AU2002308085A1 - Hevein-binding monoclonal antibodies - Google Patents

Description

HEVEIN-BINDING MONOCLONAL ANTIBODIES

Field of the Invention

This invention relates to antibody engineering technology. More particularly, the present invention relates to human IgE antibodies and derivatives thereof, which bind allergenic hevein with high affinity and specificity. The present invention also relates to processes for making and engineering such hevein-binding monoclonal antibodies and to methods for using these antibodies and derivatives thereof in the field of immunodiagnostics, enabling qualitative and quantitative determination of allergenic hevein in biological and raw material samples, as well as in immunotherapy, enabling blocking of allergenic hevein in allergic patients.

Background of the Invention

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. 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 histamines, which will elicit the signs and symptoms of an allergic reaction.

Latex allergy is a serious medical problem with an increasing number of patients (Slater, 1994, Turjanmaa et al., 1996). Latex is a complex intracellular product, a milky sap, produced by the laticiferous cells of the rubber tree, Hevea brasiliensis, which is used in a variety of everyday articles, e.g. for the production of gloves, balloons, and condoms, and in manufacturing of medical devices. Latex allergy is a serious problem especially with health-care workers, rubber industry workers and patients having undergone several surgical procedures. Latex allergy has also been reported to be associated with pollen allergies and food allergies (Nel and Gujuluva, 1998). The cross-reactivity between latex and food allergens is established as the latex-fruit syndrome that might be the consequence of hevein-like protein domains or similar epitopes (Brehler et al., 1997, Chen et al., 1998, Mikkola et al, 1998). Many latex proteins have been identified as allergens (Breiteneder and Schemer, 1998). One of the major latex allergens is hevein, which is a defence protein involved in, for instance, the inhibition of several chitin-containing fungi (Lee et al., 1991, Alenius et al, 1996, Chen et al, 1997) . Hevein is a small chitin-binding protein of 43 amino acids with four disulphide bonds. Its three-dimensional structure has been determined by X-ray diffraction andNMR (Rodriguez-Romero et al, 1991; Andersen et al, 1993).

IgE antibodies distinctively recognise allergenic epitopes, which would be useful in clinics or immunodiagnostics for detecting and determining allergen concentrations of complex materials. Further, 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.

Summary of the Invention

We describe in this application the development and characterisation of human IgE antibody fragments that bind allergenic hevein with affinity and specificity high enough to be utilised as reagents in immunoassays designed for the qualitative and quantitative measurement of hevein in biological samples and, in immunotherapy of allergic patients. Specifically, the present invention describes selection of human IgE antibodies specific to hevein by the phage display technique, and the characterisation of the binding properties of the engineered antibody fragments produced in E.coli. This invention thus provides new reagents to be utilised in different kinds of immunoassay protocols, as well as human immunotherapy. 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 hevein with affinity and specificity high enough to allow qualitative and quantitative mea- surement of hevein in biological samples, as well as their use in immunotherapy. The monovalent antibodies of the present invention demonstrate a specific binding to allergenic hevein.

Another object of the present invention is to provide cDNA clones encoding hevein-speci- fie antibody chains, as well as constructs and methods for expression of such clones to produce hevein-binding antibodies, fragments thereof or other derivatives of such antibodies.

A further object of this invention is to provide methods of using such hevein-binding antibodies, fragments thereof or other derivatives of such antibodies, or combinations of them for qualitative and quantitative measurement of hevein in biological samples. Additionally, this invention provides hevein-binding antibodies, fragments thereof or other derivatives of such antibodies, or combinations of them for immunotherapy in allergic patients.

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. Brief Description of the Drawings

The figures of the constructions are not in scale.

Figure 1 shows a schematic presentation of an intact human IgE subclass antibody, Fab fragment and single-chain antibody (scFv). The antigen-binding site is indicated by a triangle.

Figure 2 shows schematically the panning procedure.

Figure 3 shows a schematic presentation of the scFv phage display vector used for the construction of scFv phage libraries.

Figure 4 shows the deduced amino acid sequence of the heavy chain variable region of the 1A4 and 1C2 antibodies. The Complementarity Determining Regions (CDRs) are underlined. Numbering is according to Kabat (Kabat et al, 1991).

Figure 5 shows the deduced amino acid sequence of the light chain variable region of the 1A4 and 1C2 antibodies. CDRs are underlined. Numbering is according to Kabat (Kabat et al, 1991).

Figure 6a shows the curve obtained from the competitive ELISA of 1 A4 Fab fragment with human IgGl subtype whose binding to hevein has been inhibited by latex polypeptide.

Figure 6b shows the curve obtained from the competitive ELISA of 1C2 Fab fragment with human IgGl subtype whose binding to hevein has been inhibited by latex polypeptide.

Figure 7 shows the result of the competitive ELISA. The binding of 1A4 Fab fragments with human IgGl subtype to hevein is inhibited by allergenic epitopes (6-mer and 13-mer) of the hevein. Abbreviations

cDNA complementary deoxyribonucleic acid

CDR complementarity determining region DNA deoxyribonucleic acid

E. coli Escherichia coli

ΕLISA 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

GFP green fluorescent protein

IgΕ immunoglobulin Ε mRNA messenger ribonucleic acid

NMR nuclear magnetic resonance PCR polymerase chain reaction

RNA ribonucleic acid scFv single-chain antibody supK a genotype of bacterial strain carrying a glutamine-inserting amber suppressor tRNA N_H variable region of a heavy chain

N_L variable region of a light chain

Detailed Description of the Invention

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. Patent 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. Patent 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 "hevein-binding antibody" is an antibody, which specifically recognises hevein and binds to it, due to interaction mediated by its variable domains.

As examples of fragments of such antibodies falling within the scope of the invention we disclose here scFv fragments of 1A4 and 1C2 as shown in Figures 4 and 5. In one preferred embodiment, the present invention thus provides derivatives of hevein-binding antibodies, e.g. Fab fragments or scFv fragments. It will be appreciated that mutant versions of the CDR sequences or complete NL and NH sequences having one or more conservative substitutions which do not substantially affect binding capability, may alternatively be employed.

For use in immunoassay, e.g. for qualitative or quantitative determination of hevein 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 hevein 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.

In another aspect, the present invention also provides DΝA molecules encoding an antibody or antibody derivative of the invention, and fragments of such DΝAs, which encode the CDRs of the N_L and/or NH region. Such a DΝA 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 DΝA molecule of the invention, including host cells capable of expressing an antibody or anti- body 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 hevein. The human IgE scFv-phage library was constructed from mRΝAs isolated from lymphocytes of a latex-allergic patient. The variable region of the light and heavy chain cDΝAs were synthesised using human IgE-speci- fic primers for Fd cDΝAs and human kappa (K) and lambda (λ) light chains using human K and λ chain specific primers. The variable regions of the light and heavy chains were amplified by PCR using human K and λ chain specific primers for VK and Nλ cDΝAs and human IgE specific primers for N_H cDΝAs, respectively. The human IgE scFv library was constructed by cloning the variable region cDΝAs 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 recombinant hevein in solution and the binders were captured on streptavidin. The elution of phages was done with 100 mM HCI (pH 2.2) followed by immediate neutralisation with 2 M Tris solution. The phage eluate was amplified in E. coli cells. After 5 rounds of biopanning, soluble scFv fragments were produced from isolated phages. The binding specificity of the selected scFv fragments was analysed by ELISA. Several hevein-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-hevein 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 hevein-binding hybrid proteins, which retain both affinity and specificity for hevein as defined hereinbefore.

The development and characterisation of the human hevein-binding recombinant anti- bodies and their usefulness in immunoassays is now described in more detail in the following examples.

EXAMPLE 1

THE RECOMBINANT HEVEIN-SPECIFIC scFv FRAGMENT BY PHAGE DISPLAY SELECTION

In this example the human IgE scFv library was constructed and selected by allergenic hevein in order to isolate scFv fragments with affinity and specificity to hevein. Construct- ion of human IgE scFv phage library was prepared indirectly by constructing IgE Fab-ic 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

100 ml of heparinised blood was obtained from a latex-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 NH₄C1, 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 resus- pended 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 (Cεl and Cε2) and the primer of the kappa (Cκl) and lambda (Cλl) 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 (NHla-NH7a) and CεlΝotI primer. Accordingly, the kappa and lambda light chains were amplified using specific primers for variable region of the light chains (Nκla-Nκ6b and Nλla-NλlO) and CεlΝotI primer, respectively. Primers for the secondary PCR were Cκl and Nκ/λl and Cε2 for the Fd region, Nκ/λl and Cλl for the kappa light chain and Nλl A and Cκ/λl 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 DΝA fragments were purified.

The final PCR products of the different antibody fragments were pooled and digested with appropriate restriction enzymes. Digested DΝA fragments, encoding IgE Fd region and K 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 DΝAs from different libraries were isolated and used as template DΝAs 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 N_H specific primers (NH1-NH4 and NH1A). Amplification of the variable region of the light chains was done using the following primer pairs: Nκl-Nκ7, Nκ2-Nκ8, Nκ3-Nκ9, Nκ4-Nκl0, Nκ5-Vκl 1 and Nκ6-Nκl 1 for human kappa chain and Vλl-Nλ8, Nλ2-Nλ9, Nλ3-Nλ9, Vλ4-Nλ9, Nλ5-Nλl0, Nλ6-Nλl0 and Nλ7-Nλl0 for human lambda chain (see Tables III and IN). The amplified DΝA fragments were purified and digested in order to ligate into a scFv phage display vector (Fig.3). Ligation mixtures were transformed into E. coli XL-1 Blue cells resulting in the human IgΕ scFv-κ and scFv-λ libraries with approximately 10⁵ independent clones.

II. Selection of the human scFv-Iibraries

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 hevein-binding antibody frag- ments, the human IgΕ scFv-κ and scFv-λ libraries displayed on the surface of the bacterio- phage were pooled and panned using an affinity panning procedure (Fig.2). First the phage pools were allowed to react either with biotinylated, immunoreactive hevein or with a bio- tinylated control protein (background) for 1.5 h. Thereafter, the phage pools were transferred to micro titre plate wells coated with biotin binding streptavidin. After a 30-min incu- bation, the wells were washed 3 times with PBS and the binders were eluted with acidic buffer (100 mM HCI, pH 2.2), and immediately neutralised with 2M Tris solution. For the next panning round the eluted phage pools were amplified by infecting E. coli XL-1 Blue cells. Five rounds of panning were performed.

III. Characterisation of the hevein-binders

After the last panning cycle scFv phage display DΝA was isolated and transformed into E. coli HB2151 (supK) 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 ΕLISA test using hevein-coated wells to catch the hevein-specific binders and control protein wells to see non-specific binding (data not shown). Most of the clones bound with high affinity to hevein. Nineteen of the most promising clones were sequenced (Sanger et al, 1977) and two of them were selected for further characterisation (Figures 4 and 5). EXAMPLE 2

CLONING AND CHARACTERISATION OF HUMAN Fab FRAGMENTS WITH HEVEIN-BINDING SPECIFICITY

In this example the human IgE scFvs with hevein-binding specificity were converted to human Fab fragments with IgGl subtype. Due to known difficulties in forming multimers, the 1A4 and 1C2 scFvs, obtained from the scFv antibody library, were cloned and bac- terially 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 hevein-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 fragments designated to 1 A4G and 1C2G were produced and the Fab fragments were 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 Fab fragments

The characterisation of the purified 1A4G and 1C2G was performed by competitive

ELISA. First, increasing amounts of latex polypeptides, isolated from latex examination gloves according to Alenius and co-workers (1996), were incubated with the samples, 1 A4G and 1C2G, and then the reaction mixtures were applied onto microtitre plate wells coated with allergenic GFP-hevein fusion protein. Preparation of latex polypeptides have been analysed to contain high latex allergenic activity (data not shown). Figure 6 shows the result of the competitive ELISA. The binding of the 1A4G (Figure 6a) and 1C2G (Figure 6b) to hevein could be inhibited by adding increasing amounts of native hevein. IgE antibodies bind specifically to allergenic epitopes. To study the binding specificity of the 1 A4G antibody in more detail a competitive ELISA with peptides comprising the allergenic epitopes was performed (Figure 7). Banerjee and co-workers (1997) have studied the allergenic epitopes of hevein, and they found two potential allergenic epitopes, 6-mer and 13-mer. In competitive ELISA the binding of the 1A4G to the immobilised hevein was inhibited by using the peptides of the allergenic epitopes. These results obtained in different competitive ELISAs indicate that the antibodies isolated from the antibody library can bind specifically to the recombinant hevein and the native hevein as well. In addition, the preliminary results demonstrate that the 1 A4G antibody binds specifically to the allergenic epitopes of hevein.

TABLE I: Primers used for cDNA synthesis and PCR amplification of the human IgE Fd region.

Cεl: 5'- GCTGAAGGTTTTGTTGTCGACCCAGTC -3' Cε2: 5'- CACGGTGGGCGGGGTGAAGTCCC -3*

CεNotI: 5'- GAATGGTGCGGCCGCGCTGAAGGTTTTGTTGTCG -3'

VHla: 5'- ATGGCCGCAGCTCAGGTKCAGCTGGTGCAG -3*

VHlb: 5'- ATGGCCGCAGCTCAGGTCCAGCTTGTGCAG -3'

VHlc: 5'- ATGGCCGCAGCTSAGGTCCAGCTGGTACAG -3' VHld: 5'- ATGGCCGCAGCTCARATGCAGCTGGTGCAG -3'

VH2a: 5'- ATGGCCGCAGCTCAGATCACCTTGAAGGAG -3'

VH2b: 5'- ATGGCCGCAGCTCAGGTCACCTTGARGGAG -3'

VH3a: 5'- ATGGCCGCAGCTGARGTGCAGCTGGTGGAG -3'

VH3b: 5'- ATGGCCGCAGCTCAGGTGCAGCTGGTGGAG -3' VH3c: 5'- ATGGCCGCAGCTGAGGTGCAGCTGTTGGAG -3'

VH4a: 5'- ATGGCCGCAGCTCAGSTGCAGCTGCAGGAG -3'

VH4b: 5'- ATGGCCGCAGCTCAGGTGCAGCTACAGCAG -3'

VH5a: 5'- ATGGCCGCAGCTGARGTGCAGCTGGTGGAG -3'

VH6a: 5*- ATGGCCGCAGCTCAGGTACAGCTGCAGCAG -3' VH7a: 5'- ATGGCCGCAGCTCAGGTSCAGCTGGTGCAA -3'

VH1 A: 5'- TTACTCGCGGCCCAGCCGGCCATGGCCGCAGCT -3'

TABLE II: Primers used for cDNA synthesis and PCR amplification of human kappa and lambda chains.

Cκl: 5'- AGGTAGGGCGCGCCTTAACACTCTCCCCTGTTGAAGC -3' Nκla: 5'- ATGGCAGCGGCTRACATCCAGATGACCCAG -3'

Niclb: 5'- ATGGCAGCGGCTGMCATCCAGTTGACCCAG -3'

Nκlc: 5'- ATGGCAGCGGCTGCCATCCRGATGACCCAG -3'

Nκld: 5'- ATGGCAGCGGCTGTCATCTGGATGACCCAG -3'

Nκ2a: 5'- ATGGCAGCGGCTGATATTGTGATGACCCAG -3' Nκ2b: 5'- ATGGCAGCGGCTGATRTTGTGATGACTCAG -3'

Nκ3a: 5'- ATGGCAGCGGCTGAAATTGTGTTGACRCAG -3'

Nκ3b: 5'- ATGGCAGCGGCTGAAATAGTGATGACGCAG -3'

Nκ3c: 5'- ATGGCAGCGGCTGAAATTGTAATGACACAG -3'

Nκ4a: 5'- ATGGCAGCGGCTGACATCGTGATGACCCAG -3' N 5a: 5'- ATGGCAGCGGCTGAAACGACACTCACGCAG -3'

Nκ6a: 5'- ATGGCAGCGGCTGAAATTGTGCTGACTCAG -3'

Nκ6b: 5'- ATGGCAGCGGCTGATGTTGTGATGACACAG -3'

Nk/λl: 5'- TTGTTATTGCTAGCTGCACAACCAGCAATGGCAGCGGCT -3'

Cλl: 5'- AGGTAGGGCGCGCCTTATGAACATTCYGYAGGGGC -3' Nλl a: 5'- ATGGCAGCGGCTCAGTCTGTGCTGACTCAG -3'

Nλlb: 5'- ATGGCAGCGGCTCAGTCTGTGYTGACGCAG -3'

Nλlc: 5'- ATGGCAGCGGCTCAGTCTGTCGTGACGCAG -3'

Nλ2 : 5'- ATGGCAGCGGCTCAGTCTGCCCTGACTCAG -3'

Nλ3a: 5'- ATGGCAGCGGCTTCCTATGWGCTGACTCAG -3' Nλ3b: 5*- ATGGCAGCGGCTTCCTATGAGCTGACACAG -3'

Nλ3c: 5'- ATGGCAGCGGCTTCTTCTGAGCTGACTCAG -3'

Nλ3d: 5'- ATGGCAGCGGCTTCCTATGAGCTGATGCAG -3'

Nλ4 : 5'- ATGGCAGCGGCTCAGCYTGTGCTGACTCAA -3'

Nλ5 : 5'- ATGGCAGCGGCTCAGSCTGTGCTGACTCAG -3' Nλ6 : 5'- ATGGCAGCGGCTAATTTTATGCTGACTCAG -3'

Nλ7 : 5'- ATGGCAGCGGCTCAGRCTGTGGTGACTCAG -3'

Nλ8 : 5'- ATGGCAGCGGCTCAGACTGTGGTGACCCAG -3' Nλ4/9: 5'- ATGGCAGCGGCTCWGCCTGTGCTGACTCAG -3' NλlO: 5'- ATGGCAGCGGCTCAGGCAGGGCTGACTCAG -3'

TABLE III: Primers used for PCR amplification of the human variable regions of the heavy chain.

NH1: 5'- ATTTACTCGAGTGAGGAGACGGTGACCAGGGTGCC -3' NH2: 5'- ATTTACTCGAGTGAAGAGACGGTGACCATTGTCCC -3' VH3: 5'- ATTTACTCGAGTGAGGAGACGGTGACCAGGGTTCC -3' VH4: 5'- ATTTACTCGAGTGAGGAGACGGTGACCGTGGTCCC -3' VH1A: 5'- TTACTCGCGGCCCAGCCGGCCATGGCCGCAGCT -3'

TABLE IN: Primers used for PCR amplification of the human variable regions of the light chains.

Vκl: 5'- TTATAGAGCTCGACATCCAGATGACCCAGTCTCC -3' Nκ2: 5'- TTATAGAGCTCGATGTTGTGATGACTCAGTCTCC -3' Nκ3: 5'- TTATAGAGCTCGAAATTGTGTTGACGCAGTCTCC -3' Nκ4: 5'- TTATAGAGCTCGACATCGTGATGACCCAGTCTCC -3' Nκ5: 5'- TTATAGAGCTCGAAACGACACTCACGCAGTCTCC -3' Nκ6: 5'- TTATAGAGCTCGAAATTGTGCTGACTCAGTCTCC -3' Nκ7: 5'- TATAAGCGGCCGCACGTTTGATTTCCACCTTGGTCCC -3' Nκ8: 5'- TATAAGCGGCCGCACGTTTGATCTCCAGCTTGGTCCC -3* Nκ9: 5'- TATAAGCGGCCGCACGTTTGATATCCACTTTGGTCCC -3' Nκl0: 5'- TATAAGCGGCCGCACGTTTGATCTCCACCTTGGTCCC -3' V l l: 5'- TATAAGCGGCCGCACGTTTAATCTCCAGTCGTGTCCC -3' Nλl : 5'- ATTTAGAGCTCCAGTCTGTGTTGACGCAGCCGCC -3' Nλ2: 5'- ATTTAGAGCTCCAGTCTGCCCTGACTCAGCCTGC -3' Nλ3: 5'- ATTTAGAGCTCTCCTATGTGCTGACTCAGCCACC -3' Nλ4: 5'- ATTTAGAGCTCTCTTCTGAGCTGACTCAGGACCC -3' Nλ5: 5'- ATTTAGAGCTCCACGTTATACTGACTCAACCGCC -3' Nλ6: 5'- ATTTAGAGCTCCAGGCTGTGCTCACTCAGCCGTC -3' Nλ7: 5'- ATTTAGAGCTCAATTTTATGCTGACTCAGCCCCA -3' Nλ8: 5'- ATATTGCGGCCGCACCTAGGACGGTGACCTTGGTCCC -3' Nλ9: 5'- ATATTGCGGCCGCACCTAGGACGGTCAGCTTGGTCCC -3' NλlO: 5'- ATATTGCGGCCGCACCTAAAACGGTGAGCTGGGTCCC -3'

TABLE N: Primers used for PCR amplification of the human Fd regions with IgE and IgGl subtype.

5'Cε: 5'-GCTCACCGTCTCCTCAGCCTCCACACAGAGCCCATCCG-3'

3'Cε: 5'-GCATTGCATTGCGGCCGCTTAATGGTGATGGTGATGATGGCTGAAGGT TTTGTTGTCGACCC-3'

5'Cγ: 5'-GGTCACCGTCTCCTCAGCCTCCACCAAGGGCCC-3'

3'Cγ: 5'-TTTAGTTTATGCGGCCGCTTAATGGTGATGATGATGGTGACAAGATTTG GGCTCTGC-3'

5Nε: 5'-TTACTCGCGGCCCAGCCGGCCATGGCCGCAGCT-3 '

3Nε: 5'-TGAGGAGACGGTGACC-3'

5'Cκ: 5'-GGGACACGACTGGAGATTAAAACTGTGGCTGCACCATCTGTC-3'

3'Cκ: 5'-AGGTAGGGCGCGCCTTAACACTCTCCCCTGTTGAAGC-3' 5'Nκ: 5'-ATGGCAGCGGCTGAAACGACACTCACGCAG-3' and

5'-TTGTTATTGCTAGCTGCACAACCAGCAATGGCAGCGGCT-3'

3Nκ: 5'-TTTAATCTCCAGTCGTGTCCC-3'.

References

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Claims

Claims

1. A monoclonal antibody with binding specificity to allergenic hevein, or a functional fragment or derivative thereof.

2. The monoclonal antibody according to claim 1, wherein the fragment is a scFv fragment or a Fab fragment.

3. The monoclonal antibody according to claim 2, wherein the scFv fragment or the Fab fragment are derived from an antibody belonging to an IgE subclass.

4. The monoclonal antibody according to claim 2, wherein the fragment is scFv fragment 1A4 or 1C2.

5. An isolated DNA molecule encoding the monoclonal antibody or a fragment or derivative thereof according to any one of the preceding claims, and fragments of such DNA, which encode at least one antibody chain of said antibody or antibody derivative.

6. The isolated DNA molecule according to claim 5, wherein the antibody chain is the CDR of the N_L and/or N_H region.

7. The isolated DΝA molecule according to claim 5 cloned into a vector.

8. The isolated DΝA molecule according to claim 7, wherein said vector is an expression vector capable of expressing antibodies, as well as fragments and derivatives thereof as claimed in any one of claims 1 to 4.

9. A host cell containing a DΝA according to any one of claims 5 to 8.

10. The host cell according to claim 9, capable of expressing a monoclonal antibody or a fragment or derivative thereof as claimed in any one of claims 1 to 4 or at least one antibody chain of said antibody or antibody derivative.

11. The host cell according to claim 10, wherein the antibody chain is the scFv fragment as claimed in any one of claims 2 to 4.

12. A method of preparing a monoclonal antibody or a fragment or derivative thereof according to any one of claims 1 to 4, comprising the steps of

- culturing a host cell according to claim 9 capable of expressing at least one of the required antibody chains, and

- recovering said antibody or antibody fragment or derivative.

13. The method according to claim 12, further comprising the steps of

- combining component chains after the recovery step,

- introducing combined component chains into a second host cell, and

- recovering said combined component chains.

14. The method according to claim 12, further comprising the step of labelling said antibody or antibody derivative.

15. A method of preparing a monoclonal antibody or a fragment or derivative thereof according to any one of claims 1 to 4, comprising the step of

- synthetically producing at least a portion of said antibody or antibody derivative.

16. A phage or microbial cell which presents an antibody fragment according to any one of claims 2 to 4 as a fusion protein with a surface protein.

17. A method of selecting an antibody fragment according to any one of claims 2 to 4, comprising the steps selecting said antibody fragment from a display library of antibody fragments containing a phage or cell according to claim 16.

18. A method of assaying hevein in a sample, comprising the steps of

- obtaining said sample, and

- assaying for hevein by employing a monoclonal antibody or a fragment or derivative thereof according to any one of claims 1 to 4.

19. A test kit comprising an antibody or a fragment or derivative thereof according to any one of claims 1 to 4 in a suitable container for transport and storage.

20. A monoclonal antibody or a fragment or derivative thereof according to any one of claims 1 to 4 for use in immunodiagnostics.

21. A monoclonal antibody or a fragment or derivative thereof according to any one of claims 1 to 4 for use in immunotherapy.