**5. Approaches for an immunotherapy of allergy based on modified recombinant allergens**

Several *in vitro* and *in vivo* assays indicate that allergy vaccines based on recombinant allergens might have provide a safe and efficacious immunotherapy. However, recombinants with the same amino acid sequence and similar allergenic activity as the natural allergens can elicit IgEmediated side effects, which are a major risk during allergen-specific immunotherapy. To overcome this problem different approach have been designed to obtain molecules without or reduced IgE reactivity [76]. Recombinant DNA technology has allowed the rational design and production of well-defined modified allergens for this purpose. Hypo-allergens are molecules derived from wild type allergen which exhibits reduced capacity to react with IgE antibodies and low ability to induce IgE-mediated mast cells or basophile degranulation. They are designed to reduce the risk of anaphylaxis during the course of immunotherapy, are molecules with conserved T-cell epitopes that could be recognized by specific T lymphocytes to induce a protective response against wild-type allergen.

Some allergens are present in the nature as a mix of several isoforms with high structural homology but different IgE reactivity. The production by molecular cloning of natural isoforms with low IgE reactivity has been used to propose anti-allergy vaccines. Bet v 1.0401 and Bet v 1.1001 are isoforms that have lower IgE reactivity compared to the Bet v 1.0101 [77, 78]. Antibody response against Bet v 1.0401 is IgG4-specific and has low capacity to induce basophile degranulation [79].

### **Hypo-allergens obtained by site-directed mutagenesis**

The availability of multiple clones of recombinant allergens has facilitated the implementation of site directed mutagenesis to obtain modified allergens for a better immunotherapy. There are several examples of this approach that illustrate the potential use for the development of new vaccines. Mouse allergic individuals are sensitized mainly against the major allergen Mus m 1 a urinary protein belonging to the lipocalin superfamily which have typicall β-barrel fold, that can be modified by mutation in the Tyr 120 residue, [80, 81]. Two hypo-allergenic variants of this allergen; mutants 1Y120A, and Mus m 1-Y120F were expressed in *P. pastoris* with a modified fold [82]. The mutants showed low capacity to react with IgE from allergic individ‐ uals and induced lower basophil degranulation than those induced by Mus m 1. In lympho‐ proliferation assays, using cells from mouse allergic individuals the mutants induced similar lymphoproliferation to that induced by Mus m 1. In other study three variants of an allergen from Artemisia (Art v 1), C22S, C47S and C49S [83], showed low IgE reactivity and mediator release from RBL cells. In addition, the variants C49S and C22S induced significantly higher T cell proliferative response in Artemisia allergic patients compared to the obtained with rArt v 1, suggesting a potential utility for the immunotherapy of population allergic to Artemisia. Using a different approach; mutations in residues involved in IgE binding but maintaining the 3D structure of the natural allergen, Spangforth et al. showed that mutants Gln45-Ser and Pro108-Gly of allergen Bet v 1 displayed lower IgE reactivity and induced synthesis of IgG antibodies that block the IgE reactivity against the natural Bet v1 [84]. Unlike the above mentioned studies. The x-ray crystallography structures of the mutants were similar to the natural allergen, indicating that the reduced IgE reactivity is not mediated by an inadequate folding.

### **Hybrid proteins**

groups and treated for two years with rBet v 1, natural birch pollen extract, natural Bet v 1 (nBet v 1) or placebo, to compare the efficacy of each preparation for allergen-specific immu‐ notherapy. Treatment with rBet v 1 reduced symptoms of rhinoconjunctivitis and skin reactivity induced by birch pollen, and showed to be safety without serious adverse events. In contrast, one adverse event appears in the group treated with nBet v 1. Clinical improvement and reduction of sensitivity were accompanied with marked increase in Bet v 1-specific IgG1, IgG2 and IgG4 levels, which were higher in the rBet v 1-treated group than in nBet v 1-treated group. Importantly, new IgE specificities were induced in 3 patients treated with birch pollen

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

In a placebo controlled immunotherapy study, a mixture of equimolar concentration of five *Phleum pretense* allergens (Phl p 1, Phl p 2, Phl p 5a, Phl p 5b and Phl p 6) was administered via subcutaneous for 18 months in patients with grass pollen–induced allergic rhinitis, to deter‐ mining efficacy and safety [43]. The immunotherapy showed a 36.5% lower median average symptom score for active treatment compared with placebo and reduction in the need for medication. By the first and second pollen season, improvement in quality of life scores was present in the patients receiving active treatment. Active treatment induced IgG1 concentra‐ tions approximately 60-fold, peaking during the rusty 12 months of the study and IgG4 levels showed a 4000 fold increase by the end of treatment. In contrast, after immunotherapy IgE levels in the active treated group were significantly lower than placebo group. Only about 1% of recombinant grass allergen injections led to systemic reactions. This was the first clinical study of immunotherapy with a cocktail of 5 recombinant grass pollen allergens that showed its clinical efficacy, good tolerance and strong induction of allergen specific IgG antibody

**5. Approaches for an immunotherapy of allergy based on modified**

Several *in vitro* and *in vivo* assays indicate that allergy vaccines based on recombinant allergens might have provide a safe and efficacious immunotherapy. However, recombinants with the same amino acid sequence and similar allergenic activity as the natural allergens can elicit IgEmediated side effects, which are a major risk during allergen-specific immunotherapy. To overcome this problem different approach have been designed to obtain molecules without or reduced IgE reactivity [76]. Recombinant DNA technology has allowed the rational design and production of well-defined modified allergens for this purpose. Hypo-allergens are molecules derived from wild type allergen which exhibits reduced capacity to react with IgE antibodies and low ability to induce IgE-mediated mast cells or basophile degranulation. They are designed to reduce the risk of anaphylaxis during the course of immunotherapy, are molecules with conserved T-cell epitopes that could be recognized by specific T lymphocytes to induce

Some allergens are present in the nature as a mix of several isoforms with high structural homology but different IgE reactivity. The production by molecular cloning of natural isoforms

extract, but in none of rBet v 1 or nBet v 1 treated patients.

response.

Applications

298

**recombinant allergens**

a protective response against wild-type allergen.

Hybrid proteins are structures composed by two or more allergens or short portions of them in only one molecule, in this way new interaction and bonds are generated, which may alter the 3D structure and B epitopes characteristic of natural allergens. Decreasing the capacity of IgE binding and mast cell degranulation. However, if these proteins conserve the T cell epitopes, they could induce a protective response after allergen challenge. A single molecule composed by different allergen polypeptides might reduce the number of molecules to be included in the vaccine. Furthermore, hybrid molecules consisting of several copies of homologous allergens or immunologically unrelated allergens could be used for the allergy treatment in the patients who are sensitized to several allergens.

T. P. King, who constructed a molecule composed by two allergens from insect and demon‐ strated *in vitro* and mice models studies its anti-allergenic properties [85], pioneered the design of hybrid proteins for allergen immunotherapy. Gonzales-Rioja *et al*. [86] obtained by PCRbased engineering a molecule composed by two allergens from the pollen *Parietaria judaica*

(Par j 1 and Par j 2) and demonstrated an important reduction of the IgE binding capacity by skin prick test. Linhart *et al*. [87], constructed by PCR-based recombination, a nucleotide sequence coding for principal allergens of timothy grass, Phl p 1, Phl p 2, Phl p 5 and Phl p 6. The hybrid protein induced T cell proliferation similar to the equimolar mix of individual allergens, the lymphocytes secreted regulatory and Th1 cytokines, IL-10 and IFN-γ, showing capacity to induce immune deviation to a protective profile. In an allergic mouse model, exposure to hybrid molecule induced the production of IgG that blocked mast degranulation. However, this molecule was also capable to bind IgE from allergic individuals, and to induce basophile degranulation, and high percentage of individuals allergic to timothy grass were identified using this molecule, suggesting a potential as a diagnosis reagent.

protein constructed with segments derived from Phl p 2 reassembled in altered order and expressed as a trimer showed absence of IgE reactivity with sera from allergic patients. Basophile activation and skin prick tests, showed reduction of the allergenicity of this molecule compared to recombinant Phl p 2. Furthermore, IgG antibodies produced by immunized mice were able to inhibit the binding of recombinant Phl p 2 to the IgE from allergic subjects [91]. Mosaic proteins have been studied as a potential vaccine for immunotherapy of birch allergy [92] and house dust mite allergy [93]. A mosaic protein composed of reorganized segments of Bet v 1 preserved the specific T cell epitopes and showed approximately 100-fold reduced allergenic activity compared with recombinant Bet v 1 [94, 95] and induced specific IgG antibodies inhibitors of IgE reactivity to Bet v1 of sera from patients with pollen allergy [96]. The mosaic protein exhibited none IgE reactivity and lower basophile activation. Furthermore, immunization with Bet v 1 derivatives induced IgG antibodies that recognized Bet v 1 and

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Fragments of allergens or modification of these, might be poorly immunogenic because they don´t have enough T cell epitopes capable to stimulate a protective immune response. An increase of immunogenicity can be obtained when proteins are made as oligomers which enhance the number of T cell epitopes in the molecule. It has been observed that immunoge‐ nicity of Bet v1 increase when obtained as oligomer [97, 98]. By dot-blot analysis and lympho‐ proliferative responses in PBMCs from birch pollen allergic patients, trimers of re-organized segments of Bet v1 had lower capacity to bind IgE and enhanced capacity to stimulate lymphoproliferation. The CD203c expression analysis showed reduced allergenicity of these oligomers, and when administrated to mice in an immunization scheme, induced the produc‐ tion of high titer of IgG1 antibody, that inhibited human IgE binding to wild type Bet v 1 [99].

It has been suggested that administration of higher allergen doses enhances the efficacy of immunotherapy [100]. However, administration of high doses increases the risk of anaphy‐ laxis. One approach to overcome this problem is to deliver high doses of allergen to B and T cells directly, thus providing higher effective doses to stimulate a protective response, and avoiding the interaction of allergen with IgE antibodies [101]. An allergen vaccine for cat allergy composed of the major cat allergen Fel d 1 fused to the HIV-derived translocation peptide TAT was designed to mediate cytoplasmic uptake of extracellular proteins [102, 103]. Un modified version of this approach, denominated Modular Antigen Translocation (MAT) technology have been developed [104, 105], which consists of allergen fused to a peptide, to direct them to the cytosol, and a truncated human invariant chain (Ii), to target the protein to MHC class II heterodimers assembled in the endoplasmic reticulum and thus circumventing phagosomal uptake and degradation. The allergens Asp f 1, Der p 1, Bet v 1, PLA2 and Fel d 1 fused to MAT, induced lymphoproliferation of PBMCs stimulated *ex vivo* with low allergen doses, induced the secretion of Th1 type cytokines and IL-10, and inhibit the production of Th2 cytokines [105]. The cat allergen Fel d 1 fused to MAT (MAT- Fel d 1) when administered directly to the inguinal lymph nodes of allergic mice, showed capacity to stimulate the

inhibited IgE binding to Bet v 1 [92].

**5.1. Molecules to target specific compartments or receptors**

**Targeting allergens to endoplasmic reticulum**

The utility of hybrid proteins for the immunotherapy of house dust mite allergy have been studied by Asturias, *et al*. [88], who designed two hybrid proteins composed by *D. pteronyssi‐ nus* allergens Der p 1 and Der p 2. The QM1 structure was composed of almost the whole sequence of both allergens; a Der p 2-fragment from residues 5 to 123 at the N-terminus, introducing point mutations on cysteine 8 and 119 to serine to avoid the formation of a disulphide bridge, and a Der p 1-fragment from residues 4 to 222 at the C-terminus were joined. The QM2 structure, was composed with the residues 1 to 73 and 74 to 129 of Der p 2, linked to residues 5 to 222 of Der p 1. Western-blot assays with a serum pool from house dust mite allergic patients showed decrease IgE reactivity of QM1, while QM2 showed no detectable IgE binding capacity. The hypoallergenic properties of both hybrids were demonstrated by skin tests. In vitro test with sample from allergic patients QM2 induced similar lymphoproliferation that the induced by natural Der p 1 and Der p 2, whereas, QM1 induced higher proliferation. It was demonstrated that antisera raised by immunization of mice with QM1 or QM2 lead to the production of specific antibodies capable of blocking the binding of IgE reactivity to natural allergens.

Recently, a hybrid protein composed of three allergens of *Chenopodium album* pollen, in the order Che a 3-Che a 1-Che a 2, was constructed by using overlapping extension polymerase chain reaction, expressed in *E. coli* BL21-CodonPlus(DE3)-RIL, to obtain a 46 kDa protein. Sera from allergic patients showed lower IgE binding affinity to the hybrid molecule than the mixture of recombinant allergens and the *C. album* pollen extract. Most of the allergic patients showed positive skin test to a mixture of the three allergens, however, when tested with the hybrid molecule allergic patients showed negative test or highly reduced weal area compared to the mixture or to the pollen extract [89].

#### **Mosaic proteins**

Mosaic proteins are constituted by different segments of the same allergen, in different order as they are present in the native molecule, such re-arrange generate new intra-molecular interactions that alter B cell epitopes. A mosaic protein called P1m constructed with four segments of the pollen allergen Phl p 1, showed lower IgE reactivity compared to the natural allergen and was unable to induce histamine release from basophiles of allergic individuals. However, this molecule conserved capacity to induce the proliferation of PBMCs. Immunized rabbits expressed IgG antibodies that blocked the binding of Phl p 1 to the IgE and inhibit histamine release from basophiles obtained from allergic individuals [90]. Other mosaic protein constructed with segments derived from Phl p 2 reassembled in altered order and expressed as a trimer showed absence of IgE reactivity with sera from allergic patients. Basophile activation and skin prick tests, showed reduction of the allergenicity of this molecule compared to recombinant Phl p 2. Furthermore, IgG antibodies produced by immunized mice were able to inhibit the binding of recombinant Phl p 2 to the IgE from allergic subjects [91]. Mosaic proteins have been studied as a potential vaccine for immunotherapy of birch allergy [92] and house dust mite allergy [93]. A mosaic protein composed of reorganized segments of Bet v 1 preserved the specific T cell epitopes and showed approximately 100-fold reduced allergenic activity compared with recombinant Bet v 1 [94, 95] and induced specific IgG antibodies inhibitors of IgE reactivity to Bet v1 of sera from patients with pollen allergy [96]. The mosaic protein exhibited none IgE reactivity and lower basophile activation. Furthermore, immunization with Bet v 1 derivatives induced IgG antibodies that recognized Bet v 1 and inhibited IgE binding to Bet v 1 [92].

Fragments of allergens or modification of these, might be poorly immunogenic because they don´t have enough T cell epitopes capable to stimulate a protective immune response. An increase of immunogenicity can be obtained when proteins are made as oligomers which enhance the number of T cell epitopes in the molecule. It has been observed that immunoge‐ nicity of Bet v1 increase when obtained as oligomer [97, 98]. By dot-blot analysis and lympho‐ proliferative responses in PBMCs from birch pollen allergic patients, trimers of re-organized segments of Bet v1 had lower capacity to bind IgE and enhanced capacity to stimulate lymphoproliferation. The CD203c expression analysis showed reduced allergenicity of these oligomers, and when administrated to mice in an immunization scheme, induced the produc‐ tion of high titer of IgG1 antibody, that inhibited human IgE binding to wild type Bet v 1 [99].

#### **5.1. Molecules to target specific compartments or receptors**

### **Targeting allergens to endoplasmic reticulum**

(Par j 1 and Par j 2) and demonstrated an important reduction of the IgE binding capacity by skin prick test. Linhart *et al*. [87], constructed by PCR-based recombination, a nucleotide sequence coding for principal allergens of timothy grass, Phl p 1, Phl p 2, Phl p 5 and Phl p 6. The hybrid protein induced T cell proliferation similar to the equimolar mix of individual allergens, the lymphocytes secreted regulatory and Th1 cytokines, IL-10 and IFN-γ, showing capacity to induce immune deviation to a protective profile. In an allergic mouse model, exposure to hybrid molecule induced the production of IgG that blocked mast degranulation. However, this molecule was also capable to bind IgE from allergic individuals, and to induce basophile degranulation, and high percentage of individuals allergic to timothy grass were

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

The utility of hybrid proteins for the immunotherapy of house dust mite allergy have been studied by Asturias, *et al*. [88], who designed two hybrid proteins composed by *D. pteronyssi‐ nus* allergens Der p 1 and Der p 2. The QM1 structure was composed of almost the whole sequence of both allergens; a Der p 2-fragment from residues 5 to 123 at the N-terminus, introducing point mutations on cysteine 8 and 119 to serine to avoid the formation of a disulphide bridge, and a Der p 1-fragment from residues 4 to 222 at the C-terminus were joined. The QM2 structure, was composed with the residues 1 to 73 and 74 to 129 of Der p 2, linked to residues 5 to 222 of Der p 1. Western-blot assays with a serum pool from house dust mite allergic patients showed decrease IgE reactivity of QM1, while QM2 showed no detectable IgE binding capacity. The hypoallergenic properties of both hybrids were demonstrated by skin tests. In vitro test with sample from allergic patients QM2 induced similar lymphoproliferation that the induced by natural Der p 1 and Der p 2, whereas, QM1 induced higher proliferation. It was demonstrated that antisera raised by immunization of mice with QM1 or QM2 lead to the production of specific antibodies capable of blocking the binding of IgE reactivity to natural

Recently, a hybrid protein composed of three allergens of *Chenopodium album* pollen, in the order Che a 3-Che a 1-Che a 2, was constructed by using overlapping extension polymerase chain reaction, expressed in *E. coli* BL21-CodonPlus(DE3)-RIL, to obtain a 46 kDa protein. Sera from allergic patients showed lower IgE binding affinity to the hybrid molecule than the mixture of recombinant allergens and the *C. album* pollen extract. Most of the allergic patients showed positive skin test to a mixture of the three allergens, however, when tested with the hybrid molecule allergic patients showed negative test or highly reduced weal area compared

Mosaic proteins are constituted by different segments of the same allergen, in different order as they are present in the native molecule, such re-arrange generate new intra-molecular interactions that alter B cell epitopes. A mosaic protein called P1m constructed with four segments of the pollen allergen Phl p 1, showed lower IgE reactivity compared to the natural allergen and was unable to induce histamine release from basophiles of allergic individuals. However, this molecule conserved capacity to induce the proliferation of PBMCs. Immunized rabbits expressed IgG antibodies that blocked the binding of Phl p 1 to the IgE and inhibit histamine release from basophiles obtained from allergic individuals [90]. Other mosaic

identified using this molecule, suggesting a potential as a diagnosis reagent.

allergens.

Applications

300

**Mosaic proteins**

to the mixture or to the pollen extract [89].

It has been suggested that administration of higher allergen doses enhances the efficacy of immunotherapy [100]. However, administration of high doses increases the risk of anaphy‐ laxis. One approach to overcome this problem is to deliver high doses of allergen to B and T cells directly, thus providing higher effective doses to stimulate a protective response, and avoiding the interaction of allergen with IgE antibodies [101]. An allergen vaccine for cat allergy composed of the major cat allergen Fel d 1 fused to the HIV-derived translocation peptide TAT was designed to mediate cytoplasmic uptake of extracellular proteins [102, 103]. Un modified version of this approach, denominated Modular Antigen Translocation (MAT) technology have been developed [104, 105], which consists of allergen fused to a peptide, to direct them to the cytosol, and a truncated human invariant chain (Ii), to target the protein to MHC class II heterodimers assembled in the endoplasmic reticulum and thus circumventing phagosomal uptake and degradation. The allergens Asp f 1, Der p 1, Bet v 1, PLA2 and Fel d 1 fused to MAT, induced lymphoproliferation of PBMCs stimulated *ex vivo* with low allergen doses, induced the secretion of Th1 type cytokines and IL-10, and inhibit the production of Th2 cytokines [105]. The cat allergen Fel d 1 fused to MAT (MAT- Fel d 1) when administered directly to the inguinal lymph nodes of allergic mice, showed capacity to stimulate the

production of high levels of IFN-γ and reduced levels of IL-4, compared to unmodified Fel d 1. Immunized mice expressed higher levels of IgG2a and showed protection against the challenge of high doses of allergenic extract. Furthermore, MAT-Fel d 1 produced 100-fold less degranulation and histamine release from basophiles compared to unmodified Fel d 1 [106].

Th2 cytokines, in a mechanism in which the thymic stromal lymphopoietin (TSLP) cytokine seems to be involved. This cytokine was shown to maintain and polarize circulating Th2 central memory cells, including allergen-specific T cells [115]. Therefore, the usefulness of this kind

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Insect sting allergy are frequently caused by insect stings of the Apidae family (honeybees and bumblebees), those from the Vespidae family (Vespula, Dolichovespula, Vespa and Polistes genera) and, in some regions, also of the Formicidae family (ants). The sting can induce local or systemic IgE-mediated hypersensitivity reactions that can be fatal [116]. Prevalence of systemic reactions caused by insect stings are reported from 0,3% to 7,5% in the United States and Europe [117, 118]. Up to one fifth of these subjects will eventually experience severe lifethreatening reactions. Hymenoptera venoms contain protein allergens, as well as nonallergenic components, including toxins, vasoactive amines, acetylcholine, and kinins. Among the multiple allergens in Hymenoptera venoms, two allergens are importan, the phospholipase A2 from of honey bee (*Apis mellifera*) (Api m 1), and of the vespid venoms antigen 5 from

Several studies have demonstrated that immunotherapy for vespid allergy with venom extracts is clinically effective and improve the quality of life and allergic symptoms. This improvement is correlated to a significant decrease of total IgE levels, and increase in specific IgG and IgG4 levels [119]. However, severe and life-threatening anaphylactic side effects may

One of the first attempts to obtain safer methods for immunotherapy of insect allergies was made with allergen-derived peptides, containing T-cell epitopes. Peptides derived from the bee allergen Api m 1, were applied to allergic individuals in different immunotherapy schemes. *In* vitro and clinical phase trials showed that T cells from such patients showed marked responsiveness to Api m 1 after long term treatment, a shift in the pattern of cytokine

The use of recombinant venom allergens for allergen specific immunotherapy has been analyzed in animal models. Intranasal administration of the recombinant allergen from wasp venom, rVes v 5, to mice prior to sensitization with natural allergens lead to a significant reduction of the allergic reaction, reduction of specific IgE and IgG2a levels, increase of mRNA levels of IL-10 and TGF-β. Pretreatment with the whole venom was less effective and caused toxic side reactions, suggesting a favorable use of the recombinant protein [124]. Hybrid proteins composed by allergens from bee venom have shown anti-allergenic properties in *in vitro* and animal models [85]. A fusion protein composed of the two major bee venom allergens Api m 1 and Api m 2 called Api m [1/2], showed reduced IgE reactivity of Api m [1/2] compared with native allergens [125]. By the other hand, basophil degranulation and skin tests showed that this fusion protein have hypo-allergenic properties. When applied subcutaneously, mice

secretion form a Th0 to a Th1 profile and increase in specific IgG4 levels [121-123].

of preparation for allergy immunotherapy deserves further evaluation.

*Vespula vulgaris* (common wasp) denominated Ves v 5.

be induced after the administration of crude allergen extracts [120].

**6. Insect sting allergy**

### **Targeting allergens to receptors on dendritic cells**

Dendritic cells (DCs) play an important role in the initiation and maintenance of T cell response to allergens. Its role in the type of T cell response generated can be influenced by the maturation state, while mature DCs induce effector T cell responses characterized by Th1 or Th2 response [107], immature or semi-mature DCs are tolerogenic and have the ability to induce Tregs [108]. DCs express an array of Fc receptors which have the capacity to enhance allergen uptake through internalization of allergen/antibody receptors complexes. When stimulated with allergen, DCs express FcεRI, and activated a signal-transducing cascade involving immunor‐ eceptor tyrosine-based activation motif (ITAM), which result in increased production of proinflammatory cytokines and chemokines, the induction of robust proliferation of allergenspecific T cells and the development of allergic symptoms [109]. DCs also express the receptor FcγRIIb that contains immunoreceptor tyrosine-based inhibition motif (ITIM) which induces inhibitory signaling events. This receptor can co-aggregate with FcεRI that activating a signaling cascade that culminates in inhibition of FcεRI signaling. Under these assumptions, Zhu, D. *et al*. [110] designed a fusion molecule called GFD composed by a human IgG Fc fragment linked to the allergen Fel d 1 by a flexible linker, with the aim to crosslink FcγRIIb and FcεRI-bounded to the cat specific-IgE. In transgenic mice expressing human FcγRIIb and FcεRI, sensitized with high doses of Fel d 1 specific-IgE and treated with several doses of GFD, the challenge with Fel d 1 didn´t cause mast cell degranulation. A scheme of immunotherapy with high doses of GFD resulted in the inhibition of allergic response against Fel d 1, pulmo‐ nary inflammation and skin reactivity in sensitized animals. Treated mice expressed IgG1 antibodies that blocked the binding of IgE to Fel d 1. When applied to mice sensitized to Fel d 1 in a scheme of rush immunotherapy, GFD blocked acute systemic allergic reaction, mast cell degranulation, bronquial hyper-reactivity and pulmonary inflammation [111]. Recently, a fusion protein composed of Fcγ chain and the *Dermatophagoides farina*e allergen, Der f 2, was obtained and tested in a Der f 2 allergic murine model [112]. After treatment with the fusion molecule, the levels of specific IgE to Der f 2, histamine and pro-inflammatory cytokines were lowered in the Fcγ-Der f 2 treated allergic mice, compared to saline-treated allergic mice. These results suggest that specific targeting of allergens to Fcγ receptors could be used as a strategy in the development of antigen-specific immunotherapy for human allergic diseases.

A different molecular design was applied to target allergens to CD64 receptor on antigen presenting cells; a fusion protein (H22-Fel d 1) composed by Fel d 1 linked to the variable region of a monoclonal antibody anti-CD64 was designed to stimulate receptor internalization [113]. Flow cytometry analysis showed that H22-Fel d 1 binds to CD64 and reacted with IgE and IgG with similar affinity compared to native allergen. *In vitro* assays demonstrated that the fusion molecule stimulates the proliferation of T lymphocytes derived from allergic individuals and the secretion of IL-5, IL-10 and IFN-γ [114]. Although H22-Fel d 1 is responsible of a positive effect that could result in a protective response against allergen challenge, it also stimulated Th2 cytokines, in a mechanism in which the thymic stromal lymphopoietin (TSLP) cytokine seems to be involved. This cytokine was shown to maintain and polarize circulating Th2 central memory cells, including allergen-specific T cells [115]. Therefore, the usefulness of this kind of preparation for allergy immunotherapy deserves further evaluation.
