EP1082340A1 - Peptid impfstoff gegen hunde- allergie - Google Patents
Peptid impfstoff gegen hunde- allergieInfo
- Publication number
- EP1082340A1 EP1082340A1 EP00919927A EP00919927A EP1082340A1 EP 1082340 A1 EP1082340 A1 EP 1082340A1 EP 00919927 A EP00919927 A EP 00919927A EP 00919927 A EP00919927 A EP 00919927A EP 1082340 A1 EP1082340 A1 EP 1082340A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- peptide
- ige
- blank
- proline
- cysteine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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- C—CHEMISTRY; METALLURGY
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- C—CHEMISTRY; METALLURGY
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- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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 com lement. 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 defferent degrees of sensitivity to canine IgE and IgG have been identified. (DeBoer, et al . Immunology and Immunopathology 37, 183-199 (1993).) DeBoer, et al . identified several monoclonal antibodies which had cross reactivity between IgG and IgE.
- DeBoer et al . Three monoclonal antibodies (A5, D9, and B3 ) were identified by DeBoer et al . , as having some affinity for canine IgE. Of the -monoclonal antibodies identified in DeBoer et al . , antibody D9 appeared to have the greatest degree of neutralization of Prausnitz-Kustner reactivity for atopic dog serum.
- DeBoer et al proposed use of their monoclonal antibodies (MAbs) in the use of antigen-specific IgE ELISA, and for quantifying canine IgE. Additionally, they proposed use of their MAbs for immunostaining of Western Blot assays, to evaluate the molecular specificity of IgE antibodies, as well as for in vi tro studies on degranulation of mast cells.
- MAbs monoclonal antibodies
- 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 .
- T cells In contrast to the situation with T cells, it is believed that there are many B cells which express receptors (i.e., surface immunoglobulin) for self-proteins , and that the reason these cells do not produce antibodies to self-proteins is because the T cells required for the antigen presentation to the B cell are normally missing.
- receptors i.e., surface immunoglobulin
- 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 .1 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 . (Tarn, J.P., Proc. Natl . Acad. Sci . U.S.A.
- 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.
- 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 : 1 cysteine-blank- blank-proline-histidine-blank-blank-blank-cysteine .
- the cysteines may form a covalent bond through a reduction reaction, cyclizing the peptide and becoming cystine.
- a peptide in accordance with the invention can comprise from 9 to 71 amino acids.
- 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 : 2 cysteine-blank-proline-histidine- blank-proline-blank-blank-cysteine .
- the cysteines may form a covalent bond through a reduction reaction, cyclizing the peptide and becoming cystine.
- a peptide in accordance with the invention that has the amino acid sequence, or a conservative variant thereof, which comprises: SEQ ID NO : 3 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 peptide in accordance with the invention which has the amino acid sequence, or a conservative variant ther ⁇ of, which comprises: SEQ ID NO : 4 serine-alanine-cysteine- histidine-proline-histidine-1eucine-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 peptide in accordance with the invention which has the amino acid sequence, or a conservative variant thereof, which comprises: SEQ ID NO : 5 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.
- an isolated and purified peptide comprising a leucine positioned two peptide bonds away from a tyrosine- arginine pair, or a conservative variant thereof; the peptide can have the form: SEQ ID NO: 6 leucine-blank-blank-tyrosine-arginine, SEQ ID NO: 7 tyrosine-arginine-blank-blank-leucine, or, SEQ ID NO : 8 leucine-blank-blank-tyrosine-arginine-blank-blank-leucine, where blank is any amino acid. At least one blank is preferably an amino acid with an aromatic ring.
- a peptide in accordance with the invention can comprise from 5 to 71 amino acids .
- a peptide in accordance with the invention that has the amino acid sequence, or a conservative variant thereof, which comprises: SEQ ID NO : 9 threonine-leucine- leucine-glutamic acid-tyrosine-arginine-methionine.
- a peptide in accordance with the invention that has the amino acid sequence, or a conservative variant thereof, which comprises: SEQ ID NO: 10 glycine-methionine- asparagine-leucine-threonine-1ryptophan-tyrosine-arginine- glutamic acid-serine-lysine .
- a peptide in accordance with the invention, or a conservative variant thereof, can be linked to a multiply antigenic peptide which comprises multiple copies of the peptide, or various peptides in accordance with the invention; the multiply antigenic peptide can comprise multiple copies of conservative variants of peptides in accordance with the invention.
- a peptide in accordance with the invention, or a conservative variant thereof, can be linked to a plant virus particle which comprises multiple copies of the peptide, or various peptides in accordance with the invention; the plant virus particle can comprise multiple copies of conservative variants of peptides in accordance with the invention.
- a method for generating canine autoantibodies directed to the canine IgE molecule comprising providing a peptide in accordance with the invention or a conservative variant thereof, and administering the provided peptide to a dog.
- the method can further comprise a step of mixing the provided peptide with an adjuvant prior to the administering step, wherein the administering step comprises administering the mixture of the peptide and the adjuvant.
- the mixing step can comprise mixing the peptide with the adjuvant at a weight ratio of about one part peptide to five parts adjuvant.
- the method can further comprise a step of administering a booster dose of the provided peptide, following the administering step. Treatment or prophylaxis of canine allergy is accomplished by performing a method in accordance with the invention.
- an immunogenic molecule comprising a peptide in accordance with the invention or a conservative variant of a peptide in accordance with the invention linked to a carrier molecule capable of facilitating an immune response.
- the carrier molecule can be KLH, BSA, MLA, a multiply antigenic peptide, or a plant virus particle.
- peptides that specifically bind a peptide in accordance with the invention; disclosed are peptides that are raised to and specifically bind a peptide in accordance with the invention.
- 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 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
- 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. For example, 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 Seguence 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 D ⁇ A sequence of nucleotides that controls and regulates expression of structural genes when operatively linked to those genes.
- Exon A contiguous region of D ⁇ A encoding a portion of a polypeptide.
- Genome The entire D ⁇ A 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 DNA or RNA consisting of a sugar moiety (pentose) , a phosphate, and a nitrogenous heterocyclic base.
- the four DNA bases are adenine ( "A” ) , guanine ("G”), cytosine ("C”) and thymine (“T”).
- the four RNA 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 DNA sequences encapsidated in a protein envelope or coat ("capsid") .
- Plasmid An autonomous self-replicating extrachromosomal circular DNA.
- PCR Polymerase Chain Reaction
- 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 mRNA into amino acid sequences. For example, the sequence
- GCTGGTGTAAG may be translated in three reading frames or phases, each of which affords a different amino acid sequence:
- 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 Molecul e 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 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 England Biolabs PhDc7c phage display library that were bound by the monoclonal antibody 15A.2. These sequences are shown in alignment with the protein sequence of canine IgE.
- 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 data showing serum IgE concentration of immunized dogs.
- Pre-bleed data is depicted by dark speckled bar graphs
- "immunized” data is depicted by solid black bar graphs
- "boosted” data is depicted in open bar graphs
- data for samples taken 10.24.97 is depicted in lightly speckled bar graphs.
- Fig. 12 depicts the level of auto anti-IgE in dogs over time, for dogs 1 through 4.
- Data for pre-bleed samples is depicted by darkly speckled bar graphs
- data for "immunized” samples is depicted by solid black bar graphs
- data for "boosted” samples is depicted by open bar graphs
- data for samples taken 10.24.97 is depicted by lightly speckled bar graphs .
- Fig. 13 depicts the ability of immune serum, from dog 4, to inhibit conjugated 8H.8 from binding to native canine IgE immobilized on a solid phase.
- Fig. 14 depicts the ability of monoclonal antibody 8H.8 and monoclonal antibody 15A.2 to bind to purified canine IgE immobilized on a solid phase.
- Fig. 15 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. 16 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. 17 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.18 depicts data for studies that compared the binding of monoclonal 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. 19 depicts data from a study evaluating the ability of antibody 8H .8 conjugated to a signal moiety to bind to solid phases having various peptides immobilized thereon.
- Fig. 20 depicts the ability of polyclonal anti-human IgE antibodies to bind to various peptides immobilized on a solid phase. MODES FOR CARRYING OUT INVENTION
- 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 B cell.
- the serum levels of IgE are elevated in patients experiencing allergic disease.
- antibodies were generated that specifically bind to a species' IgE, and 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.
- 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").
- a self-peptide such as an IgE molecule
- 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.
- the approach followed in accordance with the present invention is the development of antibodies, either ex vivo monoclonal or in vivo 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 the 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 8H.8 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 8H.8 antibody.
- the affinity of 8H.8 for soluble canine IgE was lower than the affinity of this monoclonal for IgE on a sold phase.
- 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.
- 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
- 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 caninelgE.
- Figure 7 depicts the alignment of the 15A.2 epitope region from seven different mammals: dog, 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 phase from, a fresh overnight culture.
- 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 biotinylateed 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.
- a serial dilution of the peptide was prepared and bound to strepavidin-coated microtiter plates (5 ⁇ g strepavidin per well) . Bound peptide was detected with HRPO conjugated 15A.2 monoclonal antibody.
- Figure 9 shows the results of this experiment. This solid phase specific 15A.2 mimotope peptide bound the 15A.2 monoclonal antibody in a concentration dependent manner.
- 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.
- an auto-anti-IgE response by immunizing an animal with a peptide derived from the sequence of its own IgE, along with a peptide that might associate with an MHC molecule.
- the size and sequence of the IgE-derived peptide and/or the product of its internal processing in antigen presenting B cells may represent a unique sequence, which when associated with MHC effectively appears foreign to T cells.
- a unique auto-anti-IgE specificity may, therefore, be generated.
- the resulting immune response serves to target the IgE-expressing B cell. This targeting affects IgE synthesis by down regulating IgE synthesis from B cells, and/or targets the IgE+ B cell for destruction.
- the present invention comprises an 11 amino acid peptide with the sequence SEQ ID NO: 10 Gly-Met -Asn-Leu-Thr-Trp-Tyr-Arg-Glu-Ser-Lys .
- Alternative preferred embodiments in accordance with the invention comprise an isolated and purified peptide of five or more amino acids which when administered to a dog, either alone or with an adjuvant or carrier, induce the production of antibodies directed to that dog's IgE when in solution but with low binding affinity when the IgE is bound to mast cells.
- a native sequence of amino acids from the C-terminal region of canine IgE exon 3, of from 5 to 71 amino acids, or a conservative variant of such a sequence is used.
- Particularly preferred is an 11 amino acid stretch of exon 3, designated peptide E3a.5.
- All peptides, including native sequences and conservative variant sequences derived from peptides which bind the 8H.8 monoclonal antibody preferably contain at least one leucine positioned two peptide bonds away from a tyrosine-arginine pair, i.e., contain a sequence in the form of: SEQ ID NO : 6 L-blank-blank-Y-R, SEQ ID NO : 7 Y-R-blank-blank-L, or, SEQ ID NO: 8 L-blank-blank-YR-blank-blank-L.
- Blank can be any amino acid; preferably at least one blank in a particular sequence is an amino acid with an aromatic ring.
- a host is actively immunized against allergic manifestations by administration of a peptide containing molecule which is able to induce the formation of antibody capable of binding circulating IgE and/or down regulating the synthesis of endogenous IgE.
- This reduction in the serum level of IgE reduces, or eliminates, allergic responses in these animals.
- an amino acid sequence in accordance with the invention induced an auto-anti-IgE response in dogs when they were immunized with the sequence.
- the auto-anti-IgE response reduced the level of serum IgE measured in these dogs.
- This reduction in the serum level of IgE reduces, or eliminates, allergic responses in these animals.
- a defined auto-anti-IgE response was achieved upon administration of the peptides in accordance with the invention.
- This auto-anti-IgE response decreased the serum IgE levels in the recipient dogs.
- the identified peptide is used to induce these responses in puppies as a prophylaxis for allergy.
- these peptides are administered to any dog that is experiencing clinical manifestations of allergy.
- peptides in accordance with the invention are administered to those dogs experiencing allergic dermatitis.
- Alternative preferred embodiments in accordance with the invention comprise an isolated and purified peptide of 12 or more amino acids which when administered to a dog, either alone or with an adjuvant or carrier, induces the production of antibodies directed to that dog's IgE when in solution.
- a sequence of amino acids designated SEQ ID NO: 11 Val-Thr-Leu-Cys-Pro-Asn-Pro-His-Ile- Pro-Met-Cys, or a conservative variant of such a sequence is used.
- All peptides, including native sequences and conservative variant sequences derived from peptides that bind the 15A.2 monoclonal antibody preferably contain at least one cysteine- two peptide bonds from a proline-histidine pair, which is three spaces from a cysteine, or contain the sequence cysteine-one peptide bond from a proline-histidine pair, one peptide bond from a proline, which is two peptide bonds from a cysteine, i.e., contain a sequence in the form of: SEQ ID NO:l
- Blank can be any amino acid.
- a host is actively immunized against allergic manifestations by administration of a peptide-containing molecule that is able to induce the formation of antibody capable of binding circulating IgE and/or down regulating the synthesis of endogenous IgE. This reduction in the serum level of IgE reduces, or eliminates, allergic responses in these animals.
- a specific binding molecule such as monoclonal antibody 15A.2 or 8H.8, provides an anti-IgE moiety in dogs.
- This antibody either alone or in combination, is used as a prophylactic agent for allergy, or is used therapeutically for dogs experiencing allergic disease, and effectively reduces or eliminates IgE-mediated allergic disease.
- T Cell Dependent Generation of Antibodies As discussed below, there are mechanisms to induce antibodies that are independent of a need for T cell interactions. However, for T cell dependent mechanisms, if the patient's IgE antibody, recognized by its own B cell, should hold a foreign antigen in its binding site, then this foreign antigen will be internalized and processed in the same way that the IgE is internalized and processed. Peptides derived from this processed foreign antigen, in association with MHC, will therefore be presented on the surface of the B cell. Accordingly, a B cell reactive to a self-molecule will now also be presenting epitopes that are recognized by T cells initially responsible for mounting an immune response to a foreign antigen. These T cells will now help this auto-reactive B cell to produce auto-reactive antibodies.
- a peptide immunogen possibly a hapten, which defines an epitope recognized by a B cell
- a carrier protein for example, the epitope recognized by the B cell can be an epitope on surface-bound IgE.
- the carrier protein is KLH or BSA.
- a large carrier protein, such as KLH, provides T cell epitopes required for the generation of an immune response directed against the hapten.
- the carrier protein contains peptide epitope sequences that activate T cells; in turn, these activated T cells help to generate an immune response to the conjugated peptide.
- An alternative mechanism for inducing T cell dependent activation of B cells is to prepare a recombinant fusion protein having the epitopes recognized by the B cell, e.g. self IgE epitopes, translated along with epitopes bound by T cells.
- antibody epitopes are inserted into the coat proteins of a cow pea virus.
- the virus is thus able to express these peptides on its surfaces, and present them to the immune system in order to induce antibody production.
- a perceived advantage of this embodiment is that the plant virus proteins are recognized as foreign by the immunized animal.
- the availability of epitopes on the virus particles bound by T cells induces a T cell dependent antibody response against the inserted self-peptides of interest.
- both B and T cell responses to the recombinant virus particle containing IgE epitopes from a given species are generated. Accordingly, a particularly effective mechanism for achieving auto-immunity to IgE is provided; this autoimmunity serves to lessen or eliminate allergic symptoms.
- Multiply antigenic peptides were synthesized which contained four copies of the 11 N-terminal amino acids of canine IgE exon 3, i.e., a 4 -MAP peptide.
- a 4 -MAP peptide was synthesized which contained four copies of the 11 N-terminal amino acids of canine IgE exon 3, i.e., a 4 -MAP peptide.
- 4-MAP peptide comprising the 11 amino acid N-terminal region of exon 3 discussed herein, a T-cell independent induction of an immune response is believed to occur.
- Each 4-MAP peptide contains four identical peptide sequences of interest. This structural configuration provides for the possibility that B cells that recognize this structure may become activated in a T cell-independent fashion.
- An individual 4-MAP peptide has the ability to bind to multiple receptors on a memory B cell, and thereby to cross-link the receptors, whereupon signals that lead to the activation, maturation and elaboration of soluble auto-anti-IgE molecules from the B cell are induced without the need for T cell interactions .
- the large carrier protein such as KLH
- KLH provides T cell epitopes required for the generation of an immune response directed against the hapten.
- MAP peptides or an MLA adjuvant
- these T cell epitopes are not present.
- the resulting immune response is independent of T cells.
- Each serum sample was assayed for IgE concentration as well as auto anti-IgE activity.
- IgE concentration was measured using the Quik IgE ELISA assay. Tables 3 and 4 show results obtained from a single assay. Figure 11 graphically depicts these same results. IgE levels decreased from 50% to 71% at the time of the first bleed, which was 3 weeks post immunization ("immunized” data). Three weeks later, 3 of 4 dogs showed a slight rebound in IgE concentration, (although still depressed from pre-bleed levels) , while the serum IgE of dog #2 continued to drop ("boosted” data). The IgE concentration in all experimental animals at the time of the final bleed was significantly lower than the pre-bleed and ranged from 65%; to 82% ("10.24.97" data) .
- the 8H.8 antibody was initially raised to the exon 3a peptide; thus, the epitope bound by 8H.8 is available on the exon 3a peptide. Accordingly, administration of the exon 3a peptide in the Pmal fusion protein also served to administer the peptide sequence of the peptide E3a.5 in the Pmal form. Thus, the more significant reduction in IgE levels for dogs 3 and 4 appears to be correlated to the multiple antigenic peptide format in which the E3a.5 peptide was administered. Moreover, auto anti-IgE antibody concentration was determined by ELISA. Purified canine IgE was coated on ELISA plates and the serum from the immunized animals was incubated on the solid phase overnight.
- Auto anti-IgE antibodies were detected using an horseradish peroxidase (HRPO) labeled anti- canine IgG antibody designated 14A.3. Auto anti-IgE levels were not quantitated, but absorbance values were compared between pre-bleed samples and serum obtained from immunized animals .
- HRPO horseradish peroxidase
- Figure 12 presents absorbance values representing auto anti-IgE antibody concentration in the experimental animals.
- Positive and negative controls consisted of wells pre-incubated with 10 ⁇ g,l monoclonal antibody 14A.3 (neg. control) or 10 ng/ml monoclonal antibody 8H.8 (positive control).
- Monoclonal antibody 14A.3 is known to recognize canine IgG.
- the wells were washed and a serial, 2-fold dilution of HRPO-conjugated monoclonal antibody 8H.8 starting at l ⁇ g/ml was added to each pre-incubated well. After one hour the plates were washed and a standard ELISA performed.
- Figure 14 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 15 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 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 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 an (X) reflects 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 18 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 20 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 phaae 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.
- the anti-human IgE polyclonal antibodies bound to human IgE when immobilized on a solid phase. It was also seen that the polyclonal antiserum exhibited limited binding to peptide E3a.5 extended when the polyclonal antibodies were present at very high concentrations; this was believed to be a binding artifact due to the high antibody concentration. Thus, it was found that antibodies to Human IgE do not specifically bind to canine JgE or to peptides in accordance with the invention.
- Example 2 This Example repeats the study of Example 1, along with additional control substances.
- the 4 -MAP E3a.5 peptide formulation used in Example 1 is used along with derivatives of this peptide.
- Two peptides (E3a.5 scrambled, and TM#2 FIV) serve as the negative controls.
- This example also evaluates an extended peptide from the C-terminal region from exon 3 of the IgE heavy chain for an ability to generate an auto anti-IgE response. All peptides are administered to the dog in a 4 -MAP configuration .
- Dogs immunized with these peptides are monitored for the presence of auto anti-IgE antibodies and circulating IgE concentration. Additionally, receptor-bound IgE on mast cells is evaluated by measuring the skin test response of these animals with crosslinking antibodies.
- the E3a.5 peptide was used in conjunction with the KLH carrier molecule in order to evaluate T cell dependent immune responses to self peptides derived from IgE.
- Appropriate negative controls for the canine peptide e.g., TM#2 FIV, are also used.
- a carrier protein such as KLH
- KLH adjuvant serves to evaluate a classic T cell dependant immune response.
- This example therefore, evaluates T cell dependant immune response.
- four dogs are evaluated: two 2 dogs per immunogen, as follows:
- E3a.5-KLH molecule induces auto-anti IgE immunoglobulin production .
- the dogs to which TM#2 FIV are administered show no IgE response.
- the FIV-KLH molecule induces antibody production specific for the FIV peptide, but produces no change in serum IgE levels.
- Example 4 the E3a.5 peptide was used in conjunction with a carrier molecule in order to evaluate T cell independent immune responses to self peptides derived from IgE.
- Appropriate negative controls for the E3a.5 peptide e.g., TM#2 FIV
- a carrier protein such as KLH
- KLH provides T cell epitopes required for the generation of an immune response directed against the hapten.
- MAP peptides, or the MLA adjuvant these epitopes are not present.
- any resulting immune response is independent of helper T cells.
- This example therefore, evaluates T cell independent immune response.
- four dogs are evaluated: two 2 Dogs per immunogen, as follows:
- the E3a.5 peptide was used in conjunction with a carrier molecule derived from a plant virus protein, in order to evaluate T cell dependent immune responses to self peptides derived from IgE.
- Appropriate negative controls for the E3a.5 peptide e.g., TM#2 FIV, are also used in the same plant virus particle format.
- a carrier protein such as KLH
- KLH provides T cell epitopes required for the generation of an immune response directed against the hapten.
- the present approach for generating antibodies to peptide antigens involves placing multiple copies of a peptide in accordance with the invention on the surface of plant virus particles.
- EPICOATTM technology (Axis Genetics pic, Cambridge, England) is the presently preferred system for producing such particles.
- the EPICOATTM technology is based on chimeric virus particle (CVP technology which utilizes the recombinant genetic modification of plant viruses .
- 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 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, i.e., a peptide in accordance with the invention, in a prominent position on the virus surface, whereby up to sixty copies of the peptide are 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.
- the leaves are gently rubbed, and 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.
- CVPs Chimeric virus particles
- cow pea plants are readily grown in abundance in controlled environments, allowing generation of large quantities of CVPs containing many copies of peptides in accordance with the invention.
- T cell dependent immune response evaluates T cell dependent immune response.
- the T cell binding epitopes are provided by the viral proteins, and it is believed that these viral proteins are potent inducers of T cell dependent immunological mechanisms.
- ten dogs are evaluated: two dogs per immunogen, as follows:
- the dogs to which TM#2 FIV in CVP carrier are administered show no IgE response.
- the FIV-CVP molecule induces antibody production specific for the FIV peptide, but produces no change in serum IgE levels.
- 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 10. 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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US281761 | 1999-03-30 | ||
PCT/US2000/008523 WO2000058349A1 (en) | 1999-03-30 | 2000-03-30 | Peptide vaccine for canine allergy |
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JP4571952B2 (ja) * | 2004-02-05 | 2010-10-27 | アクセス ビジネス グループ インターナショナル リミテッド ライアビリティ カンパニー | 抗アレルギー組成物及び関連方法 |
JP2007526919A (ja) * | 2004-03-05 | 2007-09-20 | バイオ−ラッド ラボラトリーズ,インコーポレイティド | 糖タンパク質g2ペプチドを用いたhsv−2型特異的免疫検定 |
EP1872792A1 (de) | 2006-06-29 | 2008-01-02 | Biotech Tools S.A. | Methode zur Herstellung eines hydrolysierten Allergens |
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