Patent Application
20120009210
The instant application contains a Sequence Listing that has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 8, 2011, is named LNK—002CIP_Sequence_Listing.txt, and is 16,425 bytes in size.
The present invention relates to mosaic antigens reassembled from naturally-occurring allergens, in particular plant pollen allergens, more particularly grass and tree pollen allergens. The mosaic antigens described herein display reduced allergenic activity and thus are useful as allergy vaccines for the treatment of allergic disorders and sensitized allergic patients and for prophylactic vaccination.
A large percentage of the population suffers from IgE-mediated allergies. Many of those patients suffer from allergic reactions against several antigens. A high percentage of these allergic reactions are caused by plant allergens, particularly pollen from anemophilous (i.e., “wind loving”) plants. Among North American plants, the most prolific producers of allergenic pollen are weeds, primarily ragweed, though sagebrush, redroot pigweed, lamb's quarters, Russian thistle (tumbleweed), and English plantain are also important. Grasses and trees are also primary sources of allergenic pollens. Although there are more than 1,000 species of grass in North America, only a few produce highly allergenic pollen. These are mostly summer grasses, examples of which include timothy grass, Kentucky bluegrass, Johnson grass, Bermuda grass, redtop grass, orchard grass, and sweet vernal grass. Examples of trees that produce allergenic pollen include members of the birch, oak, ash, elm, hickory, pecan, box elder, and mountain cedar families. Allergy to pollens from birch and related trees (alder, hazel), are quite prevalent in Northern and Middle Europe, North America, and certain parts of Australia and Asia.
The symptoms of allergy, such as allergic rhino conjunctivitis, asthma, dermatitis, hay fever, hives and even anaphylactic shock, arise from the interaction between antibodies and allergens, more particularly IgE recognition of allergens. In particular, IgE molecules bind to an allergen, for example, a plant pollen. The tail region of the IgE molecule, i.e., the Fc part, binds to Fc receptors that are mainly located on the surface of mast cells in tissues and basophils in the blood. Antigen binding triggers the mast cells or basophils to secrete a variety of cytokines and biologically active compounds, especially histamine. These molecules cause blood vessels to dilate and become leaky which in turn helps white blood cells, antibodies and complements components to enter sites of reaction. Those molecules are on the other hand largely responsible for the symptoms of allergic reactions. There are different degrees of allergic reactions which range from slight itching of the eyes and the symptoms of a slight cold over severe pains to live-threatening symptoms like anaphylactic shock which may occur for example after the sting of a bee.
While drug therapy may reduce the symptoms of an allergic response, only allergen-specific immunotherapy (ASIT) can serve to avoid the allergic reaction and thereby effectively “treat” allergic disorders. ASIT is based on the administration of a small amount of a disease-eliciting allergen to the patient in order to induce antigen-specific nonresponsiveness. More particularly, the administration of a small amount of antigen leads to the production of allergen-recognizing IgG antibodies or “blocking antibodies”. These so-called blocking antibodies largely inhibit contact between the allergen and the IgE molecules present in the patient's body; thus, the reaction between the allergen and the mast cells mediated by IgE molecules is largely avoided.
In the field of allergen-specific immunotherapy (ASIT), different allergy vaccines have been developed. Previously, these vaccines simply consisted of small amounts of the native allergen or natural allergen extracts to be applied to the patient. However, with the development of genetic engineering, vaccines based on recombinant allergens have been produced. A major disadvantage of such allergen-containing vaccines is that the application of such vaccines causes in the patient unwanted side-effects. If, for example, the allergen against which the patient is allergic is applied subcutaneously to the patient an unwanted side-effect like itching up to anaphylactic shock can occur since the IgE antibodies present in the patient's body react with the allergen and cause the allergic reaction.
In an effort to overcome the undesired side-effects of conventional immunotherapeutic agents, hypoallergenic allergens, i.e., allergens having reduced allergenic potential as compared to their naturally-occurring counterparts, are eagerly sought.
To that end, Applicants have developed a process for the preparation of a mosaic antigen derived from a naturally-occurring allergen whereby the complete amino acid sequence of a naturally-occurring allergen (or “native allergen” or “wild-type allergen”) is reassembled in a manner in which sequences relevant to the induction of blocking IgG antibodies and the dominant T-cell epitopes are preserved while those sequences associated with IgE recognition are avoided or reduced. Thus, the present invention is directed to a method of making reassembled mosaic antigens and reassembled mosaic antigens obtained therefrom.
Accordingly, it is an object of the present invention to provide a hypoallergenic mosaic antigen assembled from all or substantially all of the amino acids of a native allergen, though with the amino acid components arranged in a different order. In one preferred embodiment, the “different order” arises from exchanging an allergen fragment that includes the naturally-occurring N-terminus with an allergen fragment that includes the naturally-occurring C-terminus. Additionally or alternatively, at least two of fragments that are in adjacent positions in the naturally-occurring allergen may be oriented into non-adjacent positions in the reassembled mosaic antigen.
Hypoallergenic mosaic antigens of the present invention are designed to retain at least one T-cell epitope specific to the naturally-occurring allergen and to be capable of inducing IgG antibodies that hinder IgE binding to the naturally-occurring allergen. Accordingly, it is an object of the present invention to provide a hypoallergenic mosaic antigen capable of inducing allergen-specific IgG antibodies that recognize the naturally-occurring allergen and inhibit recognition of the naturally-occurring allergen by serum IgE from allergic patients. In a preferred embodiment, the mosaic antigen has reduced allergenic activity as compared to the naturally-occurring allergen. For example, the IgE reactivity of the mosaic antigen have an IgE reactivity value that is no more than 10% of that obtained for the naturally-occurring allergen, preferably no more than 5% thereof.
Although the present invention is not limited to any one particular allergen or class or allergen, plant allergens, especially plant pollen allergens, are preferred. In one particularly preferred embodiment, the plant pollen allergen is a birch pollen allergen, for example the major birch pollen allergen, Bet v 1.
Mosaic antigens of the present invention may be defined in terms of their peptide (amino acid) sequences. Illustrative examples of preferred peptide sequences are set forth in SEQ ID NOs: 15, 17, and 19. However, the present invention also extends to nucleotide (DNA, RNA) sequences that code for the hypoallergenic mosaic antigens described herein. Examples of preferred nucleotide sequences include DNA sequences coding for amino acid sequences of SEQ ID NO: 15, 17, and 19, and sequences complementary thereto.
It is a further object of the present invention to provide a method of making a reassembled mosaic antigen of the present invention, the method including the steps of:
The method of the present invention involves a rational design approach that is distinct from the gene shuffling and molecular breeding techniques of the prior art, such as those described by Wallner et al. (J. Allergy Clin. Immunol., vol. 120(2), August 2007, p. 374-380) and Short (U.S. Pat. No. 6,489,145). Unlike the prior art methods, the mosaic antigens of the present invention are not the result of random shuffling and screening but rather the result of affirmative design, wherein allergen fragments are selected for reassembly according to the following criteria:
Accordingly, the method of the present invention allows for the production of mosaic antigens having a reduced or eliminated capacity to bind IgE while conserving at the same time those features of the allergen that are required to induce a T-cell mediated immune response. Thus, the reassembled mosaic antigens of the present invention are capable of inducing a strong allergen-specific IgG response, i.e., therapeutic levels of blocking IgG antibodies, while simultaneously inhibiting or suppressing IgE production. In this manner, the allergic and/or inflammatory response to the native allergen may be substantially avoided. As such, the mosaic antigens of the present invention find particular utility in the treatment of allergies and allergic disorders. Accordingly, it another object of the present invention to provide a method of treating an allergic disorder in a subject in need thereof including the step of administering to the subject a therapeutically effective amount of a mosaic antigen of the present invention or a DNA sequence coding for such an allergen.
The reassembled mosaic antigens of the present invention find utility in the treatment of an allergic disorder. Accordingly, it is yet another object of the present invention to provide methods for treating or preventing allergic disorders that include the step of administering one or more hypoallergenic mosaic antigens of the present invention to a subject in need thereof. In a preferred embodiment, the allergic disorder is a pollen allergy, more preferably birch pollen allergy, even more preferably allergic disorders caused by reaction to the major birch pollen allergen Bet v 1.
The mosaic antigens obtained according to the present invention may be easily combined with a pharmaceutically acceptable carrier, diluent and/or excipient and finished to a pharmaceutical preparation or medicament. Accordingly, is yet another object of the present invention to provide a medicament for the treatment or prevention of an allergic disorder.
The reassembled mosaic antigens of the present invention also find utility in the preparation of a vaccine for the treatment or prophylaxis of an allergic disorder. Accordingly, it is a further object of present invention to provide vaccines for the treatment of allergic disorders, more particularly vaccines that include one or more hypoallergenic mosaic antigens of the present invention. To that end, the mosaic antigens obtained according to the present invention may be combined with a suitable vaccine adjuvant and finished to a pharmaceutical acceptable vaccine preparation. A vaccine preparation of the instant invention may include further allergens, preferably wild-type allergens, especially a mixture of wild-type allergens, recombinant wild-type allergens, derivatives of wild-type protein allergens or mixtures thereof. Such mixtures may be specifically tailored for the needs (i.e., allergen profile) of a particular patient.
In addition to mosaic antigens per se, a nucleic acid coding for a mosaic antigen of the present invention or a nucleotide sequence complementary thereto may also serve as a suitable vaccine. Accordingly, it is yet another object of the present invention to provide a vaccine for the treatment or prevention of an allergic disorder comprising a nucleotide sequence coding for one or more mosaic antigen(s) of the present invention.
It will be understood by those skilled in the art that one or more aspects of this invention can meet certain objectives, while one or more other aspects can meet certain other objectives. Each objective may not apply equally, in all its respects, to every aspect of this invention. As such, the preceding and subsequently presented objects can be viewed in the alternative with respect to any one aspect of this invention.
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. However, it is to be understood that both the foregoing summary of the invention and the following detailed description are of a preferred embodiment and not restrictive of the invention or other alternate embodiments of the invention. In particular, while the invention is described herein with reference to a number of specific embodiments, it will be appreciated that the description is illustrative of the invention and is not constructed as limiting of the invention. Various modifications and applications may occur to those who are skilled in the art, without departing from the spirit and the scope of the invention, as described by the appended claims. Likewise, other objects, features, benefits and advantages of the present invention will be apparent from this summary and certain embodiments described below, and will be readily apparent to those skilled in the art. Such objects, features, benefits and advantages will be apparent from the above in conjunction with the accompanying examples, data, figures and all reasonable inferences to be drawn therefrom, alone or with consideration of the references incorporated herein.
Various aspects and applications of the present invention will become apparent to the skilled artisan upon consideration of the brief description of the tables and figures and the detailed description of the present invention and its preferred embodiments that follows:
Table 1: Characteristics of Phl p 2-derived synthetic peptides. Sequence, number of amino acids, position in the Phl p 2 allergen; molecular weight and bioelectric point of the peptides are displayed. Peptide 1 corresponds to SEQ ID NO:3, Peptide 2 corresponds to SEQ ID NO:4 and Peptide 3 corresponds to SEQ ID NO:5.
Table 2: Immediate type skin reactions to complete rPhl p 2 and to Phl p 2 mosaic (P2M). Two timothy grass pollen allergic patients (Individual 1,2) were tested for skin reactivity with P2 and P2M. The mean wheal diameters (mm) are displayed for five different concentrations of rPhl p 2 and Phl p 2 mosaic, as well as for timothy grass pollen extract and histamine.
Table 3: Inhibition of grass pollen allergic patients IgE binding to rPhl p 2 by rabbit aP2M and rabbit aP2 antibodies. The percentage inhibition of IgE binding is displayed for 5 patients.
Table 4: Inhibition of IgE binding of patients with allergy to rBet v 1 by IgG antibodies raised with CFA-adsorbed proteins. The percentage inhibition of IgE binding is displayed for 18 patients.
Table 5: Inhibition of IgE binding of patients with allergy to rBet v 1 by IgG antibodies raised with Al(OH)3-adsorbed proteins. The percentage inhibition of IgE binding is displayed for 18 patients.
Table 6: Demographic, serologic, and clinical characteristics of the individuals tested.
Table A: (Appendix) Preferred wild-type allergens to be modified in accordance with the mosaic approach of the present invention.
The present invention relates to a mosaic antigen assembled from all or substantially of the component amino acids of a naturally-occurring allergen, in particular a plant allergen, more particularly an allergen derived from tree and grass pollen. The reassembled mosaic antigens described herein display reduced allergenic activity as compared to their naturally-occurring counterparts and thus are useful as medicaments for the treatment of sensitized allergic patients as well as allergy vaccines for prophylactic vaccination. Particular embodiments of the mosaic antigen of the present invention, the therapeutic constructs associated therewith, and the methods of making and using same are described in greater detail below.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the present invention, the following definitions apply:
As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “molecule” is a reference to one or more molecules and equivalents thereof known to those skilled in the art, and so forth.
As used herein, the term “allergen” refers to a nonparasitic environmental antigen capable of stimulating a type-I hypersensitivity reaction (i.e., an IgE response) in atopic individuals.
As used herein, the phrases “naturally-occurring allergen” and “native allergen” are interchangeably used to refer to the complete wild-type form of the allergen as it is found in nature.
In the context of the present invention, the native allergen may be an indoor, animal, food or seasonal allergen. A list of illustrative allergens is found in Table A of co-pending U.S. application Ser. No. 11/720,598, published as US 2008/0286311, the contents of which are incorporated by reference herein and of which Table A is reproduced herein.
Of the indoor allergens, the major house dust mite allergens, in particular Der p 1 and Der p 2, and the major storage mite allergens, especially Lep d 2, are particularly preferred. In terms of animal allergens, the present invention contemplates mosaics of the major cat allergen, Fel d 1, as well as those derived from the major bee and wasp allergens. In terms of food allergens, olive allergens, particularly major allergens of Olea europea such as Ole e 1 are particularly preferred.
In terms of seasonal allergens, plant allergens, particularly anemophilous or wind-carried plant pollens, even more preferably grass, weed and tree pollens, are particularly preferred. Examples of preferred grass pollens include, but are not limited to, those derived from timothy grass, Kentucky bluegrass, Johnson grass, Bermuda grass, redtop grass, orchard grass, and sweet vernal grass. Of the grass pollens, the major allergens of timothy grass, especially Phl p 1 (see US 2009/0098167), Phl p 2 (see U.S. Pat. No. 7,491,396), Phl p 5, Phl p 6, Phl p 7, and Phl p 12 (see US 2008/0286311) are particularly preferred.
Examples of preferred weed pollens include, but are not limited to, those derived from ragweed, sagebrush, pigweed, tumbleweed, cockleweed, sticky-weed and Russian thistle. Of the weed pollens, the major allergens of short ragweed (Ambrosia artemisiifoli), e.g., Amb a 1, and sticky-weed (Parietaria judaica), e.g., Par j 2, are particularly preferred.
Examples of preferred tree pollens include, but are not limited to, those derived from members of the birch, oak, ash, elm, hickory, pecan, box elder, and mountain cedar families. Of the tree pollens, the birch pollen allergens, Bet v 1 and Bet v 4, are of particular interest. As noted above, the present invention relates to a hypoallergenic mosaic antigen having a rearranged amino acid sequence as compared to its naturally-occurring counterpart. In the context of the present invention, term “mosaic antigen” refers to a polypeptide allergen assembled from all or substantially all of amino acids of a naturally-occurring allergen, though arranged in a different order. The reassembled mosaic antigen of the present invention may be derived from a naturally-occurring allergen that has been cleaved into at least two, preferably at least 3 or 4, preferably non-overlapping subset components or fragments. When the amino acid sequence of the native allergen is known, it is common general knowledge of a person skilled in the art to prepare peptides of varying lengths therefrom using conventional technologies. For example, the subset peptide fragments can be prepared by chemical synthesis. Alternatively, the peptides can be readily prepared by Polymerase Chain Reaction since suitable primers can be easily synthesized when the sequence is known.
Once the native allergen has been cleaved into two, three or more fragments, those fragments can be newly assembled to provide the mosaic antigen of the present invention. The mosaic antigen is preferably “hypoallergenic”, i.e., has reduced allergenic potential as compared to the native allergen. In the context of the present invention, the term “hypoallergenic” means that the IgE reactivity of the mosaic antigen has been reduced to not more than 20%, preferably not more than 10%, even more preferably not more than 5% of an IgE reactivity value obtained for the native allergen.
In the simplest case, the naturally-occurring allergen is divided at single a cleavage site into two non-overlapping peptide fragments. In the context of the present invention, the term “cleavage site” refers to the position in the polypeptide where one fragment ends and another fragment starts. Thus, the two allergen fragments include fragment A having the N-terminus and ending at the cleavage site and fragment B starting with the cleavage site and ending with the carboxy terminus of the polypeptide. The two fragments may then be rearranged in such a manner that now fragment B represents the N-terminus and fragment A represents the C-terminus. This resulting “B-A” configuration is an example of a reassembled mosaic antigen.
The mosaic allergen of the present invention is preferably produced recombinantly, though the subset allergen fragments may also be chemically synthesized and subsequently linked together.
As noted above, the reassembled mosaic antigens of the present invention find particular utility in the treatment and prevention of allergic disorders. In the context of the present invention, the terms “allergy” and “allergic disorder” are interchangeably used to refer to any disorder that is caused by a hypersensitive reaction of the immune system, typically a type I or immediate hypersensitity, to a normally harmless environmental substance (i.e., an allergen). Examples of allergic disorders include asthma, eczema, contact dermatitis, hives, hay fever, allergic rhinitis and rhinoconjunctivitis, airborne allergies and hay fevers (such as ragweed and birch pollen allergies). The present invention is particularly suited to the treatment of allergy to airborne particles such pollens. In these cases, symptoms typically arise in areas in contact with air, such as eyes, nose and lungs. For instance, allergic rhinitis, also known as “hay fever”, causes irritation of the nose, sneezing, and itching and redness of the eyes Inhaled allergens can also lead to asthmatic symptoms, caused by narrowing of the airways (bronchoconstriction) and increased production of mucus in the lungs, shortness of breath (dyspnea), coughing and wheezing.
Although applications of the reassembled mosaic antigen of the present invention are described in detail below in the context of human therapy, one of skill in the art will readily recognize that the present invention has both human medical and veterinary applications. Accordingly, the terms “subject” and “patient” are used interchangeably herein to refer to the person or animal being treated or examined. Exemplary animals include house pets (e.g., dogs and cats), livestock (e.g., cows, horses, etc.) and zoo animals. In a preferred embodiment, the subject is a mammal, more preferably a human.
The present invention relates to hypoallergenic mosaic antigens assembled from all or substantially all of the amino acid components of a naturally-occurring allergen, though rearranged into a different order. In the context of the present invention, the mosaic antigen may be obtained by (a) cleaving the naturally-occurring allergen into at least two allergen fragments, preferably at least three non-overlapping allergen fragments; and reassembling the allergen fragments to yield an amino acid sequence that includes substantially all of the amino acids of the original naturally-occurring allergen, though arranged in a different order.
As noted above, mosaic antigens of the present invention result from the intentional selection of allergen fragments that meet certain criteria. Firstly, the allergen fragments selected for reassembly should exhibit reduced allergenic activity. The allergenic activity of the allergen fragments and/or mosaic antigen may be experimentally confirmed, for example, by reacting the peptide of interest with sera from patients that are allergic to the naturally-occurring allergen.
Accordingly, it is an important aspect of the present invention to divide the wild-type allergen into such fragments that substantially do not react with IgE antibodies. As noted above, the IgE reactivity of the mosaic antigen is preferably reduced to not more than 20%, preferably not more than 10%, even more preferably not more than 5% of an IgE reactivity value obtained for the native allergen. If a particular allergen fragment still reacts with IgE antibodies in a substantial amount, such fragment should not be used for the preparation of the mosaic antigen. It is advisable to test the fragments of the naturally occurring antigen to be used in the mosaic antigen with sera from different allergic patients since there may be variations with regard to specificity and amount of IgE concentration in each serum.
Reduced allergenic activity may also be characterized by a low ability to degranulate mast cells or basophils. Relative IgE reactivity and IgE-mediated allergenic activity may be experimentally determined using conventional assays and protocols such as those described in the Examples section herein. Examples of conventional in vitro assays suitable for assessing allergenic activity include RAST (Sampson and Albergo, J. Allergy Clin. Immunol. 74:26, 1984), ELISAs (Burks et al., N. Engl. J. Med. 314:560, 1986), immunoblotting (Burks et al., J. Allergy Clin. Immunol. 81:1135, 1988), basophil histamine release assays (Nielsen, Dan. Med. Bull. 42:455, 1995 and du Buske, Allergy Proc. 14:243, 1993) and others (Hoffmann et al., Allergy 54:446, 1999).
It is also imperative that the selected allergen fragments retain important allergen-specific T-cell epitopes. The presence of requisite T-cell epitopes may be experimentally determined, e.g., by measuring the ability of the fragment to induce a T-cell mediated immune response, or, alternatively, may be determined in silico, e.g., using known T-cell epitope motifs, such as those available in the Swiss-Prot protein database, alone or in combination with conventional mapping techniques, such as those described by Thomas Zeiler and Tuomas Virtanen in their chapter entitled “The Mapping of Human T-Cell Epitopes of Allergens” from Methods in Molecular Medicine: Allergy Methods and Protocols, Humana Press, 2008, Volume 138, pp. 51-56.
As a further selection criteria, the cleavage/fragmentation process preferably disrupts conformational IgE epitopes but preserves peptide sequences capable of focusing IgG antibodies towards the wild-type IgE epitopes. With regard to the former, IgE antibodies present in sera will react with the peptide if an IgE epitope is present on the peptide. If there are, however, no linear IgE epitopes or if conformational IgE epitopes are destroyed by separating the whole naturally occurring allergen there will be no binding of IgE with the peptide. The IgE antibodies can subsequently easily be detected by reaction with specific anti-antibodies that bind to the IgE antibody. Those anti-antibodies are usually labeled for detection.
With regard to the latter, by leaving intact portions of IgE epitopes or peptide sequences proximate to such IgE epitopes, one can substantially eliminate IgE reactivity while at the same time retain the ability to induce IgG antibodies that hinder IgE binding to the wild-type allergen (i.e., “blocking IgG antibodies”).
Bearing in mind the above-noted criteria, the optimum cleavage site(s) and resulting allergen fragments may be readily determined. For example, using sequence analysis techniques and rational design approaches that are conventional in the art, one of skill in the art can readily identify B-cell epitopes capable of inducing allergen-specific blocking IgG antibodies and major T-cell epitopes. Preferred mosaic antigens retain the ability to induce immunotherapeutic levels of allergen-specific blocking IgG antibodies and the major T-cell epitopes while simultaneously exhibiting reduced allergenic activity. When the naturally-occurring allergen is to be split into two non-overlapping fragments, only one cleavage site is required. The resulting fragments are referred to herein as “A” and “B”, wherein the A fragment includes the N-terminus and the B fragment includes the C-terminus. Following the guidance herein, the reassembled mosaic antigen will have a B-A order, wherein the B fragment that now constitutes the N-terminus and the A fragment includes the C-terminus.
However, the instant mosaic techniques of the present invention are not restricted to two fragments. In fact, the naturally-occurring allergen may divided into three (A, B, C), four (A, B, C, D), five (A, B, C, D, E), six (A, B, C, D, E, F), and indeed any number of subset peptide components. The more parts formed, the more options for providing mosaic antigen are provided. Nevertheless, for best results, it is preferable that the peptide fragments to be reassembled be of approximately equal size and be as large as possible. Each fragment should include at least 10 amino acid residues, especially at least 15 amino acid residues. The ideal fragment size may vary, ranging from as little as 10 to 40 amino acids, upwards to 100 to 120 amino acids, more preferably from 30 to 70 amino acids.
When a naturally-occurring allergen having a native order of A-B-C is to be split into three fragments, the possible mosaic antigens include: B-C-A; B-A-C; C-B-A; C-A-B and A-C-B. However, when reassembling the fragments to form the mosaic antigen, it is preferable to avoid combining fragments that are localized in adjacent positions in the naturally-occurring allergen, e.g. C, A, B. The theory is that IgE binding epitopes may be formed again on the mosaic antigen. It is, however, essential that the mosaic antigen contain substantially all amino acids of the naturally-occurring antigen. Certainly some amino acids that clearly have no functions may be deleted and other amino acids may be deleted for production reasons. Nevertheless, the mosaic antigen should maintain many amino acids as possible. However, additional amino acids may be added to the mosaic antigen to facilitate production or the expression.
The mosaic approach to hypoallergenic allergen design disclosed herein may be applied to any of a number of native allergens, though plant allergens, particularly grass and tree pollen allergens are most preferred.
In the context of grass pollen allergens, allergens of groups I and II. Preferred group II allergens are described in the following publications:
In one particularly preferred embodiment, the allergen used for the mosaic antigen is the timothy grass pollen allergen Phl p 1 or Phl p 2. The mature sequence of the timothy grass pollen allergen Phl p 1 is found in Genbank Accession Number X78813. The three-dimensional structure of Phl p 1 has been solved by X-ray crystallography and is available in the PDB (1N10). From this 3D structure, IgE and T-cell epitopes have been experimentally determined and suitable mosaic proteins have been devised. See Ball et al., “Reducing Allergenicity by Altering Allergen Fold: A Mosaic Protein of Phl p 1 for Allergy Vaccination”, Allergy 2009, vol. 64: pp. 569-580, the contents of which are incorporated herein in their entirety. In particular, Ball et al. describe a recombinant Phl p 1 mosaic, P1M, having a B-D-A-C rearrangement that, as compared to wild-type rPhl p 1 (P1):
The amino acid and nucleotide sequences for the timothy grass pollen allergen 2 are disclosed in WO 94/23035. A more detailed description of the Phl p 2 from timothy grass pollen is provided in De Marino et al., Structure (1999) Vol. 7, No. 8, p. 943-952. The Phl p 2 antigen is preferred since it reacts with serum IgE from about 70% of grass pollen allergic individuals and elicits histamine release from basophils of sensitized patients.
In the course of the present invention, it has been found that the Phl p 2 allergen is preferably split into three peptides, namely peptide 1 having amino acids 1-33, peptide 2 having amino acids 34-64 and peptide 3 having amino acids 65-96. By rearranging the peptides in the order 1, 3 and 2 a mosaic antigen is provided which can be used for hypoallergenic vaccination. This mosaic antigen has the advantage that a sufficient amount of blocking IgE antibodies is produced, but the undesired side-reactions associated with the vaccination are nearly completely avoided. The amino acid sequence of the preferred Phl p 2 mosaic antigen has SEQ ID NO:1. The DNA coding for this preferred mosaic antigen has SEQ ID NO:2.
In the context of tree pollen allergens, pollens derived from members of the birch, oak, ash, elm, hickory, pecan, box elder, and mountain cedar families. Of these, the major birch pollen allergen, Bet v 1, is of particular interest. The amino acid and nucleotide sequences for wild-type Bet v 1 and proposed mosaics thereof are set forth herein in SEQ ID Nos: 13-20.
In the course of the present invention, it has been found that the Bet v 1 allergen may be split into two or three peptides. In a first embodiment, referred to herein as Bet v 1 rs1, the native allergen is split into two peptide fragments, fragment A′ composed of amino acids 1-74 of SEQ ID NO: 13 and fragment B′ composed of amino acids 75-160 of SEQ ID NO: 13. The fragments are reassembled in a B′-A′ configuration [(75-160)-(1-74)] to give rise to Bet v 1 rs1, the amino acid and nucleotide sequences for which are set forth herein in SEQ ID NOs; 15 and 16.
In a second embodiment, referred to herein as Bet v 1 rs2, the native allergen is again split into two peptide fragments, with fragment A″ composed of amino acids 1-109 of SEQ ID NO: 13 and fragment B″ composed of amino acids 110-160 of SEQ ID NO: 13. The fragments are reassembled in a B″-A″ configuration [(110-160)-(1-109)] to give rise to Bet v 1 rs2, the amino acid and nucleotide sequences for which are set forth herein in SEQ ID NOs; 17 and 18.
In a third embodiment, referred to herein as Bet v 1 m, the native allergen is split into three peptide fragments, with fragment A composed of amino acids 1-59 of SEQ ID NO: 13, fragment B composed of amino acids 60-109, and fragment C composed of amino acids 110-160 of SEQ ID NO: 13. The fragments are reassembled in a C-B-A configuration [(110-160)-(60-109)-(1-59)] to give rise to Bet v 1 m, the amino acid and nucleotide sequences for which are set forth herein in SEQ ID NOs; 19 and 20.
In all three cases, as compared to wild-type rBet v 1, the derivatives:
As such, the mosaic antigen is able to provide the benefits of wild-type allergen vaccination, with virtually none of the undesired allergic side-reactions associated therewith.
As noted above, the reassembled hypoallergenic mosaic antigens of the present invention, being capable of inducing a strong allergen-specific IgG response, i.e., therapeutic levels of blocking IgG antibodies, while simultaneously inhibiting or suppressing IgE production, find particular utility in the treatment of allergies and allergic disorders.
Accordingly, one aspect of the present invention relates to a method of treating an allergic disorder in a subject in need thereof including the step of administering to the subject a therapeutically effective amount of a mosaic antigen of the present invention or a nucleic acid coding for such an allergen. In a preferred embodiment, the mosaic antigen is formulated for parenteral administration, more preferably for intradermal or subcutaneous injection, including, as needed, suitable pharmaceutical carrier(s), excipients(s) and diluent(s) such as are conventional in the art. The pharmaceutically formulated allergen may be singly or repeatedly administered, for example in accordance with conventional immunotherapy protocols.
Another aspect of the present invention relates to the use of a mosaic antigen in connection with the preparation of a medicament for the treatment or prevention of an allergic disorder. In the context of medicament preparation, the mosaic antigen is preferably formulated with a suitable pharmaceutical carrier and administered together with an adjuvant. Examples of suitable adjuvants include alum compositions, such aluminum hydroxide gel. Alternatively, the mosaic antigen may be covalent bound to another component that generally enhances the immunologic reaction of the body. Carbohydrate bead compositions such as described in co-pending U.S. application Ser. No. 10/510,655 filed Nov. 30, 2004, the contents of which are incorporated herein, are also contemplated.
Yet another aspect of the present invention relates to the use of a mosaic antigen of the present invention in connection with the preparation of a vaccine for the treatment or prophylaxis of an allergic disorder. To that end, a nucleic acid coding for a mosaic antigen of the present invention or a nucleotide sequence complementary thereto may serve as a DNA or RNA vaccine. Accordingly, it is yet another object of the present invention to provide a vaccine for the treatment or prevention of an allergic disorder comprising a nucleic acid coding for one or mosaic antigen(s) of the present invention. The vaccine is preferably formulated for subcutaneous administration, optionally including a pharmaceutically acceptable carrier and/or suitable vaccine adjuvant. For nucleic acid vaccines, a suitable polynucleotide sequence is inserted into the target cells. In addition to the sequence coding for the mosaic antigen, such a nucleotide vaccine may also contain regulatory elements like promoters, ribosome binding sites or termination sequences. Such nucleotide sequences are preferably incorporated into a suitable carrier that allows the nucleotide to come to the protein synthesizing machinery of the cells.
Hereinafter, the present invention is described in more detail by reference to the Examples. However, the following materials, methods and examples only illustrate aspects of the invention and in no way are intended to limit the scope of the present invention. As such, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
A. Preparation of Synthetic Phl p 2-Derived Peptides Lacking Allergenic Activity
In order to identify Phl p 2 fragments without allergenic activity, peptides, each comprising about ⅓ of the Phl p 2 protein were chemically synthesized (Table 1). The peptides had a length between 32 and 34 amino acids with molecular weights of around 3.7 kDa and together covered the complete Phl p 2 amino acid sequence.
The three peptides were synthesized using Fmoc (9-fluorenylmethoxycarbonyl)-strategy with HBTU (2-(1H-benzotriazol-1-yl) 1,1,3,3,tetramethyluronium hexafluorophosphat)-activation (0.1 mmol small-scale cycles) on the Applied Biosystems (Foster City, Calif.) peptide synthesizer Model 433A. Preloaded PEG-PS (polyethylenglycol polysterene) resins (0.15-0.2 mmol/g loading) (per Septive Biosystems, Warrington, UK) were used as solid phase to build up the peptides. Chemicals were purchased from Applied Biosystems. Coupling of amino acids was confirmed by conductivity monitoring in a feedback control system. One cysteine residue was added to each peptide at the N- or C-terminus to facilitate coupling of the peptides to carriers. Peptides were cleaved from the resins with a mixture of: 250 μl distilled water, 250 μl Triisopropylsilan (Flukan, Buchs, Switzerland), 9.5 ml TFA for 2 h and precipitated in tert-Butylmethylether (Flukan, Buchs, Switzerland). The identitiy of the peptides was checked by mass-spectrometry and they were purified to >90% purity by preparative HPLC (PiChem, Graz; Austria) (Focke M, Mahler V, Ball T., Sperr. W R, Majlesi Y, Valent P, Kraft D, Valenta R. Nonanaphylactic synthetic peptides derived from B cell epitopes of the major grass pollen allergen, Phl p 1, for allergy vaccination. FASEB J. 2001, 15: 2042-2044.
The allergenic activity of the Phl p 2-derived peptides was evaluated by comparing the IgE-reactivity of complete rPhl p 2 with the peptides by dot blot analysis (
Bound IgE antibodies were detected as described previously (Valenta R. Duchene M, Ebner C, Valent P, Sillaber C, Deviller P, Ferreira F, Tejkl M, Edelmann H, Kraft D, Scheiner O. Profilins constitute a novel family of functional plant pan-allergens. J. Exp. Med. 1992, 175: 377-385). Sera from all 35 grass pollen allergic patients showed IgE reactivity to nitrocellulose-dotted rPhl p 2 but no serum reacted with any of the three Phl p 2-derived peptides (
B. Characterization of the Recombinant Phl p 2 Mosaic Protein
A recombinant Phl p 2 mosaic protein was obtained by recombination of the three Phl p 2-derived peptides in altered sequence. This mosaic protein was created under the assumption that recombination of three non-allergenic Phl p 2 fragments in altered order will deliver a mosaic protein with disrupted three-dimensional structure and consequently reduced allergenic activity. In addition it was expected that the mosaic protein will exhibit better immunogenicity compared to the individual smaller peptide units and preserve the entire primary amino acid sequence of Phl p 2 thus containing the relevant T cell epitopes of Phl p 2.
The recombinant Phl p 2 mosaic was constructed by PCR-based gene amplification of codas coding for the three peptides in the order shown in
The cDNA coding for a his-tagged rPhl p 2 allergen was obtained by PCR using a combination of the 5′ primer P2/1 (SEQ ID NO:6) and the 3′ primer P2/7 (SEQ ID NO:12): CGC GAA TTC TCA GTG GTG GTG GTG GTG GTG CTC TTC TGG CGC GTA GGT GGC and the cDNA coding for Phl p 2 as a template.
The cDNAs coding for the his-tagged Phl p 2 mosaic and the his-tagged Phl p 2 allergen were separately ligated into Nde I/Eco RI cut plasmids pET17b (Novagen). The DNA sequences of the two plasmid constructs was confirmed by sequence analysis and the recombinant proteins were expressed in Escherichia coli BL21 (DE3) (Novagen) by induction with 0.5-mM isopropyl-β-thiogalactopyranoside at an optical density at 600 nm of 0.4 in liquid culture (LB, medium containing 100 mg/l ampicillin) for additional 4 hours at 37° C. E. coli cells from a 500 ml culture were harvested by centrifugation and prepared for purification under native (rPhl p 2) or denaturing conditions (rPhl p 2 mosaic) according to the manufacturers advice (Quiagen, Hilden, Germany). Protein samples were analyzed for purity by sodium codicil sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and protein staining (Fling S P, Gregerson D S. Peptide and protein molecular weight determination by electrophoresis using a high-molarity Tris buffer system without urea. Anal. Biochem. 1986, 155:83-88) (
C. The rPhl p 2 Mosaic Lacks IgE Reactivity and Allergenic Properties
The IgE binding capacity of purified Phl p 2 mosaic (P2M) was compared with that of Phl p 2 wild-type by dot blot experiments as described for the peptides using sera from twelve timothy grass pollen allergic patients (
Histamine released in the cell free supernatants was determined in triplicates by radioimmunoassay and is expressed as mean percentage of the total histamine content of the cells as described by Valent et al.
The strongly reduced allergenic activity of rPhl p 2 mosaic was confirmed by skin testing in grass pollen allergic patients (Table 2). SPTs (skin prick tests) were performed on the individuals' forearms. Twenty microliter aliquots containing 5 concentrations of rPhl p 2 and of Phl p 2-derived mosaic P2M (1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml) were applied. In addition, standardized skin prick solutions (timothy grass pollen extract and histamine) (Allergopharma, Reinbeck, Germany) were tested. Reactions were recorded 20 minutes after SPT by photography and by transferring the ballpoint pen-surrounded wheal area with a scotch tape to paper. The mean wheal diameter (Dm) was calculated by measuring the maximal longitudinal and transversal diameter and dividing their sum by 2 as described by Focke et al., 2001.
rPhl p 2 induced strong wheal reactions already at the lowest concentration tested, i.e., 1 μg/ml, whereas rPhl p 2 mosaic induced only mild wheal reactions at the maximal concentrations tested (i.e., 8-16 μg/ml) thus confirming the reduced allergenic activity of the mosaic protein.
D. Immunization with the rPhl p 2 Mosaic Induces IgG Antibodies that Recognize rPhl p 2 Wild-type and Inhibits Allergic Patients' IgE Binding to Phl p 2
In order to test whether immunization with Phl p 2 mosaic and Phl p 2 mosaic will induce
IgG antibodies that react with natural Phl p 2, rabbits were immunized with rPhl p 2 mosaic, KLH-coupled rPhl p 2 mosaic or rPhl p 2 using Freund's adjuvant as described by Focke et al.
The reactivity of rabbit IgG antibodies with rPhl p 2 was studied by dot blot experiments (
The rabbit anti-rPhl p 2 mosaic antiserum reacted strongly with the immunogen (rPhl p 2 mosaic) as well as with the rPhl p 2 allergen (
E. Measurement of Blocking Antibodies
It was studied whether IgG antibodies induced by immunization with the rPhl p 2 mosaic inhibit the binding of allergic patients' serum IgE to complete rPhl p 2 by ELISA competition using sera from five grass pollen allergic patients (Table 3). ELISA plates (Nunc Maxisorp, Rokslide, Denmark) were coated with rPhl p 2 (1 μg/ml) and preincubated either with a 1:100 dilution of each of the anti-Phl p 2 mosaic and anti-Phl p 2 antiserum and, for control purposes, with the corresponding preimmunsera. After washing plates were incubated with 1:3 diluted sera from five Phl p 2-sensitized grass pollen allergic patients and bound IgE antibodies were detected with alkaline phosphate conjugated monoclonal rat anti-human IgE antibody (Pharmingen, San Diego, Calif.), diluted 1:1000. The percentage inhibition of IgE binding achieved by preincubation with the anti-Phl p 2 mosaic and Phl p 2 was calculated as follows: % inhibition of IgE binding=100−ODI/ODP×100. ODI and ODP represent the extinctions after preincubation with the rabbits immune and preimmune serum, respectively as described by Focke et al., 2001.
The anti-Phl p 2 mosaic antibodies inhibited the binding of grass pollen allergic patients IgE binding to Phl p 2 (20.93% average inhibition) albeit to a lower degree as was achieved by preincubation with antibodies induced by immunization with the rPhl p 2 allergen (54.73% average inhibition).
The results of the immunization studies thus show that antibodies raised against the rPhl p 2 mosaic recognize the Phl p 2 wild-type allergen and inhibit allergic patients IgE recognition of Phl p 2.
A. Materials and Methods
Patients suffering from birch pollen allergy were characterized by case history and positive skin prick testing. Serum IgE Abs specific to birch pollen extract and rBet v 1 were determined by immuno CAP measurements (Phadia, Uppsala, Sweden) as described3 (Table 6). Control sera were taken from two non-allergic volunteers.
The plasmid pET 17b (Novagen Inc., Madison, Wis., USA), used for the expression of rBet v1 and of the rBet v 1 derivatives is as described by Hoffman-Sommergruber et al.17 The recombinant Escherichia coli-expressed (BL 21-DE3) (Stratagene, La Jolla, Calif., USA) birch pollen allergen Bet v 1 (batch # 21), was obtained from Biomay (Vienna, Austria).
Bip 1, a monoclonal antibody with specificity for the major birch pollen allergen Bet v 1, was previously described by Laffer et al.18 The mouse mAb 4A6 was raised against purified recombinant birch pollen profiling (see Widemann et al.19). Anti-IgE mAb E-124.2.8 was purchased from Immunotech (Marseille, France).
Mouse IgG mAbs against peptide 2 (mAb#2) (aa 30-59) and against peptide 6 (mAb#12) (aa 74-104) of Bet v 1 were obtained by immunization of mice using KLH-coupled synthetic peptides (peptide 2: LFPKVAPQAISSVENIEGNGGPPTIKKISF (SEQ ID NO: 21); peptide 6: EDVHTNFKYNYSVIEGGPIGDTLEKISNEIK (SEQ ID NO: 22).
Based on the Bet v 1 sequence described by Mothes et al.2, synthetic genes were generated giving rise to three different recombinant Bet v 1 derivatives: Restructured Bet v 1 #1 (Bet v 1-rs1) comprising amino acids 75-160+1-74, Restructured Bet v 1 #2 (Bet v 1 rs2) comprising amino acids 110-160+1-109, and Bet v 1 mosaic comprising amino acids 110-160+60-109+1-59 (
The synthetic genes of each of the recombinant Bet v 1 derivatives were cloned into the pET17b (Novagen Inc., Madison, Wis., USA) cloning vector via NdeI and EcoR1 restriction sites. The correct sequence of the derivative molecules was confirmed by double stranded DNA sequencing (Eurofins Medigenomix GmbH, Ebersberg, ATG Biosynthetics GmbH, Merzhausen, Germany).
Batch fermentation of E. coli BL 21 (DE3) transformed with pET-17b-Bet v 1-rs1, -rs2 or mosaic was carried out at 37° C. in a 10 L fermenter (New Brunswick, Bioflow 3000) in LB medium with the addition of 0.05% (v/v) glycerol, 0.25% (w/v) MgSO4. 7 H2O, and 0.18% Na2HPO4. 2 H2O for 8 h at 37° C. until a cell density (0D600nm) of 0.4 to 0.6 was reached. Protein expression was induced by adding 0.5 mM isopropyl-B-thiogalactopyranoside (IPTG) (Calbiochem, Merck, Darmstadt, Germany). Recombinant proteins were produced and characterized as follows:
Inclusion bodies were isolated from the cells using lysozyme (0.1 mg/g cell wet weight) (Sigma-Aldrich, St. Louis, Mo., USA) and repetitive freezing and thawing in buffer I (50 mM Tris base, 1 mM EDTA and 0.1% Triton X-100) for Bet v 1-rs1 and Bet v 1-mosaic, or buffer II (25 mM NaH2PO4, pH 7.4 and 0.1% Triton X-100) for Bet v 1-rs 2 (5 mL/g cell wet weight). NaCl was added to a final concentration of 200 mM, and the suspensions were centrifuged (10000 g for 30 min. at 4° C.) leaving the proteins containing inclusion bodies in the pellet.
Bet v 1-rs1 and Bet v 1-rs2 pellets were washed with 1% Triton X-100, 2 mM EDTA, 2 mM B-mercaptoethanol, 20 mM Tris/HCl pH 8.0 (3 times) and afterwards with 50% ethanol, 20 mM Tris/HCl pH 8.0 (2 times). The Bet v 1-mosaic pellet was washed once with 1% Triton X-100, 20 mM NaH2PO4 pH 7.4 and once with 25% ethanol, 20 mM NaH2PO4 pH 7.0. Inclusion bodies were suspended and stirred for 30 min in buffer A (6 M urea, 10 mM Tris/HCl, 1 mM EDTA, pH 7.0) in case of Bet v 1-rs1, or in buffer B (5 M urea in 20 mM sodium acetate buffer, pH 5.0) in case of Bet v 1-rs2 and mosaic. The suspensions were centrifuged (10000 g for 30 min. at 4° C.) and the final supernatant used for purification.
Recombinant Bet v 1-rs1 was first purified by anion exchange chromatography (AIEC) on a Q-Sepharose FF column (GE Healthcare, UK Limited) by applying a linear gradient from 0-250 mM NaCl in buffer A. Fractions containing Bet v 1-rs1 were dialyzed against buffer C (6 M urea, 20 mM NaH2PO4, 1.5 M NaCl, pH 4.5) and subjected to hydrophobic interaction chromatography (HIC) using a Phenyl Sepharose FF column (GE Healthcare, UK Limited) equilibrated with buffer C. Purified Bet v 1-rs1 was eluted with a linear gradient from 0% to 100% buffer D (6 M urea, 20 mM Tris base, pH 9.3) and pure fractions were dialyzed first against 6M urea and then against 1 mM acetic acid. Finally, the protein was subjected to 0.2 μm filtration and stored at −20° C.
Recombinant Bet v 1-rs2 and -mosaic were purified by cation exchange chromatography (CIEC) using a SP-Sepharose FF column (GE Healthcare, UK Limited) equilibrated with buffer B and eluted with a linear gradient from 0-400 mM NaCl in the same buffer. To the fractions containing Bet v 1-rs2 NaCl was added to a final concentration of 1.7 M. Protein was centrifuged (10000 g for 30 min at 4° C.) and the supernatant subjected to hydrophobic interaction chromatography (HIC) using a Phenyl Sepharose FF column (GE Healthcare, UK Limited), equilibrated with buffer E (6 M urea, 1.7 M NaCl in a 20 mM sodium acetate buffer, pH 5.0). Bet v 1-rs2 was then eluted with a linear gradient from 1.7-0 M NaCl in buffer E and fractions containing >90% pure rBet v 1-rs2 were dialyzed against buffer F (6 M Urea, 20 mM NaH2PO4 pH 7.0) and subjected to anion exchange chromatography (AIEC) using a Q-Sepharose FF column (GE Healthcare, UK Limited) equilibrated with the buffer F. To the fractions containing Bet v 1-mosaic urea and NaCl were added to a final concentration of 6.5 M and 3.5 M respectively. The protein was centrifuged (10000 g for 30 min at 4° C.) and the supernatant subjected to hydrophobic interaction chromatography (HIC) using a Phenyl Sepharose FF column (GE Healthcare, UK Limited), equilibrated buffer G (6.5 M urea, 3.5 M NaCl in a 20 mM sodium acetate buffer, pH 5.0). Bet v 1-mosaic was eluted with a linear gradient from 3.5-0 M NaCl in buffer G.
Finally, Bet v 1-rs2 or -mosaic fractions containing >90% purity were pooled, dialyzed against 5 mM NaH2PO4 pH 7.4 and subjected to 0.2 μm filtration and stored at −20° C.
Purified rBet v 1 and each of the rBet v 1 derivatives (5 μg protein/slot) were resolved on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence or absence of 2-mercaptoethanol36. Proteins were visualized by staining with Coomassie brilliant blue. The presence of endotoxin was determined in a limulus amebocyte lysate (LAL) chromogenic assay (QCL-1000R Chromogenic LAL Endpoint Assay, Bio-Whittaker, Walkersville, USA).
Laser desorption mass spectra of the proteins were acquired in a linear mode with a MALDI-ToF instrument (Microflex, Bruker, Billerica, Mass., USA). Samples were dissolved in 10% acetonitrile (0.1% trifluoroacetic acid), and sinapinic acid (dissolved in 60% acetonitrile, 0.1% trifluoroacetic acid) was used as a matrix. For sample preparation a 1:1 mixture of protein and matrix solution was deposited onto the target and air-dried.
CD spectra were acquired on a JASCO (Tokyo, Japan) J-810 spectropolarimeter. CD measurements were performed with purified rBet v 1 and rBet v 1 derivatives at room temperature, at protein concentrations of 0.1 mg/ml using a rectangular quartz cuvette with 0.1-cm path length. Far ultraviolet (UV) spectra were recorded in the wavelength ranges between 190 and 260 nm with a resolution of 0.5 nm at a scan speed of 50 nm/min and resulted from averaging of three measurements. The final spectra were baseline-corrected and results were expressed as the mean residue ellipticity (Θ) at a given wavelength. The secondary structure content of rBet v 1 and Bet v 1 derivative molecules was calculated using the secondary structure estimation program CDSSTR37.
For gel filtration rBet v 1 derivatives which had been stored for 22 months at −20° C. were loaded onto a Superdex™ 200 5/150 GL column (GE Healthcare, Sweden), equilibrated with 10 mM sodium phosphate buffer pH 7.4 containing 150 mM NaCl. Bovine y-globulin (158 kDa), Chicken Ovalbumin (44 kDa) and Horse Myoglobin (17 kDa) from Gel Filtration Standard (BIO-RAD, Richmond, Calif.) were used for calibration. Recombinant Bet v 1 which also had been stored for 22 months at −20° C. was applied to the column under the same conditions to show the elution characteristics of the wild-type protein. The flow rate was 0.3 ml/min. The molecular masses were calculated using linear regression of the logarithm of molecular mass versus elution volumes derived from UV measurements at 280 nm. Recombinant Bet v 1 derivatives were reapplied to gelfiltration with the Superdex™ 200 5/150 GL column to check the stability of the proteins after further storage at −20° C.
Detection of IgG Binding Capacity of rBet v 1 and rBet v 1 Derivatives:
Purified recombinant Bet v 1 and rBet v 1 derivative molecules were tested for reactivity with specific antibodies. Five μg of each protein/slot was separated by SDS-PAGE20 and blotted onto nitrocellulose21. Nitrocellulose blotted proteins were incubated either with a 1:2000 dilution of a rabbit anti-rBet v 1 or the corresponding pre-immune serum, or with a 1:1000 dilution of mouse monoclonal IgG antibodies. Bound IgG antibodies were detected with a 1:1000 dilution of 125I-labeled goat anti-rabbit antibodies or with a 1:1000 dilution of 125I-labeled goat anti-mouse antibodies (NEN Life Science Products, Inc., Boston, Mass., USA) and visualized by autoradiography9.
IgE Reactivity of Dot-Blotted rBet v 1 and Bet v 1 Derivatives:
Two μL aliquots containing 1 μg of purified rBet v 1, each of the rBet v 1 derivatives, bovine serum albumin (BSA) and human serum albumin (HSA) (negative control proteins) (Roth, Karlsruhe, Germany) were dotted onto nitrocellulose. Nitrocellulose strips were incubated with sera from nineteen birch pollen allergic individuals, two non-allergic individuals or buffer without addition of serum. Bound IgE antibodies were detected with a 1:20 dilution of 125I-labeled anti-human IgE antibodies (RAST RIA, Demeditec Diagnostics, Germany). The presence of rBet v 1 and rBet v 1 derivatives on the nitrocellulose membrane was shown with rabbit anti-rBet v 1 antibodies, which were detected with 1:1000 dilution of 125I-labeled donkey anti-rabbit antibodies (NEN Life Science Products, Inc., Boston, Mass., USA).
Rabbits were immunized twice, at study day 0 and at day 28, with 200 μg of purified rBet v 1, rBet v 1-rs1, rBet v 1-rs2, or rBet v 1-mosaic initially adsorbed to CFA (Complete Freund's adjuvant) and followed by booster injection using IFA (Incomplete Freund's adjuvant), or with 100 μg of the proteins adsorbed to Al(OH)3. Pre-immune sera were obtained from the rabbits before immunization (Charles River Breeding Laboratories, Kisslegg, Germany).
ELISA plates (Greiner, Kremsmiinster, Austria) were coated with rBet v 1, rBet v 1 derivatives or BSA (negative control) (5 μg/ml diluted in PBS) at 4° C. overnight. After washing three times with PBS-T (PBS+0.05% Tween 20) and blocking with 2% bovine serum albumin (BSA) (Roth, Karlsruhe, Germany) in PBS-T for 6 hours, plates were incubated either with rabbit antisera in five different dilutions (PBS-T, 0.5 w/vol % BSA) (1:1000, 1:5000, 1:10000, 1:100000 and 1:1000000) for antibodies generated with CFA adsorbed proteins or in eight different dilutions (1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000 and 1:64000) for antibodies induced with Al(OH)3 adsorbed proteins. Controls were performed with normal rabbit antibodies. Plates were washed five times with PBS-T and bound rabbit IgG antibodies were detected with a 1:1000 diluted anti-rabbit IgG Horseradish Peroxidase linked whole antibody from donkey (GE Healthcare, UK Limited) for 1 hour at 37° C. and 4° C. After washing with PBS-T (5 times) the color development was performed by addition of staining solution ABTS (2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt; Sigma-Aldrich, St. Louis, Mo., USA) (100 μl/well). The optical density was measured using an ELISA Reader (Dynatech, Denkendorf, Germany) at 405 nm.
The allergenic activity of allergen derivatives was compared with that of the Bet v 1 wild-type allergen using CD203c assays as follows:
Heparinized peripheral blood samples were obtained from birch pollen allergic individuals after informed consent was given. Blood aliquots (100 μl) from six patients were incubated (unique) with serial dilutions (0.005 to 50 pM) of rBet v1, an equimolar mix of the rBet v 1 fragments (F1+F2), or rBet v 1-rs 1 for 15 minutes at 37° C. Blood aliquots (100 μl) from additional four patients were incubated (unique) with serial dilutions (0.005 to 50 pM) of rBet v1, rBet v 1-rs1, rBet v 1-rs2, or rBet v 1-mosaic as described above. A monoclonal anti-IgE antibody E-124.2.8 (1 μg/ml) (Immunotech, Marseille, France) and PBS (control buffer) were used as controls. Thereafter, samples were washed in PBS containing 20 mM EDTA (Gibco, Carlsbad, Calif., USA) and cells were incubated with 10 μl of PE-conjugated CD203c mAb 97A6 (Immunotech, Marseille, France) for 15 minutes at room temperature. After erythrocyte lysis using FACS™ Lysing Solution (Becton Dickinson Biosciences, San Jose, Calif., USA), cells were washed and resuspended in PBS and then analyzed by two-color flow cytometry on a FACSScan (Becton Dickinson Biosciences, San Jose, Calif., USA) using Flowjo Software (Tree Star Inc., Ashland, Oreg., USA). Anti-IgE-induced up-regulation of CD203c was calculated from mean fluorescence intensities (MFIs) obtained with stimulated (MFIstim) and unstimulated (MFIcontrol) cells, and is expressed as stimulation index (MFIstim: MFIcontrol)3. Three patients (
The inhibition of allergic patients' IgE binding to Bet v 1 by IgG antibodies was performed using an ELISA competition assay as follows:
ELISA plates (Greiner, Kremsmiinster, Austria) were coated with 100 μl of rBet v 1 (5 μg/ml diluted in PBS) overnight at 4° C. Plates were blocked with 2% bovine serum albumin (BSA) (Roth, Karlsruhe, Germany) in PBS-T (PBS 0.05% Tween 20) for 6 hours at 4° C. overnight and then preincubated overnight at 4° C. with 1:50 dilutions (in PBS 0.5% BSA/0.05% Tween) of the rabbit sera anti-rBet v 1, anti-rBet v 1-rs1, anti-rBet v 1-rs2 or anti-rBet v 1-mosaic raised with CFA, or 1:10 dilutions for rabbit sera raised with Al(OH)3, and for control purposes by using the corresponding rabbit pre-immune sera. Plates were washed three times with PBS-T and incubated with 1:10 diluted sera from 18 birch pollen allergic patients sensitized to Bet v 1. Bound human IgE antibodies were detected using a 1:2500 diluted AP-conjugated (alkaline phosphatase) mouse monoclonal anti-human IgE antibody (BD Pharmingen, San Diego, Calif., USA). Color development was performed by addition of staining solution ABTS (2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt; Sigma-Aldrich, St. Louis, Mo., USA) (100 μl/well) and the optical density was measured in an ELISA Reader (Dynatech, Denkendorf, Germany) at 405 nm. The percentage of inhibition of IgE-binding was calculated using the OD values obtained, as follows: percent inhibition of IgE binding=100−(ODs/ODp)×100. ODs, extinction coefficient after preincubation with the rabbit serum. ODp, extinction coefficient after preincubation with the pre-immune serum.
B. Rational Construction of Hypoallergenic rBet v 1 Derivatives
It has been shown that two recombinant fragments of Bet v 1 comprising amino acids 1-74 and 75-160 preserved the Bet v 1-specific T cell epitopes but exhibited an approximately 100 fold reduced allergenic activity compared to rBet v 1 as shown in vitro by basophil activation testing and in several in vivo provocation studies9, 22-25. Each of these fragments contains a peptide defined by two monoclonal antibodies mAb#2 (aa 30-59) and mAb#12 (aa 74-104) which induced Bet v 1-specific IgG antibodies inhibiting the binding of birch pollen allergic patients IgE to Bet v 126 (
C. Characterization of the Recombinant Bet v 1 Derivatives
High level expression of the recombinant proteins yielding more than 20% of the total E. coli proteins was obtained. Each of the recombinant proteins could be purified from the inclusion body fraction of the bacteria via several chromatography steps to more than 90% purity (
Next, Bet v 1-specific antibody probes were used to test IgG reactivity of the Bet v 1 derivatives. Nitrocellulose-blotted rBet v 1 and rBet v 1 derivatives reacted with the polyclonal rabbit antibodies that had been raised against rBet v 1 (
D. The Bet v 1 Mosaics Lack IgE Reactivity and Allergenic Properties
None of the 19 birch pollen allergic patients tested exhibited any detectable IgE reactivity to the rBet v 1 derivatives whereas they showed IgE-binding to rBet v 1 (
Next, rBet v 1-rs1 was compared with rBet v 1 and an equimolar mix of rBet v 1 fragments, testing for allergenic activity using basophils from birch pollen allergic patients. rBet v 1-rs1 did not cause any up-regulation of the CD203c expression up to the maximum concentration (i.e., 50 pM) tested in the six patients whereas rBet v 1 started to induce basophil activation at 0.5 pM and each of the patients responded to 50 pM. rBet v 1-rs1 exhibited even lower allergenic activity than the rBet v 1 fragment mix which induced CD203c up-regulation at 5 pM in one patient and at 50 pM in two of the 6 patients (
E. Immunization with the rBet v 1 Mosaics Induces IgG Antibodies that Recognize Bet v 1 and Inhibits Allergic Patients'IgE Binding to Bet v 1
As shown in
The derivatives were further investigated to determine whether rBet v 1 derivative-induced rabbit IgG antibodies can inhibit the binding of patients' serum IgE to the wild-type rBet v 1 in an ELISA competition assay. In case of immunization with CFA adsorbed proteins the anti-rBet v 1-rs1 antiserum inhibited the binding of birch pollen allergic patients' IgE to rBet v 1 between 56.5% and 98% (85% mean inhibition). The anti-rBet v 1-rs2 antiserum showed inhibition rates between 59.5% and 98.5% (87% mean inhibition) and the anti-rBet v 1-mosaic inhibited between 58% and 99.5% (82% mean inhibition). Interestingly, the rabbit antiserum against rBet v 1 derivatives showed higher average inhibition rates than the anti-rBet v 1 antibodies which yielded only 62% mean inhibition of IgE binding to rBet v 1 (Table 4). Immunization with Al(OH)3 adsorbed rBet v 1 derivatives resulted also in Bet v 1-specific IgG responses which blocked allergic patients IgE binding to Bet v 1 (
The results herein confirm the utility of the inventive mosaic approach to the design of hypoallergenic allergens particularly suited to immunotherapy for the treatment and prevention of allergic disorders.
All patents and publications mentioned herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
While the invention is herein described in detail and with reference to specific embodiments thereof, it is to be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications can be made therein without departing from the spirit and scope of the invention. Other advantages and features will become apparent from the claims filed hereafter, with the scope of such claims to be determined by their reasonable equivalents, as would be understood by those skilled in the art. Thus, the invention is intended to be defined not by the above description, but by the following claims and their equivalents.
Preferred wild-type allergens to be modified in accordance with mosaic approach of the present invention:
Ambrosia artemisiifolia
Ambrosia trifida
Artemisia vulgaris
Helianthus annuus
Mercurialis annua
Chenopodium album
Salsola kali
Humulus japonicus
Parietaria judaica
Parietaria officinalis
Cynodon dactylon
Dactylis glomerata
Festuca pratensis
Holcus lanatus
Lolium perenne
Phalaris aquatica
Phleum pratense
Poa pratensis
Sorghum halepense
Phoenix dactylifera
Alnus glutinosa
Betula verrucosa
Carpinus betulus
Castanea sativa
Corylus avellana
Quercus alba
Fraxinus excelsior
Ligustrum vulgare
Olea europea
Syringa vulgaris
Plantago lanceolata
Cryptomeria japonica
Cupressus arizonica
Cupressus sempervirens
Juniperus ashei
Juniperus oxycedrus
Juniperus sabinoides
Juniperus virginiana
Platanus acerifolia
Acarus siro
Blomia tropicalis
Dermatophagoides farinae
Dermatophagoides
microceras
Dermatophagoides
pteronyssinus
Euroglyphus maynei
Glycyphagus domesticus
storage mite
Lepidoglyphus destructor
Tyrophagus putrescentiae
Bos domesticus
Canis familiaris
Equus caballus
Felis domesticus
Cavia porcellus
Mus musculus
Rattus norvegius
Alternaria alternata
Cladosporium herbarum
Aspergillus flavus
Aspergillus fumigatus
Aspergillus niger
Aspergillus oryzae
Penicillium
brevicompactum
Penicillium chrysogenum
Penicillium citrinum
Penicillium oxalicum
Fusarium culmorum
Trichophyton rubrum
Trichophyton tonsurans
Candida albicans
Candida boidinii
Psilocybe cubensis
Coprinus comatus
Rhodotorula mucilaginosa
Malassezia furfur
Malassezia sympodialis
Epicoccum purpurascens
Aedes aegyptii
Apis mellifera
Bombus pennsylvanicus
Blattella germanica
Periplaneta americana
Chironomus kiiensis
Chironomus thummi
Ctenocephalides felis felis
Thaumetopoea pityocampa
Lepisma saccharina
Dolichovespula maculate
Dolichovespula arenaria
Polistes annularies
Polistes dominulus
Polistes exclamans
Polistes fuscatus
Polistes gallicus
Polistes metricus
Vespa crabo
Vespa mandarina
Vespula flavopilosa
Vespula germanica
Vespula maculifrons
Vespula pennsylvanica
Vespula squamosa
Vespula vidua
Vespula vulgaris
Myrmecia pilosula
Solenopsis geminata
Solenopsis invicta
Solenopsis saevissima
Triatoma protracta
Gadus callarias
Salmo salar
Bos domesticus
Gallus domesticus
Metapenaeus ensis
Penaeus aztecus
Penaeus indicus
Penaeus monodon
Todarodes pacificus
Helix aspersa
Haliotis midae
Rana esculenta
Brassica juncea
Brassica napus
Brassica rapa
Hordeum vulgare
Secale cereale
Triticum aestivum
Zea mays
Oryza sativa
Apium gravaolens
Daucus carota
Corylus avellana
Malus domestica
Pyrus communis
Persea americana
Prunus armeniaca
Prunus avium
Prunus domestica
Prunus persica
Asparagus officinalis
Asparagus
Crocus sativus
Lactuca sativa
Vitis vinifera
Musa x paradisiaca
Ananas comosus
Citrus limon
Citrus sinensis
Litchi chinensis
Sinapis alba
Glycine max
Vigna radiata
Arachis hypogaea
Lens culinaris
Pisum savitum
Actinidia chinensis
Capsicum annuum
Lycopersicon esculentum
Solanum tuberosum
Bertholletia excelsa
Juglans nigra
Juglans regia
Anacardium occidentale
Ricinus communis
Sesamum indicum
Cucumis melo
Anisakis simplex
Argas reflexus
Ascaris suum
Carica papaya
Dendronephthya nipponica
Hevea brasiliensis
Homo sapiens
Triplochiton scleroxylon
| Number | Date | Country | Kind |
|---|---|---|---|
| 03.001242.1 | Jan 2003 | EP | regional |
This application is a continuation-in-part of U.S. patent application Ser. No. 12/349,614 filed Jan. 7, 2009, which is a divisional of U.S. patent application Ser. No. 10/542,735, filed Jul. 21, 2005 (now U.S. Pat. No. 7,491,396 issued Feb. 17, 2009), which, in turn, is a national stage of PCT Application No. PCT/EP03/14507, filed Dec. 18, 2003, which, in turn, claims priority to European Patent Application No. 03.001242.1 filed Jan. 21, 2003. The contents of these priority applications are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10542735 | Jul 2005 | US |
| Child | 12349614 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12349614 | Jan 2009 | US |
| Child | 13179116 | US |
