p allergens) , pear (Pyr c allergens) , pistachio (Pis v allergens) , redfish (Seb m allergens) , rice (Ory s allergens) , rye (Sec c allergens) , salmon (Sal s allergens) , scallop (Cha m allergens) , sesame (Ses I allergens) , shrimp (Met e allergens and Pen m allergens) , soybean (Gly m allergens) , swordfish (Xip g allergens) , tomato (Lyc e allergens) , walnut (Jug r allergens) , and wheat (Tri a allergens) . For a review, see Valenta et al . (2015) . The foods most often associated with food allergy in the U.S. include cow's milk, hen's egg, peanut, tree nut, wheat, soy, fish, and shellfish. The majority of allergens from these and other sources have been cloned and structurally characterized including for example Api g 1-4, Ara h 1-7, Bos d 5, Cha f 1, Cor a 1.0401, Cha m ?, Dau c 1, Gly m 1 and 3, Horn a 1, Jug r 1-2, Jug r 19 kDa, Mai d 1, Pen a 1, Pern v ?, Pers a 1, Pru av 1 and 4, Pyr c 1 and 4-5, Sal s 1, Sin a 1, and wheat glutenins and gliadins (for a review, see Lorenz et al . (2001) . Molecular cloning, characterization and sequencing of these and other allergens allows the synthesis of nested sets of overlapping peptides covering the full allergen sequence for identification of immunodominant CD4+ T cell epitopes within major, clinically relevant allergens. In a preferred embodiment, for identification of all potential T cell epitopes, allergen-specific T cell lines and clones from large patient cohorts are screened for reactivity against overlapping synthetic peptides spanning the entire sequence of the allergen molecule, each usually 15 to 20 amino acids long with overlaps from 5 amino acids upwards. Core epitopes within T cell-reactive peptides are mapped subsequently using peptide sets truncated from the N- and C-termini, typically revealing eight or nine residue core epitopes for CD4+ T cells. Optimal T cell stimulation often requires longer sequences including flanking residues to stabilize the HLA-peptide-TCR complex and improve expression of peptide on the antigen presenting cell surface. Consistent with naturally processed peptides eluted from HLA class II molecules, candidate peptides for inclusion in short allergen peptide therapy range from 12-20 residues.

Mapping of T cell epitopes can be performed using peripheral blood mononuclear cells (PBMC) from individuals with the specific allergy of interest, either directly ex vivo, or after enrichment for allergen-specificity as T cell lines (oligoclonal populations) or T cell clones (monoclonal populations) .

The most critical peptides are identified by a range of immunological assays using high-throughput methodologies including flow cytometry with dyes such as carboxyfluorescein diacetate succinimidyl ester (CFSE) to detect proliferating cells by decreased intensity of staining, cytokine capture, and fluorochrome-conjugated HLA class II-peptide tetramers (for a review, see O'Hehir et al., 2016) .

CFSE-based approaches are sensitive for detection of peptide- responsive T cells, particularly when combined with other activation markers such as CD25, but bystander proliferation may reduce specificity (Van Hemelen et al . , 2015). ELISPOT-based approaches can be used for high-throughput screening of PBMC for T cell epitope peptide recognition (e.g., Tye-Din et al . , 2010). Identified T cell epitopes are then validated by screening large patient population cohorts and using rigorous assay design and appropriate statistical methods (e.g., wok et al . , 2010).

HLA-peptide tetrameric complexes are sensitive and specific analytes for identification and characterization of allergen- specific T cells directly ex vivo, but tetramer synthesis is expensive and many HLA class II molecules are not easily isolated for use in tetramers, limiting the HLA-coverage obtainable . Alternatively, in silico algorithms consider thousands of known epitope sequences to predict CD4+ T cell epitopes by- detecting theoretical HLA class II binding motifs within protein sequences (Schulten et al., 2013) [38]. While algorithms provide preliminary guidance cost-effectively, comprehensiveness is limited and HLA-binding motif predictions require validation in functional peripheral blood T cell assays (Van Hemelen et al . , 2015) .

A catalogue of allergen T cell-reactive sites mapped to date is available from The Immune Epitope Database (IEDB) (available from: http://www.immuneepitope.org). Meta-analysis identified 1406 allergen-derived CD4+ T cell epitopes derived from human T cell reactivity (Vaughan et al . , 2010). Despite large numbers, it is estimated that this represents <17 % of all allergens in the International Union of Immunological Societies (IUIS) allergen database (available from: http://www. allergen.org).

Examples of identified T cell epitopes of food allergens include those from cow's milk ( sl -casein, β-lactoglobulin) , peanut (Ara hi and Ara h 2), hen's egg (ovomucoid (Gal d 1) and ovalbumin (Gal d 2) ) , hazelnut (Cor a 1.04), Brazil nut (Ber e 1) , beef (bovine serum albumin) , celery (Api g 1) , walnut (Jug r 2), and shrimp (Met e 1) (Bohle, 2006; Prickett et al . , 2011; Prickett et al . , 2013; Archila et al . , 2015; Wai et al . , 2015; Ramesh et al . , 2016).

T cell epitopes are found scattered throughout an allergen sequence, but consideration of collective properties of the epitopes allows ranking according to dominance to optimize peptide candidates for therapy (Schulten et al . , 2013. Important properties for the method of the present invention include donor and T cell line/clone responder frequency, patterns of reactivity, reproducibility of T cell response and, importantly, ability to induce a response in patient PBMC in large patient cohorts. The ability of T cell epitope peptide candidates for the method of the present invention to show widespread degeneracy of binding to a range of MHC class II molecules is important for targeting genetically diverse patient populations. Since human CD4+ T cells recognize allergen epitopes in the context of particular HLA class II molecules encoded by one of three highly polymorphic loci, HLA-DR, HLA-DP, or HLA-DQ, preferred HLA-binding T cell epitope prediction algorithms include those for HLA-DR, HLA- DP, or HLA-DQ.

7. Thermosensitive hydrogels for peptide immunotherapy in Phase A

In another embodiment, the present invention discloses thermosensitive hydrogels which are suitable for subcutaneous administration and sustained local delivery of hydrogel- embedded components for peptide immunotherapy in Phase A.

Preferred thermosensitive hydrogels are injectable in situ- forming gel systems which a) undergo a sol-gel-sol transition, preferably forming a free flowing sol at room temperature and a non- flowing gel at body temperature, b) can serve as depot for sufficient quantities of above listed components, c) allow the release of sufficient quantities of the embedded components over a prolonged period of at least 2 to 3 days, d) are chemically and physically compatible with all embedded components, and e) are biodegradable.

In a more preferred embodiment of the invention, biodegradable thermogelling hydrogels are used which are composed of FDA- approved biodegradable polymers . Preferred biodegradable polymers approved by the FDA and used in a clinical trial, include but are not limited to poly (D, L-lactic acid), poly (lactic-co-glycolic acid) (PLGA) , and copolymers of L- lactide and D,L-lactide. All FDA approved polymers have been studied extensively for their biocompatibility, toxicology, and degradation kinetics. Furthermore, these polymers have been shown to release embedded therapeutics for several hours up to several weeks in vivo. Useful for the method of the present invention are biodegradable thermogelling block polymers which are based on monomethoxy poly (ethylene glycol) (MPEG) including but not limited to a) diblock copolymers consisting of MPEG and poly ( ε-caprolactone) (PCL) (Hyun et al . , 2007), b) MPEG-jb- (PCL-ran-PLLA) diblock copolymers (Kang et al . , 2010), and c) diblock copolymers consisting of MPEG and PLGA (Peng et al . , 2010) . MPEG copolymers containing PCL provide the advantage that they do not create an acidic environment upon biodegradation in contrast to MPEG copolymers containing PLLA and PLGA (Hyun et al., 2007).

Preferred for the method of the present invention are biodegradable thermogelling triblock polymers including but not limited to a) PLGA-PEG-PLGA (Qiao et al . , 2005), b) PEG- PLGA-PEG (Zhang et al . , 2006a), and c) PEG-PCL-PEG (PECE) (Gong et al . , 2009). Various biodegradable thermogelling triblock polymers made up of PLGA and PEG are disclosed in patent W099/18142. At lower temperatures, hydrogen bonding between hydrophilic PEG segments of the copolymer chains and water molecules dominate in aqueous solutions, resulting in the dissolution of these copolymers in water. As the temperature increases, the hydrogen bonding becomes weaker, while hydrophobic forces of the hydrophobic segments such as PLGA segments are getting stronger, leading to sol-gel transition. PEG, PLGA and PCL are well-known, FDA-approved, biodegradable and biocompatible materials which have been widely used in the biomedical field.

Most preferred for the method of the present invention are biodegradable thermogelling PLGA-PEG-PLGA triblock polymers. Compared to other biodegradable hydrogels, injectable thermo- gelling PLGA-PEG-PLGA polymers possess several advantages including easy preparation, a formulation process which is free of harmful organic solvents (e.g., Qiao et al. 2005), application of building blocks which are approved for parenteral use in humans by the FDA, excellent biocompatibility, and well established procedures for the production of composits comprising liposomes (e.g., Xing et al., 2014). Furthermore, PLGA-PEG-PLGA hydrogels provide another important advantage in that tolerance-interfering Thl- type or Th2-type immune responses are avoided.

In another embodiment, the present invention discloses biodegradable thermogelling polymers which allow modification of their degradation kinetics. Preferred are biodegradable thermogelling polymers for the method of the present invention which maintain their structural integrity for a few days but do not remain in the body for more than a month. Therefore, biodegradable thermogelling polymers which allow modification of their degradation kinetics, are preferred for the method of the present invention. For example, PLLA segments can be incorporated into the PCL segment of MPEG-PCL copolymers, since PLLA provides better accessibility of water to the ester bonds of PLLA which enhances the hydrolytic degradation of the copolymer ( ang et al . , 2010) . In another example, the rate of PLGA-PEG-PLGA hydrogel erosion can be modified by altering the molar ratio of DL-lactide/glycolide in the PLGA segment. The DL-lactide moiety is more hydrophobic than the glycolide moiety. Therefore, by increasing the molar ratio of DL- lactide/glycolide in the PLGA segment of PLGA-PEG-PLGA triblock copolymers, more stable hydrogels are formed due to stronger hydrophobic interactions among the copolymer molecules (Qiao et al. 2005) . 8. Therapeutically effective doses of active components and adjuvants for peptide immunotherapy in Phase A

Determination of a therapeutically effective dose is well within the capability of those skilled in the art. The therapeutically effective dose can be estimated initially in animal models, usually mice, rats, rabbits, dogs, pigs, or non-human primates. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

Dosage regimens may be adjusted to provide the optimum therapeutic response. The quantity of the hydrogel-embedded components depends on the release kinetics of the depot- providing hydrogel and is adjusted to a level that guarantees the continuous release of therapeutically effective doses over a period of at least 2 to 5 days . The quantity of embedded components will vary according to factors such as the weight and the age of the individual, and the ability of the composition to induce an effective immune response in the individual .

The following data of this section provide useful guidelines for the determination of a therapeutically effective dose for those skilled in the art. 8.1. Therapeutically effective doses of CpG-ODN

CpG-ODN have been studied in various experimental models and clinical trials.

Toxicity of CpG-ODN in mice. Using CpG-ODN 1018 (5'- TGACTGTGAACGTTCGAGATGA) , Campbell et al . (2009) have demonstrated that doses up to 2.5 mg/kg (50 ^g/20 g mouse) of this B-class CpG-ODN are applicable in mice (associated with little weight loss in the range of 5%) , whereas a dose of 5 mg/kg (100 μ<3/20 g mouse) of CpG-ODN 1018 resulted in marked pulmonary inflammation in lung tissue sections from C57BL/6 mice harvested 4 days after treatment . The toxicity of CpG-ODN in mice is TLR9-dependent and mediated by TNF- . Based on the differential TLR9 expression patterns in rodents (in monocyte/macrophage lineage cells as well as in plasmacytoid DCs (pDCs) and B cells) versus humans (B cells are the principal TLR9-expressing cells) CpG-ODN elicit TNF-a- dependent toxicity in rodents but not in humans (Campbell et al . , 2009) .

Tolerance-promoting amount of CpG-ODN 1826. Allergic bronchopulmonary aspergillosis is a Th2- sustained allergic condition. In the study of Volpi et al . (2013), the effects of CpG-ODN 182 6 ( 5 ' - CCATGACGTTCCTGACGTT- 3 ' , phosphorothioate- stabilized B-class CpG-ODN) on the hypersensitivity response to Aspergillus antigens in the mouse lung was evaluated. The animals were treated intraperitoneally with 30 μg CpG-ODN 1826 (approx. 5 nmol; M approx. 6059) per mouse twice, on the same days as the first and second administration of A. fumigatus culture filtrate extract. The Th2 -dependent allergic phenotype was greatly attenuated by CpG, which enhanced the production of IL-10, a marker of protective Treg activity in Aspergillus allergy. In the study of Wingender et al . (2006) C57BL/6 mice were immunized by i.v. or s.c. (intra ear pinna) injection of 100 μg OVA alone or in combination with 50 ^g phosphorothioate- stabilized 20-mer CpG-ODN 1668 ( 5 ' -tccatgacgttcctgatgct- 3 ' ) corresponding to 7.86 nmol (MW 6364). Only the systemic application of CpG-ODN resulted in IDO-mediated suppression of T cell expansion and CTL activity in the spleen. Based on these data, induction of IDO in cells of draining lymph nodes via s.c. injection of CpG-ODN requires larger amounts of CpG- ODN than IDO induction in spleen cells via i.v. injection. However, even in spleen cells IDO induction requires i.v. injection of large amounts of CpG-ODN 1668. Smaller doses of i.v. administered CpG-ODN 1668 (0.5 μg and 5 ^g) induced innate immunity as indicated by the finding that these doses drastically reduced the load of adenovirus in the host after i.v. infection with adenovirus expressing OVA.

Tolerance-promoting amount of liposomal CpG-ODN 1826. In the study of Konur et al . (2008), weekly intradermal injection for two weeks of small unilamellar vesicles (SUV) composed of cholesterol (chol) , dilauroyl-phosphatidyl-ethanolamine (DLPE) and dioleoylphosphatidyl-serine (DOPS) at a ratio of 1:1:1, containing both 300 ^g TRP2 peptide and 5 nmol CpG-ODN 1826 (approx. 30 μg) , into the flanks of BL6 mice suppressed antigen-specific T cell responses and diminished lymphocyte numbers (see Fig.2B), whereas intradermal injection of 300 g TRP2 peptide and 5 nmol CpG-ODN 1826 (approx. 30 ^g) as aqueous solution under identical conditions increased antigen- specific T cell responses significantly. Several factors may have contributed to the immunosuppressive effects of the liposomal approach, first of all the high liposomal peptide amount, but also the increased concentration of tolerance-promoting phosphatidylserine (PS) -presenting liposomes and the amount of liposomal CpG-ODN 1826 which was sufficient to attenuate the allergic phenotype in the study of Volpi et al . (2013), and last not least the targeted delivery of the liposomes to antigen-presenting cells via presentation of PS. However, in order to generate tolerogenic effects with liposomal CpG-ODN in the presence of lower peptide quantities, the liposomal CpG-ODN content most likely needs to be increased.

Hydrogel-embedded PS- liposomes containing PO CpG-ODN. Taking the slow release of PS-liposomes from PLGA-PEG-PLGA hydrogels into consideration, administration of PLGA-PEG-PLGA hydrogel- embedded PS- liposomes containing up to 120 μg of PO CpG-ODN/20 g mouse (corresponds to 6.0 mg/kg) appears to be a tolerable dose. This would lead to the release of approx. 12 μg liposomal PO CpG-ODN within the first 12 hours (10% release) , approx. 20 μg liposomal PO CpG-ODN within 24 hours (17% release) , and approx. 40 g liposomal CpG-ODN within 48 hours (35% release) . Intracellular accumulation of phagocytosed CpG- ODN is prevented by using nuclease- susceptible PO CpG-ODN with a regular phosphodiester backbone.

8.2. Therapeutically effective doses of anionic PLGA spheres

PLGA particles have been studied in a variety of animal models in the recent past. Results obtained from these studies provide useful information for the application of plain anionic PLGA nanoparticles in humans.

Studies with plain PLGA particles. In the study of Jilek et al . (2004), 5 mg of plain biodegradable PLGA microspheres (size range: 1-10 μτη) in 100 μΐ 0.25% methocel in nanopure water were administered subcutaneously to mice once a week for three consecutive weeks. Three weeks later, mice were sensitized subcutaneously with 6 doses of 100 ng bee venom phospholipase A2 combined with 1 mg alum given at 2 -week intervals. Over a period of 8.5 months, the resulting immune responses were analyzed. The data demonstrate that plain PLGA microspheres can induce tolerance in mice for as long as 6 months post-sensitization, based on a dual mechanism that does initially rely on a TH2 to TH1 immune deviation and then on IL- 10 -mediated suppression.

Studies with allergen/antigen- loaded PLGA particles. Allergen- or antigen- loaded PLGA particles have been studied in several animal models. For example, in the study of Scholl et al . (2004) Bet v 1-sensitized mice were treated once by subcutaneous administration of PLGA nanoparticles (size range: 470-750 nm; 90% value) containing 20 μg Bet v 1 (16.5 ^g Bet v 1/mg dry weight of polymer) in 170 μΐ PBS/1% Pluronic (11.7 μg Bet v 1/100 μΐ) . Pluronic F-68 was used as stabilizer of the PLGA nanoparticles. Treated mice developed high levels of Bet v 1-specific IgG2a antibodies (Thl response) , whereas IgGl levels (Th2 marker) decreased significantly. Moreover, T cells from treated mice showed IFN-γ (Thl response) and IL-10 production (tolerance-promoting interleukin) . Hydrogel-PLGA particle compositions. US 6,287,588 Bl provides an example of a composition comprising 10 mg of PLGA microspheres loaded with Zn-human growth hormone in 100 μΐ of a 20% PLGA-PEG-PLGA hydrogel in 10 iti HEPES buffer, pH 7.0. This formulation can be injected smoothly using a 24 -gauge needle due to excellent wetting and suspending ability of the PLGA-PEG-PLGA hydrogel.

8.3. Therapeutically effective doses of calcipotriol

Calcipotriol (or calcipotriene) is a synthetic derivative of calcitriol, which has similar VDR binding properties as compared to calcitriol, but has low affinity for the vitamin D binding protein (DBP) (for a review, see Tremezaygues and Reichrath; 2011) . In vivo studies in rats showed that effects of calcipotriol on calcium metabolism are 100-200 times lower as compared to calcitriol while the tolerance-promoting effects of calcipotriol are comparable to those of calcitriol (e.g., Al-Jaderi et al . , 2013).

The half-life of calcipotriol in circulation is measured in minutes (Kragballe, 1995) . The rate of clearance (serum half- life of 4 min in rats) is approximately 140 times higher for calcipotriol than for calcitriol. Furthermore, calcipotriol is rapidly metabolized and effects of the metabolites have been demonstrated to be 100 times weaker than those of the parent compound (Kissmeyer and Binderup, 1991) .

Uptake of calcipotriol. Calcipotriol has been used clinically for more than 10 years for topical treatment of psoriasis without systemic toxicity (for a review, see Plum and DeLuca, 2010) . Clinical studies with radiolabeled ointment indicate that approximately 6% of the applied dose of calcipotriene is absorbed systemically when the ointment is applied topically to psoriasis plaques or 5% when applied to normal skin. In another study, it was found that calcipotriol permeated through all skins to only a limited extent over 2Oh after application but was efficiently retained in all skins at a level at 20h of between 40% (pig) and 60% (rat and mouse) of the applied dose (Li et al . , 2013) . Toxicity studies. Single dose toxicity studies of calcipotriol administered to rats by subcutaneous injection revealed LD50 values of 2.19 mg/kg in males (approx. 440 ^g/200-g male rat) and 2.51 mg/kg in females (approx. 380 g/150-g female rat) (Imaizumi et al . , 1996) . Rats died probably due to the circulatory and renal disturbance. According to this study, no death occurred up to a single s.c. dose of 540 Mg/kg body weight (approx. 81 g/150-g female rat) , and no loss of body weight could be observed two weeks after administration.

Additional toxicity studies have been performed with calcitriol, the dose- limiting hypercalcemic effects of which are 100-200 fold more pronounced than those of calcipotriol.

Mice can become hypercalcemic on day 2 or day 3 at calcitriol doses higher than 750 ng/mouse when administered as a single bolus i.p. (Muindi et al . , 2004). According to this study, however, calcitriol can be administered as a single bolus i.p. injection up to 500 ng/mouse (in 0.2 ml of normal saline) .

Unlike daily calcitriol treatments, single calcitriol doses pose a lower hypocalcemia risk and, therefore, allow administration of higher doses. In other animal studies, mice were treated by a single i.p. injection of 0.1 ml propylene glycol containing 300 ng calcitriol (Cantorna et al . , 1998a), or by a single i.p. injection of 0.1 ml scafflower oil containing up to 400 ng calcitriol (Nashold et al., 2013).

Animal studies . In animal studies, vitamin D molecules and analogs thereof have been used for the treatment of OVA- induced allergy in mice (Ghoreishi et al . , 2009), OVA- induced allergic asthma in mice (Taher et al . , 2008), insulin- dependent diabetes mellitus in NOD mice (Zella et al . , 2003), Lyme arthritis and collagen-induced arthritis in mice (Cantorna et al., 1998b), and experimental autoimmune encephalomyelitis (EAE) in mice (Branisteanu et al . , 1995) . In the allergy model, mice were treated on their shaved dorsal skin with 30 mg/day of calcipotriol ointment (contains 50 μg calcipotriol/g 1.5 pg calcipotriol/30 mg) (Donovex, Leo Pharma) for three days followed by transcutaneous immunization with OVA in the presence of CpG adjuvant. This treatment abolished antigen-specific CD8+ T cell priming and induced CD4+CD5+ Tregs, thereby promoting antigen- specific tolerance (Ghoreishi et al . , 2009).

Additional animal studies have been performed with calcitriol. In a murine model of allergic asthma, OVA-sensitized mice were subjected to allergen-specific immunotherapy by three s.c. injections of 100 pg OVA in the presence of 10 ng calcitriol (corresponding to approx. 1-2 μg calcipotriol) . This treatment significantly inhibited airway hyper-responsiveness and caused a significant reduction of serum OVA-specific IgE levels (Taher et al . , 2008).

In a murine model of type 1 diabetes, a diet containing 50 ng calcitriol/mouse/day (corresponding to approx. 5-10 μg calcipotriol/mouse/day) was administered three times/week. This treatment prevented diabetes onset in NOD mice as of 200 days (Zella et al . , 2003) .

In a murine arthritis model, mice received a daily diet supplemented with 20 ng calcitriol/mouse/day (corresponding to approx. 2-4 μg calcipotriol/mouse/day) . This dose was found to be effective in inhibiting the progression of arthritis without producing hypercalcemia (Cantorna et al . , 1998b).

In a murine EAE model, i.p. injection of 5 μg of calcitriol/kg body weight (200 ng calcitriol/20g mouse; corresponding to approx. 20-40 g calcipotriol/20g mouse) every 2 days prevented the appearance of paralysis in 70% of the treated mice (Branisteanu et al . , 1995)

Liposomal calcipotriol . Calcipotriol is lipophilic and is incorporated into the lipid bilayer of liposomes. Using liposomes made of dimyristoyl -phosphatidylcholine (DMPC) or egg-PC and a molar ratio of calcipotriol (MW 412.6) to lipid of 0.03 to 1, incorporation rates of more than 80% have been reported (Merz and Sternberg, 1994) . Tolerance-inducing dose of hydrogel-embedded PS-liposomes containing calcipotriol . As demonstrated in the study of Ghoreshi et al . (2009), topical treatment of mice with 1.5 g calcipotriol for three days abolished antigen-specific CD8+ T cell priming and induced CD4+CD5+ Tregs, thereby promoting antigen-specific tolerance. Based on the study of Li et al . (2013) , calcipotriol permeates through murine skins to only a limited extent over 20h after application, but is efficiently retained in murine skins at a level of 60% of the applied dose after 2Oh. Thus, transdermal uptake of less than 3 calcipotriol (1 μg over 3 days) into the skin mediated the induction of tolerance in mice. Based on the study of Ghoreshi et al. (2009), subcutaneous administration of hydrogel-embedded 20 g liposomal calcipotriol/mouse will deliver a sufficiently tolerizing quantity of liposomal calcipotriol to APC via PS-liposomes . Release studies with hydrogel-embedded PS-liposomes have demonstrated that 20 μg liposomal calcipotriol embedded in PLGA-PEG-PLGA hydrogels leads to the release of approx. 2.0 μg liposomal calcipotriol within the first 12 hours (10% release), approx. 3.4 ^g liposomal calcipotriol within 24 hours (17% release), and approx. 7.0 μg liposomal calcipotriol within 48 hours (35% release) . These amounts are equivalent to the tolerizing quantity of calcipotriol in the study of Ghoreshi et al . (2009).

On the other hand, administration of hydrogel-embedded 20 μg liposomal calcipotriol/mouse represents a tolerable dose. Calcitriol can be administered as a single bolus i.p. injection up to 500 ng/mouse (Muindi et al . , 2004). Assuming only a 100-times lower effect of calcipotriol on calcium metabolism as compared to calcitriol, 500 ng calcitriol corresponds to 50 g calcipotriol.

8.4. Therapeutically effective doses of glucocorticoids

Most preferred glucocorticoids for the method of the present invention are those which exhibit a high anti- inflammatory potency which is proportional to their glucocorticoid potency established for their capacity to elevate glycemia. Glucocorticoids with a high anti- inflammatory potency include but not limited to dexamethasone and betamethasone (for a review, see Longui, 2007) . However, glucocorticoids with a moderate anti- inflammatory potency such as prednisone, prednisolone, methylprednisolone, and triamcinolone, as well as those with a lower anti-inflammatory potency such as cortisone and hydrocortisone are also applicable for the method of the present invention. A dexamethasone dose of 0.25 mg/m²/day corresponds to 2.5 mg/m²/day of prednisolone and hydrocortisone 10 mg/m²/day (for a review, see Gupta and Bhatia, 2008) .

All of these glucocorticoids have been studied in a variety of animal models and evaluated in clinical trials. For example, in mice dexamethasone has been administered by i.p. injection at doses of 10-40 μg/20-g mouse (0.5-2.0 mg/kg) for 7 days, leading to a 30% decrease in the number of intestinal VDRs (Hirst and Feldman, 1982a) . In rats, dexamethasone has been administered at doses of 0.15-7.5 mg/150-g female rat (1.0- 50.0 mg/kg) for 7 days (Hirst and Feldman, 1982b).

In clinical trials, different glucocorticoids and varying combinations thereof have been evaluated. For example, several randomized controlled trials comparing dexamethasone with prednisolone in the treatment of acute asthma exacerbations in children have been published. One study compared emergency department (ED) treatment with an initial dose of oral prednisolone 2 mg/kg (max. 60 mg) followed by 1 mg/kg daily for four days with oral dexamethasone 0.6 mg/kg (max. 16 mg) daily for two days (Qureshi et al . , 2001) .

Another study compared ED treatment with an initial dose of oral prednisolone 1 mg/kg (max. 30 mg) followed by 1 mg/kg twice daily for five days with a single dose of oral dexamethasone 0.6 mg/kg (max. 18 mg) (Altamimi et al . , 2006) . Still another study compared ED treatment with a single dose of prednisolone 2 mg/kg (max. 80 mg) followed by 1 mg/kg (max. 30 mg) twice daily for five days with a single dose of 0.6 mg/kg oral dexamethasone (max. 16 mg) followed by one dose of 0.6 mg/kg oral dexamethasone to take the next day (Greenberg et al. , 2008) .

Liposomal dexamethasone phosphate (DexP) . In the study of Hegeman et al. (2011), liposomal DexP has been administered i.v. at a concentration of 11.2 DexP/20-g mouse (adult male C57BL/6 mice with a body weight of 20-24g) . The DexP to lipid ratio was 28 μg DexP/μιηοΙ lipid (comprising PC, cholesterol and PE at a molar ration of 55:40:5) . In another study (Anderson et al . , 2010), liposomal DexP has been administered i.v. at a concentration of 1 mg DexP/kg body weight for 3 days, corresponding to three injections of 20 μg DexP/20-g mouse. The DexP to lipid ratio was 40 μg DexP/μπιοΙ lipid (comprising DPPC, DPPG and cholesterol at a molar ration of 50:10:40) .

A more than three-fold higher amount of liposomal DexP (3.75 mg liposomal DexP/kg body weight, corresponding to 75 μg/20-g mouse or 563 μg/l50-g female rat) has been administerd i.v to rats 6, 24 and 48 hours after induction of antigen- induced arthritis (US20060147511A1) .

8.5. Therapeutically effective doses of tofacitinib

Tofacitinib has been approved by FDA to treat adults with moderately to severely active rheumatoid arthritis (RA) who have had an inadequate response to, or who are intolerant of, methotrexate .

Using the method of the present invention, the application of tofacitinib as tolerance-promoting immune modulator is restricted to a few days until its release from injected hydrogels is completed. Therefore, short-term clinical studies with tofacitinib provide valuable information about therapeutically effective doses of tofacitinib.

Phase II study of Kremer et al . (2009) over 6 weeks. Patients (n = 264) were randomized equally to receive placebo, 5 mg, 15 mg or 30 mg of tofacitinib twice daily for 6 weeks, and were followed up for an additional 6 weeks after treatment. By week 6, the American College of Rheumatology 20% improvement criteria (ACR20) response rates were 70.5%, 81.2% and 76.8% in the 5 mg, 15 mg and 30 mg twice-daily groups, respectively, compared with 29.2% in the placebo group. However, the infection rate in both the 15 mg and the 30 mg twice daily groups was 30.4% (versus 26.2% in the placebo group) . Phase II study of Fleischmann et al . (2012b) over 24 weeks. In this 24 -week, double-blind, phase lib study, patients with RA (n = 384) were randomized to receive placebo, tofacitinib at

1, 3, 5, 10 or 15 mg administered orally twice a day.

Treatment with tofacitinib at a dose of ≥3 mg twice a day resulted in a rapid response with significant efficacy compared with placebo, as indicated by the primary end point (ACR20 response at week 12), achieved in 39.2% (3 mg) , 59.2% (5 mg) , 70.5% (10 mg) and 71.9% (15 mg) in the tofacitinib group compared with 22.0% of patients receiving placebo. Preferred concentration of tofacitinib at the site of allergen or autoantigen presentation. On oral administration of tofacitinib 5 or 10 mg twice a day, serum levels of approximately 100-300 nM are achieved, and such therapeutic levels are known to last for 4-6 h ( ubo et al . , 2014) . Based on the different inhibitory potency of tofacitinib for the four members of the Janus kinase family in enzyme assays (Flanagan et al . , 2010; Meyer et al . , 2010), lower concentrations of tofacitinib inhibit signalling via JA 1 and JAK3 (IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, and IF -γ) , whereas higher concentrations of tofacitinib inhibit also signalling via JAK1 and TY 2 (IL-10, IL-12, IL-20, IL-22, IL- 23 and IFNs) , and via JAKl, JAK2 and TYK2 (IL-6, IL-11, IL-27 and G-CSF) .

For the induction of tolerance according to the method of the present invention it is important, to inhibit JAK1/JAK3- mediated signalling, but to avoid inhibition of JAK1/TYK2- mediated signalling since IL-10 is essential for the induction of tolerance .

Since partial inhibition of JAK1/TYK2 -mediated signaling is likely to occur at a concentration of 100-300 nM, for the method of the present invention a therapeutic serum level of tofacitinib of 50-100 nM is preferred.

8.6. Therapeutically effective doses of allergen-derived T cell peptides

T cell reactive sites have been mapped for many allergens including food allergens and are catalogued in The Immune Epitope Database (www.iedb.org). A meta-analysis of this database confirmed 1406 allergen-derived CD4+ T cell epitopes based on human T cell reactivity (for a review, see Prickett et al . , 2015) . Some of these T cell reactive sites (allergen- derived T cell peptides) have been evaluated for the treatment of allergy in murine and human studies which provide useful guidelines .

Murine study with food allergen-derived T cell peptides. In a murine egg allergy model (Yang et al . , 2010), BALB/c mice were sensitized by oral gavage at a dose of 1.0 mg of OVA and 10 μg of cholera toxin twice per week for a period of 4 weeks, and subsequently treated at week 6 by subcutaneous injection of 100 μg of a single OVA-derived T cell peptide or 300 g of a mixture of three OVA-derived T cell peptides (each peptide: 100 /g) in PBS three times weekly for a period of 3 weeks. After oral challenge at week 10 with 20.0 mg of OVA, analyses revealed in mice treated with the three OVA-derived peptides significantly decreased anaphylactic responses (two- to threefold difference to placebo-treated mice) , accompanied by lower serum histamine and OVA-specific IgE levels. Further analyses of splenocytes obtained from treated mice, revealed a T helper type 1-biased response (increase of IFN-γ, IL-12p40) with a diminished T helper type 2-reponse (decrease of IL-4, IL-5 and IL-13) .A similar cytokine expression profile was determined in intestinal tissues, accompanied by a pronounced mR A expression of regulatory molecules TGF-β and forkhead box transcription factor 3 (FOXP3) . Based on these data, 300 μg of subcutaneously injected T cell peptides are sufficient to induce in mice local repressive mechanisms mediated by subsets of regulatory T cells.

Human studies with allergen-derived T cell peptides. While data from human studies with food allergen-derived T cell peptides are not available, T cell peptides derived from cat allergen Fel d 1 and bee venom allergen Ap m 1 (phospholipase A2) have been used for immunotherapeutic studies.

In one study with cat-allergic subjects (Norman et al . , 1996), subcutaneously administered Fel d 1 peptides comprised an equimolar mixture of two long 27 amino acid sequences from the two chains of Fel d 1 and contained multiple T cell epitopes. After four subcutaneous injections of the peptide mixture (750 ^g/ml of each peptide) , clinical benefit was demonstrable at 6 weeks but adverse events included nasal congestion, flushing, pruritus and chest tightness for minutes to hours after peptide delivery. The possibility of retained conformational structure within the long peptides and IgE-mediated reactivity likely explained the early adverse events . In a subsequent clinical trial with allergen-derived T cell peptides, shorter peptides from allergenic proteins were used immunotherapy for bee venom allergy since they are unable to cross-link IgE and possess minimal inflammatory potential. In this study (Miiller et al . , 1998), bee venom-allergic patients were treated by subcutaneous injection with three T cell epitope peptides of the major bee venom allergen phospholipase A2 (PLA2) , each of 11-18 amino acid residues. The treatment included injection, in successive doses, of 0.1 μg of an equimolar mixture consisting of the three peptides, followed by 1 μg, 3 μg, 6 μg, 12 μg_f 25 g, 50 μg, and then three times 100 μg in weekly intervals, resulting in a cumulative dose of 397.1 μg of the peptide mixture. Consistent with linked suppression, clinical efficacy was achieved to a subsequent PLA2 challenge and live whole bee sting challenge. However, some subjects developed peptide-specific IgE and two subjects developed local erythema with occasional palmar pruritus. These findings emphasize the importance of using the shortest possible peptides comprising T cell epitopes to minimize the risk of IgE-mediated adverse events.

Another important consideration is the quantity of subcutaneously or intradermally administered T cell peptides. Since administration of T cell peptides at a concentration of 750 g caused several adverse events, including late asthma responses due to a flare of released cytokines from peptide- stimulated T cells, the quantity of T cell peptides was tenfold reduced to approx. 75 μg (for a review, see Prickett et al., 2015). Early-phase studies with short T cell peptides, typically 13-17 amino acids in length, administered at this concentration via the intradermal route into non- inflamed skin demonstrated safety and clinical efficacy (Worm et al . , 2013) .

The shortness of the peptides avoids any potential for IgE cross-linking or inflammatory cell activation and careful dose adjustment seems to avoid the late asthma response observed earlier with higher concentrations of peptides.

8.7. Therapeutically effective doses of peptide-loaded phosphatidylserine (PS) -liposomes

Various clinical studies with liposomes as drug delivery system have been performed and numerous liposomal formulations are on the market for a wide spectrum of diseases (for a review, see Goyal et al . , 2005). These applications provide valuable guidelines of therapeutically effective liposomal doses .

Animal studies with PS-liposomes . While PS- liposomes have not been evaluated in humans, PS-liposomes have been studied in a variety of animal models in the recent past. Results obtained from these studies provide useful information for the application of PS-liposomes in humans.

For example, using BALB mice (body weight approx. 20 g) , the effect of subcutaneously (s.c.) administered PS-liposomes on immune responses upon subsequent injection of ovalbumin (OVA) or keyhole limpet hemocyanine (KLH) in complete Freund' s adjuvant (CFA) has been investigated (Hoffman et al . , 2005). In this study, PS-containing liposomes comprising a 30:30:40 molar ratio of PS to PC to cholesterol, were first injected s.c. in one flank of 6-8 weeks old BALB mice (100 μΐ, corresponding to 0.5 mg of total lipid) , followed after one hour by another s.c. injection of 150 μΐ of an emulsion containing 50 μg of OVA or 150 μg of KLH in CFA in the same region. As evident from the data, subcutaneously administered PS-liposomes specifically inhibited responses to antigens. Numbers of total leukocytes and antigen-specific CD4+ T cells were reduced as well as the level of antigen-specific IgG in blood. There was also a decrease of draining lymph node tissue mass and the size of germinal centers in spleen and lymph nodes (Hoffman et al . , 2005).

Using a rat model of acute myocardial infarction (MI) in another study, the effects of intraveneously (i.v.) administered PS-liposomes on cardiac macrophages has been investigated (Harel-Adar et al., 2011). In this study, 150 μΐ of a 0.06 M solution of PS-liposomes comprising a 30:30:40 (1:1:1.33) molar ratio of PS to PC to cholesterol (9 μπιοΐ lipid or approx. 5.4 mg lipid; compare Example 1.1), were injected i.v. through the femoral vein of female Sprague- Dawley rats (body weight approx. 150 g) 48 hours after MI induction. As evident from the data, i.v. administered PS- containing liposomes promoted angiogenesis, preservation of small scars, and prevented ventricular dilatation and remodeling. Following uptake of PS- liposomes by macrophages, the cells secreted high levels of the anti- inflammatory cytokines TGF-β and IL-10 and upregulated the expression of the mannose receptor CD206, concomitant with downregulation of proinflammatory markers such as T F- and the surface marker CD86 (Harel-Adar et al . , 2011).

Loading of PS-liposomes with allergen-derived peptides. Methods for encapsulation of peptides in liposomes are known to the person skilled in the art. Various formulations of liposomes containing encapsulated peptides have been prepared (for a review, see Mohan et al . , 2015) and studied in a variety of animal models (e.g., Konur et al . , 2008; Belogurov et al. , 2013) .

Encapsulation efficiencies of peptides into liposomes depend on the hydrophilicity or hydrophobicity of the peptides, the lipid composition and the molar ratio of lipid to peptide. In one study, a peptide derived from the tyrosinase-related protein 2 (TRP2 : SVYDFFVWL) was encapsulated in liposomes at a lipid-to-TRP2 molar ratio of 20:1. Using a lipid mixture composed of cholesterol (CH) , dilauroylphosphatidyl- ethanolamine (PE) and dioleylphosphatidyl-serine (PS) at a molar ratio of 1:1:1, respectively, the encapsulation efficiency of TRP2 ranged between 20-50% (Konur et al . , 2008). In another study, three peptides derived from the myelin basic protein (MBP 46-62, MBP 124-139; MBP 147-170) were encapsulated in mannosylated liposomes (prepared from egg phosphatidyl-choline and 1% monomannosyl dioleyl glycerol) at a lipid-to-peptide weight ratio of 330:1. Using this high lipid-to-peptide ratio, the resulting small unilamellar liposomes entrapped more than 90% of the initial peptide amount (Belogurov et al . , 2013). Same considerations apply to allergen-derived T cell peptides.

Therapeutic concentrations of hydrogel-embedded PS-liposome containing encapsulated allergen-derived peptides. The effective dose of PS-liposome-encapsulated peptides in PLGA- PEG-PLGA hydrogels depends on the injection protocol in Phase A. Calculations of therapeutically effective concentrations of hydrogel-embedded liposome-encapsulated allergen-derived peptides are based on the data of clinical trials with short allergen-derived T cell peptides (for a review, see Prickett et al . , 2015). In any case, the amount of allergen-derived peptides in PLGA-PEG-PLGA hydrogel compositions needs to be higher than those in previous clinical trials with short allergen-derived T cell peptides, since the release of PS- liposomes from subcutaneously injected PLGA-PEG-PLGA hydrogels is relatively slow (approx. 10% within the first 12 hours, approx. 17% with 24 hours and approx. 35% within 48 hours) .

In one embodiment, the treatment is performed in monthly intervals by 4 to 6 subcutaneous injections of increasing doses of hydrogel-embedded PS-liposomes containing short allergen-derived T cell peptides. For example, in a 6 injection protocol the first injected hydrogel composition may contain 50 μg of liposomal T cell peptides, the second 100 μg, the third 200 g, the fourth 400 μg, and the last two also 400 μg . The 400 μg doses lead to the release of approx. 40 μg liposomal peptides within the first 12 hours, another 30 g liposomal peptides within 24 hours (total of 70 μg) , and another 70 μg liposomal peptides within 48 hours (total of 140 μg) . These concentrations are comparable to the 75 g of T cell peptides injected intradermally in the study of Worm et al . (2013), which proved to be safe and efficacious. In another embodiment, the treatment is performed in monthly- intervals by 4 to 6 subcutaneous injections of identical doses of hydrogel-embedded PS-liposomes containing short allergen- derived T cell peptides. In a preferred embodiment, each injected hydrogel composition contains an amount of 200 μg to 600 μg liposomal peptides. In a more preferred embodiment, each injected hydrogel composition contains an amount of 400 μg of liposomal peptides.

9. Pharmaceutical formulations of hydrogel compositions for peptide immunotherapy in Phase A

In another embodiment, the therapeutic compositions of the present invention are incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the therapeutic compositions of the present invention and pharmaceutically acceptable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic systems, and the like, compatible with the components of the therapeutic compositions of the present invention. The use of such media and agents for pharmaceutically active substances is well known in the art.

Solutions or suspensions used for subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates or phosphates and agents for the adjustment of toxicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. The composition should be fluid to the extent that easy syringability exists. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case dispersion and by use of surfactants. The composition must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol , phenol, ascorbic acid, thimoseral, and the like. In all cases, the composition must be sterile. Sterile injectable solutions can be prepared by filtered sterilization. The preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. 10. Tolerance- inducing strategies with allergen- derived T cell peptides in Phase A

In another embodiment, the present invention discloses several methods for the first subcutaneous hydrogel-based immunotherapeutic step with food allergen-derived T cell epitope-containing peptides (T cell peptides) in Phase A including a) PS-liposomal approaches using tolerance-promoting ODN, b) PS-liposomal approaches using PLGA spheres as alternative tolerance-promoting adjuvant, c) PS-liposomal approaches using tolerance-promoting immune modulators, d) PS- liposomal approaches using a combination of tolerance- promoting immune modulators and adjuvants, e) PS-liposomal approaches without CpG-ODN or other adjuvants, and f) non- liposomal approaches. Methods which use hydrogel-embedded PS-liposomes provide several important therapeutic advantages over currently available peptide-based techniques. Most important, peptides encapsulated in PS-liposomes are preferentially recognized and phagocytosed by antigen-presenting cells (APCs) . Phosphatidyl- L-serine residues on the surface of the PS-liposomes represent eat-me signals for dendritic cells and macrophages. As a result, hydrogel-embedded PS-liposome-encapsulated peptides are targeted directly to these cells and most efficiently presented to tolerance-promoting Tregs, whereas the uptake and subsequent presentation of non-encapsulated peptides by APCs is significantly less effective.

Furthermore, PS-liposomes are capable of inducing tolerance- promoting antigen-presenting cells (APC) including dendritic cells (DCs) and macrophages. They mimic the tolerance- promoting effects of apoptotic cells and have been shown to inhibit the maturation of dendritic cells and to enhance their secretion of anti-inflammatory cytokines.

The uptake of hydrogel-embedded PS- liposomal T cell peptides by peripheral APC is further optimized by the hydrogel- embedded find-me molecules ATP and UTP. The hydrogel-mediated sustained delivery technology of the present invention mimics the physiological role of find-me signals which are released continuously from apoptotic cells, thereby establishing a chemotactic gradient that stimulates the migration of APC to the subcutaneous injection site of the hydrogel composition. The hydrogel-mediated sustained delivery technology of the present invention provides an additional advantage in that also low molecular weight find-me molecules with a short plasma half-life such as ATP and UTP can be used for establishing a chemotactic gradient for efficient peripheral phagocytosis. Thereby, expenses for production and clinical testing can be reduced significantly. 10.1. PS-liposomal approaches using tolerance-promoting ODN as adjuvant

Method A. In one embodiment, the present invention discloses thermosensitive hydrogels for a locally restricted but sustained delivery of a) hydrogel-embedded phosphatidyl-L- serine (PS) -presenting liposomes (PS-liposomes) containing one or more food allergen-derived short T cell peptides, b) tolerance-promoting amounts of hydrogel-embedded CpG-ODN or GpC-ODN or GpG-ODN, and c) one or more hydrogel-embedded find- me signals for attracting peripheral DCs and macrophages to the site of the injected hydrogel in order to enhance phagocytosis of hydrogel-embedded components by antigen- presenting cells (APC) .

Method A provides an approach for amplified induction of tolerance using CpG-ODN or GpC-ODN or GpG-ODN as tolerance- promoting adjuvant. As described, high doses of CpG-rich oligodeoxynucleotides (CpG-ODN) lead to association of TLR9, TRIF and TRAF6, resulting in activation of the alternate (noncanonical) pathway of NF-κΒ signaling and subsequent induction of IRF3- and TGF- β-dependent immune-suppressive tryptophan catabolism (Volpi et al . , 2012; Volpi et al . , 2013) . GpC oligodeoxynucleotides (GpC-ODN) have also the ability to modulate dendritic cells (DCs) in a tolerogenic manner via TLR7/TRIF-mediated signaling events (Volpi et al . , 2012) , and GpG-ODN have been demonstrated to delay the onset and to attenuate the severity of lupus nephritis by antagonizing and blocking the activation of multiple TLRs (Graham et al . , 2010).

The addition of one of these ODN to the hydrogel composition provides the advantage that T cell peptides are presented by APC which have been effectively tolerized by two mechanisms including the tolerance-promoting effects of PS- liposomes and those of CpG-ODN or GpC-ODN or GpG-ODN. Preferred find-me molecules for Method A are ATP and/or UTP.

Method B . In another embodiment, the present invention discloses thermosensitive hydrogels for a locally restricted but sustained delivery of a) hydrogel-embedded phosphatidyl-L- serine (PS) -presenting liposomes (PS-liposomes) containing one or more food allergen-derived short T cell peptides, and tolerance-promoting amounts of hydrogel-embedded CpG-ODN or GpC-ODN or GpG-ODN, and b) one or more hydrogel-embedded find- me signals for attracting peripheral DCs and macrophages to the site of the injected hydrogel in order to enhance phagocytosis of hydrogel-embedded components by antigen- presenting cells (APC) .

Method B allows the induction of tolerance via lower amounts of CpG-ODN or Gpc-ODN or GpG-ODN due to direct targeting of dendritic cells and macrophages by the tolerance-promoting eat-me signal phosphatidyl-L-serine on the surface of the CpG- ODN- or GpC-ODN- or GpG-ODN-containing PS-liposomes . Thereby, uptake of encapsulated CpG-ODN or GpC-ODN or GpG-ODN together with encapsulated T cell peptides by antigen-presenting cells is most efficient and the intracellular concentration of CpG- ODN or GpC-ODN or GpG-ODN in APC is increased significantly. Furthermore, encapsulation of ODN in PS-liposomes allows the application of ODN with a regular phosphodiester backbone which are fully nuclease-susceptible . Thereby, intracellular accumulation of phagocytosed CpG-ODN is prevented. Preferred find-me molecules for Method B are ATP and/or UTP.

10.2. PS-liposomal approaches using PL6A spheres as alternative tolerance-promoting adjuvant Method C . In another embodiment, the present invention discloses thermosensitive hydrogels for a locally restricted but sustained delivery of a) hydrogel-embedded phosphatidyl-L- serine (PS) -presenting liposomes (PS-liposomes) containing one or more food allergen-derived short T cell peptides, b) tolerance-promoting amounts of hydrogel-embedded anionic poly (lactic-co-glycolic acid) (PLGA) spheres, and c) one or more hydrogel-embedded find-me signals for attracting peripheral DCs and macrophages to the site of the injected hydrogel in order to enhance phagocytosis of hydrogel-embedded components by antigen-presenting cells (APC) .

Method C provides an approach for amplified induction of tolerance using plain anionic PLGA spheres as tolerance- promoting adjuvant. As demonstrated in several studies, even plain PLGA spheres have the capacity to promote tolerance (for a review, see Getts et al., 2015) . For example, using a murine phospholipase A2- induced allergy model, Jilek et al . (2004) have demonstrated that plain PLGA microspheres (size range of 1-10 μπι) can induce tolerance for as long as 6 months post- sensitization. Preferred find-me molecules for Method C are ATP and/or UTP.

Similar to Method A, the addition of plain anionic PLGA spheres to the hydrogel composition provides the advantage that T cell peptides are presented by APC which have been effectively tolerized by two mechanisms including the tolerance-promoting effects of PS- liposomes and those of plain anionic PLGA spheres . The application of plain anionic PLGA spheres as alternative tolerance-promoting adjuvant provides an important advantage in that PLGA spheres are already in clinical use (for a review, see Lii et al . , 2009) .

10.3. PS-liposomal approaches using tolerance-promoting immune modulators

Method D . In another embodiment, the present invention discloses thermosensitive hydrogels for a locally restricted but sustained delivery of a) hydrogel-embedded phosphatidyl -L- serine (PS) -presenting liposomes (PS-liposomes) containing one or more food allergen-derived short T cell peptides, b) one or more hydrogel-embedded tolerance-promoting immune modulators, and c) one or more hydrogel-embedded find-me signals for attracting peripheral DCs and macrophages to the site of the injected hydrogel in order to enhance phagocytosis of hydrogel -embedded components by antigen-presenting cells (APC) . Method D provides an approach for enforced induction of tolerance using one or more tolerance-promoting immune modulators. The application of hydrogel -embedded tolerance- promoting immune modulators allows to target in addition to antigen-presenting cells (APC) including dendritic cells and macrophages also other immune cells and, thereby, to extend the tolerizing effect of such molecules. For example, dexamethasone phosphate (DexP) exerts its immunosuppressive and anti- inflammatory activity not only in dendritic cells and macrophages, but also in various other immune cells including T cells, eosinophils, mast cells, and neutrophils.

Preferred immune modulators for this approach are NF - KB inhibitors including but not limited to vitamin D3 and analogs thereof with short plasma half- lives such as calcipotriol, and glucocorticoids. NF-κΒ inhibitors are known to inhibit the maturation process of dendritic cells (DCs) , thereby generating tolerizing DCs which are capable of inducing tolerance-promoting regulatory T cells. Preferred find-me molecules for Method D are ATP and/or UTP.

Method E . In another embodiment, the present invention discloses thermosensitive hydrogels for a locally restricted but sustained delivery of a) hydrogel-embedded phosphatidyl-L- serine (PS) -presenting liposomes (PS-liposomes) containing one or more food allergen-derived short T cell peptides, and one or more tolerance-promoting immune modulators, and b) one or more hydrogel-embedded find-me signals for attracting peripheral DCs and macrophages to the site of the injected hydrogel in order to enhance phagocytosis of hydrogel-embedded components by antigen-presenting cells (APC) .

Method E allows the enforcement of tolerance with relatively low amounts of immune modulators due to direct targeting of dendritic cells and macrophages by the tolerance-promoting eat-me signal phosphatidyl-L-serine on the surface of the immune modulator-containing PS-liposomes . Thereby, uptake of liposomal immune modulators together with liposomal T cell peptides by antigen-presenting cells is most efficient and the intracellular concentration of immune modulators in APC is increased significantly. Preferred immune modulators for this approach are also NF-KB inhibitors including but not limited to vitamin D3 and analogs thereof with short plasma half- lives such as calcipotriol , and glucocorticoids . Vitamin D3 and analogs thereof are incorporated in the lipid bilayer of PS-liposomes and water- soluble glucocorticoids such as dexamethasone phosphate are encapsulated in PS-liposomes. Preferred find-me molecules for Method E are ATP and/or UTP.

10.4. PS-liposomal approaches using combinations of tolerance- promoting immune modulators and adjuvants Method F . In another embodiment, the present invention discloses thermosensitive hydrogels for a locally restricted but sustained delivery of a) hydrogel-embedded phosphatidyl-L- serine (PS) -presenting liposomes (PS-liposomes) containing one or more food allergen-derived short T cell peptides, b) one or more hydrogel-embedded tolerance-promoting immune modulators, c) tolerance-promoting amounts of hydrogel-embedded ODN (selected from CpG-ODN, GpC-ODN or GpG-ODN) , or tolerance- promoting amounts of hydrogel-embedded plain anionic PLGA spheres, and d) one or more hydrogel-embedded find-me signals for attracting peripheral DCs and macrophages to the site of the injected hydrogel in order to enhance phagocytosis of hydrogel-embedded components by antigen-presenting cells (APC) .

Method F provides an approach for most enforced induction of tolerance using one or more tolerance-promoting immune modulators in addition to plain anionic PLGA spheres or oligodeoxynucleotides with CpG or GpC or GpG motifs as tolerance-promoting adjuvant. Preferred immune modulators for this approach are also NF-κΒ inhibitors including but not limited to vitamin D3 and analogs thereof with short plasma half-lives such as calcipotriol , and glucocorticoids. Since each of the tolerance- inducing mechanisms in Method F including CpG-ODN or GpC-ODN or GpG-ODN, PS- liposomes and NF- B inhibitors targets different receptors, this approach is suited to synergistically induce tolerance in a very efficient manner. Furthermore, the application of hydrogel-embedded tolerance-promoting immune modulators allows to target also other immune cells in addition to APCs and, thereby, to extend the tolerizing effect of such molecules. Preferred find-me molecules for Method F are ATP and/or UTP.

Method G . In another embodiment, the present invention discloses thermosensitive hydrogels for a locally restricted but sustained delivery of a) hydrogel-embedded phosphatidyl-L- serine (PS) -presenting liposomes (PS- liposomes) containing one or more food allergen-derived short T cell peptides, and one or more tolerance-promoting immune modulators, b) tolerance- promoting amounts of hydrogel-embedded plain anionic PLGA spheres or tolerance-promoting amounts of hydrogel-embedded CpG-ODN or GpC-ODN or GpG-ODN, and c) one or more hydrogel- embedded find-me signals for attracting peripheral DCs and macrophages to the site of the injected hydrogel in order to enhance phagocytosis of hydrogel-embedded components by antigen-presenting cells (APC) .

Method G provides also an approach for most enforced induction of tolerance using one or more tolerance-promoting immune modulators in addition to plain anionic PLGA spheres or oligodeoxynucleotides with CpG or GpC or GpG motifs as tolerance-promoting adjuvant. Preferred immune modulators for this approach are also NF-κΒ inhibitors including but not limited to vitamin D3 and analogs thereof with short plasma half-lives such as calcipotriol , and glucocorticoids. However, in contrast to Method F, the tolerance-promoting immune modulators are incorporated into the lipid bilayer (e.g., calcipotriol) or encapsulated (e.g., dexamethasone phosphate) in PS- liposomes together with food allergen-derived T cell peptides. This allows the application of lower amounts of immune modulators since PS-liposomal immune modulators are targeted directly to APC. As a result, the intracellular concentration of immune modulators in APC is high despite low concentrations in the hydrogel composition. Preferred find-me molecules for Method G are ATP and/or UTP.

Method H. In another embodiment, the present invention discloses thermosensitive hydrogels for a locally restricted but sustained delivery of a) hydrogel -embedded phosphatidyl-L- serine (PS) -presenting liposomes (PS-liposomes) containing one or more food allergen-derived short T cell peptides, and one or more tolerance-promoting immune modulators, and tolerance- promoting amounts of hydrogel -embedded CpG-ODN or GpC-ODN or GpG-ODN, and b) one or more hydrogel-embedded find-me signals for attracting peripheral DCs and macrophages to the site of the injected hydrogel in order to enhance phagocytosis of hydrogel-embedded components by antigen-presenting cells (APC) . Method H provides another approach for most enforced induction of tolerance. In this approach hydrogel-embedded PS-liposomes contain three components, food allergen-derived short T cell peptides, tolerance-promoting immune modulators and tolerance- promoting amounts of oligodeoxynucleotides with CpG or GpC or GpG motifs. This allows direct targeting of these components to APC resulting in optimal presentation of allergen-derived T cell peptides by most efficiently tolerized APC. Preferred immune modulators for this approach are also NF-κΒ inhibitors including but not limited to vitamin D3 and analogs thereof with short plasma half- lives such as calcipotriol , and glucocorticoids. Preferred find-me molecules for Method H are ATP and/or UTP.

10.5. PS-liposomal approaches without CpG-ODN or other tolerance-promoting adjuvants

Method I . In one embodiment, the present invention discloses thermosensitive hydrogels for a locally restricted but sustained delivery of a) hydrogel -embedded phosphatidyl -L- serine (PS) -presenting liposomes (PS-liposomes) containing one or more food allergen-derived short T cell peptides, and b) one or more hydrogel-embedded find-me signals for attracting peripheral DCs and macrophages to the site of the injected hydrogel in order to enhance phagocytosis of hydrogel-embedded components by antigen-presenting cells (APC) . Method I provides the important advantage of simplicity, but is likely less effective than Methods A-H due the omission of tolerance-promoting adjuvants and immune modulators.

10.6. Non-liposomal approach

Method J . In another embodiment, the hydrogel -based compositions of Methods A-I are employed without PS-liposomes.

All components of the compositions are embedded directly in the hydrogel . While the omission of PS-liposomes eliminates a direct targeting effect to APC, Method J provides the advantage of a simplified production and clinical testing procedure.

10.7. Combinations of Methods A-J. In another embodiment, the present invention discloses combinations of Methods A-J. All combinations are based on thermosensitive hydrogels for subcutaneous injection, containing at least one or more allergen-derived T cell peptides, either as hydrogel-embedded components or as encapsulated components in hydrogel-embedded PS-liposomes. Additional hydrogel-embedded components are selected from a) one or more find-me molecules, b) one or more tolerance- promoting NF-κΒ inhibitors, either directly embedded in the hydrogel or as PS- liposomal NF-κΒ inhibitors, and c) plain anionic PLGA spheres or oligodeoxynucleotides (ODN) with CpG or GpC or GpG motifs as tolerance-promoting adjuvant, wherein the ODN are embedded in the hydrogel either directly or as PS- 1iposoma1 adjuvant .

11. Biomarkers for the induction of T cell-mediated tolerance by peptide immunotherapy in Phase Ά

In another embodiment, the present invention discloses useful biomarkers for a successful induction of T cell-mediated tolerance by peptide immunotherapy in Phase A. Although these biomarkers have been identified in a recent dose escalation study for subcutaneous auto-antigen-specific tolerance induction (Burton et al . , 2014), they provide also useful biomarkers for the induction of T cell-mediated tolerance in food-allergic patients.

Burton et al . (2014) have demonstrated that in the Tg4 T-cell receptor (TCR) transgenic murine model of experimental autoimmune encephalomyelitis (EAE) in which >90% of CD4+ T cells recognize the nine-residue N-terminal peptide of myelin basic protein (MBP Acl-9) , self-antigen-specific tolerance can be effectively induced via the subcutaneous (s.c.) route, eliciting IL-10-secreting CD4+ T cells with an anergic, regulatory phenotype. Dose escalation minimized CD4+ T-cell activation and proliferation during the early stages of immunotherapy, preventing excessive systemic cytokine release. The ability of cells to secrete IL-10, which serves as a promising mediator of effective antigen-specific immunotherapy (Sabatos-Peyton et al., 2010), and to suppress EAE increased in a dose-dependent manner. Tolerance induced by escalating dose immunotherapy was effective whether administered prophylactically or therapeutically (Burton et al . , 2014).

Furthermore, the s.c. route of administration proved to be more effective than the intranasal route, with a 1,000-fold lower dose of antigen being effective for anergy induction when compared with previous studies.

Gradual establishment of a regulatory CD4⁺ T-cell phenotype.

At each consecutive stage of the dose escalation the sequential modulation of CD4+ T-cell phenotype has been characterized. The gradual establishment of a regulatory CD4⁺ T-cell phenotype was characterized by expression of specific negative co- stimulatory molecules and transcription factors, in addition to the regulatory cytokine IL-10, all of which are used as surrogate markers for allergen/autoantigen-specific tolerance induction according to the method of the present invention. Transcription factors previously associated with IL-10 expression include Maf, Ahr and Nfil3 (Pot et al . , 2009; Motomura et al., 2011; Apetoh et al . , 2010). The induction of IL-21 expression is also noteworthy, as IL-21 contributes to the IL-27-driven production of IL-10 in murine T cells (Pot et al . , 2009) . The most notable correlation with effective immunotherapy was the induction of a set of negative co-stimulatory molecules including PD-1, LAG-3, TIM-3 and TIGIT. Some of these molecules have previously been associated with T cell exhaustion (Wherry, 2011) , while others have been described as markers of IL-10-secreting Trl cells (Gagliani et al . , 2013; Okamura et al., 2009). Burton et al. (2014) demonstrated a positive correlation between IL-10 production and the expression of LAG-3 , TIGIT, PD-1 and TIM-3. However, expression of these markers is not uniquely restricted to the IL-10+ population; only 11% of LAG-3+ cells were IL-10+ and -50% of Tim-3+ or TIGIT+ cells were IL-10+ (Burton et al . , 2014) . These results suggest that while LAG-3 and PD-1 are good markers of the anergic CD4+ T-cell population induced by escalating dose immunotherapy, TIGIT and TIM-3 are better discriminators of IL-10 -secreting cells induced by immunotherapy (Burton et al . , 2014) .

CD49b (integrin -2) was also found to correlate with IL-10 expression in CD4+ T cells from autoantigen-treated mice; however, within the LAG-3+ CD49b+ population, only 33% of cells were found to express IL-10 (Burton et al . , 2014) .

Although the necessary extent of established regulatory CD4+ T-cell phenotypes for the induction of tolerance may vary for different patients and also for different allergic and autoimmune diseases, the study of Burton et al . (2014) provides valuable cornerstones. In this study, mice were treated every 3-4 days six times s.c. with an escalating dose of autoantigen (escalating from 0.08 μg to 0.8 ^g and then to 4 x 8 μg, or escalating from 0.08 /xg to 0.8 μg and then to 8 μg and finally to 3 x 80 μg) . Induction of tolerance was associated with a percentage of CD4+ T cells expressing IL-10, c-Maf or LAG-3 in at least 50% of the cells. A rising percentage of TIGIT+ cells also accumulated during autoantigen-specific immunotherapy (20% of activated CD4⁺ T cells) . The proportion of cells expressing TIM-3 remained relatively stable throughout the treatment, while the percentage of PD-1⁺ cells increased upon initial CD4+ T-cell activation and further increased during the later stages of the treatment .

Based on these data, reasonable induction of T cell-mediated tolerance by peptide immunotherapy in Phase A is considered to be achieved if at least 50% of CD4+ T cells express IL-10, c- Maf or LAG-3 , and the percentage of PD-1⁺ cells and TIGIT+ cells in the CD4+ T cell population has increased significantly.

12. Therapeutic protocols for a combined Phase A and Phase B immunotherapy of food allergy In still another embodiment, the present invention discloses therapeutic protocols for the treatment of food allergy by combining subcutaneous hydrogel-based immunotherapy with food allergen-derived T cell epitope-containing peptides (Phase A) and OIT or sublingual immunotherapy with intact food allergens (Phase B) . Subcutaneous immunotherapy with allergen-derived peptides is performed first to decrease disease-promoting effector T cells and to increase tolerance-promoting regulatory T cells (Tregs) , thereby re-directing the T cell status towards tolerance. OIT or sublingual immunotherapy with intact food allergens is performed thereafter to induce the generation of protective allergen-specific antibodies and to further enhance the development of a tolerogenic T and B cell status. OIT or sublingual immunotherapy with intact food allergens in Phase B is performed as described in the literature including an escalating up-dosing phase and a subsequent maintenance phase (for reviews, see Uyenphuong and Burks, 2014; Vazquez-Ortiz and Turner, 2016). The combination of subcutaneous immunotherapy with food allergen-derived T cell peptides and subsequent oral (OIT) or sublingual (SLIT) immunotherapy with intact food allergens has the potential to minimize adverse reactions. Although reactions are generally mild, the risk of anaphylactic reactions for each patient is substantial, given that doses are orally administered daily over an extended period of treatment .

For sublingual immunotherapy of food allergy, the combination with a prior subcutaneous immunotherapy with food allergen- derived T cell peptides has the potential to increase the therapeutic efficacy of sublingual food allergen-specific immunotherapy significantly and, thereby, to make the SLIT approach for food allergy an attractive alternative to oral immunotherapy (OIT) . Without a robust increase of therapeutic efficacy, the SLIT approach does not appear to be suitable for the treatment of food allergy since the increase in amount of food allergen that can be tolerated following SLIT is modest and significantly lower than that achievable with OIT (for a review, see Vazquez-Ortiz and Turner, 2016) .

12.1. Escalating dose protocol in Phase A

In one embodiment, the treatment in Phase A is performed in monthly intervals by 4 to 6 subcutaneous injections of increasing doses of hydrogel-embedded PS- liposomes containing short allergen-derived T cell peptides.

For example, using a protocol according to Method A (see section 10) including 6 subcutaneous injections in intervals of 2-4 weeks, the first subcutaneously injected hydrogel composition may contain 50 μg of T cell peptides encapsulated in PS-liposomes, tolerance-promoting amounts of CpG-ODN, and sub-micromolar concentrations of ATP and UTP. Based on experimental release analyses of PS-liposomes from PLGA-PEG- PLGA hydrogel compositions, approx. 7.5 ^g of T cell peptides are released during the first 24 hours (15% release) and approx. 17.5 μg of T cell peptides during the first 48 hours (35% release) . The second hydrogel composition may contain 100 μg of T cell peptides encapsulated in PS-liposomes, CpG-ODN, ATP and UTP. The third hydrogel composition may contain 200 μg of T cell peptides encapsulated in PS-liposomes, CpG-ODN, ATP and UTP, and the fourth hydrogel composition may contain 300 μg of T cell peptides encapsulated in PS-liposomes, CpG-ODN, ATP and UTP. The last two hydrogel compositions are identical with the fourth hydrogel composition. Hydrogel compositions containing 300 μg of T cell peptides in PS-liposomes release approx. 45 g of T cell peptides during the first 24 hours and approx. 105 μg of T cell peptides during the first 48 hours.

12.2. Identical dose protocol in Phase A

In another embodiment, the treatment is performed in intervals of 2-4 weeks by 4 to 6 subcutaneous injections of identical doses of hydrogel-embedded PS-liposomes containing short allergen-derived T cell peptides. In one embodiment, each injected hydrogel composition contains an amount of 100-600 μg liposomal peptides. In a preferred embodiment, each injected hydrogel composition contains an amount of 200-400 μg of liposomal peptides. In a more preferred embodiment, each injected hydrogel composition contains an amount of 300 μg of liposomal peptides

For example, using a 6 injection protocol according to Method A (see section 10) all subcutaneously injected hydrogel compositions may contain 300 g of T cell peptides encapsulated in PS-liposomes, tolerance-promoting amounts of CpG-ODN, and sub-micromolar concentrations of ATP and UTP. Such hydrogel compositions release approx. 45 g of T cell peptides during the first 24 hours and approx. 105 μg of T cell peptides during the first 48 hours.

EXAMPLES The following examples are intended to illustrate but not limit the present invention.

EXAMPLE 1: SYNTHESIS OF PL6A-PEG-PLGA HYDROGELS

The biodegradable triblock polymer described in this example has a PLG/PEG weight ratio of 2.3 (70/30), and a lactide/glycolide molar ratio of approx. 15/1. Synthesis of the triblock copolymer is performed according to published protocols (Qiao et a.1. , 2005) .

Copolymer synthesis. Polyethylene glycol (PEG 1000) is purchased from Fluka, poly (DL-lactide) from Sigma, glycolide (1, 4-Dioxane-2, 5-dione) from Sigma, and stannous 2- ethylhexanoate from Aldrich.

A total of 25 g of DL-lactide, glycolide and PEG are used for polymerization (16.6 g DL-lactide, 0.9 g glycolide, 7.5 g PEG 1000) (PLG/PEG weight ratio of 70/30 (2.3)) . Under nitrogen atmosphere, PEG 1000 is dried under vacuum and stirring at 120°C for 2 h in a vigorously dried Erlenmeyer reaction flask. Then the reaction flask is filled with dry argon. DL-lactide and gycolide monomers are added under stirring followed by the addition of Stannous 2 -ethylhexanoate (0.2% w/w) . Then the tube is sealed under argon. The sealed flask is immersed and kept in an oil bath thermostated at 130°C. After approx. 16 h the flask is cooled to room temperature, and the product is dissolved in cold water. After completely dissolved, the copolymer solution is heated to 80°C to precipitate the copolymer and to remove the water-soluble low molecular weight copolymers and unreacted monomers. The supernatant is decanted, the precipitated copolymer is again dissolved in cold water followed by heating to induce precipitation. This process of dissolution followed by precipitation is repeated three times. Alternatively the polymer can be dissolved in acetonitrile , sterile filtered, and precipitated by mixing with sterile water and heating. Finally, the copolymer is dried under vacuum at room temperature until constant weight.

Molecular weight determination. The molecular weight of the copolymer is determined by gel permeation chromatography using polystyrene standards as described by Qiao et al . (2005) .

Figure 8 shows a representative GPC analysis of the copolymer of Example 1.

Measurement of gelation temperature. The gelation temperature is determined as described by Qiao et al . (2005) A 2 ml transparent vial is filled with 200 μΐ water solution of the copolymer (20% w/w and 25% w/w) , is placed in a water bath. The solution is heated in 1°C steps beginning at 26 °C in a thermomixing device (Eppendorf) . At each temperature step the gelation is checked by careful inversion of the tube. When the solution is not free- flowing, gelation of the solution occurred, the temperature read from the thermometer is determined as gelation temperature. Figure 9 shows the gelling temperature of the copolymer of Example 1 in dependence of the polymer concentration. EXAMPLE 2: SYNTHESIS OF PS-LIPOSOMES

This example describes the synthesis of unilamellar PS- liposomes from a lipid mixture of phosphatidyldserine (PS) (either 1, 2-dipalmitoyl-sn-glycero-3-phospho-L-serine sodium salt (Sigma-Aldrich) , l-palmitoyl-2-oleoyl-sn-3-glycerophospho -L-serine (POP-L-S) , or bovine brain phosphatidyldserin (Avanti Polar Lipids)), phosphatidylcholine (PC) (either 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DMPC; Sigma-Aldrich) , l-palmitoyl-2-oleoyl-sn-3 -glycerophosphocholine (POPC; Avanti Polar Lipids) , or egg phosphatidylcholine (egg-PC; Avanti Polar Lipids) ) , and cholesterol (CH; Avanti Polar Lipds) at a ratio of 30:30:40 (PS to PC to CH) according to Hoffmann et al. (2005) .

A chloroform/methanol (2:1, v/v) solution containing 30 pmol PS (approx. 22.7 mg) , 30 mol PC (approx. 22.0 mg) and 40 mol CH (approx. 15.5 mg) is placed in a conical flask and dried by rotary evaporation to prepare a thin lipid film. Thereafter, the flask is placed in a desiccator for at least one hour to completely remove the solvent. Then, 1.7 ml of phosphate- buffered saline (PBS) is added (approx. 35 mg total lipid/ml) and multilamellar vesicles are generated by intense vortex dispersion. For the preparation of unilamellar vesicles, the multilamellar preparation is extruded 10 times through a 1 m pore polycarbonate membrane (Nucleopore, USA) . PS- liposomes with a particle size of approx. 1 m are suitable for efficient uptake by macrophages (Harel-Adar et al., 2011). The liposome suspension is centrifuged at 5000xg for 5 minutes and the supernatant is discarded by pipetting and replaced by 1.7 ml of PBS and vortexed to resuspend the liposomes. The final liposomal suspension contains approx. 59 μπιοΐ (approx. 35 mg) of lipid/ml (59 m liposomal suspension) . Unilamellar PS-liposomes prepared by this procedure have been shown to disperse uniformly in physiological medium at a concentration of 60 mM total lipid due to repulsion forces (Harel-Adar et al._f 2011).

The degree of PS exposure on liposomes is assessed by binding of FITC-annexin V to surface-exposed PS and subsequent analysis by FACS.

EXAMPLE 3: SYNTHESIS OF PS-LIPOSOMES CONTAINING OVALBUMIN (OVA) -DERIVED PEPTIDES

This example describes the synthesis of unilamellar PS- liposomes containing three OVA-derived peptides according to the method of Example 2.

OVA-derived peptides. Three peptide sequences of OVA have been identified as immunodominant T cell determinants in the BALB/c mouse (Yang and Mine, 2008) . Subcutaneous immunotherapy with a cocktail of these peptides significantly decreased anaphylactic responses in OVA- sensitized mice upon oral challenge with a high dose of OVA (Yang et al . , 2010) .

The primary sequence of these peptides are:

AMVYLGAKDSTRTQI OVA region: 39-53 (MW 1653.9) good water solubility

SWVESQTNGIIRNVL OVA region: 147-161 (MW 1715.9) poor water solubility

(3 ) AAHAEINEAGREWG OVA region: 329-343 (MW 1522.6) good water solubility The optimal peptide length of 15 aa was determined primarily based on the binding characteristics defined for BALB/c major histocompatibility complex (MHC) (H2d) class II molecules (Zhang et al . , 2005). Synthesis . A chloroform/methanol (2:1, v/v) solution containing 30 mol phosphatidylserine (PS) (approx. 22.7 mg) , 30 pmol phosphatidylcholine (PC) (approx. 22.0 mg) and 40 μιτιοΐ cholesterol (CH) (approx. 15.5 mg) is placed in a conical flask and dried by rotary evaporation to prepare a thin lipid film. Thereafter, the flask is placed in a desiccator for at least one hour to completely remove the solvent. Then, 1.7 ml of phosphate-buffered saline (PBS) is added containing 8.5 mg of a peptide cocktail (5.0 mg/ml) comprising 3.4 mg of peptide 1 (2.0 mg/ml), 1.7 mg of peptide 2 (1.0 mg/ml), and 3.4 mg of peptide 3 (2.0 mg/ml).

Multilamellar vesicles are generated by intense vortex dispersion. For the preparation of unilamellar vesicles, the multilamellar preparation is extruded 10 times through a 1 pm pore polycarbonate membrane (Nucleopore, USA) . The liposome suspension is centrifuged at 5000xg for 5 minutes and the supernatant is discarded by pipetting and replaced by 1.7 ml of PBS and vortexed to resuspend the liposomes. Optionally, residual unincorporated peptides that have not been removed by the centrifugation step, may be removed by subsequent size exclusion chromatography on a Sephadex G50 column. Analysis of the encapsulation efficiency. The concentration of encapsulated peptides is determined after dissolution of the liposomes in 1% (v/v) Triton X-100 by a colorimetric peptide assay (Thermo Fisher Scientific) providing a linear range of

As demonstrated in a recent study (Belogurov et al . , 2013), liposomal encapsulation of three peptides derived from the myelin basic protein (MBP) at a peptide to lipid ratio /w/w) of 1:330 (0.15 mg peptides/ml per 50 mg lipid/ml) provided an encapsulation efficiency of more than 90%. Since in this example a significantly higher peptide to lipid ratio of 1:7 (5.0 mg peptide/ml per 35 mg/lipid/ml) is used, the encapsulation efficiency of the three OVA-derived peptides is significantly lower.

The final liposomal suspension contains approx. 59 pmol (approx. 35 mg) of lipid/ml (59 mM liposomal suspension) and approx. 1.5 mg/ml OVA-derived peptides (based on 30% encapsulation efficiency) , comprising approx. 600 μg/ml OVA peptide 1, approx. 300 g/ml OVA peptide 2, and approx. 600 ^g/ml OVA peptide 3.

EXAMPLE 4: SYNTHESIS OF PS-LIPOSOMES CONTAINING OVALBUMIN- DERIVED PEPTIDES AND CALCIPOTRIOL

This example describes the synthesis of unilamellar PS- liposomes containing bilayer-incorporated calcipotriol (Tocris Bioscience, UK) and encapsulated OVA-derived peptides according to the method of Example 3. Calcipotriol molecules are incorporated into the lipid bilayer and intercalate between the hydrocarbon chains of phospholipid molecules (Merz and Sternberg, 1994) . Using calcipotriol for incorporation into liposomes made of DMPPC or egg-PC in a molar ratio of calcipotriol (MW 412.6) to lipid of 0.03 to 1, incorporation rates of more than 80% have been reported (Merz and Sternberg, 1994) . Since in this example, a two- fold lower molar ratio of calcipotriol to lipid of 0.015 to 1 is used, the incorporation rate is slightly higher.

Synthesis . A chloroform/methanol (2:1, v/v) solution containing 30 pmol phosphatidylserine (PS) (approx. 22.7 mg) , 30 pmol phosphatidylcholine (PC) (approx. 22.0 mg) and 40 μτηοΐ cholesterol (CH) (approx. 15.5 mg) is placed in a conical flask, mixed with a stock solution of calcipotriol in methanol (10 mg/ml) in a molar ratio of calcipotriol to lipid of 0.015 to 1.0 (620 pg calcipotriol corresponding to approx. 1.5 μιτιοΐ) , and dried by rotary evaporation to prepare a thin lipid film. Thereafter, the flask is placed in a desiccator for at least one hour to completely remove the solvent. Then, 1.7 ml of phosphate-buffered saline (PBS) is added containing 8.5 mg of a peptide cocktail (see Example 3) comprising 3.4 mg of peptide 1 (2.0 mg/ml), 1.7 mg of peptide 2 (1.0 mg/ml), and 3.4 mg of peptide 3 (2.0 mg/ml) .

Multilamellar vesicles are generated by intense vortex dispersion. For the preparation of unilamellar vesicles, the multilamellar preparation is extruded 10 times through a 1 μιτι pore polycarbonate membrane (Nucleopore, USA) . The liposome suspension is centrifuged at 5000xg for 5 minutes and the supernatant is discarded by pipetting and replaced by 1.7 ml of PBS and vortexed to resuspend the liposomes. Optionally, residual unincorporated peptides that have not been removed by the centrifugation step, may be removed by subsequent size exclusion chromatography on a Sephadex G50 column.

Analysis of the encapsulation efficiency. The calcipotriol concentration in the liposomal suspensions is determined by UV absorption at 252 nm (molar extinction coefficient of 42,000; Plum et al., 2004) after dissolution of the liposomes in ethanol. Alternatively, the calcipotriol concentration in the liposomal suspensions can be determined by reversed phase HPLC using a C18-column and acetonitrile : water (77:23) as elution agent (Cirunay et al . , 1998). Calcipotriol is detected by UV absorption at 263 nm.

The concentration of encapsulated peptides is determined after dissolution of the liposomes in 1% (v/v) Triton X-100 by a colorimetric peptide assay (Thermo Fisher Scientific) providing a linear range of 15-1000 μg/ml .

The final liposomal suspension contains approx. 59 μπιοΐ (approx. 35 mg) of lipid/ml (59 mM liposomal suspension) , approx. 310 μg (751 nmol) calcipotriol/ml liposomal suspension (based on 85% incorporation rate), and approx. 1.5 mg OVA- derived peptides/ml (based on 30% encapsulation efficiency) , comprising approx. 600 μg/ml OVA peptide 1, approx. 300 μg/ml OVA peptide 2, and approx. 600 μg/ml OVA peptide 3.

EXAMPLE 5: SYNTHESIS OF PS-LIPOSOMES CONTAINING OVA-DERIVED PEPTIDES AND CpG-ODN This example describes the synthesis of unilamellar PS- liposomes containing encapsulated PO CpG-ODN 1826 ( 5 ' - TCCATGACGTTCCTGACGT - 2 " ; MW approx. 6059) with a natural phosphodiester backbone (PO CpG-ODN 1826) and encapsulated OVA-derived peptides according to the method of Example 3.

In the study of onur et al . (2008), 10 mg CpG-ODN 1826 (MW approx. 6059; 1.65 μπιοΐ) solved in 1.0 ml HEPES-buffer, pH 7.4, were encapsulated in small unilamellar liposomes (SUV) composed of cholesterol (chol) , dilauroyl-phosphatidyl- ethanolamine (DLPE) and dioleoyl-phosphatidylserine (DOPS) at a molar ratio of 1:1:1 (40 μπιοΐ of total lipid; ODN to lipid ratio: approx. 1:25). The final liposome preparation contained approx. 1 mg ODN (165 nmol) /40 μπιοΐ lipid (4.125 nmol ODN/1.0 μπιοΐ lipid; ODN to lipid ratio of approx. 1:25), reflecting an encapsulation efficiency in the range of 10%.

The study of Golali et al . , (2012) demonstrates that CpG-ODN 1826 with a nuclease-resistant phosphorothioate backbone (PS CpG-ODN 1826) and CpG-ODN 1826 with a natural phosphodiester backbone (PO CpG-ODN 1826) are encapsulated with comparable efficiencies in large unilamellar liposomes (composed of distearoyl-phosphatidylcholine (DSPC) and cholesterol (chol) at a molar ratio of 2:1) . Using 200 g CpG-ODN (approx. 33 nmol) per 30 μπιοΐ lipid (ODN to lipid ratio of approx. 1:1000), the encapsulation efficiencies of PO CpG-ODN 1826 and PS CpG-ODN 1826 were 9.8 ± 1.2% and 9.2 ± 0.8% (n=3), respectively.

In Example 5, the ODN to lipid ratio of approx. 1:40 (2.5 μπιοΐ CpG-ODN 1826/100 μπιοΐ lipid) is similar to the conditions applied by Konur et al . (2008) . Synthesis . A chloroform/methanol (2:1, v/v) solution containing 30 pmol phosphatidylserine (PS) (approx. 22.7 mg) , 30 μπιοΐ phosphatidylcholine (PC) (approx. 22.0 mg) and 40 μιτιοΐ cholesterol (CH) (approx. 15.5 mg) is placed in a conical flask and dried by rotary evaporation to prepare a thin lipid film. Thereafter, the flask is placed in a desiccator for at least one hour to completely remove the solvent. Then, 1.5 ml PBS containing 15.0 mg of PO CpG-ODN 1826 (approx. 2.5 μπιοΐ) and 1.7 ml PBS containing 8.5 mg of a peptide cocktail (see example 3) comprising 3.4 mg of peptide 1 (2.0 mg/ml) , 1.7 mg of peptide 2 (1.0 mg/ml), and 3.4 mg of peptide 3 (2.0 mg/ml) .

Multilamellar vesicles are generated by intense vortex dispersion. For the preparation of unilamellar vesicles, the multilamellar preparation is extruded 10 times through a 1 μπι pore polycarbonate membrane (Nucleopore, USA) . The liposome suspension is centrifuged at 5000xg for 5 minutes and the supernatant is discarded by pipetting and replaced by 1.7 ml of PBS and vortexed to resuspend the liposomes. Optionally, residual un- incorporated CPG-ODN and OVA-derived peptides that have not been removed by the centrifugation step, may be removed by subsequent size exclusion chromatography on a Sephadex G50 column.

Analysis of the encapsulation efficiency. The encapsulation efficiency of CpG-ODN 1826 is determined by generating a standard curve of free PO CpG-ODN 1826. All samples and standards contain normalized lipid amounts and the detergent C12E8 (dodecyl octaethylene glycol ether; Sigma-Aldrich) at a final concentration of 1%. Thereafter, SYBR Green I (Invitrogen) is added to the plate at a final dilution of 1:15,000 and the fluorescence quantified in a fluorescence plate reader using an excitation of 485 nra and emission of 528 nm.

The concentration of encapsulated peptides is determined after dissolution of the liposomes in 1% (v/v) Triton X-100 by a colorimetric peptide assay (Thermo Fisher Scientific) providing a linear range of 15-1000 μg/ml .

The final liposomal suspension contains approx. 59 μιηοΐ (approx. 35 mg) of lipid/ml (59 mM liposomal suspension) , approx. 0.9 mg/ml of encapsulated PO CpG-ODN 1826 (approx. 150 nmol) , corresponding to an encapsulation efficiency of approx. 10%, and approx. 1.5 mg OVA-derived peptides/ml (based on 30% encapsulation efficiency) , comprising approx. 600 μg/ml OVA peptide 1, approx. 300 g/ml OVA peptide 2, and approx. 600 μg/ml OVA peptide 3.

EXAMPLE 6: SYNTHESIS OF HYDROGEL/PS-LIPOSOME COMPOSITIONS

This example describes the synthesis and characterization of thermogelling PLGA-PEG-PLGA hydrogels containing either empty or loaded phosphatidylserine (PS) -liposomes .

Synthesis of PLGA-PEG-PLGA hydrogels. The biodegradable triblock polymer described in this example has a PLG/PEG weight ratio of 2.3 (70/30), and a lactide/glycolide molar ratio of approx. 15/1. Synthesis of the triblock copolymer is performed and characterized as described in example 1. Synthesis of empty PS-liposomes. PS-liposomes are prepared as described in Example 2. The final liposomal suspension contains approx. 59 μιτιοΐ (approx. 35 mg) of lipid/ml (59 mM liposomal suspension) .

Synthesis of PS-liposomes loaded with OVA-derived peptides. PS-liposomes loaded with OVA-derived peptides are prepared as described in Example 3. The final liposomal suspension contains approx. 59 pmol (approx. 35 mg) of lipid/ml (59 mM liposomal suspension) and approx. 1 mg OVA-derived peptides/ml (based on 40% encapsulation efficiency) .

Synthesis of PS-liposomes loaded with OVA-derived peptides and calcipotriol . PS-liposomes loaded with OVA-derived peptides and calcipotriol are prepared as described in Example 4. The final liposomal suspension contains approx. 59 ymol (approx. 35 mg) of lipid/ml (59 mM liposomal suspension) , approx. 310 g (751 nmol) calcipotriol/ml liposomal suspension (based on 85% incorporation rate) , and approx. 1 mg OVA-derived peptides/ml (based on 40% encapsulation efficiency) .

Synthesis of PS-liposomes loaded with OVA-derived peptides and PO CpG-ODN 1826. PS-liposomes loaded with OVA-derived peptides and PO CpG-ODN 1826 are prepared as described in Example 5. The final liposomal suspension contains approx. 59 μπιοΐ (approx. 35 mg) of lipid/ml (59 mM liposomal suspension) , approx. 1.5 mg encapsulated PO CpG-ODN 1826 (approx. 0.247 ^mol)/ml, corresponding to an encapsulation efficiency of approx. 10%, and approx. 1 mg OVA-derived peptides/ml (based on 40% encapsulation efficiency) . Preparation of hydrogel/PS-liposome compositions.

Different concentrations of the PLGA-PEG-PLGA copolymer of Example 1 (22.5% w/w, and 30% w/w) in water are mixed with liposomal suspensions in PBS of example 2 at a ratio of two volumes hydrogel solution to one volume of liposomal suspension. The final concentration of the hydrogel is 15% (w/w) or 20% (w/w) containing empty or loaded PS-liposomes at a concentration of approx. 20 ymol (12 mg) of lipid/ml. Liposomal OVA-derived peptides are present at a concentration of approx. 330 μg/ml₇ liposomal calcipotriol at a concentration of approx. 103 μς (250 nmol) calcipotriol/ml , and liposomal PO CpG-ODN 1826 at a concentration of approx. 500 (approx. 83 nmol) /ml.

Gelation characteristics of hydrogel/PS-liposome compositions.

The gelation temperature of hydrogel/PS-liposome compositions is determined as described by Qiao et al . (2005). Transparent vials are filled with 200 μΐ water containing different concentrations of the copolymer (22.5% w/w, and 30% w/w), cooled to 4°C and mixed with 100 μΐ PBS containing empty or loaded PS-liposomes or 100 μΐ PBS containing no liposomes. The final concentration of the copolymer is 15% (w/w) and 20% (w/w) containing liposomes at a concentration of approx. 20 pmol (12 mg) of lipid/ml. The vials are placed in a water bath and each solution is heated in 1°C steps beginning at 20 °C in a thermos-mixing device (Eppendorf) . At each temperature step the gelation is checked by careful inversion of the tube. When the solution is not free-flowing, gelation of the solution occurred and the temperature is determined as gelation temperature. Figure 10 shows the effect of PS-liposomes on the gelation characteristics of PLGA-PEG-PLGA copolymers. In vitro degradation of hydrogel/PS- liposome compositions.

The in vitro degradation behavior of hydrogel/PS-liposome compositions is evaluated by the mass loss and/or the molecular weight reduction with time upon incubation in PBS.

Samples (0.2 ml) are incubated in PBS , pH 7.4, at 37°C under mild agitation in a water bath. The solid residues are removed from the incubation medium at scheduled time intervals and lyophilized. The samples are weighted and the weight loss is calculated. For determination of the molecular weight reduction, the solid residues are solved in cold water and analyzed by gel permeation chromatography using polystyrene standards as described by Qiao et al . (2005) .

EXAMPLE 7: RELEASE OF PS-LIPOSOMES FROM GELLED HYDROGELS

This example describes the in vitro release characteristics of PS-Liposomes with encapsulated FITC-BSA from thermogelling PLGA-PEG-PLGA hydrogels.

Synthesis of thermogelling PLGA-PEG-PLGA hydrogels . The biodegradable triblock polymer described in this example has a PLG/PEG weight ratio of 2.3 (70/30), and a lactide/glycolide molar ratio of approx. 15/1. Synthesis of the triblock copolymer is performed as described in Example 1.

Synthesis of FITC-BSA-containing PS-liposomes . A chloroform/methanol (2:1, v/v) solution containing 30 mol phosphatidylserine (PS) (approx. 22.7 mg) , 30 ymol phosphatidylcholine (PC) (approx. 22.0 mg) and 40 ymol cholesterol (CH) (approx. 15.5 mg) is placed in a conical flask and dried by rotary evaporation to prepare a thin lipid film. Thereafter, the flask is placed in a desiccator for at least one hour to completely remove the solvent. Then, 1.5 ml of PBS containing 1.5 mg FITC- labeled bovine serum albumin (FITC-BSA, Sigma-Aldrich) is added and multilamellar vesicles are generated by intense vortex dispersion. For the preparation of unilamellar vesicles, the multilamellar preparation is extruded 10 times through a 1 μιτι pore polycarbonate membrane (Nucleopore, USA) . The liposome suspension is centrifuged at 5000xg for 5 minutes and the supernatant is discarded by pipetting and replaced by 1.5 ml of PBS and vortexed to resuspend the liposomes. The final liposomal suspension contains approx. 66.7 mol (40.1 mg) of lipid/1.0 ml.

The amount of encapsulated FITC-BSA in liposomes is determined by dissolving the lipid vesicles with 1% (v/v) Triton X-100 and monitoring the absorbance of FITC-BSA at 495 nm. Using the conditions of this example, the encapsulation efficacy is 22% (220 μg FITC-BSA/ml PS- liposome suspension) .

In vitro release of FITC-BSA-containing PS-liposomes from hydrogel/liposome compositions. The in vitro release of FITC- BSA-containing PS-liposomes from hydrogel/PS-liposome compositions is determined after gelling of the hydrogel/PS- liposome compositions at 37°C by monitoring the supernatant for the development of absorbance at 495 nm in the presence of Triton X-100.

Vials are filled with 200 μΐ water containing different concentrations of the copolymer (22.5% w/w, and 30% w/w) , cooled to 4°C and mixed with 100 μΐ PBS containing FITC-BSA- loaded PS- liposomes . The final concentrations of the copolymer are 15% (w/w) and 20% (w/w) containing PS-liposomes with encapsulated FITC-BSA at a concentration of 22.2 pmol lipid/ml (13.3 mg/ml) . The reaction mixtures are incubated at 37°C under mild agitation in a water bath until gelling. Thereafter, 1.7 ml PBS, pH 7.4, is added to each sample and incubation at 37°C is continued. At specified sample collection times, 0.5 ml aliquots of the supernatant are withdrawn and replaced by an identical volume of PBS, pH 7.4, to maintain release conditions. The amount of released PS-liposomes is determined by measuring encapsulated FITC-BSA via absorbance at 495 nm in the supernatant after dissolving the lipid vesicles with 1% (v/v) Triton X-100 (Cohen et al . , 1991) or by fluorescence detection in suitable detection systems.

Using the experimental conditions of this example, approx. 7% of the PS-liposomes are released from the hydrogel within the first 5 hours, approx. 15% after 24 hours and approx. 35% after 48 hours. Results are shown in figure 8.

EXAMPLE 8: RELEASE OF FIND-ME SIGNALS FROM GELLED HYDROGELS

This example describes the release of find-me signal ATP from thermogelling PLGA-PEG-PLGA hydrogels for attraction of peripheral antigen-presenting cells to the injection site of hydrogel-based compositions.

Synthesis of thermogelling PLGA-PEG-PLGA hydrogels. The biodegradable triblock polymer described in this example has a PLG/PEG weight ratio of 2.3 (70/30), and a lactide/glycolide molar ratio of approx. 15/1. The synthesis is performed as described in Example 1. In vitro release of ATP from PLGA-PEG-PLGA hydrogels. An aliquot of 20 μΐ of a 10 mM solution of ATP is combined with 160 μΐ of 25% hydrogel solution and 20 μΐ of lOx PBS (final concentration of ATP: 1 mM) . The mixture is incubated for 2 minutes at 37°C to induce gelling and overlayed with 1 ml of PBS. At frequent time points the supernatant is removed by pipetting and stored at 4°C. The removed supernatant is replaced by fresh 1 ml of PBS. After 48 hours the samples are measured at 260 nm and the amount of released ATP is calculated as percentage of a reference sample containing a concentration of ATP equaling 100% release.

Using these experimental conditions, approx. 50% of the hydrogel-embedded ATP is released within the first 5 hours in an initial burst, followed by another 10% with the next 20 hours. After 48 hours approx. 75% of the hydrogel-embedded ATP is released. Results are shown in figure 2. After an initial burst release of approx. 40% during the first 3 hours, an additional 15-20% release occurs during the next 20 hours (approx. 55% release in 24 hours) , followed by a further 15- 20% release during the next 24 hours (approx. 75% release in 48 hours) .

EXAMPLE 9: RELEASE OF LIPOSOME-EMBEDDED OLIGODEOXYNUCLEOTIDES (ODN) FROM GELLED HYDROGELS

This example describes the release of oligodeoxynucleotides complexed with a cationic liposomal preparation (Cellfectin II; Life Technologies, USA) from thermogelling PLGA-PEG-PLGA hydrogels. The liposomal preparation contains the cationic lipids tetramethyltetra-palmitylspermine and dioleoyl- phosphatidyl-ethanolamine . Synthesis of thermogelling PLGA-PEG-PLGA hydrogels. The biodegradable triblock polymer described in this example has a PLG/PEG weight ratio of 2.3 (70/30), and a lactide/glycolide molar ratio of approx. 15/1. The synthesis is performed as described in Example 1.

Complexation of ODN with Cellfectin. A 22-mer ODN (sequence: agaatttttagtgtatgtacaa) at a concentration of 1.0 mM in 200 ml PBS, pH 7.4, is mixed with 20 ml of Cellfectin II in PBS, pH 7.4 (1 mg/ml) and incubated for a 15 min incubation at room temperature .

Preparation of hydrogel-ODN/Cellfectin compositions. The PLGA- PEG-PLGA triblock copolymer of Example 1 is dissolved at room temperature in water at a concentration of 30% w/v polymer, followed by the addition of 20 ml lOxPBS and 13 ml of ODN/Cellfectin complexes to 167 ml gel solution.

Release of ODN/Cellfectin complexes from PLGA-PEG-PLGA. The formulation is placed in a 2 ml vial, incubated at 37 °C for 2 min until gelling, and 1.8 ml of PBS, pH 7.4, is added. The vial is incubated at 37°C. At specified sample collection times, the supernatant is withdrawn and replaced by an identical volume of PBS pH 7.4 to maintain release conditions.

Determination of released ODN/ Cellfectin complexes.

At indicated times samples of the supernatant of the gels are taken and analyzed for released ODNs by absorption at 260 nm and densitometric analysis (densitometric software: ImageJ, NIH Bethesda) of UV-iluminated ethidium bromide stained agarose gels (1.5% agarose gel in 0. lx TAE buffer with ethidium bromide) . Controls correspond to complete release values (100%) and dilutions thereof are used for a calibration curve . Release of ODN is depicted as percentage of total release over time. As shown in figure 3, approx. 25% of ODN/Cellfectin complexes are released from a gelled PLGA-PEG- PLGA hydrogel during the first 6 hours.

EXAMPLE 10: SYNTHESIS OF HYDROGEL COMPOSITION A FOR THE TREATMENT OF OVA- LLERGIC MICE ( (NON-LIPOSOMAL COMPOSITION A)

This example describes the synthesis of PLGA-PEG-PLGA hydrogel compositions comprising hydrogel-embedded OVA-derived peptides, hydrogel-embedded B class CpG-ODN 1826 containing a full phosphorothioate backbone, and hydrogel-embedded ATP and UTP for the treatment of OVA-sensitized mice.

OVA-derived peptides. Three peptide sequences of OVA identified as immunodominant T cell determinants in the BALB/c mouse (Yang and Mine, 2008) , are used as cocktail in this experiment. The primary sequence of these peptides are:

AMVYLGAKDSTRTQI OVA region: 39-53 (MW 1653.9) good water solubility

SWVESQTNGIIRNVL OVA region: 147-161 (MW 1715.9) poor water solubility

(3) AAHAEINEAGREWG OVA region: 329-343 (MW 1522.6) good water solubility

The optimal peptide length of 15 aa was determined on the binding characteristics defined for BALB/c major histocompatibility complex (MHC) (H2d) class II molecules (Zhang et al., 2005).

In the study of Yang et al. (2010), OVA-sensitized BALB/c mice were treated by subcutaneous injection of 300 μg of a mixture of three OVA-derived T cell peptides (each peptide: 100 ^g) in PBS three times weekly for a period of 3 weeks (900 μg of peptides/week) .

Considering the retarded release of peptides from hydrogel compositions, in this example one subcutaneous hydrogel injection per week over a period of 4 weeks is performed. Due to limited solubility of the three peptides in aqueous solutions, the hydrogel composition of each subcutaneous injection (300 μΐ of a 15% w/v PLGA-PEG-PLGA hydrogel composition in three 100 μΐ aliquots) contains 100 g of peptide 1 (40 μΐ of 2.5 mg/ml PBS), 100 μg of peptide 3 (40 μΐ of 2.5 mg/ml PBS), and 50 μg of peptide 2 (50 μΐ of 1 mg/ml PBS) .

CpG-ODN 1826. For the composition of this example, the 20-mer class B CpG-ODN 1826 (MW 6364; 5' - tccatgacgttcctgacgtt- 3 ' ) containing a full phosphorothioate backbone (specific for murine TLR9) is used.

The study of Volpi et al . (2013) provides an estimate of the tolerance-promoting amount of CpG-ODN 1826 in mice. C57BL/6 mice were sensitized by the concomitant intraperitoneal (100 μg) and subcutaneous (100 μg) administration of Aspergillus fumigatus culture filtrate extract followed 1 week later by the intranasal instillation of 20 μg of the extract. After an additional 7 days, bronchial colonization of A. fumigatus was induced by resting conidia (asexually produced spores) administered intratracheally (1 x 10⁷) , and the animals were evaluated 1 week later for parameters of allergic airway inflammation. Treatment with CpG-ODN 1826 was performed by intraperitoneal injection of 30 μg of class B CpG-ODN 1826 (approx. 4.7 nmol; MW approx. 6364) per mouse twice, on the same days as the first and second administration of Aspergillus fumigatus culture filtrate extract.

Considering the retarded release of CpG-ODN from hydrogel compositions, in this example one subcutaneous hydrogel injection per week over a period of 4 weeks is performed. The hydrogel composition of each subcutaneous injection (300 μΐ of a 15% w/v PLGA-PEG-PLGA hydrogel composition in three 100 μΐ aliquots) contains 40 μg of class B CpG-ODN 1826 (10 μΐ of a 4.0 mg/ml solution in PBS) .

ATP and UTP . For the method of the present invention it is important to restrict the concentration of released ATP and/or UTP to the nanomolar range since extracellular nucleotides at concentrations of more than 1 μΜ are considered proinflammatory ( ono and Rock, 2008) . Therefore, for therapeutic applications PLGA-PEG-PLGA hydrogels are loaded with ATP and/or UTP at a concentration of approx. 1 μΜ. Thereby, the concentration of released nucleotides will not exceed the critical limit of 1 μΜ, since within the first hour only approx. 20% of embedded nucleotides are released, followed by another 10% with the next hour and decreasing percentages during the following hours. Furthermore, triphosphate nucleotides released from the hydrogel into the extracellular space are rapidly degraded by extracellular enzymes to di- and mono-phosphate nucleotides. The hydrogel composition of each subcutaneous injection (300 μΐ of a 15% w/v PLGA-PEG-PLGA hydrogel composition in three 100 μΐ aliquots) contains 0.3 nmol ATP (5 μΐ of a 60 μΜ solution in PBS) and 0.3 nmol UTP (5 μΐ of a 60 μΜ solution in PBS) .

Synthesis of PLGA-PEG-PLGA hydrogel composition A comprising OVA-derived peptides, CpG-ODN, ATP and UTP. The PLGA-PEG-PLGA triblock copolymer of Example 1, dried under vacuum at room temperature until constant weight, is dissolved at 4°C in PBS at a concentration of 30% w/v polymer, and then mixed with OVA-derived peptides, CpG-ODN 1826, ATP and UTP. The hydrogel composition of each subcutaneous injection (300 μΐ) contains a) 150 μΐ of a 30% w/v PLGA-PEG-PLGA solution in PBS, b) 40 μΐ (100 μg) of OVA peptide 1 (2.5 mg/ml PBS), c) 50 μΐ (50 μg) of OVA peptide 2 (1 mg/ml PBS) , d) 40 μΐ (100 μg) of OVA peptide 3 (2.5 mg/ml PBS), e) 10 μΐ (40 g) of class B CpG-ODN 1826 (4.0 mg/ml PBS), f) 5 μΐ (0.3 nmol) of ATP (60 μΜ in PBS), and g) 5 μΐ (0.3 nmol) of UTP (60 μΜ in PBS) .

The final concentration of the hydrogel is 15% (w/w) containing in 300 μΐ a) 100 μg OVA peptide 1, b) 50 g OVA peptide 2, c) 100 μg OVA peptide 3, d) 40 μg of class B CpG- ODN 1826, e) 0.3 nmol ATP (1 μΜ) , and f) 0.3 nmol UTP (1 μΜ) .

EXAMPLE 11: SYNTHESIS OF HYDROGEL COMPOSITION B FOR THE TREATMENT OF OVA-ALLERGIC MICE (LIPOSOMAL PEPTIDE COMPOSITION B) This example describes the synthesis of PLGA-PEG-PLGA hydrogel compositions B comprising hydrogel-embedded PS-liposomes containing encapsulated OVA-derived peptides, hydrogel- embedded B class CpG-ODN 1826 containing a full phosphorothioate backbone, and hydrogel-embedded ATP and UTP for the treatment of OVA-sensitized mice.

OVA-derived peptides encapsulated in PS-liposomes . As described in Example 3, encapsulation of three OVA-derived peptides in PS-liposomes resulted in a final liposomal suspension which contains approx. 59 μπιοΐ (approx. 35 mg) of lipid/ml (59 m liposomal suspension) and approx. 1.5 mg/ml OVA-derived peptides (based on 30% encapsulation efficiency) , comprising 600 ^g/ml OVA peptide 1, 300 μg/ml OVA peptide 2, and 600 μg/ml OVA peptide 3.

The hydrogel composition of each subcutaneous injection (300 μΐ of a 15% w/v PLGA-PEG-PLGA hydrogel composition in three 100 μΐ aliquots) contains 130 μΐ of liposomal OVA peptides comprising approx. 4.6 mg of lipid, approx. 80 g of OVA peptide 1, approx. 40 g of OVA peptide 2, and approx. 80 g of OVA peptide 3 (total of approx. 200 μg of OVA-derived peptides) .

CpG-ODN 1826. For the composition of this example, the 20-mer class B CpG-ODN 1826 (MW 6364; 5' -tccatgacgttcctgacgtt-3' ) containing a full phosphorothioate backbone (specific for murine TLR9) is used.

The hydrogel composition of each subcutaneous injection (300 μΐ of a 15% w/v PLGA-PEG-PLGA hydrogel composition in three 100 μΐ aliquots) contains 40 μg of class B CpG-ODN 1826 (10 μΐ of a 4.0 mg/ml solution in PBS) . ATP and UTP . The hydrogel composition of each subcutaneous injection (300 μΐ of a 15% w/v PLGA-PEG-PLGA hydrogel composition in three 100 μΐ aliquots) contains 0.3 nmol ATP (5 μΐ of a 60 μΜ solution in PBS) and 0.3 nmol UTP (5 μΐ of a 60 μΜ solution in PBS) .

Synthesis of PLGA-PEG-PLGA hydrogel composition B comprising liposomal OVA-derived peptides, CpG-ODN, ATP and UTP. The PLGA-PEG-PLGA triblock copolymer of Example 1, dried under vacuum at room temperature until constant weight, is dissolved at 4°C in PBS at a concentration of 30% w/v polymer, and then mixed with liposomal OVA-derived peptides, CpG-ODN 1826, ATP and UTP. The hydrogel composition of each subcutaneous injection (300 μΐ) contains a) 150 μΐ of a 30% w/v PLGA-PEG- PLGA solution in PBS, b) 130 μΐ of liposomal OVA-derived peptides (approx. 4.6 mg of lipid, approx. 80 μg of OVA peptide 1, approx. 40 μg of OVA peptide 2, and approx. 80 μg of OVA peptide 3 (total of approx. 200 μg of OVA-derived peptides), c) 10 μΐ (40 μg) of class B CpG-ODN 1826 (4.0 mg/ml PBS), d) 5 μΐ (0.3 nmol) of ATP (60 μΜ in PBS), and e) 5 μΐ (0.3 nmol) of UTP (60 μ in PBS) .

The final concentration of the hydrogel is 15% (w/w) containing in 300 μΐ a) approx. 80 μg liposomal OVA peptide 1, b) approx. liposomal 40 μg OVA peptide 2, c) approx. liposomal 80 μg OVA peptide 3, d) 40 μg of class B CpG-ODN 1826, e) 0.3 nmol ATP (1 μΜ) , and f) 0.3 nmol UTP (1 μ ) .

The slow release of loaded PS- liposomes from gelled PLGA-PEG- PLGA hydrogels leads to the release of approx. 20 μg liposomal peptides during the first 24 hours (15% release) , and another 40 liposomal peptides during the next 24 hours (35% release) . As a result, approx. 70 ^g liposomal peptides are delivered to antigen-presenting cells in proximity of subcutaneously injected hydrogel B compositions during the first 48 hours.

EXAMPLE 12 : SYNTHESIS OF HYDROGEL COMPOSITION C FOR THE TREATMENT OF OVA-ALLERGIC MICE (LIPOSOMAL PEPTIDE-CpG ODN COMPOSITION C)

This example describes the synthesis of PLGA-PEG-PLGA hydrogel compositions C comprising hydrogel -embedded PS- liposomes containing encapsulated OVA-derived peptides and PO CpG-ODN 1826, and hydrogel -embedded ATP and UTP for the treatment of OVA- sensitized mice.

OVA-derived peptides and PO CpG-ODN 1826 encapsulated in PS- liposomes . For this example, the three OVA-derived peptides of Example 3 and PO CpG-ODN 1826 ( 5 ' -TCCATGACGTTCCTGACGTT- 3 ' ; MW approx. 6059) with a natural phosphodiester backbone of Example 5 are used.

As described in Example 5, co-encapsulation of three OVA- derived peptides and PO CpG-ODN 1826 in PS-liposomes resulted in a final liposomal suspension which contains approx. 59 ymol (approx. 35 mg) of lipid/ml (59 mM liposomal suspension) , approx. 0.9 mg/ml encapsulated PO CpG-ODN 1826 (approx. 150 nmol) , corresponding to an encapsulation efficiency of approx. 10%, and approx. 1.5 mg OVA-derived peptides/ml (based on 30% encapsulation efficiency) , comprising approx. 600 μg/ml OVA peptide 1, approx. 300 μg/ml OVA peptide 2, and approx. 600 μg/ml OVA peptide 3.

The hydrogel composition of each subcutaneous injection (300 μΐ of a 15% w/v PLGA-PEG-PLGA hydrogel composition in three 100 μΐ aliquots) contains 130 μΐ of liposomal OVA peptides comprising approx. 4.6 mg of lipid, approx. 80 μg of OVA peptide 1, approx. 40 μg of OVA peptide 2, approx. 80 μg of OVA peptide 3 (total of approx. 200 g of OVA-derived peptides) , and approx. 115 μg of encapsulated PO CpG-ODN 1826 (approx. 19 nmol) .

The slow release of loaded PS- liposomes from gelled PLGA-PEG- PLGA hydrogels leads to the release of approx. 17 μg liposomal CpG-ODN 1828 and 20 μg liposomal peptides during the first 24 hours (15% release) , and another 23 μg liposomal CpG-ODN 1828 and 40 μg liposomal peptides during the next 24 hours (35% release) . As a result, approx. 40 μg liposomal CpG-ODN 1828 and approx. 70 μg liposomal peptides are delivered to antigen- presenting cells in proximity of subcutaneously injected hydrogel C composition during the first 48 hours.

ATP and UTP . The hydrogel composition of each subcutaneous injection (300 μΐ of a 15% w/v PLGA-PEG-PLGA hydrogel composition in three 100 μΐ aliquots) contains 0.3 nmol ATP (5 μΐ of a 60 μΜ solution in PBS) and 0.3 nmol UTP (5 μΐ of a 60 μΜ solution in PBS) .

Synthesis of PLGA-PEG-PLGA hydrogel composition B comprising liposomal OVA-derived peptides, CpG-ODN, ATP and UTP . The PLGA-PEG-PLGA triblock copolymer of Example 1, dried under vacuum at room temperature until constant weight, is dissolved at 4°C in PBS at a concentration of 30% w/v polymer, and then mixed with liposomal OVA-derived peptides and CpG-ODN 1826, ATP and UTP.

The hydrogel composition of each subcutaneous injection (300 μΐ) contains a) 150 μΐ of a 30% w/v PLGA-PEG-PLGA solution in PBS, b) 130 μΐ of liposomal OVA-derived peptides and PO CpG- ODN 1826 (approx. 4.6 mg of lipid, approx. 80 μg of OVA peptide 1, approx. 40 μg of OVA peptide 2, and approx. 80 μg of OVA peptide 3 (total of approx. 200 μg of OVA-derived peptides) , and approx. 115 μg of encapsulated PO CpG-ODN 1826 (approx. 19 nmol) , c) ) 5 μΐ (0.3 nmol) of ATP (60 μΜ in PBS), and d) 5 μΐ (0.3 nmol) of UTP (60 μΜ in PBS).

The final concentration of the hydrogel is 15% (w/w) containing in 300 μΐ a) approx. 80 μg liposomal OVA peptide 1, b) approx. liposomal 40 μg OVA peptide 2, c) approx. liposomal 80 g OVA peptide 3, d) approx. liposomal 115 μg of PO CpG-ODN 1826, e) 0.3 nmol ATP (1 μΜ) , and f) 0.3 nmol UTP (1 μΜ) .

EXAMPLE 13: PEPTIDE-BASED IMMUNOTHERAPY OF MICE ORALLY SENSITZED WITH OVALBUMIN (OVA)

In this example, the therapeutic efficacy of the hydrogel compositions of Examples 10 and 11 for peptide-based immunotherapy of mice orally sensitized with OVA are evaluated.

Murine model for OVA- induced food allergy A schematic outline of this experiment is shown in Fig. 14. Female BALB/c mice (8 weeks old at starting day) are used. Animals are housed under an egg- free diet (14% protein (wheat and corn) and 3.5% fat) in a 12 h lighting cycle. Food and water are available ad libitum.

Sensitization. Following a 1-week acclimatization period, mice are sensitized by oral gavage at a dose of 1.0 mg of HPLC- purified OVA and 10 μg of cholera toxin (List Biologicals, Denver, CO, USA), twice per week at days 2 and 4 of week 2,3,4 and 5.

Specific immunotherapy is performed by 4 successive subcutaneous (SC) injections of the hydrogel compositions of Examples 10 and 11 at day 1 of week 6, 7, 8, 9.

Four groups of mice (10 mice per group) are compared.

Group I: Treatment with 300 μΐ (administration in three 100 μΐ aliquots) hydrogel composition A of Example 10 comprising a) 15% w/v PLGA-PEG-PLGA, b) 100 μg OVA peptide

1, c) 50 μg OVA peptide 2, d) 100 μg OVA peptide 3, e) 40 μg of class B CpG-ODN 1826, f) 0.3 nmol ATP, and g) 0.3 nmol UTP .

Group II: Treatment with 300 μΐ (administration in three

100 μΐ aliquots) hydrogel composition B of Example 11 comprising a) 15% w/v PLGA-PEG-PLGA, b) a) approx. 80 μg Ps-liposomal OVA peptide 1, b) approx. PS-liposomal 40 μg OVA peptide 2, c) approx. PS-liposomal 80 μg OVA peptide 3, d) 40 of class B CpG-ODN 1826, e) 0.3 nmol ATP, and f) 0.3 nmol UTP.

Group III (allergy group) : Placebo treatment with 300 μΐ 15% w/v PLGA-PEG-PLGA.

Group IV (control group) : Placebo treatment with 300 μΐ 15% w/v PLGA-PEG-PLGA.

The cha11enge is performed by oral administration of 20 mg OVA in PBS at day 1 of week 10. Control mice receive only PBS.

Analysis of anaphylactic responses are evaluated 20-40 min after the challenge by measurement of the temperature and according to a scoring system (Li et al . , 1999) as follows: 0, no symptoms; 1, scratching and rubbing around the nose and head; 2, puffiness around the eyes, pilar erecti (erection of the hair of the skin due to contraction of the tiny arrectores pilorum muscles) , reduced activity, and/or decreased activity with increased respiratory rate; 3, wheezing, labored respiration, cyanosis around the mouth and the tail; 4, no activity after prodding, or tremor and convulsion; 5, death.

As observed in a similar murine egg allergy study (Yang et al . , 2010), treatment with hydrogel-embedded or PS-liposome- encapsulated OVA-derived peptides reduces the development of hypersensitivity reactions. ELISA analyses in serum. Following the final oral challenge, whole blood is collected by cardiac puncture and two serum samples are pooled in equal volumes within each group, a total of 5 pooled serum samples per group due to limited volume in individual mouse serum for subsequent ELISA assays for the determination of histamine (R-Biopharm AG, Germany) ) , OVA-IgE and OVA-specific IgG (MD Bioproducts, Zurich, Switzerland) .

In accordance with the study of Yang et al . (2010), serum levels of histamine show a significant decrease upon treatment with hydrogel-embedded or PS- liposome-encapsulated OVA-derived peptides, whereas serum levels of OVA-specific IgE are only slightly decreased and serum levels of OVA-specific IgG are comparable to those in positive control animals. Apparently, peptide immunotherapy does not induce the production of so- called "blocking IgG' (Strait et al., 2006).

Analysis of OVA-specific IgA in fecal pellets. A mass of 1.0 g of fecal pellets are added to 7.5 ml of PBS pH 7.4 and homogenized for 2 x 30 s using a homogenizer. Samples are subsequently centrifuged at 500 g for 20 min and the supernatants are carefully passed through a 0.45 mm diameter syringe filter. Concentrations of OVA-specific IgA are determined by ELISA (Chondrex, Inc., Redmont, A USA).

The analyses show a gradual increase of OVA-specific IgA in fecal pellets upon treatment with hydrogel-embedded or PS- liposome-encapsulated OVA-derived peptides. This observation supports reports showing that low or decreased levels of allergen-specific IgA were associated with the development of food allergies (e.g., Frossard et al., 2004). Analysis of intestinal gene expression pattern by RT-PCR. Following the final oral challenge (mice are euthanized 24 h later) , mouse ileums from pre- selected groups are freshly and individually isolated (n = 6 samples per group) , and immediately placed in ice-cold RNA laters solution (Sigma- Aldrich, St. Louis, MO, USA) followed by storage at -30° C. Total RNA is extracted from individual mice (30 mg of tissue/mouse) using the spin column procedure from RNeasy kits (Qiagen Inc., Valencia, CA, USA). Total RNA integrity is assessed on 1% agarose gels, and the respective concentration and purity are determined by measuring the absorbance ratios A260/A280. Complementary DNA (cDNA) is synthesized from 1.0 mg/mouse of total RNA using Quantitect Rev kit (Qiagen Inc.), following the manufacturer's instructions.

Real-time fluorescence-monitored PCR reactions are performed using an iCycler detection system. The temperature profile is 95° C for 15 min, then 15 s at 95° C (denaturation) , 56° C for 15 s (annealing) and 72° C for 30 s (extension) , repeated for 45-50 cycles. The efficiency of all qPCR reactions (primer pairs) is between 90% and 110% as per standard PCR primer design parameters. All data are normalized to the housekeeping gene encoding for glyceraldehyde- 3 -phosphate dehydrogenase (GAPDH) and the relative mRNA expression ratios are determined as described (Livak and Schmittgen, 2001) . The sequences of the primers used for real-time RT-PCR analyses of 11-4, IL-5, IL-13, IL-12p40, IL-10, IL-18, IFN-γ, TGF- β Ι , F0XP3 and GAPDH are summarized in Table 1 (Fig. 16) .

As observed in the murine egg allergy study of Yang et al . (2010) , after treatment with hydrogel-embedded or PS-liposome- encapsulated OVA-derived peptides the intestinal expression pattern of Th2 -biased cytokines including IL-4, IL-5 and 11-13 show a significant decrease, while the expression of Thl- biased cytokines including IFN-γ, 11-12 and 11-18 is slightly elevated. Most important is the observation that the intestinal expression of TGF-β and FOXP3 is increased significantly, indicating the induction of Treg-associated responses .

Preparation of spleen cell cultures and analysis of cytokine secretion. Following the final oral challenge (mice are euthanized 24 h later) , spleens from individual mouse are aseptically removed into ice-cold RPMI-1640 medium (Gibco Invitrogen, New York, NY, USA) , containing sodium bicarbonate (1.5 g/1) , glucose (4.5 g/1) , L-glutamine (2mM) , sodium pyruvate (lmTh2 -biased cytokines including M) , penicillin (50 U/ml) and streptomycin (50 mg/ml) . Two whole spleens are pooled within each group. Single cell suspensions are prepared as described previously (Rupa et al . , 2007) and cell viability is assessed by trypan blue exclusion. Cells are cultured in 24-well plates (Corning Inc., Corning, NY, USA) at a density of 2.5 x 10⁶/ml in the absence (negative control wells) or presence of purified OVA (100 mg/ml) . Supernatants are collected after 72 h of culture in a 5% CO2 humidified incubator, and assayed for the presence of cytokines IFN-γ (Thl-biased) and IL-4 (Th2-biased) , in triplicate wells, using antibody reagents purchased from commercial kits (BD Pharmingen, Mississauga, Canada) following the manufacturer's instructions .

The results from this analysis are in accordance with those obtained from the analysis of the intestinal cytokine expression pattern. Secretion of the Th2 -biased cytokine IL-4 is reduced significantly, whereas secretion of the Thl-biased cytokine IFN-γ is slightly increased.

EXAMPLE 14: IMMUNOTHERAPY OF MICE ORALLY SENSITZED WITH OVALBUMIN (OVA) BY A COMBINATION OF OVA-DERIVED PEPTIDES AND INTACT OVA

In this example, the therapeutic efficacy of a combination of immunotherapy with OVA-derived peptides (Phase A) and subsequent oral immunotherapy with intact OVA (Phase B) is evaluated in mice orally sensitized with OVA. Phase A is performed as described in Example 13 using the hydrogel compositions of Example 10. Phase B is performed as described by Leonard et al . (2012) .

Murine model for OVA- induced food allergy

A schematic outline of this experiment is shown in Fig. 15. Female BALB/c mice (8 weeks old at starting day) are used. Animals are housed under an egg- free diet (14% protein (wheat and corn) and 3.5% fat) in a 12 h lighting cycle. Food and water are available ad libitum.

Sensitization. Following a 1-week acclimatization period, mice are sensitized by oral gavage at a dose of 1.0 mg of HPLC- purified OVA and 10 μg of cholera toxin (List Biologicals,

Denver, CO, USA) , twice per week (days 2 and 4) for a duration of 4 weeks (week 2,3,4, and 5) .

Specific immunotherapy includes Phase A and Phase B treatment. Phase A is performed by 4 successive subcutaneous (s.c.) injections of the hydrogel compositions of Example 10 at day 1 of week 6,7,8, and 9. Phase B is performed as OIT over a period 14 days (week 10 and 11) by daily gavage of increasing doses of OVA from 0.5 mg (days 1,2 of week 10), 2.5 mg (days 3,4 of week 10), 5 mg (days 5-7 of week 10), 12.5 mg (days 8,9 of week 11) , 25 mg (days 10-14 of week 11) .

Four groups of mice (10 mice per group) are compared. Group I (sensitized mice) Phase A:

Treatment with 300 μΐ (administration in three 100 μΐ aliquots) hydrogel composition A of Example 10 comprising a) 15% w/v PLGA-PEG-PLGA, b) 100 μg OVA peptide 1, c) 50 μg OVA peptide 2, d) 100 μg OVA peptide 3, e) 40 μg of class B CpG-ODN 1826, f) 0.3 nmol ATP, and g) 0.3 nmol UTP.

Phase B:

Treatment by daily gavage of increasing doses of OVA from 0.5 mg, 2.5 mg, 5 mg, 12.5 mg, and 25 mg.

Group II (sensitized mice; placebo treatment in Phase A) Phase A:

Treatment with 300 μΐ (administration in three 100 μΐ aliquots) hydrogel composition comprising 15% w/v PLGA-PEG-PLGA in PBS.

Phase B:

Treatment by daily gavage of increasing doses of OVA from 0.5 mg, 2.5 mg, 5 mg, 12.5 mg, and 25 mg. Group III (sensitized mice; placebo treatment in Phase A and Phase B)

Phase A: Treatment with 300 μΐ (administration in three 100 μΐ aliquots) hydrogel composition comprising 15% w/v PLGA-PEG-PLGA in PBS.

Phase B:

Treatment by daily gavage of PBS

Group IV (no sensitization with OVA, ; placebo treatment in Phase A and Phase B) )

Phase A:

Treatment with 300 μΐ (administration in three 100 μΐ aliquots) hydrogel composition comprising 15% w/v

PLGA-PEG-PLGA in PBS.

Phase B:

Treatment by daily gavage of PBS

The challenge is performed in by oral administration of 50 mg OVA in PBS at day 1 of week 12. Control mice (group IV) receive only PBS .

After recovery from the oral challenge, the development of systemic tolerance is analyzed by intraperitoneal injection of increasing amounts of OVA starting with 1 μg, then 10 μg and finally 100 μς. After discontinuation of OIT for 2 weeks, mice are re- challenged by oral administration of 50 mg OVA in PBS at day 1 of week 14 to assess tolerance.

After recovery from the oral re-challenge, the further development of systemic tolerance is analyzed by intraperitoneal injection of increasing amounts of OVA starting with 1 ig, then 10 pg and finally 100 pg.

Preparation of cells and tissues. Mice are euthanized one day after the final challenge (5 mice of each group after the first oral and subsequent systemic challenge and 5 mice of each group after the oral re-challenge and subsequent systemic challenge) .

Analysis of oral tolerance. Analyzed are anaphylactic responses (temperature, scoring system) after oral challenge (see Example 13) , levels of OVA- specific IgA in fecal pellets (see Example 13) , and intestinal gene expression pattern by RT-PCR of 11-4, IL-5, IL-13, IL-12p40, IL-10, IL-18, IF -γ, TGF-βΙ, F0XP3 (see Example 13) .

Furthermore, serum levels of histamine and OVA- specific immunoglobulins including IgE and IgG are determined as described in Example 13.

Analysis of systemic tolerance. Analyzed are anaphylactic responses (temperature, scoring system) after systemic (intraperitoneal) challenge with OVA, OVA-mediated activation of peripheral basophils and peritoneal mast cells after systemic (intraperitoneal) challenge with OVA, and the secretion of Th2 -biased cytokine IL-4 and Thl-biased cytokine IFN-γ in spleen cell cultures as described in Example 13.

For the analysis of OVA-mediated activation of peripheral basophils, blood is collected in heparinized tubes, diluted 1:1 with RPMI medium, and incubated at 37°C for 60 min with 10-100 pg/ml OVA or media alone. Red blood cells are lysed, and cells are fixed and stained for CD49b and IgE to detect basophils, and CD200R as an activation marker according to Leonard et al . (2012) . CD3 and CD19 are used to gate out B and T cells.

For the analysis of OVA-mediated activation of peritoneal mast cells, peritoneal lavage is collected and cells are stimulated as described for peripheral basophils and stained for ckit and IgE to detect mast cells, and CD107a (LAMP-1) as an activation marker according to Leonard et al . (2012) .

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Claims

CLAIMS :

Pharmaceutical composition for the induction of food tolerance by a combination of subcutaneous hydrogel-based immunotherapy (Phase A composition) and subsequent oral (OIT) or sublingual (SLIT) immunotherapy (Phase B composition) , wherein the Phase A composition comprises a thermogelling hydrogel for locally restricted sustained release of embedded components comprising a) one or more hydrogel-embedded food allergen-derived T cell peptides, b) a tolerance-promoting dose of synthetic oligodeoxy- nucleotides (ODN) containing one or more CpG or GpC or GpG motifs, c) one or more hydrogel-embedded find-me molecules for attracting peripheral antigen-presenting cells (APC) to the site of the administered hydrogel composition, and d) optionally one or more tolerance-promoting immune modulators, wherein the Phase B composition comprises one or more modified or non-modified food allergens including natural and recombinant food allergens or B cell epitope- containing fragments thereof .

Pharmaceutical composition according to claim 1 wherein the thermogelling hydrogel for Phase A compositions is selected from the group consisting of polyethylene, polypropylene, polyethylene oxide (PEO) , polypropylene oxide (PPO) , polyurethane, polyurea, polyamides, polycarbonates, polyaldehydes , polyorthoesters, polyiminocarbonates , poly caprolactone (PCL) , poly-D, L-lactic acid (PDLLA) , poly-L- lactic acid (PLLA) , lactides of said lactic acids, polyphosphazenes , polyglycolic acids, monomethoxypoly (ethylene glycol) (MPEG) , or copolymers or mixtures of any of the above including poly (lactic-co-glycolic acid) (PLGA) , copolymers of L-lactide and D, L-lactide, polyester copolymers, diblock copolymers consisting of MPEG and PCL, MPEG and PCL-ran-PLLA, MPEG and PLGA, PEO and PLLA, and triblock copolymers consisting of PEO-PPO-PEO, PEG-PCL-PEG, PEG-PLGA-PEG, and PLGA-PEG-PLGA, wherein the thermogelling hydrogel is a biodegradable or biostable polymer, preferably biodegradable, more preferably biodegradable and reverse thermogelling, wherein the gelling temperature is between 20°C and 40°C, preferably between 25°C and 35°C, and/or wherein 90% degradation of the polymer weight in body environment and/or 90% release of hydrogel-embedded components from the polymer is completed within 1 to 14 days, preferably within 2 to 7 days.

Pharmaceutical composition according to claims 1 and 2, wherein the food allergen-derived T cell peptides for Phase A compositions are encapsulated in hydrogel- embedded tolerance-promoting phosphatidyl -L-serine- presenting liposomes (PS-liposomes) , wherein PS- liposomes include conventional PS- liposomes, PS-ethosomes , PS- niosomes, and elastic PS-liposomes , preferably conventional PS-liposomes, wherein conventional PS- liposomes include multilamellar and large or small unilamellar PS-liposomes, preferably unilamellar PS- liposomes with a diameter of 0.5-5 μιη, more preferably unilamellar PS-liposomes with a diameter of 0.8-1.5 μπι, wherein for the preparation of conventional PS-liposomes various lipid mixtures containing phosphatidyl-L-serine (PS) , phosphatidylcholine (PC) and, optionally, cholesterol (CH) are applicable, wherein lipid mixtures comprising molar ratios of PS : PC of 30:70 up to 50:50 for PS-containing liposomes without cholesterol and molar ratios of PS:PC:CH Of 10:50:40 up to 30:30:40 for PS- containing liposomes with cholesterol are preferred.

Pharmaceutical composition according to claims 1 to 3, wherein hydrogel-embedded synthetic oligodeoxynucleotides (ODN) with one or more CpG or GpC or GpG motifs for Phase compositions are selected from a) ODN containing a fully or partially nuclease-resistant phosphorothioated backbone with one or more CpG or GpC or GpG motifs, and b) ODN containing a nuclease-susceptible natural phosphodiester backbone (PO ODN) with one or more CpG or GpC or GpG motifs, wherein PO ODN are preferred for co- encapsulation with food allergen-derived T cell peptides in hydrogel-embedded PS-liposomes .

Pharmaceutical composition according to claims 1 to 4, wherein find-me molecules for attracting peripheral APC to the site of administered hydrogel Phase A compositions are selected from lysophosphatidyl-choline (LPC) , sphingosine-1-phosphate (SIP) and the nucleotides ATP and UTP, wherein ATP and UTP are preferred find-me molecules.

Pharmaceutical composition according to claims 1 to 5, wherein optional tolerance-promoting immune modulators for Phase A compositions are selected from vitamin D3 , preferably calcidiol, vitamin D3 derivatives exhibiting a short serum half-life and comparable tolerance-promoting effects as calcitriol and calcidiol, but low effects on calcium metabolism, preferably calcipotriol, and water- soluble glucocorticoids, preferably dexamethasone phosphate, wherein lipophilic tolerance-promoting immune modulators including vitaimin D3 and derivatives thereof preferably are incorporated into the lipid layer of hydrogel-embedded PS- liposomes containing encapsulated food allergen-derived T cell peptides or both food allergen-derived T cell peptides and CpG-ODN, and wherein water-soluble glucocorticoids are provided as hydrogel- embedded immune modulators or as liposomal immune modulators encapsulated in hydrogel-embedded PS- liposomes together with food allergen-derived T cell peptides or both food allergen-derived T cell peptides and CpG-ODN.

Composition according to any of the claims 1 to 6, wherein all components of each hydrogel composition for Phase A are mixed as a single preparation prior to injection, wherein the components are mixed with each other in a therapeutically effective quantity, wherein optionally galenic compounds are additionally admixed to the preparation, and wherein the composition is galenically prepared for subcutaneous, intramuscular, or intraocular administration, preferably for subcutaneous administration.

8. Use of a Phase A composition according to one of the claims 1 to 7 for re-directing the allergic T cell status towards tolerance by decreasing disease-promoting effector T cells and increasing tolerance-promoting regulatory T cells without anaphylactic risks for food allergic patients, wherein subsequent administration of a Phase B composition according to claim 1 after sufficient modulation of CD4⁺ T-cells towards IL-10 -secreting CD4⁺ T cells with an anergic, regulatory phenotype is more efficient in inducing the generation of protective allergen-specific antibodies, and significantly less associated with the risk of adverse side effects, wherein the treatment duration of Phase B is determined individually based on the development of protective antibodies and the improvement of the clinical score upon oral food challenges.