BR102016022313A2 - PROCEDURE FOR THE PRODUCTION OF A RECOMBINANT HYPOALERGENIC ANTIGEN FOR PROPHILACTIC USE AND TREATMENT OF ALLERGIC DISEASES CAUSED BY THE ANTIGEN DROPIC BLOMIA DUST MITE - Google Patents

Abstract

The process of producing a hypoallergenic recombinant antigen for the prophylactic use and treatment of allergic diseases caused by the tropical blomy dust mite, antigen, antigen-containing formulations of the present invention relates to the design and production of a recombinant antigen derived from a hypoallergenic allergen. blomia tropicalis having potential for use in vaccine formulations for allergic diseases caused by this dust mite. more particularly the invention relates to the field of specific allergen immunotherapy for immunoglobulin and (ige) mediated allergic diseases and caused by the blomia tropicalis dust mite. more specifically, it is a mutant polypeptide derived from the block molecule whose amino acid sequences are shown in sequence: 1 characterized by a reduction in IgE reactivity in sera from allergic patients, as well as lacking the ability to activate basophils. humanized mice sensitized with sera from allergic patients. also refers to methods of producing and purifying the recombinant mutant antigen presented in seq. id: 1 in a prokaryotic expression system in e.g. coli.

Description

(54) Title: PROCESS OF PRODUCTION OF A HYPOALLERGENIC RECOMBINANT ANTIGEN FOR THE PROPHYLATIC USE AND TREATMENT OF ALLERGIC DISEASES CAUSED BY THE TROPICAL DUST MITE, ANTIGEN, FORMULATIONS CONTAINING THE ANTIGEN (51) Int. CL4: C07K A61K 39/35; A61P 37/08; C12N 15/62 (73) Holder (s): FEDERAL UNIVERSITY OF BAHIA (72) Inventor (s): NEUZA MARIA ALCÂNTARA NEVES; JUAN RICARDO URREGO ALVAREZ; EDUARDO SANTOS DA SILVA; LUIS GUSTAVO CARVALHO PACHECO; CARINA DA SILVA PINHEIRO (57) Abstract: Production process of a hypoallergenic recombinant antigen for the prophylactic use and treatment of allergic diseases caused by the tropical dust mite Blomia, antigen, formulations containing the antigen The present invention relates to the design and production of a recombinant antigen derived from an allergen of Blomia tropicalis having potential for use in vaccine formulations for allergic diseases caused by this dust mite. More particularly, the invention relates to the field of allergen specific immunotherapy for allergic diseases mediated by immunoglobulin E (IgE) and caused by the dust mite Blomia tropicalis. More specifically, it is a mutant polypeptide derived from the Blo t 2 molecule whose amino acid sequences are presented in SEQ ID NO: 1 characterized by a reduction in IgE reactivity in sera from allergic patients, in addition to not having the ability to activate basophils humanized rats sensitized with sera from allergic patients. It also refers to the methods of production and purification of the mutant recombinant antigen presented in SEQ ID No: 1 in an e (...)

MW (kDa) A.l. D.l. D.L.S. D.P.A. M.P

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TITLE: “Process of production of a hypoallergenic recombinant antigen for prophylactic use and treatment of allergic diseases caused by dust mites

Tropical blomia, antigen, formulations containing the antigen ”

Field of the Invention [001] The present invention relates to the design and production of a hypoallergenic recombinant protein derived from the sequence of the Blo t 2 allergen present in the dust mite Blomia tropicalis and the respective antigen production processes and formulations containing the antigen .

[002] More particularly the invention relates to the field of immunotherapy specific allergen of allergies mediated by immunoglobulin E (IgE) and caused by the mite Blomia tropicalis and any other cross-reactive mite Ex, Dermatophagoides pteronyssinus.

[003] More specifically the invention is a polypeptide protein derived from the sequence of Blo t 2 that has been subjected to an in-silic mutation of amino acids present on the surface of the molecule in order to reduce the ability to bind with IgE.

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Foundations of the invention [004] Allergic diseases are a public health problem that affects quality of life and can even cause the death of patients [King RM, Knibb RC, Hourihane JO: Impact of peanut allergy on quality of life, stress and anxiety in the family. Allergy 2009, 64 (3): 461-468.]. The prevalence of these diseases has increased in recent years worldwide [Isolauri E, Huurre A, Salminen S, Impivaara O: The allergy epidemic extends beyond the past few decades. Clin Exp Allergy, 2004, 34 (7): 1007-1010 .; Branum AM, Lukacs SL: Food allergy among children in the United States. Pediatrics 2009, 124 (6): 1549-1555], and it is likely that this increase is more related to environmental or lifestyle changes than to changes in the genetic profile of individuals [Devereux G, Barker RN, Seaton A: Antenatal determinants of neonatal immune responses to allergens. Clin Exp Allergy, 2002, 32 (1): 43-50]. It is estimated that more than 25% of the world population has some type of allergic disease such as asthma, renitis or atopic dermatitis. Most allergic diseases are characterized by an exacerbated immune response, in which antibody titers are increased, against substances that are generally not dangerous, called allergens. These allergenic substances are present in many sources such as dust mites, animal hair, drugs and food. [Platts-Mills TA, Woodfolk JA: Allergens and their role in the allergic immune response. Immunological reviews 2011, 242 (1): 51-68] [005] Repetitive and persistent exposure to allergens produces chronic and inflammatory inflammation associated with the high production of specific type antibodies

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3/22 immunoglobulin E (IgE) and the proliferation of CD4 + T lymphocytes responsible for the production profile of Th2 cytokine secretion [Galli SJ, Tsai M, Piliponsky AM: The development of allergic inflammation. Nature 2008, 454 (7203): 445-454]. The cytokines of the Th2 profile such as IL-4, IL-13 and IL-5 are responsible for the activation and migration of pro-inflammatory cells to the site of contact with the allergen. In summary, the immunopathogenesis of allergic diseases begins with the production of the IgE antibody by B lymphocytes, followed by the binding of this antibody to the high affinity (FceRI) and low affinity (FcsRIII) receptors of mast cells, basophils and eosinophils, triggering type I hypersensitivity. After repeated exposure of the allergen, it binds to the fixed antibodies in the pro-inflammatory cells and initiates the degranulation and release of pro-inflammatory molecules such as histamine, leukotriens, protaglandins, proteases and pro-inflammatory cytokines. These molecules cause a wide variety of symptoms, depending on the part of the body where they are released, for example, on the skin (urticaria, angiodema, eczema), gastrointestinal tract (nausea, vomiting and abdominal pain along with diarrhea) and on the respiratory system (rhinitis, cough, wheezing and asthma) and systemic ( anaphylaxis) [006] Until now, for diseases like asthma there is no treatment available on the market that is 100% effective. Current drugs are focused on controlling the symptoms of the disease, as in the case of the use of glucocorticoids, antihistamines, etc. In addition, allergic diseases represent very high costs for health systems around the world [Sicherer SH, Leung DY: Advances in allergic skin disease, anaphylaxis, and hypersensitivity reactions to foods, drugs, and insects in 2012. J Allergy Clin Immunol. 2013, 131 (1): 55-66]. For example, in the United States of America, expenses related to asthma treatment were around US $ 10,748,000 in just one

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4/22 year, and for the treatment of rhinitis it was approximately $ 1,233,000 [Gergen PJ: Understanding the economic burden of asthma. J Allergy Clin Immunol 2001, 107 (5

Suppl): S445-448.].

[007] In Brazil, asthma was the third cause of hospitalization in 2001 according to the federal government's DATASUS database, with the total cost to the public health system of US $ 35,000,000 per year for this disease [L.A. Santos, M.A. Oliveira, S.M.Direct costs of asthma in Brazil: a comparison between controlled and uncontrolled asthmatic patients. Braz. J. Med. Biol. Res (2007) 40: 943-948]. In Bahia, the total direct and indirect costs of patients with asthma, before 2006, were R $ 181,652.94 / year, that is, R $ 2,838.33 / year per patient. Currently, after the application of the Asthma and Allergic Rhinitis control program in Bahia (ProAR) [Franco R, Campos H, Sarinho EC. The cost of severe asthma for the public health system and for families. Gaz Med Bahia 2008; 78 (2): 45-51] costs were reduced to R $ 59,345 / year. Even so, the long-term goal is to try to reduce it with new therapeutic forms of intervention [Franco R, Nascimento HF, Cruz AA. Analysis of the cost effectiveness of PROAR - A model program for the control of severe asthma. Gaz Med Bahia 2008; 78 (1): 3-10.]

Dust Mite Allergy [008] More than 50% of allergic patients worldwide suffer from allergies caused by Blomia tropicalis and Dermatophagoides pteronyssinus mites [Sade K, Roitman D, Kivity S. Sensitization to Dermatophagoides, Blomia tropicalis, and other mites in atopic patients. J Asthma. 2010 Oct; 47 (8): 849-52]. Currently, several proteins from these mites

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5/22 have been identified and characterized. However, although Blomia tropicalis and Dermatophagoides pteronyssinus belong to different families (Glycyphagidae and

Pyroglyphidae respectively), they have 40% homology, which is a percentage considered low and therefore the sensitivity to these mites should be investigated separately.

[009] So far, Blomia tropicalis, only has 13 allergens recognized by the

International Union of Immunological Societies (IUIS), Blo t 5 being considered the most important allergen so far, and has been widely studied by our group [Carvalho Kdos A1, by Melo-Neto Blomia tropicalis Blo t 5 and Blo t 21 recombinant allergens might confer higher specificity to serodiagnostic assays than whole mite extract. BMC Immunol. 2013 Feb 27; 14: 11], and other groups [F. C. Yi, K. Y. Chua, Immunoglobulin E reactivity of native Blo t 5, a major allergen of Blomia tropicalis. Clin Exp Allergy 2004; 34: 1762-1767], however, little information is available about group 2 of allergens for this mite. In mites, group 2 has a lipid-binding domain, which consists of two flat beta leaves that form a hydrophobic internal cavity that can bind to lipid molecules. In the case of Dermatophagoides spp. group 2 is important, with very high reactivities in almost all populations [Jiménez, S. Puerta, L. Recombinant Allergens of Blomia tropicalis and Dermatophagoides pteronyssinus in a Tropical Environment. Allergy Clin Immunol Int

- J World Allergy Org. 2007, 19/16] It is noteworthy that the homology between Der p 2 and Blo t 2 is relatively low; however, they have 6 conserved cysteine residues that form important disulfide bonds for protein conformation and stability.

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6/22 [010] The group 2 of Blomia tropicalis allergens has several isoforms that are described at the transcriptome level, but until now, only one of them has been tested, however, this protein was obtained insoluble and with low reactivity of IgE [Christophel J, Expression of the Recombinant Group 2 Allergen from Blomia tropicalis Comparison of the Immunoreactivity of Blo t 2 and Blo t 5. J Allergy Clin Immunol. Vol.

109, Issue 1, Supplement 1, Page S135], However, further studies are needed to clarify the IgE reactivity of the allergen Blo t 2. Our group already has unpublished results that show that this allergen is a predominant protein in B. tropicalis and it seems to have an important allergenic role in the Brazilian population. According to our data, presented at the Congress of the European Academy of Allergy and Clinical Immunology (2016), at least 54% (22/44) of the sera of patients allergic to Blomia tropicalis from Salvador, Bahia, Brazil, tested, react to rBlo t 2; in addition, we demonstrated that this molecule is capable of inducing the release of beta-hexosaminidase in humanized mouse cells sensitized with serum from allergic patients [Urrego, J, Physico-chemical and immunological characterization of the recombinant Blomia tropicalis allergen Blo t 2. EAACI congress 2016. Abstract 182 http://www.professionalabstracts.com/eaaci2016/programme-eaaci2016.pdf1, therefore being a candidate to be used for diagnosis or immunotherapy for B.tropicalis mite allergy.

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Immunotherapy for allergic diseases [011] Treatment for this type of disease is very difficult, and constitutes a medical challenge, and most conventional treatments are pharmacological and used to control symptoms, without acting directly on the cause of disease. However, since 1911, Doctors Noon and Freeman have initiated a different scheme for the treatment of these diseases known as immunotherapy, which is the only causal and prophylactic treatment that can modify the natural course of the disease with recognized effectiveness [Noon L. Prophylactic inoculation against hay fever. Lancet. 1911; 1: 1572-3] [012] Allergy immunotherapy consists of administering repeated doses of an allergen (or extract) to an allergic patient to induce immunological tolerance, leading to a significant improvement in symptoms [Bousquet J, Lockey R , Malling HJ. Allergen immunotherapy: therapeutic vaccines for allergic diseases. WHO position paper. J Allergy Clin Immunol. 1998 Oct; 102 (4 Pt 1): 558-62. ]. The mechanisms of action of immunotherapy imply a modulation of the patient's immune response, modifying the predominant Th2 profile to a regulatory type response with induction of cytokines IL-10 and TGF-β. IL-10 induces an isotype change from IgE to IgG4, which acts as an IgE-blocking antibody, while TGF-β increases IgA levels. A successful immunotherapy regimen is characterized by decreased levels of IgE in addition to increased production of IgG4 [Casale TB, Stokes JR. Future forms of immunotherapy. J Allergy Clin Immunol. 2011; 127 (1): 8-15; quiz 6-7.] [013] Currently, in allergy immunotherapy, the total extract of the allergenic substance to which the individual is sensitive is administered. However, despite the benefits

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8/22 offered by immunotherapy using total extracts, there are still many disadvantages and risks of adverse reactions such as anaphylaxis, because the extracts are complex mixtures and still have undefined components that can cause new sensitizations [Durham SR, Leung DY. One hundred years of allergen immunotherapy: time to ring the changes. J Allergy Clin Immunol. 2010; 127 (1): 3-7]

Recombinant hypoallergenic allergens in immunotherapy [014] Many studies have been carried out focused on the development of recombinant allergens, in order to improve the efficacy and safety of specific immunotherapy (SIT). But SIT still has some problems, like serious side effects (for example: anaphylactic shock) that can happen due to the application of non-standard doses of allergens [Vrtala S, Focke-Tejkl M, Swoboda I, Kraft D, Valenta R: Strategies for converting allergens into hypoallergenic vaccine candidates. Methods 2004, 32: 313320]. Another disadvantage of using allergenic extracts is that they contain other potentially allergenic proteins, which can induce new sensitizations during immunotherapeutic treatment [Ferreira F, Wallner M, Breiteneder H, A H, J T, C. E: Genetic engineering of allergens for immunotherapy. Int Arch Allergy

Immunol 2002, 128: 171178]. Therefore, the need arose for the use of hypoallergenic recombinant allergens. Several clinical studies show that their use reduces the risks of anaphylaxis, and other side effects, without decreasing the effectiveness when compared to the non-hypoallergenic recombinant allergen [Linhart B, Focke-Tejkl M, Weber M, Narayanan M, Neubauer a., Mayrhofer H, Blatt K, Lupinek C,

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Valent P, Valenta R: Molecular Evolution of Hypoallergenic Hybrid Proteins for Vaccination against Grass Pollen Allergy. J. Immunol. 2015]. One explanation for this characteristic is that hypoallergens are constructed in such a way that they reduce the allergenic activity of the original antigen, by means of mutations in IgE epitopes, but maintaining the T cell epitopes, leading to the induction of immune tolerance [Banerjee S, Weber M, Blatt K, Swoboda I, Focke-Tejkl M, Valent P, Valenta R, Vrtala S: Conversion of Der p 23, a new major house dust mite allergen, into a hypoallergenic vaccine. J. Immunol. 2014,

192: 486775] [015] Hypoallergenization can be done on the basis of trial and error, testing various peptides from the respective recombinant antigens or rationally designed through in silico mutagenesis [Chen KW, Blatt K, Thomas WR, Swoboda I, Valent P, Valenta R, Vrtala S: Hypoallergenic Der p 1 / Der p 2 combination vaccines for immunotherapy of house dust mite allergy. J. Allergy Clin. Immunol. 2012, 130: 43543.e4] [Thalhamer T, Dobias H, Stepanoska T, Proll M, Stutz H, Dissertori O, Lackner P, Ferreira F, Wallner M, Thalhamer J, et al .: Designing hypoallergenic derivatives for allergy treatment by means of in silico mutation and screening. J. Allergy Clin. Immunol. 2010,

125]

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Definitions [016] The term “antigen” is defined as any foreign substance that binds to a specific antibody, or to a specific lymphocyte. The antigen may be able to induce an immune response, i.e. an immunogen. In addition, the antigen may be able to induce an allergic reaction, i.e. the allergen. Alternatively, an antigen can be a pathogen.

[017] The term "allergen" is defined as a substance capable of inducing allergy or an allergic reaction. The allergen can induce the allergy or allergic reaction by inducing the production of IgE, and later binding to this antibody linked to effector cells of allergic reactions.

[018] The term "hypoallergenic" is defined as the decrease in an allergen's ability to induce an allergic reaction. This decrease in allergic reaction may be due to a decrease in the ability to bind with IgE antibodies, and / or it may be due to other mechanisms not yet defined [019] The term “mutation”, as used herein refers to a single or multiple amino acid substitutions in a native allergen. However, it is preferred that the hypoallergenic derivative that has undergone mutagenesis comprises a low number of point mutations, in order to substantially preserve, in the derivative, epitopes of T cells of the wild type allergen.

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11/22 [020] The “Z score” of a protein: it is defined as a measure of energy that measures the stability of the molecule, the objective is not to vary the Z score much with the introduction of mutations in the amino acids on the surface of the molecule.

TECHNOLOGICAL PROSPECTION [021] The patent number WO 2012072678 A1 and title “Hypoallergenic polypeptides for the treatment of house dust mite allergy” shows the process of hypoallergenization by protein fusion having the advantages of hypoallergen containing all the T cell epitopes of the original molecule , and to induce stronger IgG4 responses. Said synthetic peptide has three fragments of the Der p 1 antigen which comprises the amino acids (aa) 1 - 84, (aa) 85 - 143 and (aa) 144 - 222 (1.1, 1 .2 and 1.3). And two fragments of the Der p 2 antigen comprising amino acids (aa) 1- 53 and (aa) 54 - 129 (2.1 and 2.2). The system was assembled in the following order 1.3, 2.2, 1.2, 2.1, and 1.1. The antigen has been shown to have reduced IgE reactivity in patients allergic to antigens from the mite Dermatophagoides, Der p 1 and Der p 2.

[022] The patent number WO 2006058359 A2 and title “Protein allergen derivatives” shows the process of hypoallergenization by fragmentation and reinversion of the sequence of two proteins of the dust mite Dermatophagoides called Der p 1 and Der p 2. Furthermore, it shows the generation of a hypoallergenic variant of the grass prophyline Thimothy Phl p 12 by DNA shuffling technology. In both cases, said molecules had the ability to significantly reduce the binding capacity of the

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12/22 molecule with IgE in addition to reduced allergic activity inducing the production of IgG antibodies with a blocking capacity to the native allergen.

[023] Patent number WO 2004065414 A1 and title “Process for the preparation of hypoallergenic mosaic antigens” shows the hypoallergenization process by evaluating IgE epitopes. However, they carry out a reorganization of the amino acid sequence so that the structure of the wild-type allergen is not maintained. These molecules are derived from the Phl p 2 allergen, from Timothy grass pollen, showing a reduction in allergic activity.

[024] Patent number WO2014045084 A1 and title “Fusion proteins with representation of different allergens: vaccine proposal for dust mite allergy” describes two fusion proteins composed of the fragments of the allergens: Blo t 5, Blo t 10, Der p 8, Block t 8, Der p 1, Der p 2 and Der p 7; and Blo t 5, Blo t 10, Blo t 12, Blo t 8 and Blo t respectively. Fusion proteins have reduced reactivity to serum IgE in allergic patients.

[025] US patent number 20070065468 A1 and title “Chimeric allergens for immunotherapy” shows a chimeric polypeptide in order to reduce the binding of specific IgE to at least two dust mite allergens. Said chimeric polypeptide comprises an amino acid sequence of at least two mite allergens (Der p 1, Der p 2 or Blo t 5), observing a reduced allergic reaction in sera of allergic patients.

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Summary of the invention [026] The invention consists of using the modeling of a hypoallergenic molecule based on an in-silic mutation method of an allergenic molecule (Blo t 2) present in the B. tropicalis dust mite. The invention has several advantages over the methods used so far for the development of recombinant hypoallergens, because it is based on new bioinformatics analyzes to predict protein stability, since there is an exchange of amino acids on the surface of the molecule. Then the mutants with the highest Z score values were selected

Detailed description of the invention

In silico mutagenesis on the surface of the Blo t 2 allergen molecule: Design of the mutant: SEQ ID NO: 1 [027] The present invention relates to the mutation of the Blo t 2 allergen to convert it into a hypoallergenic one, by exchanging the polar amino acids or loaded that are accessible on its surface, in order to change loads and decrease the connection to IgE; the process comprises the following steps:

[028] - Prediction of the three-dimensional structure of the native Blo t 2 allergen, using as a model the structure of a homologous protein with a known three-dimensional structure. The template or model was Der p 2, a dust mite allergen. D.

pteronyssinus.

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14/22 [029] - Design of the mutant with the introduction of 8 point mutations in the amino acids present on the surface of the native allergen, and the exchange was carried out by changes in the polar or charged amino acid residues in order to change the protein charges . The selected amino acids were as follows: T35K K48T H74E K81D S90E I92D T95E N99Q. Generating the sequence of claim 001: SEQ ID

NO: 1 [030] - Calculation of the score or Z score of the mutant with the eight amino acid exchanges on the surface of the Blo t 2 mutant using the ProSa2003 program developed by the C.A.M.E. (Center of Applied Molecular Engineering) at the University of Salzburg in Austria. The software is a tool for the analysis of 3D protein structures, whether they are theoretically predicted or an insilic modeling product. The program generates scores that reflect the quality of protein structures. These potentials are derived from known protein structures, capturing the most common properties of native globular proteins in terms of even atom, and protein-solvent interactions. ProsSa 2003 comes with updated knowledge-based potentials, a reinforced PDB analyzer, a command line history, and a set of new commands that support in-silico mutagenesis.

[031] - The quality of the global model calculated for the mutant allergen SEQ ID

NO: 1 was a z-score of -6.21, showing an acceptable mutant score and an optimal stability model.

Design and synthesis of the harmonized sequence of the mutant rBlo 12: SEQ ID NO: 2 [032] The sequence of Blo t 2 with the eight mutations was recoded to a

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15/22 excellent expression in E. coli. In this strategy, the frequency of the codons of the host microorganism are closely aligned with the frequencies of use of codons of the host organism of the target gene. Harmonization of mutant rBlo t 2 was done using species-specific codon frequency tables on the website http: //www.kazusa.or.ip/codon/ for Blomia tropicalis as “Target organism” and the E.coli strain B as a host organism. The harmonized sequence of mutant rBlo t 2 (SEQ ID NO: 2) was synthesized by ATG: biosynthetics (Merzhausen, Germany) in the cloning and expression vector pET 30a (+)

Usage examples [033] The following examples are for illustrative purposes only and should not be construed as limiting the claims made for this invention. Currently, there are several systems available for the production of recombinant proteins, which can also be used for the production of the hypoallergenic antigen claimed in this invention, including production systems in eukaryotic organisms and in vitro systems for cell-free production. In addition, different forms of purification of recombinant proteins can also be used to obtain the purified form of the claimed hypoallergenic antigen, including a fusion system with several partner proteins and various forms of purification by chromatography.

Example 1: Expression and purification of mutant rBlo 12: SEQ ID NO: 1 [034] The cloning vector containing the mutant rBlo t 2 sequence was transformed into a E.coli Shuffle T7 strain (New England Biolans, USA). Protein expression was performed in LB culture medium by induction with 0.4mM Isopropyl-b-DPetition 870160076103, from 12/16/2016, p. 17/33

16/22 thio-Galactopyranoside at the time the cell culture obtained an OD6oo: 0.6. The culture was maintained for 16 hours at 16 ° C and with continuous agitation at 200 g. Subsequently, the bacteria were harvested by centrifugation at 4,000 g for 20 minutes at 4 ° C; the pellet obtained was resuspended in a 50mM Tris / Cl pH solution: 8.5 and 0.5 M Urea. The bacteria were lysed for 3 cycles of freezing and thawing with liquid nitrogen and homogenized for 3 minutes with the Ultra-Turrax T25® device (IKA, Germany) at maximum speed. In addition, the sample was incubated with DNase I (1: 1000) for 2 hours at room temperature. The supernatant was collected and centrifuged at 13,000 g for 20 minutes at 4 ° C. The constitutive proteins of the bacterium were precipitated with acetic acid, until reaching pH: 4.0. The mutant rBlo t 2 was collected from the supernatant after centrifugation at 13,000 g for 20 min at 4 ° C and pre-purified in non-reducing conditions, on a HiTrap SPFF ion exchange column (GE Healthcare, Sweden). The flow-throw was concentrated and purified by size exclusion on a Superdex 75 10/300 column

GL (GE Healthcare, Sweden).

Biochemical and biophysical characterizations of recombinant allergens and hypoallergenic derivatives [035] According to the regulatory and quality control standards of medicinal products, several parameters are monitored during the production of recombinant allergens or their derivatives. In general, these parameters can be grouped into three categories: (i) process viability and efficiency, (ii) physical-chemical characteristics, and (iii) biological activity.

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Example 3: Viability and efficiency of the production process of hypoallergenic recombinant antigens [036] The parameters investigated were the yield and solubility of the recombinant proteins. The fermentation conditions were optimized so that the recombinant proteins represent at least 5% of the total cell protein. In addition, the recombinant protein was soluble in physiological buffers (Figure 1). To optimize yield and solubility, a series of procedures and conditions (protein extraction, purification steps, etc.) (figure 1) were tested, selected and standardized.

Example 4: Physico-chemical characterization of the mutant Blo 12 [037] The physicochemical parameters important for the characterization of proteins are well established in the literature. The most important are: (i) degree of purity and homogeneity and (ii) structure and stability.

[038] (i) Degree of purity: the preparation must be pure with respect to the total protein content of the bacterium and homogeneous with regard to the molecular weight of the recombinant protein. However, the protein was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). (see figure 1) [039] (ii) Structure and stability. The circular dichroism technique is commonly used to determine the secondary structure of proteins and to determine thermal stability. Thus, the samples were analyzed by this technique in the presence of sodium phosphate buffer (5 mM, pH 7.4) using the JASCO J-810 spectrophotometer (Jasco, Tokyo, Japan). Thermal denaturation was carried out varying the temperature from 20 ° C to 95 ° C and the thermal renewal evaluated from 95 ° C to 20 ° C, with a

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18/22 speed of 1 ° C / minute. In addition, the elements of the secondary structure of the mutant rBlo t 2 were determined by Fourier transformation infrared spectroscopy (FTIR). The spectrometric signals of the protein were mainly in the region corresponding to β-leaves and structural elements without order. At least 24% of the protein corresponds to β-leaves, see Figure 2

Example 5: Test of recombinant antigens for reactivity to sera from patients allergic to B. tropicalis mite [040] The recombinant antigens of rBlot 2 and the hypoallergenic mutant rBlo t 2 were subjected to the SDS - PAGE assay on acrylamide gel and then transferred to a nitrocellulose membrane (Whatman, Brentford, UK) by electrotransfer. The membranes were blocked with a 20% BSA solution and incubated with a mixture of 6 sera from patients allergic to Blomia tropicalis diluted 1:10, overnight at 4 ° C. After 3 washes, the membrane was incubated with a mouse antibody / human Anti - IgE conjugated to alkaline phosphatase (BD Biosciences, Franklin Lakes, NJ, USA) diluted at 1: 10,000 for two hours at room temperature. The band corresponding to the molecular size of the protein was visualized using NBT and BCIP as substrates for the alkaline phosphatase reaction (see Figure 3).

[041] For the ELISA assay, high-binding plates (Nunc MaxiSorp®,

Rochester, NY) were sensitized with 5Lig / inl. of the recombinant native Blo t 2 or rBlo t mutant in phosphate buffered saline (1X PBS), pH: 7.4 over night. After washing with PBS

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19/22 containing 0.05% Tween-20 (PBS-T), the plates are blocked with 200 pL of buffer

Tris-Saline containing 0.5% BSA and 0.05% Tween-20 (TBS / T / BSA) and incubated with the allergens at 4 ° C for 16 hours. Human sera were diluted 1:10 in TBS / T / BSA and incubated with mouse / anti-human IgE antibody conjugated to alkaline phosphatase (BD Biosciences, Franklin Lakes, NJ, USA) (1: 4000). After washing with TBS-T, the plates were incubated with a 100 µl solution of 10 mM p-nitrophenyl phosphate (PNPP). Absorbance was determined at 405 nm using a plate reader (Ges mBH, Tecan, Salzburg, Austria). (See figure 4, A)

Example 6: Hypoallergenic Biological Activity Test [042] The biological activity of recombinant proteins was monitored using a rat leukemic basophil cell line (RBL) that expresses the high-affinity human IgE receptor (FceRI). The cells were sensitized with sera rich in serum IgE from patients allergic to B. tropicalis and stimulated with serial dilutions of the recombinant allergens. The release of β-hexosaminidase was tested through its enzymatic function in the cleavage of the fluorogenic substrate methylumbeliferyl-N-acetyl ^ -D-glucosaminide and calculated as a percentage of the total enzyme content in cells solubilized with Triton X-100. (See figure 4, B)

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Statistical analysis [043] The normalities of the data are determined by the D'Agostino and Pearson test. Statistical analysis to compare the test under development with the

ELISA was with Student's t-test

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Brief description of the figures

Figure 1. Expression and purification of the hypoallergenic rBlo t 2 in the E.coli Shuffle T7 strain. The protein was purified under non-reducing conditions and without the use of Histidine solution. Lines: A.I., Before induction with 0.4 mM IPTG; D.I., After induction with 0.4 mM IPTG; D.L.S, After lysis of the bacterium (supernatant) with 50mM Tris / Cl pH: 8.5 and 0.5 M UREA; D.P.A, After acid precipitation with 100% acetic acid; M.P. mutant purified by ion exchange chromatography and size exclusion.

Figure 2. Measurement of the secondary structure components of the rBlo t 2 (rBlo t 2_8X) mutant by: (A). Circular dichroism (CD): The spectrum was measured at 20 ° C and 95 ° C in a 10 mm vat. The protein was diluted in 10mM PBS, pH 7.2 to a final concentration of 0.1 mg / ml. 5 measurements of the spectrum were made at a speed of 10nm / min. Then, the mean of the spectra was calculated and corrected by the buffer contribution. The mutant recombinant protein exhibited a more disordered conformation than the wild recombinant. (B). The percentage of secondary structure elements was determined by infrared spectrometry after Fourier transformation (In English: FTIR). The spectrum signals were mainly in regions corresponding to elements without order and in β-leaves with 25.4%.

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Figure 3. Immunoblot of the mutant hypoallergenic antigen of rBlo t 2 (rBlo t 2_8X) using a mixture of 10 sera from patients allergic to Blomia tropicalis. Seropositivity was determined by the ImmunoCAP assay with IgE levels above 0.35 kU / L. (THE). The transfer SDS-PAGE developed with Coomasie blue. (B) and (C), Nitrocellulose membranes transferred, blocked, incubated with sera and with an anti-human IgE mouse antibody (1: 1000) conjugated to alkaline phosphatase. The band of the wild allergen (rBlo t 2.2) was visualized using the developing substrates NBT and BCIP, showing a strong reaction. The mutant rBlo t 2_8X allergen did not show reactivity to IgE in both extract (B) and purified (C) compared to the two isoforms of the wild allergen (Blo t 2.2 and Blo t 2.5

Figure 4. (A). ELISA using sera from patients allergic to Blomia tropicalis showing the IgE reactivity of native rBlo t 2 and mutant rBlo t 2. (B). Assay for measuring the release of inflammatory mediators using humanized rat basophils (huRBL). The Blo t 2 mutant (rBlo t 2_8X) did not have the ability to degranulate the basophils compared to the native allergen (rBlo t 2).

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Claims (4)

001. HYPOALLERGENIC RECOMBINANT ANTIGEN characterized by a mutant recombinant antigen derived from the allergen Blo t 2 of the dust mite Blomia tropicalis, which has reduced allergenic activity, for use in the prophylaxis and treatment of asthma and other allergic diseases 002. HYPOALLERGENIC RECOMBINANT ANTIGEN according to claim 001, characterized by an amino acid sequence that derives from specific modifications of the existing sequences for the wild-type allergen Blo t 2 of dust mites, an antigen with 12 reported isoforms, which belongs to the group of allergens with ML lipid binding domain (Group 2), with a molecular weight of approximately 13 kDa and with approximately 142 amino acids 003. HYPOALLERGENIC RECOMBINANT ANTIGEN according to claims 001 and 002, characterized by a sequence of amino acids containing the specific mutations responsible for hypoallergenization, compared to the wild-type antigen Blo t 2, it is represented by the amino acid sequence: SEQ ID No: 1 004. PROCESS OF PRODUCTION OF THE HYPOALLERGENIC RECOMBINANT ANTIGEN according to claims 001 to 003, characterized by comprising the following steps: in-silico design of the amino acid sequence of Petition 870170059262, of 8/16/2017, p. 2/5 2/3 antigen with reduced allergenic activity; design of the plasmid containing the nucleic acid sequence encoding the recombinant antigen; method of obtaining the recombinant antigen using heterologous systems 005. METHOD according to claim 004, characterized in that the insylic design of a hypoallergen using the nucleotide sequence encoding the Blo t 2 wild-type allergen of the Blomia tropicalis dust mite as a template. The referred in-silico design involved the replacement of eight amino acid residues of the wild-type Blo t 2 molecule that led to the alteration of the surface of the protein structure, with the objective of exchanging the charges and providing the decrease in IgE binding, resulting in the coding sequence identified as SEQ ID No: 2 006. HYPOOLERGENIC RECOMBINANT ANTIGEN CODING SEQUENCE according to claims 004 and 005, characterized by the existence of specific mutations in relation to the coding sequence of the wild-type allergen Blo t 2, here identified as SEQ ID No: 2, as well as the versions reverse or complementary sequences, in addition to versions containing synonymous mutations derived from codon optimization and / or harmonization processes for recombinant production purposes 007. METHOD according to claims 004 to 006, characterized in that the expression vector design containing transcriptional unit that includes the nucleotide sequence SEQ ID No: 2, as well as its variants according to claim Petition 870170059262, of 8/16/2017, p. 3/5 3/3 006, to allow expression of the recombinant version of the hypoallergenic recombinant antigen 008. METHOD according to claims 004 to 007, characterized by the recombinant production of hypoallergenic antigen in heterologous bacterial, eukaryotic and / or in vitro cell-free systems; includes recombinant production systems that involve the fusion of the hypoallergenic recombinant antigen of SEQ ID No.1 with different partner proteins, in addition to the alteration of the SEQ ID No.2 nucleotide sequence, for purification and / or optimization of the obtaining process of the recombinant version of the hypoallergenic recombinant antigen claimed in claims 001 to 003 009. A VACCINE FOR THE IMMUNOTHERAPY OF ALLERGIC DISEASES CAUSED BY DUST MITES characterized by the use in its formulation of the hypoallergenic recombinant antigen claimed in claims 001 to 003, in which said vaccine contains the antigen in its purified form or combined with excipient agents and / or adjuvants Petition 870170059262, of 8/16/2017, p. 4/5 1/4 MW (kDa) A.l. D.l. D.L.S. D.P.A. M.P FIGURE 1 Petition 870160076103, of 12/16/2016, p. 29/33 2/4 THE. Blo t 2 8x 20 ° Blo 12 8x 95 ° absorbance units rBlo 12 rBlo 12_8x FIGURE 2 Petition 870160076103, of 12/16/2016, p. 30/33 3/4 THE. MW (kDa) 250 100 130 Blo 12.2 Blo 12.5 Blot2_SX B · purified purified Pre-purified MW (kDa) 250 100 130 Blo 12.2 Blo 12.5 Purified purified Blot2_8X Pre-purified ç. MW (kDa) 250i - 100 * 25 | fll 15 * 10 * FIGURE 3 Extra cto Blomia Slot 2.2 tropicalis. purified Blo t 2_8X Purified Petition 870160076103, of 12/16/2016, p. 31/33 4/4 Α. η- 10 Coat: 2 pg / mL Non-allergic patients (Data not shown): pure rBlo t 2: 0.012 rBlo t 2 extract: 0.021 rBlo t 2 8X: 0.002 Mean absorbance (OD ₄₀₅ ' rBlo t 2 pure: 3.18 rBlo t 2 extract: 0.83 rBlo t 2_8X: 0.041 Shuffle extract (bacteria): 0.034 Protein concentration [ng] rBlo t 2 rBlo t 2_8X FIGURE 4 Petition 870160076103, of 12/16/2016, p. 32/33 1/1