+

WO2008143594A1 - Polypeptides hypoallergéniques - Google Patents

Polypeptides hypoallergéniques Download PDF

Info

Publication number
WO2008143594A1
WO2008143594A1 PCT/SG2008/000189 SG2008000189W WO2008143594A1 WO 2008143594 A1 WO2008143594 A1 WO 2008143594A1 SG 2008000189 W SG2008000189 W SG 2008000189W WO 2008143594 A1 WO2008143594 A1 WO 2008143594A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
bio
group
sequence
variant
Prior art date
Application number
PCT/SG2008/000189
Other languages
English (en)
Inventor
Kaw Yan Chua
Tai-Huang Huang
Mandar T. Naik
Chi-Fon Chang
I-Chun Kou
Original Assignee
National University Of Singapore
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National University Of Singapore filed Critical National University Of Singapore
Publication of WO2008143594A1 publication Critical patent/WO2008143594A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • 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
    • A61K39/0003—Invertebrate antigens
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2

Definitions

  • the present invention relates to the fields of cell biology, molecular biology, microbiology and genetics. It also relates to the field of medicine, especially therapy and diagnosis.
  • Allergens from clinically important mite species have been identified and categorized into groups based on their sequence homology (Chapman et al., 2007; Thomas and Smith, 1999). Groups 1 and 2 are the major allergens of widely distributed Dermatophagoides genus of dust mites (Thomas and Smith, 1999). Recent studies revealed that Blomia tropicalis mite allergens are probably more important clinically than Dermatophagoides pteronyssinus allergens in the densely populated tropical and subtropical regions, where dual sensitization by both mite species is common (Arruda et al., 1997; Chew, 1999; Puccio, 2004).
  • Immunotherapy remains the only truly disease-modifying treatment for asthma and allergic rhinitis (Nelson, 2007).
  • specific allergen immunotherapy is an effective prophylactic treatment for atopic IgE-mediated disease, in particular for severe seasonal allergic rhinitis (Mailing, 1998; Bousquet et al. 1998; Durham et al. 1999; Walker et al. 2001).
  • Specific allergen immunotherapy has been used for more than 90 years for the management of allergic disorders, including seasonal and perennial allergic rhinitis, allergic asthma, and hymenoptera sensitivity.
  • Specific allergen immunotherapy involves the administration of incremental doses of allergen into sensitized subjects in order to achieve a state of clinical tolerance to subsequent exposure (Rolland and O'Hehir, 1998).
  • Specific allergen immunotherapy using subcutaneous injections of an increasing dose of allergen have been shown to be effective in children and adults suffering from allergic rhinitis and asthma (Abramson et al. 2000).
  • allergen immunotherapy One major problem with specific allergen immunotherapy is the concern that the administration of allergenic material may cause severe, life-threatening anaphylactic reactions (Mailing, 1998; Mailing and Weeke, 1993; Bousquet et al. 1998).
  • hypoallergenic polypeptides and methods of generating hypoallergenic allergens.
  • hypoallergenic BIo t 5 variants for safe and effective immunotherapy. It enables the rational design and testing of hypoallergenic variants of BIo t 5 and other Group 5 allergens, for the production of variants with attenuated allergenicity for use as immunotherapies.
  • a variant Group 5 polypeptide comprising a mutation corresponding to a surface residue of BIo t 5 shown in Figure 3 or at an epitope of BIo t 5 shown in Figure 5, with reference to the position numbering of a Blomia tropicalis BIo 1 5 sequence shown as SEQ ID NO: 1, or a fragment, homologue, variant or derivative thereof.
  • the polypeptide may be derivable from a parent Group 5 polypeptide.
  • the polypeptide may be derivable form a wild type Group 5 polypeptide. It may comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identical to that of a parent or wild type Group 5 polypeptide.
  • the polypeptide may comprise a triple-stranded, coiled-coil bundle. It may comprise first and third helices running parallel with each other. It may comprise a second helix antiparallel with the first and third helices.
  • the polypeptide may have reduced allergenicity compared to a parent or wild type Group 5 polypeptide.
  • the reduced allergenicity may be determined by ELISA or histamine release assay.
  • the reduced allergenicity may comprise reduced IgE binding or reduced IgE reactivity. It may comprise reduced skin reactivity. It may comprise increased IFN- ⁇ production. It may comprise increased IL-IO production. It may comprise a substantially identical or similar or enhanced ThI or T-regulatory T-cell antigenicity as compared to a parent or wild type Group 5 polypeptide. It may comprise a substantially identical or similar or reduced Th2 antigenicity as compared to a parent or wild type Group 5 polypeptide
  • the variant Group 5 polypeptide may comprise substantially identical or similar T-cell antigenicity compared to a parent or wild type Group 5 polypeptide. It may comprise both reduced allergenicity and retained T-cell antigenicity.
  • the parent or wild type polypeptide may be from mite.
  • the mite may be a house dust mite or a storage mite.
  • the parent or wild type polypeptide may be from Blomia spp, such as Blomia tropicalis. It may be from Dermatophagoides spp, Euoglyphus spp, Glycyphagus spp, Lepidoglyphus spp, Acarus spp or Tyrophagus spp.
  • the parent or wild type polypeptide may comprise a BIo t 5 sequence shown as SEQ ID NO: 1.
  • It may comprise a Der f 5 sequence (Accession Number: BAE45865), a Der p 5 sequence (Accession Number: P14004), a Lep d 5 sequence (Accession Number: Q9U5P2), a Der p 21 sequence (Accession Number:
  • the polypeptide may comprise a mutation corresponding to E9, E20, Q21, N23, H24, A25, E27, K28, E30, H31, Q32, L34, Y35, Q37, H38, Q39, D41, E42, N44, E45, N46, K47, E53, K54, 155, 156, R57, E58, D60, V61, V62, C63, A64, M65, E67, G68, A69, Q70, G71, A72, E74, R75, E76, L77, K78, R79, E91, Q94, T95, L96, K98, 199, LlOl, K102, D103, K105, E106, E108, Q109, Kl 10, Kl 12, Dl 13, Ql 15, Tl 16 and Ql 17, with reference to the position numbering
  • the polypeptide may comprise a mutation corresponding to a surface charged residue of BIo t 5 polypeptide.
  • the polypeptide may comprise a mutation at a D, E, H, R or K residue.
  • the mutation may be selected from the group consisting of: E20, H24, E27, K28, E30, H31, H38, D41, E42, E45, E53, K54, R57, E58, D60, E67, E74, R75, E76, K78, R79, E91, E92, K98, K102, D103, K105, E106, E108, Kl 10, Kl 12 or Dl 13, with reference to the position numbering of a Blomia tropicalis BIo t 5 sequence shown as SEQ ID NO: 1.
  • the polypeptide may comprise a mutation corresponding to an epitope residue of a BIo 1 5 polypeptide.
  • the mutation may comprise N46, K47, K54 or R57, with reference to the position numbering of a Blomia tropicalis BIo t 5 sequence shown as SEQ ID NO: 1.
  • the polypeptide may comprise a substitution to a residue selected from the group consisting of: F, A, L, M, I, W, P and V, such as V, A, P or I.
  • the polypeptide may comprise more than one mutated residue. It may comprise two, three or more mutated residues.
  • the mutated residues may be at one or more positions selected from E45, N46, V61 , R57 and A69. They may be at E45A, N46A, V61I, R57A or A69P.
  • the polypeptide may comprise a mutation or mutations selected from the group consisting of: E45, N46, V61, R57, A69, E45+N46, E45+V61, E45+R57, E45+A69, N46+V61, N46+R57, N46+A69, V61+R57, V61+A69, R57+A69, E45+N46+R57, E45+N46+V61, E45+N46+A69, E45+R57+A69, E45+R57+V61, E45+V61+A69, N46+A69+V61, N46A+R57A+V61I, N46+R57+A69 and R57+V61+A69.
  • the polypeptide may comprise a mutation or mutations selected from the group consisting of: E45A, N46A, V61I, R57A, A69P, E45V+N46A, E45V+V61I, E45V+R57A, E45V+A69P, N46A+V61I, N46A+R57A, N46A+A69P, V61I+R57A, V61I+A69P, R57A+A69P, E45V+N46A+R57A, E45V+N46A+V611, E45V+N46A+A69P,
  • the polypeptide may comprise a parent BIo 1 5 sequence shown as SEQ ID NO: 1 with a E45V substitution (E45V).
  • the polypeptide may comprise a parent BIo t 5 sequence shown as SEQ ID NO: 1 with a N46A substitution and a V61I substitution (N46+V61I A).
  • the polypeptide may comprise a parent BIo t 5 sequence shown as SEQ ID NO: 1 with a R57A substitution and an A69P substitution (R57A+A69P).
  • nucleic acid comprising a sequence capable of encoding a variant Group 5 polypeptide according to the 1 st aspect of the invention.
  • a nucleic acid sequence comprising a mutation at one or more residues such that the nucleic acid encodes a mutation corresponding to a surface residue of BIo t 5 shown in Figure 3 or at an epitope of BIo t 5 shown in Figure 5, with reference to the position numbering ofa Blomia tropicalis BIo t 5 sequence shown as SEQ ID NO: 1, or a fragment, homologue, variant or derivative thereof.
  • the nucleic acid sequence may be such that the polypeptide encoded by the nucleic acid comprises a feature set out above, or in which the nucleic acid comprises a mutation at one or more residues such that the nucleic acid encodes a mutation set out above.
  • a plasmid or expression vector comprising such a nucleic acid, or capable of expressing such a polypeptide.
  • a cell or host cell comprising such a nucleic acid or such a plasmid or expression vector, or capable of expressing such a polypeptide.
  • the cell or host cell may be transformed with the nucleic acid, plasmid or expression vector.
  • the present invention in a 6 th aspect, provides a method of expressing a polypeptide comprising expressing the polypeptide from a cell or host cell as set out above.
  • the polypeptide may optionally be purified as part of the method.
  • a method of altering the sequence of a polypeptide by introducing an amino acid mutation as set out above into a parent or wild type polypeptide, for example such that the altered polypeptide comprises a feature set out above.
  • an 8 th aspect of the present invention we provide a method of reducing the allergenicity of a Group 5 polypeptide, the method comprising such a method.
  • a polypeptide obtainable by such a method.
  • a polypeptide set out above a nucleic acid set out above, a plasmid or expression vector set out above, a cell or host cell set out above or a polypeptide set out above for use in a method of medical treatment, such as of allergy, for example by specific allergen immunotherapy (SIT), of an individual.
  • SIT specific allergen immunotherapy
  • a polypeptide such as a nucleic acid, such a plasmid or expression vector, such a cell or host cell or such a polypeptide in the preparation of a medicament for medical treatment, such as of allergy, for example Type I allergy, such as by specific allergen immunotherapy (SIT), of an individual.
  • SIT specific allergen immunotherapy
  • a pharmaceutical composition such as a vaccine, comprising such a polypeptide, such a nucleic acid, such a plasmid or expression vector, such a cell or host cell or such a polypeptide together with a pharmaceutically acceptable adjuvant, excipient, diluent or carrier.
  • a method for preparing a pharmaceutical composition as set out above comprising admixing such a polypeptide, such a nucleic acid, such a plasmid or expression vector, such a cell or host cell or such a polypeptide with a pharmaceutically acceptable adjuvant, excipient, diluent or carrier.
  • a pharmaceutically acceptable adjuvant, excipient, diluent or carrier there is provided, according to a 14 th aspect of the present invention, an antibody capable of specifically binding to a polypeptide as set out above.
  • a diagnostic kit comprising a polypeptide as set out above.
  • the kit may further comprise an antibody as set out above.
  • a method of assessing the relevance, safety or outcome of therapy of a subject for example comprising mixing an IgE-containing sample of the subject with a polypeptide or antibody or both, and assessing the level of IgE reactivity.
  • a method of treatment or prevention of an allergy such as Type I allergy in an individual, the method comprising administering to an individual a therapeutically effective dose of a polypeptide set out above, a nucleic acid set out above, a plasmid or expression vector set out above, a cell or host cell set out above or a polypeptide set out above.
  • a model for a Blomia tropicalis BIo 15 polypeptide or part thereof comprising a 3-D NMR structure as set out in any of Figures IB-D, Figure 3, Figure 4 or Table El.
  • FIG. 1 Primary sequences alignment of Group 5 and Group 21 allergens from different mite species, viz. Blomia tropicalis, Lepidoglyphus destructor, Dermatophagoides farinae and Dermatophagoides pteronyssinus. The sequence identities and conservative substitutions are shaded yellow and gray, respectively and the percentage identity with respect to BIo t 5 is shown at the end. The numbers on left and right sides of the sequences are the residue numbering for proteins encoded in the cDNA sequence deposited in the databases. The 17 residue leader peptide of BIo 1 5 is boxed and the residues of mature BIo 1 5 are numbered in blue on the top along with the secondary structure.
  • the critical residues of mAb 4A7 epitope are located on surfaces I and II as indicated.
  • B The NMR solution structure of BIo t 5 shown as an ensemble of 20 conformers overlaid for backbone atoms of helical residues Leu 18 to Aspl 13 on the lowest energy first conformer.
  • C The ribbon representation of the lowest energy conformer of BIo 1 5. The three helices are numbered A, B and C, and are colored red, green and purple, respectively.
  • D The surface charge representation showing the charge distribution on BIo t 5 with blue for positive and red for negative charge. The left figure is the same orientation as figures B and C, the right figure is rotated 180° on y-axis.
  • Figure 3 A helical wheel representation with lines indicating observed long range NOEs.
  • the residues on the wheel are color-coded in blue-green-yellow-red according to Wimley and White hydrophobicity scale.
  • Aromatic residue are shown in blue, hydrophobic aliphatic residues are green and their shade turn more yellowish with decreasing hydrophobic index, similarly polar residues are colored orange and finally charged residues are dark orange to red.
  • Identifiable NOEs between Helix A and Helix B are shown with black lines, between Helix B and Helix C with purple lines, and between Helix C and Helix A with cyan lines.
  • Helix B is drawn in reverse direction to indicate its anti-parallel orientation with respect to Helix A and Helix C.
  • FIG. 4 The backbone dynamics of BIo t 5.
  • A Residues which undergo ms- ⁇ s chemical exchange are shown as histogram against residue number. The relaxation data for these residues were fit with Model 3 or 4.
  • B ns-ps timescale plasticity of BIo t 5 backbone. On the top is a scatter plot of order parameter, S 2 for the BIo t 5, while at the bottom is histogram of the local root-mean-square deviations seen in BIo t 5 NMR ensemble, both are plotted as function of the residue number. A schematic representation of the secondary structure is shown above the panel.
  • C The order parameters shown in panel (A) are mapped on the BIo 1 5 backbone shown as a green sausage.
  • the radius of sausage is directly proportional to (l-S 2 ) value (i.e. wider regions indicate more flexible parts of the protein).
  • Order parameters for residues for which reliable relaxation parameters could not be derived or, which undergo more complicated motions than modeled by the five models used in Modelfree analysis, are interpolated between the neighboring residues and are colored white. Red balls indicate residues, which in addition are identified to undergo ms- ⁇ s chemical exchange and require R ex term for modeling the relaxation data.
  • FIG. 5 A histogram showing chemical shift perturbation in BIo t 5 backbone amide resonances upon Fab' complex formation. The average ( ⁇ ) and standard deviation ( ⁇ ) of this data is 0.1 lppm and 0.13ppm respectively. These are drawn over the figure as purple horizontal lines to judge statistical significance of change seen for individual residue. The major change seen for residues concluded to interact with the CDRs on Fab' fragment is colored blue and red for the epitope surfaces I and II, respectively. The data collected at 37 0 C is shown with filled bars, whereas at lower 22 0 C is shown with open symbols. Residues which could't be assigned for the bound form at both 22 0 C and 37 0 C are shown by asterisk.
  • FIG. 1 Also shown on top is a scatter plot of change in peak intensity against residue number for the cross saturation transfer experiment performed on 2 H 5 15 N-BIo t 5 bound to non-labeled Fab' (green filled circles), and the control experiment performed on free 2 H 5 15 N-BIo 1 5 (open circles).
  • the connecting line and dotted curve are shown to highlight the trend. Shown on top is strictly qualitative assessment of change in water accessibility on Fab' binding at 37 0 C using CLEANEX-PM experiment at fairly long 100ms mixing time. Red vertical bars coded for residues which can be seen in both free and Fab' bound BIo 1 5, whereas residues coded blue are seen only in the free BIo t 5.
  • GIn 117 is coded with both red and blue as only its side-chain amide is visible in the bound form spectrum.
  • Glnl 15 and Glnl 17 are colored blue to indicate water accessibility of their backbone and/or side-chain amides in both free and Fab' bound BIo t 5. Lys49 is colored black, and is water accessible in free form and potentially disappear from bound form spectrum due to rapid H/D exchange.
  • Panel (C) also shows how the side-chains of Asp46, Lys47, Lys54 and Arg57 are defined in different conformers of the NMR ensemble.
  • D An orthogonal view of panel (B).
  • Figure 6. Human IgE reactivity of BIo t 5 and BIo t 5 fragments.
  • A The human IgE epitopes of full length BIo t 5, BIo t 5]_8o and BIo 1 54 6- n 7 were evaluated with 28 BIo t 5 positive sera (•) from asthmatic children by direct ELISA. Four sera from non-atopic subjects (o) were used as control. Each dot on the figure indicates an individual serum.
  • the cutoff value (long dash line) was determined by mean plus three fold of standard deviation of the control sera.
  • the Y-axis represents ELISA titer as determined by OD 4 o 5n m readingxserum dilution factor.
  • FIG. 7 The 15 N Relaxation Data.
  • the data was acquired on 1 mM 15 N-BIo 1 5 buffered to pH 7.5 at 22 0 C.
  • the values o ⁇ R ⁇ , i? 2 , and heteronuclear NOE are plotted as a function of the residue number in the primary sequence.
  • the connecting dotted lines are drawn to guide reader's eyes.
  • the diagram on the top is a schematic representation of BIo t 5 secondary structure.
  • Average NMR longitudinal rate constant (Ri) (A), transverse relaxation rate constant (R 2 ) (B) and heteronuclear ( 1 H)- 15 N NOE ratio (C) for BIo t 5 are 1.03 ⁇ 0.14 s '1 , 18.23 ⁇ 3.19 s "1 and 0.74 ⁇ 0.31 , respectively.
  • FIG. 8 The Strip Plot. A strip plot from 3D- 15 N edited, TROSY-based NOESY spectrum acquired at 100ms mixing time on the purified 2 H, 15 N- BIo t 5 bound to the Fab'. The inter-amide NOE connectivity between the neighboring amides was used to derive the assignments of Fab' bound BIo 1 5. The skewed shapes of some peaks in this simplistic representation are a result of low S/N ratio, as can be judged from the positive (colored red) and negative (colored green) contours plotted at identical base level and slope.
  • Figure 9 The Hydrogen/Deuterium Protection Factors for Free BIo t 5 at 22 0 C.
  • the filled bars show data obtained from H/D exchange rates derived by curve fitting the peak intensities as a function of time after reconstituting lyophilized BIo t 5 in D 2 O.
  • the open bars indicate H/D exchange rates obtained from the CLEANEX-PM experiment.
  • Figure 10. The Spectrum of the Truncated Mutants. 2D-HSQC spectrum of the two overlapping Blot 5-derived peptides, (A) BIo 1 5i_ 8 o and (B) BIo 1 5 46- i ⁇ ⁇ .
  • variant Group 5 polypeptides comprise one or more mutations.
  • the mutation may be relative to a wild type or parent Group 5 polypeptide.
  • the mutation may be of a surface residue of a Group 5 polypeptide.
  • the surface residue may correspond to a surface residue of BIo 1 5, such as shown in Figure 3.
  • the mutation may be of an epitope of the Group 5 polypeptide.
  • the mutation may correspond to an epitope of BIo t 5, such as shown in Figure 5.
  • the position of the mutation may be assigned with reference to the position numbering of a relevant sequence, such as a Blomia tropicalis BIo 1 5 sequence shown as SEQ ID NO: 1.
  • the variant Group 5 polypeptide may comprise a mixture of surface residue and epitope residue mutations.
  • the Group 5 polypeptide variant may comprise any combination of one, two, three, four, five, six, seven, eight, nine, ten or more mutations at surface residues, mutations at epitope residues or both.
  • variant polypeptides with sequence alterations comprising amino acid substitutions in a Group 5 polypeptide sequence.
  • a fragment, homologue, variant or derivative of such a variant Group 5 polypeptide For the purposes of this document, the variant polypeptides are referred to for simplicity as "variant Group 5 polypeptides”.
  • the mutation may be at a relevant residue of a Group 5 polypeptide such as a wild type or parent Group 5 polypeptide.
  • the relevant residue may comprise an allergenic residue.
  • the relevant residue may comprise for example a surface residue or an epitope residue.
  • the relevant residue may be identified through various means, for example by solving the solution structure of the Group 5 polypeptide by NMR, etc and identifying surface or epitope residues.
  • the residue or residues to be mutated may be identified as a corresponding residue by comparison with a known 3D structure of a reference Group 5 polypeptide, for example a Group 5 polypeptide with sequence homology, identity or similarity with the relevant Group 5 polypeptide of interest.
  • the reference Group 5 polypeptide may comprise a Blomia tropicalis BIo t 5 polypeptide, the 3D NMR structure, surface residues and epitope of which is disclosed in this document.
  • the residue to be mutated may be one which corresponds to E9, E20, Q21, N23, H24, A25, E27, K28, E30, H31, Q32, L34, Y35, Q37, H38, Q39, D41, E42, N44, E45, N46, K42, E53, K54, 155, 156, R57, E58, D60, V61, V62, C63, A64, M65, E67, G68, A69, Q70, G71, A72, E74, R75, E76, L77, K78, R79, E91, Q94, T95, L96, K98, 199, LlOl, K102, D103, K105, E106, E108, Q109, Kl 10, Kl 12, Dl 13, Ql 15, Tl 16 or Ql 17, with reference to the position numbering of a Blomia tropicalis BIo t 5 sequence shown as SEQ ID NO: 1.
  • corresponding relevant residues may include for example a corresponding surface residue or a corresponding epitope residue. These may then modified through means known in the art (for example, PCR mutagenesis) to produce variants of the Group 5 polypeptide of interest.
  • the corresponding relevant residue may be identified, alternatively or in addition, by sequence alignments with other Group 5 family polypeptides, including Blomia tropicalis BIo t 5 . Sequence an alignment are discussed elsewhere in this document and an example is shown in Figure 1.
  • the relevant residue may be identified as one which is conserved with more than 30 %, more than 40 %, more 50%, more than 60 %, more than 70 %, more than 80 % or more than 90 % identity in members of Group 5 polypeptides chosen for alignment.
  • the residue may be conserved in all or substantially all known homologous proteins within the species from which said naturally occurring allergen originates. While sequence homology, identity and similarity are important criteria in choosing a corresponding residue, other factors may need to be taken into account.
  • Such other factors may comprise charge density of the polypeptide region at the putative corresponding residue position, neighbouring residues, alignment of neighbouring residues, size and nature of the putative corresponding residue, etc. Where no single corresponding residue may be identified by the methods taught above, it is generally possible to identify two, three, four or five such candidate corresponding residues which most closely match the sequence alignment and other factors identified above. Mutations at these positions may be generated to produce variant Group 5 polypeptides, and the properties of these variant Group 5 polypeptides may be tested to identify polypeptides of interest for future study and as candidates for therapy.
  • the mutation may comprise any change in residue identity. It may comprise a point mutation.
  • the mutation may comprise any mutation type, such as a substitution or deletion, for example, a substitution.
  • the substitution may be with another residue which does not occur in the same position.
  • the position may be the position in the amino acid sequence of a parent or wild type polypeptide.
  • the position may be the position in any homologous protein. For example, the position may be in a homologous protein within the taxonomic species from which a naturally occurring allergen originates.
  • the substitution may be to a non-polar, hydrophobic, polar or uncharged amino acid residue.
  • the non-polar or hydrophobic residue may comprise a F, A, L, M, I, W, P or V residue, such as a V, A, P or I residue.
  • the polar or uncharged residue may comprise a C, G, Q, N, S, Y or T residue.
  • the variant Group 5 polypeptide may comprise an amino acid substitution to an uncharged residue.
  • the variant Group 5 polypeptide may comprise a mutation at a surface residue.
  • a relevant residue for possible modification comprises a surface residue of the Group 5 polypeptide.
  • the surface residue may comprise a water- exposed or solvent-exposed residue.
  • the surface-exposed amino acids to be substituted in the naturally occurring allergen may have a solvent accessibility of above 20 %, such as above 30 %, above 40 % or above 50 %.
  • the relevant residue comprises a surface residue
  • the surface residue may comprise any residue in positions e, b, f, c or g.
  • the residue may comprise a residue in position e, b, f, c or g of Helix A, position b, f, c or g of Helix B, or position e, b, f, c or g of Helix C.
  • the residue may comprise a residue at position b, c or f of any of these helices.
  • the surface residue may comprise any of amino acids E, Q, N, H, A, K, L, Y, D, I, R, V, C, M, G, or T.
  • the residue may comprise a charged residue, e.g., a surface charged residue, such as H, R, K, D or E.
  • the variant Group 5 polypeptide may comprise a mutation at one or more of residues corresponding to: E20, H24, E27, K28, E30, H31, H38, D41, E42, E45, E53, K54, R57, E58, D60, E67, E74, R75, E76, K78, R79, E91, E92, K98, K102, D103, K105, E 106, El 08, Kl 10, Kl 12 or Dl 13, with reference to the position numbering of a Blomia tropicalis BIo 1 5 sequence shown as SEQ ID NO: 1.
  • the Group 5 variant polypeptide may comprise one, two, three or four or more surface residue mutations.
  • the residue may comprise a residue with a large side chain, such as I, L, V, T, M, N, Q, D, E, K, R, H, F, and Y.
  • the residue may comprise a residue with a small side chain, such as G, A, P, S, and C.
  • Such large or small side chain residues may be surface residues.
  • the variant Group 5 polypeptide may comprise a mutation at an epitope residue.
  • a relevant residue for possible modification comprises an epitope residue of the Group 5 polypeptide.
  • the epitope may comprise an antibody epitope, such as a monoclonal antibody epitope. It may comprise an epitope of antibody mAb 4A7. It may comprise an IgE epitope.
  • An epitope of a Blomia tropicalis BIo t 5 polypeptide is shown in Figure 5. The variant
  • Group 5 polypeptide may therefore comprise a mutation at an epitope residue corresponding to any of the residues of the epitope of Blomia tropicalis BIo t 5 shown in Figure 5.
  • the epitope residue may comprise a residue of Surface I. It may comprise a residue of Surface II. It may be selected from the group consisting of: N46, K47, K54 or R57, with reference to the position numbering of a Blomia tropicalis BIo t 5 sequence shown as SEQ ID NO: 1.
  • the Group 5 variant polypeptide may comprise one, two, three or four or more epitope residue mutations.
  • the variant Group 5 polypeptide may share one or more properties with a parent or wild type Group 5 polypeptide, or may have such properties reduced or enhanced as compared to the parent or wild type Group 5 polypeptide.
  • the property may comprise a physical property, such as structure. For example, it may comprise the same or similar secondary structure or tertiary structure or quaternary structure as the wild type or parent polypeptide.
  • the variant Group 5 polypeptide may comprise the same ⁇ -carbon backbone tertiary structure as the parent or wild type Group 5 polypeptide, such as a naturally occurring allergen.
  • the variant Group 5 polypeptide may comprise a triple-stranded, coiled- coil bundle. It may comprise first and third helices running parallel with each other. It may comprise a second helix running antiparallel with the first and third helices.
  • the 3 dimensional structure of the variant Group 5 polypeptide may be substantially as depicted in Figure IB- ID, Figure 3 or Figure 4. It may comprise epitope surfaces as depicted in Figure 3.
  • the variant Group 5 polypeptide may on the other hand comprise a non-helical structure.
  • the predominant secondary structure of the variant Group 5 polypeptide is not alpha-helical or the predominant tertiary structure is not a helical coiled coil, or both.
  • a non-helical polypeptide comprise a linear structure.
  • the circular diachroism trace of a non-helical polypeptide may have minimum ellipticities between the wavelengths of 200 to 240 run. Methods to determine the degree of helicity of a polypeptide, by measuring the circular dichroism molar ellipticities of the polypeptide, are known to the skilled reader.
  • the variant Group 5 polypeptide may comprise a property of a parent or wild type polypeptide which may be a biochemical property.
  • the property may comprise an antigenic property such as a T-cell antigenic property or an allergenic property.
  • the properties of a variant Group 5 polypeptide may be compared with the properties of a reference Group 5 polypeptide, such as a cognate naturally occurring polypeptide, a native polypeptide, a wild type polypeptide, or a parent polypeptide.
  • the variant Group 5 polypeptide may comprise antigenicity such as T-cell antigenicity.
  • the Group 5 polypeptide variant may comprise a T-cell epitope. T- cell epitopes are believed to be involved in initiation and perpetuation of the immune response to a protein allergen which is responsible for the clinical symptoms of allergy.
  • the variant Group 5 polypeptide may substantially be as antigenic as a reference Group 5 polypeptide. There may be some reduction in antigenicity, but this will generally be less than 90%, such as less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%. less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%. On the other hand, there may be an enhancement in antigenicity, such as 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more or 90% or more.
  • Antigenicity may in particular be assayed by measuring T-cell activation using the "Human T-CeIl Proliferation Assay" described below.
  • T-cell antigenicity may be expressed as the ability to activate T-cells, such as T-cells of particular phenotypes.
  • Phenotypes of interest include ThI , T-regulatory and Th2 phenotypes.
  • the variant Group 5 polypeptide retains substantially the T-cell antigenicity of the reference Group 5 polypeptide, e.g., a cognate wild type, native or parent Group 5 polypeptide.
  • the variant Group 5 polypeptide comprises enhanced T-cell antigenicity, or the ability to activate T-cells such as of a particular phenotype, for example within the limits set out above.
  • the T-cell antigenicity may comprise a ThI or T-regulatory T- cell antigenicity, or both.
  • the Group 5 variant polypeptide may be capable of activating T-cells that are of the ThI phenotype, or activating T-cells that are of the T-regulatory phenotype, or both.
  • the ThI T-cells may comprise IL-2 and ⁇ -IFN producing cells.
  • the T-regulatory cells may comprise ⁇ -INF or IL-10 producing cells or both.
  • the variant Group 5 polypeptide may comprise an enhanced ability to activate ThI or T-regulatory T-cells, as compared to a reference Group 5 polypeptide.
  • the variant Group 5 polypeptide may therefore be capable of activating, or have an enhanced ability to activate, a ThI response or a T-regulatory response, or both, compared to a reference Group 5 polypeptide.
  • the increase or enhancement may be within the limits set out above.
  • a ThI response or a T-regulatory response will typically result in a reduced allergic reaction.
  • the ThI or T-regulatory T-cells may be specific to the Group 5 polypeptide.
  • the variant Group 5 polypeptide may be capable of activating BIo t 5 specific T-cells that are of ThI or T- regulatory phenotypes, or both.
  • the variant Group 5 polypeptide has reduced T-cell antigenicity, or the ability to activate T-cells such as of a particular phenotype, for example within the limits set out above.
  • the T-cell antigenicity may comprise a Th2 T-cell antigenicity.
  • the Group 5 polypeptide variant may comprise substantially the same ability to activate T-cells that are of the Th2 phenotype as compared to a reference Group 5 polypeptide.
  • the Th2 T-cells may comprise IL-4, IL-5 and IL- 13 producing cells.
  • the variant Group 5 polypeptide may comprise an reduced ability or inability to activate T-cells that are of the Th2 phenotype compared to a reference Group 5 polypeptide.
  • the variant Group 5 polypeptide may therefore be incapable of or have a reduced ability to activate a Th2 response, compared to a reference Group 5 polypeptide. The reduction may be within the limits set out above.
  • the Th2 T-cells may be specific to the Group 5 polypeptide.
  • the variant Group 5 polypeptide may be substantially incapable of activating BIo t 5 specific T-cells that are of the Th2 phenotype.
  • a Th2 response will typically result in enhanced allergic reaction.
  • T-cell antigenicity may be measured as T-cell stimulating activity, e.g., the capacity of a polypeptide to interact with T cells to elicit a T-cell response.
  • T-cell antigenicity such as a T-cell assay
  • Methods of measuring T-cell antigenicity such as a T-cell assay are well known in the art, and include thymidine uptake assays. An detailed description of an example assay is set out below as the "Human T-CeIl Proliferation Assay".
  • Such assays detect thymidine incorporation as a measure of cell proliferation in the presence of a polypeptide.
  • the methods may further include cytokine profiling using ELISA, to measure the types of cytokines released in the culture supernatant produced by T cells upon activation.
  • PBMC obtained from Group 5 allergic subjects will be used for these assays with non-atopic subjects included as controls. Reference is also made to Kozutsumi et al (2007).
  • PBMC Peripheral blood mononuclear cells
  • Ficoll-Paque Plus GE Healthcare Life Sciences centrifugation at 800 g for 20 min.
  • a total of 4x10 5 PBMC are cultured in 200 ⁇ l of AIM-V medium in 96-well U bottom plates for 6 days at 37 °C in 5% CO2 incubator.
  • Group 5 variant polypeptide e.g., BIo 1 5
  • specific T cell proliferation assays e.g., BIo 1 5
  • PBMC culture is set up in triplicate in the absence or presence of 1 ⁇ M of variant Group 5 polypeptide such as BIo t 5 protein.
  • Thymidine uptake is measured by pulsing the cells with 1 ⁇ Ci per well of 3 H-thymidine (GE Healthcare Life Sciences) for 18 hours. The cultures are harvested on a FilterMat with the Mrcro96 harvester (Skatron Instruments AS, Lier, Norway). 3H-thymidine incorporation is measured using a Beckman LS6500 liquid scintilation counter (Beckman Coulter, Inc. Fullerton, CA, USA).
  • results are expressed as a stimulation index or the difference of counts-per-minute (delta cpm) of the Group 5 variant polypeptide to the non-Group 5 variant polypeptide stimulated controls (e.g., BIo t 5 to the non-Bio 1 5 stimulated controls).
  • PBMCs are cultured as the T cell proliferation assays. Cytokines in the culture supernatants are collected on day 6. The cytokine profile is detected by the Thl/Th2 cytokine kit of BDTM Cytometric Bead Array (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. The protein levels in the culture supernatants are measured for interleukin (IL)-2, IL-4, IL-5, IL-IO, tumor necrosis factor ⁇ (TNF- ⁇ ) and interferon- ⁇ (IFN-
  • the variant Group 5 polypeptide may have reduced allergenicity, i.e., is hypoallergenic, compared to a reference Group 5 polypeptide.
  • the allergenicity may be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more, as assayed by any one or more of the methods set out below, in comparison with a reference Group 5 polypeptide.
  • the reduced allergenicity may comprise reduced IgE binding or reduced IgE reactivity.
  • IgE reactivity should be taken to refer to the degree or extent of interaction between IgE and a relevant polypeptide, for example as measured in vitro. The measurement may be compared to the degree or extent of interaction or both between a reference polypeptide and IgE.
  • the method of measurement of IgE reactivity may comprise an ELISA IgE immunoassay or any other appropriate quantitative methods known in the art.
  • Reduced IgE reactivity of a subject polypeptide may comprise a reduction in IgE reactivity as measurable in a statistically significant manner (p ⁇ 0.05) in at least one immunoassay using serum from a subject allergic to a reference allergen.
  • the specific IgE binding to the mutated allergen may be reduced in comparison to naturally-occurring isoallergens or similar recombinant proteins.
  • the reduced IgE binding or reactivity may be assayed by any means known in the art, such as in an immunoassay with sera from source- specific IgE reactive allergic patients or pools thereof.
  • IgE binding and reactivity may for example be assayed by the use of a "Human Allergen specific IgE ELISA” or a “Human IgE Inhibition ELISA", as set out below.
  • Histamine or 2-(4-imidazolyl)ethylamine is a biogenic amine chemical well known to the skilled reader.
  • Reduced histamine release may comprise the reduced release of histamine from basophilic granulocytes upon stimulation by the a Group 5 polypeptide variant compared to stimulation by a reference Group 5 polypeptide.
  • Basophilic granulocytes may comprise human basophilic granulocytes.
  • the quantitative in vitro detection of the decrease in total percentage of histamine may be measured using any appropriate immunoassay, a method well known to a person skilled in the art. An example assay is set out below under "Histamine Release Assay".
  • the reduced histamine release of the mutants may arise from reduced affinity toward the specific IgE bound to the cell surface or their reduced ability to facilitate cross-linking. Histaimine release may be reduced by the percentages set out above.
  • 96-well ELISA plates are coated with 50 ⁇ l of purified allergen in a suitable buffer, such as rBlo t 5 (5 m ⁇ g/ml) in 0.1 M NaHCO 3 solution (pH 8.2) at 4°C, overnight. After washing, plates are blocked with 100 ⁇ l of 1 % BSA in PBS containing 0.05%
  • Tween-20 PBS-Tween
  • the sera of allergen sensitised subjects are diluted from 1:5 to 1:30 with blocking buffer and applied (50 m ⁇ l each with duplicates) to the treated plates. After overnight incubation at 4°C, the plates are washed and incubated with blocking buffer diluted (2000:1) biotin-conjugated monoclonal mouse anti-human IgE (clone B3102E8) (Southern
  • ELISA is performed by coating each well of the ELISA plate with 250 ng of wildtype BIo t 5 protein in 50 ⁇ l of 0.1 M sodium bicarbonate pH 8.3, and left at 4°C for overnight.
  • 5-fold serial diluted concentrations (from 100 nM to 0.00128 nM, 8 dosage points) of wildtype or mutant BIo t 5 proteins are used to absorb the diluted sera from 12 Der p 1 IgE positive subjects.
  • the dilution factor for each serum is pre-determined by direct human IgE ELISA.
  • 25 ⁇ l of the diluted serum is mixed with 25 ⁇ l of 8 different dilutions of the wildtype and mutant BIo t 5 proteins.
  • the absorbed sera are then reacted to the coated wildtype BIo t 5 protein on wells at 4
  • the bound IgE is detected by biotinylated mouse anti-human IgE (50 ⁇ l of 250 ng/ml, Southern Biotechnology, Birmingham, CA, USA) followed by ExtrAvidin-alkaline phosphatase (50 ⁇ l of 1 :2000 dilution, Sigma, St Louis, MO, USA) each for 1 hour.
  • pNPP p-nitrophenylphosphate
  • Wildtype and mutant BIo 1 5 proteins are 5-fold serial diluted in the histamine release buffer to concentrations ranging from 1000 nM to 2.048 x 10 "5 nM (12 dosage points).
  • Histamine release is performed with heparinized whole blood using histamine release kit according to the manufacturer's instruction. Briefly, 150 ⁇ l of heparinized whole-blood samples is incubated with equal volume of the various concentrations of the diluted BIo 1 5 proteins at 37°C for 1 hour. Histamine release occurs upon allergen stimulation of basophilic granulocytes depending on their sensitivity to the allergen.
  • the resultant mixture is centrifuged at 700 g for 10 minutes. After centrifugation, 100 ⁇ l of the supernatant is mixed with 100 ⁇ l of indicator buffer (provided by the kit) and 20 ⁇ l of acylation reagent and incubated at room temperature (18 to 25 °C).for 30 minutes. Then, 750 ⁇ l of assay buffer is added and vortexed on a mixer. The resultant mixtures are introduced into the respective wells (50 ⁇ l per well) of the microtiter plate. Another 50 ⁇ l of freshly prepared enzyme conjugate is added into each well, followed by 50 ⁇ l of histamine antiserum. The plate is covered with adhesive foil and is incubated for 3 hours at room temperature on a reciprocal shaker at 80 rpm.
  • the plate is washed 4 times with 250 ⁇ l of wash buffer, and the excess water is removed by tapping the inverted plate on a paper towel. After that, 100 ⁇ l of TMB substrate solution is added into each well and incubated further for 40 minutes at room temperature on an orbital shaker orbiting at 80 rpm. The reaction is stopped by adding 100 ⁇ l of TMB stop solution into each well, and the contents in the well are mixed gently by shaking the plate.
  • the optical density of each well is measured at 405 nm wavelength 15 minutes after the addition of the TMB stop solution.
  • the histamine concentrations is converted by the standard curve in the same plate.
  • Skin reactivity is the in vivo appearance of wheals on the surface of animal skin, such as in humans, exposed to an allergen.
  • Reduced skin reactivity may be defined as a reduction in diameter and/or area of visible wheals on the skin as observed when the in vivo skin prick test (SPT) is used, a method well known to a skilled person in the art.
  • SPT skin prick test
  • IFN- ⁇ or 'interferon- ⁇ ' is a cytokine well known to a skilled person and may comprise human IFN- ⁇ .
  • the increased production of IFN- ⁇ may comprise increased production of IFN- ⁇ by T-cells stimulated by a relevant polypeptide as compared to a reference polypeptide (for example a variant Group 5 polypeptide compared to a reference Group 5 polypeptide).
  • the increase may be measured with a T-cell cytokine assay, the method being well known to a person skilled in the art.
  • IL-IO or 'interleukin-10' is a cytokine well known to a skilled person in the art.
  • Increased production of IL-IO may be measured with a T-cell cytokine assay, the method being well known to a skilled person.
  • the Group 5 variant polypeptides described here are particularly suitable for the treatment or prevention of allergy, such as atopic allergy.
  • the Group 5 polypeptide variants may be used as immunotherapies, such as specific immunotherapy (SIT).
  • Group 5 variant polypeptide, nucleic acid, or a fragment, homologue, variant or derivative thereof may be used to alleviate the symptoms of allergy, or to treat allergy.
  • allergy refers to any allergic reactions such as allergic contact hypersensitivity.
  • the allergy may be to an allergen from any source, for example, a source known to induce allergenic responses in humans.
  • the allergy may be to a tree pollen allergen, a grass pollen allergen, a weed pollen allergen, a feline antigen, or a fungal allergen.
  • the allergy may be to a tree pollen allergen, for example Bet v 1 and Bet v 2 from birch tree.
  • the allergy may be to a grass pollen allergen, for example, PhI p 1 and PhI p 2 from timothy grass. It may be to a weed pollen allergen, for example, antigen E from ragweed. It may be to an animal allergen, for example, a canine or feline antigen.
  • the allergy may be to a major feline antigen, for example, FeI d 1.
  • the allergy may be to a fungal allergen, for example a major fungal allergen, for example, Asp fl, Asp f2, and Asp ⁇ from Aspergillus fumigatus.
  • the allergy is to a dust mite allergen, such as a house dust mite allergen.
  • the allergen may be derived from a mite from Family Glycyphagidae or Family Pyroglyphidae. Dust mites of Family Glycyphagidae include those in the genera Aeroglyphus, Austroglycyphagus, Blomia, Ctenoglyphus, Glycyphagus, Gohieria, Lepidoglyphus. Dust mites of Family Pyroglyphidae include those in the genera Dermatophagoides, Euroglyphus, Pyroglyphus.
  • the allergy may be to an allergen from a species in any of these genera.
  • the allergy may be to an allergen which is a group 1 allergen (Der p 1, Der f 1, BIo t
  • Allergies suitable for treatment with Group 5 variant polypeptide, nucleic acid, or a fragment, homologue, variant or derivative thereof may therefore include a seasonal respiratory allergy, allergic rhinitis, hayfever, nonallergic rhinitis, vasomotor rhinitis, irritant rhinitis, an allergy against grass pollens, tree pollens or animal danders, an allergy associated with allergic asthma, and food allergies.
  • Group 5 variant polypeptide, nucleic acid, or a fragment, homologue, variant or derivative thereof may be used to treat allergies to house dust mite ⁇ Dermatophagoides spp), such as
  • the allergens may be comprised in faeces of Dermatophagoides spp.
  • the mutation includes a number and a letter, e.g., E45, then this refers to [amino acid residue/position according to the numbering system]. Accordingly, for example, the mutation of a glutamic acid amino acid residue in position 45 is designated as E45;
  • substitution includes a letter, a number and a letter, e.g., E45V, then this refers to [original amino acid/position according to the numbering system/substituted amino acid]. Accordingly, for example, the substitution of glutamic acid with valine in position 45 is designated as E45V.
  • Multiple mutations may be designated by being separated by addition marks "+”, e.g. N46A+V61I representing mutations in position 46 and 61 substituting asparagine with alanine and valine with isoleucine respectively.
  • the reference polypeptide sequence is derived from the Blomia tropicalis sequence having UniProt or SWISS-PROT accession number 096870, but without the signal sequence MKFAIVLIAC FAASVLA (SEQ ID NO: 2)
  • a reference nucleic acid sequence of Blomia tropicalis BIo t 5 is set out below (SEQ ID NO: 3): 1 aaaacactca caatccacaa actcaaacaa caatgaagtt cgccatcgtt
  • the reference nucleic acid sequence is derived from the Blomia tropicalis sequence having UniProt or EMBL Accession Number U59102. It comprises the signal sequence.
  • Programs such as FASTA, CLUSTAL-V, CLUSTAL-W, T-Coffee, DALI (distance matrix alignment) and SSAP (sequential structure alignment program) are well known in the art, and may be employed for producing sequence alignments.
  • Figure 1 An example of such an alignment is shown as Figure 1 , and this figure may be referred to for allocation of residue numbers or corresponding amino acids in any Group 5 polypeptide. Using this numbering system originating from for example the amino acid sequence of
  • BIo 1 5 obtained from Blomia tropicalis, aligned with amino acid sequences of a number of other known Group 5 polypeptide, it is possible to indicate the position of an amino acid residue in a Group 5 polypeptide unambiguously.
  • the numbering system is also applicable to all relevant homologous sequences.
  • the position numbering may be applied to homologous sequences from other Blomia species, or homologous sequences from other organisms.
  • Blomia spp such as Blomia tropicalis, Dermatophagoides spp, Euoglyphus spp, Glycyphagus spp, Lepidoglyphus spp, Acarus spp or Tyrophagus spp.
  • homologous sequences examples include a Der f 5 sequence (Accession Number: BAE45865), a Der p 5 sequence (Accession Number: P 14004), a Lep d 5 sequence (Accession Number: Q9U5P2), a Der p 21 sequence (Accession Number: ABC73706) and a BIo 1 21 sequence (Accession Number: AY800348).
  • Such homologues have 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater homology, for example 70% or more, 80% or more, 90% or more or 95% or more homology, with the reference sequence SEQ ID NO: 1 above, or the sequences having UnitProt or SWISS-PROT accession number 096870. Sequence homology between proteins may be ascertained using well known alignment programs and hybridisation techniques described herein. Such homologous sequences, as well as the functional equivalents described below, will be referred to in this document as the "Group 5 polypeptides".
  • the numbering system used in this document makes reference to a reference sequence SEQ ID NO: 1, which is derived from the Blomia tropicalis sequence having UniProt or SWISS-PROT accession number 096870, but without the signal sequence MKF AIVLIAC FAASVLA.
  • This signal sequence is located N terminal of the reference sequence and consists of 17 amino acid residues. Accordingly, it will be trivial to identify the particular residues to be mutated or substituted in corresponding sequences comprising the signal sequence, or indeed, corresponding sequences comprising any other N- or C- terminal extensions or deletions.
  • the Group 5 polypeptide variants may be derived from a Group 5 polypeptide sequence.
  • the Group 5 polypeptide may comprise an allergen of group 5.
  • an allergen is a polypeptide that is capable of inducing IgE-mediated (atopic) allergy, i.e, it is a protein that stimulates the production of, and reacts with, antibodies (IgE) thus creating an allergic reaction (immediate-type hypersensitivity).
  • IgE antibodies
  • pollen allergens from plants, venom allergens from insects, dust-mite allergens, and animal hair allergens are examples of pollen allergens from plants, venom allergens from insects, dust-mite allergens, and animal hair allergens.
  • allergens are designated "according to the accepted taxonomic name of their source as follows: the first three letters of the genus, space, the first letter of the species, space, and an Arabic number. The numbers are assigned to the allergens in the order of their identification, and the same number is generally used to designate homologous allergens of related species.”
  • a "Group 5 polypeptide” as the term is used in this document, may comprise a polypeptide antigen that belongs to allergen group 5, under the WHO/IUIS Allergen Nomenclature.
  • a Group 5 polypeptide may encompass any Group 5 polypeptides already discovered as well as any further additions to the 'Group 5' class of polypeptides. It further comprises all naturally occurring and recombinant derivatives, isoforms and fragments of any Group 5 polypeptide.
  • the Group 5 may comprise other allergens, which do not belong in allergen group 5, but which comprise sequence identity, homology or similarity to allergen group 5 polypeptides.
  • a Group 5 polypeptide may comprise a polypeptide having at least 30%, 40%, 50%, 60%, 70% or more sequence identity to a reference Group 5 polypeptide, such as Blomia tropicalis BIo t 5.
  • the sequence identity may be 67%, as used in the WHO/IUIS Allergen Nomenclature criteria.
  • Group 5 polypeptides may be identified by database searching, for example, by running a search on Allermatch (www.allermatch.org) with a query sequence comprising a known Group 5 polypeptide such as Blomia tropicalis BIo 1 5.
  • the Group 5 polypeptide may further include any of the protein isoforms of the allergen, and may be further further include naturally-occurring and recombinant allergens.
  • Examples of Group 5 polypeptides include the following: Act d 5 (Kiwi fruit), Alt a 5 (Alternaria rot fungus), Amb a 5 (Short ragweed), Amb p 5 (Western ragweed), Amb 1 5
  • the mite may comprise Blomia spp, such as Blomia tropicalis, Blomia kulagini, Blomia tjibodas, or Blomia thori.
  • the Group 5 polypeptide may comprise a BIo 1 5 sequence such as a sequence shown as SEQ ID NO: 1.
  • the Group 5 polypeptide may comprise a a BIo t 21 sequence (such as shown in Accession Number: AY800348)
  • the mite may comprise Dermatophagoides spp, such as Dermatophagoides farinae, Dermatophagoides microceras and Dermatophagoides pteronyssinus,
  • the Group 5 polypeptide may comprise a Der f 5 sequence (such as shown in Accession Number:
  • the mite may comprise Lepidoglyphus spp, such as Lepidoglyphus destructor.
  • the Group 5 polypeptide may comprise a Lep d 5 sequence (such as shown in Accession Number: Q9U5P2).
  • the mite may comprise Acarus spp, such as Acarus siro or Acarusfarris.
  • the mite may comprise Euoglyphus spp, such as Euroglyphus maynei.
  • the mite may comprise Glycyphagus spp, such as Glycyphagus domesticus.
  • the mite may comprise a Tyrophagus spp, such as Tyrophagus putrescentiae or Tyrophagus longior.
  • the Group 5 polypeptide may comprise Blomia tropicalis BIo t 5
  • the Group 5 polypeptide variants may comprise BIo t 5 variants.
  • BIo t 5 exhibits no known enzymatic activity and its biological role in mites is unknown.
  • a recent study on BIo 1 5 paralogue protein, BIo 121 localizes both proteins to the midgut and hindgut contents of B. tropicalis and in the mite fecal particles (Gao et al., 2007).
  • BIo t 5 The full length cDNA of BIo t 5 contains a 432 base-pair open reading frame corresponding to 17 residue signal peptide followed by 117 residue mature BIo 1 5 protein (Arruda et al., 1997). Sequence homologues of BIo t 5 include other mite group 5 allergen proteins which share about 40% identity ( Figure 1). EXAMPLE VARIANT GROUP 5 POLYPEPTIDES
  • variant Group 5 polypeptides comprising one mutated residue, or more than one mutated residue, such as two, three or more mutated residues.
  • the residues may be at one or more positions selected from E45, N46, V61, R57 and A69.
  • the mutations may comprise substitutions E45A, N46A, V61I, R57A or A69P.
  • Single residue mutant Group 5 polypeptides include E45, N46, V61, R57 and A69.
  • the Blomia tropicalis BIo 15 variant may comprise a substitution E45A, N46A, V61I, R57A or A69P.
  • LQEKIIRELD VVCAMIEGAQ GALERELKRT DLNILERFNY EEAQTLSKIL LKDLKETEQK VKDIQTQ Double mutants of Group 5 polypeptides include Blomia tropicalis BIo t 5 polypeptides with two mutations at any of the following pairs of residues: E45+N46, E45+V61, E45+R57, E45+A69, N46+V61, N46+R57, N46+A69, V61+R57, V61+A69 and R57+A69.
  • the double mutants may comprise any of the following pairs of substitutions: E45V+N46A, E45V+V61I, E45V+R57A, E45V+A69P, N46A+V61I, N46A+R57A, N46A+A69P, V61I+R57A, V61I+A69P, R57A+A69P.
  • the triple mutants may comprise any of the following triplets of substitutions: E45V+N46A+R57A, E45V+N46A+V61I, E45V+N46A+A69P, E45V+R57A+A69P, E45V+R57A+V61I, E45V+V61I+A69P, N46A+A69P+V61I, N46A+R57A+V61I, N46A+R57A+V61I, N46A+R57A+A69P or R57A+V61I+A69P.
  • variant polypeptides disclosed in this document are referred to as “Group 5 variant polypeptides”.
  • Nucleic acids encoding such variant polypeptides are also disclosed and will be referred to for convenience as “Group 5 variant nucleic acids”.
  • Group 5 variant polypeptides and nucleic acids will be described in further detail below.
  • the sequences on which the Group 5 variant polypeptides and nucleic acids are basedm ay comprise polypeptides having Group 5 polypeptide activity.
  • parent polypeptides i.e. The term “parent polypeptides” should be interpreted accordingly, and taken to mean the polypeptides on which the Group 5 variant polypeptides are based. They are described in further detail below.
  • the parent polypeptide may comprise a Group 5 polypeptide sequence, as described in further detail below.
  • the parent polypeptide may be a variant of any of the wild type sequences, that is to say, the parent polypeptide may itself be engineered, or comprise a Group 5 polypeptide.
  • the variant Group 5 polypeptides described in this document may retain the features of the parent polypeptides, and additionally may have additional beneficial properties, for example, reduced allergenicity.
  • the properties of the variant Group 5 polypeptides are described in further detail below.
  • the Group 5 polypeptide mutants described here may be used for any suitable purpose. They may be used for purposes for which the parent polypeptide is suitable, or for which the parent polypeptide is unsuitable because of an undesirable property (which may be reduced or amerliorated in a the variant Group 5 polypeptide). In particular, they may be used for therapy such as immunotherapy. Uses of such variant Group 5 polypeptides are described in further detail below.
  • the Group 5 variant polypeptides may comprise one or more further mutations in addition to those positions set in this document. There may be one, two, three, four, five, six, seven or more mutations such as substitutions in addition to those already set out. Other mutations, such as deletions, insertions, substitutions, transversions, transitions and inversions, at one or more other locations, may also be included. In addition, the Group 5 variants need not have all the substitutions at the positions listed. Indeed, they may have one, two, three, four, or five substitutions missing, i.e., the wild type amino acid residue is present at such positions. FRAGMENTS, HOMOLOGUES, VARIANTS AND DERIVATIVES
  • Fragments, homologues, variants and derivatives of Group 5 variant polypeptides are also included.
  • the Group 5 variant polypeptides may be made by biochemical methods, for example, by recombinant DNA methods as known in the art. Accordingly, it will be understood that Group 5 variant polypeptides specifically include recombinant Group 5 variant polypeptides.
  • the Group 5 variant polypeptides disclosed also include homologous sequences obtained from any source, for example related viral/bacterial proteins, cellular homologues and synthetic peptides, as well as variants or derivatives thereof.
  • polypeptides also include those encoding homologues of Group 5 variant from other species including other microorganisms.
  • homologues from higher animals such as mammals (e.g. mice, rats or rabbits), especially primates, more especially humans are also included.
  • a "homologous" sequence is taken to include an amino acid sequence which is at least 15, 20, 25, 30, 40, 50, 60, 70, 80 or 90% identical, such as at least 95 or 98% identical at the amino acid level over at least 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 114 amino acids with a relevant sequence, such as a Group 5 polypeptide sequence.
  • the relevant sequence may comprise a sequence of native Group 5 shown as SEQ ID NO: 1.
  • homology should typically be considered with respect to those regions of the sequence known to be essential for protein function rather than non-essential neighbouring sequences. This is especially important when considering homologous sequences from distantly related organisms.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present document homology may be expressed in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate % homology between two or more sequences.
  • % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids). Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.
  • the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties.
  • a suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al, 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al, 1990, J. MoI.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details).
  • the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62 may be used.
  • the BLAST algorithm is employed, with parameters set to default values.
  • the BLAST algorithm is described in detail at http://www.ncbi.nih.gov/BLAST/blast help.html, which is incorporated herein by reference.
  • the search parameters are defined as follows, can be advantageously set to the defined default parameters.
  • "substantial identity" when assessed by BLAST equates to sequences which match with an EXPECT value of at least about 7, such as at least about 9 or 10 or more.
  • the default threshold for EXPECT in BLAST searching is usually 10.
  • BLAST Basic Local Alignment Search Tool
  • blastp, blastn, blastx, tblastn, and tblastx these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul (Karlin and Altschul 1990, Proc. Natl. Acad. ScL USA 87:2264-68; Karlin and Altschul, 1993, Proc. Natl. Acad. ScL USA 90:5873-7; see http://www.ncbi.nih.gov/BLAST/blast help.html) with a few enhancements.
  • the BLAST programs are tailored for sequence similarity searching, for example to identify homologues to a query sequence. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al (1994) Nature Genetics 6:119- 129.
  • blastp compares an amino acid query sequence against a protein sequence database
  • blastn compares a nucleotide query sequence against a nucleotide sequence database
  • blastx compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database
  • tblastn compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands)
  • tblastx compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • BLAST uses the following search parameters:
  • HISTOGRAM Display a histogram of scores for each search; default is yes. (See parameter H in the BLAST Manual). DESCRIPTIONS - Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter V in the manual page).
  • EXPECT The statistical significance threshold for reporting matches against database sequences; the default value is 10, such that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a match is greater than the EXPECT threshold, the match will not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. (See parameter E in the BLAST Manual). CUTOFF - Cutoff score for reporting high-scoring segment pairs. The default value is calculated from the EXPECT value (see above).
  • HSPs are reported for a database sequence only if the statistical significance ascribed to them is at least as high as would be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, leading to fewer chance matches being reported. (See parameter S in the BLAST Manual). Typically, significance thresholds can be more intuitively managed using EXPECT.
  • ALIGNMENTS Restricts database sequences to the number specified for which high-scoring segment pairs (HSPs) are reported; the default limit is 50. If more database sequences than this happen to satisfy the statistical significance threshold for reporting (see EXPECT and CUTOFF below), only the matches ascribed the greatest statistical significance are reported. (See parameter B in the BLAST Manual).
  • MATRIX - Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX.
  • the default matrix is BLOSUM62 (Henikoff & Henikoff, 1992).
  • the valid alternative choices include: PAM40, PAMl 20, PAM250 and IDENTITY.
  • No alternate scoring matrices are available for BLASTN; specifying the MATRIX directive in BLASTN requests returns an error response.
  • FILTER - Mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (1993) Computers and Chemistry 17:149-163, or segments consisting of short-periodicity internal repeats, as determined by the XNU program of Claverie & States (1993) Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman (see http://www.ncbi.nlm.nih.gov).
  • Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.
  • Low complexity sequence found by a filter program is substituted using the letter "N” in nucleotide sequence (e.g., "NNNNNNNNNNNNN”) and the letter "X” in protein sequences (e.g., "XXXXXXXXX").
  • Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs. It is not unusual for nothing at all to be masked by SEG, XNU, or both, when applied to sequences in SWISS-PROT, so filtering should not be expected to always yield an effect.
  • sequences are masked in their entirety, indicating that the statistical significance of any matches reported against the unfiltered query sequence should be suspect.
  • NCBI-gi causes NCBI gi identifiers to be shown in the output, in addition to the accession and/or locus name.
  • Sequence comparisons may be conducted using the simple BLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST. In some embodiments, no gap penalties are used when determining sequence identity.
  • % homology such as % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
  • variant or derivative in relation to the amino acid sequences disclosed here includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant amino acid sequence retains substantially the same activity as the unmodified sequence.
  • the modified sequence may comprise at least one biological activity as the unmodified sequence, such as all the biological activities of the unmodified sequence.
  • variant or derivative may comprise at least one biological activity of a relevant Group 5 polypeptide, such as a variant Group 5 polypeptide.
  • Group 5 variant polypeptides or fragments or homologues thereof may be further modified for use in the methods and compositions described here. Typically, modifications are made that maintain the biological activity of the sequence. Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided that the modified sequence retains the biological activity of the unmodified sequence. Alternatively, modifications may be made to deliberately inactivate one or more functional domains of the polypeptides described here. Functional domains of Group 5 polypeptides may include the three ⁇ helices, with a second ⁇ helix in antiparallel configuration with first and third ⁇ helices.
  • Amino acid substitutions may include the use of non-naturally occurring analogues, for example to increase blood plasma half-life of a therapeutically administered polypeptide.
  • Polypeptides also include fragments of the full length sequence of a Group 5 polypeptide. Such fragments may comprise at least one epitope. Methods of identifying epitopes are well known in the art and are described in detail in the Examples. Fragments will typically comprise at least 6 amino acids, such as at least 10, 20, 30, 50 or 100 amino acids.
  • Group 5 polypeptides, fragments, homologues, variants and derivatives are typically made by recombinant means. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis.
  • the proteins may also be produced as fusion proteins, for example to aid in extraction and purification.
  • fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences.
  • the fusion protein may be such that it does not hinder the function of the protein of interest sequence. Proteins may also be obtained by purification of cell extracts from animal cells.
  • the Group 5 variant polypeptides, variants, homologues, fragments and derivatives disclosed here may be in a substantially isolated form. It will be understood that such polypeptides may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated.
  • a Group 5 polypeptide variant, homologue, fragment or derivative may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the protein in the preparation is a protein.
  • the Group 5 variant polypeptides, variants, homologues, fragments and derivatives disclosed here may be labelled with a revealing label.
  • the revealing label may be any suitable label which allows the polypeptide, etc to be detected. Suitable labels include radioisotopes, e.g.
  • Labelled polypeptides may be used in diagnostic procedures such as immunoassays to determine the amount of a polypeptide in a sample. Polypeptides or labelled polypeptides may also be used in serological or cell-mediated immune assays for the detection of immune reactivity to said polypeptides in animals and humans using standard protocols.
  • a Group 5 variant polypeptide, variant, homologue, fragment or derivative disclosed here, optionally labelled, my also be fixed to a solid phase, for example the surface of an immunoassay well or dipstick.
  • labelled and/or immobilised polypeptides may be packaged into kits in a suitable container along with suitable reagents, controls, instructions and Ihe like.
  • Such polypeptides and kits may be used in methods of detection of antibodies to the polypeptides or their allelic or species variants by immunoassay.
  • Immunoassay methods are well known in the art and will generally comprise: (a) providing a polypeptide comprising an epitope bindable by an antibody against said protein;
  • the Group 5 variant polypeptides, variants, homologues, fragments and derivatives disclosed here may be used in in vitro or in vivo cell culture systems to study the role of their corresponding genes and homologues thereof in cell function, including their function in disease.
  • truncated or modified polypeptides may be introduced into a cell to disrupt the normal functions which occur in the cell.
  • the polypeptides may be introduced into the cell by in situ expression of the polypeptide from a recombinant expression vector (see below).
  • the expression vector optionally carries an inducible promoter to control the expression of the polypeptide.
  • host cells such as insect cells or mammalian cells
  • post-translational modifications e.g. myristolation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation
  • Such cell culture systems in which the Group 5 variant polypeptides, variants, homologues, fragments and derivatives disclosed here are expressed may be used in assay systems to identify candidate substances which interfere with or enhance the functions of the polypeptides in the cell.
  • nucleic acid encoding a Group 5 variant polypeptide, which we refer to as a "Group 5 variant nucleic acid”.
  • nucleic acids encoding variants, homologues, derivatives and fragments of Group 5 variant polypeptides, as well as fragments, homologues, derivatives and variants of Group 5 variant nucleic acids.
  • nucleic acids having sequences which correspond to or encode the alterations in the Group 5 variant polypeptide sequences.
  • the nucleic acids may be used for producing such polypeptides for the purposes described here.
  • nucleic acids capable of encoding any polypeptide sequence set out in this document The skilled person will be aware of the relationship between nucleic acid sequence and polypeptide sequence, in particular, the genetic code and the degeneracy of this code, and will be able to construct such Group 5 variant nucleic acids without difficulty. For example, he will be aware that for each amino acid substitution in the Group 5 variant polypeptide sequence, there may be one or more codons which encode the substitute amino acid.
  • one or more Group 5 variant nucleic acid sequences may be generated corresponding to that Group 5 variant polypeptide sequence.
  • the Group 5 variant polypeptide comprises more than one substitution, for example R57A+A69P
  • the corresponding Group 5 variant nucleic acids may comprise pairwise combinations of the codons which encode respectively the two amino acid changes.
  • the Group 5 variant nucleic acid sequences may be derivable from parent nucleic acids which encode any of the parent polypeptides described above.
  • parent nucleic acids may comprise wild type sequences, e.g., SEQ ID NO: 3.
  • the Group 5 variant nucleic acid may comprise a recombinant fragment of Group 5 variant nucleic acid, or any fragment, homologue, variant or derivative thereof. Fragments, homologues, variants and derivatives of each of the above sequences are also included.
  • the Group 5 variant nucleic acids may therefore comprise nucleic acids encoding wild type Group 5 polypeptides, but which encode another amino acid at the relevant position instead of the wild type amino acid residue.
  • the Group 5 variant nucleic acid sequences may also comprise wild type sequences with one or more mutations, e.g., which encode parent polypeptides described above.
  • nucleic acid sequences which are not identical to the particular Group 5 variant nucleic acid sequences, but are related to these, will also be useful for the methods and compositions described here, such as a variant, homologue, derivative or fragment of a Group 5 variant nucleic acid sequence, or a complement or a sequence capable of hybridising thereof.
  • Group 5 variant nucleic acid should be taken to include each of these entities listed above. Mutations in amino acid sequence and nucleic acid sequence may be made by any of a number of techniques, as known in the art.
  • Variant sequences may easily be made using any of the known mutagenesis techniques, for example, site directed mutagenesis using PCR with appropriate oligonucleotide primers, 5' add-on mutagenesis, mismatched primer mutagenesis, etc.
  • the Group 5 variant nucleic acid sequences may be made de novo.
  • the mutations may be introduced into parent sequences by means of PCR (polymerase chain reaction) using appropriate primers. It is therefore possible to alter the sequence of a polypeptide by introducing any desired amino acid substitutions into a parent polypeptide, such as having Group 5 polypeptide activity, such as into a Blomia tropicalis BIo t 5 sequence at amino acid or nucleic acid level, as described.
  • PCR polymerase chain reaction
  • the Group 5 variant polypeptide does not need in fact to be actually derived from a wild type polypeptide or nucleic acid sequence by, for example, step by step mutation. Rather, once the sequence of the Group 5 variant polypeptide is established, the skilled person can easily make that sequence from the wild type with all the mutations, via means known in the art, for example, using appropriate oligonucleotide primers and PCR. In fact, the Group 5 variant polypeptide can be made de novo with all its mutations, through, for example, peptide synthesis methodology.
  • the Group 5 variant polypeptides and/or nucleic acids are derived or derivable from a "precursor" sequence.
  • the term "precursor” as used herein means an polypeptide or nucleic acid that precedes the polypeptide or nucleic acid which is modified according to the methods and compositions described here.
  • the precursor may be an polypeptide or nucleic acid that is modified by mutagenesis as described elsewhere in this document.
  • the precursor may be a wild type polypeptide or nucleic acid, a variant wild type polypeptide or nucleic acid or an already mutated polypeptide or nucleic acid.
  • Polynucleotide As used here in this document, the terms “polynucleotide”, “nucleotide”, and nucleic acid are intended to be synonymous with each other. “Polynucleotide” generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
  • Group 5 variant nucleic acids, variants, fragments, derivatives and homologues may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides.
  • oligonucleotides A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.
  • variants in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.
  • variants, homologues or derivatives may code for a polypeptide having biological activity.
  • sequence homology there may be at least 50 or 75%, such as at least 85% or at least 90% homology to the sequences shown in the sequence listing herein. There may be at least 95%, such as at least 98%, homology. Nucleotide homology comparisons may be conducted as described above.
  • a sequence comparison program which may be used may comprise the GCG Wisconsin Bestfit program described above.
  • the default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch.
  • the default gap creation penalty is -50 and the default gap extension penalty is -3 for each nucleotide.
  • Nucleotide sequences that are capable of hybridising selectively to the sequences presented herein, or any variant, fragment or derivative thereof, or to the complement of any of the above.
  • Nucleotide sequences may be at least 15 nucleotides in length, such as at least 20, 30, 40 or 50 nucleotides in length.
  • hybridization shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction technologies.
  • Polynucleotides capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement will be generally at least 70%, such as at least 80 or 90% for example at least 95% or 98% homologous to the corresponding nucleotide sequences presented herein over a region of at least 20, such as at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
  • selectively hybridizable means that the polynucleotide used as a probe is used under conditions where a target polynucleotide is found to hybridize to the probe at a level significantly above background.
  • the background hybridization may occur because of other polynucleotides present, for example, in the cDNA or genomic DNA library being screening.
  • background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, such as less than 100 fold as intense as the specific interaction observed with the target DNA.
  • the intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32 P.
  • Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, VoI 152, Academic Press, San Diego CA), and confer a defined "stringency” as explained below.
  • Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5 0 C to 10 0 C below Tm; intermediate stringency at about 10 0 C to 20 0 C below Tm; and low stringency at about 20 0 C to 25 °C below Tm.
  • a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.
  • both strands of the duplex are encompassed by the methods and compositions described here.
  • the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included.
  • Polynucleotides which are not 100% homologous to the Group 5 variant sequences disclosed here but which are also included can be obtained in a number of ways. Other variants of the sequences may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. For example, Group 5 variant homologues may be identified from other individuals, or other species. Further recombinant Group 5 variant nucleic acids and polypeptides may be produced by identifying corresponding positions in the homologues, and synthesising or producing the molecule as described elsewhere in this document.
  • the collagen region, neck region and carbohydrate binding domain in such homologues may be identified, for example, by sequence gazing or computer assisted comparisons, and selected for combination into or production of a recombinant Group 5 variant.
  • other viral/bacterial, or cellular homologues of Group 5 polypeptides particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to a Group 5 nucleic acid.
  • Such homologues may be used to design non-human Group 5 nucleic acids, fragments, variants and homologues. Mutagenesis may be carried out by means known in the art to produce further variety.
  • Sequences of Group 5 homologues may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal or non-animal species, particularly microbial or fungal species, and probing such libraries with probes comprising all or part of any of the Group 5 nucleic acids, fragments, variants and homologues, or other fragments of Group 5 nucleic acids under conditions of medium to high stringency.
  • Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the Group 5 nucleic acids.
  • conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PiIeUp program is widely used.
  • the primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences. It will be appreciated by the skilled person that overall nucleotide homology between sequences from distantly related organisms is likely to be very low and thus in these situations degenerate PCR may be the method of choice rather than screening libraries with labelled fragments the Group 5 sequences. In addition, homologous sequences may be identified by searching nucleotide and/or protein databases using search algorithms such as the BLAST suite of programs.
  • polynucleotides may be obtained by site directed mutagenesis of characterised sequences, for example, Group 5 nucleic acids, or variants, homologues, derivatives or fragments thereof. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
  • the polynucleotides described here may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
  • a primer e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
  • Such primers, probes and other fragments will be at least 8, 9, 10, or 15, such as at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term "polynucleotides" as used herein.
  • Polynucleotides such as a DNA polynucleotides and probes may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the lipid targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector
  • Polynucleotides or primers may carry a revealing label. Suitable labels include radioisotopes such as 32 P or 35 S, enzyme labels, or other protein labels such as biotin. Such labels may be added to polynucleotides or primers and may be detected using by techniques known per se. Polynucleotides or primers or fragments thereof labelled or unlabeled may be used by a person skilled in the art in nucleic acid-based tests for detecting or sequencing polynucleotides in the human or animal body.
  • Such tests for detecting generally comprise bringing a biological sample containing DNA or RNA into contact with a probe comprising a polynucleotide or primer under hybridising conditions and detecting any duplex formed between the probe and nucleic acid in the sample.
  • detection may be achieved using techniques such as PCR or by immobilising the probe on a solid support, removing nucleic acid in the sample which is not hybridised to the probe, and then detecting nucleic acid which has hybridised to the probe.
  • the sample nucleic acid may be immobilised on a solid support, and the amount of probe bound to such a support can be detected. Suitable assay methods of this and other formats can be found in for example WO89/03891 and WO90/13667.
  • Tests for sequencing nucleotides involve bringing a biological sample containing target DNA or RNA into contact with a probe comprising a polynucleotide or primer under hybridising conditions and determining the sequence by, for example the Sanger dideoxy chain termination method (see Sambrook et al.).
  • Such a method generally comprises elongating, in the presence of suitable reagents, the primer by synthesis of a strand complementary to the target DNA or RNA and selectively terminating the elongation reaction at one or more of an A, C, G or TAJ residue; allowing strand elongation and termination reaction to occur; separating out according to size the elongated products to determine the sequence of the nucleotides at which selective termination has occurred.
  • suitable reagents include a DNA polymerase enzyme, the deoxynucleotides dATP, dCTP, dGTP and dTTP, a buffer and ATP. Dideoxynucleotides are used for selective termination.
  • the Group 5 variant polypeptide sequence may be used as a therapy in any form, such as in an isolated form.
  • isolated means that the sequence is at least substantially free from at least one other component with which the sequence is naturally associated in nature and as found in nature. In one aspect, the sequence is in a purified form.
  • purified means that the sequence is in a relatively pure state - e.g. at least about 90% pure, or at least about 95% pure or at least about 98% pure.
  • the variant Group 5 polypeptides may be derived from, or are variants of, another sequence, known as a "parent polypeptide" or a "parent sequence”.
  • parent polypeptide as used in this document means the polypeptide that has a close or the closest, chemical structure to the resultant variant, i.e., the variant Group 5 polypeptide or nucleic acid.
  • the parent polypeptide may be a precursor polypeptide (i.e. a polypeptide that is actually mutated) or it may be prepared de novo.
  • the parent polypeptide may be a wild type polypeptide, or it may be a wild type polypeptide comprising one or more mutations.
  • precursor means an polypeptide that precedes the polypeptide which is modified to produce the polypeptide.
  • the precursor may be an v that is modified by mutagenesis.
  • the precursor may be a wild type polypeptide, a variant wild type polypeptide or an already mutated polypeptide.
  • wild type is a term of the art understood by skilled persons and means a phenotype that is characteristic of most of the members of a species occurring naturally and contrasting with the phenotype of a mutant.
  • the wild type polypeptide is a form of the polypeptide naturally found in most members of the relevant species.
  • the relevant wild type polypeptide in relation to the variant polypeptides described here is the most closely related corresponding wild type polypeptide in terms of sequence homology.
  • this will be the corresponding wild type sequence regardless of the existence of another wild type sequence that is more closely related in terms of amino acid sequence homology.
  • the parent polypeptide may comprise a polypeptide which exhibits a Group 5 polypeptide activity such as allergenicity.
  • the allergenicity may comprise a Type I allergenicity.
  • the Group 5 polypeptide activity may comprise binding activity, such as IgE binding activity.
  • the parent polypeptide may comprise a Group 5 polypeptide itself.
  • the parent polypeptide may be a Blomia tropicalis Group 5 polypeptide such as a polypeptide having UniProt or SWISS-PROT accession number 096870 ("Blomia tropicalis BIo t 5" or "BIo t 5").
  • Other members of the Group 5 family may be used as parent polypeptides; such
  • Group 5 family members will generally be similar to, identical to, homologous to, or functionally equivalent to such a Group 5 polypeptide such as a Blomia tropicalis Group 5 polypeptide such as a polypeptide having UniProt or SWISS-PROT accession number 096870. Where sequence similarity or homology is used to identify such members, the sequence similarity or homology or identity may be 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more. Such Group 5 family members may be identified by standard methods, such as hybridisation screening of a suitable library using probes, or by genome sequence analysis.
  • Group 5 family or “Group 5 polypeptide” includes at least a Blomia tropicalis BIo 1 5 sequence (SEQ ID NO: 1), a Der f 5 sequence (Accession Number: BAE45865), a Der p 5 sequence (Accession Number: P14004), a Lep d 5 sequence (Accession Number: Q9U5P2), a Der p 21 sequence (Accession Number: ABC73706) and a BIo 1 21 sequence (Accession Number: AY800348). Other sequences of this family may be identified by the methods disclosed here.
  • functional equivalents of Blomia tropicalis BIo 1 5 and other members of the Group 5 family may also be used as starting points or parent polypeptides for the generation of Group 5 variant polypeptides as described here.
  • a "functional equivalent" of a protein means something that shares one or more of the functions of that protein. The “functional equivalent” may share substantially all the functions of that protein. Such functions may comprise biological functions, such as allergenic functions or allergenicity, for example Type I allergenicity. The functions may comprise binding functions, such as IgE binding.
  • the "functional equivalent” may comprise any activity of a Blomia tropicalis Group 5 polypeptide for example. It may be obtained from other sources.
  • the functionally equivalent polypeptide may have a different amino acid sequence but will have Group 5 polypeptide activity.
  • the functional equivalent may comprise some sequence homology to the Blomia tropicalis BIo 1 5 sequence mentioned above. Sequence homology between such sequences may be at least 20%, at least 30% or more, at least 40% or more, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%.
  • sequence homologies may be generated by any of a number of computer programs known in the art, for example BLAST or FASTA, etc.
  • a suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al, 1984, Nucleic Acids Research 12:387).
  • Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid- Chapter 18), FASTA (Atschul et al., 1990, J. MoI. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7- 60).
  • the GCG program may be used.
  • the functional equivalents will be capable of specifically hybridising to any of the sequences set out above. Methods of determining whether one sequence is capable of hybridising to another are known in the art, and are for example described in Sambrook, et al (supra) and Ausubel, F. M. et al. (supra).
  • Functional equivalents which have sequence homology to the Group 5 polypeptides disclosed above may be suitable for use as parent polypeptides. Such sequences may differ from the Group 5 polypeptide sequence at any one or more positions.
  • the variant polypeptide residues may be inserted into any of these parent sequences to generate the variant Group 5 polypeptide sequences.
  • variant Group 5 polypeptides may additionally comprise one or more mutations, as set out above, corresponding mutations may be made in the nucleic acid sequences of the functional equivalents of a Group 5 polypeptide and Group 5 family member in order that they may be used as starting points or parent polypeptides for the generation of Group 5 variant polypeptides as described here.
  • polypeptides are suitable for use in the applications described herein, in particular, as therapies such as immunotherapies, etc.
  • the parent polypeptides may be modified at the amino acid level or the nucleic acid level to generate the Group 5 variant sequences described here. Therefore, we provide for the generation of Group 5 variant polypeptides by introducing one or more corresponding codon changes in the nucleotide sequence encoding a Group 5 polypeptide.
  • the nucleic acid numbering may be made with reference to the position numbering of a Blomia tropicalis BIo t 5 nucleotide sequence shown as SEQ ID NO: 3. Alternatively, or in addition, reference may be made to the sequence with EMBL Accession Number U59102. However, as with amino acid residue numbering, the residue numbering of this sequence is to be used only for reference purposes only. In particular, it will be appreciated that the above codon changes can be made in any Group 5 family nucleic acid sequence.
  • the parent polypeptide may comprise the "complete" polypeptide, i.e., in its entire length as it occurs in nature (or as mutated), or it may comprise a truncated form thereof.
  • the Group 5 variant derived from such may accordingly be so truncated, or be "full-length".
  • the truncation may be at the N-terminal end or the C-terminal end.
  • the parent polypeptide or Group 5 variant may lack one or more portions, such as sub-sequences, signal sequences, domains or moieties, whether active or not etc.
  • the parent polypeptide or the Group 5 variant polypeptide may lack a signal sequence, as described above.
  • the parent polypeptide or the Group 5 variant may lack one or more catalytic or binding domains.
  • nucleic acid molecules described here may be provided in the form of vectors.
  • Vectors comprising such nucleic acid include plasmids, phasmids, cosmids, viruses (including bacteriophages), YACs, PACs, etc. They will usually include an origin of replication and may include one or more selectable markers e.g. drug resistance markers and/or markers enabling growth on a particular medium.
  • a vector may include a marker that is inactivated when a nucleic acid molecule, such as the ones described here, is inserted into the vector.
  • a further marker may be provided that is different from the marker that is inactivated (e.g. it encodes a different type of drug resistance).
  • Vectors may include one or more regions necessary for transcription of RNA encoding a polypeptide. Such vectors are often referred to as expression vectors. They will usually contain a promoter and may contain additional regulatory regions - e.g. operator sequences, enhancer sequences, etc. Translation can be provided by a host cell or by a cell free expression system.
  • Vectors need not be used for expression. They may be provided for maintaining a given nucleic acid sequence, for replicating that sequence, for manipulating, it or for transferring it between different locations (e.g. between different organisms).
  • nucleic acid molecules may be incorporated into high capacity vectors (e.g. cosmids, phasmids, YACs or PACs). Smaller nucleic acid molecules may be incorporated into a wide variety of vectors.
  • Group 5 variant polynucleotides can be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate the nucleic acid in a compatible host cell.
  • we provide a method of making polynucleotides by introducing a polynucleotide into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell.
  • Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.
  • a polynucleotide in a vector may be operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
  • the term "operably linked” means that the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
  • the control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.
  • Vectors may be transformed or transfected into a suitable host cell as described below to provide for expression of a protein. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein. Vectors will be chosen that are compatible with the host cell used.
  • the vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used, for example, to transfect or transform a host cell.
  • Control sequences operably linked to sequences encoding the polypeptide include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in.
  • the term promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.
  • the promoter is typically selected from promoters which are functional in mammalian cells, although prokaryotic promoters and promoters functional in other eukaryotic cells, such as insect cells, may be used.
  • the promoter is typically derived from promoter sequences of viral or eukaryotic genes.
  • it may be a promoter derived from the genome of a cell in which expression is to occur.
  • eukaryotic promoters they may be promoters that function in a ubiquitous manner (such as promoters of ⁇ -actin, ⁇ -actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase). They may also be promoters that respond to specific stimuli, for example promoters that bind steroid hormone receptors.
  • Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter.
  • MMLV LTR Moloney murine leukaemia virus long terminal repeat
  • RSV rous sarcoma virus
  • CMV human cytomegalovirus
  • the promoters may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated.
  • any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences.
  • Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
  • Polynucleotides may also be inserted into the vectors described above in an antisense orientation to provide for the production of antisense RNA.
  • Antisense RNA or other antisense polynucleotides may also be produced by synthetic means.
  • Such antisense polynucleotides may be used in a method of controlling the levels of RNAs transcribed from genes comprising any one of the polynucleotides described here.
  • HOST CELLS Vectors and polynucleotides or nucleic acids comprising or encoding variant Group 5 nucleic acids, fragments, homologues, variants or derivatives thereof may be introduced into host cells for the purpose of replicating the vectors/polynucleotides and/or expressing the polypeptides encoded by the polynucleotides.
  • the polypeptides may be produced using prokaryotic cells as host cells, eukaryotic cells, for example yeast, insect or mammalian cells, in particular mammalian cells, may be used.
  • Vectors/polynucleotides may be introduced into suitable host cells using a variety of techniques known in the art, such as transfection, transformation and electroporation. Where vectors/polynucleotides are to be administered to animals, several techniques are known in the art, for example infection with recombinant viral vectors such as retroviruses, herpes simplex viruses and adenoviruses, direct injection of nucleic acids and biolistic transformation.
  • retroviruses such as retroviruses, herpes simplex viruses and adenoviruses
  • a cell capable of expressing a variant Group 5 polypeptide described here can be cultured and used to provide the variant Group 5 polypeptide, which can then be purified.
  • the cell may be used in therapy for the same purposes as the Group 5 variant polypeptide.
  • cells may be provided from a patient (e.g. via a biopsy), transfected with a nucleic acid molecule or vector and, if desired, cultured in vitro, prior to being returned to the patient (e.g. by injection). The cells can then produce the Group 5 variant polypeptide in vivo.
  • the cells may comprise a regulatable promoter enabling transcription to be controlled via administration of one or more regulator molecules. If desired, the promoter may be tissue specific.
  • Expression is not however essential since the cells may be provided simply for maintaining a given nucleic acid sequence, for replicating the sequence, for manipulating it, etc.
  • Such cells may be provided in any appropriate form. For example, they may be provided in isolated form, in culture, in stored form, etc. Storage may, for example, involve cryopreservation, buffering, sterile conditions, etc.
  • Such cells may be provided by gene cloning techniques, by stem cell technology or by any other means. They may be part of a tissue or an organ, which may itself be provided in any of the forms discussed above. The cell, tissue or organ may be stored and used later for implantation, if desired. Techniques for providing tissues or organs, include stem cell technology, the provision of cells tissues or organs from transgenic animals, retroviral and non-retroviral techniques for introducing nucleic acids, etc.
  • cells may be provided together with other material to aid the structure or function or of an implant.
  • scaffolds may be provided to hold cells in position, to provide mechanical strength, etc.
  • These may be in the form of matrixes of biodegradable or non-biodegradable material.
  • WO95/01810 describes various materials that can be used for this purpose.
  • Host cells comprising polynucleotides may be used to express polypeptides, such as Group 5 polypeptides, fragments, homologues, variants or derivatives thereof.
  • Host cells may be cultured under suitable conditions which allow expression of the proteins. Expression of the polypeptides may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG.
  • an inducer substance for example dexamethasone or IPTG.
  • Polypeptides can be extracted from host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption. Polypeptides may also be produced recombinantly in an in vitro cell-free system, such as the TnTTM (Promega) rabbit reticulocyte system.
  • the Group 5 variant polypeptides and nucleic acids may be produced by any means known in the art. Specifically, they may be expressed from expression systems, which may be in vitro or in vivo in nature. Specifically, we describe plasmids and expression vectors comprising Group 5 nucleic acid sequences, which may be capable of expressing Group 5 variant polypeptides. Cells and host cells which comprise and may be transformed with such Group 5 nucleic acids, plasmids and vectors are also disclosed, and it should be made clear that these are also encompassed in this document.
  • a variant Group 5 polypeptide may be produced by methods known in the art.
  • the host cell may be cultured a medium and caused to express the polypeptide.
  • the polypeptide may be secreted, or it may be intracellular.
  • the polypeptide is isolated or purified from the culture by for example fractionation or affinity purification in the case of a secreted protein, with a further step of cell lysis to release intracellular proteins if necessary.
  • Methods of cell culture, expression and purification are well known to a person skilled in the art and may be carried out by standard techniques, for example as set out in Sambrook et al. ANIMALS
  • transgenic animals such as non-human transgenic animals. Such animals may be useful for producing the particular Group 5 variant polypeptides described here (e.g. via secretion in milk, as described herein). Alternatively, they may be useful as test animals for analysing the effect(s) of such Group 5 variant polypeptides. Techniques for producing transgenic animals are well known and are described e.g. in
  • a nucleic acid encoding a Group 5 variant polypeptide of interest may be microinjected into a pronucleus of a fertilised oocyte.
  • the oocyte may then be allowed to develop in a pseudopregnant female foster animal.
  • the animal resulting from development of the oocyte can be tested (e.g. with antibodies) to determine whether or not it expresses the particular polypeptide.
  • it can be tested with a probe to determine if it has a transgene (even if there is no expression).
  • a transgenic animal can be used as a founder animal, which may be bred from in order to produce further transgenic animals. Two transgenic animals may be crossed. For example, in some cases transgenic animals may be haploid for a given gene and it may be desired to try to provide a diploid offspring via crossing.
  • a transgenic animal may be cloned, e.g. by using the procedures set out in WO97/07668 and WO97/07699 (see also Nature 385:810-813 (1997)).
  • a quiescent cell can be provided and combined with an oocyte from which the nucleus has been removed combined. This can be achieved using electrical discharges.
  • the resultant cell can be allowed to develop in culture and can then be transferred to a pseudopregnant female.
  • a moiety comprising a Group 5 variant polypeptide, a Group 5 variant nucleic acid, a vector comprising Group 5 variant, a cell expressing Group 5 variant, an Group 5 variant binding agent, a moiety identified/identifiable by a screen as described here, when used as an analytical tool or when present in a system suitable for analysis, especially high throughput analysis.
  • Such an analytical tool or system is useful for a plethora of different purposes. These include diagnosis, forensic science, screening, the identification or characterisation of individuals or populations, preventative medicine, etc.
  • a library will generally comprise a plurality of heterologous moieties. Such libraries may comprise at least 100, at least 10,000, at least 1,000,000, or at least 1,000,000,000 heterologous moieties. Desirably a moiety is provided at a predetermined position within a library. In some cases a plurality of moieties may be present within a library at predetermined positions. A predetermined position may be assigned spatial co-ordinates. These may be stored or processed in a computer in order to assist in analysis.
  • the array may comprise a regular array.
  • the array may have a predetermined pattern. It may have a grid-like pattern.
  • Such arrays may comprise at least 100, at least 10,000, at least 1,000,000, or at least 1,000,000,000 components.
  • a library or array may include naturally occurring moieties, non-naturally occurring moieties, or a mixture of naturally occurring and non-naturally occurring moieties.
  • the moieties may provided in solution, on beads, on chips (see e.g. Fodor (1993) Nature 364:555- 556), on bacteria (see e.g. US Patent 5223409), on spores (see e.g. US Patent 5223409), on 'phage (see e.g. Scott and Smith (1990) Science 249:386-90 and US Patent 5223409), etc.
  • Such Group 5 variant moieties may be immobilised upon a surface, if desired.
  • one or more nucleic acid molecules may be immobilised upon a surface (e.g.
  • the surface of a bead or a chip may, for example, be silicon, glass, quartz, a membrane, etc.
  • Techniques for immobilising nucleic acid molecules upon a surface are known and are disclosed, for example, in EP-A-0487104, WO96/04404, WO90/02205, WO96/12014, WO98/44151. In some cases they may include a step of nucleic acid amplification, and may involve PCR.
  • Immobilisation is not however essential, even if moieties are to be used in high throughput analysis.
  • moieties may be provided in wells, channels, grooves or other containment means.
  • an array or in immobilised or non-immobilised form it is often desirable to locate the position of one or more moieties being analysed or being used in analysis. This can be done by assigning it spatial co-ordinates, which may be provided, stored or processed or provided by a computer. In some cases the location may be determined by a sensor (e.g. a CCD device), which may be operatively linked with a computer.
  • a sensor e.g. a CCD device
  • any of the Group 5 variant nucleic acids disclosed here may be administered to an individual in the form of a DNA vaccine.
  • DNA vaccines are known in the art, and are described in detail in, for example, WO03012117, WO03007986, etc.
  • the Group 5 variant may be administered to an individual in the form of a DNA vaccine.
  • a DNA encoding the Group 5 variant for example, a Group 5 variant nucleic acid as disclosed here, may be in any form, for example in the form of a cloned plasmid DNA or a synthetic oligonucleotide.
  • the DNA may be delivered together with a cytokine, for example, IL-2, and / or other co-stimulatory molecules.
  • the cytokines and / or co-stimulatory molecules may themselves be delivered in the form of plasmid or oligonucleotie DNA.
  • ISS immunostimulatory DNA sequences
  • ANTIBODIES We also provide monoclonal or polyclonal antibodies to polypeptides or fragments thereof. Thus, we further provide a process for the production of monoclonal or polyclonal antibodies to an Group 5 variant polypeptide, fragment, homologue, variant or derivative thereof If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide bearing an epitope(s) from a polypeptide. Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an epitope from a polypeptide contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography.
  • a selected mammal e.g., mouse, rabbit, goat, horse, etc.
  • Serum from the immunised animal is collected and treated according to known procedures.
  • serum containing polyclonal antibodies to an epitope from a polypeptide
  • Monoclonal antibodies directed against epitopes in the polypeptides can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.
  • Panels of monoclonal antibodies produced against epitopes in the polypeptides can be screened for various properties; i.e., for isotype and epitope affinity.
  • An alternative technique involves screening phage display libraries where, for example the phage express scFv fragments on the surface of their coat with a large variety of complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the term "antibody”, unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies (scFv). Furthermore, the antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400.
  • Antibodies may be used in method of detecting polypeptides present in biological samples by a method which comprises: (a) providing an antibody; (b) incubating a biological sample with said antibody under conditions which allow for the formation of an antibody- antigen complex; and (c) determining whether antibody-antigen complex comprising said antibody is formed.
  • Suitable samples include extracts tissues such as brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle and bone tissues or from neoplastic growths derived from such tissues.
  • Antibodies may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.
  • Variant Group 5 polypeptides may be produced in large amounts at low cost in a bioactive form, allowing the development of Group 5 variant polypeptides containing formulations by aerosolisation, nebulisation, intranasal or intratracheal administration.
  • compositions comprising the Group 5 variant polypeptide or nucleic acid may be administered alone, the active ingredient may be formulated as a pharmaceutical formulation.
  • pharmaceutical compositions comprising Group 5 variant polypeptide or nucleic acid, or a fragment, homologue, variant or derivative thereof.
  • Such pharmaceutical compositions are useful for delivery of Group 5 variant polypeptide, nucleic acid, fragment, homologue, variant or derivative thereof to an individual for the treatment or alleviation of symptoms as described.
  • the composition may include the Group 5 variant polypeptide, nucleic acid, fragment, homologue, variant or derivative thereof, a structurally related compound, or an acidic salt thereof.
  • the pharmaceutical formulations comprise an effective amount of Group 5 variant polypeptide, nucleic acid, fragment, homologue, variant or derivative thereof, together with one or more pharmaceutically-acceptable carriers.
  • An "effective amount" of an Group 5 variant polypeptide, nucleic acid fragment, homologue, variant or derivative thereof is the amount sufficient to alleviate at least one symptom of a disease as described, such as atopic allergy.
  • the effective amount will vary depending upon the particular disease or syndrome to be treated or alleviated, as well as other factors including the age and weight of the patient, how advanced the disease etc state is, the general health of the patient, the severity of the symptoms, and whether the Group 5 variant polypeptide, nucleic acid, fragment, homologue, variant or derivative thereof is being administered alone or in combination with other therapies.
  • Suitable pharmaceutically acceptable carriers are well known in the art and vary with the desired form and mode of administration of the pharmaceutical formulation. For example, they can include diluents or excipients such as fillers, binders, wetting agents, disintegrators, surface-active agents, lubricants and the like.
  • the carrier is a solid, a liquid or a vaporizable carrier, or a combination thereof.
  • Each carrier should be “acceptable” in the sense of being compatible with the other ingredients in the formulation and not injurious to the patient.
  • the carrier should be biologically acceptable without eliciting an adverse reaction (e.g. immune response) when administered to the host.
  • compositions disclosed here include those suitable for topical and oral administration, with topical formulations being for example used where the tissue affected is primarily the skin or epidermis (for example, psoriasis, eczema and other epidermal diseases).
  • the topical formulations include those pharmaceutical forms in which the composition is applied externally by direct contact with the skin surface to be treated.
  • a conventional pharmaceutical form for topical application includes a soak, an ointment, a cream, a lotion, a paste, a gel, a stick, a spray, an aerosol, a bath oil, a solution and the like.
  • Topical therapy is delivered by various vehicles, the choice of vehicle can be important and generally is related to whether an acute or chronic disease is to be treated.
  • compositions for topical application include shampoos, soaps, shake lotions, and the like, particularly those formulated to leave a residue on the underlying skin, such as the scalp (Arndt et al, in Dermatology In General Medicine 2:2838 (1993)).
  • the concentration of the Group 5 variant polypeptide, nucleic acid, fragment, homologue, variant or derivative thereof composition in the topical formulation is in an amount of about 0.5 to 50% by weight of the composition, such as about 1 to 30%, about 2- 20% or about 5-10%.
  • the concentration used can be in the upper portion of the range initially, as treatment continues, the concentration can be lowered or the application of the formulation may be less frequent.
  • Topical applications are often applied twice daily. However, once-daily application of a larger dose or more frequent applications of a smaller dose may be effective.
  • the stratum corneum may act as a reservoir and allow gradual penetration of a drug into the viable skin layers over a prolonged period of time.
  • a sufficient amount of active ingredient must penetrate a patient's skin in order to obtain a desired pharmacological effect. It is generally understood that the absorption of drug into the skin is a function of the nature of the drug, the behaviour of the vehicle, and the skin. Three major variables account for differences in the rate of absorption or flux of different topical drugs or the same drug in different vehicles; the concentration of drug in the vehicle, the partition coefficient of drug between the stratum corneum and the vehicle and the diffusion coefficient of drug in the stratum corneum. To be effective for treatment, a drug must cross the stratum corneum which is responsible for the barrier function of the skin. In general, a topical formulation which exerts a high in vitro skin penetration is effective in vivo. Ostrenga et al (J. Pharm. ScL, 60:1175-1179 (1971) demonstrated that in vivo efficacy of topically applied steroids was proportional to the steroid penetration rate into dermatomed human skin in vitro.
  • a skin penetration enhancer which is dermatologically acceptable and compatible with the agent can be incorporated into the formulation to increase the penetration of the active compound(s) from the skin surface into epidermal keratinocytes.
  • a skin enhancer which increases the absorption of the active compound(s) into the skin reduces the amount of agent needed for an effective treatment and provides for a longer lasting effect of the formulation.
  • Skin penetration enhancers are well known in the art. For example, dimethyl sulfoxide (U.S. Pat. No. 3,711,602); oleic acid, 1,2-butanediol surfactant (Cooper, J. Pharm.
  • a long acting form of agent or composition may be administered using formulations known in the art, such as polymers.
  • the agent can be incorporated into a dermal patch (Junginger, H. E., in Acta Pharmaceutica Nordica 4:117 (1992); Thacharodi et al, in Biomaterials 16:145-148 (1995); Niedner R., in Hautier 39:761- 766 (1988)) or a bandage according to methods known in the arts, to increase the efficiency of delivery of the drug to the areas to be treated.
  • the topical formulations can have additional excipients for example; preservatives such as methylparaben, benzyl alcohol, sorbic acid or quaternary ammonium compound; stabilizers such as EDTA, antioxidants such as butylated hydroxytoluene or butylated hydroxanisole, and buffers such as citrate and phosphate.
  • preservatives such as methylparaben, benzyl alcohol, sorbic acid or quaternary ammonium compound
  • stabilizers such as EDTA, antioxidants such as butylated hydroxytoluene or butylated hydroxanisole, and buffers such as citrate and phosphate.
  • the pharmaceutical composition can be administered in an oral formulation in the form of tablets, capsules or solutions.
  • An effective amount of the oral formulation is administered to patients 1 to 3 times daily until the symptoms of the disease alleviated.
  • the effective amount of agent depends on the age, weight and condition of a patient.
  • the daily oral dose of agent is less than 1200 mg, and more than 100 mg.
  • the daily oral dose may be about 300-600 mg.
  • Oral formulations are conveniently presented in a unit dosage form and may be prepared by any method known in the art of pharmacy.
  • the composition may be formulated together with a suitable pharmaceutically acceptable carrier into any desired dosage form. Typical unit dosage forms include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories.
  • the formulations are prepared by uniformly and intimately bringing into association the agent composition with liquid carriers or finely divided solid carriers or both, and as necessary, shaping the product.
  • the active ingredient can be incorporated into a variety of basic materials in the form of a liquid, powder, tablets or capsules to give an effective amount of active ingredient to treat the disease.
  • therapeutic agents suitable for use herein are any compatible drugs that are effective for the intended purpose, or drugs that are complementary to the agent formulation.
  • the formulation utilized in a combination therapy may be administered simultaneously, or sequentially with other treatment, such that a combined effect is achieved. Examples
  • BIo t 5 is over-expressed in E. coli as glutathione S-transferase fusion protein and purified as described elsewhere (Arruda et al., 1997). Recombinant BIo t 5 is 119 amino acid protein ( ⁇ 14kDa) with a small non-native Gly-Ser tag at the N-terminus after thrombin cleavage. BIo t 5 samples with 2 H, 13 C and 15 N isotope enrichment and the combinations thereof, for the NMR studies are prepared by established protocol (Marley et al., 2001).
  • NMR data is acquired in Shigemi tube on ImM BIo 1 5 buffered at pH 7.5 in 50 mM potassium phosphate, 100 mM NaCl, 2 mM EDTA, 0.001% (w/v) NaN 3 and minimum 10% (v/v) D 2 O.
  • Truncated BIo t 5 mutants, BIo t 5I -80 and BIo t 54 6 -11 7 are generated by PCR method, and are expressed and purified similar to the full length BIo t 5. The purity of all protein samples is ascertained by gel electrophoresis and mass spectroscopy.
  • the aliphatic side-chain resonances are primarily assigned using HCCH-COSY and HCCH-TOCSY experiments, with the help of complimentary HCC(CO)NH, (H)CC(CO)NH, HBHA(CO)NH and (H)CCH-TOCSY experiments.
  • 15 N-edited TOCSY-HSQC acquired with 60ms mixing time is used to resolve ambiguity due to the spectral overlap.
  • the aromatic side- chains are assigned using proton homo-nuclear TOCSY and NOESY experiments and verified with 13 C-HMQC, (HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE experiments.
  • One hundred random conformers are annealed in 10000 steps for seven cycles and 20 structures with lowest target function after the final cycle are selected for further analysis with the programs MoIMoI, version 2K.2 (Koradi et al., 1996), Prosa2003 (Sippl, 1993), Aqua, version 3.2 and Procheck-NMR, version 3.5.4 (Laskowski et al., 1996).
  • the steady-state heteronuclear ( 1 H)- 15 N NOE experiment is carried out in an interleaved manner, with and without proton saturation and repeated twice.
  • the NOE effect is calculated as error weighed average ratio of peak intensities, with error estimates obtained by propagating the base-plane noise. Diffusion Tensor Optimization and Modelfree Analysis
  • the relaxation data are then interpreted using the extended Lipari-Szabo Modelfree formalism to gain insights into the magnitudes and time scales of the backbone motion (Clore et al., 1990; Lipari and Szabo, 1982a, b).
  • the analysis considered five semi-empirical forms of the spectral density function with each form composed of terms describing the motion of the N-H bond vector.
  • the internal motion is assumed to be occurring on two different, fast and slow time scales characterized by the effective correlation times, ⁇ and ⁇ s (where ⁇ / « ⁇ s « ⁇ m ), and the square of order parameters, S" 2 / and S 2 ,.
  • the analysis also accounts for the line broadening due to chemical exchange, R ex .
  • the relaxation data is fitted against five semi-empirical models using the program Modelfree version 4.1 (Mandel et al., 1995). For each model, 500 randomly distributed data sets are generated and the model selection is done using a statistical testing protocol described by Mandel et al. Initially, models are selected at a fixed diffusion tensor by comparing the sum-squared error of the optimal fit with the 0.05 critical value of the distribution and wherever applicable, by F-test comparisons to the 0.2 critical value of the distribution.
  • Figure 7 shows the backbone amide N spin relaxation parameters for BIo t 5 at 600MHz.
  • Figure 8 shows a strip plot from 3D-15N edited, TROSY-based NOESY spectrum acquired at 100ms mixing time on the purified 2 H, 15 N- BIo t 5 bound to the Fab'.
  • mAb monoclonal antibody
  • 4A7 recognizing several isoform of BIo t 5
  • mAb monoclonal antibody
  • the mAb used in current study is purified from mouse ascites fluid using Protein-G affinity column and characterized by gel electrophoresis and bicinchoninic acid assay. Dimer F(ab') 2 is obtained by digesting it with 20:1 pepsin for 2 hours at pH 4.0 and 37 0 C. Undigested intact mAb is separated from F(ab') 2 using protein-A affinity column and digested again.
  • the F(ab') 2 either free or as a complex with BIo t 5 is subjected to mild reduction using 0.1 mM ⁇ -mercaptoethanol at pH 8.5. This preferentially broke the inter-domain disulfide bridge resulting in Fab' or Fab'-Blo t 5 complex, respectively, which is further purified on FPLC using Sephadex-200 column.
  • NMR data is acquired at 37 0 C on 0.2 mM Fab'-Blo t 5 complex buffered at pH 7.5 in 50 mM potassium phosphate, 100 mM NaCl, 0.1 mM ⁇ -mercaptoethanol, 2 mM EDTA, 0.001% (w/v) NaN 3 and minimum 5% (v/v) D 2 O.
  • the human IgE ELISA are carried out on 0.5 ⁇ M of BIo t 5 or its truncated mutants with 50 ⁇ l per well described previously (Kuo et al., 2003).
  • BIo t 5 positive sera are diluted from 1 :10 to 1:40 and incubated with allergens coated wells at 4°C for 16 hours.
  • final concentrations of 1 ⁇ M of inhibitors are pre-incubated with individual serum at 4°C for 16 hours.
  • the pre-absorbed sera are then reacted with plate bound full length BIo t 5.
  • plate bound full length BIo 1 5 or BIo t 5 46- i ⁇ ⁇ are incubated with serial diluted mAb 4A7 or isotype control for 1 hour and washed before incubated with sera.
  • the plate bound IgE are developed as the direct ELISA described above.
  • the coordinates and the experimental restraints for the BIo 1 5 structure are available from the Protein Data Bank under accession code 2JMH.
  • the NMR chemical shift assignments were deposited in the Biological Magnetic Resonance Bank with accession code 7276.
  • Group 5 allergen proteins from different mites share high sequence homology (Figure 1); amongst these Dermatophagoides pteronyssinus mite allergen, Der p 5 is known to undergo acid induced irreversible polymerization (Liaw et al., 2001).
  • a multifaceted approach including analytical ultracentrifugation, small angle X-ray scattering, gel filtration, cross- linking and NMR is used to confirm monometric state of BIo 1 5 under our experimental conditions (data not shown). Further circular dichroism spectrum of BIo t 5 showed it is mainly a helical protein with thermal melting point of 59 0 C (data not shown).
  • the structure of BIo t 5 is determined using distance and angle restrains derived using solution NMR methodology and is represented as 20 conformer ensemble in Figure IB. It is calculated by using total 1371 or on an average 12 experimental restrains per residue as described in Table El .
  • First sixteen residues from the N-terminus region of the protein are poorly defined due to intrinsic disorder (vide infra); but the remaining structure shows high precision with root-mean-square deviation of 0.40 ⁇ 0.06A for the backbone atoms. None of the conformers in the ensemble violates experimentally derived distance or torsion angle restrains by more than 0.1 A and 5°, respectively, and 92.40 ⁇ 1.07% of residues fall in energetically most favored regions of Ramachandran plot.
  • BIo t 5 is a triple stranded coiled-coil bundle with first helix (Helix A; residue 18 to 46) and third helix (Helix C; residue 84 tol 13) in parallel arrangement, while the second helix (Helix B, residue 49 to 77) running anti-parallel to the other two helices. All three helices of BIo t 5 appear slightly tilted with inter-helical angles of approximately 20° and are connected with sharp turns. Coiled-coil proteins can be readily identified by the presence of heptad repeat- a seven residue pattern denoted by (abcdefg) n .
  • Each helix in BIo t 5 contains minimum twenty- eight residues or four heptad repeats as predicted previously from the primary sequence (Liaw et al., 2001), although the heptad positions for second helix in the structure are different
  • a simplistic helical wheel representation of identified long range NOE patterns is shown in Figure 3, with residues color coded using Wimley and White hydrophobicity scale (Wimley and White, 1996).
  • Most of the 34 hydrophobic residues in BIo 1 5 occupy heptad position a and d, and internalize against position d and a, respectively from the neighboring coils.
  • the analysis considered five semi- empirical forms of the spectral density function with each form composed of terms describing the motion of the N-H bond vector (Mandel et al., 1995).
  • the internal motion is assumed to be occurring on two different, fast and slow time scales characterized by effective correlation times, Tf and ⁇ s (where ⁇ / « ⁇ s « ⁇ m ), and the square of order parameters, S *2 / and S 2 S -
  • S 2 S 2 ⁇ S 2 S (0 ⁇ S" 2 ⁇ 1) and corresponds to the spatial restriction of the N-H bond vector.
  • the analysis also accounts for line broadening due to chemical exchange, R ex .
  • Modelfree analysis effectively elucidates both, ns-ps timescale mobility of the protein backbone as well as slow ms- ⁇ s conformational exchange. It must be emphasized that motional anisotropy also contributes to the R2 values and if wrongly characterized can cause erroneous interpretation of /? ex (Tjandra et al., 1995b).
  • Model 1 (S 2 -only) is an appropriate fit for 45 residues, 13 residues fit to model 2 (S 2 and ⁇ e ), 1 residue fits to model 3 (S 2 and R ex ), 6 fit to model 4 (S 2 , ⁇ e , and R ex ), and 12 residues fit to model 5 (S 2 /, S 2 S , and ⁇ e ).
  • the sequence variation of the generalized order parameter, S 12 , and the chemical exchange rates, R ex are plotted in Figure 4A and 4B, respectively.
  • the average order parameter for BIo t 5 is 0.88 ⁇ 0.14; and if only residues located in regions of regular secondary structure are considered, the average order parameter is 0.943 ⁇ 0.030.
  • the order parameters are mapped as radius of a green sausage on the BIo t 5 backbone in Figure 4C.
  • the protein has a long and floppy N-terminus, where the order parameter sharply drops below 0.5.
  • NOEs no long range NOEs could be identified between the N-terminus and the helical structured regions and hence it is poorly defined in the structure (Figure IB and histogram in Figure 5A).
  • These N-terminus residues also have chemical shifts very similar to the random coil (Wishart et al., 1992) and very low amide protection factors
  • the N-terminus closely resembles random coil, which does not interact with the structured regions of BIo 1 5, but potentially impart a favorable entropy contribution to the protein stability.
  • the order parameter also drops, though to much lesser extent around the two inter-helical turns and the C-terminus. The motional plasticity exhibited by the turns probably is essential to hold the rigid helical assembly.
  • BIo t 5 specific monoclonal antibody (mAb), 4A7( ⁇ 150 kDa) used in this study is purified from mouse ascites fluid (Yi et al., 2005). To facilitate NMR study, its proteolytically cleaved Fab' fragment is isolated. Sandwich ELISA assay on F(ab') 2 fragment and intact mAb showed identical binding ability for BIo 1 5, confirming the integrity of the Fab' and ruling out non-specific damage to the CDR (data not shown).
  • Figure 6A shows the absolute compounded difference for the backbone amide chemical shifts between free and Fab'-bound BIo 1 5. Most of the amides, except those belonging to residues concluded as the epitope regions, show uniform chemical shift perturbation with 10% trimmed average of 0.9ppm, and indicates BIo t 5 retains its triple helical fold in Fab'-bound state.
  • Epitope surface-I comprises of residues Leu43-Lys47 of the C-terminal end of Helix A
  • epitope surface-II comprises of residues Lys54-Arg57 located in Helix B. These two epitope surfaces are separated by 6 residues in sequence.
  • the most perturbed residues i.e. residues with ⁇ > 0.24ppm or one standard deviations from the average ⁇ , are Leu43, Asn46, Lys47, Lys54, Ile55, Ile56 and Arg57.
  • the two epitope surfaces are located at the C-terminal end of the helix bundle with surface I and II positioned on opposite faces of the molecule ( Figure 6B and D).
  • the shortest distance between backbone amides of the two surfaces is from Lys47 to Lys54 (11.6 ⁇ 0.4 A) and if the side-chains of these lysines are taken into account, the distance between the two N ⁇ atoms is 19.9 ⁇ 1.1 A.
  • Strikingly residues between surface-I to surface-II, namely Ser48, Glu50, Leu51, Gln52 and Glu53 show very insignificant chemical shift change.
  • epitope surface-I perturbation does not appear to be a secondary effect of binding on epitope surface-II, and these residues probably are approached by a different CDRs on the Fab' fragment.
  • a probable scenario is that the two epitope surfaces are separately recognized by the heavy chain (V H ) and light chain variable regions (V L ) and the BIo t 5 molecule is being held on the shallow cleft formed by the two chains.
  • Hydrogen/Deuterium (H/D) exchange rates provide wealth of information about water accessibility of different backbone amides and can be used to probe changes in local conformation. This experiment has previously been used for epitope mapping of major allergen Der p 2 (Mueller et al., 2001). We calculated H/D protection factors for BIo t 5 at 22 0 C and the procedure and results are as follow.
  • the backbone amide hydrogen-deuterium exchange rates are calculated from series of 1 H- 15 N HSQC spectrum acquired after reconstituting lyophilized 15 N- BIo t 5 in D 2 O.
  • the CLEANEX-PM experiment is used to confirm and compliment these rates using mixing time of 5 to 20ms. These two experimental approaches are useful for the slow and the fast H/D exchange regimes, respectively. No reliable data could be obtained for residues with intermediate exchange or where the peaks overlapped.
  • qualitative estimate of the water accessibility is obtained from a spectrum recoded at long 100ms mixing time. A change in the peak intensity of this experiment after Fab' binding indicates a possible change in the water accessibility of that particular amide.
  • Figure 9 shows Hydrogen/Deuterium Protection Factors for Free BIo t 5 at 22 * C.
  • CLEANEX-PM experiment was also used to deduce protection factors for the randomized N-terminus region.
  • the H/D exchange rate is known to increase with temperature and appears to be the main reason for the disappearance of N-terminus resonances between Glnl-Hisl6, and Lys49 and Thr80 from the two inter-helical turns at 37 0 C.
  • Unfortunately quantitative analysis of the Fab' fragment bound BIo 1 5 at 37 0 C is hindered due to approximately 3 hours of acquisition time required to collect meaningful 2D- spectrum on the 0.2 mM sample used in our study.
  • the localization of human IgE epitopes in BIo t 5 is performed using two overlapping Blot 5-derived peptides, BIo 1 5 I-80 and BIo 1 5 46- i i 7 .
  • the peptides are designed to keep the helical elements intact so as they either begin or terminate at the inter-helical turn positions of the full-length protein, and retained the key residues comprising the two epitope surfaces for mAb 4A7 determined by NMR.
  • Peptide BIo 1 5i -8 o contained helices A and B, while peptide BIo t 5 46- n 7 contained helices B and C.
  • BIo t 5 46- n 7 resembles an extensively studied truncated form of BIo 1 5 designated as BtM, except for the first three N-terminal residues (Caraballo et al., 1998).
  • Figure 10 shows the 2D-HSQC spectrum of the two overlapping Blot 5-derived peptides, (A) BIo 1 5 i -80 and (B) BIo 1 546- n 7 .
  • IgE reactivity of these peptides is tested against 28 BIo t 5 specific IgE positive human sera and is compared to the full-length protein by direct and cross inhibition IgE ELISA.
  • the BIo t 54 6- ⁇ 7 bound substantial but lower amount of BIo 1 5 specific IgE as compared to that of full length BIo 1 5 (p - 0.05) ( Figure 6A).
  • the antigen pre-absorption study showed the significant lower inhibitory capability of BIo t 54 6- ⁇ 7 to plate bound BIo t 5 as compared to that of full length BIo t 5 (p ⁇ 10 "5 ).
  • the maximum inhibition is less than 50% with two exceptions.
  • the inhibition capability of BIo t 5i-8o to BIo t 5 is even lower than that of BIo t 5 46- ⁇ 7 with a maximum inhibition of less than 20% (p ⁇ 10 "5 ) ( Figure 6B).
  • Peptide BIo 1 5i -80 is found to be mis-folded at room temperature and explains the major role played by helix C in the structural assembly of BIo t 5.
  • Peptide BIo 1 54 6- ii 7 appears to be highly heat resistant with a melting point > 8O 0 C and has a heavily perturbed HSQC spectrum compared to that of BIo t 5. It also elutes earlier than BIo t 5 in gel filtration and probably exists as a dimer.
  • the hydrophobic core of BIo t 5 is mainly made up of Crick's knob-in-hole type of packing; a hydrophobic side chain or knob is buried against the hole formed by four or more side chains on the neighboring helices.
  • Such packing can be further categorized depending on how the buried knob approaches the hole, and if it's an isolated one-way (Type-2) or a complementary burial with the knob itself acting as hole (Type-4).
  • a cyclically complementary Type-4 daisy chain arrangement can arise out of the hydrophobic burial in an interleaved fashion (Walshaw and Woolfson, 2001).
  • the BIo t 5 structure has one such daisy chain arrangement formed by Leu36, Leu59, and ThrlO7 and their neighbors, from Helices A, B, and C, respectively.
  • the rest of the core is mainly made up of isolated or Type-2 packing.
  • Helix C appears to be the best packed among the three, utilizing either one-way or complementary burial of its side chain knobs against the side chain holes in either of the two neighboring helices.
  • Helix C preferentially partners Helix A in the beginning, with side chains of Ala93, LeulOO, and ThrlO7 buried deeply in Helix A holes, and with Helix B toward its end, with LeulO4, GlulO8, and VaIl 11 buried in Helix B.
  • all three helices are necessary for the structural integrity of BIo t 5 and demonstrate the limitation of mapping the conformational B epitopes with overlapping peptides ( Figure 6).
  • Glu45Ser mutation was shown to completely abolish mAb binding and to reduce the polyclonal human IgE reactivity by 50% (Spangfort et al., 2003).
  • Hyal a 9 residue continuous epitope was identified against mAb 21El 1.
  • the conformational epitope is made up of Argl38 and a linear array of residues between Hisl41 and Argl48. It is located at the tip of a helix-turn-helix motif that protrudes from the protein core and fits into a pocket formed by the six CDRs of Fab (Padavattan et al., 2007).
  • mAb BV16 could inhibit 40% of the binding to Bet v 1-specific human IgE (Mirza et al., 2000), whereas mAb 21El 1 could inhibit up to 57% of IgE binding to Hyal (Padavattan et al., 2007).
  • mAb 4A7 inhibited 45% of the BIo t 5-specific human IgE binding ( Figure 6C), and its epitope was mapped on two surfaces connected by a turn, with the critical residues Asn46 and Lys47 on surface I and Lys54 and Arg57 on surface II.
  • the group 5 allergen from D was mapped on two surfaces connected by a turn, with the critical residues Asn46 and Lys47 on surface I and Lys54 and Arg57 on surface II.
  • Example 14 B Cell Epitopes of Other Mite Allergens Only limited structural information is available regarding the conformal antibody epitopes of the dust mite allergens.
  • the mAb epitopes of Der p 2 were predicted by NMR by using the H/D exchange experiments and were confirmed by mutagenesis (Mueller et al., 2001). The extensive study suggested that the probable epitope residues include Arg31, Lys33, Asn93, Lys97, and Ile96 along with other residues.
  • the epitopes determined are discontinuous and invariably contain charged residues, with a notable proportion of lysine and arginine.
  • Our experimental data are more stringent in mapping the epitope as compared to the published reports, partially due to the elipsoid shape of BIo 15, but it is very likely that the proposed experiments will work equally well for the epitope mapping of other more globular allergens.
  • Example 15 Discussion Here, we have solved the three-dimensional structure of the major allergen BIo 1 5 and have mapped critical residues of the mAb 4A7 epitope by NMR. This discontinuous epitope is also shown to overlap with the human IgE epitope(s) of BIo t 5.
  • Example 16 Immunotherapy: Design of a Hypoallergenic BIo 1 5 Protein
  • hypoallergenic BIo 1 5 proteins are generated by systemic site-directed mutagenesis on the surface residues.
  • the scanning of the critical residues for human IgE epitopes is started with the charged residues and the residues with large side chains. Single mutation and multiple mutations are to be generated to assess their contribution to human IgE binding.
  • the residues with small side chains are examined with the same procedure as well.
  • the single, double and triple mutation mutants are generated and assessed by direct human IgE ELISA, human IgE inhibition ELISA, and histamine release assay, as described in the Examples below. They are also evaluated by NMR analysis.
  • the wildtype BIo t 5, the single mutation BIo 1 5 proteins (E45V, N46A, K47A, K47P, K49A, and R57A) and the double mutation BIo t 5 proteins (N46A+R57A, K47A+R57A, K49A+R57A, N46A+V61I, and R57A+A69P) are coated on ELISA plates and reacted with 1 :5 diluted serum of subject 8 and pool sera from 5 subjects, using the protocols described above under "Human BIo 1 5 specific IgE ELISA” and "Human IgE Inhibition Enzyme- Linked Immunosorbant Assay (ELISA)". The results are shown in Figure 11. The E45V is less reactive with IgE from subject 8.
  • the N46A+V61I, and R57A+A69P are less reactive with the IgE of the pool sera.
  • PROCHECK a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283-291.
  • Socket a program for identifying and analysing coiled-coil motifs within protein structures. J MoI Biol 307, 1427-1450.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Insects & Arthropods (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pulmonology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Polypeptide de groupe 5 variant comprenant une mutation correspondant à un résidu de surface de blot 5 présenté en figure 3 ou une mutation sur un épitope de blot 5 présenté en figure 5, en référence à la numérotation des positions de la séquence de blot 5 Blomia tropicalis présentée en tant SEQ ID NO: 1. L'invention concerne aussi un fragment, un homologue, un variant ou un dérivé du polypeptide précité.
PCT/SG2008/000189 2007-05-17 2008-05-17 Polypeptides hypoallergéniques WO2008143594A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US93063507P 2007-05-17 2007-05-17
US60/930,635 2007-05-17

Publications (1)

Publication Number Publication Date
WO2008143594A1 true WO2008143594A1 (fr) 2008-11-27

Family

ID=40032172

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SG2008/000189 WO2008143594A1 (fr) 2007-05-17 2008-05-17 Polypeptides hypoallergéniques

Country Status (1)

Country Link
WO (1) WO2008143594A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002006323A1 (fr) * 2000-07-18 2002-01-24 National University Of Singapore Proteines immunogenes bt5 derivees d'allergenes d'acariens domestiques
WO2006132607A1 (fr) * 2005-06-10 2006-12-14 National University Of Singapore Allergenes mutants

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002006323A1 (fr) * 2000-07-18 2002-01-24 National University Of Singapore Proteines immunogenes bt5 derivees d'allergenes d'acariens domestiques
WO2006132607A1 (fr) * 2005-06-10 2006-12-14 National University Of Singapore Allergenes mutants

Similar Documents

Publication Publication Date Title
Chan et al. Nuclear magnetic resonance structure-based epitope mapping and modulation of dust mite group 13 allergen as a hypoallergen
Swoboda et al. Recombinant carp parvalbumin, the major cross-reactive fish allergen: a tool for diagnosis and therapy of fish allergy
Neudecker et al. Mutational epitope analysis of Pru av 1 and Api g 1, the major allergens of cherry (Prunus avium) and celery (Apium graveolens): correlating IgE reactivity with three-dimensional structure
Fletcher et al. Structure of a soluble, glycosylated form of the human complement regulatory protein CD59
Gibbs et al. The cysteine-rich secretory protein domain of Tpx-1 is related to ion channel toxins and regulates ryanodine receptor Ca2+ signaling
Findeisen et al. RFamide peptides: structure, function, mechanisms and pharmaceutical potential
Gieras et al. Mapping of conformational IgE epitopes with peptide-specific monoclonal antibodies reveals simultaneous binding of different IgE antibodies to a surface patch on the major birch pollen allergen, Bet v 1
Flicker et al. A human monoclonal IgE antibody defines a highly allergenic fragment of the major timothy grass pollen allergen, Phl p 5: molecular, immunological, and structural characterization of the epitope-containing domain
Naik et al. Roles of structure and structural dynamics in the antibody recognition of the allergen proteins: an NMR study on Blomia tropicalis major allergen
Kurnellas et al. Chaperone activity of small heat shock proteins underlies therapeutic efficacy in experimental autoimmune encephalomyelitis
De Marino et al. An immunoglobulin-like fold in a major plant allergen: the solution structure of Phl p 2 from timothy grass pollen
Hill et al. Solution structure of the sodium channel antagonist conotoxin GS: a new molecular caliper for probing sodium channel geometry
Cantillo et al. Tropomyosins in mosquito and house dust mite cross‐react at the humoral and cellular level
Cui et al. Molecular cloning, expression, sequence analyses of dust mite allergen Der f 6 and its IgE-binding reactivity with mite allergic asthma patients in southeast China
Pantoja-Uceda et al. Solution structure and stability against digestion of rproBnIb, a recombinant 2S albumin from rapeseed: relationship to its allergenic properties
Machado et al. Conformational dynamics of a cysteine-stabilized plant defensin reveals an evolutionary mechanism to expose hydrophobic residues
Ahumada et al. Identification and physicochemical characterization of a new allergen from Ascaris lumbricoides
Mei et al. Reducing allergenicity to arginine kinase from mud crab using site-directed mutagenesis and peptide aptamers
Cabanos et al. Expression, purification, cross-reactivity and homology modeling of peanut profilin
Kaur et al. Rational design and computational evaluation of a multi-epitope vaccine for monkeypox virus: Insights into binding stability and immunological memory
Jiao et al. Purification and characterization of enolase as a novel allergen in Platanus acerifolia pollen
EP2664624A1 (fr) Variantes d'allergène
Palladino et al. Neuroglobin–prion protein interaction: what's the function?
Lee et al. Protein sequence analysis of a novel 103‐kDa Dermatophagoides pteronyssinus mite allergen and prevalence of serum immunoglobulin E reactivity to rDer p 11 in allergic adult patients
Soh et al. Two distinct conformations in Bet v 2 determine its proteolytic resistance to cathepsin S

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08754026

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08754026

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载