Vaccine

专利摘要:
The present invention relates to isolated polypeptides useful for immunization against self-antigens. In particular, the present invention relates to self-proteins capable of producing self-antibodies when administered in vivo. The present invention relates in particular to making human cytokines immunogenic in humans. The present invention also relates to pharmaceutical compositions comprising such compounds and to the use of such compounds in the medical field and to methods of making such compounds.
公开号:KR20030081490A
申请号:KR10-2003-7011573
申请日:2002-03-01
公开日:2003-10-17
发明作者:클레어 애쉬만；제임스 스코트 크로웨；조나단 헨리 엘리스；앨런 피터 루이스
申请人:글락소 그룹 리미티드；
IPC主号:

专利说明:

Vaccine {VACCINE}
[2] Asthma is a chronic lung disease caused by inflammation of the lower respiratory tract and is characterized by recurrent dyspnea. The patient's airway is sensitive and swelling or inflammation to some extent, even if there are no symptoms. Inflammation narrows the airway, reducing the inflow and outflow of airflow into the lungs, resulting in shortness of breath, causing wheezing, and chest compressions and coughing. Asthma is triggered by hypersensitivity to allergens (eg dust mites, pollen, mold), irritants (eg smoke, vapor, strong odors), respiratory infections, exercise and dry weather. These factors cause the airways and the lining of the airways to swell and even become inflamed, after which breathing becomes difficult and difficult, mucus obstructs the airways until symptoms of asthma and muscles around the airways are tightened.
[3] COPD is a generic term used to describe a respiratory disease that exhibits symptoms similar to asthma and is treated with the same medications used to treat asthma. COPD is characterized by chronic progressive, mainly irreversible airflow obstruction. Individual factors contributing to the progression of this disease are unknown, but smoking is believed to account for 90%. Symptoms include cough, chronic bronchitis, dyspnea and respiratory infusion. Eventually, this disease will result in serious disability and death.
[4] Due to various problems related to the production, administration and tolerance of monoclonal antibodies, there is a growing interest in how to build patient-specific immune systems for inducing endogenous antibodies with suitable specificity by vaccination. However, mammals generally do not contain high titers of antibodies to self-proteins present in serum because the immune system has homeostatic mechanisms that inhibit their formation. The importance of this tolerance mechanism has been demonstrated by diseases such as myasthenia gravis, in which auto-antibodies are induced by the nicotine acetylcholine receptors in skeletal muscle, causing asthenia and fatigue (Drachman, 1994, N Engl J Med 330: 1797-1810). ). Therefore, there is a need for a vaccine method that can avoid antibody tolerance mechanisms without inducing self-antibody-mediated pathology.
[5] Many methods have been devised to destroy B cell tolerance without causing unacceptable autoimmune toxicity. However, all of these have significant disadvantages.
[6] One technique involves chemically crosslinking a self-protein (or peptide derived therefrom) to a highly immunogenic carrier protein such as keyhole limpet haemocyanin (Antibodies: A laboratory manual "Harlow, E and Lane D. 1988, Cold Spring Harbor Press) This method is a modification of the widely used hapten-carrier system to produce antibodies against poorly immunogenic targets such as low molecular weight chemical compounds. The fusion process can destroy potentially valuable epitopes, and many of the induced antibody responses are derived from carrier proteins, in addition, these methods are only applicable to protein vaccination and are incompatible with nucleic acid immunogens.
[7] Modifications of the carrier protein technique include constructing genes encoding fusion proteins including both carrier proteins (eg, hepatitis B core protein) and self-proteins (The core antigen of hepatitis B virus). as a carrier for immunogenic peptides ", Biological Chemistry. 380 (3): 277-83, 1999). The fusion gene may be administered directly as part of a nucleic acid vaccine. Alternatively, it may be used in a suitable host cell in vitro. The gene product can be expressed and purified and delivered as a conventional vaccine with or without an adjuvant, but fusing the macrocarrier protein to the self-protein may press or distort the structure of the self-protein, The efficiency is reduced in inducing cross-reactivity of the antibody with the molecule, and many antibodies, like conventional crosslinked carrier systems, Response is induced against the carrier portion of the fusion, wherein - the carrier reaction may limit the effectiveness of subsequent booster dose of vaccine or increase the chance of allergic or anaphylactic reaction.
[8] More elaborate methods have been described by Dalum et al. Wherein one class II MHC-restricted epitope is inserted into a target molecule. Using these methods, they used ubiquitin (Dalum et al, 1996, J Immunol 157: 4796-4804; Dalum et al, 1997, Mol Immunol 34: 1113-1120) and cytokine TNF (Dalum et al, 1999, Nature Biotech 17 : 666-669). As a result, all T cell help arises from the sequences of these single epitopes or junctions. This method may be used in subjects with a suitable MHC class II haplotype for the designed vaccine or in a lucky subject with a class II molecule capable of actually binding to a conjugated epitope, but humans may be of any normal type, such as a typical population. In non-negative communities, the vaccine will not be effective in much of the population. In addition, because the inserted epitopes are typically derived from completely unrelated proteins such as ovabumin or lysozyme, the additional sequence to some extent interferes with the folding of the target protein, thus allowing the adoption of the fully natural conformation of the target protein. Will be suppressed.
[9] In contrast, the present invention provides a number of potential T cell epitopes that retain the target molecule in a conformation close to the native form. Due to these properties, the vaccine of the present invention acts as an effective immunogen in complex, non-inbred communities such as those composed of human patients. This property is achieved by mutating the self-protein, resulting in a sequence that can be found in similar proteins at that point.
[10] Many recent documents have identified an important role for the Th2 cytokine IL-13 in inducing pathology in the ovalbumin model of allergic asthma (Wills-Karp et al, 1998; Grunig et al, 1998). In this study, mice that had previously sensitized to ovalbumin were injected with soluble IL-13 receptors that bind to and neutralize IL-13. The treatment group completely eliminated airway hyperresponsiveness to acetylcholine. Treated mice reversed goblet cell metastasis as seen in the control group. In supplementary experiments, lung IL-13 levels were elevated by overexpression in transgenic mice or by installing proteins into the airways of wild type mice. In both settings, airway hypersensitivity, eosinophil invasion, and increased mucus production were observed (Zhu et al, 1999). These data show that IL-13 activity is sufficient and necessary to elicit several major clinical and pathological characteristics of allergic asthma in well-identified models.
[11] Thus, a vaccine capable of inducing a neutralizing response to IL-13 will constitute a useful therapeutic for the treatment of allergic asthma in humans. It will also be applied to the treatment of certain parasitic infection related diseases (Brombacher, 2000) and diseases associated with IL-13 production in fibrosis (Chiaramonte et al, 1999), such as chronic obstructive pulmonary disease. The present invention will meet this need.
[12] Thus, the concepts and theories of the present invention are described in connection with IL-13, but can be applied to all mammalian self-proteins having similar proteins in the second species.
[13] Summary of the Invention
[14] The present invention provides an isolated polypeptide that is 30% to less than 100% identical to a human protein.
[15] (a) contains one or more mutations that characterize similar proteins other than humans;
[16] (b) can produce antibodies in humans;
[17] (c) the antibody is structurally similar to a human protein sufficient to bind both a human protein and a polypeptide;
[18] (d) provides a polypeptide that is not an antibody.
[19] As such, the invention provides, in one embodiment, a protein having a B cell epitope from a mammalian self-antigen, and a mutation resulting in a similar protein sequence from a second mammalian species, wherein the protein is a B-cell epitope. In derived species, B-cell epitopes can elicit an immune response that recognizes the native protein from which it is derived.
[20] Preferably, the sequence of the analogous protein is five, more preferably more than eight consecutive amino acids. Thus, the proteins of the invention contain sequences identical to analogous sequences in five, preferably at least eight consecutive amino acids. In an alternative embodiment, there is provided a protein having a B cell epitope of its own protein, which is implanted by substitution into a framework of similar proteins from a second mammalian species, which protein is B-cell in the species from which the B cell epitope is derived. It can trigger an immune response that recognizes the natural protein from which the epitope is derived.
[21] It will be appreciated that the proteins of the invention are not antibodies.
[22] The resulting immune response is preferably an antibody response, most preferably a neutralizing antibody response.
[23] In general, mutations are preferably introduced into the surface unexposed areas of the molecule so that the surface exposed areas are preserved. Since the immune system can utilize surface exposed areas, these areas often contain B-cell epitopes. Accordingly, the present invention provides a protein comprising a conserved surface exposed area of a magnetic protein and wherein the mutation has been introduced into a non-surface exposed area, wherein the mutation is immune to self-protein in the species from which the magnetic protein is derived. Sequences of similar proteins are generated to trigger reactions.
[24] The magnetic protein is preferably a human protein, but may be a protein from any mammal which is desired to elicit an autoimmune response. The immune response is preferably specific for the natural proteins and immunogens of the invention. Such proteins have minimal crossreactivity or neutralizing capacity for other magnetic proteins.
[25] The magnetic antigen is preferably a cytokine, more preferably a four helix cytokine, more preferably IL-4 or IL-13, most preferably IL-13. Thus, in a preferred embodiment of the present invention there is provided a chimeric protein comprising B cell epitopes from human IL-13 provided in the murine IL-13 backbone. Such constructs can elicit specific anti IL-13 antibody responses in humans. This construct is shown in FIG. 9 (seq: ID No 21 and 22). Similarly, IL-4 constructs comprising human IL-surface regions and murine frameworks are shown in FIG. 13 (Seq ID: No 25).
[26] In addition, the present invention,
[27] An expression vector comprising a polynucleotide of the invention and capable of expressing a polypeptide of the invention;
[28] A host cell comprising the expression vector of the invention;
[29] A method of producing a polypeptide of the invention, the method comprising maintaining the host cell of the invention under conditions suitable for expressing the polypeptide of the invention and isolating said polypeptide; And
[30] Providing a vaccine composition comprising a polypeptide or polynucleotide of the invention and a pharmaceutically acceptable carrier.
[31] In another aspect, the invention provides a method of designing and preparing a polypeptide according to the invention,
[32] 1. identifying one or more regions of a magnetic protein, typically a human protein, wherein an antibody response is desired,
[33] 2. identifying the amino acid sequence of his protein,
[34] 3. Confirming by recombinant DNA techniques the amino acid sequence of the analogous protein construct of the chimeric molecule containing at least one target region identified in step 1, wherein the amino acid sequence is obtained from the sequence identified in step 2, wherein the mutant protein is self Providing a method comprising identifying an amino acid from the sequence (s) identified in step 3 sufficient to fold the resulting protein into a form similar to that of its protein so as to elicit an immune response that recognizes the protein. do.
[1] The present invention relates to an isolated polypeptide useful for immunity to self-antigens. In particular, the present invention relates to self-proteins which, when administered in vivo, can produce self-antibodies. The present invention relates in particular to methods for making human cytokines immunogenic in humans. The invention also relates to pharmaceutical compositions comprising such compounds and to the use of such compounds in medicaments and methods of making such compounds.
[35] GST = glutathione S-transferase, rmIL-13 = recombinant mouse IL-13, rhIL-13 = recombinant human IL-13, cIL-13 = chimeric IL-13
[36] 1 depicts the sequence of mouse chimeric IL-13 vaccine constructs. The underlined amino acid symbols represent the sequence of human IL-13, and the unmarked symbols are from murine IL-13.
[37] 2 shows analysis of GST cIL-13 by 4-20% Tris-glycine SDS-PAGE gel (Novex), stained for total protein with Coomassie Blue.
[38] 3 shows western blot analysis of GST-cIL-13.
[39] 4 shows ELISA analysis of the interaction of cIL-13 and GST-cIL-13 with anti-mIL-13 polyclonal antibodies, anti-hIL-13 polyclonal antibodies and anti-GST polyclonal antibodies. do.
[40] 5 shows an ELISA analysis of the interaction of cIL-13 and GST-cIL-13 with the mIL-13 receptor, mIL-13Rα1 and mIL-13Rα2.
[41] 6 shows anti-phospho-STAT6 western blot of A549 lysate.
[42] 7 shows antibody responses induced by immunization with GST-cIL-13 (mouse F5) or cIL-13 (mouse E5).
[43] 8 shows anti-phospho-STAT6 western blot analysis of A549 lysate.
[44] 9 shows a chimeric IL-13 vaccine for use in humans. The underlined amino acid symbols represent the sequences found in murine IL-13 and the symbols without any indication are from human IL-13.
[45] 10 depicts anti-mouse IL-13 antibody profile after administration of various adjuvants with cIL-13.
[46] 11 shows the serum neutralizing capacity of mice after administration of cIL-13.
[47] 12 depicts another cIL-13 used as a mouse immunogen.
[48] 13 depicts chimeric IL-4 used in human anti IL-4 vaccine.
[49] Throughout the specification and the appended claims, unless the context requires otherwise, the terms "comprise" or its derivative forms, such as "comprising" and the like, should be interpreted as comprehensive, ie, using these terms. Doing so implies that it may include an integer or element that is not explicitly mentioned.
[50] As described herein, the present invention relates to isolated polypeptides and isolated polynucleotides. In the context of the present invention, the term “isolated” means that the polypeptide or polynucleotide is not present in its natural state, at least to some extent, or as long as it is synthetically produced by, for example, recombinant or mechanical synthesis. It is intended to mean no. Thus, the term “isolated” means that the polypeptide or polynucleotide is a suspension of cells, cells or cell fragments, proteins, peptides, expression vectors, organic or inorganic solvents, or other biological or non-biological agents as appropriate. Includes the possibility of coexistence with a substance, except where the polynucleotide is present in a state found in nature.
[51] An advantage of the present invention is that the polypeptide of the present invention allows for folding the region of a self protein, eg, a human protein, in which an antibody response is desired, in a form that is sufficiently different from human protein but very similar to human protein to provide good T cell help. It contains together with a region that is characteristic of similar proteins optimized by evolution. This may allow the production of antibodies that recognize their antigens. Typically, the generated immune response includes generating a neutralizing antibody response.
[52] The human protein according to the invention may be a full-length protein encoded by the human genome or a domain or sub-unit of a full-length protein encoded by the human genome. If it is desired to produce neutralizing antibodies against the functional domain- or receptor binding domain of a self antigen, chimeric antigens comprising only these regions can be prepared. Thus, the exposed regions of these domains, or B cell epitopes of these domains, are conserved, and mutations of similar proteins are introduced into portions or surface exposed domains other than the B cell epitopes.
[53] The term "protein" is intended to include shorter sequences of amino acid residues that may be referred to, for example, as peptides such as neuropeptides. Human proteins will typically be subject to post-translational changes such as glycosylation, proteolytic cleavage, phosphorylation, and other changes well known to those skilled in the art. The human protein is preferably a cytokine, hormone, growth factor or extracellular protein, more preferably a four helix cytokine, most preferably IL-13. Cytokines include, for example, IL1, IL2, IL3, IL-4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL20, IL21, IL25, TNF, TGF, GMCSF, MCSF and OSM. Quadruple cytokines include IL2, IL3, IL-4, IL5, IL13, GMCSF and MCSF. Hormones include, for example, luteinizing hormone (LH), follicle stimulating hormone (FSH), chorionic gonadotropin (CG), VGF, GHrelin, auguuti, auguti-related proteins and neuropeptide Y. have. Growth factors include, for example, VEGF. Extracellular proteins include, for example, APP or B-amyloid.
[54] Similar proteins are proteins that are orthologous or paralogous to self-proteins, such as human proteins, which can be identified by blood relatives to the common ancestors of various organisms. It is thus believed that this performs a similar conserved function in various organisms. Therefore, the orthologus gene is a gene similar in sequence derived from one ancestor gene, an equivalent gene in various species, and has evolved according to characteristics from a common ancestor. In particular, in humans, orthologous proteins are structurally equivalent molecules in mammals other than humans. Paralogue proteins are proteins that appear in more than one copy in an organism given by replication (Venter, Science; 1336, vol 291; 2001), ie homologous sequences diversified by gene replication (shared common evolutionary ancestors). to be. Preferably, the analogous protein is an orthologue. Orthologous proteins typically have the same name as human proteins and typically perform the same function, for example murine IL-13 is an ortholog for human IL-13. Analogous proteins are typically mammals or birds, for example bovine animals, sheep, rodents such as murines, pigs, monkeys, felines, canines or humans. Preferably, the analogous protein is murine. Thus, in the context of the present invention, murine IL-13 is a similar (and orthologous) protein to human IL-13. Similarly, monkey IL-4 is a similar (and orthologous) protein to human IL-4.
[55] Polypeptides of the invention preferably comprise two, three, four, five, six, seven, eight, nine, ten, eleven or more mutations that characterize similar proteins. More preferably, the polypeptide comprises three or more mutations. Each mutation can be characterized by the same or different analogous proteins. Thus, the first mutation can be characteristic of murine analogs, and the second mutation can be characteristic of monkey analogs. According to one feature, the polypeptide comprises three or more mutations, each of which is characterized by a variety of analogs. However, preferably, each mutation is characterized by the same analog. Mutations are changes in the amino acid sequence of a protein and include, for example, deletions, insertions, and substitutions. Preferably, the mutation is a substitution. Preferably, more than one amino acid is substituted in each surface unexposed region.
[56] A mutation that characterizes a similar protein is a mutation that results in a sequence of a human protein that is closer in identity to the sequence of the similar protein after the mutation is performed on the human protein. For example, if the human sequence is ProProArgVal and the murine analog has the sequence ProProTyrVal, the mutation that characterizes the analogous protein is the substitution of Tyr with Arg. Preferably, the mutation is not performed at residues that are surface residues in the active protein that are naturally folded in aqueous solution under physiological conditions. Such surface residues, particularly those which form a loop structure, are often B cell epitopes, and it is preferred that all of these regions are conserved. These introduced mutations have the function of destroying the tolerance of self-proteins and are immunogenic in species from which unmutated proteins are derived.
[57] In one embodiment, the polypeptides of the invention preferably have at least 30% and less than 100% identity with the human protein over the full length of the human protein. Preferably the polypeptide is at least 40% identical to the human protein, for example at least 50%. More preferably the polypeptide is at least 60% identical to the human protein, for example at least 70%. Most preferably the polypeptide is at least 85% identical to human protein, for example about 90%. Such proteins can give rise to human immune half that recognizes human proteins.
[58] For example, the UWGCG package provides a BESTFIT program that can be used to calculate homology (eg can be used with default settings) (see Devereus et al. (1984) Nucleic Acids Research 12, p387-). 395). The PILEUP and BLAST algorithms are described, for example, as described in Altschul (1993) J. Mol, Evol. 36, 290-300; Altschul et al (1990) J. Mol. Biol. 215: 403-10. It can be used to calculate homologous or alignment sequences (typically on their default settings).
[59] Software for performing BLAST analysis is publicly available through the National Biological Information Center ( http://www.ncbi.nlm.nih.gov/ ). This algorithm first identifies a high number of sequence pairs (HSPs) by identifying short words of length W among query sequences that match or meet a quantity threshold score T when aligned with words of equal length in the database sequence. It includes checking. T means neighboring word score limits (Altschul et al, 1990). This initial neighboring word hit acts as a seed to initiate a search to find HSPs containing them. Such word hits extend in both directions along each sequence as long as the cumulative alignment value can increase. The extension of the word hit in each direction is such that the accumulated alignment value falls by an amount X from the maximum value; The cumulative level drops below zero due to the accumulation of one or more negative level residue alignments; It stops when the end of each sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and alignment speed. The BLAST program is a code length (W) 11, BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919), by default when the program is used for polynucleotides. A comparison of alignment (B) 50, expected value (E) 10, M = 5, N = 4, and both strands is used.
[60] The BLAST algorithm performs a statistical analysis of the similarity between the two sequences (Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)), which provides an indication of the probability by which two nucleotide or amino acid sequences may coincide. For example, a sequence may be different if the minimum sum probability in the comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, most preferably less than about 0.001. It is considered analogous to the sequence.
[61] Successful design of polypeptides according to the present invention, for example, by demonstrating that, when expressed in a suitable host cell, the polypeptide adopts a form sufficiently similar to that of its own protein, resulting in an antibody having cross reactivity with the native magnetic protein. Can be proved. This may be due to immunological techniques such as binding of monoclonal or polyclonal antibodies in ELISA, or physiochemical techniques such as circular dichroism, or crystallographic techniques such as X-ray crystallography or computer modeling, or many well known to those skilled in the art. Can be presented using other methods.
[62] Further confirmation of successful design can be accomplished by administering the polypeptide produced in itself by appropriate vaccination methods and observing whether an antibody is capable of binding to the protein. Such binding can be assessed using ELISA techniques using recombinant or purified natural proteins or through bioassays to examine the effect of the protein on sensitive cells or tissues. Particularly preferred assessments are to observe a causal relationship with respect to the activity of the protein in an intact host and to determine whether the presence of the antibody induced by the method of the present invention modulates that phenomenon. As such, the proteins of the invention will be able to produce antibodies against natural antigens in species from which the natural protein is derived.
[63] Polypeptides of the present invention may be added with desired properties (e.g., addition of sequence tags to facilitate purification or increase immunogenicity), undesirable properties (e.g. unnecessary action activity at the receptor) or membranes To remove the transmembrane domain, it may be further modified by mutations, eg, substitution, insertion, or deletion of amino acids. In particular, the present invention specifically contemplates fusion partners that facilitate purification, such as GST expression partners or polyhistidine tags that enhance expression.
[64] In a preferred embodiment, human IL-13 is provided having one or more of the following mutations or conservative substitutions thereof characteristic for mouse IL-13. The numbering below means IL-13 expressed with the signal sequence of IL-13 of E. coli.
[65] R → K at position 30
[66] V → S at position 37
[67] Y → F at position 63
[68] A → V at position 65
[69] E → D at position 68
[70] E → Y at position 80
[71] K → R at position 81
[72] M → I at position 85
[73] G → H at position 87
[74] Q → H at position 113
[75] V → I at position 115
[76] D → K at position 117
[77] More preferably human IL-13 comprises two or more, preferably at least 3, 4, 5, 6 or more mutations or conservative substitutions thereof. Preferably all 12 mutations are present.
[78] "Conservative substitutions" means that one amino acid is substituted with another amino acid having similar properties such that one skilled in the art of peptide chemistry can expect that the secondary structural and numerical properties of the polypeptide will not substantially change.
[79] For example, certain amino acids may be substituted with other amino acids in the protein structure without significant loss of mutual binding capacity with structures such as, for example, the antigen binding region of an antibody or the binding region of a substrate molecule. Since defining the biologically active activity of a protein is because the defining of the protein is the mutual ability and properties of the protein, certain amino acid sequence substitutions can be made in the protein sequence and, of course, the DNA coding sequence on which it is based, and nevertheless similar Proteins with properties are obtained. Thus, various modifications can be made to the peptide sequence of the disclosed composition or the corresponding DNA sequence encoding the peptide, without significant loss of bioavailability or activity.
[80] In order to effect such alterations, hydropathic indexes of amino acids can be considered. The importance of amino acid hydropathic indices in contributing interactive biological functions to proteins is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). The relative hydropathy properties of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. The hydropathic index of each amino acid has been determined based on its hydrophobicity and charge properties (Kyte and Doolittle, 1982). These values are as follows: isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8); Cysteine / cystine (+2.5); Methionine (+1.9), alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).
[81] It is known that certain amino acids may be substituted by other amino acids having similar hydropathic indices or values, which still produce proteins with similar biological activity, for example, still producing proteins that are biologically equivalent in functionality. Known in In order to make such a change, substitution of amino acids having a hydropathic index within ± 2 is preferable, substitution of amino acids having a hydropathic index within ± 1 is particularly preferable, and substitution of amino acids having a hydropathic index within ± 0.5 is more preferable. Particularly preferred. It is also understood in the art that substitution of analogous amino acids can be effectively performed based on hydrophilicity. U.S. Patent No. 4,554,101, the entire contents of which are hereby incorporated by reference in its entirety, describes that the average value of the maximum local hydrophilicity of a protein determined by the hydrophilicity of adjacent amino acids correlates with the biological properties of the protein. have.
[82] As detailed in US Pat. No. 4,554,101, the following hydrophilicity levels have been assigned to amino acid residues: arginine (+3.0); Lysine (+3.0); Aspartate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1), alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). It is well known in the art that amino acids can be substituted with other amino acids having similar hydrophilicity and are still biologically equivalent, in particular immunologically equivalent proteins. In such a modification, substitution of amino acids having hydrophilicity within ± 2 is preferred, substitution of amino acids having hydrophilicity within ± 1 is particularly preferred, and substitution of amino acids having hydrophilicity within ± 0.5 is more particularly preferred.
[83] Thus, as outlined above, amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. Examples of substitutions that take various of these properties into consideration are well known to those skilled in the art and include: arginine and lysine; Glutamate and aspartate; Serine and threonine; Glutamine and asparagine; And valine, leucine, and isoleucine. These are the preferred conservative substitutions.
[84] Amino acid substitutions may also be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphoteric properties of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; Positively charged amino acids include lysine and arginine; Amino acids having uncharged polar head groups with similar hydrophilicity include leucine, isoleucine, and valine; Glycine and alanine; Asparagine and glutamine; And serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative alterations include (1) alanine, proline, glycine, glutamine, asparagine, serine, threonine; (2) cysteine, serine, tyrosine, threonine; (3) valine, isoleucine, leucine, methionine, alanine, phenylalanine; (3) valine, isosocin, leucine, methionine, alanine, phenylalanine; (4) lysine, arginine, histidine, and (5) phenylalanine, tyrosine, tryptophan, histidine.
[85] In a preferred embodiment, the mutated IL-13 of the present invention comprises variants thereof comprising one or more of the following sequences or conservative substitutions:
[86] L K E L I E E L S N; (SEQ ID No 1)
[87] F C V A L D S L: (SEQ ID No 2)
[88] A I Y R T Q R I L H G; (SEQ ID No 3)
[89] K I E V A H F I T K L L; (SEQ ID No 4).
[90] Polypeptides of the invention are encoded by the polynucleotides of the invention. Those skilled in the art will be able to readily determine the polypeptide sequence encoding the polypeptide using the genetic code. Once the required nucleic acid sequences are determined, polynucleotides having the desired sequences can be prepared as described in the Examples. Those skilled in the art can easily adapt and apply any necessary parameters such as primers and PCR conditions. It will also be appreciated by those skilled in the art that, due to the degeneracy of the genetic code, there may be one or more polynucleotides that encode the polypeptides of the invention.
[91] Polynucleotides of the invention are typically RNA, for example mRAN or DNA, for example genomic DNA, cDNA or synthetic DNA. Preferably the polynucleotide is DNA. Particularly preferably cDNA.
[92] The invention further provides an expression vector, which is a nucleic acid construct comprising a polynucleotide of the invention. In addition, nucleic acid constructs will include polyadenylation signals that may be necessary to enable expression of proteins in appropriate initiators, promoters, enhancers and other elements such as mammalian cells and are placed in the correct orientation for this purpose.
[93] Promoters may be eukaryotic promoters such as CD68 promoter, Gal1, Gal10 or NMT1 promoter, prokaryotic promoters such as Tac, Trc or Lac, or viral promoters such as cytomegalovirus promoter, SV40 promoter, polyhedrin promoter, P10 Promoter, or respiratory syncytial virus LTR promoter. It is preferred that the promoter is a viral promoter. In particular, it is preferred that the promoter is a cytomegalovirus immediate early promoter optionally comprising exon 1 from the HCMV IE gene.
[94] Transcriptional regulatory elements include enhancers such as hepatitis B surface antigen 3 ′ untranslated region, CMV enhancer; Introns such as CD68 introns, or CMV introns A, or regulatory regions, such as CMV 5 ′ untranslated regions.
[95] It is preferred that the polynucleotide is operably linked to a promoter on the nucleic acid construct such that upon insertion of the construct into a mammalian cell, the polynucleotide is expressed to produce the encoded polypeptide. The backbone of the nucleic acid construct may be RNA or DNA, for example plasmid DNA, viral DNA, bacterial DNA, bacterial artificial chromosomal DNA, yeast artificial chromosomal DNA, synthetic DNA. The nucleic acid construct may also be an artificial nucleic acid such as phosphorothioate RNA or DNA. Preferably the construct is DNA. Plasmid DNA is particularly preferred.
[96] The invention further provides host cells comprising the expression vector of the invention. Such cells include prokaryotic cells such as transient or preferably stable higher eukaryotic cell lines, lower eukaryotic cells such as yeast, or bacterial cells, such as mammalian cells or insect cells, for example using a baculovirus expression system. . Specific exemplary cells that can be modified by insertion of a vector encoding a polypeptide of the invention include mammalian HEK293T, CHO, HeLa, NSO and COS cells. Preferably the cell line chosen will be a cell line that not only is stable but also allows mature glycosylation of the polypeptide. Expression can be in transformed oocytes. The polypeptides of the invention can be expressed in cells of non-transgenic animals, preferably mice, or in milk of large mammals such as goats, sheep and cattle. Animals other than transformed humans expressing polypeptides of the invention are included within the scope of the invention. The polypeptide of the present invention can also be expressed in Xenopus laevis oocytes.
[97] The invention also relates to a pharmaceutical composition or vaccine composition comprising a therapeutically effective amount of a nucleic acid construct or polypeptide of the invention, optionally in combination with a pharmaceutically acceptable carrier, which preferably comprises phosphate buffered saline (PBS), saline And pharmaceutically acceptable excipients such as dextrose, water, glycerol, ethanol, liposomes or combinations thereof. The vaccine composition optionally comprises a therapeutically effective amount of a nucleic acid construct of the invention formulated on metal beads, preferably gold beads. The vaccine composition of the present invention may also comprise an adjuvant such as, for example, those specified, imiquimod, tucaresol or alum.
[98] Protein adjuvant formulations are preferred because they induce high titer antibody responses.
[99] Preferably the adjuvant is taken simultaneously with the present invention and formulated together in a preferred embodiment. Adjuvants contemplated in the present invention include the following, and this list is by no means exhaustive and does not exclude other agents: synthetic imidazoquinolines such as imiquimod [S-26308, R-837 Harrison, et al. 'Reduction of recurrent HSV disease using imiquimod alone or combined with a glycoprotein vaccine', Vaccine 19: 1820-1826, (2001)); And resiquimod [S-28463, R-848], Vasilakos, et al. 'Adjuvant activites of immune response modifier R-848; Comparison with CpG ODN', Cellular immunology 204: 64-74 (2000) ), Schiff bases of carbonyl and amines that are structurally expressed on antigen presenting cells and T-cell surfaces, such as Tucarazole (Rhodes, J, et al.'Therapeutic potentiation of the immune system by constimulatory Schiff-). base-forming drugs', Nature 377: 71-75 (1995)), cytokines, chemokines and co-stimulatory molecules, Th1 inducers such as interferon gamma, IL-2, IL-12, IL-15 and IL-18 , Th2 inducers such as IL-4, IL-5, IL-6, IL-10 and IL-13, and other chemokine and co-stimulatory genes such as MCP-1, MIP-1 alpha, MIP-1 beta , RANTES, TCA-3, CD80, CD86 and CD40L, other immunostimulatory target ligands such as CTLA-4 and L-selectin, apoptosis stimulating proteins and peptides such as Fas, (49), adjuvants based on synthetic lipids Burnt, Vaxfectin (Reyes et al., 'Vaxfectin enhances antigen specific antibody titres and maintains Th1 type immune responses to plasmid DNA immunization', Vaccine 19: 3778-3786), squalene, alpha-tocopherol, polysorbate 80, DOPC and Cholesterol, Endotoxin, [LPS], Butler, B., 'Endotoxin,' Toll-like receptor 4, and the afferent limb of innate immunity ', Current Opinion in Microbiology 3: 23-30 (2000); CpG oligo- and di-nucleotides, Sato, Y. et al., 'Immunostimulatory DNA sequences necessary for effective intradermal gene immunization', Science 273 (5273): 352-354 (1996), Hemi, H. (Hemmi, H.) et al., 'A Toll-like receptor recognizes bacterial DNA', Nature 408: 740-745 (2000)] and other possible ligands for initiating Toll receptors to produce Th1-induced cytokines such as synthetic Mycobacterial lipoprotein, mycobacterial protein p19, peptidoglycan, tycoic acid and lipids A.
[100] Specific preferred adjuvants that induce a predominant Th1-type response are, for example, lipid A derivatives such as monophosphoryl lipid A, or preferably 3-de-O-acylated monophosphoryl lipid A. MPL Adjuvant is commercially available from Corixa Corporation (Seattle, WA; see, US Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (where CpG dinucleotides are not methylated) also induce a predominant Th1 response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and US Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also disclosed in Sato et al., Science 273: 352, 1996. Another preferred adjuvant is QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; Or comprises a saponin or derivative thereof, such as a pillar or jipso Kane catapult rhodium quinone containing quinoa (Gypsophila or Chenopodium quinoa) saponin, Quill A (Quil A).
[101] The invention also provides a method for treating or preventing an IL-13 mediated disease, any indication or disease associated therewith, comprising administering an effective amount of a protein, polynucleotide, vector or pharmaceutical composition according to the invention. . Administration of the pharmaceutical composition may take the form of one or more individual doses, eg, according to a “prime-boost” therapeutic vaccination regimen. In certain cases it will be desirable to incorporate "prime" vaccination into plasmid-derived vectors via particle mediated DNA delivery of polynucleotides according to the invention, and administering recombinant viral vectors comprising the same polynucleotide sequence. To "boost" or stimulate the protein in the adjuvant. Conversely, a first sensitization is made using a viral vector or protein formulation, typically a protein formulated with an adjuvant, and stimulated with a DNA vaccine according to the invention.
[102] For the treatment of self-antigens such as IL-13 mediated diseases, it is preferred that the adjuvant be the preferred inducer of the Th1 response. In particular, the adjuvant comprises an immunostimulatory CpG oligonucleotide as described in (WO96102555). Typical immunostimulatory oligonucleotides will have a length of 8-100 bases and include the general formula X ₁ CpGX ₂ , wherein X ₁ and X ₂ are nucleotide bases and C and G are not methylated.
[103] Preferred oligonucleotides for use in the adjuvant or vaccine of the invention preferably contain two or more dinucleotide CpG motifs isolated by three or more, more preferably six or more nucleotides. Oligonucleotides of the present invention are typically deoxynutotide. In a preferred embodiment, the oligonucleotides, although the phosphorodiester and other internucleotide bonds are within the scope of the present invention, include oligonucleotides having mixed internucleotide bonds, such as mixed phosphorothioate / phosphodiester, The internucleotide of the nucleotide is phosphorodithioate, or more preferably a phosphorothioate bond. Other internucleotide bonds that stabilize oligonucleotides can be used. Processes for preparing phosphorothioate oligonucleotides or phosphorodithioates are described in US Pat. Nos. 5,666,153, 5,278,302 and WO 95/26204.
[104] Examples of preferred oligonucleotides have the following sequence. Such sequences preferably contain phosphorothioate modified internucleotide bonds.
[105]
[106] Another CpG oligonucleotide may comprise a preferred sequence with discrete deletions or additions thereto. CpG oligonucleotides used in the present invention can be synthesized by any method known in the art (eg EP468520). Preferably, such oligonucleotides can be synthesized by using an automated synthesizer. Adjuvant formulations containing the CpG oligonucleotide can be purchased from Qiagen under the trade name "ImmunEasy".
[107] The composition of the present invention can be used for prophylaxis and treatment. The present invention provides a polypeptide or polynucleotide according to the present invention for use as a drug. The invention also provides the use of a polypeptide or polyoligonucleotide of the invention in the manufacture of a medicament for the treatment of allergies, respiratory diseases such as asthma and COPD, parasitic infection related diseases, fibrosis or cirrhosis.
[108] The invention also provides a method of inoculation comprising administering an effective amount of a vaccine composition of the invention to a patient and eliciting an immune response against the vaccine composition.
[109] The invention also provides a vaccine composition as described herein for use in vaccination of mammals for IL-13 mediated diseases such as allergies, respiratory diseases, parasitic infection related diseases, fibrosis and cirrhosis. Respiratory diseases include, for example, asthma, such as allergic asthma, and chronic obstructive pulmonary disease (COPD). In particular, vaccine compositions capable of inducing neutralization against IL-13 may thus be useful for the treatment of human asthma, in particular allergic asthma. Such vaccine compositions also include certain parasitic infection-related diseases (Brombacher, 2000 Bioessays 22: 646-656) and diseases in which IL-13 production is associated with fibrosis [Chiaramonte et al, 1999, J Clin Inv 104: 777 -785, eg, for the treatment of chronic obstructive pulmonary disease (COPD) and cirrhosis of the liver.
[110] Vaccine compositions of the invention can be administered in a variety of ways, including, for example, intramucosal administration such as oral and intranasal administration; It may be administered by intrapulmonary, intramuscular, subcutaneous or intradermal route. If the antigen is to be administered as a protein based vaccine, the vaccine will typically be formulated with an adjuvant and will be suspended in water for injection prior to use. Such compositions may be administered to a subject as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic. Typically, such compositions may be administered intramucosally, but other routes of administration are possible.
[111] One technique for intradermal administration includes particle bombardment (known as the 'gene gun' technique and described in US Pat. No. 53,71015). Proteins may be formulated with sugars to form small particles, or DNA encoding antigens may be coated on inert particles (eg, gold beads), as they pass through the recipient's surface (eg, skin), for example For example, it is accelerated at a speed sufficient to pass by the ejection means operating under high pressure from the projection device. (Particles coated with the nucleic acid vaccine construct of the present invention and particles per protein are within the scope of the present invention, and devices loaded with such particles are also within the scope of the present invention). Other methods of integrating nucleic acid particle constructs or compositions containing such constructs to recipients include ultrasound, electrical stimulation, electroporation, and microseeding as described in US Pat. No. 5,697,901.
[112] Nucleic acid constructs of the invention can also be administered by special delivery vector means useful for gene therapy. Gene therapy methods are described, for example, in Verme et al., Nature 1997, 389: 239-242. Both viral and non-viral systems can be used. Viral based systems include retroviral based systems, lentiviral based systems, adenovirus based systems, adeno-associated virus based systems, herpes virus based systems and vaccinia-virus based systems. Non-viral based systems include direct administration of nucleic acids and liposome based systems. For example, the vector can be encapsulated by liposomes or encapsulated in polylactide co-glycolide (PLG) particles.
[113] Nucleic acid constructs of the invention can also be administered by transformed host cell means. Such cells include cells harvested from a subject. Nucleic acid vaccine constructs are introduced into cells harvested in vitro, and the transformed cells can later be introduced into a subject. Nucleic acid constructs of the invention can be integrated into nucleic acids present in cells by homologous recombination methods. The transformed cells can be grown in vitro, if desired, and one or more resulting cells can be used in the present invention. The cells may be provided to the appropriate site of the patient by known surgical or microsurgical methods (eg, transplantation, microinjection, etc.). Suitable cells include dendritic cells.
[114] The amount of vaccine composition delivered depends on the species and body weight of the mammal to be immunized, the disease state treated / prevented, the vaccination protocol adopted (ie, single versus repeated administration), the route of administration, and the potency and dose of the adjuvant compound selected. It can be very diverse. Based on this diversity, the physician or veterinarian will be able to determine the appropriate dose level, for example if the vaccine is a nucleic acid, the dose may be a nucleic acid construct of 0.5 to 5 μg / kg or a composition containing it. In particular, the dosage may vary depending on the route of administration. For example, when using intradermal administration on gold beads, the total dose will preferably be between 1 μg and 10 ng, particularly preferably the total dose will be between 10 μg and 1 ng. When the nucleic acid construct is administered directly, the total dose is generally higher, for example 50 μg to 1 mg or more. The dose is an example of an average case.
[115] In protein vaccines, the amount of protein in each vaccine dosage form is selected in an amount that induces an immune protective response without significant adverse effects in a typical vaccine. Such amounts will vary depending on which immunogen is used and how it is present. In general, each dose is expected to contain 1 to 1000 μg of protein, preferably 1 to 500 μg, more preferably 1 to 100 μg, most preferably 1 to 50 μg. The optimal amount for a particular vaccine can be confirmed by standard studies, including the observation of the appropriate immune response in the subject to be vaccinated. After the initial inoculation, the subject may receive one or several booster immunity at sufficient intervals. Such vaccine formulations may be for first inoculation regimens or for booster inoculation regimens; For example, it may be administered systemically via the transdermal, subcutaneous or intramuscular route, or applied to the mucosal surface via, for example, the nasal or oral route.
[116] Of course, there may be individual cases where a higher or lower dose range is advantageous, even if such is within the scope of the present invention.
[117] The vaccine composition may be administered on a one-time basis or administered repeatedly, for example, for about 1 day to about 18 months, preferably for one month, for example, 1 to 7 times, preferably 1 to 4 times. have. Such administration may optionally be continued at regular intervals of 1 to 12 months for the remaining life of the patient. In one embodiment, the patient will be inoculated with different forms of antigen at the first booster inoculation. For example, the antigen will be administered first as a DNA based vaccine and then as a protein adjuvant base formulation. However, once again, such treatment regimens may include the size and species of the animal involved, the amount of nucleic acid vaccine and / or protein composition administered, the route of administration, the efficacy and amount of any adjuvant compound used, the skilled veterinarian or It will vary depending on any other factors that may be apparent to the physician.
[118] The following examples illustrate the theory of the present invention in non-human mice, where the protein is a murine with mutations in human protein properties, but the result is that the protein is B cell epitope from humans with mutations in mouse characteristics, or other It is illustrated to facilitate the prediction of the treatment of humans with similar proteins.
[119] Throughout the following examples of the present invention, a wide variety of known and practiced techniques in molecular and cellular biology are used. Practical details of these are described [Sambrook et al., 1989, 2 nd edition. Cold Spring Harbor Press: New York]. The amino acid sequence or representation may be given in single letter code or three letter code. The acronym 'h' means protein or gene from human, 'm' means protein or gene from murine, and 'c' means chimeric construct. 'r' is used to indicate recombinant protein.
[120] 1. Design of Vaccines Against Murine IL-13
[121] IL-13 belongs to the family of four helix cytokine folds defined by SCOP (Murzin et al., 1995, J Mol Biol 247: 536-540). Each member of this fold superfamily is structurally related but difficult to align at the sequence level. The 3D structure of IL-13 has not yet been determined, but the structure for many other quad helix cytokines has been determined. A number of sequences representing the folds that determine protein multiple sequence alignments for the IL-13 orthologue and also the structure of one or more members have been determined (IL-4, GM-CSF, IL-5 and IL-2) Protein multiple sequence alignments for different cytokines were determined. Secondary structure predictions include DSC (King and Sternberg, 1996, Prot Sci 5: 2298-2310), SIMPA96 (Levin, 1997, Prot Eng 7: 771-776) and Pred2ary (Chandonia and Karplus, 1995). , Prot Sci 4: 275-285] for IL-13 protein multiple sequence alignment. Individual cytokine protein multiple sequence alignments are arranged relative to each other using sequence information and structure information (from known crystal structures and secondary structure predictions).
[122] Antigenic sites, in particular B-cell epitopes, are predicted for murine IL-13 using Chameleon software (Oxford Molecular) and mapped to IL-4 using protein multi-sequence alignments so that they are IL I got the idea that it can be structurally located on -13. From this assay, the exposed areas that were potentially antigenic and involved in receptor binding were selected.
[123] From this model, a chimeric IL-13 sequence was designed, wherein the sequence of the predicted antigenic loop was obtained from murine IL-13, and the sequence of this predicted structure (mainly helical) region was obtained from human IL-13. The purpose of this design is to identify target epitopes from murine IL-13 for the possibility that neutralizing antibodies can be produced and, despite structural similarity to natural proteins, allow for the presence of one or more CD4 T helper epitopes. To have them present on a framework containing sufficient sequence modifications to the (murine) proteins. Nucleic acid and protein sequences selected for this example of the chimeric IL-13 vaccine are shown in FIG. 1 (SEQ ID NOs 19 and 20). The underlined sequences correspond to the sequences identified in human orthologs. Twelve amino acids were substituted to achieve the sequence in FIG. 1. It should be understood that by degeneracy of the genetic code, multiple possible nucleic acid sequences can encode the same protein. It will also be appreciated that there are other possible chimeric IL-13 vaccine designs with other orthologous variations in the unexposed areas within the scope of the present invention.
[124] 1.2 Preparation of Chimeric IL-13
[125] Chimeric IL-13 (cIL-13) DNA sequences were synthesized from a series of partially overlapping DNA oligonucleotides, and the sequences cIL-13-1 to cIL-13-6 are shown in Table 1. This oligo was annealed and formed cIL13 DNA by PCR, where the specifications of the cycle were 1 minute at 94 ° C and then 30 cycles at 94 ° C for 25 cycles, 1 minute at 55 ° C and 2 minutes at 72 ° C. It was. After 7 min at 72 ° C., it was cooled to 4 ° C. when complete. The reaction product contained a band of predicted size, 361 base pairs, which was subcloned into the T / A cloning vector pCR2.1 (Invitrogen, Groningen, The Netherlands) to convert pCR2.1-cIL-13. Formed. The digested fragments of BamH1 and Xho1 cIL-13 from pCR2.1-cIL-13 then form BamH1 and Xho1 in pGEX4T3 (Amersham Pharmacia, Bucks Amersham, UK) forming pGEX4T3-cIL-13 / 1. Subcloned into site. In sequencing the pGEX4T3-cIL-13 / 1 construct, we found an extra 39 base pairs of DNA sequence (derived from the pCR2.1 vector) between the sequences for GST and CIL-13. To correct this, we repeated PCR for cIL-13, primers CIL-13Fnew and cIL-13R using pGEX4T3-cIL-13 / 1. The PCR product obtained was then cloned back into pGEX4T3 using BamH1 and Xho1 restriction sites to form the expression vector pGEX4T3-cIL-13. The sequence of these constructs was confirmed by dideoxy terminator sequencing. This vector encodes a gene fusion protein consisting of glutathione-S-transferase and cIL-13 (GST-cIL-13). Two portions of the protein are linked by short spacers containing the recognition site for thrombin. Such fusion proteins can be readily purified by glutathione sepharose affinity chromatography and then used directly, or a preparation of free cIL-13 can be produced by cleavage with thrombin.
[126] Table 1. Oligonucleotides Used to Construct Chimeric IL-13
[127]
[128]
[129] The pGEX4T3-cIL-13 expression vector was transformed with E. coli BLR strain (Novagen, supplied by Cambridge Biosciences, Cambridge, UK). Expression of GST-cIL-13 was induced by adding 0.5 mM of IPTG to the culture in logarithmic growth phase at 37 ° C. for 4 hours. This bacterium is then collected by centrifugation and the methods described above for the purification of similar GST-human IL-13 fusion proteins (McKenzie et al, 1993, Proc Natn Acad Sci 90: 3735-3739) from them. GST-cIL-13 was purified.
[130] Characterization of cIL-13 Properties
[131] Samples of purified GST-cIL-13 were assayed by SDS-PAGE electrophoresis. 2, it can be seen that the purified protein contains a protein of the size predicted for GST-cIL-13. Low bands indicate a small amount of GST, which is caused by partial separation of the fusion protein during manufacture.
[132] To confirm that the purified protein is GST-cIL-13, the samples were separated by SDS-PAGE and blotted onto PVDF membranes, followed by Western blotting to the presence of IL-13 and GST immunoactivity. Assayed for. Since cIL-13 contains sequences derived from both human and murine IL-13, it was predicted that it would be recognized by the specific antiserum designated in human IL-13 or mouse IL-13. The blot was made with 3% fetal bovine serum albumin (BSA) in TBS (50 mM Trisma hydrochloride, 138 mM sodium chloride, 2.7 mM potassium chloride, pH 8.0) containing 0.05% Tween-20 (TBST). Block overnight at 4 ° C., incubate with shaking the primary antibody for 1 hour at room temperature and wash 4 times with TBST. Secondary antibodies were added for 1 hour at room temperature with shaking, followed by 4 washes and color development using SuperSignal Chemiluminescent Reagent (Pierce, Rockfor, Illinois, USA).
[133] 3 (Legend below) shows the results of this analysis, indicating that the purified protein is recognized by antibodies to human IL-13, mouse IL-13 and GST, thereby confirming the predicted structure.
[134]
[135] The primary antibodies used in this experiment were as follows: anti-hIL-13 [catalog number AF-213-NA, R & D Systems, Oxford Abingdon, UK, 1 μg / ml usage]; anti-mIL-13 [Catalog number AF-413-NA, manufactured by R & D Systems, Inc. 1 μg / ml)]; And anti-GST [Catalog No. 27-4590D, Pharmacia Products, Used 1/200]. Secondary antibodies used in this experiment were: HRP-conjugated anti-chlorine IgG (catalog number A-5420, Sigma-Aldrich Company Limited, Dorsett Pulley, UK, usage 1 / 40,000).
[136] The protein sample was GST-cIL-13 prepared as described in Example 2; Recombinant human IL-13 (rhIL-13) [Catalog No. CH1-013, Cambridge Biosciences, Cambridge, UK]; Recombinant mouse IL-13 (rmIL-13) [Catalog No. 413-ML-025, available from R & D Systems]; ^And GST prepared from E. coli transformed with empty pGEX4T3 vector as described above (Sambrook et al, 1989, 2nd edition, Cold Spring Harbor Press: New York).
[137] 1.3 Confirmation of Chimera IL-13
[138] Samples of GST-cIL-13 and cIL-13 (formed from GST-cIL-13 by thrombin cleavage) to confirm that GST-cIL-13 takes a conformation similar to that of native IL-13 in solution. Was assayed by ELISA. 96-well Maxisorp plates [Life Technology Limited, Paisley, UK] were overnight at 4 ° C. in GIL in cIL-13, GST-cIL-13, mIL-13, hIL-13 or carbonate-bicarbonate buffer. Coated. This plate was then blocked with 3% BSA / TBST for 1 hour at RT, washed three times in TBST, then incubated primary antibody for one hour at room temperature and then washed three times in TBST. Secondary antibody was added for 1 hour, washed three times in TBST and developed for 30 minutes using 0-phenylenediamine dihydrochloride peroxidase substrate (OPD, Sigma Aldrich). Primary and secondary antibodies used in this experiment are as described above. As shown in FIG. 4, GST-cIL-3 and cIL-13 were specifically recognized by antibodies against human IL-13 and mouse IL-13. From these data, it was confirmed that the chimerism treatment did not crudely alter the protein conformation.
[139] 1.4 Binding of chimeric IL-13 to receptors
[140] ELISA was set up to determine whether cIL-13 can bind to any of the known mouse IL-13 receptors (mIL-13R1 or mIL-13R2). 96 wells Maxithorb plates were coated overnight at 4 ° C. with anti-human IgG in carbonate-bicarbonate buffer (Catalog No. I-3382, Sigma Aldrich). Plates were then blocked for 1 hour at room temperature with 3% BSA / TBST, washed three times in TBST, then mIL-13R1-Fc or mIL-13R2-Fc (catalog numbers 491-IR-200 and 539-, respectively). IR-100, from R & D Systems) was incubated at room temperature for 1 hour. After washing, the plates were incubated with dilution of mIL-13 or cIL-13 or GST-cIL-13 for 1 hour at room temperature and again biotinylated anti-mIL-13 (catalog number BAF413, manufactured by Al & D. Systems). Incubated with). After further washing and incubating with streptavidin conjugated horseradish peroxidase, the plates were developed for 30 minutes using 0-phenylenediamine dihydrochloride peroxidase substrate. As indicated in FIG. 5, it can be seen that both cIL-13 and GST-cIL-13 can bind to one of the mIL-13 receptors. In addition, it was confirmed from these data that chimeric formation did not crudely alter protein morphology.
[141] 1.5 Bioactivity of Chimeric IL-13
[142] The bioactivity of GST-cIL-13 was assessed by the ability of these proteins to phosphorylate STAT6 in human lung fibroblast line A549. These cells express human type-2 IL-4 receptors that respond to both IL-4 and IL-13. Stimulation of these cells with hIL-4, hIL-13 or mIL-13 was plated into 60 mm tissue culture dishes (Life Technologies, Inc.) in RPMI (Life Technologies, Inc.) and with 70% of the confluence. Phosphorylation of the grown signaling protein STAT 6.5 × 10 ⁵ A549 cells was induced. The cells were then incubated with 2 to 150 ng / ml cytokine and the cIL-13 was purified at 37 ° C. for 15 minutes. Because the presence of a GST fusion partner can alter the cytokine bioactivity, chimeric IL-13 was assayed as a GST-cIL-13 fusion protein and free cIL-13 was not fused by thrombin isolation. As a control, rmIL-13 and GST were also tested. Cell lysates were then prepared and subjected to western blotting for the presence of phospho-STAT6 using rabbit anti-phospho-STAT6 polyclonal antibodies (NEB, Hertz-Hitskin, catalog number 9361S). Assay. The blot was blocked overnight at 5% BSA / TBST (BSA is A-7906 made from Sigma Aldrich, where the primary antibody is 0.1% Tween-20 specific for phospho), and the primary antibody is 1 It was added at 1/1000 h at rt for 3 h and then washed three times with TBST. Anti-rabbit HRP conjugated secondary antibody (A-4914, Sigma Aldrich) was added at 1/5000 for 1 hour at room temperature, washed four times with TBST, and then HRP chemiluminescent substrate ECL reagent (Amersham Pharmacia). Product). The results of this experiment are shown in FIG.
[143] Each lane was loaded with the following protein:
[144]
[145]
[146] As the recombinant protein reagent, the one described in FIG. 3 was used.
[147] A549 cells were treated with 50 or 10 ng / ml (but not 2 ng / ml) of rmIL-13 to induce phosphorylation of STAT6 showing bioactivity. Treatment of A549 cells with 50 ng / ml (but not 10 or 2 ng / ml) of cIL-13 induced phosphorylation of STAT6 showing bioactivity. Similarly, 150 ng / ml GST-cIL-13 (which corresponds to approximately 50 ng / ml cIL-13 on a molar basis) shows bioactivity, while 30 and 6 ng / ml do not. Thus, cIL-13 acts as an agonist at these receptors, but exhibited approximately five times less bioactivity than mIL-13 under these experimental conditions.
[148] 1.6 Immunization with cIL-13
[149] The formation of autoantibodies against mouse IL-13 was induced in Balb / c mice using cIL-13 and GST-cIL-13 as immunogens. The base of the 6-8 week old female mouse tail was injected subcutaneously (sc) with approximately 30 μg of protein dissolved in Freund's complete adjuvant (CFA). Three booster immunizations were then performed at the same site, each consisting of approximately 10 μg protein in incomplete Freund's adjuvant for boost. Each treatment group included 5 animals, which were immunized according to the protocol of Table 2 below.
[150] TABLE 2
[151] groupImmunization ASaline Control in CFA / IFA s / c B30/10 μg GST in CFA / IFA s / c CUnimmunized Pure Mice D30/10 μg GST-hIL-13 in CFA / IFA s / c E30/10 μg cIL-13 in CFA / IFA s / c F30/10 μg GST-cIL-13 in CFA / IFA s / c
[152] Dayprocess -12Pre-bleeding 0Primary immunization 14First boost immunization 27Tail blood 42Tail blood 49Second boost immunization 70Tail blood 97Tail blood 99Third boost immunization 113Tail blood 140Tail blood
[153] Serum samples were obtained by venepuncture of the tail vein at the time points specified in Table 2. After clarification by centrifugation, the samples were ELISA assayed for the presence of specific IgG responses to mouse IL-13, human IL-13 and GST. Animals in groups A to D did not have anti-mouse IL-13 antibodies at any time. All animals in groups B, D and F showed a strong IgG response to GST (animals in group E also showed a strong antibody response to GST, which was applied to the cIL-13 sample used to immunize these mice). Due to the presence of residual GST). Anti-mouse IL-13 antibody response was induced in all 5 of 5 animals in group F and in 4 of 5 animals in group E. 7A and 7B show immunological analysis for one of animals in group F and one of animals in group E (7b) (gst-cIL-13 and cIL-13 immunized, respectively). These results suggest that immunization with GST-cIL-13 or cIL-13 can disrupt tolerance to mIL-13 to produce mouse anti-mIL-13 antibodies.
[154] Sera from two mice (F1d70 and F5d97) with strong anti-mIL-13 IgG responses were tested for the bioactive neutralization capacity of rmIL-13 in the A549 / Phospo-STAT6 assay. Prior to incubation for 15 minutes at 37 ° C. with A549 cells, 20 ng / ml or 10 ng / ml of rmIL-13 (R & D System) was incubated with 1% serum in serum-free RPMI tissue medium for 15 minutes at room temperature. Cell lysates were prepared and analyzed by Western blot in the presence of phospho-STAT6 as described above. As a negative control, anti-hIL-13 serum was obtained from Balb / c mice immunized with GST-hIL-13 and had a strong anti-hIL-13 IgG response by ELISA, but no anti-mIL-13 antibody. Turned out to be. As a positive control, neutralized anti-mIL-13 antibody (R & D Systems, catalog number AF-413-NA) was added to normal mouse serum to a final concentration of 1 μg.
[155] The results of this experiment are shown in FIG. 8 and the following were tested:
[156] laneCytokineAntibodies One20ng / ml rmIL-13Normal mouse serum 210ng / ml rmIL-13Normal mouse serum 30ng / ml rmIL-13Normal mouse serum 420ng / ml rmIL-13Serum Sample F1d70 510ng / ml rmIL-13Serum Sample F1d70 60ng / ml rmIL-13Serum Sample F1d70 720ng / ml rmIL-13Anti-hIL-13 mouse serum 810ng / ml rmIL-13Anti-hIL-13 mouse serum 90ng / ml rmIL-13Anti-hIL-13 mouse serum 10Molecular weight markers- 110ng / ml rmIL-13Normal mouse serum + anti-mIL-13 1220ng / ml rmIL-13Serum Sample F5d97 1310ng / ml rmIL-13Serum Sample F5d97 140ng / ml rmIL-13Serum Sample F5d97 1520ng / ml rmIL-13Normal mouse serum + anti-mIL-13 1610ng / ml rmIL-13Normal mouse serum + anti-mIL-13
[157] Immunization with chimeric IL-13 immunogens of the present invention is in a form comparable to exogenous anti-murine IL-13 antibodies (lanes 15, 16), with mouse IL-13 antibodies (lanes, 4, 5, 12, 13) Induces the production of autoantibodies against mouse IL-13, which can neutralize bioactivity. This activity is absent in normal mouse serum (lanes 1 and 2) and in antibodies from animals immunized with GST-hIL-13 (lanes 7, 8) (lanes 7, 8).
[158] The data provide a basis for treating mammals with IL-13 dependent disease by inducing external neutralization of antibody activity by vaccination with cIL-13.
[159] 1.7 Alternative Structure
[160] 1.7.1 6 his tagged cIL-13 design
[161] The protein produced by bacteria, GST-cIL-13, is insoluble and requires in vitro solubilization and refolding. Size exclusion chromatography indicates that the refolding process produces several differentially folded forms, which may produce irrelevant antibodies in which the rate of immune response does not bind pure mouse IL-13. Imply that it is derived from the form.
[162] Therefore, such candidates may not produce the most potent neutralizing anti mouse IL-13 antibody response possible.
[163] For this reason, 6 his-cIL-13 is cloned into a mammalian expression vector such that the mammalian expressed 6 his-cIL-13 is soluble and does not require refolding in vitro.
[164] 1.7.2 FIG. 12 (SEQ ID NOs 23 and 24) shows vaccine antigens with different similar mutations. A protein sequence numbered according to the scheme wherein the glycine residue in the sequence “GPVPR” sequence is residue 1. The single-stranded sequence corresponds to the spiral region predicted from the calibrated structural model. Thick underlined residues indicate the point of incorporation in the mutant mouse sequence.
[165] 11 Mouse Leu is replaced by Val (rat).
[166] 21 Mouse Ser replaced by Thr (non-ortho).
[167] 63 mouse Thr replaced by Phe (non-ortho).
[168] 71 Mouse Gly replaced with Ala (Dog / Pig / Cow)
[169] 100 Mouse Ser replaced by Thr.
[170] 104 Mouse Gln is replaced with Asn (non-orthotype).
[171] 108 mouse His is replaced with Arg (non-orthotype).
[172] 1.8 Application to Human Therapy
[173] Figure 9 depicts one possible vaccine antigen according to the invention induced in the production of human anti-human IL-13 antibodies. This would be useful for the treatment of diseases characterized by excessive or inappropriate IL-13, for example asthma. The sequence corresponding to mouse IL-13 is underlined. The structure comprises 12 amino acid substitutions similar to murine IL-13. These are:
[174] R → K at position 30
[175] V → S at position 37
[176] Y → F in position 63
[177] A → V at position 65
[178] E → D at position 68
[179] E → Y at position 80
[180] K → R at position 81
[181] M → I at position 85
[182] G → H at position 87
[183] Q → H at position 113
[184] V → I at position 115
[185] D → K at position 117
[186] Figure 13 (SEQ ID NO 25) shows one possible vaccine for humans based on chimeric IL-4. This is an example of a chimeric human IL-4 vaccine protein. The underlined amino acid residues are those derived from mouse IL-4 comprising an alpha-helix structural region, wherein amino acid 21 is incorporated into the first helix. Unmarked symbols indicate amino acid residues derived from human IL-4. The location of the alpha-helix region was obtained from the literature (Zuegg, J et al (2001) Immunol and Cell Biol 79: 332-339).
[187] Example 2: Immune Response to gst-cIL-13 is Specific to Mouse IL-13 and Does Not Cross React with Mouse IL-4
[188] Because mouse IL-13 is structurally similar to mouse IL-4, serum from GST-cIL-13 immunized mice (appearing to contain high titer anti-mouse IL-13 autoantibodies) is anti-mouse IL- 4 ELISA and in vitro mIL-4 neutralizing bioassay were used for cross reactivity to mouse IL-4.
[189] 2.1 anti-mouse IL-4 ELISA.
[190] 96-well maxifov plates were coated overnight at 4 ° C. with anti-mouse IL-4 monoclonal antibody (Cat. No. MAB404, R + D Systems) in sodium bicarbonate buffer. The plates were then blocked with 3% BSA / TBST for 1 hour at room temperature and washed three times with TBST and 1 at room temperature with mouse IL-4 (Cat. No. 404-ML-005, R + D Systems). Incubate for hours. After washing, plates were incubated with mouse serum for 1 hour at room temperature, washed again and incubated with HRP conjugated anti-mouse IgG polyclonal antibody (Cat. No. A-9309, SIGMA). After further washing, the plates were developed for 30 minutes with O-phenylenediamine dihydrochloride peroxidase substrate.
[191] Anti-mouse IL-4 antibody levels in serum were expressed as endpoint titers. Endpoint titers are defined as serum dilutions that are equivalent to twice the ELISA background reading.
[192] mouse Anti-mouse IL-4 antibody Terminal titer Anti-mouse IL-13 antibody Terminal titerC2 (serum samples taken at 125 days post 4 X GST-cIL-13 vaccine)1/9001/80000
[193] Very low levels of mouse IL-4 cross-reactivity were detected in these serum samples. In contrast, much higher anti-mouse IL-13 antibody endpoint titers were previously measured in these serum samples using anti-mouse IL-13 antibody ELISA. The level of mouse IL-4 cross-reactivity measured by this ELISA was not predicted to have mouse IL-4 neutralizing effect in vivo. These serum samples were evaluated for mouse IL-4 neutralizing capacity in the mouse IL-4 biological assay in vivo.
[194] 2.2 In Vitro Mouse IL-4 Neutralizing Biological Assay
[195] Mouse IL-4 stimulates proliferation of CTLL cells in vitro. Therefore, an assay was developed to assess the mouse IL-4 neutralizing capacity of serum from mice vaccinated with this GST-clL-13 vaccine in these cells.
[196] To determine the ability of mouse serum to neutralize the bioactivity of recombinant mouse IL-4 on mouse CTLL cells (Cat. No. 87031904, ECACC), 3ng / ml recombinant mouse IL-4 was prepared in 96-well tissue culture plates (Invitrogen). Incubated with various concentrations of serum at 37 ° C. for 1 h. After this preincubation, CTLL cells were added. Assay mixtures containing various serum dilutions, recombinant mouse IL-4 and CTLL cells were incubated at 37 ° C. for 70 hours in a humidified CO ₂ incubator. MTT substrate (Cat. No. G4000, Promega) was added during the last 4 hours of incubation, after which the acid solution was added to stop the reaction and dissolve the metabolized blue formagen product. The absorbance of the solution in each well was read with a 96-well plate reader at 570 nm wavelength.
[197] It should be noted that this assay can only measure mouse IL-4 neutralizing capacity at serum dilution of 1/100 or greater. Serum dilution below 1/100 induces nonspecific proliferative effects in CTLL cells.
[198] The ability of the serum to neutralize mouse IL-4 bioactivity was expressed as the serum dilution (= ND ₅₀ ) needed to neutralize the bioactivity of a predetermined amount of mouse IL-4 by 50%. The greater the serum sample dilution, the stronger the neutralization capacity.
[199] The highest concentration of mouse C2 serum tested was 1/100 dilution. Since this did not neutralize the bioactivity of 3ng / ml mouse IL-4 by 50%, ND ₅₀ was expressed at a dilution of <1/100.
[200] mouse Mouse IL-4 neutralizing capacity (ND ₅₀ ) Mouse IL-13 Neutralization (ND ₅₀ )C2 (serum samples taken at 125 days post 4 X GST-cIL-13 vaccine)<1/1001/5300
[201] Mouse IL-4 neutralizing ability was not detected in the dilution of the sera tested in these serum samples. In contrast (as assessed for mouse IL-13 neutralization capacity), these serum samples strongly neutralized mouse IL-13 bioactivity.
[202] These data demonstrate that there is no association of mouse IL-4 neutralizing ability, although very low levels of mouse IL-4 cross-reactivity in serum can be measured by anti-mouse IL-4 antibody ELISA.
[203] 2.3 New Mouse IL-13 Neutralization Biological Assay to Assess Mouse IL-13 Neutralization Capacity of Mouse Serum Samples
[204] Conventional GST-clL-13 bioactivity and mouse IL-13 neutralization data were calculated using STAT-6 phosphorylation readout in A549 cells. Such an assay is cumbersome and cannot readily yield quantitative data. Mouse IL-13 stimulates proliferation of TF-1 cells in vitro. Therefore, an assay was developed to assess the mouse IL-13 neutralization capacity of serum from mice vaccinated with GST-clL-13 in these cells.
[205] 2.4 In Vitro Mouse IL-13 Neutralization Biological Assay
[206] To determine the ability of mouse serum to neutralize the bioactivity of recombinant mouse IL-13 on human TF-1 cells (obtained in-house), 5 ng / ml recombinant mouse IL-13 was added to 1 well in 96 well tissue culture plates (Invitrogen). Incubated with various concentrations of serum at 37 ° C. for hours. After this preincubation, TF-1 cells were added. Assay mixtures containing various serum dilutions, recombinant mouse IL-13 and TF-1 cells were incubated at 37 ° C. for 70 hours in a humidified CO ₂ incubator. MTT substrate (Cat. No. G4000, Promega) was added during the last 4 hours of incubation, after which the acid solution was added to stop the reaction and dissolve the metabolized blue formagen product. The absorbance of the solution in each well was read with a 96-well plate reader at 570 nm wavelength.
[207] It should be noted that this assay can only measure mouse IL-13 neutralization at serum dilutions greater than 1/100. Serum dilution below 1/100 induces nonspecific proliferative effects in TF-1 cells.
[208] The ability of the serum to neutralize mouse IL-13 bioactivity was expressed as the serum dilution (= ND ₅₀ ) required to neutralize the bioactive activity of a predetermined amount of mouse IL-13 by 50%. The greater the serum sample dilution, the stronger the neutralization capacity.
[209] Mouse IL-13 neutralizing ability of serum obtained from mice immunized with GST-clL-13 was measured by the above method. A potent IL-13 neutralization reaction occurred as indicated below.
[210] Mice (serum samples taken 125 days after 4 × GST-cIL-13 vaccine) Mouse IL-13 Neutralization (ND ₅₀ ) C11/1250 C21/5230 C31/523 C41/417 C51/1670
[211] 2.5 Measurement of Mouse IL-13 Neutralization Levels Required for Efficacy in the 'Ovalbumin Challenge' Mouse Asthma Model
[212] To standardize the required efficacy of IL-13 autoantibodies for the treatment of asthma, mice were treated with varying doses of rabbit anti-mouse IL-13 polyclonal antibody during the Ovalbumin challenge in the Ovalbumin Challenge mouse asthma model. (Passively administered by intraperitoneal injection). Model parameters such as airway hyperresponsiveness (AHR), goblet cell metaplasia (GCM) and pulmonary inflammatory cell content were measured at the end of the experiment. Efficacy in this model was related to mouse IL-13 neutralization levels obtained in mouse serum. Mouse neutralization biological assay was used to measure mouse IL-13 neutralization levels in serum samples.
[213] Treatment group (manual dose of rabbit anti-mouse IL-13 antibody) Mouse IL-13 Neutralization (ND ₅₀ ) Capacity1/4100 High capacity1/2670 Medium capacity1/476 Minimum capacity1/207
[214] All treatment groups receiving up to three doses of antibody were similar. All three groups showed equivalent or better (in case of AHR) or better (in case of GCM) gold standard treatments (dexamethasone, 3 x 1.5 mg / kg administration by intraperitoneal route) used in this model. The lowest dose of antibody administered was about half the efficacy of dexamethasone and the 'untreated' positive control.
[215] Thus, the IL-13 neutralization level obtained in the 'medium dose' treatment group represents the efficacy threshold required for IL-13 auto vaccines in this animal model. Efficacy threshold is defined as the minimum level of IL-13 neutralization in mouse serum that is required to show 100% effect in the asthma model. Therefore, 1 × ED ₁₀₀ corresponds to ND ₅₀ of 1/476.
[216] Importance of defined efficacy threshold
[217] The level of IL-13 neutralization required for effects in the 'Ovalbumin Challenge' mouse asthma model was defined above. The level of GST-clL-13 induced IL-13 neutralization in mice C1-3 and C5 exceeds the efficacy threshold required for effect in the asthma model. These results are shown in FIG.
[218] Thus, the GSL-cIL-13 vaccine is expected to show efficacy in the mouse asthma model.
[219] Example 3: Immunogenicity Profile of GST-clL-13 in Combination with Various Adjuvant
[220] 3.1 Immunization Protocol
[221] GST-clL-13 was used as an immunogen to induce the formation of auto-antibodies against mouse IL-13 in Balb / c mice. Female mice aged 6 to 8 weeks were administered once with about 100 μg protein in an adjuvant. Thereafter, booster immunization consisting of 50 μg protein in the adjuvant was performed four times. Each treatment group included 5 animals, which were immunized according to the protocol in the table below.
[222] Serum samples were obtained by venipuncture the tail vein at the designated timepoint. After sorting by centrifugation, samples were assayed by ELISA for the presence of specific IgG responses to mouse IL-13.
[223] groupImmunization AGST-cIL-13 i / m in AS03 BGST-cIL-13 i / p in alum CGST-cIL-13 i / m in 'Easy' DGST-cIL-13 s / c in CFA / IFA EGST-cIL-13 s / c in PBS FNo immunization
[224] Dayprocess -7Pre-bleeding 0Primary immunization 21First boost immunization 35Tail blood 49Second boost immunization 63Tail blood 77Third boost immunization 92Tail blood 106Fourth boost immunization 125Tail blood
[225] 3.2 Immunogen + Adjuvant Formulations.
[226] Preparation of Emulsion Adjuvant AS03
[227] Tween 80 was dissolved in phosphate buffered saline (PBS) to prepare a 2% solution in PBS. To provide a 100 ml 2-fold emulsion, 5 g of DL alpha tocopherol and 5 ml of squalene were vortexed to complete mixing. 90 ml of PBS / Twin solution was added and mixed thoroughly. The emulsion obtained was then passed through a syringe and finally microfluidized using an M110S microfluidics device. The oil droplets obtained had a size of about 180 nm.
[228] The adjuvant was mixed 1: 1 with the protein solution, vortex briefly (10 seconds at medium speed) and incubated for 10 minutes at room temperature on an orbital shaker. Vortex briefly (ie 2 × 50 μl per mouse, one injection in each quadriceps) before injection and administration of a total of 100 μl suspension per mouse via the intramuscular route at two different locations. New ones were prepared before each immunization.
[229] alum
[230] Provided by SIGMA (Cat. No. A-1577). Alum suspension in 2 mg / ml PBS was prepared. The adjuvant was mixed 1: 1 with the protein solution, vortex briefly and incubated with gentle shaking for 10 minutes at room temperature. A total of 100 μl of suspension per mouse was vortexed briefly before intraperitoneal (i / p) injection and administration. New ones were prepared before each immunization.
[231] CpG-ImmunEasy
[232] Supplied by Qiagen (Cat. No. 303101). The adjuvant storage container was mixed in a gentle vortex and the adjuvant was mixed 1: 1 with the protein by gently pipetting up and down five times. Incubate at room temperature for 15 minutes. The mixture was pipetted up and down five times gently and a 100 μl suspension per mouse was administered by intramuscular route at two separate locations (ie 2 × 50 μl per mouse, one injection in each quadriceps). New ones were prepared before each immunization.
[233] CFA / IFA
[234] Supplied from Sigma (Cat. Nos. F-5881, F-5506). Formulated 1: 1 with premixed main CFA or IFA for boost. Samples were rotationally mixed to ensure a uniform white suspension with CFA / IFA. Store on ice for at least 30 minutes prior to use and thoroughly mix thoroughly before administration.
[235] 3.3 Anti-Mouse IL-13 Antibody Responses
[236] Anti-mouse IL-13 Antibody Detection ELISA was used to observe the anti-mouse IL-13 antibody response in serum samples.
[237] 96-well Maxisorp plates were coated overnight at 4 ° C. with anti-mouse IL-13 monoclonal antibody (Cat. No. MAB, R + D system) in carbonate-bicarbonate buffer. Plates were blocked for 1 hour at room temperature with 3% BSA / TBST, washed three times in TBST and incubated with mouse IL-13 (Cat. No. 413-ML-025, R + D system) for 1 hour at room temperature. It was. After washing, incubated with mouse serum for 1 hour at room temperature, washed again, and incubated with HRP conjugated anti-mouse IgG polyclonal antibody (SIGMA, Cat. No. A9309). After further washing the plate, the plate was expressed for 30 minutes with 0-phenylenediamine dihydrochloride peroxidase substrate.
[238] Levels of anti-mouse IL-13 antibody in serum were expressed as endpoint titers. Endpoint titer is defined as the degree of dilution of serum corresponding to doubling the ELISA background reading.
[239] mouse Anti-mouse IL-13 Antibody Endpoint Titers AS03 alum CpG CFA / IFA One1/8751/72501/675001/6750 21/92501/8001/800001/975 31/1601/90001/540001/6000 41/90001/65001/625001/16000 51/36001/100001/775001/31000
[240] FIG. 10 depicts anti-mouse IL-13 antibody profile at day 125 in various treatment groups for serum samples diluted at 1/100.
[241] All five mice immunized with GST-clL-13 in combination with CpG adjuvant showed a strong anti-mouse IL-13 self-antibody response. This is in contrast to other adjuvants, where the response is not very consistent across groups in other adjuvants, and some mice have very weak responses.
[242] These results indicate that the CpG adjuvant is more effective in exhibiting consistent and high titer anti-mouse IL-13 self-antibody responses compared to other adjuvants tested.
[243] These serum samples were analyzed for IL-13 neutralizing capacity in an in vitro IL-13 neutralizing bioassay.
[244] 3.4 IL-13 Neutralization Capacity.
[245] In order to measure the ability of mouse serum to neutralize the bioactivity of recombinant mouse IL-13 against human TF-1 cells (ATCC Cat. No. CRL-2003), 5 ng / ml recombinant mouse IL-13 was tested in 96-well tissue. The culture plates (Gibco BRL) were incubated with serum at various concentrations for 1 hour at 37 ° C. Following the preculture period, TF-1 cells were added. Assay mixtures containing various serum dilutions, recombinant mouse IL-13 and TF-1 cells were incubated at 37 ° C. for 70 hours in a humidified CO ₂ incubator. MTT substrate (Cat. No. G4000, Promega) was added during the last 4 hours of incubation, the reaction was terminated with acid solution and the metabolized blue formazan product was dissolved. Absorption of the solution in each well was read at 570 nm wavelength in a 96-well plate reader.
[246] Note that this assay can only measure mouse IL-13 neutralization in serum dilution levels greater than 1/100. Serum dilution below 1/100 induces a non-specific proliferative effect in TF-1 cells.
[247] The ability of the serum to neutralize mouse IL-13 bioactivity is expressed as the degree of dilution of the serum required to neutralize the bioactivity of 5 ng / ml mouse IL-13 by 50% (= ND ₅₀ ). The more dilute serum samples are required, the stronger the neutralization capacity.
[248] The highest concentration of mouse D5 serum tested was 1/100 dilution. This does not neutralize the bioactivity of 5ng / ml mouse IL-13 by 50%, so ND ₅₀ is expressed as a <1/100 dilution.
[249] Mice (serum samples taken at day 125) Mouse IL-13 Neutralization (ND ₅₀ ) C11/1250 C21/5230 C31/523 C41/417 C51/1670 D5<1/100
[250] Serum samples at day 125 taken from all five mice immunized with GST-clL-13 in combination with a CpG adjuvant can potently neutralize the bioactivity of mouse Il-13 in an in vitro bioassay. In contrast, serum samples at day 125 taken from mouse D5 (immunized with GST-clL-13 in CFA / IFA) were unable to neutralize the bioactivity of mouse IL-13 at all dilutions tested.
[251] These results indicate that CpG adjuvant is more effective in increasing neutralizing anti-mouse IL-13 self-antibody response compared to other adjuvants tested.

权利要求:
Claims (25)
[1" claim-type="Currently amended] An isolated protein that is at least 30% less than 100% identical to a human protein, wherein the protein is not an antibody,
(a) contains one or more mutations that are characteristic of similar non-human proteins,
(b) can produce antibodies in humans,
(c) A protein structurally similar to a human protein that is sufficient for the antibody to bind to both a human protein and a polypeptide.
[2" claim-type="Currently amended] Similar proteins of a second mammalian species, with B-cell epitopes from mammalian self-antigens, so that the protein can elicit an immune response that recognizes the native protein from which the B-cell epitope is derived from the species from which the B-cell epitope is derived Proteins with mutations resulting in the sequence of.
[3" claim-type="Currently amended] Self-transplanted by substitution into a framework of similar proteins from a second mammalian species such that the protein can elicit an immune response that recognizes the native protein from which the B-cell epitope is derived from the species from which the B-cell epitope is derived. Proteins with B-cell epitopes of proteins.
[4" claim-type="Currently amended] 4. A method according to any one of claims 1 to 3, comprising a conserved surface region introduced into an area that is not surface exposed, wherein said mutation results in an immune response against the magnetic protein in the species from which the protein is derived. A protein characterized by causing a sequence of analogous proteins to cause.
[5" claim-type="Currently amended] The protein according to any one of claims 1 to 4, wherein the immune response is a neutralizing antibody response.
[6" claim-type="Currently amended] 6. The protein of claim 1, wherein the human protein or B-cell epitope is derived from a cytokine. 7.
[7" claim-type="Currently amended] 7. The cytokine of claim 6 which is a four helix cytokine.
[8" claim-type="Currently amended] 8. The cytokine according to claim 7, which is IL-4 or IL-13.
[9" claim-type="Currently amended] Mutated human IL-13 having one or more of the following substitutions or a substitution comprising a conservative substitution thereof:
R → K at No. 30
V → S at 37
Y → F at 63
A to V at 65
E → D at 68
E → Y at 80
K → R at 81
M → I at 85
G → H at 87
Q → H at 113
V → I at 115
D → K at 117
[10" claim-type="Currently amended] 10. The mutated human IL-13 of claim 9 having a plurality of substitutions described in claim 9.
[11" claim-type="Currently amended] The mutated human IL-13 of claim 9 or 10, having one or more of the following sequences or variants of such sequences comprising one or more conservative substitutions:

[12" claim-type="Currently amended] Mutated human IL-13 shown in FIG. 9.
[13" claim-type="Currently amended] A polynucleotide encoding a protein according to any one of claims 1 to 12.
[14" claim-type="Currently amended] The polynucleotide of claim 13, wherein the DNA is operably linked to a promoter.
[15" claim-type="Currently amended] A vector comprising the polynucleotide of claim 13 or 14.
[16" claim-type="Currently amended] A host transformed with the polynucleotide of claim 13 or 14 or the vector of claim 15.
[17" claim-type="Currently amended] A pharmaceutical composition comprising a protein, polynucleotide, or vector according to any one of claims 1 to 15 together with a pharmaceutically acceptable carrier or excipient.
[18" claim-type="Currently amended] 18. The pharmaceutical composition of claim 17, further comprising an adjuvant.
[19" claim-type="Currently amended] A pharmaceutical composition according to claim 18 comprising a protein according to any one of claims 1 to 12 and an immunostimulatory oligonucleotide.
[20" claim-type="Currently amended] The pharmaceutical composition of claim 19, wherein the immunostimulatory oligonucleotide is selected from the group:

[21" claim-type="Currently amended] A protein, polynucleotide, vector, host or composition according to any one of claims 1 to 20 for use in medicine.
[22" claim-type="Currently amended] Use of a protein according to any one of claims 1 to 12 for the manufacture of a medicament for the treatment of an IL-13 mediated disease.
[23" claim-type="Currently amended] Use according to claim 22, which is used to treat asthma.
[24" claim-type="Currently amended] A method of treating or preventing an IL-13 mediated disease, comprising administering to a patient in need thereof a safe and effective amount of a composition according to any one of claims 17 to 20.
[25" claim-type="Currently amended] 1. Identifying one or more regions of the human, typically human, protein in which the antibody response is desired.
2. Identifying the amino acid sequence of his protein.
3. Confirming by recombinant DNA techniques the amino acid sequence of the analogous protein construct of the chimeric molecule containing at least one target region identified in step 1, wherein the amino acid sequence is obtained from the sequence identified in step 2, wherein the mutant protein is self Identifying an amino acid from the sequence (s) identified in step 3 sufficient to be able to fold the resulting protein into a form similar to that of its protein so as to elicit an immune response that recognizes the protein. A method for preparing a protein according to any one of claims 12-12.

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同族专利:

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公开号 | 公开日

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AU2002233560B2|2006-09-07|

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HU0303372A2|2004-01-28|

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WO2002070711A1|2002-09-12|

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ZA200306647B|2004-11-26|

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CA2439628A1|2002-09-12|

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JP2005502314A|2005-01-27|

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NO20033882L|2003-10-31|

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JP4238031B2|2009-03-11|

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MXPA03007915A|2003-12-04|

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PL365066A1|2004-12-27|

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NZ527873A|2005-12-23|

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US20050260216A1|2005-11-24|

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NO20033882D0|2003-09-02|

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CN1543504A|2004-11-03|

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EP1368477A1|2003-12-10|

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US20030194391A1|2003-10-16|

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US20050186209A1|2005-08-25|

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HU0303372A3|2008-06-30|

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BR0207819A|2004-03-02|

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CZ20032373A3|2004-01-14|

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IL157498D0|2004-03-28|

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GB0105360D0|2001-04-18|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2001-03-03|Priority to GBGB0105360.2A

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2001-03-03|Priority to GB0105360.2

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2002-03-01|Application filed by 글락소 그룹 리미티드

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2002-03-01|Priority to PCT/GB2002/000900

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2003-10-17|Publication of KR20030081490A

优先权:

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申请号 | 申请日 | 专利标题

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GBGB0105360.2A|GB0105360D0|2001-03-03|2001-03-03|Chimaeric immunogens|

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GB0105360.2|2001-03-03|

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PCT/GB2002/000900|WO2002070711A1|2001-03-03|2002-03-01|Vaccine|