JP2002516634A - Methods of identifying antibodies and peptides useful in the treatment of septic shock and experimental arthritis and uses thereof - Google Patents

Abstract

  (57) [Summary] The present invention teaches the use of antibodies raised against Group A mucopeptides to treat septic shock and rheumatoid arthritis, and the identification of specific peptides that can be used in such treatments. The invention encompasses methods of using antibodies raised against the group A mucopeptides (including use as a means of identifying a particular peptide), the particular peptide itself, and methods of using the peptide. I do. In some embodiments, the particular peptide functions as a mimetic for these antibodies. Such peptides can be administered to an animal as a subject to block the microbial effects that cause rheumatoid arthritis through tumor necrosis factor. Alternatively, certain peptides of the invention can be used to immunize an individual susceptible to rheumatoid arthritis to prevent further progression of the disease. In yet another aspect, the peptide functions as a binding partner for these antibodies.

Description

DETAILED DESCRIPTION OF THE INVENTION Antibodies and peptides useful in treating septic shock and experimental arthritis Method for identification and use thereof                                Field of the invention   The present invention is useful in treating septic shock, experimental arthritis and related disorders A novel peptide identification method. Some of these new peptides are anti-glu Screening for its Affinity or Mimeticity to Anti-A Mucopeptide Antibodies Is done. The role of these novel peptides and related antibodies in septic shock Disclose therapeutic use.                                Background of the Invention   Years of research have revealed the basic structure of many Gram-positive and Gram-negative organisms. I made it clear. In connection with the Gram-positive bacteria, the skeleton of these organisms It is a polysaccharide-peptide complex called a peptide complex (FIG. 1). Basic repetition The repeat unit is the alternating mochi of neuraminic acid-N-acetylglucosamine (NAM-NAG) molecule. Where the interpeptide cross-links vary from organism to organism.   The gram-negative bacteria are a common sugar, commonly known as lipid A. Has lipids on which various oligosaccharides and polysaccharides depend on the bacteria Has been added. The common structure of lipid A is shown in FIG. Lipid A and And the mucopeptide complex, when injected into an animal, are each independently extremely potent. And elicit strong and harmful responses. The most severe of these responses is often It is often the production of large amounts of tumor necrosis factor (TNFα).   At this time, the main way to block this TNF induction is with respect to the TNF molecule itself. Administer the antibody or use a soluble TNF receptor molecule to Competitive inhibition of TNF binding to vesicles. Both of these methods, Will block the release of cytokines. Models with restricted epitope specificity Noclonal antibodies have proven unsuccessful in this regard [Fishe r et al., Crit. Care Med., 1994, 21: 318-3272; McCloskey et al., Ann. Intem. Med., 121: 1-5] . F (ab)_TwoPreliminary attempts using anti-TNF polyclonal antibodies have shown Although considered expensive, such a method is effective Before we can show that we need to do some further work.   As described above, Lipid A and the mucopeptide conjugate may cause the significantly deleterious response. The mechanism by which monocytes are initiated has not yet been elucidated, but many researchers We have agreed that the above cell surface receptors need to be involved. This receptor Searching for one or more identification methods is an extremely active area of research. In the past few years In particular, Morrison and his colleagues have identified a 70 kDa protein on monocytes. However, this is thought to be associated with lipopolysaccharide (LPS) [Dziarski, J.B. Chem., 1994, 266: 4719-4725: Lei et al., Int. Rev. Immunol., 1990, 6: 223-235; Lei et al. J. Immunol., 1991, 147: 1925-1932; Rabin et al., J. Infect. Dis., 1993, 168: 135-142]. This monoclonal antibody, which is reactive with the 70 kDa protein, produces TNFα by monocytes. Has been reported to be responsible for this 70 kDa Protein reinforces the proposal that it may be the LPS receptor (Morrison et al., J.I. nfect. Dis., 1990, 162: 1063-1068). Apart from this putative identification of the receptor, Little is known about immunologically important mechanisms.   Thus, how Lipid A and the mucopeptide conjugate could have their deleterious effects There is a need to better understand the mechanisms involved in triggering. More practical In some aspects, alternatives to blocking the induction of TNFα during septic shock There is great demand. Therefore, the presence of lipid A and the mucopeptide complex There is also great demand for the design of new drugs that can neutralize harmful effects. You.   Citation of any reference herein is made by those references to the present invention. Note that this does not mean that it is recognized as "known art" .                                Summary of the Invention   In the broadest aspect of the invention, the invention relates to septic shock and rheumatism. For treating inflammatory diseases such as osteoarthritis, Crohn's disease, psoriasis, and the like. Group A mucope to identify specific peptides that can be used in various therapies The use of antibodies raised against the peptide is taught. In some embodiments, the identification Will act as mimics of these antibodies. In another aspect, the These peptides will function as binding partners for these antibodies.   One aspect of the present invention relates to common gram-positive and gram-negative bacteria. It is derived from the assumption that there is a structural motif. The present invention supports this premise. Pioneer the flow and, consequently, identify treatments for septic shock and related disorders Introduce new methods.   One of the features of the present invention resides in both lipid A and group A mucopeptides. Generating and identifying antibodies that bind to common motifs. Departure One related aspect of Ming is to generate and characterize peptides that mimic these antibodies. Is to do. These peptides bind to LPS and the group A mucopeptide. By acting on the bacterial drug and its receptor. Inhibits binding to As a result, this is a consequence of TNFα induction and septic shock. The onset can be prevented.   Accordingly, the present invention includes a method for obtaining such inhibitory peptides. A certain form In certain embodiments, the particular peptide is transferred to a phage library and Obtained by contacting the rear. The phage library comprises a set of phages. Each phage contains a random DNA sequence, which sequence Encodes the peptide of The DNA sequence of the phage library is expressed and the species Each random peptide is generated. The random peptide is the mucopepti And its ability to bind to the mucopeptide complex Sorted out based on strength. Purifying a phage containing the candidate peptide, The DNA encoding the peptide is sequenced. Therefore, the amino acid of this candidate peptide The acid sequence can be deduced.   The candidate peptides are synthesized from their respective amino acid sequences. This synthesis, for example, Performed by standard genetic engineering techniques or, more preferably, by solid phase peptide synthesis. Wear. Next, these synthetic candidate peptides are contacted with lipid A, It is further characterized by its ability to bind to A. Thus, lipid A and the lipid A Peptides that bind to both Group A mucopeptides can be obtained. In addition, These selected peptides are compared to the group A mucopeptide and the anti-group A Contact with mucopeptide antibody. Inhibitory peptide is used to form the antigen-antibody complex From the selected peptides based on their ability to inhibit the.   In certain embodiments described above, to obtain a series of random peptides, This combination was also produced by chemical synthesis. It is also possible. Furthermore, this combination of random peptides It can also be obtained by a combination of two techniques. Combination of random peptides For all of these embodiments provided by the present invention, using Library or chemical synthesis can be used.   In another embodiment of the present invention, the inhibitory peptide further comprises a tumor necrosis factor α (T NFα) is selected based on its ability to inhibit bacterial-mediated production. this The method comprises combining the inhibitory peptide with a mononuclear cell sensitized with LPS or the mucopeptide. Including the additional step of contacting. Preferred of the method for identifying the inhibitory peptide In one aspect, the inhibitory peptide comprises the group A mucopeptide and the LP It binds to common structural elements that are present in both S molecules.   The inhibitory peptides obtained by the methods described herein also form part of the present invention. To achieve. These inhibitory peptides have the following characteristics: Loop A binds both the mucopeptide and the LPS molecule, and further comprises anti-group A Inhibits the binding of the mucopeptide antibody to its group A mucopeptide antigen.   In one preferred embodiment, the inhibitory peptide further comprises a tumor cell in the target cell. Inhibits bacterial-mediated production of tumor necrosis factor. The target cell is In response to stimulation with group A mucopeptide and / or the LPS molecule, It can be a mononuclear cell or any other cell that secretes cytokines. More preferred embodiment Wherein the mononuclear cell is a human mononuclear cell.   The invention also relates to septic shock, rheumatoid arthritis, Crohn's disease, psoriasis and Peptides of the Invention and Pharmaceutically Acceptable for the Treatment of Experimental and Experimental Arthritis Pharmaceutical compositions, which include the carriers described above.   Administering to a mammal a therapeutically effective amount of such a pharmaceutical composition. A method of treating septic shock in a mammal is also included in the present invention. Septic In a preferred embodiment for treating shock or rheumatoid arthritis, The mammal is a human.   The invention also provides a therapeutically effective amount of an inhibitory peptide of the invention and a pharmaceutically acceptable amount. Experimental administration comprising administering to a subject animal a pharmacological composition comprising a carrier to be administered. It also includes a method for treating severe arthritis. The present invention also provides therapeutically effective amounts of the instant invention. A pharmaceutical composition comprising a light inhibitory peptide and a pharmaceutically acceptable carrier. A similar method of treating rheumatoid arthritis by administration is also contemplated. ing.   A related aspect of the invention is the method of the invention, wherein the LPS-mediated induction of TNFα in a mononuclear cell is Adding antibodies prepared against pure Group A mucopeptides to cells Thus, it relates to a method of blocking. In a preferred embodiment, the mononuclear cells are human It is a mononuclear cell.   Another aspect of the present invention provides that the mucopeptide conjugate is used for treating rheumatoid arthritis. It is guided on the premise that it elicits a comparable immune response. The present invention Aspects include a method of identifying an antigenic peptide. Special in this way One embodiment is to administer the selected peptide to an animal and then subject the animal to an experimental procedure. Administering to the animal a group A mucopeptide capable of inducing arthritis; The group wherein the selected peptide induced experimental arthritis in the animal. A based on the ability to elicit a protective antibody response to mucopeptide Identifying the peptide, including steps. In one particular embodiment of this type, the animal is Rats, and the amount of Group A mucopeptide administered was 1 g of body weight of the rats. Equivalent to the group A cell fragment containing 15-30 μg rhamnose per The dose, i.e. a dose equal to 3-6 mg rhamnose per 200 g rat. ( The amount of the mucopeptide is typically determined by the amount of rhamnow measured for the mucopeptide. Expressed as a function of the quantity of Because the rhamnose content of the mucopeptide Is relatively constant and can be easily measured). Prefer this type In a preferred embodiment, 125-250 μg of the selected peptide per gram of body weight of the rat. Dose of 25 to 50 mg of peptide selected per rat, i.e. 200 g of rat. The animals are administered prior to administration of the Group A mucopeptide.   The selected peptide binds to an anti-group A mucopeptide antibody and Selection is based on the ability to inhibit binding to Group A mucopeptides. This One related method for selecting such peptides is a series of random peptides. And an anti-group A mucopeptide antibody and contacting the candidate peptide The candidate peptide is selected based on its ability to bind to a loop A mucopeptide antibody. Including the step of performing The selected peptide is then recovered and / or If not, the above-mentioned screen for identifying and identifying the antigenic peptide of the present invention is used. Can be used in leaning assays.   In connection with the earlier aspects of the invention, the series of random peptides is Obtained from Ibrary. According to one particular aspect, the selected peptide is a peptide. Obtained by contacting a large library with an acceptable bacterium . This phage library contains a series of phages, each of which is And a random DNA sequence encoding a particular peptide. This phage live Expression of the rally DNA sequence produces a variety of random peptides. The selected peptide can be recovered as follows: The phage encoding the selected peptide is purified and the selected peptide is The sequence of the DNA to be loaded can be determined. Then, with respect to the selected peptide, Can be deduced from the DNA sequence. Alternatively, Combinatorial libraries can also be used.   Next, the selected peptide can be synthesized from its respective amino acid sequence. You. This synthesis can be performed, for example, using standard genetic engineering techniques or, more preferably, solid phase peptides. It can be carried out by a tide synthesis method.   The antigenic peptide obtained by the method described herein is also a part of the present invention. Is configured. These inhibitory peptides have the following characteristics: Binds to anti-group A mucopeptide antibodies and these Induces protective antibody response in animals against experimental arthritis induced by tide I do. The present invention also includes the antigenic peptide of the present invention and a pharmaceutically acceptable carrier And pharmacological compositions.   The present invention also relates to a method for producing arthritis in animals that have been challenged to produce experimental arthritis. Methods for analyzing the ability to prevent the development of experimental arthritis are also included. In one aspect In addition, the method includes the step of providing the animal with the group A muco prior to the onset of experimental arthritis. Antibody prepared against the peptide is administered to prevent the development of experimental arthritis as a result Stopping. In another embodiment, preventing the development of experimental arthritis The method comprises the step of providing the animal with the antigenic peptide of the invention before experimental arthritis develops. And administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier.   The invention also includes a method of treating experimental arthritis in an animal. In one aspect The method further comprises the step of providing the animal with the group A mucope after the onset of experimental arthritis. Administering antibodies prepared against the peptide, thereby reducing experimental arthritis in the animal. Including improving. In a related aspect, the invention relates to the development of experimental arthritis. After the disease, the animal contains the antigenic peptide of the present invention and a pharmaceutically acceptable carrier. Administering said pharmacological composition and thereby ameliorating experimental arthritis in said animal It also includes the law.   By making changes in the amino acid sequences of the antigenic and inhibitory peptides, Anti-group A mucopeptide antibodies and their anti-group A mucopeptides It is also possible to increase the binding affinity. For example, a slight change is Substitution of an acidic amino acid for another amino acid, such as glutamine for aspartic acid Includes acid substitution. Greater changes can be planned, such as a single Replace Nitin with Arginine or replace d-amino acid with its corresponding 1-amino acid It is also possible to substitute for an acid. To optimize certain properties of these peptides All such substitutions of are included in the present invention.   These and other aspects of the invention refer to the following drawings and detailed description. This will be better understood by reference.                             BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 relates to the prior art, and shows the mucope of the gram-positive bacterial cell wall. 1 shows the structure of a peptide complex. Polysaccharide-peptide of Gram-positive bacterial cell wall FIG. 2 is a schematic diagram of a composite.   FIG. 2 relates to the prior art and shows the sugar content of the gram-negative bacterial cell wall. It is a figure which shows a lipid (lipid A) typically.   FIG. 3 compares the structures of the mucopeptide and the glycolipid complex. . The structures of the peptidoglycan (mucopeptide complex) and lipid A were compared. of It is.   FIG. 4 shows the results prepared for pure Group A Streptococcus mucopeptide. Of TNFα by bacterial cell wall components in the presence or absence of antibodies Indicates induction. The three-dimensional bar graph shows the pure Group A Streptococcus mucope Antibodies prepared against peptides in the presence (stippled bar) or absence (shaded bars) of antibody In a single addition (FIG.4A) or 20 ng of the mucopeptide complex Was generated in human mononuclear cells (y-axis) by a single addition of LPS (Figure 4B). 3 shows a comparison of TNFα (expressed in ng).                             DETAILED DESCRIPTION OF THE INVENTION   The present invention provides for the treatment of septic shock, Crohn's disease, psoriasis, and rheumatoid arthritis. Using antibodies raised against said group A mucopeptides for treatment As well as compositions. These antibodies can be used in these treatments By identifying certain peptides, they can be used directly or indirectly. it can.   Whether the above 70 kDa protein is the LPS receptor that has been sought for a long time, The Streptococcus mucopeptide complex binds the LPS to the 70 kDa protein. The fact that the interaction can be inhibited is due to the fact that the LPS and LPS have some structural similarities. And is consistent with the mucopeptide complex. Therefore, the present invention relates to the pure group A comprising the use of an antibody prepared against the Streptococcus mucopeptide. The body is able to block the induction of TNFα in human mononuclear cells, and this ability Group A mucopeptide or gram-negative cells obtained from gram-positive bacteria It is irrelevant to which of the LPS obtained from the product was sensitized (see FIG. 4). No. Looking at the three-dimensional structure of both groups, as shown in Fig. 3, there is a remarkable class in the skeleton. Similarity exists. This similarity is further present in both Gram-positive and Gram-negative organisms. Existing structural elements bind to receptors present in human mononuclear cells, and It suggests that production of TNFα can be induced.   The present invention relates to the common A present on the group A mucopeptide and the LPS molecule. To aid in the identification of novel peptides that will bind to structural elements of -Describes a novel method using mucopeptide antibodies. These peptides are Similar to the binding domain in the anti-mucopeptide antibody, and Binding of the cell receptor to the common structural elements present in Can be interrupted. This results in the TN by the target cells Inhibits Fα production. Therefore, the present invention utilizes this novel method to Shock, Crohn's disease, psoriasis and experimental arthritis (animals with rheumatoid arthritis) Model), and the study of various diseases. For those skilled in the art, Diseases are merely exemplary and have other related mechanisms. Will also understand that the teachings of the present invention can be successfully treated .   As used herein, the terms “mucopeptide complex”, “group A mucopeptide”, “ "Peptidoglycan" and "polysaccharide-peptide conjugate" are used interchangeably, 1 depicts an integral aspect of the cell wall of Gram-positive bacteria. This one aspect Elicit an immune response in mammals that can cause septic shock You.   As used herein, the terms "lipid A", "glycolipid", "lipopolysaccharide" or "LPS" Used interchangeably and represents an integral aspect of the cell wall of Gram-negative bacteria You. Each of these integral aspects involves mammals that can cause septic shock. Elicit an immune response.   The term `` inhibitory peptide '' as used herein has the following characteristics: It binds to the group A mucopeptide, binds to the LPS molecule, and Inhibits the binding of the Group A mucopeptide antibody to the Group A mucopeptide.   The term `` antigenic peptide '' as used herein has the following characteristics: Does not bind to the group A mucopeptide, but does not bind to the anti-group A mucopeptide. Joints that bind to antibodies and are induced by group A mucopeptides A protective antibody response to inflammation is elicited in the animal.   As used herein, the term “common structural element” refers to “common structural motif” Used interchangeably, at least one binding partner has an affinity that can withstand comparison At least two other, distinct, including such common structural motifs Three-dimensional structural similarity that allows selective binding to molecules Represent.   As used herein, the term "experimental arthritis" refers to the occurrence of redness and swelling of an animal's feet. Has clinical symptoms, including: Such symptoms may include observing the diameter of the foot and / or Can be tracked by measuring the diameter. These symptoms, over time, Increase or decrease. Increased foot size is a sign of arthritis.                                   antibody   According to the present invention, mucopeptides prepared from natural sources or recombinant technologies Mucopeptides produced by more or chemical synthesis, as well as fusion proteins , Its fragments or other derivatives or analogs can be used as immunogens. Can be used to produce an antibody that recognizes the mucopeptide and LPS. You. Similarly, LPs obtained from natural sources or obtained by chemical synthesis S, as well as fragments or other derivatives or analogs thereof, Can be used as an immunogen to produce antibodies that discriminate between both antibodies and LPS. Such antibodies can be polyclonal, monoclonal, chimeric, single chain, Fragments, and Fab expression libraries.   If the molecule can specifically interact with an antigen recognition molecule of the immune system, For example, an immunoglobulin (antibody) or T cell antigen receptor is "antigenic." Anti A native polypeptide has at least about 5, and preferably at least about 10, Contains amino acids. The antigenic portion of a molecule is responsible for recognizing antibodies or T cell receptors. May be a portion of the molecule that is immunodominant for By conjugating with a carrier molecule for immunization, it is possible to produce antibodies against the molecule. Can be used for A molecule that is antigenic is itself immunogenic, i.e., without a carrier. It need not be capable of eliciting an immune response.   An “antibody” is any immunoglobulin that binds to a specific epitope. Includes the body and its fragments. This term is used for polyclonal, monochrome And chimeric antibodies (which are disclosed in U.S. Pat.Nos. 4,816,397 and 4,816,567). And Fab, F (ab ')._TwoAnd F (v) -containing antibodies (single-chain (Including antibodies). Accordingly, species such as those used herein The term “antibody molecule” in its various grammatical forms refers to whole immunoglobulin molecules and Means both immunologically active portions of immunoglobulin molecules, including body binding sites Shall be. "Antibody binding sites" refer to heavy and light chains that specifically bind to an antigen. chain It is a structural part of an antibody molecule consisting of a variable region as well as a hypervariable region.   Exemplary antibodies are intact immunoglobulin molecules, substantially intact immunoglobulins Molecules, and Fab, F (ab ') in the art_TwoAnd these known as F (v) A part of an immunoglobulin molecule, including a paratope, Minutes are preferred for use in the treatment methods described herein.   Fab and F (ab ') of antibody molecule_TwoThe portion is a substantially intact antibody, known in the art. Prepared by the proteolytic reaction of papain and pepsin, respectively, on the molecule It is. In this regard, for example, U.S. Patent No. 4,342, issued to Theofilopolous et al. See No. 566. Some of the Fab 'antibody molecules are also well known and combine two heavy chain portions The reaction of the disulfide bond with mercaptoethanol The alkylation of protein mercaptans with reagents such as iodoacetamide Therefore, F (ab ')_TwoGenerated from parts. In the present invention, an antibody comprising a complete antibody molecule Is preferred.   The term “monoclonal antibody” in its various grammatical forms refers to a specific antigen An antibody having only one species of responsive, antibody binding site. Therefore, the thing A clonal antibody typically contains a single antibody against any antigen with which it immunoreacts. Shows binding affinity. Monoclonal antibodies can therefore be used in multiple antibody binding sites. Antibody molecules, each of which is immunospecific for various antigens. And include, for example, bispecific (chimeric) monoclonal antibodies.   The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. Means things. An adjuvant can function as a tissue depot, where the depot is the antigen depot. Slowly releases and also acts as a lymphoid activator, causing the immune response to be non-specific (Hood et al., Immunology, p. 384, 2nd edition, Benjamin / Cummings, Menlo, CA Park (1984)). Often primary sensitization with antigen alone, in the absence of adjuvant Will not be able to elicit a humoral or cellular immune response. Aju Bunt is complete Freund's adjuvant, incomplete Freund's adjuvant, sapo Inorganic gels such as nin and aluminum hydroxide, surfactants such as lysolecithin, pull Ronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, Keyhole limpet hemocyanin, dinitrophenol, and extremely Human adjuvants such as BCG (Bacille Calmett-Guerin) e-Guerin)) and Corynebacterium parvum But not limited to these. Preferably, the adjuvant is pharmaceutically acceptable Things.   Various procedures known in the art may be used to perform the mucopeptide and LPS or fragmentation thereof. For the production of polyclonal antibodies that cross-react with antibodies, derivatives or analogs. Can be used. In order to produce antibodies, various host animals are treated with the mucopeptide or LPS or its derivatives (e.g., fragments or fusion proteins) can be injected. Wherein the host is a rabbit, mouse, rat, sheep, Including, but not limited to, goats. In one embodiment, the mucopeptide or Converts LPS or a fragment thereof to an immunogenic carrier such as bovine serum albumin (BS A) or keyhole limpet hemocyanin (KLH). Various horse mackerel Using a conjugate to enhance the immune response, depending on the species of the host; Examples of such adjuvants include Freund (complete and incomplete), aluminum hydroxide and the like. Inorganic gels, surfactants such as lysolecithin, pluronic polyols, polya Neon, peptide, oil emulsion, keyhole limpet hemocyanin, Dinitrophenol, and very useful human adjuvants such as BCG (Bacyl Bacille Calmette-Guerin) and Corynebacterium pal Bam (Corynebacterium parvum), but is not limited thereto.   The mucopeptide, LPSN or a fragment, analog or derivative thereof To prepare monoclonal antibodies, use continuous cell lines in culture. Alternatively, any technique for producing antibody molecules can be used. Examples are Kohler et al., Natur e, 1975, 256: Hybridoma technology and trio first developed by 495-497 Technology, human B-cell hybridoma technology (Kozbor et al., Immunology Today, 1983, 4: 72) and EBV-hybridoma technology (Co le et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, (Alan R. Ijss, Inc. , (1985)). Immortalized antibody-producing cell lines other than fusion Techniques, such as direct transformation of B lymphocytes with cancer DNA, or Epsch Transformation by Epstein-Barr virus It can be produced by fection or the like. For this, for example, statements such as M. Schreier Contributions: Hybridoma Techniques (1980); Hammerling et al .; Monodonal Antibodies, 19 80; and U.S. Pat.Nos. 4,341,761, 4,399,121, 4,427,783, 4, No. 444,887, No. 4,451,570, No. 4,466,917, No. 4,472,500, No. 4,491, See 632 and 4,493,890.   In one of the attendant aspects of the invention, monoclonal antibodies are It can be produced in sterile animals using TC / US90 / 02545). According to the present invention, human Antibodies can be used, which can be performed using human hybridomas (Cote et al., Proc. Natl. Ac. ad.Sci. USA, 1983, 80: 2026-2030) Alternatively, in vitro, human B-cells are (Cole et al., Supra, 1985). fact According to the present invention, a mouse antibody component specific for the mucopeptide or LPS Genes from offspring together with genes from human antibody molecules with appropriate biological activity Technology developed to produce "chimeric antibodies" by splicing (Morrison et al., J. Bacteriol., 1984, 159-870; Neuberger et al., Nature, 1984, 312: 604- 608; Takeda et al., Nature, 1985, 314: 452-454). Such antibodies are Within the scope of the invention. Such human or humanized chimeric antibodies can be used to treat human diseases. Or disorders (discussed below). I mean, A human or humanized antibody is a heterologous antibody that elicits an immune response, especially an allergic response. Is completely different.   According to the present invention, a technique for producing a single-chain antibody (U.S. Pat.No.4,946,778) is employed. Thus, mucopeptide / LPS-specific single chain antibodies can be produced. Another of the present invention One embodiment is a technique described for the construction of a Fab expression library (Huse et al., Sci. , 1989, 246: 1275-1281), and the mucopeptide and LPS Rapid production of monoclonal Fab fragments with defined specificity for pitopes And allows easy identification.   Antibody fragments which contain the idiotype of the antibody molecule can be produced by known techniques. Wear. For example, such fragments are produced by pepsin digestion of the antibody molecule. F (ab ')_TwoFragment: the F (ab ')_TwoDisulfide bridge of fragment A Fab ′ fragment that can be produced by reducing I And Fab fragments that can be produced by treatment with a reducing agent, Not limited to these.   In the production of antibodies, screening for a predetermined antibody is performed by a technique known in the art, For example, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), Sandwich ”immunoassay, immunoradiometric assay, gel diffusion sedimentation reaction, immunity Diffusion assays, in situ immunoassays (e.g., colloidal gold, Enzymatic or radioisotope labeling), Western blot, precipitation, aggregation Assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, Immunofluorescence assay, protein A assay, immunoelectrophoresis assay, etc. Can be achieved. In one aspect, the antibody binding detects a label on the primary antibody Is detected by In another embodiment, the primary antibody is a secondary antibody. Or by detecting the binding of the reagent or reagent to the primary antibody. even more In embodiments, the secondary antibody is labeled. Detect binding in immunoassays Many means for doing so are known and are within the scope of the present invention. For example, the mucope To select antibodies that recognize a specific epitope common to peptides and LPS, The resulting hybridoma may be a mucopeptide or Products that bind to fragments of LPS can be assayed.   The antibodies described above are relevant to the location and activity of the mucopeptide or LPS in the art. , Such as Western blotting, the mucopeptide in situ Or LPS imaging, measuring its level in appropriate physiological samples, etc., smell Can be used.   In one particular embodiment, offsets or enhances the activity of the mucopeptide and LPS Antibodies can be generated. Such antibodies are used to identify ligands. You can test using the method described below.   In one particular embodiment, the antibody is a rabbit that is predicted by the sequence of the mucopeptide. It is expressed by immunization with the envisioned synthetic peptide. Synthetic pepti Is conjugated with a carrier such as KLH hemocyanin or BSA and carbodiimide. Be used in Freund's adjuvant to immunize rabbits Can be. This expressed peptide can be prepared in large quantities and Can be used to immunize rabbits in Reunion adjuvant You.   In another particular embodiment, the mucopeptide is used to immunize a chicken. The chicken anti-mucopeptide antibody from egg yolk, e.g., mucopepti It is recovered by affinity purification on a column. This affinity Tea-purified antibodies can be screened by subsequent purification on LPS-columns. it can. Preferably, chickens used in immunization contain a particular pathogen. (SPF) conditions.   In another embodiment, the mucopeptide is used for immunization of a rabbit and the obtained Prior to further use, the polyclonal antibody to be used is conjugated to a mucopeptide-column or LPS. -Immunopurify on the column. The purified antibodies are particularly useful in semi-quantitative assays. It is for. The monoclonal antibody generated against the mucopeptide or LPS Panels for various properties, i.e., isoforms, epitopes, affinity, etc. You can lean. Of particular interest is neutralizing the activity of the mucopeptide or LPS And a monoclonal antibody that binds to the TNF receptor. Such a monk Lonal antibodies can be easily identified by the assays described herein. . Antibodies with high affinity are particularly useful in this neutralization method.   Preferably, the anti-modulator for use in the diagnostic and therapeutic methods of the invention The antibodies are affinity generated polyclonal antibodies. More preferably The antibody is a monoclonal antibody (mAb). Further, the anti-muco used herein Peptides or anti-LPS molecules are Fab, Fab ', F (ab')_TwoOr F (v) part The shape is preferably                                 peptide   The term “peptide” is used in its broadest sense and refers to two or more subunits. Amino acid, amino acid analog compound, or peptidomimetic You. The subunits can be linked by peptide bonds. In another aspect In some cases, the subunit is linked by another bond, such as an ester or ether bond. May be combined. As used herein, the term "amino acid" refers to natural and amino acids. And / or non-natural or synthetic amino acids, glycine and D or L Includes optical isomers as well as amino acid analogs and peptidomimetics.   Phage method (Scott & Smith, 1990, Science, 249: 386-390 (1990); Cwirla et al., Proc. Natl. Acad. Sci., 87: 6378-6382 (1990); Devhn et al., Science, 249: 404-406 (1990)). Use a very large library (10⁶-Ten⁸Individual chemical entities) Can be. The second method mainly uses a chemical method. The Geysen method (Geysen et al., Molecular Immunology, 1986, 23: 709-715; Geysen et al., J. Im. munologic Method, 1987, 102: 259-274) and Fodor et al. (Science, 1991, 251: 7). 67-773). Furka et al. (14^th International Congress of Biochemistry, 1988, Vol.5, Abstract FR: 013; Furka, Int.J.Peptide Protein Res., 1991,37: 487-493) Houghton (U.S. Pat.No. 4,631,211 issued December 1986) and Rutter et al. (19 U.S. Patent No. 5,010,175, issued April 23, 1991) discloses the production of peptide mixtures. Manufacturing method, which can be tested as described here. You. Such peptides can be modified by chemical synthesis, as described below. Therefore, it can be further purified.   In another aspect, synthetic libraries (Needels et al., Proc. Natl. Acad. Sci. USA, 1993, 90: 10700-4; Ohlmeyer et al., Proc. Natl. Acad. Sci. USA, 1993, 90: 10922-1092. 6; Lam et al., International Patent Publication No. WO 92/00252; Kocis et al., International Patent Publication No. WO 94/28028, The entirety of each of these documents is referred to as the present invention). Available for training. Phases such as solid phase method, liquid phase method, and peptide condensation technology Synthetic peptides prepared using known techniques, or any combination thereof, As well as non-natural amino acids. Amino used for peptide synthesis The acid was prepared according to the original solid phase method by Merrifield (1963, J. Am. Chem. Soc., 85: 2149-2154). Standard deprotection, neutralization, coupling and washing protocols Boc (N^α-Amino protected N^α-t-butyloxycarbonyl) amino acid resin, or first Described by Carpino & Han (1972, J. Org.Chem., 37: 3403-3409) Unstable N^α-Amino-protected 9-fluorenylmethoxycarbonyl (Fmoc) amino It can be an acid. Fmoc and BocN^α-Amino protected amino acids have implemented this technology Fluka, Bachem, Advanced Chemtech mtech), Sigma, Cambridge Research Bar Iochemical (Cambridge Research Biochemical), Bachem, or Penic Can be obtained from Peninsula Labs or other chemical companies. You. Further, the method of the present invention can be used with other N^α-Use protecting groups You can also. Solid phase peptide synthesis can be accomplished by techniques familiar to those skilled in the art, Such techniques are described, for example, in Stewart & Young, 1984, Solid Phase Synthetic. sis), 2nd edition, Pierce Chemical Company, Rockford, IL; elds & Noble, 1990, Int. Pept. Protein Res., 35: 161-214, and For example, it is possible to use an automatic synthesizer such as that marketed by ABS. You. Thus, the peptides of the present invention may have D-amino acids to have special properties. , D- and L-amino acid combinations, and various "designer" amino acids Acid (e.g., β-methyl amino acid, C_α-Methyl amino acid, and N_α-Methylami Acid). Synthetic amino acids are ornithine instead of lysine, Fluorophenylalanine instead of phenylalanine, and leucine or Contains norleucine instead of isoleucine. Furthermore, for specific coupling stages Assigning specific amino acids, α-helix, β-turn, β sheet, γ-turns and cyclic peptides can be produced.   In one aspect of the invention, the peptide has a specific amino acid at its C-terminus. Which can include CO_TwoH or CONH_TwoIncorporate side chains to free glycine or Or serves as a glycine-amide group. Another way to look at this particular residue The method functions as a D or L amino acid similar to the side chain consisting of the linker, Or binding to the beads. In one embodiment, the quasi-free C-terminus The residue may be of the D or L optical conformation, and in another embodiment Can use a racemic mixture of D- and L-isomers. In ancillary aspects In this case, the peptide may include a pyroglutamic acid residue as the N-terminal residue. it can.   In a further aspect, a peptide that provides useful chemical and structural properties. Would select the subunit. For example, a peptide containing D-amino acids can be Will resist L-amino acid specific proteases in vivo. Furthermore, the present invention , Preparation of Peptides with Even More Defined Structural Properties and Peptidomimetics of Use, as well as peptidomimetic linkages to prepare peptides with novel properties, For example, ester bonds are also contemplated. In another embodiment, the peptide is reduced Peptide bond, ie, R₁-CH_Two-NH-R_Two, Where R₁and R_TwoIs an amino acid residue or sequence. The reduced peptide bond is a dipeptide Can be introduced as a subunit. Such a molecule is a peptide bond Resistant to hydrolysis, eg, protease activity. Such a peptide Have unique functions and are resistant to metabolic disruption or protease activity And a ligand having an extended half-life in vivo, e.g. You. In addition, in some systems, the tethered peptides exhibit high functional activity (Hruby, 1982, Life Science, 31: 189-199: Hruby et al., 1990, Biochem. .J., 268: 249-262), the present invention incorporates a random sequence at every other position, Provided are methods for producing a constrained peptide. 1. Bound and cyclic peptides   A constrained cyclic or rigid peptide can be prepared synthetically. However And at least one amino acid or amino acid in the sequence of the peptide Insert analogs, which bind, cyclize or stiffen the peptide after processing For this purpose, a crosslinkable structure is formed by providing a chemical functional group capable of crosslinking. Cyclization is It would be advantageous if a fold-inducing amino acid was incorporated. Peptide crosslinking Examples of amino acids that allow for cysteine to form disulfide bonds, lacto Chelating the aspartic acid and the transition metal to form And a chelating agent forming a crosslinked structure, for example, γ-carboxyglutamic acid (Gla) (B achem). Protected γ-carboxyglutamic acid is available from Zee-Cheng & Olson (1 980, Biophys. Biochem. Res.Commun., 94: 1128-1132). Well, it can be prepared. The peptide sequence has at least two crosslinkable Peptides containing amino acids can be treated, for example, by oxidation of cysteine residues to give Form sulfides or add metal ions to form chelates, Peptide that is crosslinked and constrained, cyclized, or rigidified Can be formed. 2. Non-classical amino acids that induce conformational distortion   The following non-classical amino acids are incorporated into the peptide to provide F: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al., 1991 , J. Am. Chem. Soc., 113: 2275-2283); (2S, 3S) -methylphenylalanine, (2S, 3R)- Methylphenylalanine, (2R, 3S) -methylphenylalanine and (2R, 3R) -methyl Luphenylalanine (Kazmierski & Hruby, 1991, Tetrahedron Lett.); 2-aminote Trahydronaphthalene-2-carboxylic acid (Landis, 1989, Ph.D. Thesis, University of Arizona); hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate ( Miyake et al., 1989, J. Takeda Res.Labs., 43: 53-76); β-carboline (D and L) (Kazmi erski, 1988, Ph.D. Thesis, University of Arizona): HIC (histidine isoquinoline) Carboxylic acid) (Zechel et al., 1991, Int. J. Pep. Protein Res., 43); and HIC (histidine Cyclic urea) (Dharanipragada) can be introduced.   The following amino acid analogs and peptidomimetics can be incorporated into peptides and identified Secondary structure: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), β-turn induced Dipeptide analogs (Kemp et al., 1985, J. Org. Chem., 50: 5834-5838); β-sheet induced Analogs (Kemp et al., 1988, Tetrahedron Lett., 29: 5081-5082); β-turn-induced analogs (Kem p et al., 1988, TetFabedron Lett., 29: 5057-5060); α-helix-induced analogs (Kemp et al., 1988, Tetrahedron Lett., 29: 4935-4938); γ-turn induced analog (Kemp et al., 1989, J.O. rg. Chem., 54: 109-115); and analogs given by the following references: Nagai & Sato, 1985, Tetrahedron Lett., 26: 647-650; Dimaio et al., 1989, J. Chem. Soc. Perkin.  Trans.P. 1687; and Gly-Ala turn analogs (K, ahn et al., 1989, Tetrahedron Lett., 3 0: 2317); amide bond isostere (Jones et al., 1988, Tetrahedron Lett., 29: 3853-3856); tretrazol (Zabrocki et al., 1988, J. Am. Chem. Soc., 1 10: 5875.5880); DTC (Samanen et al., 1990, Int.J.Pep.Protein Res., 35: 501-509); and And Olson et al., 1990, J. Am. Chem. Soc., 112: 323-333 and Garvey et al., 1990, J. Org. Chem. ., 56: 436. β-turn and β-bulge conformationally restricted Mimetics and peptides containing them were awarded to Kahn on August 8, 1995. No. 5,440,013. 3. Derivatized and denatured peptides   Furthermore, the present invention provides for denaturation or derivatization of the peptide of the present invention. Pep Tide denaturation is well known to those skilled in the art, and includes phosphorylation, carboxymethylation, And acylation. Denaturation must be performed by chemical or enzymatic means Can be.   In another aspect, the glycosylated or fatty acylated peptide derivative is Can be prepared. The preparation of glycosylated or fatty acylated peptides is described below. Well-known in the art, as exemplified by the following documents:  There are two main sets of peptide-carbohydrate linkages. First, the ether bond is Attaches the hydroxyl group of serine or threonine to the hydroxyl group of a sugar . Second, the amide bond can be used to attach the carboxyl group of glutamate or aspartate. Binds to the amino group on the sugar. In particular, References 1 and 2 above disclose peptide- It discloses the preparation of carbohydrate ethers and amides. Acetal and Ketter The linkage can also link the carbohydrate to the peptide.   Fatty acyl peptide derivatives can also be produced. For example, a free amino group (N-terminal or Or lysyl) can be acylated, e.g., myristoylated, Not limited. In another embodiment, the structure: (CH_Two)_nCH_ThreeAn aliphatic side chain with Included amino acids can be incorporated into the peptide. Used in the present invention This and other peptide-fatty acid conjugates suitable for U.K. Patent GB-880916 2.4, International Patent Application PCT / AU89 / 00166 and Reference 5 above.                          Genetic manipulation of the peptide   In accordance with the present invention, conventional molecular biology, microbiology, and Recombinant DNA technology can be used. Such techniques are well documented in the literature. Described. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: Real Laboratory Manual (Molecular Cloning: A Laboratory Manual), 2nd edition (1989), Cold Spring Harbor, NY, published by Ludo Spring Harbor Laboratory Here, "Sambrook et al., 1989"); DNA cloning: a practical method (DNA Cloning: A Practical Approach), Vol.I, II (D.N.Glover, 1985); Oligonucleotide synthesis (Ohgonudeotide Synthesis) (edited by M.J.Gait (1985); nucleic acid hybridization (Nu cleic Acid Hybridization) (B.D.Hames & S.J.fliggins eds. (1984)); Culture of animal cells Yoyo (Animal Cell Culture) (R.I.Freshney ed. (1986)); immobilized cells and enzymes (Immob ilized Cells And Enzymes) (IRL Press (1986)); B. Perbal, Molecular Cloning A Practical Guide to Molecular Cloning (1984); FM.Ausu bel et al. (eds.), General Protocols in Molecular Biology (Current Protocols in Mo lecular Biology), published by John Wheelie & Sons (1994).   The term “sequence similarity” and all of its grammatical forms are common to the degree of identity or commonality. Correspondence between nucleic acid and amino acid sequences of proteins that have different evolutionary origins (See Reeck et al., Supra). However, common uses and In the request, the term "homology" means that when modified by an adverb such as "highly" , Meaning sequence similarity and not a common evolutionary origin.   In one particular embodiment, at least about 50% ( Preferably, at least about 75% and most preferably at least about 90 or 95% ), The two DNA sequences are "substantially homologous" or Is "substantially similar."   Similarly, in one particular embodiment, the two amino acid sequences are greater than 50% or "Substantially homologous" if greater than about 80% of the amino acids are similar (functionally equivalent) Or "substantially similar". Preferably, the similar or homologous sequence is, for example, GC G (Genetics Computer Group, Program Manual for the GCG Package, version 7, Madison, Wisconsin) Using the pileup program Identified by aligning.   The term "corresponding to" is used herein to measure the similarity or homology. Irrespective of whether the exact position is the same or different, Is used to refer to homologous sequences. Thus, the term "corresponding" Gender, not the number of the amino acid residue or nucleotide base.   The identification of a particular DNA encoding a particular long peptide of the invention can be accomplished in a number of ways. Can be achieved. For example, a certain amount of the DNA portion or its fragment If the DNA fragment is available, can be purified and labeled, the resulting DNA fragment Screening by hybridization of nucleic acids with labeled probes Kirun (Benton & Davis, 1977, science, 196: 180: Grunstein & Hogness, 1975, Proc. N ad.Acad.Sci.U.S.A., 72: 3961). For example, corresponding to the amino acid sequence of the peptide Prepare a set of oligonucleotides to use as probes for DNA Can be. These DNA fragments with substantial homology to the probe Will be hybridized. The higher the homology, the stricter the hybrid Conditions can be used.Expression of antigenic and inhibitory peptides and their analogs   Certain peptides, their derivatives or analogs or functionally active derivatives The nucleotide sequence to be loaded is converted into a suitable expression vector, that is, the inserted peptide. -Insertion into a vector containing the necessary elements for the transcription and translation of the coding sequence. Can be. Such elements are referred to herein as "promoters." Therefore, the present invention The nucleic acid encoding the peptide of the present invention has a promoter and function in the expression vector of the present invention. Combined as possible. The expression vector also preferably includes an origin of replication.   Provides necessary transcription and translation signals to recombinant expression vectors You.   Potential host vector systems include viruses (e.g., vaccinia virus, adenovirus). Infected cell lines, viruses (e.g., baculovirus) Infected insect cell lines, microorganisms such as yeast, including yeast vectors, or bacteria Bacteria, DNA, plasmid DNA, or cosmid DNA Including, but not limited to. The expression elements of a vector depend on its length and specificity. Fluctuate in sex. Depending on the host vector system used, some suitable transcription Also, any one of the translation elements can be used.   Cells containing the vector containing the peptide DNA, in a suitable cell culture medium, Culture under conditions that result in expression of the peptide by the cells.   Any of several methods for inserting DNA fragments into such vectors Used to generate the appropriate transcription / translation control signals and the coding sequence for the peptide. Expression vectors containing nucleic acids can be constructed. These methods are Toro DNA and synthetic techniques can be included.   Expression of the peptide may be by any promoter / enhancer element known in the art. These regulatory elements are functional in the host selected for expression. Needs to be A promoter that can be used to control the expression of the peptide nucleic acid is , SV40 early promoter region (Benoist & Chambon, 1981, Nature, 290: 304-310); Promoter contained in the 3 'long terminal repeat of the Rous sarcoma virus (Y amamoto et al., 1980, Cell, 22: 787-797); herpes thymidine kinase promoter (Wagne r et al., 1981, Proc. Natl. Acad. Sci. U.S.A., 78: 1441-1445); metallothionein gene Regulatory sequences (Brinster et al., 1982, Nature, 296: 39-42); prokaryotic expression vectors, such as β- Lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci.U.S.A., 75: 3727-3731); or a tac promoter (DeBoer et al., 1983, Proc. ad.Sci.U.S.A., 80: 21-25), but is not limited thereto. Also, "Recombinant Useful proteins from recombinant bacteria) See also the article entitled, Scientific American, 1980, 242: 74-79. When. Also, yeast or other fungi such as the Gal 4 promoter, ADC (alcohol Dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, Alkaline phosphatase promoter, and tissue specific and trans It also includes animal transcription control regions used in transgenic animals.   Expression vector containing nucleic acid encoding antigenic or inhibitory peptide of the invention One can be identified by four general methods: (a) a given plasmid PCR amplification of DNA or specific mRNA; (b) nucleic acid hybridization; (c) selection And (d) expression of the inserted sequence. In the first method, the nucleic acid is amplified by PCR, and the amplified product is detected. Is possible. In the second method, a foreign gene inserted into an expression vector is used. The presence of the offspring is determined using a probe containing a sequence homologous to the inserted marker gene. Can be detected by hybridizing the nucleic acid. In the third method, The recombinant vector / host system is generated by the inserted foreign gene in the vector. , Certain “selection marker” gene functions (eg, β-galactosidase activity, thymidine Kinase activity, antibiotic resistance, transformed phenotype, baculovirus Identification and selection based on the presence or absence of occlusion bodies in can do. In another example, the nucleic acid encoding the peptide is A set containing the peptide insert inserted into the “selection marker” gene sequence of the vector; The recombinant can be identified by the absence of the selectable marker gene function. You. In the fourth method, a recombinant expression vector is prepared by the method described herein. The activity, biochemical or immunology of the peptide expressed by said recombinant Can be identified by assaying for specific characteristics.   A wide range of host / expression vector combinations can be used to express the DNA sequences of the present invention. It can be used at the time. Useful expression vectors include, for example, chromosomes, non-chromosomes and It may be composed of segments of a synthetic DNA sequence. A suitable vector is Derivatives of SV40 and known bacterial plasmids such as E. coli plasmid co l El, pCR1, pBR322, pMal-C2, pET, pGEX (Smith et al., 1988, Gene, 67: 31-40), pMB9 And its derivatives, plasmids such as RP4; phage DNA's such as NM989, etc. Numerous derivatives of phage λ and other phage DNA such as M13 and filament Single-stranded phage DNA; yeast plasmid such as 2μ plasmid or its derivative Vectors useful in eukaryotic cells, for example, useful in insects or mammals Vector; a vector derived from a combination of plasmid and phage DNA, for example, Plasmids that have been modified to utilize page DNA or other expression control sequences; etc. Is included.   For example, in baculovirus expression systems, non-fusion transfer vectors, such as (but PVL941 (BamH1 cloning site; Summers), pVL139 3 (BamH1, SmaI, XbaI, EcoRI, NotI, XmaIII, BglII, and PstI cloning Site; Invitrogen), pVL392 (BglII, PstI, NotI, XmaIII, EcoRI, XbaI, SmaI And BamH1 cloning site; Summers & Invitrogen), and pBlueBacIII (BamH1, BglII, PstI, NcoI, and HindIII cloning sites, blue / white recombination Transferable vectors, such as (provided that (Not limited to these) pAc700 (BamH1 and KpnI cloning sites, where the B The amH1 recognition site starts at the start codon; Summers), pAc701 and pAc702 (pAc7 PAc360 (36 base pairs downstream of the polyhedral start codon) BamH1 cloning site; Invitrogen (195)); and pBlueBacHisA, B, C (Ba Three variants with mH1, BglII, PstI, NcoI, and HindIII cloning sites Reading frame, N-terminal peptide for ProBond purification, and puller Blue / white recombinant screening; Invitrogen (220)) Wear.   Once a particular synthetic DNA molecule has been identified and isolated, several known in the art This can be propagated using methods. Once the appropriate host system and growth If conditions are set, the recombinant expression vector can be propagated and prepared in large quantities. Can be. As explained above, the expression vectors that can be used are the following vectors Or derivatives thereof (but not limited to, but only a few examples) : Human or animal viruses, such as vaccinia virus or adenovirus Insect viruses, such as baculovirus; yeast vectors; bacteriophage Vector (eg, λ); and plasmid and cosmid DNA vectors.   Vectors can be prepared using methods known in the art, such as transfection, Poration, microinjection, transduction, cell fusion, DEAE dextran, phosphate calcium Precipitation, lipofection (lysosomal fusion), use of a gene gun, or D Use of NA vector transporters (e.g., Wu et al., 1992, J. Biol. Chem., 267: 963-967; Wu &  Wu, 1988, J. Biol. Chem., 263: 14621-14624; Hartmut et al., Dated March 15, 1990. (See Canadian Patent Application No. 2,012,311) to introduce into the predetermined host cells. I do.                                   Administration   According to the present invention, the component (s) of the therapeutic composition of the present invention comprises a parenteral, transmucosal Can be introduced by a membrane, for example, oral, nasal, or rectal, or transdermal routes You. Preferably, administration is parenteral, such as intravenous injection, and intraarterial, intramuscular, transdermal. Intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial (but not limited to) .   In another embodiment, the therapeutic compound releases into vesicles, especially liposomes (Eg, Langer, Science, 1990, 249: 1527-1533; Treat et al., Infectious Liposomes in the Therapy of Infecti ous Disease and Cancer), Lopez-Berestein and Fidler, Liss: NY, pp. 353-365 ( 1989); see Lopez-Berestein, ibid., Pp. 317-327, generally the latter literature. See). In order to reduce its systematic side effects, this should And a preferred method for introducing antibodies.   In yet another embodiment, the therapeutic compound is released as a controlled release system. Can be. For example, the peptides can be injected intravenously, implantable pumps, transdermal patches. The administration can be carried out using liposomes, liposomes or other modes of administration. In one aspect Alternatively, a pump can be used (Langer, supra, Sefton, CRC Crit. Ref. .Biomed.Eng., 1987, 14: 201; Buchwald et al., Surgery, 1980, 88: 507; Saudek et al., N. Engl. . J. Med., 1989, 321: 574). In another embodiment, the polymer Materials can be used (Medical Applications of Controlled Release) Controlled Release), Langer & Wise (ed.), CRCPress: Boca Raton, Florida, (1 974); Controlled Drug Bioavailabihty, Drug Product Design & Performance, Smolen & Ball (eds.), Wiley: NY (1984); Ranger & Peppas, J. Macromol. Sci. Rev. Macr Chem., 1983, 23:61; see also Levy et al., Science, 1985, 228: 190; Duri. ng et al., Ann. Neurol., 1989, 25: 351; see also Howard et al., J. Neurosur., 1989, 71: 105. thing). In yet another aspect, the controlled release system comprises the therapeutic target, ie, Place it near the brain so that only a small portion of the systemic dose is needed (E.g., Goodson, Medical Applications for Controlled Release)  of Controlled Release), supra, 1984, Vol. 2, pp. 115-138).   Other controlled release systems are reviewed by Langer (Science, 1990, 249: 1527-153). Discussed in 3).   In a further aspect, the invention encodes either the antigenic or inhibitory peptide of the invention. Recombinant cells transformed with nucleic acids that express these peptides at high levels Can be transplanted into a subject in need of these peptides. Preferably, Autologous cells transformed with these peptides are transplanted to prevent rejection. is there Alternatively, non-autologous cells that produce soluble factors can be prevented from immune recognition and rejection. Techniques for blocking within the polymer matrix to be terminated can also be utilized.   Thus, the therapeutic peptides of the present invention can be administered intravenously, intraarterially, intraperitoneally, intramuscularly, or otherwise. Or it can be released via the subcutaneous route of administration. Alternatively, these peptides It can also be formulated and administered by nasal or oral routes of administration. These peptides A regular supply of therapeutically effective at predetermined intervals, for example, every day, every 12 hours, etc. Providing a dose (ie, a dose effective to induce metabolic changes in the subject) Guaranteed by. These parameters are the severity of the disease state to be treated in the subject, It will depend on other functions, weight, age, gender, and other criteria. This They can be readily determined by those skilled in the art according to standard good medical practice. it can.   Subjects whose administration of these peptides is an effective therapeutic regimen are preferably humans However, any animal may be used. Thus, as will be readily appreciated by those skilled in the art. In addition, the pharmaceutical compositions and methods of the present invention can be administered to any animal, particularly a mammal. Particularly suitable for are livestock animals such as cats or dogs, cattle, horses, Farm animals such as goats, sheep and pigs, wildlife (whether true wildlife or Zoo animals), research animals such as mice, rats, rabbits, goats, Birds, such as azaleas, pigs, dogs, cats, etc., such as chickens, turkeys, canaries, etc. , Veterinary use).Nasal release : The present invention includes the inhibitory or antigenic peptide (or derivative thereof) Nasal administration of the pharmaceutical composition is also contemplated. For nasal administration, administer the therapeutic product to the nose After that, the peptide can be passed directly into the bloodstream without having to deposit the product in the lungs. And enable. Formulations for nasal release may be dextran or cyclodextran. And those containing.   For nasal administration, a useful device is a small rigid bottle, which contains A dose-dose spray device is installed. In one embodiment, the metered dose Introduces the peptide solution into a defined volume chamber. The chamber is Aerosol formulation by forming a spray when the liquid in it is compressed Has an opening dimensioned to be an aerosol. Compress the chamber and Administer the peptide. In one particular embodiment, the chamber is a piston device is there. Such devices are commercially available.   Alternatively, such devices form a spray when crushed To have an opening sized to make the aerosol formulation an aerosol. Plastic skies bottle. The opening is usually at the top of the bottle The top is generally tapered, partially conforming to the nasal cavity, Enables effective administration of the aerosol formulation. Preferably, the nasal inhaler is To administer a weighed dose of drug, the metered amount of the aerosol formulation give.   The term "mucosal penetration enhancer" refers to ketamine, such as, but not limited to, bile salts. Increase the transmucosal penetration rate of fatty acids, surfactants or alcohols or A reagent that facilitates penetration. In a specific embodiment, the penetration enhancer comprises call Sodium, sodium dodecyl sulfate, sodium deoxycholate, tauro Deoxycholate, sodium glycocholate, dimethyl sulfoxide or May be ethanol. Suitable penetration enhancers are also glycyrrhizic acid (Kowarsk U.S. Pat.No. 5,112,804 to U.S. Pat. Are preferably nonionic surfactants such as nonoxynol-9, laureth -9, poloxamer-124, octoxynol-9, or lauramide-DEA (to Stoltz And EP 0,242,643 B1).   Various as well as numerous methods for transdermally administering drugs, such as transdermal patches, It is known in the art. Transdermal patches are available, for example, from Rolando et al., April 18, 1995. U.S. Pat.No. 5,407,713, issued to Fallon et al., Issued Oct. 4, 1004. U.S. Pat. No. 5,352,456 issued to D'Angelo et al. On Aug. 9, 1994 U.S. Pat. No. 5,336,168, issued Aug. 9, 1994 to Sibalis et al. U.S. Patent No. 5,290,561, issued March 1, 1994 to Farhadieh et al., Tucker U.S. Pat.No. 5,254,346, issued Oct. 19, 1993, Berger et al., Nov. 1992. U.S. Pat.No. 5,164,189, granted on Nov. 17, 1992, granted to Sibalis et al. On Nov. 17, 1992 U.S. Patent No. 5,163,899 issued to Sibalis et al., Issued on February 18, 1992. Nos. 5,088,977 and 5,087,240, granted to Benecke et al. On April 16, 1991 US Patent No. 5,008,110 and US Patent No. 5,008,110 issued May 1, 1990 to Sibalis et al. No. 4,921,475. The entire disclosure of each of these is described by the present invention The reference.   The transdermal route of administration may be a skin penetration enhancer, such as the aforementioned U.S. Patent No. 5,164,189, U.S. Pat. No. 5,008,110 mentioned above and U.S. Pat. Enhancers as described in National Patent No. 4,879,119. Disclosure of each of these The whole matter is referred to the present invention.   The invention is further improved by reference to the following non-limiting examples. As can be appreciated, these examples are given by way of illustration of the present invention.Example Materials and methods Affinity purified anti-mucopeptide antibodies   The anti-mucopeptide antibody was affinity purified as follows. 5ml Rabbit anti-mucopeptide antibody was first passed through a Protein A Sepharose column, The antibody fraction bound to the column is dissolved in a 0.05 M sodium acetate buffer (pH 2.5). Issued. The volume of the eluate was adjusted to the original volume of the serum (5 ml) and the pH was adjusted to 5N N The pH was adjusted to 7.2 with aOH solution. Then 5 mg of Streptococcus mucopeptide (Dry weight) was mixed with 0.5 ml of anti-mucopeptide purified antibody, then at 37 ° C for 2-3 hours Gently swirled for a while. This sample was stored at a low temperature of 4 ° C. overnight. next day, The sample was centrifuged at 14000 RPM for 15-20 minutes. The resulting supernatant was removed and the resulting pellet was reconstituted with 0.5 ml of anti-mucopeptide purified antibody. Resuspended in the body and the above incubation procedure was repeated. Repeat this step It was repeated as the third time.   Following these immersions, the pellet was washed with 1 ml of 0.05 M sodium acetate buffer. (PH 2.5), gently swirl at room temperature for 1 hour, and then Was centrifuged. The pH of the supernatant was brought back to 7.2 by adding a 5N NaOH solution. Then use a sonicated mucopeptide preparation at a concentration of 1 ug / well. And tested by ELISA assay. The ELISA titer is the original absorbed Compare the volume to the non-absorbed antibody and determine if the absorbed antibody is the original unabsorbed It was adjusted to have the same titer as the anti-mucopeptide antibody.   Next, the biological activity of the affinity-purified antibody was determined using the human Tested using a mononuclear cell preparation. Then, the affinity-purified anti- -A systematic dilution of the mucopeptide antibody is added to the wells, then at a concentration of 100 μg / ml Starting with the different concentrations of LPS and mucope of the Streptococcus cell wall The peptide preparation was added. The control sample does not contain the antibody Cells. This cell suspension is_Two24 hours at 37 ° C in an incubator And then harvesting the resulting supernatant following centrifugation. And tested for TNFα production. Phytohema as an incidental control Glutinine-stimulated cells were tested to determine in the affinity-purified antibody preparation Excluded the possibility of containing non-specific inhibitors. This TNFα assay is Standard capture from Endogen (Cambridge, MA) capture) was performed using an ELISA kit.                                 Example 1 Identification of peptides useful for the treatment of septic shock   The anti-mucopeptide antibody induces TNF production by both mucopeptide and LPS The fact that they block both indicates that there is a common epitope in both structures. I'm sorry. This further indicates that the binding domain on some anti-mucopeptides This suggests that these compounds have the correct sequence for binding to both. this The binding sequences are arranged as follows. The phage library is host E. coli. coli, a random peptide sequence with about 10-15 amino acids Created to produce (Parmley & Smith, Gene, 1988, 73: 305-318; Scott & Sm ith, Science, 1990, 249: 386-249). Specifically, the phage library is low It can be mixed with the allowed E. coli at low dilution in LB agar, This can be poured on an LB agar plate. The plate is kept at 37 ° C for a predetermined period. After incubation, small clear plaques of the E. coli flora are formed, Means growth of active phage and lysis of the E. coli. For these phages A typical one is to place a drying filter on the agar plate, Can be absorbed by Ron filters. Mark the filter for orientation And remove and place in cleaning solution to block any remaining absorption sites. it can. The filter is then placed in a solution containing the radioactive mucopeptide complex. You can enter. After the specified incubation period, the filter Can be washed extensively and grown for autoradiography. The Identify plaques containing the phage that are associated with the mucopeptide complex be able to. These phages were further cloned and then as before. Can be tested again for its ability to bind to the mucopeptide complex. You. Once the phage is purified, the binding sequence contained in the phage can be Can be determined by conventional DNA sequencing techniques. Once the DNA sequence If known, synthetic peptides exhibiting these sequences can be produced. These peptides are ( 1) its ability to bind to the mucopeptide complex, (2) its ability to bind to lipid A And (3) the ability to inhibit the anti-mucopeptide antibody. Wear.   A single peptide giving a strong positive result indicates a single dominant binding peptide . Isolation of a large number of peptides requires that the peptides have a common sequence, for example, Whether the library has produced exactly the same peptide more than once Or several different phages have similar reactivity.   Synthesizing the peptide shown to be positive by ELISA, and Of the anti-mucopeptide on the induction of TNF by LPS or mucopeptide Can be assayed to test if it can be blocked. The effective Peptides have been used in animal models of shock and in some cases Can be synthesized in large quantities for use in humans to prevent shock Wear. The production of synthetic peptides requires relatively little effort and is easy to produce. The performance can be adjusted, and a large amount of crystals can be produced at a considerably low cost. Mass production Malaria, a similar combination of synthetic peptides that have recently been very successful (Patarroyo, Vaccine, 1990, 10: 175-1) 78) and has been shown to be quite safe to use in humans.                                 Example 2 Anti-group A mucopeptide antibodies and peptides in the treatment of experimental arthritis Use of   The disease rheumatoid arthritis has an unknown etiology but involves target organs and joints It is a disease that is expected to be an autoimmune disease. Many experimental arthritis (Table 1), the Streptococcus induction model is as follows: It is preferable for the reason. First, unlike other models, the ultrasonically treated cell wall A single administration of the preparation induces arthritis and then increases or decreases over the life of the animal. Return. Second, it eventually shows a striking resemblance to human disease. Finally, this is a cell The model is based on the absence of Streptococcus antigen. Recipes that have not yet been subjected to experimentation by passive administration of donor lymphocytes D Can be transferred to TNF is important in disease induction and experimental models. Plays an important role, and the disease is immediately associated with the onset of the soluble TNF receptor component. It can be blocked by injecting a child.         Table 1: Characteristics of rheumatoid arthritis and animal models of arthritis ND: means not measured.   Is the inducer primarily a Streptococcus mucopeptide-polysaccharide conjugate? Thus, the research method of the model proceeds by two types of studies. The first way is , Induction of experimental arthritis in rats. Before or after the onset of arthritis At selected time points, animals are injected with the anti-mucopeptide antibody. This injection Is performed prior to the disease, the anti-mucopeptide antibody will Protected. When injected after the onset of the disease, the symptoms and severity of the disease Be improved. Since the antibody confers passive protection, the above-described peptide may When induced by the V. or I.P. route simultaneously with the mucopeptide conjugate, the The onset of arthritis can be prevented.   The second method is based on previous interesting observations in this model (J Anusz et al., J. Exp. Med., 1984, 160: 1360-1366). Enzymes (phage lysin, mutanola The enzymatic digestion of the mucopeptide-polysaccharide complex into small pieces. When the fragments are injected, the recipient animal Become insensitive to the induction of arthritis by the mucopeptide. Therefore, These injected fragments induce classic arthritis by the large complex. It is considered to protect against fire. This defense is a response that triggers the arthritis. Through the induction of anti-mucopeptide antibody production against Can be.   These experiments indicate that this protection is due to the injection of the available anti-mucopeptide antibody. Or the fragment obtained by enzymatic degradation of the mucopeptide complex. Injection of the antibody induced after entry will result in unexperimented movement by the antibody. It can be transmitted to objects passively. Protection against induced arthritis is Once it has been shown that it can be done through the body, these antibodies Peptides of the mucopeptide fragment that will elicit a strong protective antibody response It can be used to identify mimitopes. The runs discussed herein Utilizing a dam peptide phage library, the protective antibody is Can be used to identify peptides that mimic the fragment structure of the peptide complex. These peptides have the following characteristics: (1) they are the anti-mucopeptide React with the body, (2) they do not bind to the mucopeptide complex, and (3) they Protects against arthritic diseases induced by mucopeptide complexes in animals Does not generate a neutral antibody response. Using a similar method successfully, Neisseria Mengete Mimics the structure of group C polysaccharide capsular antigen of Neisseria mengitidis Peptide (Westerink & Giardina, Microb.Pathog., 1992, 12: 19-26 ). These peptides are useful for eliciting a protective antibody response against experimental arthritis. Can be used.   Various forms of human arthritis (such as Crohn's disease and psoriatic arthritis) all lead to this disease. It is thought that they have a common antigen that makes them appear. Against all these antigens Identification of common epitopes or mimitopes has improved many of these arthritis Or the prevention of its onset.   The invention is not limited in scope by the specific embodiments described herein. Not. In fact, those skilled in the art will recognize, in addition to those set forth herein, Various improvements of the present invention will become apparent from the accompanying drawings. Such breaks Good is considered to fall within the scope of the present invention.   In addition, any bases given for nucleic acids or polyclonal peptides Size or amino acid size, and all molecular weight or molecular mass values This value is described for convenience of explanation.   All documents cited above are incorporated herein by reference in their entirety. It is.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 29/00 A61P 31/04 31/04 C07K 14/47 C07K 14/47 16/28 16/28 A61K 37 / 02 (31) Priority claim number 09 / 097,838 (32) Priority date June 16, 1998 (1998.16.16) (33) Priority claim country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI) , CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU , TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE , GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW

Claims (1)

[Claims] 1. A method for identifying an inhibitory peptide, comprising selecting a selected peptide from a Group A muco Contacting the peptide with an anti-group A mucopeptide antibody, And the anti-group A mucopeptide antibody of the selected peptide Being selected based on their ability to inhibit binding to the group A mucopeptide. The above method, characterized in that: 2. The selected peptide is present in the group A mucopeptide and LPS molecule. 2. The method according to claim 1, wherein the method is coupled to existing common structural elements. 3. The selected peptide is subjected to the following steps: (a) contacting the group A mucopeptide with various random peptides, A candidate peptide that binds to the group A mucopeptide from various random peptides Selecting the candidate peptide based on the ability of the peptide, and (b) contacting the candidate peptide with the LPS molecule, and contacting the candidate peptide with the LPS molecule. Selecting a peptide to be selected based on its binding capacity for 3. The method according to claim 2, wherein said method is selected by a method comprising: 4. The claimed claim, wherein the various random peptides are obtained from a phage library. The method of claim 3, wherein the method comprises: 5. Claims including a step of recovering the inhibitory peptide including the following steps: Method according to item 4:   A. Purifying the phage encoding the inhibitory peptide,   B. DNA encoding the inhibitory peptide contained in the purified phage Sequencing the sequence and thereby deducing the amino acid sequence of the inhibitory peptide , and   C. Chemically synthesizing the inhibitory peptide based on the amino acid sequence. 6. The following features, obtained by the method according to claim 1, include:   (i) binding to the group A mucopeptide,   (ii) binding to the LPS molecule,   (iii) inhibiting an anti-group A mucopeptide antibody, An inhibitory peptide, characterized by having: 7. In addition, it inhibits bacterial-mediated production of tumor necrosis factor in turkey cells The inhibitory peptide according to claim 6, wherein 8. The inhibitory cell according to claim 7, wherein the target cell is a mononuclear cell. Puchido. 9. 9. The composition according to claim 8, further comprising a pharmaceutically acceptable carrier. Pharmacological composition for treating septic shock. Ten. A mammal is administered a therapeutically effective amount of the pharmaceutical composition of claim 9. A method of treating septic shock in a mammal, comprising the step of administering. 11． 9. The composition according to claim 8, further comprising a pharmaceutically acceptable carrier. A pharmacological composition for treating experimental arthritis, characterized in that: 12． A method for blocking the induction of TNFα in mononuclear cells, comprising the steps of: Adding an antibody prepared against the peptide to the mononuclear cells, Stimulating with the LPS molecule. 13. 13. The method according to claim 12, wherein said mononuclear cells are human cells. 14. A method of preventing the onset of arthritis in an animal, the method comprising: An antibody prepared against the pA mucopeptide is administered to the animal and The above method, wherein the onset of arthritis is prevented. 15． A method of treating arthritis in an animal suffering from arthritis, the method comprising: Thereafter, the animal is administered with the pharmacological composition according to claim 11, wherein the animal The above method, wherein the arthritis is ameliorated. 16. (a) administering the selected peptide to the animal, (b) administering a fixed amount of a group A mucopeptide to the animal, wherein Quantitative group A mucopeptide induces experimental arthritis in the animal Can, and (c) the experimental relationship in the animal induced by the group A mucopeptide. The ability of the selected peptide to elicit a protective antibody response against arthritis Identifying the antigenic peptide based on the selected peptide. Is based on its ability to bind anti-group A mucopeptide antibodies and its ability to bind Selected on the basis of non-binding to a mucopeptide. A method for identifying a progenic peptide. 17． The selected peptide is subjected to the following steps:   A. Contacting anti-group A mucopeptide antibody with various random peptides And wherein, from the various random peptides, the group A mucopeptide The candidate peptide is selected based on the ability of the candidate peptide to bind to the antibody ,   B. Contacting the candidate peptide with the group A mucopeptide, wherein That the candidate peptide is non-binding to the group A mucopeptide. On the basis of which peptides to select are selected, and   C. Recovering the selected peptide; 17. The method according to claim 16, wherein the method is selected by a method comprising: 18． The claimed claim, wherein the various random peptides are obtained from a phage library. The method of claim 17, wherein the method comprises: 19. Recovering the selected peptide,   (i) purifying the phage encoding the selected peptide,   (ii) encoding the selected peptide contained in the purified phage Sequencing the DNA sequence, thereby determining the amino acid sequence of the selected peptide. And the process of   (iii) chemically synthesizing the selected peptide based on the amino acid sequence 19. The method according to claim 18, comprising a step. 20. An antigenic peptide obtained by the method according to claim 16, The following features:   (i) does not bind to the group A mucopeptide,   (ii) binding to an anti-group A mucopeptide antibody, and   (iii) Protection against experimental arthritis induced by group A mucopeptide Elicit a sexual antibody response in the animal, The antigenic peptide described above, wherein twenty one. An antigenic peptide according to claim 20, and a pharmaceutically acceptable carrier. A pharmacological composition for treating experimental arthritis, characterized in that: twenty two. Experimental arthritis in animals induced to develop experimental arthritis A method for preventing the onset of pharmacology, wherein the pharmacological composition according to claim 21 is subjected to an experiment. Prior to the onset of experimental arthritis, administering to the animal The above method comprising the step of preventing the onset of flame. twenty three. Experimental arthritis in animals induced to have experimental arthritis A method of improving action, comprising the steps of claim 21 after the onset of experimental arthritis. Administering the described pharmacological composition to the animal to produce the experimental arthritis in the animal. The method as described above, comprising the step of improving the use.