Patent Grant
9261511
This invention relates to protein engineering technology. More particularly, the present invention relates to human IgE antibodies and derivatives thereof, which bind non-continuous allergenic epitope, a planar surface with the area of 600-900 Å2, e.g. IgE antibodies binding to bovine milk β-lactoglobulin with high affinity and specificity. The present invention also relates to processes for making and engineering such allergen-binding monoclonal antibodies with Type I interaction and to methods for using these antibodies and derivatives thereof in the field of immunodiagnostics, enabling qualitative and quantitative determination and removal of allergenic substances in biological and raw material samples, as well as the construction of focused IgE libraries towards allergens, enabling the development of allergen-specific antibodies. In immunotherapy, the present invention enables blocking the Type I surface interaction of allergenic substances by modifying amino acid residues of allergens. Hypoallergen variant can be obtained by mutating some (1-5) amino acid residues on the planar (flat) epitope surface with bulky residues (such as Arg, Tyr, Lys, Trp). The mutated residues are those which side chains are pointing outside towards solvent thus causing minimal change to the basic structure of the allergen. The purpose of the mutagenesis is to modify the flat surface to convex surface which prevent the binding of IgE-antibodies. The resulting modified allergen can be used to evoke tolerance against particular allergens in allergic patients. The present invention allows the development of human IgE VH-region derived antibodies for those therapeutic and diagnostic targets where the binding specificity is towards areas of protein structures that are not located on the protruding regions of the surface. The invention also provides means for screening or molecular modelling of substances capable of blocking the binding of an antibody to the Type I allergenic epitope. In this invention, the development, characterisation and structure determination of the human IgE antibody fragment and derivatives thereof that binds allergenic β-lactoglobulin with affinity and specificity high enough to be utilised as reagents in immunoassays are also described.
Almost 20% of the population world-wide are suffering from allergy. Consequently, it is a health problem of increasing seriousness. Allergy is a hypersensitivity reaction against substances in air, food or water, which are normally harmless (Corry and Kheradmand, 1999). A new and foreign external agent triggers an allergic reaction, which aims at disposal of that agent from the body. In IgE-mediated allergic reactions, also called immediate or type I hypersensitivity reactions, under the first exposure of a foreign substance, allergen, to the body, IgE-bearing B-cells begin to produce soluble IgE molecules which will then bind to high-affinity IgE receptors present on the surface of a wide variety of cells, most importantly to mast cells and basophils. If the same foreign substance is encountered again, the cross-linking of the receptor-bound IgE molecules by the allergen occurs, resulting in cellular activation followed by the release of toxic products such as histamine, which will elicit the signs and symptoms of an allergic reaction.
Cow's milk allergy (CMA) is a most common cause of clinically important adverse food reactions with infants and children during the first 2 years of life (Savilahti, 1981; Host and Halken, 1990; Saarinen et al., 1999). It is characterized by a strong IgE response to milk proteins and clinical symptoms in skin and gastrointestinal tract such as atopic eczema, vomiting and diarrhoea (Vaarala et al., 1995; Saarinen, 2000). Symptoms in respiratory ducts and anaphylactic shock are also possible (Host and Halken, 1990; Schrander et al., 1993; Hill et al., 1999; Vanto et al., 1999; Saarinen, 2000). CMA is a serious problem with children, because milk is an important source of energy (up to 50%) for young children and is not very easily replaceable with non-dairy products. Nearly 85% of the milk allergic children will outgrow of their allergy by the age of 3, but remission of CMA may occur in up to one-third of older children (Sampson and Scanlon, 1989)
One of the major allergens in cow's milk is β-lactoglobulin, which belongs to the protein family known as lipocalins. Lipocalins consists a group of a small ligand binding proteins, mostly respiratory allergens such as Mus m1, Rat n1 (mouse and rat urinary proteins) and a German cockroach allergen Bla g4 (Rouvinen et al. 2001). β-lactoglobulin occurs naturally in the form of a 36 kD dimer with each subunit corresponding 162 amino acids. Totally six genetic variants of the β-lactoglobulin has been identified based on the sequence differences. The most prevalent variants A and B differ only at the position 64 (Asp→Gly) and 118 (Val→Ala) (Godovac-Zimmermann and Braunitzer, 1987). The 3D-structure of the β-lactoglobulin has been determined by X-ray diffraction (Sawyer L. et al, 1985, Brownlow, S. et al, 1997)
IgE antibodies distinctively recognise allergenic epitopes, which would be useful in clinics and immunodiagnostics for detecting and determining allergen concentrations of complex materials. Further, according to this invention, allergenic epitopes are usually different from the immunogenic epitopes of proteins. This fact has hampered the production of monoclonal antibodies capable of specific binding of allergenic epitopes by conventional methodology such as hybridoma technology. It has been recently shown that the development of allergen-specific IgE antibodies is possible by the phage display technology (Steinberger et al., 1996). This methodology is giving new tools to produce allergen-specific recombinant antibodies that can be produced in consistent quality for clinical and diagnostic applications.
The technical problem to which the present invention is related is the detection of actual binding sites of IgE antibodies in allergenic polypeptides and use of this information, e.g., to modify these polypeptides to decrease their allergenicity. Previous solutions for this problem are disclosed in U.S. Patent Application No. 2003/0175312 (Holm et al.), WO 03/096869 (Alk Abello A/S) and Jenkins et al. 2005 (J. Allergy Clin. Immunol. 115:163-170). In these documents, it is described that the putative IgE binding sites in allergenic polypeptides may be detected by sequence analysis of conserved surface structures of allergenic polypeptides. Further, in US 2005/0181446 (Roggen et al.) and Hantusch et al. 2004 (J. Allergy Clin. Immunol.) a peptide-scan approach is used to find IgE binding epitopes. However, none of these documents discloses the method of the present invention wherein an IgE binding site on an allergenic polypeptide is found based on the experimental 3D and molecular modelling data of a novel type of IgE epitope having essentially planar or flat nature. MacCallum et al. 1996 (J. Mol. Biol. 262:732-745) disclose the presence of planar surfaces on antibodies, but teach only modification of antibody structures not antigen structures. Further, the disclosure of MacCallum et al. is directed to antibodies and different kinds of antigens, such as carbohydrates and peptides, in general and does not teach anything particular on the binding between IgE antibodies and allergenic polypeptides or the surface structures of these polypeptides.
The present invention relates to human IgE antibodies and derivatives thereof, which bind to non-continuous allergenic epitope, a planar Type I surface with the area of 600-900 Å2, e.g. to IgE antibodies binding to bovine milk β-lactoglobulin with high affinity and specificity. The present invention also enables blocking the Type I surface interaction of allergenic substances by modifying amino acid residues of said surface structure or by producing a mimetope binding said surface.
We also describe in this application the development, characterisation and structure determination of the human IgE antibody fragment and derivatives thereof that binds allergenic β-lactoglobulin with affinity and specificity high enough to be utilised as reagents in immunoassays designed for the qualitative and quantitative measurement of β-lactoglobulin in biological samples, in removal of the β-lactoglobulin, in immunotherapy of allergic patients and in the construction of focused antibody libraries based on the structural data. Specifically, the present invention describes selection of human IgE antibodies specific to β-lactoglobulin by the phage display technique, the characterisation of the binding properties of the engineered antibody fragments produced in E. coli, and structure determination of the antibody-allergen immunocomplex.
This invention thus provides new reagents to be utilised in different kinds of immunoassay protocols, as well as in human immunotherapy and construction of focused antibody libraries. The invention also permits guaranteed continuous supply of these specific reagents of uniform quality, eliminating inherent batch-to-batch variation of polyclonal antisera. These advantageous effects permit the manufacture of new, specific and economical immunodiagnostic assays of uniform quality.
Consequently, one specific object of the present invention is to provide human IgE monoclonal antibodies, fragments thereof, or other derivatives of such antibodies, which bind β-lactoglobulin with affinity and specificity high enough to allow qualitative and quantitative measurement of β-lactoglobulin in biological samples, as well as their use in immunotherapy. The monovalent antibodies of the present invention demonstrate a specific binding to allergenic β-lactoglobulin.
Another object of the present invention is to provide cDNA clones encoding β-lactoglobulin-specific antibody chains, as well as constructs and methods for expression of such clones to produce β-lactoglobulin-binding antibodies, fragments thereof or other derivatives of such antibodies.
A further object of this invention is to provide methods of using such β-lactoglobulin-binding antibodies, fragments thereof or other derivatives of such antibodies, or combinations of them for qualitative and quantitative measurement of β-lactoglobulin in biological samples. Additionally, this invention provides β-lactoglobulin-binding antibodies, fragments thereof or other derivatives of such antibodies, or combinations of them for immunotherapy in allergic patients.
A further object of this invention is to provide methods of using structural data obtained for constructing focused IgE antibody libraries towards allergens for diagnostics and human IgE VH-region derived antibody libraries for therapeutic and diagnostic targets where the binding specificity is towards areas of protein structures that are not located on the protruding regions of the surface.
Other objects, features and advantages of the present invention will be become apparent from the following drawings and detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given for illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The figures of the constructions are not in scale.
cDNA complementary deoxyribonucleic acid
CDR complementarity determining region
DNA deoxyribonucleic acid
E. coli Escherichia coli
ELISA enzyme-linked immunosorbent assay
Fab fragment with specific antigen binding
Fd variable and first constant domain of a heavy chain
Fv variable regions of an antibody with specific antigen binding
IgE immunoglobulin E
mRNA messenger ribonucleic acid
NMR nuclear magnetic resonance
PCR polymerase chain reaction
RNA ribonucleic acid
scFv single-chain antibody
supE− a genotype of bacterial strain carrying a glutamine-inserting amber suppressor tRNA
VH variable region of a heavy chain
VL variable region of a light chain
The following definitions are provided for some terms used in this specification. The terms, “immunoglobulin”, “heavy chain”, “light chain” and “Fab” are used in the same way as in the European Patent Application No. 0125023.
“Antibody” in its various grammatical forms is used herein as a collective noun that refers to a population of immunoglobulin molecules and/or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site or a paratope.
An “antigen-binding site”, a “paratope”, is the structural portion of an antibody molecule that specifically binds an antigen.
Exemplary antibodies are those portions of an immunoglobulin molecule that contain the paratope, including those portions known as Fab and Fv.
“Fab” (fragment with specific antigen binding), a portion of antibodies can be prepared by the proteolytic reaction of papain on substantially intact antibodies by methods that are well known. See for example, U.S. Pat. No. 4,342,566. Fab fragments can also be produced by recombinant methods, which are well known to those skilled in the art. See, for example, U.S. Pat. No. 4,949,778.
“Domain” is used to describe an independently folding part of a protein. General structural definitions for domain borders in natural proteins are given in Argos, 1988.
A “variable domain” or “Fv” is used to describe those regions of the immunoglobulin molecule, which are responsible for antigen or hapten binding. Usually these consist of approximately the first 100 amino acids of the N-termini of the light and the heavy chain of the immunoglobulin molecule.
“Single-chain antibody” (scFv) is used to define a molecule in which the variable domains of the heavy and light chain of an antibody are joined together via a linker peptide to form a continuous amino acid chain synthesised from a single mRNA molecule (transcript).
“Linker” or “linker peptide” is used to describe an amino acid sequence that extends between adjacent domains in a natural or engineered protein.
A “β-lactoglobulin-binding antibody” is an antibody, which specifically recognises β-lactoglobulin and binds to it, due to interaction mediated by its variable domains. As used herein, the term “specifically binding” or “specifically recognizing” or the expression “having binding specificity to an allergenic epitope of β-lactoglobulin” refers to a low background and high affinity binding between an antibody or a fragment or derivative thereof and its target molecule (i.e. lack of non-specific binding). One of the embodiments of the present invention is a monoclonal antibody having binding specificity to an allergenic epitope of β-lactoglobulin (SEQ ID NO:8), or a functional fragment or derivative thereof having said specificity.
The term “planar (or flat) surface” refers to the surface structure as defined in Example 8.
As examples of fragments of such antibodies falling within the scope of the invention we disclose here scFv fragments of D1 IgE fab as shown in
For use in immunoassay, e.g. for qualitative or quantitative determination of β-lactoglobulin in biological samples, antibodies and antibody derivatives of the invention may be labelled. For these purposes, any type of label conventionally employed for antibody labelling is acceptable.
For use in immunotherapy, e.g. for blocking allergenic β-lactoglobulin in allergic patients, antibodies and antibody derivatives of the invention may be labelled. For these purposes, any pharmaceutically acceptable label conventionally employed for antibody labelling is appropriate (see, e.g., US 2007/0003579).
In another aspect, the present invention also provides DNA molecules encoding an antibody or antibody derivative of the invention, and fragments of such DNAs, which encode the CDRs of the VL and/or VH region. Such a DNA may be cloned in a vector, more particularly, for example, an expression vector which is capable of directing expression of antibody derivatives of the invention, or at least one antibody chain or a part of one antibody chain.
In a further aspect of the invention, host cells are provided, selected from bacterial cells, yeast cells, fungal cells, insect cells, plant cells and mammalian cells, containing a DNA molecule of the invention, including host cells capable of expressing an antibody or antibody derivative of the invention. Thus, antibody derivatives of the invention may be prepared by culturing host cells of the invention expressing the required antibody chain(s), and either directly recovering the desired protein or, if necessary, initially recovering and combining individual chains.
The above-indicated scFv fragments were obtained by biopanning of a human IgE scFv-phage library using allergenic recombinant β-lactoglobulin. The human IgE scFv-phage library was constructed from mRNAs isolated from lymphocytes of a milk-allergic patient. The variable region of the light and heavy chain cDNAs were synthesised using human IgE-specific primers for Fd cDNAs and human kappa (κ) and lambda (λ) light chains using human κ and λ chain specific primers. The variable regions of the light and heavy chains were amplified by PCR using human κ and λ chain specific primers for Vκ and Vλ cDNAs and human IgE specific primers for VH cDNAs, respectively. The human IgE scFv library was constructed by cloning the variable region cDNAs into a scFv phage display vector using restriction sites introduced into the PCR primers.
The human IgE scFv library was selected by phage display using a panning procedure. The human IgE scFv phage library was screened by a biotinylated allergenic native β-lactoglobulin in solution and the binders were captured on streptavidin. The elution of phages was done with 100 μM non biotinylated native β-lactoglobulin AB dimer. The phage eluate was amplified in E. coli cells. After 2 rounds of biopanning, soluble scFv fragments were produced from isolated phages. The binding specificity of the selected scFv fragments was analysed by ELISA. Several β-lactoglobulin-specific scFv fragment clones were obtained.
As described herein, the phage display technique is an efficient and feasible approach to develop human IgE recombinant anti-β-lactoglobulin antibodies for diagnostic and therapeutic applications.
While one successful selection strategy for obtaining antibody fragments of the invention has been described, numerous variations, by which antibody fragments of the invention may be obtained, will be apparent to those skilled in the art. It may prove possible to select scFv fragments of the invention directly from a phage or microbial display library of scFv fragment or its derivatives. A phage or microbial cell, which presents a scFv fragment or other antibody fragment of the invention as a fusion protein with a surface protein, represents a still further aspect of the invention.
While microbial expression of antibodies and antibody derivatives of the invention offers means for efficient and economical production of highly specific reagents of uniform quality suitable for use in immunodiagnostic assays and immunotherapy, alternatively it may prove possible to produce such a reagent, or at least a portion thereof, synthetically. By applying conventional genetic engineering techniques, initially obtained antibody fragments of the invention may be altered, e.g. new sequences linked, without substantially altering the binding characteristics. Such techniques may be employed to produce novel β-lactoglobulin-binding hybrid proteins, which retain both affinity and specificity for β-lactoglobulin as defined hereinbefore.
Planar Surface of Allergens and Production and Use of Hypoallergens
The present invention enables blocking the Type I surface interaction of allergenic substances by modifying amino acid residues on non-continuous allergenic epitope, i.e. a planar surface with the area of 600-900 Å2 on the allergenic substance (see
Hypoallergen variant can be obtained by mutating some (1-5) amino acid residues on the planar (flat) epitope surface with bulky residues (e.g. Arg, Tyr, Lys, Trp can be mutated to Ala). The mutated residues are those which side chains are pointing outside towards solvent thus causing minimal change to the basic structure of the allergen. The purpose of the mutagenesis is to modify the flat surface to convex surface which prevent the binding of IgE-antibodies. The effect of the mutation on the planar surface can be seen as lower affinity of the allergen specific IgE-antibody towards the modified allergen, preferably the mutation decreases the affinity of the specific antibody at least tenfold, more preferably more than tenfold. The resulting modified allergen can be used to evoke tolerance against particular allergens in allergic patients. Thus, the present invention provides a modified allergen carrying the type I planar epitope which has been distorted by the directed introduction of one or several mutations thereby decreasing the affinity towards the recombinant IgE molecule at least tenfold, preferably more than tenfold.
The present invention also provides a method to create tolerance in a patient for a specific allergen with a planar allergenic epitope comprising the steps of
The present invention also provides a method for the isolation of recombinant IgE monoclonal antibodies comprising the steps of
The present invention further provides a method for producing a modified allergenic polypeptide, the method comprising the steps of (a) modifying nucleic acid sequence encoding said polypeptide so that in the polypeptide expressed from the modified nucleic acid the structure of allergenic epitope of said polypeptide is altered, and (b) expressing or producing the modified allergenic polypeptide from the modified nucleic acid. Preferably step (b) comprises the steps of expressing said modified nucleic acid in a suitable host in a culture system and isolating said modified polypeptide from the culture, or producing synthetically of said modified polypeptide. Preferably said modified allergenic polypeptide is β-lactoglobulin, and/or said allergenic epitope is the planar surface as defined above, more preferably planar surface is defined by the structure or 3D-coordinates of β-lactoglobulin amino acids Val43-Lys47 and Leu57-Gln59 and/or amino acids around these amino acids, in an antibody-β-lactoglobulin immunocomplex. Said allergenic epitope can also be the epitope defined by structure coordinates of β-lactoglobulin amino acids Trp19 and Tyr20 from beta-strand A and Glu44 from beta-strand B in an antibody-β-lactoglobulin immunocomplex.
The present invention further provides a method for identifying a molecule binding to an allergenic epitope of an allergen; comprising the steps of: (a) contacting a particle, such as a virus particle, comprising the allergenic epitope and a candidate binder molecule; (b) isolating those candidate binder molecules which were able to bind to said allergenic epitope. Preferably said allergen is β-lactoglobulin, said molecule is a peptide, and said allergenic epitope is the planar surface as defined above; more preferably planar surface is defined by the structure or 3D-coordinates of β-lactoglobulin amino acids Val43-Lys47, and Leu57-Gln59 and/or amino acids around these amino acids in an antibody-β-lactoglobulin immunocomplex. A good approach in this method is the use of affinity chromatography.
Crystallographic and in Silico Screening
The three-dimensional structure of the allergenic epitope of β-lactoglobulin is defined by a set of structure coordinates as set forth below. The term “structure coordinates” refers to Cartesian coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of the allergenic epitope of β-lactoglobulin in crystal form of an antibody-allergen immunocomplex. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are then used to establish the positions of the individual atoms of the allergenic epitope of β-lactoglobulin.
Those of skill in the art will understand that a set of structure coordinates for a protein or a protein-complex or a portion thereof, is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates will have little effect on overall shape.
The variations in coordinates discussed above may be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth below could be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
Alternatively, modifications in the crystal structure due to mutations, additions, substitutions, and/or deletions of amino acids, or other changes in any of the components that make up the crystal could also account for variations in structure coordinates. If such variations are within an acceptable standard error as compared to the original coordinates, the resulting three-dimensional shape is considered to be the same.
Various computational analyses are therefore necessary to determine whether a molecule or molecular complex or a portion thereof is sufficiently similar to all or parts of the allergenic epitope of β-lactoglobulin described herein as to be considered the same. Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., San Diego, Calif.) version 4.1, and as described in the accompanying User's Guide.
Once the structure coordinates of a protein crystal have been determined they are useful in solving the structures of other crystals, especially crystals of other similar proteins.
Thus, in accordance with the present invention, the structure coordinates of the allergenic epitope of β-lactoglobulin, and portions thereof is stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery and x-ray crystallographic analysis or protein crystal.
Accordingly, in one embodiment of this invention is provided a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth below.
For the first time, the present invention permits the use of structure-based or rational drug design techniques to design, select, and synthesize chemical entities, including inhibitory compounds that are capable of binding to the allergenic epitope of β-lactoglobulin, or any portion thereof.
Those of skill in the art will realize that association of natural ligands or substrates with the binding pockets of their corresponding receptors or enzymes is the basis of many biological mechanisms of action. The term “binding site”, as used herein, refers to a region of a molecule or molecular complex that, as a result of its shape, favourably associates with another chemical entity or compound. Similarly, many drugs exert their biological effects through association with the binding pockets of receptors and enzymes. Such associations may occur with all or any parts of the binding pockets. An understanding of such associations will help lead to the design of molecules such as drugs having more favourable associations with their target receptor or enzyme, and thus, improved biological effects. Therefore, this information is valuable in designing potential ligands or inhibitors of receptors or enzymes.
The term “associating with” or “interacting with” refers to a condition of proximity between chemical entities or compounds, or portions thereof. The association or interaction may be non-covalent, wherein the juxtaposition is energetically favoured by hydrogen bonding or van der Waals or electrostatic interactions, or it may be covalent.
In iterative molecular design, crystals of a series of protein/compound complexes are obtained and then the three-dimensional structures of each complex are solved. Such an approach provides insight into the association between the proteins and compounds of each complex. This is accomplished by selecting compounds with inhibitory activity, obtaining crystals of this new protein/compound complex, solving the three-dimensional structure of the complex, and comparing the associations between the new protein/compound complex and previously solved protein/compound complexes. By observing how changes in the compound affected the protein/compound associations, these associations may be optimized.
In some cases, iterative molecular design is carried out by forming successive protein-compound complexes and then crystallizing each new complex. Alternatively, a pre-formed protein crystal is soaked in the presence of an inhibitor, thereby forming a protein/compound complex and obviating the need to crystallize each individual protein/compound complex. Advantageously, the allergenic epitope of β-lactoglobulin crystals, may be soaked in the presence of a compound or compounds, such as antibodies, to provide β-lactoglobulin/antibody crystal complexes.
As used herein, the term “soaked” refers to a process in which the crystal is transferred to a solution containing the compound of interest.
The Storage Medium
The storage medium in which the atomic co-ordinates are provided is preferably random access memory (RAM), but may also be read-only memory (ROM e.g. CDROM), or a diskette. The storage medium may be local to the computer, or may be remote (e.g. a networked storage medium, including the internet).
The invention also provides a computer-readable medium for a computer, characterised in that the medium contains atomic co-ordinates of the allergenic epitope of β-lactoglobulin.
The atomic co-ordinates are preferably those set forth below, or variants thereof.
Any suitable computer can be used in the present invention.
Molecular Modelling Techniques
Molecular modelling techniques can be applied to the atomic co-ordinates of the allergenic epitope of β-lactoglobulin to derive a range of 3D models and to investigate the structure of ligand binding sites. A variety of molecular modelling methods are available to the skilled person for use according to the invention.
At the simplest level, visual inspection of a computer model of the allergenic epitope of β-lactoglobulin can be used, in association with manual docking of models of functional groups into its binding sites.
Software for implementing molecular modelling techniques may also be used. These molecular modelling techniques allow the construction of structural models that can be used for in silico drug design and modelling.
De Novo Compound Design
The molecular modelling steps used in the methods of the invention may use the atomic co-ordinates of the allergenic epitope of β-lactoglobulin, and models derived therefrom, to determine binding surfaces.
This preferably reveals van der Waals contacts, electrostatic interactions, and/or hydrogen bonding opportunities.
These binding surfaces will typically be used by grid-based techniques (e.g. GRID [Goodford (1985) J. Med. Chem. 28: 849-857], CERIUS2) and/or multiple copy simultaneous search (MCSS) techniques to map favourable interaction positions for functional groups. This preferably reveals positions in the allergenic epitope of β-lactoglobulin for interactions such as, but not limited to, those with protons, hydroxyl groups, amine groups, hydrophobic groups (e.g. methyl, ethyl, benzyl) and/or divalent cations.
Once functional groups or small molecule fragments which can interact with specific sites in the binding surface of the allergenic epitope of β-lactoglobulin have been identified, they can be linked in a single compound using either bridging fragments with the correct size and geometry or frameworks which can support the functional groups at favourable orientations, thereby providing a compound according to the invention. Whilst linking of functional groups in this way can be done manually, perhaps with the help of software such as QUANTA or SYBYL, the following software may be used for assistance: HOOK [Available from Molecular Simulations Inc], which links multiple functional groups with molecular templates taken from a database, and/or CAVEAT [Lauri & Bartlett (1994) Comp. Aided Mol. Design. 8: 51-66], which designs linking units to constrain acyclic molecules.
Docking
Compounds in known in silico libraries can also be screened for their ability to interact with the allergenic epitope of β-lactoglobulin by using their respective atomic co-ordinates in automated docking algorithms.
Suitable docking algorithms include: DOCK [Kuntz et al. (1982) J. Mol. Biol. 161: 269-288], AUTODOCK [Available from Oxford Molecular], MOE-DOCK [Available from Chemical Computing Group Inc.] or FLEXX [Available from Tripos Inc.]. Docking algorithms can also be used to verify interactions with ligands designed de novo.
Focused IgE-Antibody Library Towards Allergens
The amino acid sequence comparison of published IgE sequences reveals that the light chains of the known IgE antibodies binding to diverse groups of allergens are strikingly conserved (see Table VII). This gives tools to construct focused allergen specific libraries that can be utilised for the isolation of allergen specific antibodies applicable in the diagnosis of allergens. The conserved light chain sequence information is used to construct a limited pool of light chains or a single light chain with the characteristic amino acid sequences identified in the IgE antibodies. This light chain sequence information is combined with a diverse pool of IgE heavy chain genes isolated from lymphocytes of several allergic patients. The resulting antibody phage display library, in either scFv or Fab display format, is used to select allergen specific IgE antibodies essentially as described in Example 1/II and Hoogenboom et al. (1998).
Human Antibody (scFv, Fab or Whole Antibody) Libraries Containing the Human IgE VH-Regions
The IgE VH-region of the D1 IgE Fab and especially the HCDR3 loop are structurally different when compared to IgG antibodies. It is forming a loop structure that is recognizing a cleft on the BLG-allergen structure. Based on this observation it should be possible to develop human IgE VH-region containing antibodies for those therapeutic targets where the binding specificity is required towards protein structures that are not exposed on the surface, e.g., substrate binding sites of enzymes and drug resistance pumps (De Genst et al. 2006). A diverse IgE VH-pool from human lymphocytes is used as a building block to construct a functional human antibody library in a scFv, Fab or whole antibody format. Resulting libraries are selected against therapeutic targets requiring specific recognition of cleft structures.
The development and characterisation of the human β-lactoglobulin-binding recombinant antibodies and their usefulness in immunoassays is now described in more detail in the following examples.
In this example the human IgE scFv library was constructed and selected by allergenic β-lactoglobulin in order to isolate scFv fragments with affinity and specificity to β-lactoglobulin (BLG). Construction of human IgE scFv phage library was prepared indirectly by constructing IgE Fab-κ and Fab-λ, libraries first, and then the particular library DNAs were used for PCR amplification of variable domains of heavy and light chains.
I. Construction of the Human IgE scFv Phage Libraries
50 ml of heparinised blood was obtained from a milk-allergic patient. Lymphocytes were isolated according to an Ig-Prime kit protocol (Novagen). Per 10 ml of blood 30 ml of lysis buffer (155 mM NH4Cl, 10 mM NH4HCO3, 0.1 mM EDTA, pH 7.4) was added and incubated on ice for 15 min with shaking occasionally. After centrifugation at 450 g for 10 min the lymphocytes, i.e. the white blood cell pellet, were collected. The pellet was washed twice with lysis buffer and after the final centrifugation the lymphocyte pellet was resuspended in D-solution. Lymphocyte RNAs were isolated using Promega's RNAgents Total RNA Isolation kit according to the manufacturer's protocol. The first strand cDNA synthesis was carried out using Promega's Reverse Transcription system kit. For the synthesis of Fd-fragment cDNA and light chain cDNAs the primers of the constant region of the epsilon (ε) chain (Cε1) and the primer of the kappa (Cκ1) and lambda (Cλ1) chain were used, respectively. Primers used for the cDNA synthesis and PCR amplifications of human IgE Fd region and light chains are showed in Table I and Table II.
PCR amplifications were carried out in two steps: a primary PCR for amplifying Fd and light chains from cDNA templates and a secondary PCR for adding restriction sites to the 5′-end of the DNA fragments obtained after a primary PCR. First the Fd region was amplified by PCR using the primers specific for the variable region of the heavy chains (VH1a-VH7a) and Cε1primer. Accordingly, the kappa and lambda light chains were amplified using specific primers for variable region of the light chains (Vκ1a-Vκ6b and Vλ1a-Vλ10) and Cκ/λ1 primer, respectively. Primers for the secondary PCR were Cκ1 and Vκ/λ1 and Cκ for the kappa light region, Vκ/λ1 and Cλ1 for the kappa light chain and Vλ1A and Cκ/λ1 for the lambda light chain. The primary PCR amplification was done at the following conditions: 1 cycle of 3 min at 93° C. for denaturation, 7 cycles of 1 min at 93° C., 30 s at 63° C. and 50 s at 58° C. for annealing and 1 min at 72° C. for elongation, 23 cycles of 1 min at 93° C., 30 s at 63° C. and 1 min at 72° C. followed by 1 cycle of 10 min at 72° C. For the secondary PCR the amplification conditions were as follows: 1 cycle of 3 min at 95° C. for denaturation, 25 cycles of 1.5 min at 94° C., 1 min at 65° C. for annealing and 1.5 min at 72° C. for elongation followed by 1 cycle of 10 min at 72° C. Between the primary and the secondary PCR and after the secondary PCR the amplified DNA fragments were purified.
The final PCR products of the different antibody fragments were pooled and digested with appropriate restriction enzymes. Digested DNA fragments, encoding IgE Fd region and κ and λ light chains, were ligated into a phagemid vector and transformed into E. coli XL-1 Blue cells to yield an Fab-κ and Fab-λ libraries of 106 independent clones. To avoid possible problems on the expression of Fab fragments on a phage particle an antibody library in scFv format was constructed. Phagemid DNAs from different libraries were isolated and used as template DNAs for amplifying the variable regions of the human IgE heavy and human light chains in order to construct human IgE scFv-κ and scFv-λ libraries.
PCR amplification of the variable region of the heavy chain was carried out using human VH specific primers (VH1-VH4 and VH1A). Amplification of the variable region of the light chains was done using the following primer pairs: Vκ1-Vκ7, Vκ2-Vκ8, Vκ3-Vκ9, Vκ-4-Vκ10, Vκ5-Vκ11 and Vκ6-Vκ11 for human kappa chain and Vλ1-Vλ8, Vλ2-Vλ9, Vλ3-Vλ9, Vλ4-Vλ9, Vλ5-Vλ10, Vλ6-Vλ10 and Vλ7-Vλ10 for human lambda chain (see Tables III and IV). The amplified DNA fragments were purified and digested in order to ligate into a scFv phage display vector (
II. Selection of the Human scFv-Libraries
The human scFv-κ and scFv-λ, libraries were selected by the phage display technique (McCafferty et al., 1990, Barbas et al., 1991). To isolate β-lactoglobulin-binding antibody fragments, the human IgE scFv-κ and scFv-λ, libraries displayed on the surface of the bacteriophage were panned using an affinity panning procedure (
III. Characterisation of the β-Lactoglobulin-Binders
After the last panning cycle scFv phage display DNA was isolated and transformed into E. coli HB2151 (supE−) cells in order to express soluble scFv fragments. Between the scFv sequence and the phage gene III sequence the scFv phage display vector contains TAG-amber stop codon which will be translated as glutamate in E. coli strains with supE+ genotype but as a stop codon in E. coli strains with supE− genotype. Sixty-two individual clones were grown in a small scale to produce soluble scFv fragments for preliminary characterisation. Clones were analysed on ELISA test using β-lactoglobulin-coated wells to catch the β-lactoglobulin-specific binders and control protein wells to see non-specific binding (data not shown). Most of the clones bound with high affinity to β-lactoglobulin. Clones were analyzed first by DNA-fingerprinting and six of the clones were sequenced (Sanger et al., 1977). Finally, one of the clones was selected for further characterisation (
In this example the human IgE scFvs with β-lactoglobulin-binding specificity was converted to human Fab fragments with IgG1 subtype. Due to known difficulties in forming multimers, the D1 scFv, obtained from the scFv antibody library, was cloned and bacterially expressed as Fab fragments (Holliger et al., 1993, Desplancq et al., 1994). The resulting antibody fragments were further characterised by a competitive ELISA.
I. Cloning of the Human Fab Fragments with β-Lactoglobulin-Binding Specificity
The Fd regions were amplified by overlapping PCR. The primers used for the PCR are given in Table V.
The resulting cDNAs of the Fd region and light chains were cloned into the bacterial expression vector, pKKtac and then transformed into E. coli RV308. Soluble Fab fragment designated to D1 IgE Fab was produced by fermentation (Nevanen et al, 2001) and the Fab fragment was purified by an introduced C-terminal hexahistidinyl tag on a Sepharose column with immobilised nickel to a substantial purity (data not shown).
II. Characterisation of the Human IgE Fab Fragments
The characterisation of the purified D1 IgE Fab was performed by competitive ELISA. First, increasing amounts of the soluble, non-biotinylated β-lactoglobulin was incubated with the D1 IgE Fab, and then the reaction mixtures were applied onto Streptavidin microtitre plate wells coated with allergenic, biotinylated β-lactoglobulin.
To study if the D1 IgE Fab is able to bind β-lactoglobulin from milk samples, the immunoprecipitation assay was performed (
To study if the D1 IgE Fab recognizes the same allergenic epitope as the IgE antibodies from patient serum, the biotinylated β-lactoglobulin was first immobilised to a microtitre plate wells coated with streptavidin. The patient serum samples were incubated in the wells together with increasing concentrations of D1 IgE Fab and the amount of the bound patient serum IgE was detected with alkaline phosphatase labelled secondary antibody, which specifically recognizes the human IgE isotype. A slight inhibition can be seen in the case of each patient tested, suggesting that the epitope which D1 IgE Fab recognizes is the same as the IgE from the patient serum. The reason why the binding of the patient serum IgE is not totally blocked, might be that the β-lactoglobulin harbours a multiple IgE-epitopes.
Crystallization and data collection Microcrystals (about 70×50×50 μm) of BLG-D1 IgE Fab were obtained with vapour diffusion method by mixing 2 μl of D1/Fab solution (concentration 1.4 mg/ml in 20 mM phosphate buffer, pH 7.0), 1 μl BLG solution (2 mg/ml in pure water), 0.5 μl of n-dodecyl-β-D-maltoside solution, and 2.5 μl of reservoir solution (14% (w/v) polyethylene glycol 3350, 0.1 M BTP (1,3-bis[tris(hydroxymethyl)methylamino]propane-hydrochloric acid) buffer, pH 5.5). The diffraction data set was collected from single crystal at the beamline ID29 in ESRF (wavelength 1.000 Å) at 100 K. The crystal belonged to the space group P212121 with unit-cell dimensions a=67.0, b=100.6, c=168.1 Å. The data set was collected at 2.8 Å resolution.
The structure was solved with the molecular replacement method using Molrep program implemented in CCP4 program package. BLG monomer (PDB code 1B8E) and Fab fragment of IgG antibody against GP41 of HW virus (1DFB) (identity 92% for light and 79% for heavy chain) were used as search models. The final structure contained one dimer of BLG complexed with two Fab fragments. Model building and refinement were done with the programs O and CNS. Because of low number of reflections restraints were used to keep both Fab/D1 fragments and BLG monomers similar. BLG exist in two isomers, the electron density suggested that we have glycine at position 64 and alanine at position 118. No water molecules were added but the elongated electron density in the lipid binding cavity of BLG was modelled as an n-dodecyl-β-D-maltoside. The final structure has an R value 24.5% and an Rfree value of 29.9%. 83.5% of the residues are in the most favoured regions and 0.6% of the residues in the disallowed regions in the Ramachandran plot. All figures were generated with Pymol (Delano, W. L. The PyMol Molecular Graphics System, http://www.pymol.org).
Inhibition of the D1 IgE Fab binding to BLG was carried out using a short peptide, KRVG that is the longest linear BLG binding peptide in the HCDR3. In competitive ELISA the biotinylated AB dimer of BLG was immobilised onto streptavidin-coated microtitre wells. First the peptide (an inhibitor) was dissolved into 0.5% BSA-PBS and then different amounts of it were incubated with the immobilised BLG. After that the wells were either washed trice with PBS or not. D1 IgE Fab was added and followed by the washings with PBS. The bound antibody was detected with AFOS-conjugated goat anti-human kappa antibody. After addition of the substrate, p-nitrophenylphosphatase, the absorbance values were read at 405 nm. The results are shown in
In this example we have used commercial AMIRA program (with AmiraMol module) to calculate the solvent excluded surface (probe radius 10 Å). The surfaces are coloured according to the Gaussian curvature which is the product of the two principal curvatures. It is negative in surface areas with hyperbolic geometry (convex-concave, like near saddle points) and positive in areas with elliptic geometry (strictly convex or strictly concave).
We have also used AMIRA program to calculate molecular interface area (cutoff 3 Å). The program shows a surface which is located exactly in the middle between two proteins. In
It is thus possible to calculate the molecular surface by using a large probe value (preferably 8-12 Å) for allergens if their three-dimensional structure is available. Such molecular surfaces can be rotated and looked in all directions and with the aid of curvature coloured surfaces a large flat area (600-900 Å2) can be identified. In
Based on the D1 IgE and the BLG immunocomplex structure, mutations were designed to the flat surface epitope on the BLG in order to produce hypoallergenic variants. Two different recombinant BLG (rBLG) mutants, T18Y and T18Y/E45Y/L57Y, were constructed (Table IX). The cDNAs encoding the rBLG and its mutants were cloned into bacterial expression vector, produced in Escherichia coli cells, chromatographically purified to a substantial purity and finally their properties were characterised.
I. Cloning of the Recombinant BLGs
The bovine recombinant BLG (rBLG) cDNA was purchased from GenScript Corporation (USA) in vector pUC57 and it contained the restriction sites of SfiI/NcoI at the 5′end and HindIII at the 3′end (Table X). The rBLG cDNA was cloned into pKKTac bacterial expression vector with the fusion of the Ervinia carotovora's pectate lyase (pelB) signal sequence (Takkinen et al., 1991) as an SfiI-HindIII fragment. The hexa histidinyl (His6) tag was introduced into 3′end of the rBLG cDNA by PCR amplification using primers 1 and 2 (Table X). Phusion DNA polymerase (Finnzymes) was used in all PCR amplifications. The amplified cDNA of rBLG-His6 was digested with SfiI and HindIII (New England Biolabs) and cloned into pKKTac expression vector. Escherichia coli XL-1 Blue was used as a host strain to construct the recombinant BLG (rBLG) and its mutants.
Two different rBLG mutants, T18Y and T18Y/E45Y/L57Y (Table IX), were cloned into pKKtac vector. The cDNAs of the rBLG-His6 T18Y and T18Y/E45Y/L57Y mutant were amplified with PCR using mismatch primers 2, 3, 4 and 5 (Table X) and the original rBLG cDNA in pUC57 vector as a template. The cDNA encoding the T18Y mutant was amplified using primers 2 and 3 and the amplified cDNA was digested with StuI and HindIII (New England Biolabs) and cloned into the pKKtac/rBLG-His6 vector (see above). The cDNA encoding the T18Y/E45Y/L57Y mutant was amplified in two steps using overlapping primers. First, the cDNA fragment of 27-165 bp was amplified using primers 3 and 4 and the cDNA fragment of 147-530 bp with the 2 and 5. Then the resulting DNA fragments were combined by overlapping PCR amplification. The primer 4 and 5 have an overlapping sequence. Finally the cDNA encoding the T18Y/E45Y/L57Y mutant was digested with StuI and HindIII and cloned into pKKTac/rBLGhis expression vector.
The DNA sequences of the rBLG-His6 and its mutants were verified by DNA sequencing (ABI 3100 Genetic Analyzer, Applied Biosystems).
II. Production of the Recombinant BLGs
The rBLG-His6 and its mutants were transformed into E. coli RV308 (ATCC 31608) strain for the bacterial expression of the rBLGs. Single colonies of each clone were inoculated into 3 ml LB, 100 μg/ml ampicillin and 1% glucose and cultivated for 16 h at +37° C. with 220 rpm shaking. Then the cultivations were 1:50 diluted into 3 ml LB with ampicillin and cultivated 3 hours at +37° C. After that the protein expression was induced by the addition of IPTG to a final concentration of 1 mM and cells were cultivated for 16 h at +30° C. with 220 rpm shaking. Then the cells were harvested and the supernatants were stored for later use. The periplasmic fraction of the cells was isolated by a freeze-thaw method (Boer et al., 2007). Briefly, cells were resuspended in 20% sucrose, 30 mM Tris, 1 mM EDTA (pH 8.0) and then incubated 5 min in dry ice-ethanol bath followed by the resuspension in 5 mM MgSO4 and incubation for 5 min at +37° C., and this freezing and thawing step was repeated trice. The supernatant and the periplasmic fractions were analysed by western blotting. First the samples were run on a 15% SDS-PAGE gel (with β-mercaptoethanol) and then the proteins were transferred onto the nitrocellulose filter. The rBLGs were detected using rabbit anti-BLG antibody (Mäkinen-Kiljunen and Palosuo, 1992) followed by AFOS-conjugated goat anti-rabbit antibody (Bio-Rad).
During the bacterial production the recombinant BLGs were secreted into the periplasmic space with almost no leakage into culture medium. For the large scale production of the rBLGs the cells containing the rBLG-His6 and its mutants in pKKTac vector in E. coli RV308 strain were inoculated TB medium containing 100 μg/ml ampicillin, 1% glucose. The cells were cultivated for 16 h at +37° C. with 220 rpm shaking. Then the cell cultures were 1:50 diluted into TB medium with 100 μg/ml ampicillin. The cells were grown at +37° C. with 220 rpm shaking until the OD600 was 4 and IPTG was added to a final concentration of 0.1 mM. The induction of the cells was carried out for 6 h at +28° C. with 220 rpm shaking. Then the cells were harvested by centrifugation with 4000×g for 15 min at +4° C. The periplasmic fractions containing the recombinant BLGs were isolated by freeze-thaw method as above.
III. Purification of the rBLGs
The purification of recombinant BLGs was performed using immobilised metal affinity chromatography (IMAC) as described earlier (Porath and Olin, 1983). Briefly, periplasmic fractions containing the rBLGs were 1:2 diluted with the binding buffer (10 mM Hepes, 1M NaCl, 10% Glyserol, 1 mM imidazole, pH 7.4) and incubated with Ni2+-loaded Chelating Sepharose (Pharmacia) for 16 h at +4° C. The column matrix with bound rBLGs was loaded into the column with gravity flow and washed stepwise with 1 mM, 10 mM, 20 mM and 50 mM imidazole in the binding buffer. Finally, the rBLGs were eluted with 75 mM, 100 mM, 200 mM and 5×500 mM imidazole in the binding buffer and 2 ml fractions were collected. The eluted fractions were analysed on 15% SDS-PAGE gel (with β-mercaptoethanol). The fractions containing the desired proteins were pooled and the IMAC-purification was repeated in a smaller scale. After the second IMAC-purification the fractions were analysed again on a SDS-PAGE gel (
IV. Circular Dichroism Measurements
For circular dichroism (CD) measurements the buffer of all rBLGs was exchanged into 5 mM Hepes (pH 7.4) using Econo Pac 10DG desalting columns (Bio-Rad) with the cut of 6000 Da. Far-UV spectrum of the native BLG (nBLG, Sigma), rBLG-His6 and the rBLG-His 6 mutants was measured with Jasco J-715 spectropolarimeter at +20° C. controlled with a Peltier thermostat (Jasco PTC-348WI) using a 1-mm quartz cell. The concentrations of the proteins were 1 mg/ml for nBLG, 0.25 mg/ml for rBLG-His6, 1.3 mg/ml for rBLG-His6 T18Y and 0.93 mg/ml for rBLG-His6 T18Y/E45Y/L57Y mutant. The CD-spectra shown are averages of three measurements (
V. Characterisation of the D1 IgE Fab Binding to rBLGs by ELISA
First the rBLGs were biotinylated. The biotinylation of the rBLGs was performed with Sulfo-NHS-LC-biotin (Pierce) in a molar ratio of 2 mol biotin:1 mol protein in 10 mM Hepes, 0.9% NaCl for 30 min at RT with a gentle shaking. The unreacted biotin was removed using Econo Pac 10 DG desalting columns (Bio-Rad). The incorporation of the biotin to the rBLGs was analysed by western blotting using SA-AFOS detection.
Then 1 μg biotinylated nBLG, rBLG-His6, rBLG-His6 T18Y mutants in 110 μl 0.5% BSA/PBS were immobilised onto the streptavidin microtitre wells (Roche) for 1 h at RT. After that 100 μl 1:15000 diluted anti-BLG D1 Fab (1.6 mg/ml) in 0.5% BSA, PBS was added to the washed wells. After a 1-h incubation the wells were washed three times with PBS. The detection of the BLGs was carried out using AFOS-conjugated goat anti-human kappa antibody (Southern Biotech). Then p-nitrophenylphosphate substrate (Sigma) was added to the wells (2 mg/ml in diethanolamine buffer). The absorbance at the wavelength 405 nm was measured after 20 minutes of adding the substrate (
The ELISA analysis with the serum samples (1:8 dilution in 0.5% BSA, PBS) was performed as above except the bound IgE from allergic patient serum was detected with AFOS-conjugated goat anti-human IgE (Southern Biotech). The absorbances were measured at 405 nm after a 2-h incubation of adding the substrate (
VI. Analysis of the Binding Kinetics
The association and dissociation constants of the D1 IgE Fab to nBLG, rBLG-His6 and its mutants were measured by BIAcore. The biotinylated BLGs were immobilised in HBS buffer (10 mM Hepes, 0.15M NaCl, 3.4 mM EDTA, 0.005% BIAcore P20 surfactant, pH 7.4) and at a concentration of 1 μg/ml onto the streptavidin biosensor chip resulting in a surface of approximately 400-500 RU. The biotinylated nBLG was immobilised only with 200 RU onto the surface of the SA-chip. The binding kinetics of the purified D1 IgE Fab was analysed at a flow rate of 30 μl/min with the concentrations 138.9 nM, 69.6 nM, 34.8 nM, 17.4 nM, 8.7 nM, 4.3 nM, 2.2 and 1.1 nM. Regeneration of the BLG surface was performed with 100 μM nBLG (Sigma). Binding curves of the 69.6 nM D1 IgE Fab solution are shown in
TABLE I: Primers used for eDNA synthesis and PCR amplification of the human IgE Fd region.
TABLE II: Primers used for cDNA synthesis and PCR amplification of human kappa and lambda chains.
TABLE III: Primers used for PCR amplification of the human variable regions of the heavy chain.
TABLE IV: Primers used for PCR amplification of the human variable regions of the light chains.
TABLE V: Primers used for PCR amplification of the human Fd regions with IgE and IgG1 subtype.
QQSYSTP--RT
QQRSNWP-PLT
SQSIGN------YLNWY
LLIYAASSLQS
QQSNRTP--ITF
SQTFNN------YLNWY
LLIYAASTLRR
QQSYSTP--LTF
SRTIYN------YLNWY
LLIHAASTLQD
QQSHGTP--LTF
SQSISS------YLNWY
LLIYAASSLQS
QQSHSTP--YTF
SHSISN------YLNWY
LLIYAASSLQS
SQSILG------YLNWY
LLIYAASTLQS
QQSYITP--RTF
SQGISS------WLAWY
LLIYSASSLQS
QQANSFP--YTF
SQSVSS-----SYLAWY
LLIYGASSRAT
QQYGSSP--LTF
SQSISS------YLNWY
LLIYAASSLQS
QQSYSTP--RTF
SQGISS------RLAWY
LLIYAASSLQS
QQYHSYP--WTF
GCC ATG GCC CTG ATT GTG
AAT TTC CAG ATC GCC TTC CGG GGT CGG TTT CAG
GGC GAT CTG GAA ATT
CTG CAG AAA TGG G
| Number | Date | Country | Kind |
|---|---|---|---|
| 20075059 | Jan 2007 | FI | national |
This application is the National Phase of PCT/FI2008/050026 filed on Jan. 29, 2008, which claims priority under 35 U.S.C. 119(e) to U.S. Provisional Application No. 60/887,862 filed on Feb. 2, 2007, and under 35 U.S.C. 119(a) to Patent Application No. 20075059 filed in Finland on Jan. 29, 2007, all of which are hereby expressly incorporated by reference into the present application.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FI2008/050026 | 1/29/2008 | WO | 00 | 10/27/2009 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2008/092992 | 8/7/2008 | WO | A |
| Number | Name | Date | Kind |
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| 20030175312 | Holm et al. | Sep 2003 | A1 |
| 20030207336 | Jardieu et al. | Nov 2003 | A1 |
| 20050181446 | Roggen et al. | Aug 2005 | A1 |
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| 1 512 695 | Mar 2005 | EP |
| 8-70866 | Mar 1996 | JP |
| WO-03054216 | Jul 2003 | WO |
| WO-03096869 | Nov 2003 | WO |
| WO-2006125201 | Nov 2006 | WO |
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| Number | Date | Country | |
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| 20100086552 A1 | Apr 2010 | US |
| Number | Date | Country | |
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| 60887862 | Feb 2007 | US |
