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WO2000058365A1 - Proteines de liaison specifiques pour le traitement d'allergies - Google Patents

Proteines de liaison specifiques pour le traitement d'allergies Download PDF

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Publication number
WO2000058365A1
WO2000058365A1 PCT/US2000/008347 US0008347W WO0058365A1 WO 2000058365 A1 WO2000058365 A1 WO 2000058365A1 US 0008347 W US0008347 W US 0008347W WO 0058365 A1 WO0058365 A1 WO 0058365A1
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Prior art keywords
binding protein
administering
blank
antibody
peptide
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PCT/US2000/008347
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English (en)
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WO2000058365A9 (fr
Inventor
Robert Lawton
Brion Mermer
Greg Francoeur
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Idexx Laboratories, Inc.
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Priority claimed from US09/281,760 external-priority patent/US6734287B1/en
Application filed by Idexx Laboratories, Inc. filed Critical Idexx Laboratories, Inc.
Priority to AU40449/00A priority Critical patent/AU757334B2/en
Priority to CA2329148A priority patent/CA2329148C/fr
Priority to JP2000608657A priority patent/JP2002539818A/ja
Priority to EP00919828A priority patent/EP1082346A1/fr
Publication of WO2000058365A1 publication Critical patent/WO2000058365A1/fr
Publication of WO2000058365A9 publication Critical patent/WO2000058365A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • C07K16/4291—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig against IgE
    • 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

Definitions

  • This invention concerns peptides. More particularly, the invention concerns compositions for administration to dogs, which actively provide immunity to the dog's immunoglobulin E molecules .
  • Atopy is a condition where a dog is allergic to inhalants such as pollens, molds or microscopic mites such as are found in house dust .
  • Antibody molecules play a role in allergic manifestations. In mammals, antibody molecules are classified into various isotypes referred to as IgA, IgD, IgE, IgG, and IgM. Antibody molecules consist of both heavy and light chain components. The heavy chains of molecules of a given isotype have extensive regions of amino acid sequence homology, and conversely have regions of difference from antibodies belonging to other isotypes. The shared regions of the heavy chains provide members of each isotype with common abilities to bind to certain cell surface receptors or to other macromolecules, such as complement. These heavy chain regions, therefore, serve to activate particular immune effector functions. Accordingly, separation of antibody molecules into isotypes serves to separate the antibodies according to a set of effector functions that they commonly activate.
  • IgE immunoglobulin E
  • IgE molecules bind to mast cells and basophils. This binding occurs when the Fc region of the IgE molecule is bound to Fc receptors on the mast cells. When such bound IgE antibodies then bind to an allergen, the allergen cross-links multiple IgE antibodies on the cell surface. This cross- linking mediates Type I immediate hypersensitivity reactions and causes release of histamines and other molecules that produce symptoms associated with allergy. Monoclonal antibodies having different degrees of sensitivity to canine IgE and IgG have been identified. (DeBoer, et al . Immunology and I munopathology 37, 183-199 (1993) .) DeBoer, et al .
  • MAbs monoclonal antibodies
  • IgE ELISA antigen-specific IgE ELISA
  • MAbs monoclonal antibodies
  • MAbs for immunostaining of Western Blot assays, to evaluate the molecular specificity of IgE antibodies, as well as for in vitro studies on degranulation of mast cells.
  • Hill and DeBoer ELISA was used to establish the total amount of IgE in canine serum in an effort to diagnose canine allergy.
  • the quantity of IgE determined to exist in canine circulation was of no use whatsoever in the diagnosis of allergy in dogs. (See, e.g., Abstract and Discussion Sections of Hill and DeBoer)
  • This finding was in direct contrast to the situation in human immunology.
  • This divergent diagnostic result based on levels of IgE in humans compared to such levels in dogs, points out the difficulty of any attempt to correlate data between animals of two different genera. This difficulty is further exacerbated by the fact that dogs can be allergic to a different set of antigens than humans are. Fleas, for instance, are a severe problem for dogs, but not humans.
  • genomic sequences encoding human and murine IgE heavy chain constant region are known (For example, see Ishida et al . , "The Nucleotide Sequence of the Mouse Immunoglobulin E Gene: Comparison with the Human Epsilon Gene Sequence", EMBO Journal 1,1117-1123 (1982). A comparison of the human and murine genes shows that they possess 60% homology within exons, and 45-50% homology within introns, with various insertions and deletions.
  • IgE is believed to mediate allergic symptoms, it may be desirable to decrease IgE levels as a mechanism for alleviating allergic symptoms.
  • a patient's own IgE molecules are self-proteins, and immune responses to such proteins are usually suppressed.
  • the suppression of immune responses to self-proteins, i.e., tolerance to self-antigens is hypothesized to occur in a number of ways.
  • T cells directed to self-antigens involves an induction of "clonal deletion" of such T cells in the thymus, whereby T cell receptors which might recognize self-peptides in association with MHC molecules are eliminated, and only those which recognize foreign peptide and MHC molecules are allowed to expand.
  • suppressor T cells may also exist which prevent the induction of immune responses to self-proteins .
  • a B cell which recognizes epitopes (antigen-binding sites) on a patient's own IgE antibodies is capable of generating antibodies, generally IgG, directed to this self- antigen, i.e., IgE.
  • IgG immunoglobulin
  • the existence of such B cells therefore, presents a unique opportunity to induce the production of auto-antibody responses.
  • the hypothesis regarding "antigen presentation" involves: the recognition of antigen by surface immunoglobulin on the B cell, the internalization and processing of this antigen, the association of peptides derived from the antigen with MHC molecules expressed on the surface of the B cell, and then, the recognition of the associated antigen peptide and MHC molecules by a particular T cell.
  • T-cell :B-cell interaction then leads to signal transduction in both cells and the synthesis and elaboration of soluble cytokines which eventually result in antibody production by the B cell.
  • an antigen is foreign does an immune response occur; otherwise the internalization and processing of self-proteins would regularly lead to the presentation of self peptide-MHC complexes to T cells and thereby lead to autoimmune antibodies.
  • the immune system in order to induce an antibody response to a self-peptide, such as IgE, the immune system must be manipulated so as to allow an auto-reactive B cell to become an antibody-secreting B cell. There is an unmet need to manipulate the immune system in this way, particularly in the context of allergic disease.
  • MAPs multiple antigenic peptides
  • Dr. James Tarn introduced by Dr. James Tarn (Tarn, J.P., (1988) Proc . Natl . Acad. Sci. U.S.A. 85 , 5409-5413)
  • the MAP approach is an improved alternative to the conventional technique of conjugating a peptide antigen to a protein carrier.
  • One of the primary limitations associated with the use of protein carriers is the large mass of the carrier relative to the attached peptide antigen. This relative size disparity may result in a low ratio of anti-peptide antibodies compared to anti-carrier antibodies.
  • MAPs typically have 4 or 8 peptide arms branching out from a lysine core matrix as depicted in Fig.l A-B.
  • the peptide antigen is conjugated to each arm.
  • This design maximizes the concentration of the antigen for a specific immunogenic response.
  • the central lysine core of the MAP-peptide has been shown to be non- immunogenic .
  • MAP-peptides are a direct response to the antigen.
  • Accurate knowledge of the chemical composition, structure, and quantity of the peptide prior to immunization is possible by directly synthesizing the antigen onto the branching lysine core.
  • the MAP approach removes the need to conjugate peptides to carrier proteins, which may alter the antigenic determinants, chemical ambiguity is eliminated.
  • MAPs are believed to induce antibody responses of high purity, increased avidity, accurate chemical definition, and improved safety.
  • Fmoc MAP resins (available from Applied Biosystems, Foster City, Calif.) are Fmoc-compatible resins connected to a small core matrix of branching lysine residues.
  • the core matrix comprises several levels of lysine residues attached to the previous lysine at both the N- ⁇ and N- ⁇ amino groups, as depicted in Figure 1A-B.
  • MAP-peptides used in experimental vaccine design have elicited high titers of anti-peptide antibodies that recognize the native protein.
  • Proc. Natl. Acad. Sci. U.S.A. 85 5409-5413 / Posnett, D.N., McGrath, H., and Tarn, J.P., (1988) J. Biol. Chem. 263 , 1719-1725; Auriault, C, Wolowczuk, I., Gras-Masse, H. , Maguerite, M. , Boulanger, D., Capron, A., and Tartar, A., (1991) Peptide Res.4 , 6-11).
  • EPICOATTM technology (Axis Genetics pic, Cambridge, England) is one such example.
  • the EPICOATTM technology is based on chimeric virus particle (CVP) technology that utilizes the recombinant genetic modification of plant viruses.
  • the EPICOATTM technology involves insertion of a small portion of a foreign protein (a peptide) into a plant virus in such a way that multiple copies of the peptide are displayed on the surface of the virus particle.
  • the EPICOATTM technology is currently based on the cow pea mosaic virus (CPMV) a plant virus that infects the cow pea plant, also known as the "black-eyed" bean.
  • CPMV cow pea mosaic virus
  • the unmodified CPMV particle is icosahedral, and about twenty-eight nanometers (nm.) in diameter.
  • CPMV particles are composed of two proteins, referred to as the large and small coat proteins. Studies have revealed a site within the small coat protein which allows presentation of a foreign peptide in a prominent position on the virus surface, whereby up to sixty copies of a particular peptide can be presented on each virus particle.
  • DNA copies of the plant virus's genetic material are used.
  • a minute quantity of the DNA encoding the virus protein, including the inserted foreign peptide, is applied to the leaves of young cow pea plant, along with an abrasive powder.
  • DNA enters the leaves and utilizes the plant's own cellular mechanisms to initiate generation of functional virus particles.
  • the virus replicates within the inoculated leaves and spreads throughout the growing plant .
  • leaf material containing large quantities of the virus is harvested.
  • Chimeric virus particles (CVPs) are isolated by centrifugation and selective precipitation of homogenized plant material. Between 1 to 2 grams of CVPs can be obtained per kilogram fresh weight of leaf material. Cow pea plants are readily grown in abundance in controlled environments, allowing generation of large quantities of CVPs.
  • a specific binding protein which specifically binds to native canine free or B cell-bound IgE exon 3, and which does not bind to IgE exon 3 when the IgE is bound to receptor on a mast cell.
  • the surface-bound IgE can be IgE expressed on the surface of a canine B cell.
  • a specific binding protein which specifically binds to an isolated and purified peptide comprising a leucine positioned two peptide bonds away from a tyrosine-arginine pair; e.g., SEQ ID NO : 1 leucine-blank-blank- tyrosine-arginine, SEQ ID NO : 2 tyrosine-arginine-blank-blank- leucine, or SEQ ID NO : 3 leucine-blank-blank-tyrosine-arginine- blank-blank- leucine .
  • the peptide can consist of from 5 to 71 amino acids.
  • an antibody which binds to a defined epitope and which is raised to an isolated and purified peptide comprising an amino acid sequence, or a conservative variant thereof, which comprises: SEQ ID NO : 4 Thr-Leu-Leu-Glu-Tyr-Arg- Met; also disclosed is a recombinant binding molecule which specifically binds to the defined epitope bound by the antibody.
  • an antibody which binds to a defined epitope and which is raised to an isolated and purified peptide comprising an amino acid sequence, or a conservative variant thereof, which comprises: SEQ ID NO : 5 Gly-Met-Asn-Leu-Thr-Trp- Tyr-Arg-Glu-Ser-Lys; also disclosed is a recombinant binding molecule which specifically binds to the defined epitope bound by the antibody.
  • a specific binding protein which is raised to a multiply antigenic peptide comprising multiple copies of an isolated and purified peptide which comprises a leucine positioned two peptide bonds away from a tyrosine- arginine pair; the specific binding protein specifically binds to a defined epitope.
  • the isolated and purified peptide can be from 5-71 amino acids.
  • a recombinant binding molecule which specifically binds to the defined epitope bound by the binding protein.
  • a specific binding protein which specifically binds to an isolated and purified peptide comprising a cysteine positioned two peptide bonds away from a proline-histidine pair positioned three peptide bonds from a cysteine.
  • the peptide can have the form: SEQ ID NO: 6 cysteine-blandk-blank-proline-histidine-blank-blank-blank- cysteine.
  • the cysteines may form a covalent bond through a reduction reaction, cyclizing the peptide and becoming cystine.
  • the peptide can comprise from 9 to 71 amino acids.
  • an antibody that binds to a defined epitope and which is raised to an isolated and purified peptide that has the amino acid sequence, or a conservative variant thereof, which comprises: SEQ ID NO : 7 serine-valine-threonine- 1eucine-cysteine-proline-asparagine-proline-histidine- isoleucine-proline-methionine-cysteine-glycine-glycine- glycine.
  • the cysteines may form a covalent bond through a reduction reaction, cyclizing the peptide and becoming cystine.
  • a recombinant binding molecule that specifically binds to the defined epitope bound by the antibody.
  • an antibody that binds to a defined epitope and which is raised to an isolated and purified peptide that has the amino acid sequence, or a conservative variant thereof, which comprises: SEQ ID NO : 8 serine-alanine-cysteine- proline-asparagine-proline-histidine-asparagine-proline- tyrosine-cysteine-glycine-glycine-glycine .
  • the cysteines may form a covalent bond through a reduction reaction, cyclizing the peptide changing the amino acid to cystine.
  • a recombinant binding molecule that specifically binds to the defined epitope bound by the antibody.
  • a specific binding protein that specifically binds to an isolated and purified peptide comprising a cysteine positioned one peptide bond from a proline-histidine pair, positioned one peptide bond from a proline, positioned two peptide bonds from a cysteine;
  • the peptide can have the form: SEQ ID NO : 9 cysteine-blank-proline-histidine-blank- proline-blank-blank-cysteine .
  • the cysteines may form a convalent bond through a reduction reaction, cyclizing the peptide and becoming cystine.
  • the peptide can comprise from 9 to 71 amino acids.
  • an antibody that binds to a defined epitope and which is raised to an isolated and purified peptide that has the amino acid sequence, or a conservative variant thereof, which comprises: SEQ ID NO: 10 serine-alanine- cysteine-histidine-proline-histidine-leucine-proline-lysine- serine-cysteine-glycine-glycine-glycine .
  • the cysteines may form a covalent bond through a reduction reaction, cyclizing the peptide and becoming cystine.
  • a recombinant binding molecule that specifically binds to the defined epitope bound by the antibody.
  • Specific binding proteins in accordance with the invention can be antibodies, either polyclonal or monoclonal.
  • the monoclonal antibody can be 8H.8 or 15A.2.
  • the antibody in accordance with the invention will bind a defined epitope; also disclosed are recombinant binding molecules which specifically binds to the defined epitope bound by an antibody in accordance with the invention
  • a method for treatment or prophylaxis for canine allergy comprising: providing a specific binding molecule in accordance with the invention; and, administering the provided binding molecule to a dog.
  • the method can comprise a step of mixing the provided binding molecule with a diluent prior to the administering step, and wherein the administering step comprises administering the mixture of the molecule and the diluent .
  • a step of administering a booster dose of the binding molecule can follow the administering step.
  • cDNA clone A duplex DNA sequence representing an RNA, carried in a cloning vector.
  • Cloning The selection and propagation of a single DNA species .
  • Cloning Vector A plasmid, phage DNA or other DNA sequence, able, to replicate in a host cell and capable of carrying exogenously added DNA sequence for purposes of amplification or expression of the added DNA sequence.
  • Codon A triplet of nucleotides that represents an amino acid or termination signal.
  • Conservative variants Conservative variants of nucleotide sequences include nucleotide substitutions that do not result in changes in the amino acid sequence encoded by such nucleotides, as well as nucleotide substitutions that result in conservative amino acid substitutions, e.g., amino acid substitutions which do not substantially affect the character of the polypeptide translated from said nucleotides. For example, the character of a peptide derived from IgE is not substantially affected if the substitutions do not preclude specific binding of the peptide to canine IgE receptor or other canine IgE binding ligands.
  • amino acid sequences include amino acid substitutions or deletions that do not substantially affect the character of the variant polypeptide relative to the starting peptide.
  • polypeptide character is not substantially affected if the substitutions or deletions do not preclude specific binding of the variant peptide to a specific binding partner of the starting peptide.
  • mimotope refers to a conservative variant of an amino acid sequence, to which antibody specificity has been raised.
  • the mimotope comprises a variant of the epitope of the starting peptide such that it is able to bind antibodies that cross-react with the original epitope.
  • DNA Sequence A linear series of nucleotides connected one to the other by phosphodiester bonds between the 3 ' and 5 ' carbons of adjacent pentoses.
  • Expression The process undergone by a structural gene to produce a polypeptide. It is a combination of transcription and translation.
  • Expression Control Sequence A DNA sequence of nucleotides that controls and regulates expression of structural genes when operatively linked to those genes.
  • Exon A contiguous region of DNA encoding a portion of a polypeptide. Reference to any exon, e.g. "DNA sequence of exon 6", refers to the complete exon or any portion thereof.
  • Genome The entire DNA of a substance. It includes inter alia the structural genes encoding for the polypeptides of the substance, as well as operator, promoter and ribosome binding and interaction sequences such as the Shine-Dalgarno sequences .
  • Nucleotide A monomeric unit of D ⁇ A or R ⁇ A consisting of a sugar moiety (pentose) , a phosphate, and a nitrogenous heterocyclic base.
  • the four D ⁇ A bases are adenine ("A"), guanine ("G”), cytosine ("C”) and thymine (“T”).
  • the four R ⁇ A bases are A, G, C and uracil ("U") .
  • a and G are purines, and C, T, and U are pyrimidines .
  • Phage or Bacteriophage Bacterial virus, many of which include D ⁇ A sequences encapsidated in a protein envelope or coat (“capsid”) .
  • Plasmid An autonomous self-replicating extrachromosomal circular D ⁇ A.
  • Polymerase Chain Reaction (PCR) A method of amplifying a target D ⁇ A sequence contained in a mixture of D ⁇ A sequences, by using oligonucleotide primers that flank the target D ⁇ A sequence for repeated cycles of D ⁇ A synthesis of the target D ⁇ A sequence .
  • Polypeptide A linear series of amino acids connected one to the other by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids.
  • Reading Frame The grouping of codons during translation of mR ⁇ A into amino acid sequences.
  • the sequence GCTGGTGTAAG may be translated in three reading frames or phases, each of which affords a different amino acid sequence: GCT GGT TGT AAG-Ala-Gly-Cys-Lys G CTG GTT GTA AG-Leu-Val -Val GC TGG TTG TAA A-Trp-Leu- (STOP) .
  • Recombinant DNA Molecule A hybrid DNA sequence comprising at least two nucleotide sequences, the first sequence not normally being found together in nature with the second.
  • Specific binding Binding of one substance to another at greater binding affinity than background binding. Two substances that exhibit specific binding are referred to as specific binding partners, or as a specific binding pair. An antibody and its antigen are one example of a specific binding pair.
  • Specific Binding Molecule A molecule that exhibits specific binding to its corresponding binding partner to form a specific binding pair.
  • this definition of specific binding molecule covers monoclonal and polyclonal antibodies, antigen-binding fragments of these antibodies, hybrid antibodies, single-chain antibodies, and recombinant molecules capable of specific binding to a ligand.
  • Structural Gene A DNA sequence that encodes through its template or messenger RNA (“mRNAII) a sequence of amino acids characteristic of a specific polypeptide.
  • mRNAII messenger RNA
  • Fig. 1A depicts the core structure of a MAP protein with four arms, i.e., a 4-MAP protein
  • Fig. IB depicts the core structure of a MAP protein with eight arms, i.e., an 8 -MAP protein.
  • Fig.2 depicts data comparing the binding characteristics of several monoclonal antibodies to surface bound IgE (e.g., analogous to IgE expressed on the surface of B cells) ; IgE receptor (e.g., analogous to the Fc receptor on mast cells); and to a combination of IgE when bound by the IgE receptor.
  • IgE surface bound IgE
  • IgE receptor e.g., analogous to the Fc receptor on mast cells
  • Fig. 3 depicts the ability of recombinant exon 3 to inhibit conjugated antibody 8H.8 or conjugated antibody 15A.2 from binding to an IgE solid phase.
  • Data for 8H.8 is depicted by speckled bar graphs
  • data for 15A.2 is depicted by solid black bar graphs .
  • Fig. 4 depicts the ability of soluble, purified canine
  • IgE to inhibit monoclonal antibody 8H.8 (2.5 ⁇ g/ml) or monoclonal antibody 15A.2 (2.5 ⁇ g/ml) from binding to a canine
  • IgE solid phase Data for 8H.8 is depicted by speckled bar graphs; data for 15A.2 is depicted by solid black bar graphs.
  • Fig. 5 depicts the ability of monoclonal antibody 15A.2 or monoclonal antibody 8H.8 to inhibit IgE from binding to recombinant IgE receptor immobilized on a solid phase as detected by monoclonal antibody D9.
  • Fig. 6 depicts the amino acid sequences of the New
  • Fig. 7 depicts the alignment of the 15A.2 binding region from seven different mammals: dog, human, green monkey, cat, swine , mouse and horse .
  • Fig. 8 depicts the ability of phage displaying 15A.2 mimotope peptides to inhibit canine IgE from binding the 15A.2 monoclonal antibody on the solid phase.
  • Fig. 9 depicts the ability of a specific 15A.2 mimotope peptide sequence attached to the solid phase to be able to bind the 15A.2 mimotope monoclonal antibody.
  • Fig. 10A and 10B depict the ability of a 15A.2 mimotope peptide to be able to prevent the 15A.2 monoclonal antibody from binding canine IgE on the solid phase.
  • Fig. 11 depicts the ability of monclonal antibody 8H.8 and monoclonal antibody 15A.2 to bind to purified canine IgE immobilized on a solid phase.
  • Fig. 12 depicts data from a study evaluating the ability of antibody 15A.2 conjugated to a signal moiety to bind to solid phases having various peptides immobilized thereon.
  • Fig. 13 depicts data from a study evaluating the ability of monoclonal antibody 14K2, known to bind with canine IgE exon 4, to bind with various peptides immobilized on a solid phase .
  • Fig. 14 depicts data from a study evaluating the ability of monoclonal antibody 15A.2, known to bind to canine IgE exon 3, to bind to various peptides immobilized on a solid phase.
  • Fig. 15 depicts data for studies that compared the binding of monclonal antibody 8H.8, or a polyclonal antibody raised to recombinant IgE (Re-hu IgE) , to bind to a solid phase having peptide E3a.5 or substituted peptide E3a.5 (designated peptide S3a.5) immobilized thereon.
  • Fig. 16 depicts data from a study evaluating the ability of antibody 8H.8 conjugated to a signal moiety to bind to solid phases having various peptide immobilized thereon.
  • Fig. 17 depicts the ability of polyclonal anti-human IgE antibodies to bind to various peptides immobilized on a solid phase .
  • a hypothesis in accordance with the present invention is that anti- IgE monoclonal antibodies affect IgE levels by directly binding with the resident circulating IgE, after which the bound complex is removed from the circulation.
  • anti- IgE monoclonal antibodies affect IgE levels by interfering with the production of new IgE by B-cells.
  • Memory B-cells cells that upon interacting with antigen and T-cell become antibody producing cells (i.e., "memory IgE B-cells"), are involved in replenishing serum IgE, and these cells contain IgE as their cell surface receptors.
  • anti- IgE monoclonal antibodies bind to the IgE on these antibody producing cells and interfere with their function, such as by down-regulation or by one or more mechanisms which lead to cell destruction.
  • Two general ways have been proposed for how the binding of a monoclonal antibody to a memory B cell might interfere with production of IgE.
  • activation of a memory B cell may also require the involvement of a surface IgE-binding factor designated CD23. Binding of certain monoclonal antibodies to the cell surface IgE may preclude CD23 binding, and may thus lead to an inability to mount an IgE response.
  • IgE antibody production by a memory IgE B cell may be reduced or eliminated by binding of a monoclonal antibody that is directed against, or blocks, the receptor for the IgE molecule which is located on the surface of the memory
  • the serum levels of IgE are elevated in patients experiencing allergic disease.
  • antibodies were generated that specifically bind to a species
  • IgE IgE
  • these antibodies were administered to a patient who is a member of the species ("passive immunization"), that patient's serum levels of IgE declined.
  • the specific binding proteins in accordance with the invention are administered in pharmacologically accepted dosages, and can be administered with suitable biocompatible diluents.
  • a method and related compositions that serve to induce a response to a self-peptide , such as an IgE molecule, whereby the immune system is manipulated so as to allow an auto-reactive B cell to become an antibody-secreting B cell (“active immunization").
  • Receptors are present on mast cells that bind to IgE from the circulation.
  • the IgE molecules can become crosslinked.
  • the mast cell is induced to release histamine. Histamine is an agent that induces the manifestation of allergic symptoms.
  • cross-linking occurs by binding of the IgE molecules to an antigen, e.g., an allergen.
  • an antigen e.g., an allergen.
  • a monoclonal antibody that binds to IgE could serve as a means for cross-linking IgE which has become bound to the surface of mast cells.
  • the first is to produce monoclonal antibodies that are directed to an epitope of an IgE molecule that is only accessible when the IgE is in circulation. Since the monoclonal antibody can only bind to IgE when it is in circulation, it will not be able to bind to IgE that has become bound to the surface of a mast cell.
  • Stanworth are disclosed to cross-link IgE on mast cells, but in a manner that does not induce histamine release.
  • Stanworth has disclosed an epitope of the human IgE molecule which must be available or accessible after IgE molecules have become cross-linked on the surface of a mast cell, in order for the mast cell to release histamine.
  • Stanworth disclosed an antibody that binds to that particular epitope. Accordingly, monoclonal antibodies that are directed to this IgE epitope will serve to cross-link the IgE, but will not permit the mast cell to release histamine.
  • the approach followed in accordance with the present invention is the development of antibodies, either ex vivo monoclonal or in vi vo anti-self antibodies, directed to epitopes which are accessible on circulating IgE but which are not accessible when such IgE becomes bound to a mast cell.
  • Monoclonal antibody 15A.2 binds to canine IgE.
  • the 15A.2 monoclonal antibody was derived by immunizing mice with affinity-purified canine IgE.
  • the epitope bound by 15A.2 is conformational, not linear.
  • 15A.2 did not bind to the IgE receptor. Nor did it bind to IgE when bound to receptor.
  • 15A.2 exhibited affinity for free IgE. It binds to recombinant canine exon 3 fusion proteins in an enzyme linked immunosorbent assay (ELISA) and by Western blot, but not to other recombinant canine IgE fusion proteins.
  • ELISA enzyme linked immunosorbent assay
  • Monoclonal antibody 8H.8 also binds to canine IgE.
  • the 8H8 monoclonal antibody was derived by immunizing mice with a shortened version of exon 3 of the canine IgE molecule, designated exon 3a.
  • Exon 3a contains the C-terminal 71 amino acids of the full length exon 3. Previous studies (data not presented herein) had shown that immunizing mice with the full length exon 3 did not generate antibodies having specificity such as that ultimately found with the 8H8 antibody.
  • 15A.2 has a higher affinity for soluble native IgE
  • 8H.8 had a low affinity for soluble native IgE
  • a labeled antibody D9 was used as a means for detection in the studies depicted in Figure 5.
  • the monoclonal antibody D9 - is known to bind to IgE when bound to receptor.
  • the native IgE was present at a concentration of 2.5 micrograms per milliliter.
  • 15A.2 was effective at inhibiting binding of the native IgE to recombinant IgE receptor at antibody concentrations as low as 190 nanograms per milliliter.
  • 3,125 nanograms per milliliter of 8H.8 were necessary to inhibit the binding of native IgE to the recombinant IgE receptor.
  • 8H.8 had a lower affinity for the soluble IgE. It appears, therefore, that when a sufficient concentration of either 8H.8 or 15A.2 antibodies is provided, that the antibodies impair the binding of soluble IgE to the IgE receptor.
  • the region within native IgE recognized by 8H.8 might be partially hidden, particularly when the IgE is in serum.
  • a partially sequestered amino acid sequence of IgE may not be readily exposed to the native canine immune system and its normal protective mechanisms.
  • the full length exon 3 did not lead to the generation of antibodies having 8H.8-like specificity, the full length sequence might contain suppressor peptides capable of down- regulating or eliminating an auto-anti-IgE response which is specific against the epitope recognized by the 8H.8 antibody. Either mechanism could serve to avoid production of an autoimmune response to self-antigens .
  • 8H.8 antibody when used as an allergy therapeutic in dogs, would preferentially bind to IgE on memory B cells rather than to IgE in solution.
  • 8H.8 has a low binding affinity for IgE when bound by Fc receptor on mast cells.
  • Phage display technology was used to map the epitope bound by the 8H.8 antibody.
  • a preferred method for performing phage display technology is accomplished by use of a Ph.D. Phage Display Peptide Library Kit (New England BioLabs, Beverly Mass.) . It was found that a 7 amino acid peptide (Thr-Leu-Leu-Glu-Tyr-Arg-Met) (SEQ ID NO: 4) inhibited monoclonal antibody 8H.8 from competitive binding to either native or recombinant canine IgE. This sequence contained 6 amino acids common in form and/or spacing to the C-terminal region of exon 3.
  • E3a.5 An 11 amino acid peptide synthesized from this region (Gly-Met-Asn-Leu-Thr-Trp-Tyr-Arg-Glu-Ser-Lys) (SEQ ID NO: 5) designated E3a.5 also inhibited monoclonal antibody 8H8 from competitive binding to native or recombinant IgE.
  • IgE recognized by 15A.2 might serve as an immunogen to induce auto-anti-IgE responses and might generate antibodies capable of binding to IgE+ B cells and of down regulating IgE synthesis.
  • 15A.2 antibody when used as an allergy therapeutic in dogs, would prevent IgE from binding to mast cells and potentially affect the synthesis of IgE by B cells.
  • Phage display technology was used to map the epitope bound by the 15A.2 antibody. As noted above, a preferred method for performing phage display technology is accomplished by use of a Ph.D. Phage Display Peptide Library Kit (New England BioLabs, Beverly Mass.) .
  • Figure 6 depicts the amino acid sequences of the PhDc7c library that were bound by the monoclonal antibody 15A.2. These sequences are shown in alignment with the protein sequence of canine IgE.
  • Figure 7 depicts the alignment of the 15A.2 epitope from seven different mammals: dogs, human, green monkey, cat, swine, mouse and horse .
  • Figure 8 portrays the ability of different phage displaying 15A.2 mimotope peptides to inhibit canine IgE from binding the 15A.2 monoclonal antibody on the solid phase.
  • 466 ⁇ l of biotinylated 15A.2 antibody (10 ⁇ g/ml) were mixed with 466 ⁇ l of phage from a fresh overnight culture.
  • the antibodies and phage were allowed to bind for 2 % hours.
  • the wells were then coated with strepavidin.
  • the plates were washed three times with standard wash buffer to remove loosely bound material.
  • a serial dilution of canine IgE was prepared, starting with the concentration of 1 ⁇ g/100 ⁇ l of PBS, o.l% Tween. 100 ⁇ l of dilution IgE was added per well and allowed to bind for 10 minutes. The competition reaction was stopped by washing the plates five times with wash solution. The plate was then developed with HRPO linked D9 monoclonal antibody, which binds to domain 4 of IgE, to visualize the IgE in the solid phase. Loss of signal indicates the phage successfully repelled canine IgE competition. Table 1 shows the concentrations of the reactants at the various points on the x-axis of figure 8.
  • Figure 9 displays the ability of 15A.2 monoclonal antibody to bind a synthetic peptide.
  • the peptide SEQ ID NO: 12 Ser-Val-Thr-Leu-Cys-Pro-Asn-Pro-His-Ile-Pro-Met-Cys-Gly- Gly-Gly-Lys was synthesized and biotinylated on the epsilon carbon of the Lys residue. This sequence corresponds to the isolate M13-48.
  • the peptide was treated in a manner to promote reduction and cyclization of the cysteines to form a cyclic peptide.
  • Figures 10A and 10B depict the ability of a 15A.2 mimotope peptide to prevent the 15A.2 monoclonal antibody from binding canine IgE on the solid phase. Plates were coated with canine IgE at a concentration of 1 ⁇ g/ml. A serial dilution of the synthetic peptide was added to HRPO conjugated 15A.2 monoclonal antibody (final concentration 1 ⁇ g/ml) and allowed to bind for two hours. 100 ⁇ l of the 15A.2/peptide mixture was added to the wells and allowed to bind 1 hour. The reaction was stopped by washing the plates six times with wash buffer. The plates were developed with HRPO substrate, the reactions stopped with stop mix, and the plates were measured for O.D. using a plate reader.
  • this invention also comprises a specific binding molecule that specifically binds a ligand of the type bound by 15A.2 or 8H.8, as well as the therapeutic or prophylactic use thereof.
  • the invention comprises a monoclonal antibody specific for a conservative variant of a sequence bound by 15A.2, for a conservative variant of a sequence bound by 8H.8, or for a native sequence of canine IgE up to 100 amino acids from the C terminal portion of exon 3.
  • the specific binding molecules of the invention can be used in a method in accordance with the invention as a treatment for or as prophylaxis for allergy symptoms, i.e., passive immunization.
  • administration to a dog of either an antibody in accordance with the invention, such as 15A.2 or 8H.8, or a peptide in accordance with the invention, such as the 7, 11 or 12 amino acid peptide leads to a diminution of further IgE production. This may occur, for example, by the 15A.2 or 8H.8 antibody binding to the memory B cell and preventing further IgE production.
  • a peptide its administration would lead to production of antibodies of comparable specificity and effect as 15A.2 or 8H.8.
  • FIG. 11 depicts results of the binding study where antibody 8H.8 and antibody 15A.2 were separately assayed to determine their ability to bind to a solid phase having affinity purified canine IgE immobilized thereon. These results showed that those 8H.8 and 15A.2 bind to native IgE when immobilized on a solid phase. The results from these studies suggest that the binding of each of these antibodies to the surface bound IgE was of comparable affinity. To determine whether the affinity of binding of each antibody was, in fact, comparable, further studies were performed.
  • Figure 12 depicts results of the study where antibody 8H.8 conjugated to a signal moiety was reacted at various concentration with solid phases having various peptides immobilized thereon.
  • the data depicted by ( ⁇ ) reflects a solid phase having canine IgE immobilized thereon; data depicted by ( ⁇ ) depicts solid phase having E3a.5 immobilized thereon; data depicted by ( ⁇ ) depicts solid phase have E3a.5 extended data for a solid phase having an E3a.5 scrambled peptide immobilized thereon, data depicted by (*) depicts data for solid phase having the seven amino acid peptide identified by phage display technology to which SH8 binds immobilized thereon.
  • 8H.8 binds to each solid phase with the exception solid phase having the E3a.5 scrambled peptide.
  • Monoclonal antibody 14K2 is known to bind exclusive to canine IgE exon 4. This monoclonal antibody was reacted with various solid phases having different peptides immobilized thereon.
  • the data depicted by ( ⁇ ) reflects a solid phase having canine IgE immobilized thereon; data depicted by ( ⁇ ) depicts solid phase having E3a.5 immobilized thereon; data depicted by ( ⁇ ) depicts solid phase have E3a.5 extended peptide immobilized thereon; data depicted by (X) depicts data for a solid phase having an E3a.5 scrambled peptide immobilized thereon; and data depicted by (*) depicts data for solid phase having the seven amino acid peptide identified by phage display technology to which 8H.8 binds immobilized thereon.
  • the data depicted by ( ⁇ ) reflects a solid phase having canine IgE immobilized thereon; data depicted by ( ⁇ ) depicts solid phase having E3a.5 immobilized thereon; data depicted by ( ⁇ ) depicts solid phase have E3a.5 extended peptide immobilized thereon, data depicted by an (X) reflects data for a solid phase having an E3a.5 scrambled peptide immobilized thereon, data depicted by (*) depicts data for solid phase having the seven amino acid peptide identified by phage display technology to which 8H.8 binds immobilized thereon.
  • Figure 15 depicts data for studies that compare the binding of 8H.8 or polyclonal antibodies raised to recombinant human IgE, to peptide E3a.5 or to peptide E3a.5 with an amino acid substitution making the peptide more analogous to a sequence in the human genome which encodes human IgE, designated peptide S3a.5.
  • Data depicted by ( ⁇ ) reflects data for a solid phase having canine IgE immobilized thereon; data depicted by ( ⁇ ) reflects data for a solid phase having a peptide E3a.5 extended immobilized thereon; data depicted by ( ⁇ ) reflects data for a solid phase having human IgE immobilized thereon and, data depicted by an (X) reflects data for a solid phase having a 8H.8 peptide immobilized thereon.
  • 8H.8 binds to canine IgE, peptide E3a.5 extended or the seven amino acid peptide identified by phage display technology as the binding epitope for 8H.8, when each of these peptides were immobilized on a solid phase. Additionally, it is seen that 8H.8 does not bind to human IgE.
  • Figure 17 depicts data for studies which compare the ability of polyclonal anti human IgE antibodies ability to bind to solid phases having various peptides immobilized thereon.
  • Data depicted by ( ⁇ ) reflects data for a solid phase having canine IgE immobilized thereon; data depicted by ( ⁇ ) reflects data for a solid phase having a peptide E3a.5 extended immobilized thereon; data depicted by ( ⁇ ) reflects data for a solid phase having human IgE immobilized thereon and, data depicted by an (X) reflects data for a solid phase having a 8H.8 peptide immobilized thereon.
  • amino acid sequences for the epitope bound by 8H.8, the 11 amino acid sequence used in the immunization studies presented herein, as well as analogous sequences from feline and Human IgE sequences are set forth in Table 2. Additionally, a substituted canine sequence was prepared having an amino acid substitution which made the peptide more closely analogous to the human peptide.

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Abstract

Cette invention concerne des protéines de liaison qui se lient spécifiquement l un peptide isolé et purifié comprenant une leucine positionnées entre duc liaisons peptidiques à l'écart d'une paire tyrosine-arginine. Parmi les autres protéines de liaison selon l'invention figure celles qui se lient spécifiquement à une peptide isolée et purifiée avec la séquence cystéine-blanc-blanc-proline-histidine-blanc-blanc-blanc-cystéine (SEQ ID NO:6) ou cystéine-blanc-proline-histidine-blanc-proline-blanc-blanc-cystéine (SEQ ID NO:9).
PCT/US2000/008347 1999-03-30 2000-03-30 Proteines de liaison specifiques pour le traitement d'allergies WO2000058365A1 (fr)

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AU40449/00A AU757334B2 (en) 1999-03-30 2000-03-30 Specific binding proteins for treating canine allergy
CA2329148A CA2329148C (fr) 1999-03-30 2000-03-30 Proteines de liaison specifiques pour le traitement d'allergies
JP2000608657A JP2002539818A (ja) 1999-03-30 2000-03-30 イヌのアレルギーを治療するための特異的結合蛋白
EP00919828A EP1082346A1 (fr) 1999-03-30 2000-03-30 Proteines de liaison specifiques pour le traitement d'allergies

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JP2006151880A (ja) * 2004-11-30 2006-06-15 Asahi Breweries Ltd 抗イヌＩｇＥモノクローナル抗体、その製造法および用途
SG10201400388QA (en) * 2008-12-09 2014-05-29 Pfizer Vaccines Llc IgE CH3 peptide vaccine

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US7393529B2 (en) 1998-04-09 2008-07-01 Idexx Laboratories, Inc. Methods and compositions for inhibiting binding of IgE to a high affinity receptor
US7931898B2 (en) 1998-04-09 2011-04-26 Idexx Laboratories, Inc. Methods and compositions for inhibiting binding of IgE to a high affinity receptor
US8252907B2 (en) 1998-04-09 2012-08-28 Idexx Laboratories, Inc. Methods and compositions for inhibiting binding of IgE to a high affinity receptor

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