Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/35—Allergens
  • A61K39/36—Allergens from pollen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

• the present invention relates to non-anaphylactic, i.e. hypoallergemc, forms of the major timothy grass pollen allergen Phi p 6 and the use of the forms for hyposensitization and for diagnosis.
• the invention also relates to a method for hyposensitization of a mammalian individual, typically a human individual, suffering from type I allergy against the Phi p 6 allergen.
• Type I allergy is a genetically determined hypersensitivity disease that affects more than 20% of the population in industrialized countries (1).
• allergic patients produce IgE antibodies against ;?er se innocuous, mostly air-born proteins from pollen, mites, moulds and animal hair/dander.
• the symptoms of Type I allergy result from allergen-induced crosslinking of effector cell (mast cell, basophil)-bound IgE antibodies and subsequent release of inflammatory mediators (2). Since approximately 40% of allergic individuals suffer symptoms following contact with grass pollen, research has concentrated on the characterization of relevant grass pollen allergens by protein and immunochemical methods (3). While groups of major allergens have been identified as cross-reactive moieties that occur in most grass species (4), nothing was known concerning their nature and biological functions.
• grass pollen allergens belong to a family of cell wall-loosening proteins (expansins) (8) and grass group 5 allergens may possess RNAse activity (9) has restimulated ideas that the biological function of a given protein may be related to its allergenicity.
• major grass pollen allergens can either become attached to small sized particles (e. g., group 1 allergens to diesel exhaust (10)) or may become airborn as small pollen subcompartments (e. g., group 5 allergens in amyloplasts (11)) would provide a possible mechanism of how certain allergens may be able to reach the deep airways of patients and to elicit allergic asthma.
• T-cell epitopes harbour a great variety of different T-cell epitopes (Ebner et al., J. Immunol 150 (1993) 1047-1054; Joost-van-Neerven et al., J. Immunol. 151 (1993) 2326-2335; and Schenket al, J. Allergy Clin. Immunol. 96 (1995) 986-996) which may overlap with continuous IgE-epitopes. To prevent crosslinking of effector cell (mast cell, basophil) bound IgE and mediator release, T-cell epitopes and IgE epitopes need to be dissected.
• Vrtala et al., J. Clin. Invest. 99(7) 1673-1681 (1997) and WO 99/16467 disclose a novel strategy of reducing the anaphylactic activity of the major birch allergen Bet v 1 by disrupting the three dimensional structure by expressing two parts of the Bet v 1 cDNA representing amino acids 1-74 and 75-160 in Escherichia coli. In contrast to the complete recombinant Bet v 1, the recombinant fragments showed almost no allergenicity. Both non-anaphylactic fragments induced proliferation of human Bet v 1- specific T cell clones, indicating that they harboured all dominant T cell epitopes and therefore could be used for safe and specific T cell immunotherapy.
• Bet v 1 allergen possesses discontinuous (i.e. conformational) IgE epitopes rather than continuous IgE epitopes as is the case for many other allergens.
• the major timothy grass pollen allergen Phi p 6 contains continuous (sequential) IgE epitopes and would therefore not be susceptible to the above fragmentation strategy to reduce anaphylactic activity as outlined above.
• Phi p 6 deletion variants may be constructed by genetic (recombinant) or synthetic fragmentation, which fragments may be used for specific immunotherapy of grass pollen allergy with reduced anaphylactic side effects.
• Such fragments with strongly reduced anaphylactic ability will below be referred to as non- anaphy lactic or hypoallergemc.
• a hypoallergemc immunogenic molecule derived from the Phi p 6 allergen, wherein the Phi p 6 molecule has an N-terminal and/or C-terminal deletion which makes the molecule at least substantially lack IgE binding capacity.
• the N-terminal or C-terminal deletion may be a terminal truncation of the allergen.
• the deletion may also be internal within the N-terminal or C-terminal part of the allergen, respectively.
• allergen molecule fragments may be produced by recombinant DNA techniques or peptide synthetic chemistry as is per se well known to the skilled person.
• the present invention provides a hypoallergemc immunogenic combination of molecules derived from the Phi p 6 allergen, comprising (i) a Phi p 6 molecule having an N-terminal deletion which makes the molecule at least substantially lack IgE binding capacity, and (ii) a Phi p 6 molecule having a C-terminal deletion which makes the molecule at least substantially lack IgE binding capacity, which two molecules together encompass the complete amino acid sequence of the Phi p 6 allergen.
• the respective sequences of the two Phi p 6 molecules may overlap or be contiguous.
• the sizes of the N-terminal and C-terminal deletions of the Phi p 6 allergen necessary for the fragments to be useful for the purposes of the invention, i.e. that the fragments are (i) immunogenic and (ii) non-IgE reactive, may readily be determined by the skilled person.
• the lack or presence of IgE binding ability of a particular N- terminal or C-terminally deletion molecule may easily be determined, the lack of IgE reactivity indicating that the molecule may be applied without or with low risk of inducing anaphylactic side effects.
• Immunogenic activity of the molecules may be determined by their capability of being recognized by a polyclonal antiserum to the complete Phi p 6 allergen. In this way fragments and fragment combinations, respectively, may be selected which have a very high likelyhood of being capable of eliciting immune responses which protect against the complete allergen.
• a third aspect of the invention is a specific hyposensitization therapy.
• Such therapy may be performed as known in the art for protein allergens and encompasses administering repeatedly to the mammal, typically a human individual, suffering from type I allergy against the allergen an immunogen that is capable of raising an IgG immune response against the allergen.
• the immunogen may be admixed with suitable adjuvants such as aluminium oxide.
• Administration may be done systemically, for instance by injection, infusion, etc, but also the oral route has been suggested in order to expose the intestinal part of the immune system. See also Norman PS, "Current status of immunotherapy for allergies and anaphylactic reactions" Adv. Internal. Medicine 41 (1996) 681-713.
• the immunogen to be administered may be an immunogenic molecule according to the first aspect of the invention, or a mixture of such molecules, preferably, the above-mentioned hypoallergemc immunogenic combination of molecules derived from the Phi p 6 allergen according to the second aspect of the invention, i.e. (i) a Phi p 6 molecule having an N-terminal deletion which makes the molecule at least substantially lack IgE binding capacity, and (ii) a Phi p 6 molecule having a C-terminal deletion which makes the molecule at least substantially lack IgE binding capacity, which two molecules together encompass the complete amino acid sequence of Phi p 6. More specifically, the immunogens may be used to induce antibody responses in a patient, and/or to elicit T cell response, and/or to modulate antibody and T cell repsonse to induce tolerance.
• a fourth aspect of the present invention provides the use the immunogen according to the first aspect, or the combination of immunogens according to the second aspect, as an antigen in an immunoassay for detecting specific antibodies of the IgA, IgD, IgE, IgG or IgM class directed against the Phi p 6 allergen from which the immunogen(s) derive.
• Appropriate assay variants involve formation of a ternary immune complex between the immunogen, sample antibody and an antibody directed against the Ig-class of interest.
• the sample may be any Ig-containing biological fluids, for instance a blood derived sample (serum, plasma, whole blood), CSF, etc.
• the hypoallergemc fragments may be used for diagnostic monitoring (e.g. IgG measurements, measurement of T cell responses) during therapy when inducing a new immune response against the fragments.
• a rabbit anti-rPhl p 6 antiserum was raised against purified, recombinant Phi p 6 using Freunds adjuvans (Charles River, Kissleg, Germany).
• Recombinant timothy grass pollen allergens, rPhl p 1, rPhl p 2 and rPhl p 5 were purified as described (13).
• Recombinant timothy grass pollen profilin was purified by poly (L-proline) affinity chromatography (6).
• the sequences of Hoi 1 5 and Hor v 5 allergens were retrieved from the EMBL database. Multiple sequence alignment was produced with ClustalW (19) and edited by hand. The GDE sequence editor (S. Smith, Harvard University, Cambridge, MA) and COLORMASK (J. Thompson, EMBL, Heidelberg, Germany) were used to colour conserved residues with related properties (19). Protein secondary structure and surface accessibility predictions were done with the PHD program on the EMBL PredictProtein server (20).
• the IgE binding capacity of phage clones expressing Phi p 6 isoforms and fragments was investigated by a plaque lift assay (21).
• the DNA coding for the mature Phi p 6 allergen was PCR-amplified from the clone 142 DNA, subcloned into the Ndel/Eco R I site of pET-17b.
• Recombinant Phi p 6 was expressed in E. coli BL 21 (DE 3) in liquid culture. Cells were suspended in 25 mM Imidazole, pH 7.4, 0.1% Triton X-100 and lysed by addition of lysozyme (20 ⁇ g/g cells) for 30 minutes at room temperature as well as by freeze-thawing cycles.
• DNA was digested with DNAse I (0.1 mg/g cell pellet) for 20 minutes at room temperature.
• the protein extract was centrifuged for 20 min at lO.OOOxg (Sorvall RC5C; SS34 rotor) to remove insoluble materials.
• rPhl p 6 was enriched in a precipitate obtained by addition of ammonium sulfate (40-60% w/v).
• the precipitate was dissolved in lOmM Tris pH 6, dialyzed against this buffer and after centrifugation (20min, lO.OOOg, Sorvall RC5C; SS34 rotor) was applied to a diethylaminoethyl cellulose- Sepharose column (Pharmacia).
• MALDI-TOF Microx assisted laser desorption and ionisation - time of flight
• CD circular dichroism
• the reversibility of the unfolding process was checked by measuring the restoration of the CD signal upon cooling (50°C/h) to the starting temperature (20°C).
• ⁇ u was assumed to be equal to ⁇ 220 at 85°C and ⁇ N to 0220 at 20°C.
• Granulocytes were isolated from heparinized blood samples of grass pollen allergic individuals containing rPhl p 6-reactive IgE antibodies by dextran sedimentation (22). Cells were incubated with increasing concentrations of purified rPhl p 5, rPhl p 6, and with an anti-human IgE antibody (E124.2.8 D ⁇ 2,
• Phlp 6-r elated allergens Analysis of the presence of Phlp 6-r elated allergens in other grass species and tissue-specific expression of Phlp 6.
• Protein extracts from pollens, leaves and roots were obtained by homogenizing the tissues in SDS-sample buffer (24). Insoluble materials were removed by centrifuging the extracts (lO.OOOxg, 20 min; Sorvall RC5C, SS34 rotor). Protein extracts were separated by 14% SDS-PAGE (25) and blotted onto nitrocellulose (26). Nitrocellulose strips were probed with a rabbit anti-celery profilin antiserum, RP1, (12), the rabbit anti-rPhl p 6 antiserum and the latter rabbits preimmune serum. Bound rabbit antibodies were detected with a 1 :1000 diluted 125 ⁇ _ ⁇ a b e ⁇ ec ⁇ donkey anti-rabbit Ig antiserum (Amersham).
• Timothy grass pollen grains were unhydrously fixed as described (27). Ultrathin sections were incubated with equal concentrations of either rabbit anti-rPhl p 6 Ig (Ig: protein G-purified immunoglobulin fraction) or preimmune Ig. Bound rabbit antibodies were detected with goat anti-rabbit IgG antibodies coupled to 10 nm colloidal gold particles (Piano, Wetzlar, Germany) (27).
• hypoallergemc Phlp 6 (Phleum pratense deletion variants.
• N-terminal and C-terminal Phi p 6 deletion variants were generated to represent aa
• cDNAs coding for Phi p 6 aa 1-57 and Phi p 6 aa 31-110 were obtained by PCR amplification of the Phi p 6 cDNA (clone #142) using the following oligonucleotide primers:
• PCR-products were cut with Nde I/Kpn I (aa 1 -57) or with Nde I/Eco R I (aa 31 - 110), purified by preparative agarose gel electrophoresis, subcloned into plasmid pET-17b
• Phlp 6 deletion variants in E. coli and testing of their IgE-binding capacity Recombinant Phi p 6 aa 1-57 and Phi p 6 aa 31-110 were expressed in E. coli Bl 21 (DE 3) by induction with 0.5 mM isopropyl- ⁇ -thiogalactopyranoside at an OD600 of 0.8 in liquid culture for 5 h at 37°C. Equal amounts of rPhl p 6, rPhl p 6 aa 1-57 and rPhl p 6 aa 31-110 were separated by SDS-PAGE and blotted onto nitrocellulose.
• Nitrocellulose strips were incubated with serum IgE from allergic individuals, nonallergic control persons, with a rabbit anti-Phi p 6 antiserum and a rabbit preimmunserum. Bound
• IgE antibodies were detected with l ⁇ I-labeled anti-human IgE antibodies and bound rabbit antibodies with 125j_ ⁇ a beled donkey anti-rabbit antibodies.
• Phi p 6 (clone 142) protein which starts with a glycine residue and shows a high content of alanine residues (20.9%).
• the computer-aided secondary structure analysis of Phi p 6 indicates a predominant helical content and the calculation of solvent accessibility predicts that many of the N-terminal amino acids are solvent exposed while most of the C-terminal amino acids appeared buried.
• a search for sequence motifs revealed the presence of one potential N- linked glycosylation site (NAS: aa 15-17), one N-terminal myristoylation site (GKAT: aa 1-4), two cAMP-dependent protein kinase phosphorylation sites (KATT: aa 2-5; KYKT: aa 33-36) and two peroxisomal targeting sequences (GKA: aa 1-3; SKA: aa 54-56).
• the deduced Phi p 6 amino acid sequence displayed identity with a recently submitted Phi p 6 sequence (15) and similarities with the N- terminal portions of group 5 grass pollen allergens. However, Phi p 6 specific IgE shows little or no crossreactivity with group 5 allergens.
• Figure 1A shows a multiple sequence alignment, secondary structure and solvent accessibility prediction of Phi p 6 variants and group 5 allergens.
• the Phlp 6 N-terminus is relevant for IgE binding.
• the far-ultraviolet CD spectrum of purified rPhl p 6 indicates that the protein contains a considerable amount of alpha-helical secondary structure.
• the spectrum is characterized by two broad minima at 208nm and 220nm and a maximum at 191nm.
• the secondary structure prediction (37) is in good agreement with the CD measurements as it indicates predominant alpha helical secondary structure content.
• the unfolding transition of rPhl p 6 is monophasic and highly cooperative with a melting point of 61 °C. At 85°C, rPhl p 6 assumes a random coil conformation, with a typical minimum at 200nm. rPhl p 6 shows a high degree of folding reversibility, evident from the cooling curve profile ( Figure 2C) and the far- UV spectrum recorded at 20°C after cooling from 85°C ( Figure 2B).
• Phlp 6 induces dose dependent basophil histamine release and immediate type skin reactions in grass pollen allergic patients.
• rPhl p 6 induced specific and dose-dependent histamine release from basophils of a grass pollen allergic patient ( Figure 3 A).
• rPhl p 5 which represents a highly active grass pollen allergen (14, Valenta and Flicker, unpublished data) induced maximal release already at a lower concentration compared to rPhl p 6.
• rPhl p 6 rPhl p 5 and timothy grass pollen extract induced immediate type skin reactions (Table 1 ; Figure 3B). While no reactions to sodium chloride were observed, histamine induced wheal reactions in all individuals tested (Table 1; Figure 3B).
• Group 6 allergens represent pollen-specific proteins. While major groups of grass pollen allergens occur in pollens of most grass species (4), group 6 allergens were reported to occur exclusively in timothy grass (Phleum pratense) pollen (15). A rabbit anti-rPhl p 6 antiserum cross-reacted with group 5 allergens in nitrocellulose blotted pollen extracts from various monocots (Phleum pratense, Lolium perenne, Secale cereale, Triticum sativum, Avena sativa, Phragmites communis) between 25-28 kDa ( Figure 4A, lanes 2).
• Phi p 6 or Phi p 6- related allergens at 11 kDa were detected exclusively in pollens from Phleum pratense and Poapratensis. Although a putative N-glycosylation site was found in the aminoacid sequence deduced from the Phi p 6 cDNA sequence, comparable molecular weights observed for natural and recombinant Phi p 6 exclude heavy glycosylation of natural Phi p 6 ( Figures 4A, 2A). Rabbit anti-rPhl p 6 antibodies strongly reacted with Phi p 6 at 11 kDa in nitrocellulose-blotted timothy grass pollen but not with leaf or root extracts (Figure 4B, lanes 2). Profilin was detected in all three tissues at approximately 14 kDa ( Figure 4B, lanes 1).
• rabbit anti-rPhl p 6 antibodies bound to the numerous polysaccharide (P-) particles which fill much of the interior of a mature timothy grass pollen grain (Figure 4C).
• P- polysaccharide
• Figure 4C The greatest accumulation of gold particles was observed on sectioned surfaces of the P-particles indicating that Phi p 6 is present on rather than in the P-particles.
• Little (cytosol, exine) or no (mitochondria, intine) anti-rPhl p 6 immunoreactivity was observed in other parts of the pollen grain. Likewise almost no gold particles were detected in the amyloplasts.
• rPhlp 6 derivatives have a greatly reduced capacity to induce histamine release
• Granulocytes from a patient allergic to grass pollen were incubated with various concentrations of purified rPhl p 6, rPhl p 6 aa 1-57, rPhl p 6 aa 31-110, rPhl p 6 aa 1-33 or an anti-IgE mAb (E124.2.8 D ⁇ 2, Immunotech, Marseilles, France). Histamine released into the supernatant was measured by RIA (Immunotech)( Figure 6).
• rPhl p 6 induced a specific and dose-dependent histamine release from basophils of a patient allergic to grass pollen, whereas rPhl p 6-derivatives aa 1-57 and aa 31-110 did not induce any histamine release up to a concentration of 1 ⁇ g/ml.
• Phi p 6 aa 1-33 induced a 50% release of histamine at a concentration of 1 ⁇ g/ml, which represents an approximately 1000 fold reduction of histamine release compared to complete rPhl p 6.
• Phi p 6 represents a 11.8 kDa protein allergen which is recognized by IgE antibodies of 75% of grass pollen allergic patients.
• the prevalence of IgE recognition of rPhl p 6 is thus in accordance with that reported earlier for natural Phi p 6 and indicates that carbohydrate moieties do not play a relevant role in IgE recognition of Phi p 6 (28, 29).
• Phi p 6 In agreement with peptide sequence data obtained for natural Phi p 6 we found that the deduced amino acid sequence of rPhl p 6 shows a high degree of sequence homology with the N-terminal portions of group 5 grass pollen allergens, a family of 25-35 kDa major grass pollen allergens (29, 14). Due to the presence of an N- terminal hydrophobic leader peptide, Phi p 6 represents an independent allergen, rather than a group 5 allergen fragment. In agreement with the proposal of other authors who analyzed a Phi p 6 encoding cDNA clone (15) we suggest that group 5 and group 6 allergens may have evolved from common ancestor genes similar as has been described for group 1 and group 2/3 grass pollen allergens (30).
• Phi p 6 in E. coli yielded large amounts of soluble and folded recombinant protein which contained almost exclusive alpha helical secondary structure.
• the alpha helical fold of Phi p 6 is a further confirmation that there are no common structural features which predispose a certain protein to behave as an allergen. While Phi p 6 is very likely an all alpha helical protein, Bet v 1, the major birch pollen allergen (31) and Bet v 2, birch profilin (32) have a mixed alpha beta fold.
• rPhl p 6 shares with other immunologically unrelated pollen allergens (e.
• Phi p 6 the remarkable intrinsic tendency to refold into a stable conformation after denaturation.
• Another feature that is shared by Phi p 6 and other important plant allergens is its high expression in pollen tissue. The fact that most of the plant allergens characterized so far are predominantly expressed in mature pollen may therefore be interpreted as a footprint of sensitization via the respiratory tract (34). By immunogold electron microscopy, Phi p 6 was primarily localized on the P-particles of mature pollen.
• P-particles are small polysaccharide-containing bodies which represent up to 30%) of the contents of the dormant pollen grain and, during pollen germination transfer material into the growing pollen-tube wall (35, 36).
• the occurence of Phi p 6 on the P-particles may be of clinical relevance as P- particles could act as small-sized ( ⁇ 2.5 micron) and therefore respirable allergen- carriers that bring Phi p 6 in immediate contact with the bronchial mucosa.
• FIGURE 1 IgE reactivity of rPhl p 6 isoforms and fragments.
• Nitrocellulose filters containing proteins from recombinant ⁇ gt 11 phage expressing two Phi p 6 isoforms (cl42, c223), Phi p 6 fragments (cl21, cl46, cl71, c233) and for control purposes, ⁇ gtl 1 wild type phage (0) were probed with serum IgE from 9 grass pollen-allergic patients (1-9) and from one non-allergic individual (10).
• FIGURE 2 Purity of recombinant timothy grass pollen allergens. Coomassie brilliant blue-stained SDS-PAGE containing purified, recombinant timothy grass pollen allergens (Phi p 1, Phi p 2, Phi p 5, Phi p 6, timothy grass pollen profilin) and natural timothy grass pollen extract (Timothy). (M) Molecular weight marker.
• B C Circular dichroism analysis.
• B Far-UV circular dichroism spectra of rPhl p 6, expressed as mean residue ellipticity ([ ⁇ ]) (y-axis), were recorded in the wavelength range displayed on the x-axis at 20°C (continuous line), 85°C (dotted line) and at 20°C after cooling from 85°C (dashed line).
• C Thermal denaturation and cooling of purified rPhl p 6 monitored at 220 nm (x-axis: temperature in °C; y-axis: apparent fraction of the folded protein).
• FIGURE 3 rPhl p 6 induces basophil histamine release.
• Granulocytes from a grass pollen allergic patient were incubated with various concentrations (x-axis) of purified, recombinant Phi p 6 (triangles), Phi p 5 (points) or a monoclonal anti-IgE antibody (squares). The percentage of histamine released into the supernatant is displayed on the y-axis. Results represent the means (+/- SD) of triplicate determinations.
• B Induction of immediate type skin reactions with rPhl p 6 in sensitized allergic patients.
• FIGURE 4 Tissue-specific expression of Phi p 6.
• A Nitrocellulose-blotted grass pollen extracts from various monocots were probed with rabbit preimmune Ig (lanes 1) or rabbit anti-rPhl p 6 Ig (lanes 2).
• B Comparable amounts of nitrocellulose blotted protein extracts from timothy grass pollen, leaves and roots were incubated with rabbit anti -profilin Ig (lanes 1), rabbit anti-Phl p 6 Ig (lanes 2) or rabbit preimmune Ig (lanes 3).
• FIGURE 5 Reduced IgE binding capacity of Phi p 6 deletion variants
• Bound IgE antibodies were detected with 125 ⁇ _ ⁇ a b e ⁇ e anti -human IgE antibodies, bound rabbit antibodies with 125j_ ⁇ a b e ⁇ e d donkey anti -rabbit antibodies and visualized by autoradiography.
• FIGURE 6 Granulocytes from a patient allergic to grass pollen were incubated with various concentrations (1, 10 ⁇ 1, 10"2, 10 " ⁇ , 10 5, and 10 ⁇ 7 ⁇ g/ml) of purified rPhl p 6 (points), rPhl p 6 aa 1-57 (up triangles), rPhl p 6 aa 31 -110 (down triangles), rPhl p 6 aa 1-33 (rhombus) or an anti-IgE mAb (squares). Histamine released into the supernatant was measured by RIA and is displayed on the y-axis. Results represent the means of triplicate determinations.
• Dimeric immunoglobulin E serves as a unit signal for mast cell degranulation. Proc. Natl. Acad. Sci.
• Valenta and S. Spitzauer. 1998. IgE antibodies to recombinant pollen allergens (Phi p 1, Phi p 2, Phi p 5, and Bet v 2) account for a high percentage of grass pollen-specific IgE. J. Allergy Clin. Immunol. 101:258. 5. Valenta, R., and D. Kraft. 1995. Recombinant allergens for diagnosis and therapy of allergic diseases. Curr. Opin. Immunol. 7:751. 6. Valenta, R., M. Duchene, K. Pettenburger, C. Sillaber, P. Valent, P.
• Phi p 5b in timothy grass is a novel pollen RNase. FEBS Lett. 363:6.
• Interleukin 3 activates human blood basophils via high-affinity 15 binding sites. Proc. Natl. Acad. Sci. USA 86:5542.
• Bip 1 a monoclonal antibody that modulates IgE binding to birch pollen allergen, Bet v 1. J. Immunol. 157:4943.

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