Peptide microarrays and novel biomarkers for celiac disease

Abstract

Biomarkers as tools for accurate, high-fidelity, and inexpensive early detection of autoimmune diseases such as celiac disease. The present disclosure relates generally to biomarkers and peptide arrays, and more particularly to methods of using peptide arrays to identify biomarkers for autoimmune diseases such as celiac disease. Additionally, a set of novel biomarkers for celiac disease with high sensitivity and specificity is disclosed, as well as methods of treatment using the novel biomarkers.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 048,537, filed September 10, 2014, the disclosure of which is incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy, created on September 10, 2015, is named 30699_PCT_Sequence_Listing.txt and is 32,748 bytes in size. [Background technology]

[0003] background The development of accurate, inexpensive, and high-fidelity tools for the discovery of biomarkers for routine diagnostic assays to detect the presence of autoimmune diseases is crucial to meeting the clinical demand for early detection of disease to develop preventive strategies. The detection of antibodies that correlate with autoimmune disease through binding to biomarkers is one of the major approaches for the diagnosis of many diseases, including autoimmune disorders, infectious diseases, and cancer. 1-3 Indeed, the development of antibody-based diagnostic assays has been a major driver for disease diagnosis and treatment, but only a small number of biomarkers have been identified as effective disease markers. 1,4 Biomarker development faces many challenges, particularly antibody heterogeneity, variability in host responses, and reagents, to name a few. However, improved methods for biomarker discovery, and improved biomarkers for diagnosis, are needed to improve the identification and treatment of patient populations.

[0004] One such autoimmune disease is celiac disease. Celiac disease, also known as coeliac disease or coeliac sprue, affects approximately 1% of the population in Europe and North America. Many affected individuals are unaware of their condition, but this clinical oversight is now being corrected through increased clinical awareness. A gluten-free diet is the only current treatment for celiac disease. However, because regular consumption of as little as 50 mg of gluten (equivalent to 1 / 100 of a standard slice of bread) can damage the small intestine, chronic inflammation of the small intestine is common even in subjects on a gluten-free diet. Persistent small intestinal inflammation has been shown to increase the risk of cancer, osteoporosis, and death. Maintaining a gluten-free diet is challenging because gluten is so widely used in commercial soups, sauces, and ice cream. Therefore, novel epitopes for the diagnosis and treatment of celiac disease are needed. Summary of the Invention

[0005] overview

[0003] Provided herein is a novel polypeptide array for detecting or diagnosing celiac disease in a subject. In one embodiment, an array of features attached to a surface at positionally defined locations is provided, wherein the features comprise at least one engineered polypeptide chain comprising at least two epitope sequences from a bioactive polypeptide that generates an immune response in a subject with celiac disease, and the polypeptide chain further comprises at least one randomly generated polypeptide sequence. In one embodiment, the bioactive polypeptide is selected from the group consisting of alpha gliadin, beta gliadin, gamma gliadin, and omega gliadin. In one embodiment, the at least one engineered polypeptide chain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 sequences selected from the group consisting of SEQ ID NOs: 1-127, or any one or more biologically active fragments or variants thereof.

[0006] In some embodiments, the features are 6-15 amino acids in length. In one embodiment, the features are 12 amino acids in length. In some embodiments, the features attached to the surface of the array are configured to have at least 90% sensitivity and 90% specificity for detecting celiac disorder after contacting the features with a sample from a subject suspected of having celiac disorder. In some embodiments, each of the at least two discontinuous epitopes consists of three amino acids. In some embodiments, each of the at least two discontinuous epitopes consists of 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids. In one embodiment, each of the at least two discontinuous epitopes consists of three amino acids that have at least 20% sensitivity for binding to antibodies in celiac-positive samples, and the peptide chain is 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length.

[0007] In some embodiments, the array further comprises at least 10,000 features, each feature attached to the surface of the array at a different positionally defined location, the positionally defined location of each feature corresponding to the positionally defined location of a pillar, and the top surface of each pillar having a size of at least 1 μm 2 In some embodiments, each feature comprises a different engineered peptide chain compared to other features, each feature comprises at least 500 identical full-length peptide chains, wherein each identical full-length peptide chain has an engineered full-length that is at least 7 amino acids in length, and the purity of each feature in terms of the proportion of full-length engineered peptide chains is the proportion F of full-length engineered peptide chains of each feature that have an engineered sequence, and the length N of the engineered full-length sequence is F=10. (N+1)·log(E / 100%) where the average coupling efficiency E is at least 98.5% for coupling of each amino acid in the engineered sequence, the sequence length N is at least 7 amino acids in length, and the fraction of engineered peptide chains that are less than full length is equal to (1-F).

[0008] In some embodiments, the surface of the array comprises a substrate, the substrate comprising: a planar layer having an upper surface and a lower surface; and a plurality of pillars operably coupled to the layer at positionally defined locations, wherein each pillar has a planar surface extending from the layer, the distance between the surface of each pillar and the upper surface of the layer is between 1,000 and 5,000 angstroms, and the plurality of pillars are 10,000 / cm 2 It exists at a density exceeding

[0009] Also provided herein is a method for identifying novel epitopes for binding to antibodies associated with an autoimmune disorder, the method comprising the following steps: synthesizing a plurality of polypeptides on a first array, wherein the plurality of polypeptides comprise overlapping polypeptide sequences from a protein that may contain epitopes that bind to antibodies associated with an immune disorder; contacting the first array with a first sample from a subject having an immune disorder; determining which of the overlapping polypeptide sequences bound to the antibody from the first sample to generate binding data; analyzing the binding data to identify multiple contiguous epitopes in the protein; and further analyzing each of the multiple contiguous epitopes to identify pairs of multiple discontinuous epitopes that have the highest sensitivity (false positive rate) for binding to antibodies from the sample, thereby identifying novel epitopes for binding to antibodies associated with an autoimmune disorder.

[0010] In some embodiments, the method for identifying novel epitopes for binding to antibodies associated with autoimmune disorders further comprises the steps of: synthesizing a plurality of synthetic polypeptides on a second array, each synthetic polypeptide comprising at least two of the plurality of discontinuous epitopes, and each synthetic polypeptide further comprising a random polypeptide sequence; contacting the second array with a second sample from a subject having an immune disorder; determining the sensitivity (false positive rate) and specificity (false negative rate) of binding of antibodies from the second sample to each of the plurality of synthetic polypeptides; identifying synthetic polypeptides with the highest sensitivity and / or specificity for binding to antibodies associated with immune disorders, thereby identifying better novel epitopes for binding to antibodies associated with autoimmune disorders.

[0011] In some embodiments, the plurality of polypeptides comprises deamidated polypeptide sequences derived from a protein. In one embodiment, the plurality of polypeptides is 6-15 amino acids in length. In one embodiment, the autoimmune disorder is celiac disease. In one embodiment, the antibody from the first or second sample is an IgA antibody or an IgG antibody. In one embodiment, the synthetic polypeptide is 6-15 amino acids in length. In one embodiment, the synthetic polypeptide is 12 amino acids in length. In some embodiments, each of the plurality of contiguous epitopes binds to an antibody in at least 20%, 30%, 40%, or 50% of samples containing an autoimmune disorder.

[0012] Also provided herein are arrays of surface-mounted features at positionally defined locations, wherein the features comprise at least one novel epitope identified by the methods disclosed herein.

[0013] Also provided herein is a method for identifying an autoimmune disorder in a subject, the method comprising: contacting a sample from the subject with one or more arrays of the embodiments described herein; and analyzing binding of antibodies in the sample to features on the array to determine whether the subject has an autoimmune disorder. In some embodiments, the autoimmune disorder is celiac disease. In some embodiments, the method comprises a sensitivity of detection of the autoimmune disorder (false positive rate) of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the method comprises a specificity of detection of the autoimmune disorder (false negative rate) of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the method comprises a sensitivity of detecting a Marsh classification of celiac disorder in a subject that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0014] Also provided herein are compositions comprising one or more isolated polypeptides comprising a sequence selected from the group consisting of SEQ ID NOs: 1-127, or any one or more biologically active fragments or variants thereof.

[0015] Also disclosed are substantially purified and / or recombinant polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-127, any one or more biologically active fragments or variants thereof.

[0016] Disclosed is a method of treating a celiac disorder or a celiac-related disorder in a patient, the method comprising administering to the patient a formulation comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-127, or any one or more biologically active fragments or variants thereof.

[0017] Disclosed are biomarkers for celiac disease comprising a polypeptide epitope of a celiac antibody, wherein the polypeptide epitope is selected from the group consisting of SEQ ID NOs: 1-127, or any one or more biologically active fragments or variants thereof.

[0018] In some embodiments, the disclosed methods for synthesizing arrays involve global deprotection with selective activation, which offers the advantages of higher fidelity of peptide synthesis and significantly reduced turnaround times for each step. Thus, in some embodiments, this combination of high-fidelity synthesis and shorter processing times is believed to result in much higher yields and the ability to produce large numbers of chips very inexpensively with the extremely high fidelity required for diagnostic tests.

[0019] Celiac disease, a representative autoimmune disease, is an excellent model for investigating the early stages of disease development because the target protein gluten, a storage protein derived from wheat, barley, and rye, is well known to be immunogenic. 10-12 The pathology of celiac disease appears to be T cell mediated. 13 Diagnosis relies on the presence of autoreactive antibodies. The most common antibodies currently in use are directed against host proteins such as tissue transglutaminase (tTG) and endomysium. 10 , which are thought to serve as markers of autoimmunity rather than as being involved in the initiation of disease as a result of a response to gluten proteins. 14 Furthermore, the production of these antibodies can be used to predict the stage and severity of the disease and to monitor dietary compliance; however, they lack the ability to distinguish between clinically relevant phenotypes. Gliadin-specific antibodies are an example that have not demonstrated sufficient sensitivity and specificity for the diagnosis of CD. Furthermore, it remains unclear how gliadin-specific antibodies and epitopes contribute to the pathogenesis of celiac disease, especially in the early stages of onset. 15-18 .

[0020] Understanding how pathogenic epitopes are recognized by B cells and lead to celiac disease may be useful for elucidating the mechanisms of disease initiation and for developing better clinical tools. It is thought that such epitopes in gliadin peptides are modified by transglutaminase, rendering them more immunogenic to the host. To demonstrate the potential of this novel technology for identifying biomarkers for CD diagnosis, we synthesized continuous gliadin epitopes with post-translationally modified peptide sequences, discontinuous peptide sequences combined with gliadin peptide sequences, and random 3-mer or 6-mer peptide sequences. Herein, we describe a method for identifying biomarkers for CD diagnosis using a novel platform and technology involving a semiconductor mass manufacturing process for generating continuous and discontinuous peptide sequences derived from established antigens in autoimmune diseases. [The present invention 1001] 1. An array of surface-mounted features at positionally defined locations, the features comprising at least one engineered polypeptide chain comprising at least two epitope sequences from a bioactive polypeptide that generates an immune response in a subject with celiac disease, the polypeptide chain further comprising at least one randomly generated polypeptide sequence. [The present invention 1002] 1. The biologically active polypeptide, comprising: α-gliadin, β-gliadin, γ-gliadin, ω-gliadin, and other wheat-related proteins or peptides 1001. An array of the present invention selected from the group consisting of: [The present invention 1003] 1001. The array of the present invention, wherein at least one engineered polypeptide chain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 sequences selected from the group consisting of SEQ ID NOs: 1-127. [The present invention 1004] 1001. The array of the present invention, wherein the features are 6 to 15 amino acids in length. [The present invention 1005] Array of the present invention 1001, wherein the features are 12 amino acids in length. [The present invention 1006] An array of the present invention 1001, wherein the features attached to the surface of the array are configured to have at least 90% sensitivity and 90% specificity for detecting celiac disorder after contacting the features with a sample from a subject suspected of having celiac disorder. [The present invention 1007] 1001. An array of the present invention, wherein each of the at least two discontinuous epitopes consists of three amino acids. [The present invention 1008] 1001. The array of the present invention, wherein each of the at least two discontinuous epitopes consists of 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids. [The present invention 1009] 1001. An array of the present invention, wherein each of the at least two discontinuous epitopes consists of 3 amino acids with at least 20% sensitivity for binding to antibodies in celiac positive samples, and the peptide chain is 12 amino acids in length. [The present invention 1010] At least 10,000 features, each feature attached to the surface of the array at a different positionally defined location, the positionally defined location of each feature corresponding to the positionally defined location of a pillar, and the top surface of each pillar having a size of at least 1 μm 2 Feature 1001. The array of the present invention further comprising: [The present invention 1011] each feature comprises a different engineered peptide chain compared to other features, each feature comprises at least 500 identical full-length peptide chains, wherein each identical full-length peptide chain has an engineered full length that is at least 7 amino acids in length, and the purity of each feature in terms of the proportion of full-length engineered peptide chains is F, the proportion of full-length engineered peptide chains of each feature having an engineered sequence, where N is the length of the engineered full-length sequence, and F=10 (N+1)·log(E / 100%) wherein the average coupling efficiency E is at least 98.5% for coupling of each amino acid in the engineered sequence, the sequence length N is at least 7 amino acids in length, and the percentage of engineered peptide chains that are less than full length is equal to (1-F). [The present invention 1012] The surface comprises a substrate, the substrate comprising: a planar layer having an upper surface and a lower surface; and a plurality of pillars operatively coupled to the layer at positionally defined locations, each pillar having a planar surface extending from the layer, the distance between the surface of each pillar and the top surface of the layer being 1,000 to 5,000 angstroms, and the plurality of pillars having a surface area of ​​10,000 / cm 2 Pillars exist at a density exceeding Array of the present invention 1010 comprising: [The present invention 1013] 1. A method for identifying novel epitopes for binding to antibodies associated with autoimmune disorders, the method comprising the steps of: synthesizing a plurality of polypeptides on a first array, the plurality of polypeptides comprising overlapping polypeptide sequences derived from proteins that may contain epitopes that bind to antibodies associated with immune disorders; contacting the first array with a first sample from a subject having the immune disorder; determining which of the overlapping polypeptide sequences bound to antibodies from the first sample to generate binding data; analyzing the binding data to identify multiple contiguous epitopes in the protein; Further analyzing each of the plurality of continuous epitopes to identify pairs of discontinuous epitopes that are most sensitive for binding to the antibody from the sample, thereby identifying novel epitopes for binding to the antibody associated with the autoimmune disorder. [The present invention 1014] The following stages: synthesizing a plurality of synthetic polypeptides on a second array, each synthetic polypeptide comprising at least two of the plurality of discontinuous epitopes, and each synthetic polypeptide further comprising a random polypeptide sequence; contacting the second array with a second sample from a subject with the immune disorder; determining the sensitivity and specificity of binding of antibodies from said second sample to each of said plurality of synthetic polypeptides; and identifying synthetic polypeptides with the highest sensitivity and / or specificity for binding to the antibodies associated with said immune disorder, thereby identifying novel, better epitopes for binding to said antibodies associated with said autoimmune disorder. The method of the present invention 1013 further comprises: [The present invention 1015] 1015. The method of claim 1013 or 1014, wherein the plurality of polypeptides comprises a deamidated polypeptide sequence derived from a protein. [The present invention 1016] 15. The method of claim 1013 or 1014, wherein the plurality of polypeptides are 6 to 15 amino acids in length. [The present invention 1017] The method of any one of claims 1013 to 1014, wherein the autoimmune disorder is celiac disease. [The present invention 1018] 1015. The method of claim 1013 or 1014, wherein the antibody from the first or second sample is an IgA antibody or an IgG antibody. [The present invention 1019] 1014. The method of claim 10, wherein the synthetic polypeptide is 6 to 15 amino acids in length. [The present invention 1020] 1014. The method of claim 10, wherein the synthetic polypeptide is 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. [The present invention 1021] The method of claim 1013, wherein each of the plurality of continuous epitopes is bound by the antibody in at least 20%, 30%, 40%, or 50% of the samples containing the autoimmune disorder. [The present invention 1022] An array of features attached to a surface at positionally defined locations, said features comprising at least one novel epitope identified by any of the methods of inventions 1013-1021. [The present invention 1023] 1. A method for identifying an autoimmune disorder in a subject, the method comprising: contacting a sample from the subject with any of the arrays 1001 to 1012 and 1022 of the present invention; and Analyzing binding of antibodies in the sample to features on the array to determine whether the subject has the autoimmune disorder. [The present invention 1024] The method of claim 1023, wherein the autoimmune disorder is celiac disease. [The present invention 1025] The method of the present invention 1023, comprising a sensitivity for detecting an autoimmune disorder of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. [The present invention 1026] The method of the present invention 1023, comprising a specificity for detecting an autoimmune disorder of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. [The present invention 1027] 1023. The method of the present invention, comprising a sensitivity for detecting a March classification of celiac disorder in said subject of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. [The present invention 1028] A substantially purified and / or recombinant peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-127, or any one or more biologically active fragments or variants thereof. [The present invention 1029] 1. A method of treating a celiac disorder or a celiac-related disorder in a patient, comprising administering to the patient a formulation comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-127, or any one or more biologically active fragments or variants thereof. [The present invention 1030] A method for determining the degree of celiac disorder or celiac-related disorder in a patient, comprising measuring the reactivity of a serum sample from the patient contacted with a formulation comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-127, or any one or more biologically active fragments or variants thereof. [The present invention 1031] A biomarker for celiac disease comprising a polypeptide epitope of a celiac antibody, wherein the polypeptide epitope is selected from the group consisting of SEQ ID NOs: 1-127, or any one or more biologically active fragments or variants thereof. [The present invention 1032] An agent comprising one or more peptides of the present invention. [Brief explanation of the drawings]

[0021] [Figure 1A] 1 illustrates a proposed scheme for peptide synthesis on an array, according to one embodiment: (A) An array was designed with 2.1 million overlapping peptides, 12 amino acids in length, covering the entire antigen sequence with a lateral shift of 2 amino acids. [Figure 1B] (B) Examples of one-at-a-time and two-at-a-time deamidation of native gliadin sequences. [Figure 2]Figure 1 shows a flow diagram for biomarker selection, training set, and validation set analysis, according to one embodiment. Deamidated sequences of α, β, γ, or ω gliadin were synthesized on a 2.2M peptide microarray. A set of samples was run to determine the key biomarkers that were most significant in distinguishing between positive and negative cases. Key subsequences were identified, and a matrix was created to combine the best combinations of 3-mers with random 3-mers and 6-mers in silico. These sequences were then synthesized on a 110k peptide microarray with improved sensitivity and specificity and validated using a blinded set. [Figure 3A] 1A illustrates the preparation of a wafer substrate according to one embodiment. (A) [Figure 3B] (B) shows a pillar substrate according to one embodiment. [Figure 3C] (C) shows the roughness measured by AFM and calculated density of the substrate, according to one embodiment. [Figure 4] 1 shows peptide array synthesis according to one embodiment. [Figure 5A] 1 shows fluorescein results for (A) LKWLDSFTEQ (SEQ ID NO:128), according to one embodiment. [Figure 5B] According to one embodiment, (B) results for DKYYEPHLERA (SEQ ID NO:129) are shown. [Figure 5C] (C) Mass spectrometry analysis of peptide purity according to one embodiment. [Figure 6A] 1 illustrates a celiac subsequence matrix according to one embodiment. The 3-mer subsequences with the highest frequency of occurrence among sequences with high sensitivity and specificity in IgG and IgA were determined, and the best combinations of subsequences were plotted as a matrix table. These sequences were combined with random 3-mers and 6-mers to form new sequences. [Figure 6B]1 illustrates a celiac subsequence matrix according to one embodiment. The 3-mer subsequences with the highest frequency of occurrence among sequences with high sensitivity and specificity in IgG and IgA were determined, and the best combinations of subsequences were plotted as a matrix table. These sequences were combined with random 3-mers and 6-mers to form new sequences. [Figure 7] 1 illustrates a receiver operating characteristic curve for deamidated gliadin-derived peptides (DGPs), according to one embodiment. This ROC curve serves as an example of one of the synthetic deamidated gliadin-derived peptides with a high AUC of 0.99. The ROC curve is plotted based on 1 - specificity and sensitivity under each threshold for each sequence. [Figure 8] 1 shows a heat map of duodenal pathology according to Marsh classification in one embodiment.This heat map shows two clusters, that is, the peptides identified in the set have high and low antibody binding intensity.Furthermore, in the validation cohort, 33 patients with CD autoimmunity who are subsequently diagnosed with CD by blood sampling also show high binding intensity, which is similar to the high-intensity group in the training set. [Figure 9] Illustrates error bars based on duodenal pathology according to Marsh classification in one embodiment.Graphical representation of the obtained data is displayed using error bars.The error bars for each sample across the entire cohort are displayed together with the mean value in peptide units across the entire epitope set and its corresponding 95% confidence interval [CI]. [Figure 10A]

[0023] Figure 1 illustrates a heat map of antibody binding intensities in validation set samples, according to one embodiment. (A) The heat map shows antibody binding data for a novel peptide set with high significance values ​​for differentiating celiac-positive cases from controls and disease controls. Fluorescence binding intensities were normalized using a threshold value for each peptide and then converted to antibody binding units. [Figure 10B](A) A heat map of antibody binding intensities in validation set samples is shown, according to one embodiment. (B) The heat map shows antibody binding data for a novel peptide set with high significance in distinguishing celiac-positive cases from controls and disease controls. Fluorescence binding intensities were normalized using a threshold value for each peptide and then converted to antibody binding units. [Figure 10C] According to one embodiment, a heatmap of antibody binding intensity in validation set samples is shown (C), which shows the natural subgrouping of CD positive and negative cases in the validation cohort based on the Vibrant Analyzer clustering algorithm. DETAILED DESCRIPTION OF THE INVENTION

[0022] Those skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be used without departing from the principles of the invention described herein.

[0023] Detailed Description Terms and Definitions Terms used in the claims and this specification, unless otherwise specified, are defined as set forth below.

[0024] As used herein, the term "wafer" refers to a thin piece of semiconductor material, such as silicon or germanium crystal, commonly used in the manufacture of integrated circuits. Wafers may come in a variety of sizes, e.g., measuring from 25.4 mm (1 inch) to 300 mm (11.8 inches) along one dimension, and having thicknesses, e.g., from 275 μm to 775 μm.

[0025] As used herein, the terms "photoresist" or "resist" or "photoactive material" refer to a photosensitive material that changes its solubility in solution when exposed to ultraviolet or deep ultraviolet radiation. Photoresists are organic or inorganic compounds that are typically divided into two types: positive resists and negative resists. A positive resist is a type of photoresist in which the portions of the photoresist that are exposed to light become soluble in a photoresist developer. The portions of the photoresist that are not exposed to light remain insoluble in the photoresist developer. A negative resist is a type of photoresist in which the portions of the photoresist that are exposed to light become insoluble in a photoresist developer. The portions of the photoresist that are not exposed to light are dissolved by the photoresist developer.

[0026] As used herein, the term "photomask" or "reticle" or "mask" refers to an opaque plate with a transparent pattern or holes that allow light to pass through. In a typical exposure process, the pattern on the photomask is transferred to a photoresist.

[0027] As used herein, the term "coupling molecule" or "monomer molecule" includes any natural or artificially synthesized amino acid whose amino group is protected by a fluorenylmethoxycarbonyl group or a t-butoxycarbonyl group. These amino acids may optionally have their side chains protected. Examples of coupling molecules include Boc-Gly-Oh, Fmoc-Trp-Oh, etc. Other examples are described below.

[0028] As used herein, the terms "coupling" or "coupling process" or "coupling step" refer to a process of forming a bond between two or more molecules, such as linking molecules or coupling molecules. The bond may be a covalent bond, such as a peptide bond. A peptide bond may be a chemical bond formed between two molecules when a carboxyl group of one coupling molecule reacts with an amino group of another coupling molecule, releasing a molecule of water (HO). This is a dehydration synthesis reaction (also known as a condensation reaction) and typically occurs between amino acids. The resulting CO-NH bond is called a peptide bond, and the resulting molecule is an amide.

[0029] As used herein, the terms "biomolecule," "polypeptide," "peptide," or "protein" are used interchangeably to describe a chain or polymer of amino acids linked by bonds. Thus, the term "peptide," as used herein, includes dipeptides, tripeptides, oligopeptides, and polypeptides. The term "peptide" is not limited to any particular number of amino acids. In some embodiments, a peptide contains about 2 to about 50 amino acids, about 5 to about 40 amino acids, about 5 to about 20 amino acids, or about 7 to about 15 amino acids. A molecule, such as a protein or polypeptide, including an enzyme, may be a "native" or "wild-type" molecule, meaning that it occurs naturally in nature, or it may be a "mutant," "variant," "derivative," or "modification," meaning that it is made, modified, derived, or in some way different or changed from a native molecule or from another molecule, such as a mutant.

[0030] As used herein, the term "linker molecule" or "spacer molecule" includes any molecule that does not add any function to the resulting peptide, but separates and extends the peptide from the support, thereby increasing the distance between the substrate surface and the growing peptide. This generally reduces steric hindrance for reactions involving the peptide (including unimolecular folding and multimolecular binding reactions), thereby improving the performance of assays for measuring one or more aspects of peptide function.

[0031] As used herein, the term "developer" refers to a solution that selectively dissolves either exposed or unexposed material. Typically, developers are water-based solutions with trace amounts of base added. An example includes tetramethylammonium hydroxide in a water-based developer. Developers are used for initial pattern definition using commercially available photoresists. The use of developers is described in Example 1 below.

[0032] As used herein, the term "protecting group" includes groups introduced into molecules by chemical modification of functional groups for the purpose of achieving chemoselectivity in subsequent chemical reactions. Chemoselectivity refers to directing a chemical reaction along a desired pathway to obtain a preselected product compared to another. For example, the use of tboc as a protecting group allows for chemoselectivity in peptide synthesis, where the protecting group is selectively removed using a photomask and a photoacid generator, leading to a predetermined peptide coupling reaction occurring at a location defined by the photomask.

[0033] As used herein, the term "microarray" refers to a substrate on which various probe molecules of proteins or specific DNA-binding sequences are immobilized at discrete locations in an ordered manner, thereby forming a microscopic array.

[0034] As used herein, the term "microarray system" refers to a system that typically consists of a formally arranged array of biomolecular probes on a flat solid surface such as a glass, plastic, or silicon chip, plus the equipment required to handle the samples (automated robotic devices), the equipment required to read the reporter molecules (scanners), and the equipment required to analyze the data (bioinformatics tools).

[0035] As used herein, the terms "patterned area" or "pattern" or "location" refer to areas on a substrate on which different features are grown. These patterns can be defined using a photomask.

[0036] As used herein, the term "derivatization" refers to the process of chemically modifying a surface to make it suitable for the synthesis of biomolecules. Typically, derivatization involves the following steps: rendering the substrate hydrophilic, adding aminosilane groups, and adding linker molecules.

[0037] As used herein, the term "capping" or "capping process" or "capping step" refers to the addition of a molecule that prevents further reaction of the molecule to which it is attached. For example, amino groups are typically capped with an acetic anhydride molecule to prevent further formation of peptide bonds.

[0038] As used herein, the term "diffusion" refers to the spread of a chemical substance from an area of ​​higher concentration to an area of ​​lower concentration through random motion.

[0039] As used herein, the term "dye molecule" refers to a dye, which is typically a colored substance that can be bound to a substrate. The dye molecule can be useful for detecting binding between a feature on an array and a molecule of interest.

[0040] As used herein, the terms "immunological binding" and "immunological binding properties" refer to the types of non-covalent interactions that occur between an immunoglobulin molecule (or a variant thereof, such as an scFv) and an antigen for which the immunoglobulin is specific.

[0041] As used herein, the term "biological sample" refers to a sample derived from biological tissue or biological fluid that can be assayed for an analyte of interest. Such samples include, but are not limited to, sputum, amniotic fluid, blood, blood cells (e.g., white blood cells), tissue biopsy or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, and cells derived therefrom. Biological samples can also include tissue sections, such as frozen sections taken for histological purposes. While samples are typically taken from human patients, assays can be used to detect an analyte of interest in samples from any organism (e.g., mammals, bacteria, viruses, algae, or yeast) or mammals, such as dogs, cats, sheep, cows, and pigs. Samples may be pretreated or concentrated as needed by dilution into an appropriate buffer.

[0042] As used herein, the term "assay" refers to a type of biochemical test that measures the presence or concentration of a substance of interest in a solution that may contain a complex mixture of substances.

[0043] The term "subject" specifically refers to an individual, patient, target, host, or recipient, regardless of whether the subject is a human or a non-human animal, including mammals and even avian species. The term "subject" therefore includes humans, non-human primates (e.g., gorillas, marmosets, African green monkeys), livestock (e.g., sheep, cows, pigs, horses, donkeys, goats), laboratory test animals (e.g., rats, mice, rabbits, guinea pigs, hamsters), companion animals (e.g., dogs, cats), captive wild animals (e.g., foxes, deer, game animals), and avian species, including poultry birds (e.g., chickens, ducks, geese, turkeys). However, the preferred subject is a human.

[0044] The term "antigen," as used herein, refers to a molecule that elicits an immune response by a subject's immune system, such as the production of antibodies by the immune system and / or activation of the cellular immune side of the immune system (e.g., activation of phagocytes, natural killer cells, and antigen-specific cytotoxic T lymphocytes, accompanied by the release of various cytokines in response to the antigen). Antigens can be exogenous, endogenous, or autoantigens. Exogenous antigens are those that enter the body from the outside through inhalation, ingestion, or injection. Endogenous antigens are those produced within previously normal cells as a result of normal cellular metabolism or due to viral or intracellular bacterial infection. Autoantigens are normal proteins or protein complexes present in the host's body, but which can stimulate an immune response.

[0045] As used herein, the term "epitope" or "immunoreactive region" refers to a unique molecular surface feature of an antigen that can be bound by a component of the adaptive immune system, e.g., an antibody or T-cell receptor. An antigenic molecule may display several surface features that can serve as a point of interaction with a specific antibody. Any such unique molecular feature may constitute an epitope. Thus, an antibody has the capacity to be bound by several unique antibodies, each of which is specific for a particular epitope.

[0046] As used herein, the term "antibody" or "immunoglobulin molecule" refers to a molecule that is naturally secreted by a particular type of cell of the immune system, namely, B cells. There are five different natural isotypes of antibodies: IgA, IgM, IgG, IgD, and IgE.

[0047] As used herein, the term "immune-related molecule" refers to a biological molecule that is involved in the activation or regulation of an immune response. These include, for example, antibodies, T-cell receptors, or MHC complexes (e.g., human leukocyte antigens).

[0048] As used herein, the term "inflammatory response molecule" refers to a molecule that signals or mediates an inflammatory response, e.g., cytokines such as interleukins and tumor necrosis factors. Inflammatory response molecules include, for example, proinflammatory molecules.

[0049] As used herein, the term "autoimmune disorder" refers to any of a large group of diseases characterized by abnormal functioning of the subject's immune system, causing it to damage the subject's own tissues. Celiac disorder, lupus erythematosus, and rheumatoid arthritis are examples of autoimmune disorders. Autoimmune disorders can also be induced by environmental factors.

[0050] The term "percent identity" or "sequence identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by manual alignment and visual evaluation. Depending on the application, the "percent identity" may exist over a region of the compared sequences, e.g., over a functional domain, or alternatively, over the full length of the two sequences being compared.

[0051] For sequence comparison, typically, one sequence is used as a reference sequence to be compared with test sequence.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and if necessary, coordinates of subsequence are designated, and the parameters of sequence algorithm program are designated.Then, sequence comparison algorithm calculates the sequence identity of test sequence based on reference sequence according to the designated program parameters.

[0052] Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (for an overview, see Ausubel et al., supra).

[0053] One example of an algorithm that is suitable for determining sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. Identity scores can be calculated using the default values ​​for this program, available at the National Center for Biotechnology Information website as of the priority date of this application.

[0054] As used herein, the term "biologically active fragment" or variations thereof refers to a polypeptide capable of generating a substantially equal or greater T cell response in a gluten-sensitive subject than the polypeptide (e.g., gliadin) from which it is derived. In another embodiment, the biologically active fragment is capable of generating a T cell response in a gluten-sensitive subject that is at least 50%, more preferably at least 75%, of the T cell response of the peptide from which it is derived. In one embodiment, the biologically active fragment is 14, 13, 12, 11, 10, 9, or 8 amino acids in length, but not less than 7 amino acids in length. Deletions and / or additions at either end of any peptide are also specifically contemplated. Examples of biologically active fragments disclosed herein include SEQ ID NOs: 1-127.

[0055] The term "celiac disease" refers to a chronic inflammatory disease of the small intestine. The disease encompasses a range of conditions, including severe forms characterized by flattened small intestinal mucosa (hyperplastic villous atrophy), characterized by varying degrees of gluten sensitivity, and other forms characterized by milder symptoms including fatigue, chronic diarrhea, malabsorption of nutrients, weight loss, abdominal distension, anemia, and a substantially increased risk of developing osteoporosis and intestinal malignancies (lymphomas and carcinomas).

[0056] The term "gluten sensitivity" refers to a condition in which a subject is exposed to gluten or its peptide fragments and exhibits one or more symptoms of celiac disease or an inappropriate T cell response. In subjects who are not gluten sensitive, the T cell response caused by gluten ingestion is minimal or non-existent. In contrast, in subjects who are gluten sensitive, the T cell response caused by gluten ingestion is inappropriate CD4 + A T cell-mediated immune response is observed.

[0057] The terms "immune tolerance," "immunological tolerance," "tolerance," or "desensitizing" are defined herein as reducing the subject's immunological reactivity to gluten, thereby rendering a sensitized or hypersensitive subject less sensitive, insensitive, or non-responsive to gluten. Immune tolerance can occur, for example, by exposure of mucosal surfaces to tolerogenic antigenic fragments of gluten, as defined herein. Mucosal administration of both high and low doses of antigen can result in immune tolerance, which reduces the immune response to subsequent systemic administration of the antigen. There may be at least two mechanisms of immune tolerance. Tolerance to high doses of antigen appears to occur through the inactivation or clonal deletion of Th1 and Th2 cells. In contrast, tolerance to low doses of antigen leads to bystander immune suppression mediated by the stimulation of Treg cells to produce inhibitory cytokines such as interleukin-4 (IL-4), interleukin-10 (IL-10), and TGFβ.

[0058] The term "inducing immune tolerance," as used herein, refers to bringing about, causing, or eliciting immune tolerance to gluten in a subject who is sensitive to gluten.

[0059] The term "hypersensitive" is defined herein as having an abnormal physiological sensitivity to gluten.

[0060] The term "anergy" refers to a state of reversible unresponsiveness or hyporesponsiveness of T cells (or B cells) to antigens.

[0061] As used herein, "Treg" refers to a subclass of T cells whose primary role is to terminate T cell-mediated immunity during an immune response and to suppress autoreactive T cells that have escaped negative selection in the thymus. As used herein, a "Treg response" refers to a T cell subclass that expresses the forkhead family transcription factor FOXP3 (forkhead box p3) and / or the MHC class II-associated protein LAG-3, and / or expresses high levels of the IL-2 receptor alpha chain (CD25), CD4 + or CD8 + It is characterized by the differentiation and expansion of a population of Treg cells. It also has MHC class I-restricted CD8 + There is also a small population of Treg cells that express FOXP3. The presence of Treg cells in the peripheral circulation or spleen is related to CD4 + / CD25 + It can be determined by analyzing expression.This is conveniently achieved by using flow cytometry.In addition, Treg cells can also be quantified by determining the level of FOXP3 mRNA in mononuclear cells from peripheral blood or spleen by quantitative reverse transcriptase polymerase chain reaction (PCR).In addition, the induction of Treg response in vivo can also be evaluated by measuring Treg-related cytokines from mononuclear lymphocytes from peripheral blood or lymph node.Treg cells typically show higher expression levels of anti-inflammatory cytokines such as IL-10 and TGFβ, and the presence of these mediators can be determined by methods known in the art, such as flow cytometry, immunohistochemical staining or ELISA.

[0062] The term "T cell stimulatory peptide" or "stimulatory peptide" refers to a peptide or epitope that is capable of activating T cells.

[0063] The terms "activate" or "activating" or "activation" in relation to T cells refer to the binding of a costimulatory molecule by a T cell, followed by presentation of an epitope by an MHC molecule on one cell to an appropriate T cell receptor on a second (T) cell, thereby eliciting a "T cell response."

[0064] As used herein, a "toxic peptide" refers to a peptide that stimulates T cell activation in a subject.

[0065] The term "expansion," as used herein, refers to the proliferation and amplification of a T cell population following T cell activation.

[0066] The term "immunodominant" refers to the subunit (epitope) of a peptide that is most easily recognized by the immune system and therefore most influences the specificity of the induced immune response, such as a T cell response. "Immunodominant" is sometimes used interchangeably with "dominant" herein.

[0067] As used herein, the term "modulating a T cell response" refers to regulating or adjusting the T cell response in a subject sensitive to gluten, resulting in a decreased or smaller T cell response to gluten.

[0068] As used herein, "modifying cytokine secretion" refers to altering or changing the secretion of cytokines by a gluten-sensitive subject to some degree, resulting in a decrease or lessened gluten sensitivity in the subject. The term encompasses both increased secretion of a particular cytokine or combination of cytokines, and decreased secretion of a particular cytokine or combination of cytokines.

[0069] As used herein, "epitope" refers to a portion of an antigen or peptide that is recognized by the immune system, e.g., T-cell receptors or major histocompatibility complex (MHC) class I or class II, antibodies, B-cell receptors, and that is sufficient for high-affinity binding. Generally, linear epitopes for recognition will be at least about 3 amino acids in length, and may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acids in length or longer.

[0070] The term "polyepitope" refers to the presence of two or more epitopes (peptides) linked together in a single polypeptide chain.

[0071] As used herein, "antigen" and "immunogen" and variations thereof are generally used interchangeably and refer to epitope-containing structures recognized by the immune system.

[0072] The term "gluten" or "gluten proteins" encompasses alpha (α), beta (β), gamma (γ) and omega (ω) gliadins and low and high molecular weight (LMW and HMW) glutenins in wheat, B, C and D hordeins in barley, β, γ and ω secalins in rye, and optionally avenins in oats. A "gluten peptide" is a peptide derived from or encompassed by one or more of the gluten proteins.

[0073] The term "gliadin" refers to the aqueous alcohol-soluble fraction of gluten, particularly, but not exclusively, gluten derived from wheat, such as bread wheat (Triticum aestivum).

[0074] The term "glutenin" refers to the aqueous alcohol insoluble fraction of gluten, particularly, but not exclusively, gluten derived from wheat, such as bread wheat.

[0075] As used herein, "hordein" or "barley hordein" refers to gluten derived from barley (Hordein vulgare).

[0076] As used herein, "secalin" or "rye secalin" refers to gluten derived from rye (Secale cerale).

[0077] As used herein, "avedin" or "oat avedin" refers to gluten derived from oat (Avena sativa). The terms "human leukocyte antigen" and "HLA" are defined herein as the genetic fingerprint on human white blood cells and platelets, composed of proteins that play a critical role in activating the body's immune system to respond to foreign organisms. In humans and other animals, HLA is also referred to as the "major histocompatibility complex" (MHC).

[0078] Tissue "transglutaminase" is a critical factor in celiac disease because it promotes gluten-specific T cell responses. Tissue transglutaminase causes selective deamidation of gluten, which in turn leads to the generation of a series of gluten peptides that bind with high affinity to HLA-DQ2 or HLA-DQ8 molecules. The resulting HLA-DQ2 (DQ8)-gluten peptide interaction triggers a proinflammatory CD4 T cell response. Thus, the term "deamidation" refers to the conversion of glutamine to glutamic acid or asparagine to aspartic acid. As used herein, deamidation specifically refers to the conversion of glutamine to glutamic acid in gluten, a process that enhances the propensity of gluten peptides to activate T cells.

[0079] As used herein, the term "agent" refers to a collection of peptides and / or polynucleotides. The peptides and / or polynucleotides may be in the same composition (such as a vaccine), in different compositions, or a combination thereof (e.g., a first and a second peptide as defined herein are in one composition, and a third in another). If in different compositions, they are preferably considered to be in close proximity, such as in a kit. Thus, the methods of the present invention contemplate providing (e.g., administering to a subject) the individual component peptides and / or polynucleotides of the agent of the present invention in a single composition (vaccine), or in different compositions, sequentially, or in a combination thereof.

[0080] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0081] peptide The present disclosure includes the following peptides and modifications thereof. Some embodiments include novel and selective polyepitope-containing peptides that are agents or vaccines for the treatment and diagnosis of celiac disease. In some embodiments, the polyepitope-containing peptides are antigens that modulate T cell responses in subjects sensitive to gluten or with celiac disease. Examples of these polyepitope-containing, celiac-active peptides, which may be amidated at the C-terminus, are provided in Tables 1 and 2.

[0082] Table 1: IgG antibody assay (Set No. 3) TIFF2026035691000002.tif139144TIFF2026035691000003.tif195145

[0083] Table 2: IgA antibody assay (Set No. 4) TIFF2026035691000004.tif15144TIFF2026035691000005.tif223145TIFF2026035691000006.tif222145TIFF2026035691000007.tif29145

[0084] Disclosed herein are methods for identifying novel biologically active sequences and uses of such biologically active sequences. Uses of arrays or preparations containing the novel biologically active sequences disclosed herein can include research applications, therapeutic purposes, medical diagnostics, and / or stratification of one or more patients or subjects.

[0085] Biologically active variants include peptides that differ from a given peptide by one or more amino acids, also known in the art as homologs. For example, variants can include one or more amino acid substitutions in any one or more of the peptides. As used herein, "substituted" or "substitution" includes substitutions, replacements, additions, insertions, deletions, and / or deletions of amino acid residues (so that variants can be fragments). In particular, this refers to peptides with conservative substitutions that do not eliminate or significantly reduce their use in the methods of the present invention. Preferably, a biologically active variant can elicit a substantially equal or greater T cell response in a gluten-sensitive subject than the peptide from which it is derived. In another embodiment, a biologically active variant can elicit a T cell response in a gluten-sensitive subject that is at least 50%, more preferably at least 75%, of the T cell response in a gluten-sensitive subject than the peptide from which it is derived.

[0086] Biologically active variants of the peptides can be identified by altering the sequence of each peptide and then assaying the resulting peptides for their ability to stimulate an immune response, eg, the production of T cells.

[0087] In one embodiment, no more than five, more preferably no more than four, more preferably no more than three, more preferably no more than two, and even more preferably only one amino acid differs (by substitution, deletion or addition) in a given peptide compared to the peptide sequences defined herein.

[0088] In one alternative embodiment, the identity between specific sequence (variant) and reference sequence (peptide as defined herein) is at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% or more, for example, at least about 96%, 97%, 98%, 99% or more.Identity can be determined using readily available software packages, for example, BLAST (www.ncbi.nlm.nih.gov / ) and GAP. Naturally occurring amino acids include alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), hydroxyproline (O and / or Hyp), isodityrosine (IDT), and di-isodityrosine (di-IDT). Hydroxyproline, isodityrosine, and di-isodityrosine are formed post-translationally. The use of natural amino acids, particularly the 20 genetically encoded amino acids, is particularly contemplated.

[0089] Substitutions may be conservative amino acid substitutions, where the substituted amino acid has similar structural or chemical properties to the corresponding amino acid in the reference sequence, or may be non-conservative amino acid substitutions, so long as the desired activity is maintained.

[0090] By way of example, conservative amino acid substitutions include the substitution of one aliphatic or hydrophobic amino acid, such as alanine, valine, leucine, and isoleucine, for another; the substitution of one hydroxyl-containing amino acid, such as serine and threonine, for another; the substitution of one acidic residue, such as glutamic acid or aspartic acid, for another, the substitution of one amide-containing residue, such as asparagine and glutamine, for another; the substitution of one aromatic residue, such as phenylalanine and tyrosine, for another; the substitution of one basic residue, such as lysine, arginine, and histidine, for another; and the substitution of one small amino acid, such as alanine, serine, threonine, methionine, and glycine, for another.

[0091] Peptide variants can be generated by mutagenesis or other chemical methods. Alanine scanning is a useful method for identifying important amino acids. In this method, amino acid residues are replaced with Ala and their effect on the activity of the peptide is determined. For example, cysteine ​​residues can be replaced to minimize dimer formation through disulfide bonds. Each amino acid residue of the peptide is analyzed in this manner to determine the important regions of the peptide. The means for preparing such peptides are well understood in the art.

[0092] In addition to naturally occurring amino acids, non-naturally occurring or modified amino acids are also contemplated and are within the scope of the present invention. Indeed, as used herein, "amino acid" refers to naturally occurring amino acids, non-naturally occurring amino acids, and amino acid analogs, as well as their respective D or L stereoisomers.

[0093] The phrases "protecting group" and "blocking group," as used herein, refer to modifications to peptides that protect them from undesired chemical reactions, particularly in vivo. Examples of such protecting groups include esters of carboxylic acids and boronic acids, ethers of alcohols and acetals, and ketals of aldehydes and ketones. Examples of suitable groups include acyl protecting groups, such as furoyl, formyl, adipyl, azelayl, suberyl, dansyl, acetyl, theyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups, such as benzyloxycarbonyl (Cbz); aliphatic urethane protecting groups, such as t-butoxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (FMOC); pyroglutamic acid and amidation. Many other modifications that result in increased potency, extended activity, ease of purification, and / or increased half-life will be known to those skilled in the art.

[0094] In one embodiment, one of the glutamic acid residues of one or more peptides can be generated by tTG activity on the peptide. In an alternative embodiment, this reaction can occur in vivo after administration.

[0095] The peptides may contain one or more modifications, which may be natural post-translational modifications or artificial modifications. Modifications may result in chemical moieties (typically, for example, by replacing hydrogen in a C-H bond) such as amino, acetyl, acyl, carboxy, hydroxy, or halogen (e.g., fluorine) groups, or carbohydrate groups. Typically, modifications are present at the N-terminus or C-terminus. Furthermore, one or more of the peptides may be PEGylated, where PEG (polyethyleneoxy group) provides extended life in the bloodstream. One or more of the peptides may also be combined with other proteins as fusion proteins or chimeric proteins for targeting to specific sites on target cells.

[0096] Peptide variants can be obtained in which the peptide is chemically modified at the level of the amino acid side chain, amino acid chirality, and / or peptide backbone.

[0097] Certain peptides described herein may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such forms as being within its scope, including cis- (Z) and trans- (E) isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures. Additional asymmetric carbon atoms may be present in substituents, such as alkyl groups. All such isomers, as well as mixtures thereof, are intended to be encompassed by the present invention.

[0098] In another example, to prevent cleavage by peptidases, any one or more of the peptides may contain non-cleavable peptide bonds in place of particularly sensitive peptide bonds to yield more stable peptides. Such non-cleavable peptide bonds may include β-amino acids.

[0099] In certain embodiments, any one or more of the peptides may contain a functional group, e.g., in place of a fragile peptide bond, which facilitates inhibition of serine-, cysteine-, or aspartic acid-type proteases, as appropriate. For example, the present invention includes peptidyl diketones or peptidyl ketoesters, peptide haloalkyl ketones, peptide sulfonyl fluorides, peptidyl boronic acids, peptide epoxides, peptidyl diazomethanes, peptidyl phosphonates, isocoumarins, benzoxazin-4-ones, carbamates, isocyanates, isatoic anhydrides, and the like. Such functional groups are present in other peptide molecules, and general routes for their synthesis are known.

[0100] The variant may be a mimetic. The term "mimetic" is intended to refer to a substance that has a certain degree of chemical similarity to the molecule it mimics and retains the specific activity of interest (e.g., inducing tolerance). The rationale behind the use of peptidomimetics is that the peptide backbone of a protein primarily serves to orient amino acid side chains to facilitate molecular interactions, such as those between T cells and MHC peptides, antibodies and antigens, enzymes and substrates or scaffold proteins, etc. Peptidomimetics are designed to enable molecular interactions similar to those of natural molecules. Mimetics include olefins, phosphonates, aza-amino acid analogs, and the like. Those skilled in the art will readily understand methods for designing peptide mimetics and may utilize them to design mimetics of the peptides defined herein.

[0101] Peptides can also be analyzed by hydrophilicity analysis, which can be used to identify hydrophobic and hydrophilic regions of the peptide, thereby aiding in the design of peptides for experimental manipulation in binding experiments, antibody synthesis, etc. Secondary structural analysis can also be performed to identify regions of the peptide that adopt specific structural motifs. Manipulation, translation, secondary structure prediction, hydrophilicity and hydrophobicity profiles, open reading frame prediction and plotting, and sequence homology determination can be accomplished using computer software programs available in the art. Other methods of structural analysis can also be used, including, but not limited to, X-ray crystallography, mass spectrometry and gas chromatography, computer modeling, optical rotatory dispersion (ORD), or circular dichroism (CD).

[0102] The peptide, fragment, or variant may be in the form of a salt, preferably a pharmaceutically acceptable salt. "Pharmaceutically acceptable salt form" includes conventional non-toxic salts or quaternary ammonium salts of peptides, such as those derived from non-toxic organic or inorganic acids. Conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfonic acid, phosphoric acid, and nitric acid; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, and isothioic acid.

[0103] Peptides can be provided in an agent or vaccine as separate peptides or linked, for example, in a polyepitope structure. In one embodiment, peptides can be presented in a single polypeptide chain (polyepitope chain), i.e., in a linear or cyclic configuration. In another embodiment, peptides can be presented in a multiple antigen presentation system, particularly based on a dendrimer backbone such as polylysine. The polylysine backbone results in a non-linear, branched arrangement of epitopes. This system has the advantage over polyepitope chains in that the peptides do not interfere with each other or are less likely to be cleaved into cryptic epitopes, thereby inducing a complete T cell response.

[0104] Conjugates One or more of the peptides can be conjugated with a compound using standard methods. Examples of compounds that can be conjugated with the peptide include, but are not limited to, radioisotopes, fluorescent labels, chemiluminescent compounds, enzyme labels, free radicals, avidin-biotin labels, bacteriophage labels, compounds that increase the half-life of the peptide in a subject, adjuvants, MHC molecules or fragments thereof.

[0105] These compounds may facilitate detection and / or isolation of the conjugated peptide or increase its immunogenicity.

[0106] "Conjugated," as used herein, means coupled via a covalent or non-covalent bond. Although covalent bonds are preferred, the compound may also be linked to the peptide via non-covalent complexation, for example, via hydrogen bonding or electrostatic interactions, hydrophobic interactions, etc.

[0107] Typical radioisotopes include: 3 H, 125 I, 131 I, 32 P, 35 S, 14 C. 51 Cr, 36 Cl, 57 Co, 58 Co, 59 Fe, 75 Se, and 152 Contains Eu.

[0108] Typical fluorescent labels include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine.

[0109] Typical chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters. Typical bioluminescent compounds include luciferin, luciferase, and aequorin.

[0110] Typical enzyme labels include alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, glucose oxidase, and peroxidase.

[0111] In one embodiment, a non-specific linker is used between the compound and the peptide to which it is conjugated. Such a linker does not contribute to the activity of the peptide. Instead, the linker can serve as a spacer between the peptide and the functional moiety. Uses of a linker include immobilizing the peptide for purposes such as aiding in purification or detection. Alternatively, the linker may enable the attachment of a compound to the peptide, allowing for specific spatial or temporal delivery of the peptide to a specific target, such as a cell or tissue. When used as a vaccine, one or more of the peptides may be coupled to a linker that acts as a spacer between the peptide and the immunogenic carrier, or that allows for improved coupling between the peptide and the immunogenic carrier, preventing the formation of cryptic epitopes.

[0112] In one embodiment, one or more of the peptides are covalently coupled to an adjuvant (immunogenic carrier protein), such as diphtheria toxoid (DT), keyhole limpet hemocyanin (KLH), tetanus toxoid (TT), or influenza virus nucleoprotein (NP). A non-specific linker may be present between the peptide and the immunogenic carrier, and is preferably linked to or co-synthesized with the peptide to facilitate coupling to the immunogenic carrier and / or to serve as a spacer between the peptide and the immunogenic carrier.

[0113] When used as a diagnostic agent, one or more of the peptides are preferably conjugated to an immunogenic carrier not previously used for vaccination, which prevents the diagnostic agent from reacting with antibodies formed against the carrier fraction of the vaccine when monitoring for successful vaccination.

[0114] In one embodiment, the compound is an MHC class II molecule or a peptide-binding fragment thereof. The MHC class II molecule can be purified from a biological sample. Alternatively, the MHC class II molecule can be recombinantly produced. The peptide-binding fragment of the MHC class II molecule can be obtained, for example, by enzymatic cleavage of purified or recombinant intact molecules. Alternatively, the peptide-binding fragment can be recombinantly produced. In a preferred embodiment, the compound is a recombinant two-domain MHC class II molecule.

[0115] In its most basic form, a two-domain MHC class II molecule comprises the α1 and β1 domains of a mammalian MHC class II molecule, wherein the amino terminus of the α1 domain is covalently linked to the carboxy terminus of the β1 domain, and the polypeptide does not comprise either the α2 or β2 domain. The two-domain MHC class II molecule is bound to a peptide, as defined herein, by covalent or non-covalent interactions. In some embodiments, the peptide is covalently linked to the amino terminus of the β1 domain of the class II molecule. The two-domain MHC class II molecule may also comprise a detectable label, such as a fluorescent label or a toxin. When a detectable label or toxin is to be covalently linked to an MHC molecule in a specified manner (i.e., rather than randomly attached), it will generally be linked to the carboxy terminus of the molecule to minimize interference with the peptide antigen linked to the amino terminus.

[0116] Two-domain MHC class II molecules can be used in vitro to detect and quantify T cells and to regulate T cell function. That is, such molecules loaded with a selected peptide can be used to detect, monitor, and quantify a population of T cells specific for that peptide. Two-domain MHC class II molecule / peptide conjugates can also be used to induce anergy in gluten-specific T cells to alleviate symptoms associated with celiac disease. Alternatively, such molecules can be conjugated with toxins to more directly kill disease-causing T cells. Suitable toxins include protein toxins (e.g., ricin, diphtheria, and pseudomonas toxins), chemotherapeutic agents (e.g., doxorubicin, daunorubicin, methotrexate, cytotoxins, and antisense RNA), antibodies against cytotoxic T cell surface molecules, lipases, and radioisotopes that emit "hard" radiation, such as beta rays.

[0117] antigen presenting cells The agents and / or peptides defined herein can be delivered by loading the APC with, for example, the first, second and third peptides, one or more biologically active fragments or variants thereof, and / or polynucleotides encoding one or more thereof.

[0118] Preferably, APC is selected from the group consisting of dendritic cells, macrophages, B lymphocytes and liver sinusoidal endothelial cells, and expresses MHC class II molecules with the same MHC phenotype as the subject.For example, APC can express HLA-DQ2 (for example, HLA DQA1*05 and HLA DQB1*02) and / or HLA DQ8.The APC used for this purpose can be isolated from the subject that will be administered after loading, or they can be obtained from the subject that is allotype-matched.

[0119] " Loading " APC means that APC is incubated with or transfected by peptide, one or more biologically active fragments or variants thereof, or the polynucleotide encoding one or more thereof.The loading of APC can be achieved by using conventional nucleic acid transfection methods, such as lipid-mediated transfection, electroporation, and calcium phosphate transfection.

[0120] Peptide production The peptides can be prepared in any suitable manner, for example, they can be produced recombinantly and / or synthetically.

[0121] Peptides can be synthesized by standard chemical techniques, including synthesis by automated procedures using commercially available peptide synthesizers. Generally, peptide analogs are prepared by solid-phase peptide synthesis, which may involve coupling each protected amino acid residue to a resin support, preferably 4-methylbenzhydrylamine resin, via activation with dicyclohexylcarbodiimide to yield peptides with a C-terminal amide. Alternatively, chloromethyl resin (Merrifield resin) can be used to yield peptides with a free carboxylic acid at the C-terminus. After the final residue is attached, the protected peptide-resin is treated with hydrogen fluoride to cleave the peptide from the resin and deprotect side chain functional groups. The crude product can be further purified by gel filtration, high-pressure liquid chromatography (HPLC), partition chromatography, or ion-exchange chromatography.

[0122] If desired, and as outlined above, various groups can be introduced into the agent peptide during synthesis or expression that allow for conjugation to other molecules or surfaces, for example, cysteine ​​can be used to create thioesters, histidine to link metal ion complexes, carboxyl groups to form amides or esters, amino groups to form amides, etc.

[0123] Peptides can also be produced using cell-free translation systems. Standard translation systems, such as reticulocyte lysate and wheat germ extract, use RNA as a template. On the other hand, "coupled" and "ligated" systems begin with a DNA template, which is transcribed into RNA and subsequently translated.

[0124] Alternatively, the peptides can be produced by transfecting a host cell with an expression vector containing a polynucleotide encoding one or more peptides.

[0125] For recombinant production, a recombinant construct containing a sequence encoding one or more of the peptides is introduced into host cells by conventional methods such as calcium phosphate transfection, DEAE-dextrin-mediated transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

[0126] One or more of the peptides may be expressed using conventional techniques under the control of a suitable promoter in a suitable host cell, such as, for example, mammalian cells (e.g., COS, CHO, BHK, 293 HEK, VERO, HeLa, HepG2, MDCK, W138, or NIH 3T3 cells), yeast (e.g., Saccharomyces or Pichia), bacteria (e.g., E. coli, P. pastoris, or B. subtilis), insect cells (e.g., baculovirus in Sf9 cells), or other cells. After transfection of a suitable host strain and growth of the host strain to an appropriate cell density, the cells are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification of the peptide or its variants.

[0127] Suitable expression vectors include, for example, chromosomal, non-chromosomal, and synthetic polynucleotides, such as derivatives of SV40, bacterial plasmids, phage DNA, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA, such as vaccinia virus, adenovirus, adeno-associated virus, lentivirus, canarypox virus, fowlpox virus, pseudorabies virus, baculovirus, herpes virus, and retrovirus, etc. Polynucleotides can be introduced into expression vectors by conventional procedures known in the art.

[0128] The polynucleotide encoding one or more peptides can be operably linked to an expression control sequence, i.e., a promoter that directs mRNA synthesis.Representative examples of such promoters include LTR or SV40 promoter, lac or trp of E. coli, phage lambda PL promoter, and other promoters known to control gene expression in prokaryotic or eukaryotic cells or viruses.The expression vector can also include a ribosome binding site for translation initiation and a transcription terminator.

[0129] The expression vector may also contain an origin of replication and a selection marker, such as the ampicillin resistance gene of E. coli, to allow selection of transformed cells, i.e., cells that express the heterologous polynucleotide. A nucleic acid molecule encoding one or more of the peptides may be incorporated into the vector in frame with translation initiation and termination sequences.

[0130] One or more of the peptides can be recovered and purified from the recombinant cell culture (i.e., from the cells or culture medium) by well-known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, lectin chromatography, and HPLC. If the peptide is denatured during isolation and / or purification, well-known techniques for refolding proteins can be used to regenerate the active configuration.

[0131] It is preferred to use recombinant techniques to produce glycosylated peptides. It is preferred to use mammalian cells such as COS-7 and Hep-G2 cells in recombinant procedures to produce glycosylated peptides.

[0132] Peptides can also be prepared by cleavage of longer peptides, particularly from food extracts.

[0133] Pharmaceutically acceptable salts of peptides can be synthesized from peptides containing a basic or acidic moiety by conventional chemical methods. Generally, salts are prepared by reacting the free base or free acid with a stoichiometric or excess amount of an inorganic or organic acid or base that forms the desired salt in a suitable solvent.

[0134] Methods for identifying biologically active sequences This specification discloses novel epitopes that are generated by the novel epitope discovery and generation method disclosed herein.In one embodiment, the method for generating novel epitope sequences involves searching for continuous epitope sequences on polypeptides that can bind to antibodies or induce immune responses in individuals.Once epitope sequences are discovered, they are recombined with random sequences or other discovered epitope sequences to generate new synthetic polypeptide sequences that have greater sensitivity and specificity for binding to antibodies associated with autoimmune disorders than native epitopes alone.In a preferred embodiment, the process of generating and screening sequences is carried out on a peptide array that is configured to contact with a sample.

[0135] In some embodiments, as illustrated in FIG. 2, a method for identifying novel epitopes comprises the steps of: 1) generating a first plurality of overlapping polypeptide fragments, each comprising a portion of a native active protein or polypeptide that exhibits biological activity; 2) determining the specificity and sensitivity of antibodies correlated with an autoimmune disorder for each polypeptide fragment by contacting an array comprising the polypeptide fragments with a sample from a subject having an autoimmune disorder; and 3) identifying polypeptide fragments that exceed a predetermined threshold for binding sensitivity and / or specificity, or those with the greatest sensitivity and / or specificity values ​​among the collection of polypeptide fragments. selecting; 4) identifying the occurrence of epitope sequences within the polypeptide fragments from the polypeptide fragments identified in step 3; 5) generating a second plurality of synthetic polypeptides, each comprising at least two of the epitope sequences from step 4 and, optionally, at least one random polypeptide sequence; 6) determining the specificity and sensitivity of each of the synthetic polypeptides generated in step 5 by contacting an array comprising the synthetic polypeptide fragments with a sample from a subject with an immune disorder; and 7) selecting synthetic polypeptides from step 6 that exceed thresholds for specificity and sensitivity for use as biomarkers for autoimmune disorders. Optionally, steps 5 through 7 can be repeated to further refine the sensitivity and / or specificity of the synthetic polypeptides for binding antibodies associated with autoimmune disorders. This method results in the generation of a plurality of novel bioactive polypeptides useful for the diagnosis and treatment of autoimmune disorders (e.g., celiac disease).

[0136] In one embodiment, the autoimmune disorder is celiac disease. In one embodiment, the biologically active protein is gliadin. In one embodiment, the gliadin is α-gliadin, β-gliadin, γ-gliadin, or ω-gliadin.

[0137] Identification of epitopes in antigens As disclosed herein, methods are provided for identifying epitopes on bioactive proteins, such as gliadin, and used to generate novel bioactive polypeptide sequences for use in diagnosing and treating autoimmune diseases. In one embodiment, a full-length bioactive polypeptide sequence is divided into overlapping polypeptide fragments of distinct lengths. In one embodiment, each polypeptide fragment is 6-15 amino acids in length. In one embodiment, each polypeptide fragment is 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In a preferred embodiment, each polypeptide fragment is 12 amino acids in length. The amount of overlap between polypeptide fragments of a full-length bioactive polypeptide can be determined by the polypeptide fragment step size, which refers to the distance between the N-terminal or C-terminal amino acids of each polypeptide fragment as determined by the full-length bioactive polypeptide. A diagram of an embodiment in which the step size is 2 amino acids is shown in Figure 1, where the polypeptide fragments are 12 amino acids in length. This results in an overlap of 10 amino acids between adjacent polypeptide fragments. This overlap allows for more accurate determination of the active epitope sequence on the biologically active polypeptide sequence.In some embodiments, the step size can be different, for example, the step size can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids.In one preferred embodiment, the step size is 2 amino acids.Also, to improve accuracy, the step size of 1 amino acid can be used, but at the expense of needing to generate more fragment polypeptides.

[0138] Based on the scheme for generating polypeptide fragments discussed above, fragment polypeptides are synthesized on an array for screening against samples containing antibodies correlated with autoimmune disorders. Antibody binding to the fragment polypeptides on the array is detected via a secondary antibody, although other detection methods known to those skilled in the art are considered sufficient. Information regarding the binding of each polypeptide fragment to antibodies in samples from subjects identified as having or not having an autoimmune disorder is compared to determine the sensitivity and specificity of each peptide. Overlapping regions allow for the identification of epitope sequences. In one embodiment, the identified epitopes are 3-11 amino acids in length. In one embodiment, each identified epitope is 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids in length. In one preferred embodiment, each epitope is limited to 3 amino acids in length. In some embodiments, pairs of epitopes are identified from among the polypeptide fragments that exceed a threshold for specificity and / or sensitivity of binding to autoimmune-positive samples. These epitope pairs are then used to create synthetic sequences, as described below.

[0139] Creation of novel biologically active sequences The epitopes identified from the native bioactive polypeptides are used to generate and synthesize novel synthetic bioactive polypeptide sequences on an array for further screening. In one embodiment, each novel synthetic bioactive polypeptide contains at least one epitope identified by the method disclosed herein. In another embodiment, each novel synthetic bioactive polypeptide contains at least two epitopes identified by the method disclosed herein. In some embodiments, each novel synthetic bioactive polypeptide contains two, three, four, or five epitopes identified by the method described herein. In some embodiments, each novel synthetic bioactive polypeptide contains at least one or at least two epitope sequences plus a randomly generated polypeptide sequence. In some embodiments, the randomly generated sequence is 3, 6, 9, or 12 amino acids in length. In a preferred embodiment, each novel synthetic bioactive polypeptide sequence contains two 3-amino acid epitope sequences identified by the method disclosed herein and at least one randomly generated polypeptide sequence, so that a novel synthetic bioactive polypeptide sequence of 12 amino acids is generated. In one embodiment, the novel synthetic bioactive polypeptide sequence is selected from SEQ ID NOs:1-127. In one embodiment, a plurality of novel synthetic bioactive polypeptide sequences are synthesized on an array for contact with a sample to determine the sensitivity and specificity of each novel synthetic bioactive polypeptide sequence for detecting the sample having an autoimmune disorder. In one embodiment, novel synthetic bioactive polypeptides with high sensitivity and / or specificity for detecting an autoimmune disorder are selected for further modification of random polypeptide sequences around epitopes contained therein for screening on another polypeptide array. The methods described herein result in the generation of bioactive polypeptide sequences that act as epitopes for binding to antibodies associated with autoimmune diseases with high sensitivity and / or specificity.

[0140] In one embodiment, a polypeptide array is produced having a plurality of synthetic bioactive polypeptide sequences provided herein. In one embodiment, the array has at least 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 novel synthetic bioactive polypeptide sequences produced by the methods disclosed herein. In one embodiment, the array has at least 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 polypeptides having a sequence selected from the group consisting of SEQ ID NOs: 1-127. In one embodiment, the polypeptide array has a sensitivity for detecting an autoimmune disorder of greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% in subjects suspected of having the disorder. In one embodiment, the polypeptide array has a specificity for detecting an autoimmune disorder in subjects suspected of having an autoimmune disorder of greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0141] Bioactive Sequences and Methods of Therapeutic Use Vaccines and Administration The present invention also provides vaccines comprising the first, second, and third peptides, one or more biologically active fragments or variants thereof, and / or polynucleotides encoding one or more thereof. Also provided are vaccines comprising the peptides of the invention and / or the polynucleotides of the invention.

[0142] As used herein, the term "vaccine" refers to a composition that can be administered to a gluten-sensitive subject to modulate the subject's response to gluten. The vaccine can reduce the subject's immunological reactivity to gluten. Preferably, the vaccine induces tolerance to gluten.

[0143] Administration of the vaccine to a subject may induce the expression of gluten-specific effector T cell populations, e.g., gluten-specific CD4 + Tolerance can be induced by clonal deletion of T cells or by inactivation (anergy) of the T cells such that they become less responsive, or preferably unresponsive, to subsequent exposure to gluten (or peptides thereof).

[0144] Alternatively, or additionally, administration of the vaccine can alter the subject's cytokine secretion profile (e.g., resulting in a decrease in IL-4, IL-2, TNFα and / or IFNγ, and / or an increase in IL-10). The vaccine can induce a subpopulation of suppressor T cells, e.g., Treg cells, to produce IL-10 and / or TGFβ, thereby suppressing gluten-specific effector T cells.

[0145] The vaccines of the present invention can be used for the prophylactic treatment of subjects who may develop sensitivity to gluten, e.g., those diagnosed as carrying the HLA-DQ2 and / or HLA-DQ8 genes, and / or for the ongoing treatment of subjects who are sensitive to gluten, e.g., those with celiac disease. There is a substantial amount of animal data supporting the prophylactic activity of immunodominant peptides against a variety of autoimmune and model immune conditions, such as experimental allergic encephalitis.

[0146] As used herein, the term "treatment" includes arresting, inhibiting, slowing, or reversing the progression of a disease or condition, or ameliorating or preventing the clinical symptoms of a disease (e.g., celiac disease) or condition.

[0147] The amount of vaccine (or agent, peptide, polynucleotide, and / or APC) to be administered is referred to as an "effective amount." The term "effective amount" means an amount sufficient to produce the desired therapeutic or physiological effect when administered under appropriate or sufficient conditions. Single or multiple doses can be administered. Undesirable effects, e.g., side effects, sometimes appear along with the desired therapeutic effect; therefore, the practicing physician balances the potential benefits with the potential risks in determining what is an appropriate "effective amount." The exact amount required will vary from subject to subject, depending on the species, age, size, and general condition of the subject, the mode of administration, and the like. For this reason, it may be impossible to specify an exact "effective amount." However, one of ordinary skill in the art can determine an appropriate "effective amount" in any individual case using only routine experimentation.

[0148] The vaccine (or agent, peptide, polynucleotide and / or APC) modifies the T cell response in the subject to wheat, barley and rye, preferably wheat, barley, rye and oats, as represented by gliadin, secalin, hordein, glutenin and optionally avedin proteins, such that a subject treated according to the present invention can preferably eat at least wheat, rye, barley and optionally oats without significant T cell responses that would normally lead to symptoms of celiac disease.

[0149] The individual components of the agent of the present invention can be administered in the same composition, in different compositions, or combinations thereof (e.g., a first and second peptide as defined herein in one composition and a third peptide in another composition). If in different compositions, they can be administered simultaneously or sequentially.

[0150] The active substance or vaccine may include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to molecular entities and compositions that do not produce allergic, toxic, or other adverse reactions when administered to a subject, particularly a mammal, more particularly a human. Pharmaceutically acceptable carriers may be solid or liquid. Useful examples of pharmaceutically acceptable carriers include, but are not limited to, diluents, excipients, solvents, surfactants, suspending agents, buffers, lubricants, adjuvants, vehicles, emulsifiers, absorbents, dispersion media, coating agents, stabilizers, protective colloids, adhesives, thickeners, thixotropic agents, penetration agents, sequestering agents, isotonic agents, and absorption delaying agents, which do not affect the activity of the active substance of the present invention.

[0151] Carrier can be any of the conventionally used ones, and is limited only by chemical and physical considerations such as solubility and non-reactivity with active agent, and route of administration.Suitable carrier for the present invention includes conventionally used ones, such as water, saline, aqueous dextrose, lactose, Ringer's solution, buffer solution, hyaluronan, glycol, starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, etc.Also, liposome can be used as carrier.

[0152] Techniques for preparing pharmaceutical compositions are generally known in the art and are exemplified by Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980.

[0153] The term "adjuvant" generally refers to an immunostimulatory substance designed to enhance the immunogenicity of one or more peptides as defined herein. Preferably, the adjuvant does not generate a Th1 response and further promotes immune tolerance and / or reduces inflammation. Suitable adjuvants include 1) aluminum-based inorganic acid salt adjuvants, such as Al(OH)3 gel or aluminum phosphate, but may also be calcium, iron, or zinc salts; and 2) dexamethasone (Kang et al., 2008).

[0154] Administration may be oral, topical (transdermal), parenteral, by inhalation spray, or rectal in a unit dosage formulation containing a conventional non-toxic pharmaceutically acceptable carrier. The term "parenteral," as used herein, includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, subconjunctival, intracavity, transdermal, and subcutaneous injection, by aerosol for administration to the lungs or nasal cavity, or by infusion, for example, by osmotic pump.

[0155] The active compound of the present invention may be in a form suitable for oral use, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use can be prepared according to methods known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents and preservatives to provide pharmaceutically elegant and palatable preparations.

[0156] tablet Tablets containing the active ingredient mixed with pharmaceutically acceptable excipients can also be manufactured by known methods. The excipients used can be, for example, (1) inert diluents such as calcium carbonate, lactose, calcium phosphate, or sodium phosphate; (2) granulating and disintegrating agents such as cornstarch or alginic acid; (3) binders such as starch, gelatin, or gum arabic; and (4) lubricants such as magnesium stearate, stearic acid, or talc. The tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a longer-lasting effect. For example, time-delay materials such as glyceryl monostearate or glyceryl distearate can be used. They can also be coated to form osmotic therapeutic tablets for controlled release.

[0157] In some cases, preparations for oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, etc. They may also be in the form of soft gelatin capsules in which the active ingredient is mixed with an aqueous or oily medium, such as peanut oil, liquid paraffin, or olive oil.

[0158] aqueous suspension Aqueous suspensions normally contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions, which may include: (1) suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia; or (2) dispersing or wetting agents, such as C2-C6 18PEG esters of fatty acids, Tween 80 or polyethylene oxide sorbitan monolaurate, Brij or polyoxyethylene alcohol, Triton-X or polyethylene glycol p-isooctylphenyl ether, Triton-N, and Triton A-20 or polymers with 4-(1,1,3,3-tetramethylbutyl)phenol, formaldehyde and oxirane, DECON, Tris or 2-amino-2-hydroxymethyl-1,3-propanediol and Cremophor EL, etc.

[0159] The aqueous suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate; one or more coloring agents; one or more flavoring agents; and one or more sweetening agents, such as sucrose, aspartame, or saccharin.

[0160] oily suspension Oily suspension can be prepared by suspending active ingredient in vegetable oil, such as peanut oil, olive oil, sesame oil or coconut oil, fish oil containing omega-3 fatty acid, or mineral oil such as liquid paraffin.Oily suspension can contain thickening agent, such as beeswax, solid paraffin or cetyl alcohol.Sweetener and flavoring agent can be added to obtain a palatable oral preparation.These compositions can be preserved by adding antioxidants such as ascorbic acid.

[0161] Dispersible powders and granules Dispersible powders and granules are suitable for preparing aqueous suspensions.They provide the active ingredient in a mixture with dispersing or wetting agent, suspending agent and one or more preservatives.Suitable dispersing or wetting agent and suspending agent are exemplified by those already mentioned above.Additional excipients, such as the above-mentioned sweeteners, flavorings and coloring agents, can also be present.

[0162] emulsion The pharmaceutical composition may also be in the form of an oil-in-water emulsion.The oil phase may be a vegetable oil such as olive oil or peanut oil, a mineral oil such as liquid paraffin, or a mixture thereof.Suitable emulsifiers include gum arabic, gum tragacanth, soybean, lecithin, polyoxyethylene oxide sorbitan monolaurate (Tween 80).The emulsion may contain sweeteners and flavoring agents.

[0163] Syrups and elixirs Syrups and elixirs may be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol, aspartame or sucrose, and such formulations may also contain a demulcent, a preservative, a flavoring and a coloring agent.

[0164] Injection The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents as described above. Sterile injectable preparations can be suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable carriers that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland fixed oil can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid can also be used in the preparation of injectables.

[0165] Compositions suitable for parenteral administration include, but are not limited to, aqueous and non-aqueous sterile injection solutions. Suitable delivery mechanisms for subcutaneous administration include, but are not limited to, implants, depots, needles, capsules, and osmotic pumps.

[0166] Sustained Release Compositions Sustained-release compositions can also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers, which are in the form of shaped articles, such as films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid, copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene vinyl acetate and lactic acid-glycolic acid can release molecules for over 100 days, while certain hydrogels release proteins for shorter periods.

[0167] The active agent may be encapsulated, for example, in microcapsule formulations prepared by conventional techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions.

[0168] Microencapsulation for sustained release has been successfully performed on human growth hormone (rhGH), interferon (rhIFN), interleukin-2, and MN rgp120. Sustained-release formulations of these proteins were developed using PLGA polymer due to its biocompatibility and wide range of biodegradable properties. PLGA, a degradation product of lactic acid-glycolic acid, is rapidly eliminated in the human body. Furthermore, the degradability of this polymer can be tailored from months to years, depending on its molecular weight and composition.

[0169] gene therapy In a further embodiment, a polynucleotide encoding one or more of the peptides defined herein is inserted into a recombinant expression vector for administration to a subject.

[0170] The term "recombinant expression vector" refers to a plasmid, virus, or other vehicle known in the art that has been manipulated by the insertion or incorporation of a nucleic acid encoding one or more peptides. Such expression vectors contain a promoter sequence that promotes efficient transcription of the inserted gene sequence in the host. Expression vectors typically contain an origin of replication, a promoter, and specific genes that allow for phenotypic selection of transformed cells.

[0171] In one embodiment, the viral vector is derived from an adeno-associated virus (AAV) and contains a constitutive or regulatable promoter capable of inducing sufficient levels of expression of the peptides defined herein. Preferably, the viral vector contains an AAV inverted terminal repeat sequence, such as that described in WO 93 / 24641. In a preferred embodiment, the viral vector contains the polynucleotide sequence of the pTR-UF5 plasmid. The pTR-UF5 plasmid is a modified version of the pTR.sub.BS-UF / UF1 / UF2 / UFB series of plasmids (Zolotukiin et al., 1996; Klein et al., 1998).

[0172] Promoters useful for the subject invention include, for example, the cytomegalovirus immediate early promoter (CMV), the human elongation factor 1-alpha promoter (EF1), the small nuclear RNA promoters (U1a and U1b), the alpha-myosin heavy chain promoter, the simian virus 40 promoter (SV40), the Rous sarcoma virus promoter (RSV), the adenovirus major late promoter, the beta-actin promoter, and hybrid regulatory elements containing the CMV enhancer / beta-actin promoter. These promoters have been shown to be active in a wide range of mammalian cells.

[0173] The promoter is operably linked to a heterologous polynucleotide encoding one or more peptides defined herein. By "operably linked," it is intended that the promoter element is positioned relative to the coding sequence in such a way that it can effect expression of the coding sequence.

[0174] Also contemplated for use with the vectors of the present invention are inducible and cell-type specific promoters, such as the Tet-inducible promoter (Clontech, Palo Alto, Calif.) and the VP16-LexA promoter (Nettelbeck et al., 1998).

[0175] Transcriptional enhancer elements that can function to increase the level of transcription from a given promoter can also be included in the vector. Enhancers are generally positioned in either the 3' or 5' orientation relative to the promoter sequence. In addition to natural enhancers, synthetic enhancers can be used in the present invention, including muscle-specific elements, serum response factor binding elements (SREs), myocyte-specific enhancer factor-1 (MEF-1), myocyte-specific enhancer factor-2 (MEF-2), transcriptional enhancer factor-1 (TEF-1), and SP-1 (Li et al., 1999; Deshpande et al., 1997; Stewart et al., 1996; Mitchell and Tjian, 1989; Briggs et al., 1986; Pitluk et al., 1991). A synthetic enhancer randomly assembled from elements derived from Spc5-12 can also be used in the vector.

[0176] Gene therapy can be performed by ex vivo or in vivo treatment of patient's cells or tissues. The vector is introduced into suitable cells, cell lines, or tissues using methods known in the art. Viral particles and vectors can be introduced into cells or tissues in vitro or in vivo. Methods envisioned include transfection, transduction, injection, and inhalation. For example, the vector can be introduced into cells using liposomes containing the desired vector, by direct transfection with the vector alone, by electroporation, or by particle bombardment.

[0177] dosage Formulation of active substances into unit dosage forms is particularly useful for ease of administration and uniformity of dosage. As used herein, "unit dosage form" refers to a physically discrete unit suitable for unit administration to the subject to be treated. Each unit contains a predetermined amount of active agent calculated to produce a desired therapeutic effect, together with the necessary pharmaceutical carrier. The details of the unit dosage form are determined and depend on the specific characteristics of the active agent and the specific therapeutic effect to be achieved, as well as the inherent limitations in the technical field of compounding such active agents for the treatment of the subject. Alternatively, the composition may be prepared in the form of a multiple dosage form.

[0178] Examples of dosage units include sealed ampoules and vials which may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.

[0179] The agent or vaccine can also be included in a container, pack, or dispenser together with instructions for administration.

[0180] The actual amount (or dose or dosage) administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated. Prescribing treatment, e.g., determining dosage, timing, frequency, etc., is within the responsibility of a general physician or specialist (including a human physician, veterinarian, or medical scientist) and typically takes into account the disorder to be treated, the condition of the subject, the site of delivery, the method of administration, and other factors known to physicians. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences, 18th Ed. (1990), Mack Publishing, Company, Easton, Pa., USA). The dosage, frequency of administration, duration, route of administration, and the need for maintenance therapy can be based on standards for other peptide immunotherapeutics.

[0181] Effective amounts can be measured in ng / kg to g / kg body weight per minute, hour, day, week or month.

[0182] With in vivo administration of the agents or vaccines of the invention, typical dosages are from about 10 ng / kg to 100 mg / kg of mammalian body weight per day, or more, preferably from about 1 μg / kg / day to 10 mg / kg / day, and may vary depending on the route of administration. Guidance as to particular dosages and routes of delivery is provided in the literature.

[0183] Toxicity and therapeutic efficacy of an agent or vaccine can be determined by standard pharmaceutical procedures in cell cultures or experimental animals to determine the IC50 and maximum tolerated dose. The data obtained from these cell culture assays and animal studies can be used to establish appropriate ranges for humans.

[0184] Diagnostic and therapeutic effectiveness The peptides defined herein are also useful as diagnostic agents.

[0185] In one example, gluten tolerance is assayed by measuring the IL-10 and / or TGFβ secreted by stimulated cells, such as Treg cells, exposed to the peptides defined herein.Treg cells are characterized by the ability to produce large amounts of IL-10 and TGFβ.IL-10 is thought to be one of the main cytokines involved in immunosuppression, and the target of suppression is thought to be the transcriptional regulation of IL-2 in effector cells.

[0186] In another example, gluten tolerance can be assessed by stimulating cells, such as gluten-specific CD4 + Assayed by measuring IFNγ secreted from T cells.

[0187] Diagnostic tests may be performed in vitro using whole blood or cells isolated and / or fractionated therefrom.

[0188] In one example, cells are pre-exposed to one or more of the peptides (alone or conjugated to MHC molecules or fragments thereof, or APCs loaded with the peptides). In another example, cells are stimulated in vitro by co-incubation with the peptides (alone or conjugated to MHC molecules or fragments thereof, or APCs loaded with the peptides).

[0189] Direct T cell-mediated effects of agents can be monitored by functional assays utilizing cells isolated from peripheral blood or tissues (e.g., small intestine). The effect of peptide administration downstream of cognate T cells can be assayed using immune cell types, tissues, and biological fluids (e.g., plasma, intestinal secretions, urine, or feces).

[0190] In general, the biological effects of peptides recognized by cognate T cells are either proinflammatory or tolerogenic, depending on the dosing regimen, the mode of administration, and whether the peptide is modified or co-administered with another compound with immunological properties, such as an adjuvant. These and other peptides selected for use in peptide-based therapeutic vaccines are generally short (less than 29 amino acids), water-soluble, lack innate immune effects, and are recognized by a significant proportion of pathogenic T cells. Observations in animal models of T cell-mediated diseases and other human diseases indicate that activation of cognate T cells occurs after the first administration. However, repeated administration of the agent is expected to induce T cell anergy and / or tolerance. Ongoing regular peptide administration is expected to maintain tolerance to gluten, suppress inflammation in the small intestine, and systemically inhibit proinflammatory gluten-specific T cells.

[0191] Therefore, a key marker for successful treatment is the absence of inflammation in the small intestine after planned gluten ingestion. Surrogate immune markers that may predict whether intestinal tissue is normal or inflammatory after gluten ingestion include a wide range of assays that utilize pure or crude mixtures of immune cells, biological fluids, or tissue samples to measure soluble or cell-bound proteins or small molecules associated with immune activation, inflammation, or tolerance. These assays are well known to immunologists, immunohistologists, and physicians familiar with immune diseases, particularly celiac disease, in rodents and humans. More specifically, markers for assessing celiac disease and gluten-induced immune activity include small intestinal histology, serum IgA and IgG-specific gliadin (proteins or peptides), and various host proteins, including tTG.

[0192] Generic and specific markers of immunity in celiac disease that may be potentially adapted for monitoring peptide immunotherapy for celiac disease or for diagnosing celiac disease include: (a) CD4 isolated from blood or tissue + The direct effects of peptides on T cells may be due to peptide-stimulated cytokine release, T cell proliferation ex vivo / in vitro, or altered CD4 + It can be monitored by determination of T cell markers. (b) individual CD4 specific for peptides or gluten +The frequency and phenotype of T cells can generally be assessed by direct cell counting, for example, by FACS analysis. It is generally known that oral ingestion of gluten in patients with celiac disease who normally follow a gluten-free diet stimulates peptide- and gluten-specific T cells. Clinical tests, such as gluten challenge tests, can be used to evaluate T cells induced in blood or other tissues. The phenotype of isolated T cells can then be assessed fresh or after short-term in vitro expansion. T cell assays can rely on MHC-peptide complexes, antigen-stimulated intracellular cytokines, or other cell surface markers induced on antigen-activated T cells. CD4 + The functional status of T cells correlates with the presence of various cell surface and intracellular markers, such as activation markers including CD25 and CD69, or markers of "tolerance" and regulatory T cell function, such as GITR and FOXP3. The production of cytokines such as IFNγ, IL-4, IL-5, and IL-13, as well as IL-17, are considered proinflammatory in the context of classical Th1, Th2, or Th17 proinflammatory immune responses. In contrast, the secretion of IL-10 and TGFβ is associated with tolerogenic immune responses. Markers of proinflammatory immune responses are predicted to be reduced and / or tolerogenic immune responses are predicted to be enhanced. (c) CD4 +The effect of peptides on T cells can also be measured using a mixture of cells, such as whole blood, PBMCs, mononuclear cells isolated from tissues, or tissues incubated with the peptide. Individual or multiple proteins or RNA encoding related immune or disease-related proteins, such as cytokines and chemokines, may be evaluated after short-term incubation with the peptide. Assays such as IFNγ ELISpot using PBMCs before and / or after administering gluten or its peptides to patients, or multiplexed assays of chemokines and cytokines using PBMCs, can detect the biological effects of peptide-specific T cells from patients. The therapeutic effect of the peptide is indicated by a shift from markers associated with proinflammatory immune responses to markers associated with immune tolerance (e.g., IL-10), and a general decrease in proinflammatory markers such as IFNγ. (d) The effects of peptides on tissues can be practical. Functional assays can take the form of direct application of peptides to the skin to assess delayed-type hypersensitivity, as in the Mantoux test for tuberculosis, which involves intradermal application of PPD (purified protein derivative) and evaluation of the diameter of the reddening at the injection site 24-72 hours later. Peptides can also be applied to other mucosal and cutaneous sites for assessment in the same manner. Clinically, immune responses stimulated by both peptides and grain-derived proteins are important in celiac disease. For example, immunotherapy with selected peptides is expected to not only result in suppression of immune responses stimulated by T cells specific to the peptide, but also in "tolerance" to infectious and pro-inflammatory immune responses to peptides derived from other glutens and to gluten itself. Thus, the effects of peptide therapy can also be monitored in celiac disease by substituting gluten from various grains (wheat, rye, barley) for peptides in the above assays. Indeed, peptide therapy for cat-sensitive asthma has been monitored by such skin tests utilizing the whole protein antigen from which the therapeutic peptides are derived (Oldfield et al., 2002). (e) Finally, the clinical efficacy of peptide immunotherapy is assessed by histological examination of tissues exposed to gluten. This is typically the small intestine, but in experimental settings, oral and rectal mucosa are also evaluated, and in principle, other sites, such as the esophagus and colon, can also be assessed. Tissue from these sites can be obtained by direct visualization, typically endoscopic biopsy. Direct endoscopic visualization has also been used to diagnose celiac disease by mucosal appearance. Villous atrophy can be assessed by standard endoscopy and magnifying capsule endoscopy. Thus, the tolerogenic effect of peptides can be conveniently assessed by detecting microscopic tissue damage in the gastrointestinal tract. (f) Immunoglobulins specific for the peptide or other gluten peptides or autoantigens associated with celiac disease may be markers of gluten immunity with respect to disease activity and opsonizing activity that may compromise the therapeutic efficacy of the peptide itself. (g) The presence of markers associated with anaphylaxis, such as peptide- or gluten-specific IgE, or histamine release by peripheral blood basophils, can also be used to predict complications of peptide immunotherapy and the need to adjust or stop treatment.

[0193] Food Testing The present invention also provides a method for determining whether a composition or food can cause celiac disease, comprising detecting the presence of the agent of the present invention, the peptide of the present invention, and / or the polynucleotide of the present invention in a composition or food sample. Typically, this can be done by using a binding assay in which one or more compounds that specifically bind to one or more peptides defined herein are contacted with the composition, and the formation of a peptide / compound complex is detected to confirm the presence of the peptide. In one example, the compound is an antibody. Any suitable format of binding assay can be used. Typically, the assay utilizes a monoclonal antibody against a gluten peptide in a non-competitive sandwich-type ELISA. The food sample is first extracted, optionally diluted, and then tested in the assay.

[0194] The composition or food typically comprises a material from a plant that expresses gluten. Such a material may be a part of the plant, such as a harvested product (e.g., a seed). The material may also be a processed product of the plant material, such as flour or a food product containing gluten. Processing and testing of food materials in suitable binding assays is routine (see, for example, Kricka, 1998). The composition or food material may be treated with tTG before contacting with the compound.

[0195] In one embodiment, the composition or food material is contacted with at least 2, 3, 5, 10 or more antibodies specific for the peptides defined herein in deamidated and / or non-deamidated form. Preferably, the antibodies are directed against protease-resistant sequences, allowing the detection of α, β, γ and ω gliadins and LMW and HMW glutenins in wheat, B, C and D hordeins in barley, β, γ and ω secalins in rye, and optionally avenins in oats.

[0196] Antibodies directed against the peptides / epitopes defined herein may be provided in the form of a kit for use in assays for the detection and / or quantification of gluten in food products.

[0197] Protease identification The present invention also provides a method for identifying a protease capable of cleaving a peptide defined herein, comprising contacting the peptide with a protease under conditions for specific cleavage of the peptide to generate proteolytic products, and detecting the proteolytic products. In one example, the proteolytic products are detected using, for example, SDS-PAGE, HPLC, ELISA, or Western blot. In a further example, the peptide is fused to a fluorescent donor and a quenching acceptor to enable intramolecular resonance energy transfer between the fluorescent donor and the quenching acceptor. Upon cleavage, the donor and the acceptor separate, allowing detection of the donor's fluorescence emission. Typically, the peptide separates the fluorescent donor and the quenching acceptor by a distance of less than about 100 angstroms. The fluorescent donor can be attached to the C-terminus of the peptide, and the quenching acceptor can be attached to the N-terminus of the peptide, or vice versa.

[0198] Methods of using arrays with biologically active sequences Any of the arrays described herein can be used as a research tool or for research applications. In one aspect, the array can be used for high-throughput screening assays. For example, an enzyme substrate (i.e., a peptide on a peptide array described herein) can be tested by subjecting the array to an enzyme and identifying the presence or absence of the enzyme substrate on the array, for example, by detecting a change in at least one feature of the array.

[0199] Arrays can also be used in screening assays for ligand binding to ascertain substrate specificity, or to identify peptides that inhibit or activate proteins. Labeling techniques, protease assays, and binding assays useful for carrying out these methods are generally well known to those of skill in the art.

[0200] In some embodiments, arrays can be used to present known protein sequences as overlapping peptide sequences. For example, the amino acid sequence of a known protein can be divided into overlapping sequence segments of any length and any suitable overlapping frame, and peptides corresponding to each sequence segment can be synthesized in situ as disclosed herein. The individual peptide segments thus synthesized can be arranged starting from the amino terminus of the known protein.

[0201] In some embodiments, the array is used in a method in which the antigen presentation of the array comprises at least one region in which the entire antigen sequence of a known protein is covered by epitope sliding.The immunoreactive region of the antigen is determined by contacting one or more clinical samples on the array or a plurality of different arrays, and the set of peptide sequences required to present the known protein antigen is reduced.

[0202] In some embodiments, sample is applied to the array with a plurality of random peptides.Can be screened and BLAST searched for random peptides to determine the homologous domain with predetermined antigen sequence, for example, with 90% or more identity.Then, in some aspects, can be synthesized and used to identify the potential marker and / or cause of the disease of interest.

[0203] In some embodiments, the array is used for high-throughput screening of one or more genetic factors. Proteins associated with genes can be potential antigens, and antibodies against these proteins can be used to infer the relationship between genes and diseases.

[0204] In another example, the array can be used to identify one or more biomarkers. Biomarkers can be used in the diagnosis, prognosis, treatment, and management of diseases. Biomarkers can be expressed, absent, or at different levels in an individual depending on the disease state, disease stage, and response to disease treatment. Biomarkers can be, for example, DNA, RNA, proteins (e.g., enzymes such as kinases), sugars, salts, fats, lipids, or ions.

[0205] Arrays can also be used for therapeutic purposes, such as identifying one or more bioactive substances. A method for identifying a bioactive substance can include applying multiple test compounds to the array and identifying at least one test compound as a bioactive substance. The test compound can be a small molecule, an aptamer, an oligonucleotide, a chemical, a natural extract, a peptide, a protein, an antibody fragment, an antibody-like molecule, or an antibody. The bioactive substance can also be a therapeutic agent or a modulator of a therapeutic target. Therapeutic targets can include phosphatases, proteases, ligases, signaling molecules, transcription factors, protein transporters, protein sorters, cell surface receptors, secreted factors, and cytoskeletal proteins.

[0206] In another aspect, the array can be used to identify therapeutic drug candidates. For example, if one or more epitopes for a specific antibody are determined by an assay (e.g., a binding assay such as ELISA), those epitopes can be used to develop drugs (e.g., monoclonal neutralizing antibodies) against the target antibody in a disease.

[0207] In one aspect, an array for use in medical diagnosis is also provided.The array can be used to determine the response to the administration of a drug or a vaccine.For example, the response of an individual to a vaccine can be determined by detecting the antibody level of the individual using an array with peptides that present the epitopes recognized by the antibodies produced by the induced immune response.Another diagnostic use is to test an individual for the presence of biomarkers.Here, a sample is taken from a subject and tested for the presence of one or more biomarkers.

[0208] Arrays can also be used to stratify patient populations based on the presence or absence of biomarkers that indicate the likelihood that a subject will respond to a therapeutic treatment. Arrays can be used to identify known biomarkers to determine appropriate treatment groups. For example, a sample from a subject with a certain condition can be applied to the array. Binding to the array can indicate the presence of a biomarker for that condition. Previous studies may show that biomarkers are associated with positive outcomes after treatment, while the absence of a biomarker is associated with negative or neutral outcomes after treatment. Because a patient has a biomarker, the patient can also be stratified by a medical professional into treatment groups.

[0209] In some embodiments, a method of detecting the presence or absence of a protein of interest in a sample may include obtaining an array disclosed herein and contacting it with a sample that may contain the protein of interest; and determining whether the protein of interest is present in the sample by detecting the presence or absence of binding to one or more features of the array.

[0210] In some embodiments, a method for identifying a vaccine candidate can include obtaining an array as disclosed herein in contact with a sample obtained from a subject previously administered the vaccine candidate, the sample comprising a plurality of antibodies; and determining the binding specificity of the plurality of antibodies to one or more features of the array. In some embodiments, the features comprise a plurality of distinct, nested, overlapping peptide chains comprising subsequences derived from a source protein having a known sequence.

[0211] In one embodiment, a method for diagnosing and treating autoimmune disorders is provided. In one embodiment, the use of a peptide chip to detect multiple antibodies in serum samples is provided. In some embodiments, this method is performed in a single assay. In some embodiments, this method is performed on a single peptide chip. In one embodiment, this method provides the ability to detect multiple chemokines derived from autoimmune disorders. In one embodiment, this method provides the ability to identify the subtype and severity of autoimmune disorders.

[0212] In one embodiment, the diagnostic method using the peptide chip comprises: 2 has a reproducibility of greater than 0.95. In some embodiments, the method of diagnosing an autoimmune disorder using a peptide chip has a specificity of greater than 0.99 and / or a sensitivity of greater than 0.99.

[0213] In one embodiment, the autoimmune disorder is celiac disease. In another embodiment, the autoimmune disorder is lupus erythematosus. In another embodiment, the autoimmune disorder is rheumatoid arthritis.

[0214] The peptide arrays disclosed herein can be used to identify epitopes associated with autoimmune diseases. In one embodiment, the epitope is a B cell epitope, a T cell epitope, or an epitope associated with an inflammatory response (e.g., TNF). Epitopes associated with an inflammatory response can be identified by the present invention using a cytokine assay. In one embodiment, the peptide sequence identified by the cytokine assay can be used in an immunosuppressive vaccine. In other embodiments, the peptide sequence can be used as part of a peptide array to identify the presence of inflammatory molecules in subjects suspected of having an inflammatory disorder, such as an autoimmune disorder. In one embodiment, the peptide array can be used to identify B cell epitopes. In this embodiment, epitopes that bind to antibodies from a sample associated with an autoimmune disorder are identified. These peptides are then used in another peptide array useful for diagnosing autoimmune disorders. In one embodiment, diagnosing an autoimmune disorder includes identifying the subtype of the autoimmune disorder. In some embodiments, the identified B cell epitopes are used to measure a patient's response to treatment for an autoimmune disorder. In one embodiment, T cell epitopes can be identified by the present invention using an MHC complex assay (e.g., a human leukocyte antigen assay). Epitopes identified to interact with MHC complexes in subjects identified as having an autoimmune disorder can be used to treat the autoimmune disorder. Such peptides can be useful in vaccines or other drugs for modulating T cells. A flowchart depicting the identification of epitope sequences and their use according to some embodiments of the present invention is shown in Figure 3.

[0215] In some embodiments, the invention involves bioinformatics analysis of data, e.g., to identify informative subsequences, followed by synthesis and testing of synthetic peptide sequences useful for diagnosing a condition. These bioinformatics methods are implemented, in part, using computers to accomplish one or more of the following steps: 1) generating subsequences from longer sequences; 2) tabulating and ranking the occurrence of subsequences in positive hits from samples bound to arrays of tiled natural peptide sequences; 3) analyzing hits against arrays containing synthetic sequences that contain informative subsequences.

[0216] As will be apparent from the discussion below, unless otherwise specifically indicated, discussions throughout this specification using terms such as "processing" or "calculating" or "figuring out" or "determining" or "displaying" or "analyzing" or "comparing" or "identifying" refer to the operation and processing of a computer system or similar electronic computing device that manipulates and transforms data represented as physical (electronic) quantities in the registers or memory of the computer system into other data also represented as physical (electronic) quantities in the registers and memory of the computer system, and into other data also represented as physical quantities in the memory or registers of the computer system or other such information storage, transmission or display device.

[0217] The present invention also relates to a system apparatus for carrying out the operations herein. This apparatus may be specially constructed for the required purposes, or it may comprise a general-purpose computer selectively activated or reconfigured by a computer program stored on the computer. Such a computer program may be stored on a computer-readable storage medium, such as any type of disk, including but not limited to, floppy disks, optical disks, CD-ROMs, and magneto-optical disks, read-only memory (ROM), random access memory (RAM), EPROM, EEPROM, magnetic or optical cards, or any type of medium suitable for storing electronic instructions, each connected to the computer's system bus.

[0218] Various general-purpose systems may be used with programs in accordance with the teachings herein, or it may prove convenient to construct more specialized apparatus to perform the required method steps. The required structure for a variety of these systems will appear from the description below. In addition, the present invention is not described with reference to any particular programming language. It will be understood that a variety of programming languages ​​can be used to implement the teachings of the present invention as described herein.

[0219] composition compound Disclosed herein are formulations, such as photoactive formulations (e.g., photoresist formulations), coupling formulations, and linker formulations. These formulations may be useful, for example, in fabricating and / or using the substrates and / or peptide arrays disclosed herein. Generally, the components of each formulation disclosed herein are soluble in water at room temperature (approximately 25°C).

[0220] photoactive formulation Photoactive formulations are disclosed herein. In one aspect, the photoactive formulation can include a chemically amplified resist formulation. In chemically amplified (CA) resists, a primary photochemical event generates a mobile catalyst, which then induces a cascade of species that leads to secondary catalytic events within a radius of 5-25 nm, typically during a subsequent post-exposure bake (PEB). Such chemical amplification can thus achieve a total quantum yield (number of species reactions divided by the number of absorbed photons) of up to several hundred. CA resists typically contain a small amount (approximately 1-5 wt%) of a radiation-sensitive catalyst precursor, such as a photoacid generator (PAG); multiple chemical groups that can react by elimination, addition, or rearrangement in the presence of the catalyst; a polymer matrix that can disperse the other components into a smooth, transparent film; and optional additives that improve performance or processability, such as surfactants, photosensitizers, and etch resists.

[0221] In one aspect, the photoactive coupling formulation can include a photoactive compound. The photoactive compound can include a photobase generator or a photoacid generator. Exposure of the photoactive compound to electromagnetic radiation is a primary photochemical event in which a compound is generated, which then induces a secondary material transformation reaction within a diffusion-limited radius. The photoactive coupling formulation can include a photoactive compound containing a radiation-sensitive catalyst precursor, such as a photoacid generator (PAG); multiple chemical groups that can react by elimination, addition, or rearrangement in the presence of a catalyst; and optional additives that improve performance or processability, such as surfactants, photosensitizers, and etch resists.

[0222] In some embodiments, the photoactive coupling formulation comprises a photobase generator and a photosensitizer in a polymer matrix dispersed in a solvent. In some embodiments, the polymer in the composition of the photoresist is generally inert and non-crosslinkable, while the photoactive compound readily generates a sufficient amount of photobase upon exposure to electromagnetic radiation to cause the desired reaction to produce the product in acceptable yield.

[0223] In some embodiments, the photoactive formulation is not chemically amplified, i.e., all generated acid is consumed during the reaction (e.g., all tboc is deprotected and acid is consumed during the reaction). A tboc-protected amino acid can be added with the photoresist formulation to verify whether chemical amplification has occurred. In some embodiments, a photosensitizer is optional when using 248 nm.

[0224] In some embodiments, the photoactive formulation comprises a water-soluble photoacid generator and a water-soluble photosensitizer in a polymer matrix dispersed in water. In some embodiments, the polymer in the composition of the photoresist is generally inert and non-crosslinkable, but the photoreactive component will readily generate sufficient amounts of photoacid upon exposure to a deep UV irradiation tool to undergo the desired reaction to produce acceptable product yields.

[0225] In some embodiments, the photoactive formulation can include various components, such as a water-soluble photosensitizer, a water-soluble photoactive compound, a water-soluble polymer, and a solvent. Specific examples of photoactive formulations are shown in Table 1.

[0226] Photosensitizers are generally added to the formulation to increase the sensitivity of the photoacid generator and shift the absorption spectrum of the formulation towards the deep UV (248 nm). In some embodiments, the water-soluble photosensitizer may be a thioxanthenone. In some embodiments, the general thioxanthenone structure is: This is shown in TIFF2026035691000008.tif24128.

[0227] In some embodiments, the A, R1, R2, and R3 groups of the thioxanthenone structure above are: It could be TIFF2026035691000009.tif54128.

[0228] In some embodiments, the water-soluble photosensitizer can be about 0.5-5% by weight of the total formulation concentration, or about less than 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or less by weight of the total formulation concentration. The amount may be, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0% or more by weight.

[0229] In some embodiments, the water-soluble photoactive compound may be a photoacid generator (PAG) or a photobase generator (PBG). The photoacid generator (or PAG) is a cationic photoinitiator. A photoinitiator is a compound added to a formulation to convert absorbed light energy, specifically UV or visible light, into chemical energy in the form of initiating species, such as free radicals or cations. Cationic photoinitiators are widely used in photolithography. The ability of some types of cationic photoinitiators to function as potential photochemical sources of very strong protonic or Lewis acids is generally the basis for their use in optical imaging applications. In some embodiments, the photoacid generator is a water-soluble iodonium salt, a water-soluble polonium salt, or a water-soluble sulfonium salt. In some embodiments, the photoacid generator is (4-methoxyphenyl)phenyliodonium or trifluoromethanesulfonate. In some embodiments, the photoacid generator is (2,4-dihydroxyphenyl)dimethylsulfonium triflate or (4-methoxyphenyl)dimethylsulfonium triflate, as shown below: The file is TIFF2026035691000010.tif22128.

[0230] In some embodiments, the photoacid generator is iodonium and sulfonium salts of triflates, phosphates, and / or antimonates, 1,3-bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane, or 1,3-bis[1-(9-fluorenylmethoxycarbonyl)-4-piperidyl]propane. In some embodiments, the photoacid generator is present in an amount of about 0.5 to 5% by weight of the total formulation. In some embodiments, the photoacid generator is present in an amount of about less than 0.1 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, or less of the total formulation concentration. %, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or greater than 5.0 wt%.

[0231] In some embodiments, the water-soluble polymer is a water-soluble, non-crosslinked, inert polymer. In some embodiments, the water-soluble polymer is polyvinylpyrrolidone. The general structure of polyvinylpyrrolidone is as follows: TIFF2026035691000011.tif28128, where n is any positive integer greater than 1.

[0232] In some embodiments, the water-soluble polymer is a polymer of vinylpyrrolidone. In some embodiments, the water-soluble polymer is polyvinylpyrrolidone. Polyvinylpyrrolidone is soluble in water and other polar solvents. In the dry state, it is a thin, flaky powder and generally readily absorbs up to 40% of its weight in atmospheric water. In the dissolved state, it has excellent wettability and readily forms a film.

[0233] In some embodiments, the water-soluble polymer is about 0.5-5% by weight of the total formulation. In some embodiments, the water-soluble polymer is about less than 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, or 2.3% by weight of the total formulation. , 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or greater than 5.0 wt%.

[0234] In some embodiments, the solvent is water, ethyl lactate, or a combination thereof. In some embodiments, the ethyl lactate can dissolve in water at a concentration of more than 50% to form a solvent. In some embodiments, the solvent can be about 10% propylene glycol methyl ether acetate (PGMEA) and about 90% deionized water. In some embodiments, the solvent can contain up to about 20% PGMEA.

[0235] In some embodiments, the solvent is about 80-90% by weight of the total formulation concentration, hi some embodiments, the solvent is less than 70%, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 88, 89, 90, 95, 108, 107, 108, 107, 108, 107, 108, 107, 108, 107, 108, 107, 108, 107, 108, 107, 108, 107, 108, 107, 108, 107, 107, 108, 107% by weight of the total formulation concentration, or greater than 99% by weight.

[0236] The photoactivatable coupling formulation includes a coupling molecule. The coupling molecule may include an amino acid. In some cases, all of the peptides on the arrays described herein are composed of natural amino acids. In other cases, the peptides on the arrays described herein are composed of a combination of natural and unnatural amino acids. In other cases, the peptides on the arrays may be composed exclusively of unnatural amino acids. Unnatural amino acids include peptidomimetics and D-amino acids. The R group may be one found in natural amino acids or may be a group similar in size to the R group of a natural amino acid. Additionally, unnatural amino acids such as β-alanine, phenylglycine, homoarginine, aminobutyric acid, aminohexanoic acid, aminoisobutyric acid, butylglycine, citrulline, cyclohexylalanine, diaminopropionic acid, hydroxyproline, norleucine, norvaline, ornithine, penicillamine, pyroglutamic acid, sarcosine, and thienylalanine can also be incorporated. These and other natural and unnatural amino acids are available, for example, from EMD Biosciences, Inc., San Diego, Calif. In some embodiments, the coupling molecule comprises a natural or artificial amino acid or polypeptide. Examples of coupling molecules include Boc-glycine-OH and Boc-histidine-OH. In some embodiments, the artificial amino acid is a D-amino acid. In some embodiments, the coupling molecule is 1-2% by weight of the total formulation concentration. In some embodiments, the coupling molecule is about 0.5-5% by weight of the total formulation concentration.In some embodiments, the coupling molecule is less than about 0.1 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, 5.1 wt%, 5.2 wt%, 5.3 wt%, 5.4 wt%, 5.5 wt%, 5.6 wt%, 5.7 wt%, 5.8 wt%, 5.9 wt%, 6.0 wt%, 6.1 wt%, 6.2 wt%, 6.3 wt%, 6.4 wt%, 6.5 wt%, 6.6 wt%, 6.7 wt%, 6.8 wt%, 6.9 wt%, 7.0 wt%, 7. The concentration of the coupling molecule is 4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5.0% by weight, or more than 5.0% by weight. In some embodiments, the coupling molecule comprises a protected group, for example, a group protected via t-Boc or F-Moc chemistry. In most cases, increasing the concentration of the coupling molecule provides the best performance.

[0237] In some embodiments, the formulation may include a t-Boc group to aid in chemical amplification of the initiator acid generated during post-exposure baking. Thus, the formulation may include a t-Boc-protected amino acid, for example, to enhance chemical amplification during post-exposure baking. In some embodiments, the t-Boc-protected amino acid will comprise about 0.5-1 wt. % of the formulation. In some embodiments, the protected amino acid is about 0.5-5 wt. % of the total formulation concentration. In some embodiments, the protected amino acid is less than about 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.10%, 3.11%, 3.12%, 3.13%, 3.14%, 3.15%, 3.16%, 3.17%, 3.18%, 3.19%, 3.20%, 3.21%, 3.22%, 3.23%, 3.24%, 3.25%, 3.26%, 3.27%, 3.28%, 3.29%, 3.30%, 3.31%, 3.32%, 3.33%, 3.34%, 3.35%, 3.36%, 3.37%, 3.38%, 3.39%, 3.40%, 3.41%, 3.42%, 3.43%, 3.44%, 3.45%, 3.46%, 3.47%, 3.48%, 3.49%, 3.50%, 3.51%, 3.52%, 3.53%, 3.54%, 3.55%, 3.56%, 3.57%, 3.58%, 3.59%, 3.60%, 3.61%, 3.62%, 3.63%, 3.64%, 3.65%, 3.66%, 3 0.4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5.0% by weight or more than 5.0% by weight.

[0238] In some embodiments, the coupling reagent is a carbodiimide or triazole. In some embodiments, the coupling reagent is N-hydroxysuccinimide (NHS). In some embodiments, the coupling reagent is 2-4 wt% of the total formulation concentration. In some embodiments, the coupling reagent is about 0.5-5 wt% of the total formulation concentration. In some embodiments, the coupling reagent is less than 0.1 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 2.10 wt%, 2.20 wt%, 2.30 wt%, 2.40 wt%, 2.50 wt%, 2.60 wt%, 2.70 wt%, 2.80 wt%, 2.90 wt%, 2.90 wt%, 2.1 ...10 wt%, 2.10 wt%, 2.10 wt%, 4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5.0% by weight or more than 5.0% by weight.

[0239] In any of the above combinations, the formulation is completely strippable with water, even after light exposure and baking. Thus, in some embodiments, water alone is used to wash off the photoactive coupling formulation after light exposure and baking.

[0240] Carboxylic Acid Activated Formulation Disclosed herein is an activation formulation for activating carboxylic acids so that the carboxylic acids react with free amino groups of biomolecules, such as amino acids, peptides, or polypeptides. The activation formulation may include components such as a carboxylic acid group-activating compound and a solvent. In some embodiments, the carboxylic acid group-activating compound is a carbodiimide or a carbodiimide precursor. In some embodiments, the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some embodiments, the carboxylic acid group-activating compound is N-hydroxysuccinimide [NHS]. In some embodiments, the carboxylic acid group-activating compound is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [EDC], N-hydroxysuccinimide [NHS], 1,3-diisopropylcarbodiimide [DIC], hydroxybenzotriazole [HOBt], 1-hydroxy-7-azabenzotriazole [HOAt], (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) [HATU], benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate [PyBOP], and N,N-diisopropylethylamine [DIEA]. In some embodiments, the solvent is water. In some embodiments, the solvent is N-methylpyrrolidone [NMP]. In some embodiments, the carboxylic acid group-activating compound converts a carboxylic acid to a carbonyl group (i.e., carboxylic acid group activation). In some embodiments, after exposure to the activating formulation, the carboxylic acid groups are activated for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, or 60 minutes.

[0241] In some embodiments, the activation formulation comprises 4 wt% 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2 wt% N-hydroxysuccinimide [NHS] dissolved in deionized water. In some embodiments, the activation formulation comprises 4 wt% 1,3-diisopropylcarbodiimide [DIC] and 2 wt% hydroxybenzotriazole [HOBt] dissolved in NMP. In some embodiments, the activation formulation comprises 4 wt% (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) [HATU] and 2 wt% N,N-diisopropylethylamine [DIEA] dissolved in NMP. In some embodiments, the activation formulation comprises 4 wt% benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate [PyBOP] and 2 wt% DIEA dissolved in NMP.

[0242] In some embodiments, the carboxylic acid group activating compound is a carbodiimide precursor.In one aspect, the carbodiimide precursor is converted into a carbodiimide by exposure to radiation, for example, ultraviolet light.In one embodiment, the carbodiimide precursor is a thione.The carbodiimide precursor may also be called a photoactivated carbodiimide.In one embodiment, the photoactivated carbodiimide is used to achieve site-specific activation of the carboxylic acid group on the array by spatially controlling the exposure of the photoactivated carbodiimide solution to electromagnetic radiation of a preferred activation wavelength.In some embodiments, the preferred activation wavelength is 248 nm.

[0243] In some embodiments, the carbodiimide precursor is a thione that is converted to a carbodiimide through photoactivation. In one aspect, the thione is converted to a hydroxymethylphenylcarbodiimide after exposure to electromagnetic radiation. In some embodiments, the thione is 4,5-dihydro-4-(hydroxymethyl)-1-phenyl-1H-tetrazole-5-thione, 1-(3-(dimethylamino)propyl)-4-ethyl-1,4-dihydro-5H-tetrazole-5-thione, 1,4-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-1,4-dihydro-5H-tetrazole-5-thione, 4-cyclohexyl-1H-tetrazole-5(4H)-thione, or 1-phenyl-4-(piperidinomethyl)tetrazole-5(4H)-thione, etc.

[0244] In some embodiments, the activation solution comprises a carbodiimide precursor, a solvent, and a polymer. In one embodiment, the carbodiimide precursor is 4,5-dihydro-4-(hydroxymethyl)-1-phenyl-1H-tetrazole-5-thione, 1-(3-(dimethylamino)propyl)-4-ethyl-1,4-dihydro-5H-tetrazole-5-thione, or 1,4-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-1,4-dihydro-5H-tetrazole-5-thione. In some embodiments, the carbodiimide precursor is present in the activation solution at a concentration of 2.5% by weight. In some embodiments, the carbodiimide precursor is present in the activation solution at 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, 5.1 wt%, 5.1 wt%, 5.2 wt%, 5.3 wt%, 5.4 wt%, 5.5 wt%, 5.6 wt%, 5.7 wt%, 5.8 wt%, 5.9 wt%, 6.0 wt%, 6.1 wt%, 6.2 wt%, 6.3 wt%, 6.4 wt%, 6.5 wt%, 6.6 wt%, 6.7 wt%, 6.8 wt%, 6.9 wt%, 7. %, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or 5.0 wt%.

[0245] In some embodiments, the solvent is water. In some embodiments, the solvent is about 80-90% by weight of the total formulation. In some embodiments, the solvent is about less than 70%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% by weight of the total formulation.

[0246] In some embodiments, the polymer is polyvinylpyrrolidone and / or polyvinyl alcohol. In some embodiments, the polymer is about 0.5-5% by weight of the total formulation. In some embodiments, the polymer is less than 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or less by weight of the total formulation. %, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or greater than 5.0 wt%.

[0247] In some embodiments, the coupling reagent is a carbodiimide. In some embodiments, the coupling reagent is a triazole. In some embodiments, the coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some embodiments, the coupling reagent is about 0.5-5% by weight of the total formulation. In some embodiments, the coupling reagent is less than 0.1% by weight, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9 ...0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 3.1%. 4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5.0% by weight or more than 5.0% by weight.

[0248] Linker Formulations Linker formulations are also disclosed herein. The linker formulations may include components such as a solvent, a water-soluble polymer, a water-soluble linker molecule, and a water-soluble coupling reagent. In some embodiments, the polymer is 1% by weight polyvinyl alcohol and 2.5% by weight polyvinylpyrrolidone, the linker molecule is 1.25% by weight polyethylene oxide, the coupling reagent is 1% by weight 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the solvent includes water. In some embodiments, the polymer is 0.5-5% by weight polyvinyl alcohol and 0.5-5% by weight polyvinylpyrrolidone, the linker molecule is 0.5-5% by weight polyethylene oxide, the coupling reagent is 0.5-5% by weight 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the solvent includes water.

[0249] In some embodiments, the solvent is water, an organic solvent, or a combination thereof. In some embodiments, the organic solvent is N-methylpyrrolidone, dimethylformamide, dichloromethane, dimethyl sulfoxide, or a combination thereof. In some embodiments, the solvent is about 80-90% by weight of the total formulation concentration. In some embodiments, the solvent is about less than 70%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% by weight of the total formulation concentration.

[0250] In some embodiments, the polymer is polyvinylpyrrolidone and / or polyvinyl alcohol. The general structure of polyvinyl alcohol is as follows: TIFF2026035691000012.tif23128, where n is any positive integer greater than 1.

[0251] In some embodiments, the water soluble polymer is about 0.5-5% by weight of the total formulation concentration. In some embodiments, the water soluble polymer is about less than 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%. 4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5.0% by weight or more than 5.0% by weight.

[0252] In some embodiments, the coupling reagent is a water-soluble carbodiimide. In some embodiments, the coupling reagent is a water-soluble triazole. In some embodiments, the coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some embodiments, the coupling reagent is a water-soluble polymer at about 0.5-5% by weight of the total formulation concentration. In some embodiments, the water-soluble polymer is at about less than 0.1% by weight, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6. 4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5.0% by weight or more than 5.0% by weight.

[0253] A linker molecule may be inserted between the surface disclosed herein and the peptide being synthesized via a coupling molecule. The linker molecule does not necessarily provide functionality, such as molecular recognition, to the resulting peptide, but it can extend the distance between the surface and the peptide to increase the exposure of the peptide's functional region on the surface. In some embodiments, the linker may be about 4 to about 40 atoms long to provide exposure. The linker molecule may be, for example, arylacetylene, ethylene glycol oligomer [PEG] containing 2 to 10 monomer units, diamine, diacid, amino acid, or combinations thereof. Examples of diamines include ethylenediamine and diaminopropane. Alternatively, the linker may be the same molecular type as the molecule being synthesized (e.g., the nascent polymer or various coupling molecules), for example, a polymer of polypeptide and amino acid derivatives, such as aminohexanoic acid. In some embodiments, the linker molecule is a molecule having a carboxyl group at one end of the molecule and a protecting group at the second end of the molecule. In some embodiments, the protecting group is a t-Boc protecting group or an F-Moc protecting group. In some embodiments, the linker molecule is or comprises an arylacetylene, polyethylene glycol, nascent polypeptide, diamine, diacid, peptide, or a combination thereof, hi some embodiments, the linker molecule is about 0.5-5% by weight of the total formulation concentration.In some embodiments, the linker molecule is less than about 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.1% by weight of the total formulation concentration. %, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or greater than 5.0 wt%.

[0254] The non-bonded portion of the linker molecule, or the free end of the linker molecule, may have a reactive functional group that is blocked, protected, or otherwise rendered unavailable for reaction by a removable protecting group, such as t-Boc or F-Moc, as described above. Protecting groups may be attached to the monomer, polymer, or linker molecule to protect the reactive functional group therein. Protecting groups that can be used include acid-labile and base-labile protecting groups. For example, peptide amine groups may be protected with t-butoxycarbonyl [t-BOC or BOC] or benzyloxycarbonyl [CBZ], both of which are acid-labile, or with 9-fluorenylmethoxycarbonyl [FMOC], which is base-labile.

[0255] Additional protecting groups that may be used include acid labile groups such as tert-amyloxycarbonyl, adamantyloxycarbonyl, 1-methylcyclobutyloxycarbonyl, 2-(p-biphenyl)propyl(2)oxycarbonyl, 2-(p-phenylazophenylyl)propyl(2)oxycarbonyl, α,α-dimethyl-3,5-dimethyloxybenzyloxycarbonyl, 2-phenylpropyl(2)oxycarbonyl, 4-methyloxybenzyloxycarbonyl, furfuryloxycarbonyl, triphenylmethyl(trityl), p-toluenesulfenylaminocarbonyl, dimethylphosphinothioyl, diphenylphosphinothioyl, 2-benzoyl-1-methylvinyl, o-nitrophenylsulfenyl, and 1-naphthylidene for protecting amino moieties; Group-labile groups include 9-fluorenylmethyloxycarbonyl, methylsulfonylethyloxycarbonyl, and 5-benzisoazoylmethyleneoxycarbonyl; groups for protecting amino moieties that are unstable when reduced: dithiasuccinoyl, p-toluenesulfonyl, and piperidino-oxycarbonyl; groups for protecting amino moieties that are unstable when oxidized: (ethylthio)carbonyl; groups for protecting amino moieties that are unstable to a wide variety of reagents, with the appropriate agent indicated in parentheses after the group: phthaloyl (hydrazine), trifluoroacetyl (piperidine), and chloroacetyl (2-aminothiophenol); acid-labile groups for protecting carboxylic acids: tert-butyl ester; and acid-labile groups for protecting hydroxyl groups: dimethyltrityl. (See also Greene, TW, Protective Groups in Organic Synthesis, Wiley-Interscience, NY, (1981)).

[0256] Coupling Compounds Also disclosed are coupling formulations. In some embodiments, the coupling formulations can include components such as a solvent, a water-soluble polymer, a water-soluble coupling molecule, a water-soluble neutralizing reagent, and a water-soluble coupling reagent.

[0257] In some embodiments, the solvent is water, an organic solvent, or a combination thereof. In some embodiments, the organic solvent is N-methylpyrrolidone, dimethylformamide, or a combination thereof.

[0258] In some embodiments, the polymer is water-soluble vinylpyrrolidone or water-soluble vinyl alcohol. In some embodiments, the polymer is 2.5-5% by weight of the total formulation. In some embodiments, the polymer is about 0.5-5% by weight of the total formulation. In some embodiments, the polymer is less than 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4% by weight of the total formulation. %, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or greater than 5.0 wt%.

[0259] In some embodiments, the neutralizing reagent may comprise Hunig's base, the structure of which is as follows: The file is TIFF2026035691000013.tif24128.

[0260] In some embodiments, the neutralizing reagent is 1-2% by weight of the total formulation concentration. In some embodiments, the neutralizing reagent is about 0.5-5% by weight of the total formulation concentration. In some embodiments, the neutralizing reagent is less than about 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or less by weight of the total formulation concentration. %, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or greater than 5.0 wt%.

[0261] Coupling molecules can include amino acids. In some embodiments, all of the peptides on the arrays described herein are composed of natural amino acids. In other cases, the peptides on the arrays described herein can be composed of a combination of natural and unnatural amino acids. In other cases, the peptides on the arrays can be composed exclusively of unnatural amino acids. Unnatural amino acids include peptidomimetics and D-amino acids. The R group can be one found in natural amino acids or can be a group similar in size to the R group of a natural amino acid. Additionally, unnatural amino acids such as β-alanine, phenylglycine, homoarginine, aminobutyric acid, aminohexanoic acid, aminoisobutyric acid, butylglycine, citrulline, cyclohexylalanine, diaminopropionic acid, hydroxyproline, norleucine, norvaline, ornithine, penicillamine, pyroglutamic acid, sarcosine, and thienylalanine can also be incorporated. These and other natural and unnatural amino acids are available, for example, from EMD Biosciences, Inc., San Diego, Calif. In some embodiments, the coupling molecule comprises a natural or artificial amino acid or polypeptide. Examples of coupling molecules include Boc-glycine-OH and Boc-histidine-OH. In some embodiments, the artificial amino acid is a D-amino acid. In some embodiments, the coupling molecule is 1-2% by weight of the total formulation concentration. In some embodiments, the coupling molecule is about 0.5-5% by weight of the total formulation concentration.In some embodiments, the coupling molecule is less than about 0.1 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, 5.1 wt%, 5.2 wt%, 5.3 wt%, 5.4 wt%, 5.5 wt%, 5.6 wt%, 5.7 wt%, 5.8 wt%, 5.9 wt%, 6.0 wt%, 6.1 wt%, 6.2 wt%, 6.3 wt%, 6.4 wt%, 6.5 wt%, 6.6 wt%, 6.7 wt%, 6.8 wt%, 6.9 wt%, 7.0 wt%, 7. The concentration of the coupling molecule is 4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5.0% by weight, or more than 5.0% by weight. In some embodiments, the coupling molecule comprises a protected group, for example, a group protected via t-Boc or F-Moc chemistry. In most cases, increasing the concentration of the coupling molecule provides the best performance.

[0262] In some embodiments, the coupling reagent is a water-soluble carbodiimide or a water-soluble triazole. In some embodiments, the coupling reagent is 2-4 wt% of the total formulation concentration. In some embodiments, the coupling reagent is about 0.5-5 wt% of the total formulation concentration. In some embodiments, the coupling reagent is less than 0.1 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 2.10 wt%, 2.11 wt%, 2.12 wt%, 2.13 wt%, 2.14 wt%, 2.15 wt%, 2.16 wt%, 2.17 wt%, 2.18 wt%, 2.19 ... 4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5.0% by weight or more than 5.0% by weight.

[0263] In any of the above combinations, the formulation can be completely stripped off with water.

[0264] substrate Substrates are also disclosed herein. In some embodiments, the substrate surface is planar (i.e., two-dimensional). In some embodiments, the substrate surface is functionalized with free carboxylic acid groups. In some embodiments, the substrate surface is functionalized with free amine groups. A surface functionalized with free amine groups can be converted to free carboxylic acid groups by activating and reacting the carboxylic acid groups of a molecule containing at least two free carboxylic acid groups (e.g., using a carbodiimide to convert the carboxylic acid groups to carbonyl groups) and reacting the molecule with the free amine groups bound to the surface of the substrate. In some embodiments, the molecule containing multiple carboxylic acid groups is succinic anhydride, polyethylene glycol diacid, benzene-1,3,5-tricarboxylic acid, benzenehexacarboxylic acid, or carboxymethyl dextran.

[0265] In some embodiments, the substrate can include a porous layer (i.e., a three-dimensional layer) that includes functional groups for binding with the first monomer building block. In some embodiments, the substrate surface includes pillars for peptide attachment or synthesis. In some embodiments, a porous layer is added on top of the pillars.

[0266] Pillar Substrate In some embodiments, the substrate can include a planar layer having an upper surface and a lower surface; and a plurality of pillars operably coupled to the layer at positionally defined locations, wherein each pillar has a planar surface extending from the layer, the distance between the surface of each pillar and the upper surface of the layer is about 1,000-5,000 angstroms, and the plurality of pillars has a density of approximately 10,000 / cm 2 An example of a substrate is shown in Figures 3B and 3C.

[0267] In some embodiments, the distance between the surface of each pillar and the top surface of the layer may be between approximately less than 1,000 angstroms, less than 2,000 angstroms, less than 3,000 angstroms, less than 3,500 angstroms, less than 4,500 angstroms, less than 5,000 angstroms, or more than 5,000 angstroms (or any integer value therebetween).

[0268] In some embodiments, the surface of each pillar is parallel to the top surface of the layer. In some embodiments, the surface of each pillar is substantially parallel to the top surface of the layer.

[0269] In some embodiments, the plurality of pillars is greater than 500 / cm 2 Super, 1,000 / cm 2 Super, 2,000 / cm 2 Super, 3,000 / cm 2 Super, 4,000 / cm 2 Super, 5,000 / cm 2 Super, 6,000 / cm 2 Super, 7,000 / cm 2 Super, 8,000 / cm 2 Super, 9,000 / cm 2 Super, 10,000 / cm 2 Super, 11,000 / cm 2 Over 12,000 / cm 2 In some embodiments, the plurality of pillars are present at a density of greater than 10,000 / cm (or any integer value therebetween). 2 In some embodiments, the plurality of pillars is present at a density of greater than about 10,000 / cm 2 ~About 2.5 million / cm 2 In some embodiments, the plurality of pillars are present at a density of 2.5 million / cm (or any integer value therebetween). 2 It exists at a density exceeding

[0270] In some embodiments, the surface area of ​​each pillar surface is at least 1 μm 2 In some embodiments, the surface area of ​​each pillar surface is at least 0.1 μm 2 , 0.5 μm2 , 12 μm 2 , 3 μm 2 , 4 μm 2 , 5 μm 2 , 6 μm 2 , 7 μm 2 , 8 μm 2 , 9 μm 2 , 10 μm 2 , 15 μm 2 , 20 μm 2 , 25 μm 2 , 30 μm 2 , 35 μm 2 , 40 μm 2 , 45 μm 2 , or 50 μm 2 (or any integer value therebetween). In some embodiments, the surface area of ​​each pillar surface is 10,000 μm 2 In some embodiments, the surface area of ​​each pillar surface is less than 500 μm 2 Less than 1,000 μm 2 Less than 2,000 μm 2 Less than 3,000 μm 2 Less than 4,000 μm 2 Less than 5,000 μm 2 Less than 6,000 μm 2 Less than 7,000 μm 2 Less than 8,000 μm 2 Less than 9,000 μm 2 Less than 10,000 μm 2 Less than 11,000 μm 2 Less than or equal to 12,000 μm 2 has a total area of ​​less than (or any integer value in between).

[0271] In some embodiments, the distance between the surface of each pillar and the lower surface of the layer is between 2,000 and 7,000 angstroms, hi some embodiments, the distance between the surface of each pillar and the lower surface of the layer is less than about 500 angstroms, less than 1,000 angstroms, less than 2,000 angstroms, less than 3,000 angstroms, less than 4,000 angstroms, less than 5,000 angstroms, less than 6,000 angstroms, less than 7,000 angstroms, less than 8,000 angstroms, less than 9,000 angstroms, less than 10,000 angstroms, less than 11,000 angstroms, less than 12,000 angstroms, or more than 12,000 angstroms (or any integer value therebetween). In some embodiments, the distance between the surface of each pillar and the underside of the layer is 7,000, 3,000, 4,000, 5,000, 6,000, or 7,000 angstroms (or any integer value therebetween).

[0272] In some embodiments, the layer is 1,000 to 2,000 angstroms thick, hi some embodiments, the layer is approximately less than 500 angstroms, less than 1,000 angstroms, less than 2,000 angstroms, less than 3,000 angstroms, less than 4,000 angstroms, less than 5,000 angstroms, less than 6,000 angstroms, less than 7,000 angstroms, less than 8,000 angstroms, less than 9,000 angstroms, less than 10,000 angstroms, less than 11,000 angstroms, less than 12,000 angstroms, or more than 12,000 angstroms thick (or any integer value therebetween).

[0273] In some embodiments, the center of each pillar is at least 2,000 angstroms from the center of any other pillar. In some embodiments, the center of each pillar is at least about 500 angstroms, 1,000 angstroms, 2,000 angstroms, 3,000 angstroms, or 4,000 angstroms (or any integer value therebetween) from the center of any other pillar. In some embodiments, the center of each pillar is at least about 2 μm to 200 μm from the center of any other pillar.

[0274] In some embodiments, the planar layer comprises a metal. In some embodiments, the metal is chromium. In some embodiments, the metal is chromium, titanium, aluminum, tungsten, gold, silver, tin, lead, thallium, indium, or a combination thereof. In some embodiments, the layer is at least 98.5-99% metal. In some embodiments, the layer is 100% metal. In some embodiments, the layer is at least approximately greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 98.5%, or greater than 99% metal. In some embodiments, the layer is a homogeneous layer of metal.

[0275] In some embodiments, the planar layer comprises silicon, silicon dioxide, silicon nitride, etc. In some embodiments, at least one or each pillar comprises silicon. In some embodiments, at least one or each pillar comprises silicon dioxide or silicon nitride. In some embodiments, at least one or each pillar is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, or 99% silicon dioxide.

[0276] In some embodiments, the substrate may comprise a linker molecule having a free amino terminus attached to the surface of each pillar. In some embodiments, the substrate may comprise a linker molecule having a free amino terminus attached to the surface of at least one pillar. In some embodiments, the substrate may comprise a linker molecule having a protecting group attached to the surface of each pillar. In some embodiments, the substrate may comprise a linker molecule having a protecting group attached to the surface of at least one pillar. In some embodiments, the substrate may comprise a coupling molecule attached to the surface of at least one pillar. In some embodiments, the substrate may comprise a coupling molecule attached to the surface of each pillar. In some embodiments, the substrate may comprise a water-soluble polymer in contact with the surface of at least one of the pillars. In some embodiments, the substrate may comprise a water-soluble polymer in gelatinous form in contact with the surface of at least one of the pillars. In some embodiments, the substrate may comprise a water-soluble polymer in solid form in contact with the surface of at least one of the pillars.

[0277] In some embodiments, at least one surface of the pillar substrate is derivatized. In some embodiments, the substrate can include a polymer chain attached to the surface of at least one of the pillars. In some embodiments, the polymer chain includes a peptide chain. In some embodiments, the attachment to the surface of the at least one pillar is via a covalent bond.

[0278] In some embodiments, the surface of each pillar is square or rectangular in shape. In some embodiments, the substrate can be coupled to a silicon dioxide layer. The silicon dioxide layer can be about 0.5 μm to 3 μm thick. In some embodiments, the substrate can be coupled to a wafer, e.g., a silicon wafer. The silicon dioxide layer can be about 700 μm to 750 μm thick.

[0279] In some embodiments, the substrate can include a porous layer that includes functional groups for binding with the first monomer component.

[0280] Porous layer substrate Porous layers that can be used are permeable polymeric materials with a porous structure that can have functional groups (either native to the constituent polymer or introduced into the porous layer) for attachment of the first peptide component. The functional groups can include free carboxylic acid groups or free amino groups. For example, the porous layer can be composed of porous silicon with functional groups attached to its surface for attachment of the polymer component. In another example, the porous layer can include a cross-linked polymeric material. In some embodiments, the porous layer can be made of polystyrene, sucrose, dextran, polyacryloylmorpholine, polyacrylate, polymethylacrylate, polyacrylamide, polyacrylolpyrrolidone, polyvinyl acetate, polyethylene glycol, agarose, Sepharose, other traditional chromatography-type materials, and derivatives and mixtures thereof. In some embodiments, the material constituting the porous layer is selected from poly(vinyl alcohol), dextran, sodium alginate, poly(aspartic acid), poly(ethylene glycol), poly(ethylene oxide), poly(vinylpyrrolidone), poly(acrylic acid), poly(acrylic acid)-sodium salt, poly(acrylamide), poly(N-isopropylacrylamide), poly(hydroxyethyl acrylate), poly(acrylic acid), poly(sodium styrenesulfonate), poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polysaccharides, and cellulose derivatives. Preferably, the porous layer has a porosity of 10 to 80%. In one embodiment, the thickness of the porous layer ranges from 0.01 μm to approximately 1,000 μm. The pore size contained in the porous layer can range from 2 nm to approximately 100 μm.

[0281] According to another aspect of the present invention, there is provided a substrate comprising a porous polymeric material having a porosity of 10-80%, wherein reactive groups are chemically bonded to the pore surface and adapted for use in interaction with a reactive species, such as a deprotected monomer building block or polymer chain, e.g., by chemical bonding. In one embodiment, the reactive group is a carboxylic acid group. The carboxylic acid group is free to bond with, for example, an unprotected amine group of a peptide or polypeptide. In another embodiment, the reactive group is an amino group, free to bond with, for example, an unprotected carboxylic acid group of a peptide or polypeptide.

[0282] In one embodiment, the porous layer is in contact with a support layer. The support layer may comprise, for example, metal, plastic, silicon, silicon oxide, or silicon nitride. In another embodiment, the porous layer may be in contact with a patterned surface, such as the top surface of the pillar substrate described above.

[0283] array Arrays are also disclosed herein. In some embodiments, the arrays can be two-dimensional arrays. In some embodiments, the two-dimensional arrays can include features attached to a surface at positionally defined locations, each of which includes a collection of peptide chains of a determinable sequence and an intended length, and in each feature, the percentage of peptide chains in the collection that have the intended length is characterized by an average coupling efficiency at each coupling step of greater than about 98%.

[0284] In some embodiments, the surface of the array is functionalized with free carboxylic acids. In some embodiments, the free carboxylic acids are activated to bind to amine groups, e.g., during polypeptide synthesis on the array surface. In some embodiments, the surface density of free carboxylic acid groups on the array is greater than 10 / cm 2 , 100 / cm 2 , 1,000 / cm 2 , 10,000 / cm2 , 100,000 / cm 2 , 1,000,000 / cm 2 , or 10,000,000 / cm 2 Exceeds.

[0285] In some embodiments, the array may be a three-dimensional array, for example, a porous array with features attached to the surface of the porous array. In some embodiments, the surface of the porous array includes an outer surface and a surface that defines a pore volume within the porous array. In some embodiments, the three-dimensional array may include features attached to the surface at positionally defined locations, each of which includes an assembly of peptide chains of a determinable sequence and an intended length. In one embodiment, the proportion of peptide chains in the assembly that have the intended length in each feature is characterized by an average coupling efficiency at each coupling step of greater than about 98%.

[0286] In some embodiments, the average coupling efficiency at each coupling step is at least 98.5%. In some embodiments, the average coupling efficiency at each coupling step is at least 99%. In some embodiments, the average coupling efficiency at each coupling step is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%. In some embodiments, the coupling efficiency is substantially constant and greater than 98% in each coupling cycle. In some embodiments, the average coupling efficiency is greater than 98% for each coupling step used to synthesize a 4-mer, 5-mer, 6-mer, 7-mer, or longer polypeptide. In some embodiments, coupling efficiency is substantially constant and greater than 98% for each coupling step used to synthesize a 4-mer or 5-mer or 6-mer or 7-mer or longer polypeptide.

[0287] In some embodiments, the surface comprises a substrate as disclosed herein. In some embodiments, the surface is a rigid or semi-rigid material or materials. In some embodiments, the surface can be substantially planar, although in some embodiments, it may be desirable to substantially separate synthesis regions for different molecules or features, for example, by wells, raised areas, pins, pillars, etched grooves, etc. In certain embodiments, the surface can be porous. Surface materials can include, for example, silicon, biocompatible polymers such as poly(methyl methacrylate) [PMMA] and polydimethylsiloxane [PDMS], glass, SiO2 (e.g., thermally oxidized silicon wafers such as those used in the semiconductor industry), quartz, silicon nitride, functionalized glass, gold, platinum, and aluminum. Functionalized surfaces include, for example, amino-functionalized glass, carboxy-functionalized glass, and hydroxy-functionalized glass. In addition, the surface can optionally be coated with one or more layers to provide a second surface for molecular attachment or functionalization, increased or decreased reactivity, binding detection, or other special applications. The surface material and / or layer can be porous or non-porous. For example, the surface may be composed of porous silicon. Additionally, the surface may be a silicon wafer or chip, such as those used in semiconductor device manufacturing. In the case of a wafer or chip, multiple arrays can be synthesized on the wafer.

[0288] In some embodiments, each peptide chain is 5-60 amino acids in length. In some embodiments, each peptide chain is at least 5 amino acids in length. In some embodiments, each peptide chain is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length. In some embodiments, each peptide chain is less than 5 amino acids, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 amino acids, or more than 60 amino acids in length. In some embodiments, each peptide chain comprises one or more L-amino acids. In some embodiments, each peptide chain comprises one or more D-amino acids. In some embodiments, each peptide chain comprises one or more naturally occurring amino acids. In some embodiments, each peptide chain comprises one or more synthetic amino acids.

[0289] In some embodiments, the array can comprise at least 1,000 different peptide chains attached to a surface. In some embodiments, the array can comprise at least 10,000 different peptide chains attached to a surface. In some embodiments, the array can comprise at least 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, or more than 10,000 (or any integer value in between) different peptide chains attached to a surface.

[0290] In some embodiments, the array has a surface area of ​​1 cm 2 In some embodiments, the array can comprise a peptide density of at least 1,000 peptide chains attached per cm. In some embodiments, the array can comprise at least 10,000 peptide chains / cm. 2In some embodiments, the array comprises at least 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 1000, or more than 10,000 peptide chains / cm 2 (or any integer in between).

[0291] In some embodiments, each of the positionally defined locations is a different known location that is physically separate from each of the other positionally defined locations. In some embodiments, each of the positionally defined locations is a positionally identifiable location. In some embodiments, each determinable sequence is a known sequence. In some embodiments, each determinable sequence is a unique sequence.

[0292] In some embodiments, the features are covalently attached to the surface, hi some embodiments, the peptide chains are attached to the surface via linker or coupling molecules.

[0293] In some embodiments, a feature comprises a plurality of distinct, nested, overlapping peptide chains comprising subsequences from a source protein having a known sequence. In some embodiments, each peptide chain in the plurality is substantially the same length. In some embodiments, each peptide chain in the plurality is the same length. In some embodiments, each peptide chain in the plurality is at least 5 amino acids in length. In some embodiments, each peptide chain in the plurality is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length. In some embodiments, each peptide chain of the plurality is less than 5 amino acids, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 amino acids, or more than 60 amino acids in length. In some embodiments, at least one peptide chain of the plurality is at least 5 amino acids in length. In some embodiments, at least one peptide chain of the plurality is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length. In some embodiments, at least one peptide chain of the plurality is less than 5 amino acids, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or more than 60 amino acids in length. In some embodiments, each polypeptide in a feature is substantially the same length. In some embodiments, each polypeptide in a feature is the same length. In some embodiments, a feature comprises multiple peptide chains, each having a random, determinable amino acid sequence.

[0294] Methods for fabricating arrays Also disclosed herein is a method for fabricating an array. In some embodiments, the array disclosed herein can be synthesized in situ on a surface, for example, on a substrate disclosed herein. In some cases, the array is fabricated using photolithography. For example, a mask can be used to control the irradiation or exposure of specific locations on a surface on which a linker molecule with a protecting group is prepared. In the exposed locations, the protecting group is removed, newly exposing one or more reactive moieties on the linker. The surface is then contacted with a solution containing a coupling molecule. The coupling molecule can have at least one site that reacts with the newly exposed reactive moiety on the linker and at least one second reactive site that is protected by one or more protecting groups. The desired coupling molecule is then coupled to the unprotected linker molecule. This process can be repeated to synthesize multiple features at specific or topologically defined locations on a surface (see, e.g., U.S. Pat. No. 5,143,854 to Pirrung et al.; U.S. Patent Application Publication Nos. 2007 / 0154946 (filed December 29, 2005), 2007 / 0122841 (filed November 30, 2005), 2007 / 0122842 (filed March 30, 2006), 2008 / 0108149 (filed October 23, 2006), and 2010 / 0093554 (filed June 2, 2008), each of which is incorporated herein by reference).

[0295] In some embodiments, a method for producing a two-dimensional array of features can include the steps of obtaining a surface; and attaching features to the surface, each feature comprising a collection of peptide chains of determinable sequence and intended length, wherein the proportion of peptide chains in the population having the intended length in each individual feature is characterized by an average coupling efficiency of at least about 98% at each coupling step. In some embodiments, the features are attached to the surface using a coupling formulation comprising a solvent, a water-soluble polymer, a water-soluble coupling molecule, a water-soluble neutralizing reagent, and a water-soluble coupling reagent. In some embodiments, the features are attached to the surface using a coupling formulation disclosed herein. In some embodiments, the coupling formulation is stripped off using water.

[0296] In some embodiments, a method for fabricating a two-dimensional array of features can include the steps of: obtaining a substrate comprising a planar layer comprising a metal and having an upper surface and a lower surface; and a plurality of pillars operably coupled to the layer at positionally defined locations, each pillar having a planar surface extending from the layer, the distance between a surface of each pillar and a top surface of the layer being between about 1,000 and 5,000 angstroms, and the plurality of pillars having a density of about 10,000 / cm 2and coupling features to a plurality of pillars through a series of coupling reactions, each of the features comprising an assembly of peptide chains of determinable sequence and intended length, wherein the proportion of peptide chains within the assembly having the intended length for each feature is characterized by an average coupling efficiency of at least about 98% or about 98.5% for each coupling step. In some embodiments, the coupling efficiency is substantially constant for each coupling cycle and is greater than 98% or greater than 98.5%. In some embodiments, the average coupling efficiency is greater than 98% or greater than 98.5% for each coupling step used to synthesize a 4-mer, 5-mer, 6-mer, 7-mer, or longer polypeptide. In some embodiments, the coupling efficiency is substantially constant and is greater than 98% or greater than 98.5% for each coupling step used to synthesize a 4-mer, 5-mer, 6-mer, 7-mer, or longer polypeptide. The coupling steps are used for synthesis. In some embodiments, the features are coupled to the pillars using a coupling formulation comprising a solvent, a water-soluble polymer, a water-soluble coupling molecule, a water-soluble neutralizing reagent, and a water-soluble coupling reagent. In some embodiments, the features are coupled using a coupling formulation disclosed herein. In some embodiments, the coupling formulation is stripped off using water. In some embodiments, the surface of each pillar is parallel to the top surface of the layer. In some embodiments, the surface of each pillar is substantially parallel to the top surface of the layer.

[0297] In some embodiments, a method of preparing a substrate for attachment of features includes obtaining a substrate comprising a planar layer comprising a metal and having an upper surface and a lower surface, and a plurality of pillars operably coupled to the layer at positionally defined locations, each pillar having a planar surface extending from the layer, the distance between a surface of each pillar and the upper surface of the layer being between about 1,000 and 5,000 angstroms, and the plurality of pillars having a density of about 10,000 / cm 2 and attaching one or more linker molecules to the plurality of pillars. In some embodiments, the linker molecules are attached using a linker formulation comprising a solvent, a water-soluble polymer, a water-soluble linker molecule, and a water-soluble coupling reagent. In some embodiments, the linker molecules are attached using a linker formulation disclosed herein. In some embodiments, the linker molecule comprises a protecting group. In some embodiments, the surface of each pillar is parallel to the top surface of the layer. In some embodiments, the surface of each pillar is substantially parallel to the top surface of the layer.

[0298] In some embodiments, a method of preparing a surface for attaching features can include obtaining a surface and attaching a linker molecule to the surface using a linker formulation comprising a solvent, a water-soluble polymer, a water-soluble linker molecule, and a water-soluble coupling reagent. In some embodiments, the linker molecule comprises a protecting group.

[0299] In some embodiments, a method of attaching a coupling reagent to a substrate comprises obtaining a substrate comprising a planar layer comprising a metal and having an upper surface and a lower surface, and a plurality of pillars operably coupled to the layer at positionally defined locations, each pillar having a planar surface extending from the layer, the distance between the surface of each pillar and the upper surface of the layer being between 1,000 and 5,000 angstroms, a linker molecule attached to the surface of each pillar, and the plurality of pillars having a density of 10,000 / cm 2and attaching the coupling reagent to one or more linker molecules. In some embodiments, the coupling reagent is attached to one or more linker molecules using a coupling formulation comprising a solvent, a water-soluble polymer, a water-soluble coupling molecule, a water-soluble neutralizing reagent, and a water-soluble coupling reagent. In some embodiments, the coupling reagent is attached to one or more linker molecules using a coupling formulation disclosed herein. In some embodiments, at least one linker molecule is a deprotected linker molecule. In some embodiments, the coupling reagent is an amino acid. In some embodiments, the coupling reagent comprises a protecting molecule. In some embodiments, the coupling formulation is stripped off using water. In some embodiments, the surface of each pillar is parallel to the top surface of the layer. In some embodiments, the surface of each pillar is substantially parallel to the top surface of the layer.

[0300] In some embodiments, a method for attaching a coupling reagent to a surface can include obtaining a surface having a linker molecule attached thereto, and attaching the coupling reagent to the linker molecule using a coupling formulation comprising a solvent, a water-soluble polymer, a water-soluble coupling molecule, a water-soluble neutralizing reagent, and a water-soluble coupling reagent. In some embodiments, the linker molecule is a deprotected linker molecule. In some embodiments, the coupling reagent is an amino acid. In some embodiments, the coupling reagent comprises a protecting molecule. In some embodiments, the coupling formulation is stripped off using water.

[0301] In some embodiments, a method for producing a three-dimensional (e.g., porous) array of features can include the steps of: obtaining a porous layer attached to a surface; and attaching features to the porous layer, each of the features comprising an assembly of peptide chains of determinable sequence and intended length, wherein the proportion of peptide chains in the assembly having the intended length in each individual feature is characterized by an average coupling efficiency of at least about 98.5% at each coupling step. In some embodiments, the features are attached to the surface using a photoactive coupling formulation comprising a photoactive compound, a coupling molecule, a coupling reagent, a polymer, and a solvent. In some embodiments, the features are attached to the surface using a photoactive coupling formulation disclosed herein. In some embodiments, the photoactive coupling formulation is peeled off using water.

[0302] In some embodiments, a process for fabricating an array is described herein. A surface containing attached carboxylic acid groups is provided. The surface is contacted with a photoactive coupling solution containing a photoactive compound, a coupling molecule, a coupling reagent, a polymer, and a solvent. The surface is exposed to ultraviolet light in a deep ultraviolet scanner tool according to a pattern defined by a photomask, and the locations exposed to the ultraviolet light undergo photobase generation due to the presence of a photobase generator in the photoactive coupling solution. To generate sufficient photobase, the exposure energy is 1 mJ / cm. 2 ~100mJ / cm 2 It may be.

[0303] After exposure, the surface is post-baked in a post-exposure bake module. The post-exposure bake serves as a chemical amplification step. The bake step amplifies the initially generated photobase and also increases its rate of diffusion into the substrate. The post-baking temperature, which is at least 60 seconds but typically no more than 120 seconds, can vary from 75°C to 115°C depending on the thickness of the porous surface. The free carboxylic acid group is coupled to the deprotected amine group of the free peptide or polypeptide, resulting in coupling of the free peptide or polypeptide to the surface-attached carboxylic acid group. The surface can be a porous surface. Synthesis of the peptide coupled to the surface-attached carboxylic acid group occurs in an N→C synthesis direction, with the amine group of the free peptide bonding to the surface-attached carboxylic acid group of the substrate. Alternatively, a diamine linker can be attached to the free carboxylic acid group to direct synthesis in a C→N direction, in which case the carboxylic acid group of the free peptide bonds to the surface-attached amine group of the substrate.

[0304] The photoactive coupling solution can now be stripped off. In some embodiments, a method for completely stripping the photoresist with deionized (DI) water is provided herein. This process is accomplished in a development module. The wafer is spun on a vacuum chuck for, for example, 60 to 90 seconds, and deionized water is dispensed through a nozzle for approximately 30 seconds.

[0305] The photoactive coupling formulation can be applied to the surface in a coupling rotation module. The coupling rotation module can typically have 20 or more nozzles that dispense the photoactive coupling formulation. These nozzles can be configured to dispense the photoactive coupling formulation by pressurizing cylinders holding these solutions or by a pump that dispenses the required amount. In some embodiments, a pump is employed to dispense 5-8 cc of the photoactive coupling formulation onto the substrate. The substrate is spun on a vacuum chuck for 15-30 seconds, and the photoactive coupling formulation is dispensed. The rotation speed can be set at 2000-2500 rpm.

[0306] Optionally, a cap film solution coat is applied to the surface to prevent unreacted amino groups on the substrate from reacting with adjacent coupling molecules. The cap film coat solution can be prepared with a solvent, a polymer, and a coupling molecule as follows: The solvent can be an organic solvent such as N-methylpyrrolidone, dimethylformamide, or a combination thereof. The capping molecule is typically acetic anhydride, and the polymer can be polyvinylpyrrolidone, polyvinyl alcohol, polymethyl methacrylate, poly(methylisopropenyl)ketone, or poly(2-methylpentene-1-sulfone). In some embodiments, the capping molecule is ethanolamine.

[0307] This process is carried out in a capping spin module. The capping spin module can include one nozzle that can be configured to dispense the cap film coat solution onto the substrate. This solution can be dispensed by pressurizing a cylinder that stores the cap film coat solution or by a pump that dispenses the exact amount needed. In some embodiments, a pump is used to dispense approximately 5-8 cc of cap coat solution onto the substrate. The substrate is spun on a vacuum chuck for 15-30 seconds, and the coupling formulation is dispensed. The spin speed can be set to 2000-2500 rpm.

[0308] The substrate with capping solution is baked in cap baking module.Capping baking module is a hot plate specially assembled to receive wafer immediately after applying capping film coat.In some embodiments, the present invention provides a method of spin-coating capping coat solution on hot plate baking, so as to significantly accelerate capping reaction.Hot plate baking generally reduces the capping time for amino acid to less than 2 minutes.

[0309] The by-products of the capping reaction are stripped in a stripping module. The stripping module can include several nozzles, typically up to 10, assembled to dispense organic solvents such as acetone, isopropyl alcohol, N-methylpyrrolidone, dimethylformamide, and DI water. In some embodiments, the nozzles can be designated for acetone, followed by isopropyl alcohol, to be dispensed onto the spinning wafer. The spinning speed is set to 2000-2500 rpm for approximately 20 seconds.

[0310] This entire cycle can be repeated as desired, each time with a different coupling molecule, to obtain the desired sequence.

[0311] In some embodiments, polypeptides are synthesized in the N→C direction using an array containing a surface of free carboxylic acids. In one embodiment, the carboxylic acids on the surface of the substrate are activated (e.g., converted to carbonyls) to couple them to free amine groups on amino acids. In one embodiment, activation of the carboxylic acids on the surface groups can be performed by adding a solution containing carbodiimide or succinimide to the surface of the array. In some embodiments, carboxylic acids can be activated by adding a solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [EDC], N-hydroxysuccinimide [NHS], 1,3-diisopropylcarbodiimide [DIC], hydroxybenzotriazole [HOBt], 1-hydroxy-7-azabenzotriazole [HOAt], (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) [HATU], benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate [PyBOP], or N,N-diisopropylethylamine [DIEA] to the surface of the array. The activation solution is washed away, and the surface of the array is prepared for the addition of an amino acid layer (i.e., one amino acid for each activated carboxylic acid group). The carboxylic acid groups remain activated for up to 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours.

[0312] Addition of a solution containing amino acids with free amine groups to the activated carboxylic acid surface of the array results in the attachment of a single amino acid to each carboxylic acid group. In some embodiments, the amino acids include amino acids with protected amine groups. Using a photosensitive chemical reaction, a reticle can be used to remove protecting groups from the amine groups of selected amino acids at site-specific locations. For example, Fmoc-protected amino acids are mixed in a solution containing a photobase. When the solution on the array is exposed to light of a specific frequency at site-specific locations, the photobase releases a base that deprotects the amino acid, resulting in coupling of the amino acid to the activated carboxylic acid groups on the surface of the array. Another method of generating a base involves using a protected base that is subsequently unprotected by a photoacid released by a photoacid generator when exposed to light. In some embodiments, the protected base is N-Boc-piperidine or 1,4-bis(N-Boc)-piperazine.

[0313] After the completed amino acid layer is coupled, any remaining uncoupled activated carboxylic acids are capped to prevent nonspecific binding of amino acids in subsequent synthesis steps. The steps of activation, addition of amino acid layers, and capping are repeated as necessary to synthesize the desired polypeptide at a specific location on the array.

[0314] In some embodiments, peptides synthesized in the N→C direction may be capped with a diamine molecule to enhance the binding properties of the selected polypeptide sequence to a biological molecule, e.g., an antibody. In other embodiments, peptides synthesized in the C→N direction may be capped with a dicarboxylic acid molecule to enhance the binding properties of the selected sequence to a biological molecule.

[0315] During parallel synthesis of polypeptides on the array surface, the methods described herein ensure complete activation of carboxylic acids present on the array surface. Due to the long-term stability of activated esters, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more coupling cycles may be completed after a single activation step (e.g., to couple an entire layer of 2-25 or more different amino acids at different locations on the array). Because coupling occurs during hard baking (heating on a hot plate at 85-90°C for 90 seconds immediately after coating) and there is excess amino acid in solution, complete 100% deprotection of Fmoc-protected amino acids may not be required to achieve extremely high coupling yields. After addition and capping of all amino acids, all free activated carboxylic acids are coupled or capped, thus achieving high efficiency and accuracy of polypeptide synthesis.

[0316] How to use arrays Also disclosed herein are methods of using the substrates, formulations, and / or arrays. Uses of the arrays disclosed herein can include research applications, therapeutic purposes, medical diagnostics, and / or stratification of one or more patients.

[0317] Any of the arrays described herein can be used as a research tool or for research purposes. In one aspect, the array can be used for high-throughput screening assays. For example, an enzyme substrate (i.e., a peptide on a peptide array described herein) can be tested by subjecting the array to an enzyme and identifying the presence or absence of the enzyme substrate on the array, for example, by detecting at least one change in the features of the array.

[0318] Arrays can also be used in screening assays for ligand binding to ascertain substrate specificity, or to identify peptides that inhibit or activate proteins. Labeling techniques, protease assays, and binding assays useful for carrying out these methods are generally well known to those of skill in the art.

[0319] In some embodiments, arrays can be used to present known protein sequences as overlapping peptide sequences. For example, the amino acid sequence of a known protein can be divided into overlapping sequence segments of any length and any suitable overlapping frame, and peptides corresponding to each sequence segment can be synthesized in situ as disclosed herein. The individual peptide segments thus synthesized can be arranged starting from the amino terminus of the known protein.

[0320] In some embodiments, the array is used in a method in which the antigen presentation of the array comprises at least one region in which the entire antigen sequence of a known protein is covered by epitope sliding.The immunoreactive region of the antigen is determined by contacting one or more clinical samples on the array or a plurality of different arrays, and the set of peptide sequences required to present the known protein antigen is reduced.

[0321] In some embodiments, sample is applied to the array with a plurality of random peptides.Can be screened and BLAST searched for random peptides to determine the homologous domain with predetermined antigen sequence, for example, have 90% or more identity.Then, in some aspects, can synthesize and use the whole antigen sequence to identify the potential marker and / or cause of the disease of interest.

[0322] In some embodiments, the array is used for high-throughput screening of one or more genetic factors. Proteins associated with genes can be potential antigens, and antibodies against these proteins can be used to infer the relationship between genes and diseases.

[0323] In another example, the array can be used to identify one or more biomarkers. Biomarkers can be used in the diagnosis, prognosis, treatment, and management of diseases. Biomarkers can be expressed, absent, or at different levels in an individual depending on the disease state, disease stage, and response to disease treatment. Biomarkers can be, for example, DNA, RNA, proteins (e.g., enzymes such as kinases), sugars, salts, fats, lipids, or ions.

[0324] Arrays can also be used for therapeutic purposes, such as identifying one or more bioactive substances. A method for identifying a bioactive substance can include applying multiple test compounds to the array and identifying at least one test compound as a bioactive substance. The test compound can be a small molecule, an aptamer, an oligonucleotide, a chemical, a natural extract, a peptide, a protein, an antibody fragment, an antibody-like molecule, or an antibody. The bioactive substance can also be a therapeutic agent or a modulator of a therapeutic target. Therapeutic targets can include phosphatases, proteases, ligases, signaling molecules, transcription factors, protein transporters, protein sorters, cell surface receptors, secreted factors, and cytoskeletal proteins.

[0325] In another aspect, the array can be used to identify therapeutic drug candidates. For example, if one or more epitopes for a specific antibody are determined by an assay (e.g., a binding assay such as ELISA), those epitopes can be used to develop drugs (e.g., monoclonal neutralizing antibodies) against the target antibody in a disease.

[0326] In one aspect, an array for use in medical diagnosis is also provided.The array can be used to determine the response to the administration of a drug or a vaccine.For example, the response of an individual to a vaccine can be determined by detecting the antibody level of the individual using an array with peptides that present the epitopes recognized by the antibodies produced by the induced immune response.Another diagnostic use is to test an individual for the presence of biomarkers.Here, a sample is taken from a subject and tested for the presence of one or more biomarkers.

[0327] Arrays can also be used to stratify patient populations based on the presence or absence of biomarkers that indicate the likelihood that a subject will respond to a therapeutic treatment. Arrays can be used to identify known biomarkers to determine appropriate treatment groups. For example, a sample from a subject with a certain condition can be applied to the array. Binding to the array can indicate the presence of a biomarker for that condition. Previous studies may show that biomarkers are associated with positive outcomes after treatment, while the absence of a biomarker is associated with negative or neutral outcomes after treatment. Because a patient has a biomarker, the patient can also be stratified by a medical professional into treatment groups.

[0328] In some embodiments, a method for detecting the presence or absence of a protein of interest (e.g., an antibody) in a sample can include obtaining an array disclosed herein and contacting it with a sample that may contain the protein of interest; and determining whether the protein of interest is present in the sample by detecting the presence or absence of binding to one or more features of the array. In some embodiments, the protein of interest can be obtained from a body fluid, such as amniotic fluid, aqueous humor, vitreous humor, bile, serum, breast milk, cerebrospinal fluid, earwax, chyle, endolymph, perilymph, feces, female vaginal fluid, gastric acid, gastric juice, lymph, mucus, ascites, pleural fluid, pus, saliva, sebum, semen, sweat, synovial fluid, tears, vaginal secretions, vomit, or urine.

[0329] In some embodiments, a method for identifying a vaccine candidate can include obtaining an array as disclosed herein in contact with a sample obtained from a subject previously administered the vaccine candidate, the sample comprising a plurality of antibodies; and determining the binding specificity of the plurality of antibodies to one or more features of the array. In some embodiments, the features comprise a plurality of distinct, nested, overlapping peptide chains comprising subsequences derived from a source protein having a known sequence. [Example]

[0330] The following examples illustrate methods for identifying biomarkers for celiac disease. The biomarkers include a set of peptides derived from known antigens in celiac disease, including, but not limited to, α-, β-, γ-, and ω-gliadins, tissue transglutaminase (tTG), and deamidated versions thereof. The method involves synthesizing a peptide library of 12-mer peptides based on these known celiac antigens. In some embodiments, the sequences of the 12-mer peptides were identified by shifting the amino acid sequence of the known celiac antigen by either two or three amino acids at a time. Figure 1A illustrates the identification of 12-mer sequences by shifting two amino acids along the α / β-gliadin sequence. Figure 1B illustrates the deamination of one or two glutamines at a time in a 12-mer peptide to increase the size of the peptide library. The peptide library was then synthesized on a microarray, as described in more detail below, and was found to be significantly superior to other conventional peptide synthesis approaches. Coupling yields during synthesis on the array were continuously monitored for peptide yield, purity, and sequence fidelity using fluorescence, mass spectrometry, and monoclonal antibody-binding substrate assays.To identify B-cell epitope-based biomarkers for native and deamidated gliadin-derived peptides (GPs), peptide microarrays containing 2.1 million different peptides from a peptide library of GPs, including triplicate repeats of each peptide, were synthesized, extracted, and placed on top of a 96-pillar plate.

[0331] Example 1: Preparation of wafer substrate High-grade 300 mm silicon wafers with p-type boron, (1,0,0) orientation, 1–5 Ohm / cm, and 725 μm thickness were obtained from Process Specialties. A 1000 Å thermal oxide was deposited on the wafers by dry oxidation at 1000 °C for 2 hours in a furnace under a pure oxygen atmosphere. Commercially available photoresist P5107 was spin-coated onto the wafers using a Sokudo RF3S Coat / Develop Track at 2000 rpm for 40 seconds. The wafers were exposed to light at 248 nm using an inverse zero layer mask with a Nikon NSR S205 KrF Scanner. This was followed by a 90-second post-exposure bake at 110 °C, followed by development using developer NMD-3 at 2.38% (TOK America). Oxide etching was performed by wet oxide etching of the wafer using buffered hydrofluoric acid, prepared by mixing 5 parts 40% by weight ammonium fluoride (Sigma) with 1 part 49% by weight hydrofluoric acid (Sigma) for 1 minute. The wafer was then stripped using a Nanostrip (CyanTek) for 24 hours and finally rinsed in DI water and sonicated for 10 minutes in DI water. This process, illustrated in Figure 3A, resulted in a substrate with feature regions measuring 1000 Å in height, containing thermal oxide, and non-feature regions containing silicon.

[0332] A DI 5000 AFM system was used to measure the substrate roughness and calculate the density. Figure 3B shows the pillars and their dimensions formed after the above process and shown in Figure 3A. Figure 3C shows the root-mean-square (RMS) roughness of the substrate. The density of the substrate was calculated to be approximately 100-150 pM.

[0333] Example 2: Derivatization of the wafer surface The wafer was thoroughly rinsed with deionized water for 5 minutes and spin-coated with a solution containing 1.25% (v / v) 3-aminopropyltriethoxysilane [APTES] (Sigma-Aldrich) in N-methylpyrrolidone [NMP] (BDH) at room temperature for 15 minutes. The wafer was cured at 120°C for 60 minutes under a N2 atmosphere. The wafer was then spin-coated with a coupling solution containing 2% by weight Fmoc-Gly-OH (Anaspec), 2% by weight HOBt (Anaspec), and 2% by weight N,N'-diisopropylcarbodiimide [DIC] (Sigma-Aldrich) in NMP and baked at 60°C for 5 minutes. This allowed for coupling of Fmoc-glycine with the free amines present in the APTES. The wafer was then rinsed with NMP and subsequently capped with 50% (v / v) acetic anhydride mixed with 50% NMP to cap any remaining uncoupled free amines. The wafer was stripped with acetone (BDH) and isopropyl alcohol [IPA] (BDH). The Fmoc protection of glycine was removed by spin-coating the wafer with 5% (v / v) piperidine (Sigma-Aldrich) in NMP and baking at 80°C for 300 seconds. The linker Fmoc-(PEG)4-COOH (Anaspec) was then coupled to the wafer surface using a coupling solution containing 2% (wt) linker, 2% (wt) HOBt (Anaspec), and 2% (wt) N,N'-diisopropylcarbodiimide [DIC] in NMP, followed by baking at 90°C for 120 seconds. The wafer was then rinsed with NMP and subsequently capped with 50% (v / v) acetic anhydride mixed with 50% NMP to cap any remaining uncoupled free amines. To complete the surface derivatization process, the wafer was stripped with acetone and IPA.

[0334] Example 3: Peptide array synthesis The steps taken to synthesize peptides on the array are illustrated in Figure 4 and described in detail above.

[0335] Activation solution: The amino acid activation solution was prepared as follows: 1 wt% poly(methyl methacrylate) [PMMA] (Polysciences) was dissolved in N-methylpyrrolidone by ultrasonication for 10 minutes. 2 wt% Fmoc-amino acid (Anaspec) was then added to the solution, followed by 2 wt% HOBt (Anaspec). 1 wt% tetrazolethione was then added to the solution. The solution was then filtered using a 0.05 μm filter.

[0336] Mechanism of carbodiimide formation: Coupling of photoactivated carbodiimides is as follows: I went to TIFF2026035691000014.tif28128.

[0337] Tetrazolethione was used, which, upon exposure to 248 nm, undergoes a ring-opening mechanism to release a carbodiimide and activate the carboxylic acid group of the amino acid coupled to the wafer. The addition of HOBt or HOAt resulted in the formation of -OBt or -OAt esters. Thus, tetrazolethione at 248 nm was used to photoactivate the amino acid to form a stable ester for efficient coupling.

[0338] Amino acid coupling: A base resist solution containing 1 wt% polymer and 3 wt% piperidine dissolved in NMP was spin-coated onto the wafer at 3000 rpm for 30 seconds and soft-baked on a hotplate at 65°C for 1 minute. The wafer was then baked at 80°C for 300 seconds. Fmoc protection was removed from all features, leaving unprotected amine groups. The incoming amino acid activation solution was spin-coated onto the wafer at 3000 rpm for 30 seconds and soft-baked on a hotplate at 65°C for 1 minute. The wafer was then exposed to the incoming amino acid at a dose of 120 mJ / cm.2 The wafer was exposed to light using a reticle that exposed the desired features to be coupled, followed by a soft bake at 85°C for 90 seconds on a hot plate. As described above, the tetrazolethione releases carbodiimide upon exposure, achieving selective activation of amino acids in the exposed features. Therefore, the input Fmoc-protected amino acids present in the activation solution are activated and coupled to unprotected amines present on the wafer in the same step as the coupling of one layer of amino acids is completed. Each coupling layer contains a reticle for each input Fmoc amino acid to be coupled, which exposes features independently of other reticles used for the same layer. After coupling of all amino acids for a particular layer, the wafer is then spin-coated with a solution of 50% by weight NMP and 50% by weight acetic anhydride to cap the remaining unprotected amines on the wafer to which no amino acids for that particular layer have been coupled. To remove the substrate resist present on the surface after each step, the wafer is stripped in acetone and IPA. This entire process was repeated for each individual coupling layer of amino acids that were planned to be coupled to complete the synthesis of the peptide chain that was attached to the array surface.

[0339] Side-chain deprotection: After peptide synthesis was completed, any remaining side-chain protection present on any coupled amino acids was removed to allow for peptide biological activity. The side-chain deprotection solution was prepared by mixing 95 wt% trifluoroacetic acid [TFA] (Sigma-Aldrich) and 5 wt% DI water. The wafer was allowed to react with the side-chain deprotection solution for 90 minutes. This step was followed by sequential washing of the wafer with TFA (5 minutes), IPA (5 minutes), and NMP (5 minutes), neutralization with 5 wt% DIEA (Alfa Aesar) in NMP (5 minutes), and then sequential washing of the wafer with NMP (5 minutes) and IPA (5 minutes).

[0340] Example 4: Purity analysis of synthesized peptides Mass Spectrometry: The peptide LKWLDSFTEQ (SEQ ID NO: 128, which is equivalent to 1 in 24598PCT) was synthesized and cleaved from the wafer substrate as described in Examples 1-3. The peptide was dissolved in 20-70% ceric ammonium nitrate [CAN] for 1.75 minutes and loaded onto a Phenomenex Luna column at 1.5 ml / min at 35°C. The peptide mass was measured and matched the expected mass, as shown in Figure 5A.

[0341] Fluorescein Quality Control: As a second control for the peptide synthesis process, quality control of the final product fluorescein was performed. The final amino acid in each peptide sequence was deprotected with base (10% (v / v) piperidine in NMP) for 20 minutes and coupled for 30 minutes in a solution containing 1% (wt) 5(6)-FAM (Anaspec), 2% (wt) DIC, and 2% (wt) HOBt dissolved in NMP. This was followed by successive washing steps: NMP (5 minutes), ethanol (5 minutes), a mixture of 50% (wt) EDA (Sigma-Aldrich) and 50% (wt) ethanol for 30 minutes, ethanol for 15 minutes, and IPA for 5 minutes. Individual and total coupling yields for each amino acid coupling step were determined based on the fluorescent signal of the probe, as described in more detail in International Patent Application No. PCT / US2013 / 062773, the disclosure of which is incorporated herein by reference in its entirety. Examples for peptides LKWLDSFTEQ (SEQ ID NO: 128) and DKYYEPHLERA (SEQ ID NO: 129) are shown in Figures 5B and 5C.

[0342] Example 5: Celiac Disease Sample Assay To discover novel biomarkers for the diagnosis of celiac disease (CD), serum was collected from three sources: a cohort collected as part of a previous study at Mayo Clinic; 2(48 CD cases and 50 controls), a cohort from ARUP Labs (42 CD cases and 29 controls), and a commercially obtained cohort (12 rheumatoid arthritis (RA) cases and 7 systemic lupus erythematosus (SLE) cases). Previous studies of CD seropositivity in the community 19 Additional sera from a validation cohort (306 seropositive CD cases and 1590 controls) collected in [1] were also used to evaluate the diagnostic utility of peptide sets identified from the cohort or newly developed. Tables 3 and 4 show the demographic characteristics of the study population. All samples were handled by standard procedures and stored at -80°C. All samples were probed with a 1:101 primary antibody dilution and a 1:2000 secondary antibody dilution and scanned on a Nikon Total Solution Platform consisting of a Hamilton fluidics station and a Gen 2 microarray fluorescent scanner.

[0343] Table 3. Clinical characteristics of the study population TIFF2026035691000015.tif51128

[0344] (Table 4) TIFF2026035691000016.tif49128

[0345] First, 188 serum samples from a set of 90 untreated CD patients and 98 controls were analyzed for IgG and IgA reactivity to GPs attached to the microarray surface to determine the most frequently occurring 3-mer amino acid subsequences among the most active GPs in celiac samples. Two distinct consensus GP sets (gliadin-derived peptide sequences) were identified to distinguish CD from controls in this cohort, with peptide set 1 having 80% sensitivity and 85% specificity for IgG reactivity, while peptide set 2 had 86% sensitivity and 89% specificity for IgA reactivity, as shown in Table 5.

[0346] Table 5. GP sets identified for the diagnosis of CD in the training cohort. TIFF2026035691000017.tif51155* Celiac positive, positive was defined by the current standard diagnosis of CD, consisting of CD serology and / or duodenal biopsy.

[0347] Example 6: Creation of novel synthetic biomarkers for CD diagnosis As shown in Figures 6A and 6B, a matrix table was created showing the percentage occurrence of 3-mer subsequences containing the most active GPs in celiac samples. To improve the diagnostic accuracy of CD, 3-mer subsequences with high occurrences in this matrix table were combined with other frequently occurring 3-mer sequences, random 3-mers, or random 6-mer peptides from the matrix table to determine new sequence sets consisting of 6-mer, 9-mer, 12-mer, and 15-mer peptides, respectively. To evaluate the accuracy of the newly randomized peptide biomarker sequences (6-mer to 15-mer) for CD diagnosis, we used random forest (RF), a statistical algorithm that evaluates the significance of each biomarker and votes for multiple classes in a decision tree to classify samples. 20The newly identified and RF-verified peptide sequences were then synthesized on a 110k peptide microarray with triplicates. Of these newly randomized peptide sequences, 127 different randomized peptides (SEQ ID NOs: 1-127) demonstrated significantly improved sensitivity (IgG = 97% or IgA = 99%) and specificity (98% or 100%) (p<0.001) for CD diagnosis compared with peptide sets 1 and 2 from Example 5 when using the current standard CD serology test with an ELISA kit (Table 6). Tables 1 and 2 list the 127 peptides (SEQ ID NOs: 1-127) divided into sets 3 and 4 based on peptide activity in either the IgG or IgA assay, respectively. The area under the receiver operating characteristic (ROC) curve cross-validated using these 127 peptide sequences for predicting CD autoimmunity (CDA) by IgA reactivity was 0.99, as shown in Figure 7.

[0348] Example 7: Relationship between immune reactivity of novel B cell epitopes and CD severity To assess the correlation between duodenal pathology of CD and immunoreactivity to the identified peptide sets, sera from 48 clinically proven CD cases were used. While none of the peptides in Sets 1 and 2 from Example 5 were able to categorize serum samples based on the severity of intestinal disease, 127 newly randomized peptides (SEQ ID NOs: 1-127) were able to distinguish between severe and less severe CD cases, as determined by the Marsh scoring method for small intestinal pathology, as illustrated in Figures 8 and 9.

[0349] Table 6. Discriminatory power of novel peptide sets of discontinuous B cell epitopes for the diagnosis of CD. TIFF2026035691000018.tif64149* Celiac positive, where positivity is defined by the current standard of diagnosis of CD, which consists of CD serology and / or duodenal biopsy; +, tTG = anti-tissue transglutaminase-IgA antibody against tissue transglutaminase in an enzyme-linked immunosorbent assay (ELISA) using a human recombinant antigen manufactured by Inova Diagnostics, San Diego CA; ++, DGP = deamidated gliadin-derived peptide-IgA antibody against deamidated gliadin peptide in an enzyme-linked immunosorbent assay (ELISA) using a human recombinant antigen manufactured by Inova Diagnostics, San Diego CA

[0350] Example 8: Evaluation of novel B cell epitopes To validate the discriminatory power of the novel peptides (SEQ ID NOs: 1-127) in the training cohort, serum from a population cohort of 1,896 subjects was assayed in a blinded study. This cohort consisted of 306 CDA subjects and 1,590 controls who underwent current standard CD serology testing. CDA is defined by the presence of both tissue transglutaminase (tTG) IgA and endomysial antibodies (EMA), which are highly predictive of biopsy-proven celiac disease. Of these 306 subjects (CDA), 33 individuals were subsequently diagnosed with CD during follow-up. Two novel synthetic peptide sets, #3 and #4, containing discontinuous gliadin sequences, demonstrated high accuracy for distinguishing CDA cases from controls, achieving 99% sensitivity and 100% specificity (Table 6). In addition, as illustrated in Figures 10A-10C, CDA cases could also be distinguished from less reactive or more reactive groups based on antibody binding intensity, with sera from these 33 subjects who subsequently developed clinically detectable CD all showing higher intensity than those who subsequently did not develop detectable CD.

[0351] Random forest (RF) was used to assess the classification accuracy of the training and validation sets of selected peptide biomarkers. 20The newly identified peptide biomarkers (SEQ ID NOs: 1-127) were used to diagnose a training set (n = 188; 90 CDA cases and 98 controls), resulting in an overall accuracy of 98.9% (out-of-bag error 1.1%, positive predictive value (PPV) 100%, and negative predictive value (NPV) 98.2%). When the same set of biomarkers was used to classify a validation set of sera (n = 1896; 306 CDA cases and 1590 controls), which were not involved in the biomarker selection process at all, RF was able to distinguish CDA samples from controls with equal accuracy (prediction error 1.1%, PPV 100.0%, and NPV 98.2%). These autoantibody biomarkers (SEQ ID NOs: 1-127) were used to simultaneously classify all CD and control samples (n=2084; 396 CD cases, 1688 controls) by RF, and they again demonstrated 99.1% sensitivity and 100% specificity.

[0352] In CD, certain B cell epitopes of gliadin have been shown to be linear. 15 It is well recognized that antibodies that recognize three-dimensional structures are unlikely to have affinity for T cell receptors when presented by antigen-presenting cells (APCs). Because the immune response that typifies CD may require proteins derived from cereals such as wheat, we used the power of this novel ultra-throughput platform to systematically search for both linear, contiguous, and even discontinuous peptides derived from all known proteins. The novel method identified a set of novel epitopes, including a set composed of discontinuous peptide sequences derived from deamidated gliadin, which were recognized by circulating antibodies found in the sera of CD patients and showed high sensitivity and specificity for distinguishing CD from controls. The known 33-mer gliadin sequence 21These distinct 9-mer to 15-mer sequences represent novel and previously unidentified B-cell epitopes of gliadin. The identified peptides were subjected to rigorous testing for significance and predictive value by testing them against a validation cohort derived from community-based samples uninvolved in their selection, along with specific seropositive and other biopsy-positive samples. Validation demonstrated high accuracy for disease diagnosis and severity detection. For example, two seronegative samples from the training cohort and six seronegative samples from the validation cohort were captured, thereby increasing sensitivity and specificity to 99% and 100%, respectively. Furthermore, specific sequences were identified that allowed for subgrouping by severity, which was not possible with conventional antibodies.

[0353] The presently presented platform for high-throughput, high-volume manufacturing of ultra-high-density peptide microarrays for biomarker discovery provides an efficient method for mapping antigens and identifying novel epitopes through the combination of immunopotentiating sequences and random peptides. Using a peptide microarray based on 2.1 million 9-mer to 15-mer peptides, each overlapping by three or six amino acids, covering immunogenic proteins at extremely high densities, maximizing the ability to identify informative peptides, we demonstrated the effectiveness and utility of this technology to identify novel epitopes that were previously unknown but recognized by patients with autoimmune diseases. Benefits of this method include the development of more accurate diagnostic tests that can be incorporated into a panel of tests for autoimmune diseases, including celiac disease. Furthermore, by designing peptide microarrays with single-amino acid shifts, we assessed the contribution of individual amino acids of the antigen to antibody binding, achieving higher mapping resolution for the target antigen.

[0354] All previous microarray in situ synthesis methods based on photolithography 5,22-24The method described herein is based on a global deprotection step followed by selective activation, which provides two advantages: 1) much higher fidelity in peptide synthesis, and 2) a significant reduction in the time required for each step. This allows for a significantly larger number of steps, as many as 400, in the synthesis of peptide microarrays, resulting in significantly less yield loss. In some embodiments, the combination of high fidelity and shorter reaction times results in much higher yields and the ability to fabricate large numbers of chips. Other advantages include cost savings due to the high fidelity required for diagnostic testing. The method described herein utilizes state-of-the-art 248 nm semiconductor lithography semiconductor tools on a proven 300 mm silicon wafer platform. In some embodiments, the extremely high density of the microarrays enables not only the molecular diversity required for biomarker discovery, but also large-scale biomarker validation. This method reduces the chip size to 0.5x0.5mm, which is compatible with all diagnostic well plate formats, such as 96-well, 384-well, and 1396-well. 2 The ability to scale down to a size that is well suited for mass production of routine diagnostic tests allows for smaller sample sizes to be used in routine diagnostic tests.

[0355] The method disclosed herein, by using the disclosed peptide microarrays, provides a non-invasive, widely available, low-cost, and versatile method that is highly suitable for routine medical testing purposes and represents a powerful new tool for biomarker discovery.

[0356] While the present invention has been shown and described in detail with reference to preferred and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

[0357] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0358] It should be noted that the language used herein has been chosen primarily for ease of reading and instruction, and not for the purpose of precisely delineating or limiting the subject matter of the invention. Accordingly, the disclosure of the present invention is intended to be illustrative, but not limiting, of the scope of the invention.

[0359] References TIFF2026035691000019.tif205146TIFF2026035691000020.tif54145

[0360] Sequence information SEQUENCE LISTING <110> VIBRANT HOLDINGS, LLC <120> PEPTIDE MICROARRAYS AND NOVEL BIOMARKERS FOR CELIAC DISEASE <150> US 62 / 048,537 <151> 2014-09-10 <160> 129 <170> PatentIn version 3.5 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 1 Arg Arg Gly Gln Pro Phe Trp Gln Pro Glu Leu Thr 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 2 Val Val Asp Pro Glu Gln Pro Gln Gln Asp Cys Thr 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 3 Gly Gln Pro Phe Gln Pro Glu Gln Pro Trp Leu Thr 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 4 Gly Gln Pro Phe Trp Leu Thr Gln Pro Glu Gln Pro 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 5 Thr Ala Thr Val Val Asp Pro Glu Gln Pro Gln Gln 1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 6 Tyr Pro Glu Gln Pro Glu Gln Pro Gly Ser Ser Glu 1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 7 Arg Ala Asn His Leu Asn Gln Pro Glu Gln Pro Phe 1 5 10 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 8 Gln Pro Phe Trp Gln Pro Glu Gln Pro Phe Leu Thr 1 5 10 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 9 Leu His Phe Pro Glu Gln Pro Glu Gly Arg Asn Tyr 1 5 10 <210> 10 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 10 Asn Gln Pro Glu Gln Pro Phe Pro Leu Pro Val Ala 1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 11 Arg Gly Gln Pro Phe Gln Pro Glu Gln Pro Phe Trp 1 5 10 <210> 12 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 12 Thr Arg Pro Asp Leu Glu Gln Pro Phe Pro Gln Pro 1 5 10 <210> 13 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 13 His Phe Pro Glu Gln Pro Glu Gly Arg Asn Tyr Glu 1 5 10 <210> 14 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 14 Val Val Arg Arg Gly Gln Pro Phe Trp Gln Pro Glu 1 5 10 <210> 15 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 15 Gly Gln Pro Phe Trp Leu Gln Pro Glu Gln Pro Thr 1 5 10 <210> 16 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 16 Arg Gly Gln Pro Phe Trp Gln Pro Glu Leu Thr Leu 1 5 10 <210> 17 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 17 Arg Gly Gln Pro Phe Trp Leu Thr Leu Gln Pro Glu 1 5 10 <210> 18 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 18 Arg Gly Gln Pro Phe Trp Leu Gln Pro Glu Thr Leu 1 5 10 <210> 19 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 19 Phe Pro Glu Gln Pro Glu Gly Arg Asn Tyr Glu Ala 1 5 10 <210> 20 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 20 Leu Val Val Asn Phe Pro Glu Gln Pro Glu Ser Asp 1 5 10 <210> 21 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 21 Glu Gln Pro Glu Gln Pro Phe Ser Asn Leu Ile Lys 1 5 10 <210> 22 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 22 Val Arg Arg Gly Gln Pro Phe Gln Pro Glu Trp Leu 1 5 10 <210> 23 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 23 Gly Gln Pro Phe Trp Leu Thr Gln Pro Glu Gln Leu 1 5 10 <210> 24 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 24 Arg Arg Gly Gln Pro Phe Trp Leu Gln Pro Glu Thr 1 5 10 <210> 25 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 25 Phe Pro Glu Gln Pro Glu Asp Gly Ile Leu Asp Ile 1 5 10 <210> 26 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 26 Val Arg Arg Gly Gln Pro Phe Gln Pro Glu Gln Pro 1 5 10 <210> 27 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 27 Gly Gln Pro Phe Trp Leu Gln Pro Glu Gln Pro Phe 1 5 10 <210> 28 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 28 Gly Gln Pro Phe Trp Leu Thr Leu Gln Pro Glu Gln 1 5 10 <210> 29 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 29 Glu Asn Pro Glu Gln Pro Glu Gln Pro Phe Ile Lys 1 5 10 <210> 30 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 30 Arg Gly Gln Pro Phe Trp Gln Pro Glu Gln Leu Thr 1 5 10 <210> 31 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 31 His Lys Leu Val Val Asn Phe Pro Glu Gln Pro Glu 1 5 10 <210> 32 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 32 Arg Gly Gln Pro Phe Trp Leu Thr Gln Pro Glu Gln 1 5 10 <210> 33 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 33 Thr Gln Pro Glu Gln Pro Phe Val Glu Ile Pro Asp 1 5 10 <210> 34 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 34 Met Asn Met Gln Pro Glu Gln Pro Phe Gly Ser Asp 1 5 10 <210> 35 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 35 Thr Tyr Lys Tyr Pro Glu Gln Pro Glu Gln Pro Gly 1 5 10 <210> 36 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 36 Trp Asn Phe Gly Gln Phe Pro Glu Gln Pro Glu Asp 1 5 10 <210> 37 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 37 Gly Gln Pro Phe Trp Leu Gln Pro Glu Thr Leu His 1 5 10 <210> 38 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 38 Leu Thr Leu His Phe Pro Glu Gln Pro Glu Gly Arg 1 5 10 <210> 39 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 39 Asn Phe Pro Glu Gln Pro Glu Ser Asp Lys Leu Lys 1 5 10 <210> 40 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 40 Val Asn Phe Pro Glu Gln Pro Glu Ser Asp Lys Leu 1 5 10 <210> 41 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 41 Thr Leu His Phe Pro Glu Gln Pro Glu Gly Arg Asn 1 5 10 <210> 42 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 42 Leu Tyr Leu Glu Asn Pro Glu Gln Pro Glu Gln Pro 1 5 10 <210> 43 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 43 Ala Val Glu Glu Gln Pro Glu Gln Pro Gly Asp Trp 1 5 10 <210> 44 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 44 Gln Phe Pro Glu Gln Pro Glu Asp Gly Ile Leu Asp 1 5 10 <210> 45 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 45 Gln Pro Phe Trp Leu Gln Pro Glu Gln Pro Thr Leu 1 5 10 <210> 46 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 46 Phe Pro Glu Gln Pro Glu Ser Asp Lys Leu Lys Ala 1 5 10 <210> 47 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 47 Gly Gln Pro Phe Gln Pro Glu Gln Pro Phe Trp Leu 1 5 10 <210> 48 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 48 Lys Ala Arg Phe Pro Gln Pro Glu Gln Leu Arg Asp 1 5 10 <210> 49 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 49 Pro Glu Gln Pro Glu Gln Pro Ile Lys Ile Arg Ile 1 5 10 <210> 50 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 50 Ala Leu Asp Pro Thr Pro Gln Pro Glu Gln Pro Phe 1 5 10 <210> 51 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 51 Leu Val Val Arg Arg Gly Gln Pro Phe Gln Pro Glu 1 5 10 <210> 52 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 52 Phe Ala Ala Val Ala Gln Pro Glu Gln Pro Phe Cys 1 5 10 <210> 53 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 53 Gly Gln Pro Phe Trp Leu Gln Pro Glu Gln Thr Leu 1 5 10 <210> 54 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 54 Tyr Val Leu Thr Pro Glu Gln Pro Phe Pro Gln Gln 1 5 10 <210> 55 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 55 Lys Ala Arg Phe Pro Gln Pro Glu Gln Pro Phe Leu 1 5 10 <210> 56 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 56 Gln Pro Phe Trp Leu Thr Leu His Phe Gln Pro Glu 1 5 10 <210> 57 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 57 Glu Gln Pro Phe Pro Gln Pro Phe Trp Leu Thr Leu 1 5 10 <210> 58 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 58 Arg Arg Gly Gln Pro Phe Trp Gln Pro Glu Gln Pro 1 5 10 <210> 59 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 59 Arg Gly Gln Pro Phe Gln Pro Glu Trp Leu Thr Leu 1 5 10 <210> 60 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 60 Gln Glu Gln Pro Glu Gln Pro Ala Gly Thr Lys Ala 1 5 10 <210> 61 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 61 Ser Gln Pro Glu Gln Pro Phe Gly Met Val Asn Cys 1 5 10 <210> 62 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 62 Val Arg Arg Gly Pro Glu Gln Pro Phe Pro Gln Pro 1 5 10 <210> 63 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 63 Gly Gln Pro Phe Trp Gln Pro Glu Leu Thr Leu His 1 5 10 <210> 64 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 64 Leu Glu Gln Pro Glu Gln Pro Phe Ser Glu Lys Ser 1 5 10 <210> 65 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 65 Val Arg Arg Gly Gln Pro Phe Trp Leu Gln Pro Glu 1 5 10 <210> 66 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 66 Gln Pro Phe Gln Pro Glu Gln Pro Trp Leu Thr Leu 1 5 10 <210> 67 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 67 Phe Gly Gln Phe Pro Glu Gln Pro Glu Asp Gly Ile 1 5 10 <210> 68 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 68 Val Arg Arg Gly Gln Pro Phe Trp Gln Pro Glu Leu 1 5 10 <210> 69 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 69 Arg Asp Leu Tyr Leu Glu Gln Pro Glu Gln Pro Phe 1 5 10 <210> 70 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 70 Gln Pro Phe Gln Pro Glu Gln Trp Leu Thr Leu His 1 5 10 <210> 71 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 71 Asn Pro Glu Gln Pro Glu Gln Pro Ile Lys Ile Arg 1 5 10 <210> 72 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 72 Val Arg Arg Gly Gln Pro Phe Gln Pro Glu Gln Trp 1 5 10 <210> 73 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 73 Tyr Lys Tyr Pro Glu Gln Pro Glu Gln Pro Phe Gly 1 5 10 <210> 74 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 74 Gly Pro Glu Gln Pro Phe Pro Gln Pro Phe Trp Leu 1 5 10 <210> 75 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 75 Gln Pro Phe Trp Leu Gln Pro Glu Gln Thr Leu His 1 5 10 <210> 76 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 76 Val Val Asp Trp Ile Gln Pro Glu Gln Pro Gln Gln 1 5 10 <210> 77 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 77 Pro Glu Gln Pro Phe Pro Gln Gln Asp Asp Gly Ser 1 5 10 <210> 78 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 78 Arg Arg Gly Gln Pro Phe Gln Pro Glu Gln Trp Leu 1 5 10 <210> 79 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 79 Asn Gly Ile Leu Gly Pro Glu Gln Pro Glu Gln Cys 1 5 10 <210> 80 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 80 Val Val Asn Phe Pro Glu Gln Pro Glu Ser Asp Lys 1 5 10 <210> 81 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 81 Gly Gln Pro Phe Gln Pro Glu Trp Leu Thr Leu His 1 5 10 <210> 82 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 82 Gln Pro Glu Gln Pro Phe Val Asp Gln Gln Asp Cys 1 5 10 <210> 83 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 83 His Thr Tyr Lys Tyr Pro Glu Gln Pro Glu Gln Pro 1 5 10 <210> 84 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 84 Arg Arg Gly Gln Pro Phe Gln Pro Glu Trp Leu Thr 1 5 10 <210> 85 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 85 Gly Gln Pro Phe Trp Leu Thr Gln Pro Glu Leu His 1 5 10 <210> 86 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 86 Glu Asn Pro Glu Gln Pro Glu Gln Ile Lys Ile Arg 1 5 10 <210> 87 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 87 Gly Gln Pro Phe Trp Gln Pro Glu Gln Pro Leu Thr 1 5 10 <210> 88 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 88 Gly Gln Pro Phe Trp Leu Thr Leu Gln Pro Glu His 1 5 10 <210> 89 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 89 Trp Leu Thr Leu His Phe Pro Glu Gln Pro Glu Gly 1 5 10 <210> 90 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 90 Gln Pro Phe Trp Leu Thr Leu His Gln Pro Glu Gln 1 5 10 <210> 91 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 91 Arg Gly Gln Pro Phe Trp Gln Pro Glu Gln Pro Phe 1 5 10 <210> 92 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 92 Gln Pro Glu Gln Pro Gln Gln Asp Cys Thr Leu Ser 1 5 10 <210> 93 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 93 Arg Arg Gly Glu Gln Pro Phe Pro Gln Pro Phe Trp 1 5 10 <210> 94 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 94 Val Leu Thr Gln Pro Glu Gln Pro Gln Gln Gly Phe 1 5 10 <210> 95 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 95 Gln Pro Phe Trp Leu Gln Pro Glu Gln Pro Phe Thr 1 5 10 <210> 96 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 96 Gln Pro Phe Trp Gln Pro Glu Leu Thr Leu His Phe 1 5 10 <210> 97 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 97 Phe Trp Leu Thr Leu His Phe Pro Glu Gln Pro Glu 1 5 10 <210> 98 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 98 Arg Arg Gly Gln Pro Phe Trp Leu Thr Gln Pro Glu 1 5 10 <210> 99 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 99 Thr Tyr Lys Tyr Pro Glu Gln Pro Glu Gln Gly Ser 1 5 10 <210> 100 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 100 Gly Ile Leu Gly Pro Glu Gln Pro Glu Gln Pro Phe 1 5 10 <210> 101 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 101 Asp Leu Glu Gln Pro Phe Pro Gln Pro Gly Tyr Glu 1 5 10 <210> 102 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 102 Gly Gln Pro Phe Trp Gln Pro Glu Gln Pro Phe Leu 1 5 10 <210> 103 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 103 Gly Gln Phe Pro Glu Gln Pro Glu Asp Gly Ile Leu 1 5 10 <210> 104 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 104 Arg Gly Gln Pro Phe Trp Leu Gln Pro Glu Gln Thr 1 5 10 <210> 105 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 105 Asp Trp Ile Pro Glu Gln Pro Phe Pro Gln Gln Asp 1 5 10 <210> 106 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 106 Arg Gly Gln Pro Phe Trp Leu Gln Pro Glu Gln Pro 1 5 10 <210> 107 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 107 Lys Leu Val Val Asn Phe Pro Glu Gln Pro Glu Ser 1 5 10 <210> 108 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 108 Asn Phe Gly Gln Phe Pro Glu Gln Pro Glu Asp Gly 1 5 10 <210> 109 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 109 Arg Phe Pro Gln Pro Glu Gln Pro Leu Arg Asp Ala 1 5 10 <210> 110 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 110 Tyr Lys Tyr Pro Glu Gln Pro Glu Gln Gly Ser Ser 1 5 10 <210> 111 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 111 Leu Asn Leu Glu Gln Pro Glu Gln Pro Phe Pro Phe 1 5 10 <210> 112 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 112 Leu Gly Pro Glu Gln Pro Glu Gln Pro Phe Cys Gly 1 5 10 <210> 113 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 113 Ala Gly Thr Lys Ala Arg Phe Pro Gln Pro Glu Gln 1 5 10 <210> 114 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 114 Tyr Lys Tyr Pro Glu Gln Pro Glu Gln Pro Gly Ser 1 5 10 <210> 115 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 115 Lys Arg Gln Pro Glu Gln Pro Phe Lys Leu Val Ala 1 5 10 <210> 116 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 116 Arg Arg Gly Pro Glu Gln Pro Phe Pro Gln Pro Phe 1 5 10 <210> 117 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 117 Gly Ser Ser Glu Glu Arg Glu Gln Pro Glu Gln Pro 1 5 10 <210> 118 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 118 Gln Pro Phe Gln Pro Glu Gln Pro Phe Trp Leu Thr 1 5 10 <210> 119 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 119 Pro Glu Gln Pro Glu Gln Pro Gly Ser Ser Glu Glu 1 5 10 <210> 120 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 120 Val Asp Trp Ile Gln Pro Glu Gln Pro Gln Gln Asp 1 5 10 <210> 121 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 121 Leu Glu Asn Pro Glu Gln Pro Glu Gln Ile Lys Ile 1 5 10 <210> 122 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 122 Gly Thr Lys Ala Arg Phe Pro Gln Pro Glu Gln Leu 1 5 10 <210> 123 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 123 Asn Pro Glu Gln Pro Glu Gln Pro Phe Ile Lys Ile 1 5 10 <210> 124 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 124 Gln Pro Phe Trp Gln Pro Glu Gln Leu Thr Leu His 1 5 10 <210> 125 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 125 Arg Gly Gln Pro Phe Trp Leu Thr Gln Pro Glu Leu 1 5 10 <210> 126 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 126 Glu Gln Pro Glu Gln Pro Glu Val Lys Val Arg Met 1 5 10 <210> 127 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 127 Val Arg Arg Gly Glu Gln Pro Phe Pro Gln Pro Phe 1 5 10 <210> 128 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 128 Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu 1 5 10 <210> 129 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 129 Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp 1 5 10

Claims

1. An array of features attached to a surface at a positionally defined location, The feature is, (a) at least two discontinuous epitope sequences derived from a bioactive polypeptide that elicits an immune response in subjects with celiac disease, and (b) At least one randomly generated polypeptide sequence It comprises at least one manipulated polypeptide chain including, The array wherein the polypeptide chain comprises one or more sequences selected from the group consisting of SEQ ID NOs: 108, 44, 1-29, 31-43, 45-107, 109-123, and 125-127.

2. (a) The bioactive polypeptide is selected from the group consisting of α-gliadin, β-gliadin, γ-gliadin, ω-gliadin, and other wheat-related proteins or peptides; (b) The feature includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 sequences selected from the group consisting of SEQ ID NO: 1 to 127; (c) The feature is 7 to 15 amino acids long; (d) Whether the feature has a length of 12 amino acids; (e) The feature attached to the surface of the array is configured to have at least 90% sensitivity and 90% specificity with respect to the detection of celiac defects after contact between the feature and a sample from an object suspected of having celiac defects; (f) Each of the at least two discontinuous epitopes consists of three amino acids; (g) Each of the at least two discontinuous epitopes consists of 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids; (h) Each of the at least two discontinuous epitopes consists of three amino acids having at least 20% sensitivity for binding to an antibody in a celiac-positive sample, and the peptide chain is 12 amino acids long; or (i) The array further comprises at least 10,000 features, each feature mounted on the surface of the array at a positionally defined location, the positionally defined location of each feature corresponds to a positionally defined location of a pillar, and the size of the top surface of each pillar is at least 1 μm 2 And; Optional, (A) Each feature contains a different manipulated peptide chain compared to other features, each feature contains at least 500 identical full-length peptide chains, where each identical full-length peptide chain has a manipulated full length of at least 7 amino acids, and the purity of each feature with respect to the proportion of full-length manipulated peptide chains is F, the proportion of full-length manipulated peptide chains in each feature having the manipulated sequence, and the length N of the manipulated full-length sequence is F = 10 （N＋1）・log（E／100％） Characterized by, where the mean coupling efficiency E is at least 98.5% with respect to the coupling of each amino acid in the manipulated sequence, the sequence length N is at least 7 amino acids long, and the proportion of the manipulated peptide chain that is less than the full length is equal to (1-F); or (B) The surface includes a substrate, the substrate includes a planar layer having an upper and lower surface, and a plurality of pillars functionally coupled to the layer at positionally defined locations, each pillar having a plane extending from the layer, the distance between the surface of each pillar and the upper surface of the layer being 1,000 to 5,000 angstroms, and the plurality of pillars being 10,000 / cm 2 It exists at a density exceeding that, The array according to claim 1.

3. A method for identifying autoimmune disorders in a subject, The step of bringing a sample derived from the target into contact with the array described in claim 1 or 2(i)(B); and A step to determine whether the subject has an autoimmune disorder by analyzing the binding of antibodies in the sample to the feature on the array. The method, including the method described above.

4. (a) Whether the autoimmune disorder is celiac disease; (b) The method includes a sensitivity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% for detecting autoimmune disorders; (c) The method comprises specificity for detecting autoimmune disorders, which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; or (d) The method includes the sensitivity of detecting the March classification of celiac disorder in subjects where the percentage is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The method according to claim 3.

5. A substantially purified peptide and / or recombinant peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 108, 44, 1-29, 31-43, 45-107, 109-123, and 125-127, wherein the biologically active fragment or variant thereof contains at least 90% sequence identity with respect to any one of SEQ ID NO: 108, 44, 1-29, 31-43, 45-107, 109-123, and 125-127.

6. A method for determining the degree of celiac disorder or celiac-related disorder in a patient, the method comprising the step of measuring the reactivity of a serum sample of the patient that has been contacted with a formulation containing an amino acid sequence or a biologically active fragment or variant thereof selected from the group consisting of SEQ ID NO: 108, 44, 1-29, 31-43, 45-107, 109-123, and 125-127, wherein the biologically active fragment or variant contains at least 90% sequence identity to any one of SEQ ID NO: 108, 44, 1-29, 31-43, 45-107, 109-123, and 125-127.

7. A biomarker for celiac disease comprising a polypeptide epitope of a celiac antibody, wherein the polypeptide epitope is selected from the group consisting of SEQ ID NO: 108, 44, 1-29, 31-43, 45-107, 109-123, and 125-127 or its biologically active fragment or variant, and the biologically active fragment or variant contains at least 90% sequence identity with any one of SEQ ID NO: 108, 44, 1-29, 31-43, 45-107, 109-123, and 125-127.

8. An agent comprising one or more peptides as described in claim 5.

Images