tTG-DGP BIOMARKERS FOR MONITORING CELIAC DISEASE

Abstract

SOLUTION: The invention relates to a field of identification of a biomarker of a celiac disease (CeD), more particularly, the invention relates to a field of identification of a neo epitope derived from a tTG-DGP composite as a biomarker for diagnosis of the CeD and for determining a healing state of a patient who is diagnosis to suffer from the CeD. The identified neo epitope for tTG-DGP composite body identifies the CeD with diagnosis accuracy equal to or more than that of a clinically available blood serum inspection. Furthermore, these neo peptides identify the healing state of the treated CeD patient with accuracy and singularity significantly greater than those of current blood serum inspections.EFFECT: Therefore, as an effect, these neo epitopes can be used as an index of continuous mucus membrane damage of a treated CeD patient, and thereby, enabling avoiding expensive and invasive intestine biopsy.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 742,863, filed October 8, 2018, the entire contents of which are incorporated by reference herein. [Background technology]

[0002] background Celiac disease (CeD) has autoimmune features, including elevated levels of antibodies against the autoantigen tissue transglutaminase (tTG) that revert to normal upon consumption of a gluten-free diet (GFD). 1 Although a GFD is an effective treatment for CeD, patients with CeD often have difficulty adhering to the GFD, which can lead to the progression of intestinal damage. Several studies have demonstrated that persistent mucosal damage in patients with CeD mucosa who have completed treatment is associated with lymphoproliferative malignancies, bone disease, and 34,35 And possibly excess mortality 36,37 As with any chronic disease, disease monitoring is necessary for patients with CeD mucosa who have completed treatment.

[0003] Serological testing for CeD is widely available and is considered an effective first step in the diagnosis and monitoring of CeD. 5,21-25 Currently, the major serological markers of CeD are antibodies against tTG and tTG-deamidated gliadin peptides (GPs). 4,5 Recent European guidelines suggest that adequate and strongly positive serological tests for CeD, such as tests for tTG-IgA or endomysial antibodies, are sufficient to confirm CeD; therefore, small bowel biopsy may be unnecessary to diagnose CeD in this subgroup. 26 However, results of serological tests vary widely across settings and populations. 5,23,25Furthermore, little correlation has been found between the healing state of the intestinal mucosa in patients treated with CeD. 40,41 Notably, the positive predictive value of CeD serological tests is relatively small due to the low prevalence of CeD. In addition, tTG-IgA testing is not effective in diagnosing CeD in patients with selective IgA deficiency, which is more associated with CeD than the general population. A recent meta-analysis reported that serological tests for CeD, such as tests for tTG-IgA and endomysial antibodies, have low sensitivity (<50%) compared with follow-up biopsy for detecting persistent villous atrophy in CeD patients who adhere to a GFD. Compared with tTG-IgA, deamidated gliadin peptide (DGP)-IgA has been shown to successfully identify healing status in treated CeD patients, although the sensitivity and specificity of DGP-IgA are suboptimal. Due to this variability in CeD serology, small intestinal biopsy is still considered the most reliable method for diagnosing CeD and verifying intestinal healing. 14 However, biopsies are invasive and costly, so there is a need for more accurate non-invasive markers for monitoring CeD. Summary of the Invention

[0004] overview The present invention relates to the field of identification of biomarkers for CeD. More specifically, the present invention relates to the field of identification of neoepitopes derived from the tTG-DGP complex as biomarkers for the diagnosis of CeD and for determining the healing status of patients diagnosed with CeD. The identified neoepitopes of the tTG-DGP complex identify CeD with diagnostic accuracy equal to or greater than that of clinically available serological tests. Furthermore, these neopeptides identify the healing status of treated CeD patients with much higher sensitivity and specificity than current serological tests. Thus, these neoepitopes can be used as indicators of persistent mucosal damage in treated CeD patients, thereby avoiding costly and invasive intestinal biopsies.

[0005] In one embodiment, the invention provides an array comprising an array surface and at least two peptide probes, each of which comprises a binding motif selected from the group consisting of SEQ ID NOs: 1-172. The peptide probes extend from the array surface.

[0006] The array surface can include any type of surface. For example, in some embodiments, the array surface can be a solid surface. In such embodiments, the solid surface can be a microparticle.

[0007] In certain embodiments, the at least two peptide probes are capable of binding to an antibody associated with celiac disease. In some embodiments, the at least two peptide probes can further comprise a label.

[0008] In another aspect, the invention provides an array of features attached to a surface at spatially defined locations, the features comprising at least one engineered polypeptide chain, the engineered polypeptide chain comprising at least two epitope sequences derived from a bioactive polypeptide and at least one epitope sequence derived from a protein, the bioactive polypeptide generating an immune response in a subject suffering from celiac disease, the protein binding to an antibody in a subject suffering from celiac disease.

[0009] The bioactive polypeptide can be selected from the group consisting of alpha gliadin, beta gliadin, gamma gliadin, omega gliadin, and other wheat-related proteins or peptides. The protein that binds to the antibodies of a subject suffering from celiac disease can be tissue transglutaminase (tTG). In certain embodiments, the engineered polypeptide chain can comprise 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: 1-172. In further embodiments, the engineered polypeptide chain can further comprise at least one randomly generated polypeptide sequence.

[0010] The features attached to the surface at the positionally defined locations can be any amino acid length. In particular embodiments, the features attached to the surface at the positionally defined locations can be 6-15 amino acids long. In even more particular embodiments, the features attached to the surface at the positionally defined locations can be 12 amino acids long.

[0011] Similarly, the epitope sequence comprising the surface-attached feature can be any length of amino acids.For example, in some embodiments, each of the at least two epitope sequences derived from the biologically active polypeptide can be composed of 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids.In more specific embodiments, each of the at least two epitope sequences derived from the biologically active polypeptide can be composed of 3 amino acids.

[0012] In certain embodiments, the features attached to the surface can be configured to have at least 90% sensitivity and 90% specificity for detecting celiac disease after contacting the features with a sample from a subject suspected of having celiac disease. In a further embodiment, where at least one engineered polypeptide chain is 12 amino acids in length and each of the at least two epitope sequences from the biologically active polypeptide comprising the polypeptide chain consists of 3 amino acids, each of the at least two epitope sequences from the biologically active polypeptide can have at least 20% sensitivity for binding to an antibody in a celiac disease positive sample.

[0013] In certain embodiments, the array of features can include at least 10,000 features, in which each feature is attached to the surface of the array at a different, positionally defined location that corresponds to a positionally defined location of a pillar, and the top surface of each pillar is at least 1 μm 2 In such an embodiment, each feature of the array can further comprise a different engineered peptide chain compared to other features of the array. Additionally, each feature comprises at least 500 identical full-length peptide chains, each identical full-length peptide chain having an engineered full-length that is at least 7 amino acids long. In such an embodiment, the purity of each feature with respect to the proportion of full-length engineered peptide chains can be the proportion F of full-length engineered peptide chains of each feature, where the full-length engineered peptide chains have an engineered sequence and an engineered full-length sequence length N, and the proportion F is greater than or equal to F=10. (N+1)·log(E / 100%)wherein the average coupling efficiency E is at least 98.5% for coupling of each amino acid of the engineered sequence, and the sequence length N is at least 7 amino acids long, and the percentage of engineered peptide chains that are less than full length is equal to (1-F). In yet another embodiment, the surface of the array can be a substrate, and the substrate can include a planar layer having an upper surface and a lower surface. The substrate can also have a plurality of pillars operatively coupled to the layer at positionally defined locations. Each pillar can have a planar surface extending therefrom, such that 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 has a surface area of ​​at least 10,000 / cm. 2 It will exist at a density exceeding

[0014] In yet another aspect, the present invention provides a method for detecting a curative state in a subject suffering from celiac disease, comprising obtaining a sample from the subject, the sample comprising, in part, a subject antibody, contacting an array of synthetic polypeptides with the subject sample, identifying an antibody binding intensity value for each synthetic polypeptide in the array, and determining a curative state in the subject based on the identified antibody binding intensity value for each synthetic polypeptide in the array. In such an embodiment, each synthetic polypeptide in the array comprises at least two epitope sequences derived from a biologically active polypeptide that generates an immune response in a subject suffering from celiac disease and at least one epitope sequence derived from a protein that binds to the subject antibody.

[0015] The bioactive polypeptide can be selected from the group consisting of alpha gliadin, beta gliadin, gamma gliadin, and omega gliadin. Furthermore, at least one of the two epitope sequences derived from the bioactive polypeptide can comprise a deamidated polypeptide sequence. In addition, the at least two epitope sequences derived from the bioactive polypeptide can be discontinuous in the bioactive polypeptide. In certain embodiments, each of the at least two epitope sequences derived from the bioactive polypeptide can be 3 amino acids in length.

[0016] The protein that binds to the subject antibody can, in certain embodiments, include tissue transglutaminase. The subject antibody can be an IgA and / or IgG antibody. Alternatively, in some embodiments, the subject can be IgA deficient. The subject can, in certain embodiments, adhere to a gluten-free diet.

[0017] The synthetic polypeptides in the array can include one or more of the sequences selected from the group consisting of SEQ ID NOs: 1-172. In some embodiments, each synthetic polypeptide can further include at least one randomly generated polypeptide sequence. Furthermore, each synthetic polypeptide can be 12 amino acids in length. In certain embodiments, the synthetic polypeptides of the array can be configured to have at least 90% sensitivity and 90% specificity for detecting celiac disease after contacting the microarray with a subject sample. In another embodiment, the synthetic polypeptides of the array can be configured to have at least 80% sensitivity and 90% specificity for detecting a cured state in a subject suffering from celiac disease and adhering to a gluten-free diet. The array of synthetic polypeptides can be a fluorescent array.

[0018] In certain embodiments, the array can be imaged after contacting the array with the target sample to identify the antibody binding intensity value for each of the synthetic polypeptides in the array.In addition, the fluorescence emission value for each of the synthetic polypeptides can be identified.Then, the antibody binding intensity value for each of the synthetic polypeptides can be identified based on the identified fluorescence emission value.

[0019] In yet another aspect, the present invention provides a method for identifying synthetic polypeptides for detecting a cured state of a subject suffering from celiac disease. The method includes estimating antibody binding strengths of an array of synthetic tTG-DGP neoepitopes, such that antibody binding strength values ​​are associated with corresponding peptide sequences. The method further includes removing background noise using background normalization modeling performed by an expectation maximization algorithm. The method further includes applying vector machine modeling to a training set of peptides to build a hyperplane and maximize the difference in training data between two classes (celiac disease and non-celiac disease) to determine a set of disease-related peptide sequences of the tTG-DGP complex. The method then includes determining the sensitivity and specificity of each peptide identified as a disease-related peptide sequence of the tTG-DGP complex, and identifying a further set of immunogenic epitopes of the tTG-DGP complex based on the sensitivity, specificity, and predictability of celiac disease associated with the peptides of the set.

[0020] In yet another aspect, the present invention provides an array for detecting a curative state in a subject suffering from celiac disease. In some embodiments, the array comprises a synthetic polypeptide identified according to the method for identifying a synthetic polypeptide described above. In another embodiment, the array comprises one or more sequences selected from the group consisting of SEQ ID NOs: 1-172.

[0021] In yet another aspect, the invention provides an array of features attached to a surface at positionally defined locations. In such an embodiment, the array can include synthetic polypeptides identified according to the method for identifying synthetic polypeptides described above. In another embodiment, the array can include one or more sequences selected from the group consisting of SEQ ID NOs: 1-172.

[0022] In yet another aspect, the invention provides a method of identifying an autoimmune disorder in a subject, the method comprising contacting a sample from the subject with any of the arrays disclosed above, and analyzing binding of antibodies in the sample to features on the array to determine whether the subject has an autoimmune disorder.

[0023] In some embodiments, the autoimmune disease can be celiac disease. In some embodiments, the method can provide a detection sensitivity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% for autoimmune disorders. Additionally, in some embodiments, the method can provide a detection specificity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% for autoimmune diseases.

[0024] In yet another aspect, the present invention provides a substantially purified and / or recombinant peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-172, or any one or more biologically active fragments or variants thereof.

[0025] In yet another aspect, the invention provides 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.

[0026] In yet another aspect, the invention provides a method for determining the extent of a celiac disorder or a celiac-related disorder in a patient, the method 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: 1-172, or any one or more biologically active fragments or variants thereof.

[0027] In yet another aspect, the present invention provides a biomarker for celiac disease comprising a polypeptide epitope of a celiac antibody, the polypeptide epitope being selected from the group consisting of SEQ ID NOs: 1-172, or any one or more biologically active fragments or variants thereof.

[0028] In yet another aspect, the invention provides an agent comprising one or more of the biomarkers for celiac disease set forth above. [The present invention 1001] 1. An array comprising an array surface and at least two peptide probes, each of the at least two peptide probes comprising a binding motif selected from the group consisting of SEQ ID NOs: 1-172, and the at least two peptide probes extending from the array surface. [The present invention 1002] 1001. The array of claim 10, wherein said at least two peptide probes are capable of binding to an antibody associated with celiac disease. [The present invention 1003] 1001. The array of claim 1001, wherein the array surface is a solid surface. [The present invention 1004] 10. The array of claim 10, wherein the solid surface is a microparticle. [The present invention 1005] 1001. The array of claim 1001, wherein said at least two peptide probes further comprise a label. [The present invention 1006] An array of features attached to a surface at positionally defined locations, the features comprising at least one engineered polypeptide chain comprising at least two epitope sequences derived from a biologically active polypeptide that generates an immune response in a subject suffering from celiac disease, and at least one epitope sequence derived from a protein that binds to an antibody of the subject suffering from celiac disease. [The present invention 1007] 1006. The array of claim 10, wherein said biologically active polypeptide is selected from the group consisting of alpha gliadin, beta gliadin, gamma gliadin, omega gliadin, and other wheat-related proteins or peptides. [The present invention 1008] 1006. The array of claim 1006, wherein said protein that binds to an antibody of said subject suffering from celiac disease comprises tissue transglutaminase. [The present invention 1009] 10. The array of claim 10, wherein said at least one polypeptide chain further comprises at least one randomly generated polypeptide sequence. [The present invention 1010] 1006. The array of the present invention, wherein said 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: 1-172. [The present invention 1011] 1006. The array of claim 1006, wherein said features are 6 to 15 amino acids in length. [The present invention 1012] The array of claim 1006, wherein said features are 12 amino acids in length. [The present invention 1013] An array of the present invention 1006, wherein the feature elements attached to the surface of the array are configured to have at least 90% sensitivity and 90% specificity for detecting celiac disease after contacting a sample derived from a subject suspected of having celiac disease with the feature elements. [The present invention 1014] 1006. The array of claim 10, wherein each of said at least two epitope sequences derived from said biologically active polypeptide consists of three amino acids. [The present invention 1015] Array according to the present invention 1006, wherein each of said at least two epitope sequences derived from said biologically active polypeptide consists of 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids. [The present invention 1016] The array of the present invention 1006, wherein each of the at least two epitope sequences derived from the biologically active polypeptide consists of three amino acids and has a sensitivity of at least 20% for binding to an antibody in a celiac positive sample, and the peptide chain is 12 amino acids in length. [The present invention 1017] and at least 10,000 features, each feature attached to a surface of the array at a different positionally defined location, each feature corresponding to a positionally defined location of a pillar, each pillar having a top surface of at least 1 μm. 2 The array of the present invention 1006 is sized [The present invention 1018] Each feature comprises a different engineered peptide chain compared to other features, each feature comprises at least 500 identical full-length peptide chains, and each identical full-length peptide chain has an engineered full-length that is at least 7 amino acids long, and the purity of each feature in terms of the proportion of full-length engineered peptide chains is a proportion F of said full-length engineered peptide chains of each feature, said full-length engineered peptide chains having an engineered sequence and an engineered full-length sequence length N, and said proportion F is F=10 (N+1)·log(E / 100%) Arrays according to the invention, characterized in that the average coupling efficiency E is at least 98.5% for coupling of each amino acid of the engineered sequence, and the sequence length N is at least 7 amino acids long, and the percentage of engineered peptide chains that are less than full length is equal to (1-F). [The present invention 1019] 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 the positionally defined locations, each pillar having a planar surface extending from the layer, a distance between a surface of each pillar and a top surface of the layer being between 1,000 and 5,000 angstroms, and the plurality of pillars having a surface roughness of less than 10,000 / cm 2 Arrays of the present invention are present at densities of greater than 10. [The present invention 1020] A method for detecting a cured state in a subject suffering from celiac disease, comprising: obtaining a sample from the subject, the subject sample partially comprising a subject antibody; contacting an array of synthetic polypeptides with said subject sample, each synthetic polypeptide comprising at least two epitope sequences derived from a biologically active polypeptide that generates an immune response in a subject suffering from celiac disease and at least one epitope sequence derived from a protein that binds to an antibody of said subject; identifying an antibody binding intensity value for each of the synthetic polypeptides in the array; and Determining a therapeutic status of the subject based on the identified antibody binding intensity values ​​for each of the synthetic polypeptides in the array. [The present invention 1021] The method of claim 1020, wherein at least one of said at least two epitope sequences derived from said biologically active polypeptide comprises a deamidated polypeptide sequence. [The present invention 1022] The method of claim 1020, wherein said protein that binds to said target antibody is tissue transglutaminase. [The present invention 1023] The method of claim 1020, wherein said target antibody is an IgA or IgG antibody. [The present invention 1024] The method of claim 1020, wherein said bioactive polypeptide is selected from the group consisting of alpha gliadin, beta gliadin, gamma gliadin, and omega gliadin. [The present invention 1025] The method of claim 1020, wherein said at least two epitope sequences derived from said biologically active polypeptide are discontinuous in said biologically active polypeptide. [The present invention 1026] The method of claim 1020, wherein each of said at least two epitope sequences derived from said biologically active polypeptide is 3 amino acids in length. [The present invention 1027] The method of claim 1020, wherein each synthetic polypeptide further comprises at least one randomly generated polypeptide sequence. [The present invention 1028] The method of claim 1020, wherein said synthetic polypeptide is 12 amino acids in length. [The present invention 1029] The method of the present invention 1020, wherein the synthetic polypeptides of the array are configured to have at least 90% sensitivity and 90% specificity for detecting celiac disease after contacting the microarray with the subject sample. [The present invention 1030] The method of claim 1020, wherein the array comprises a fluorescent array. [The present invention 1031] identifying an antibody binding intensity value for each of said synthetic polypeptides in said array; imaging the array after contacting the array with the sample of interest; identifying a fluorescence emission value for each of said synthetic polypeptides; and identifying an antibody binding intensity value for each of said synthetic polypeptides based on the identified fluorescence emission values. The method of the present invention 1020, comprising: [The present invention 1032] The method of claim 1020, wherein said synthetic polypeptides in said array comprise one or more of the sequences selected from the group consisting of SEQ ID NOs: 1-172. [The present invention 1033] The method of claim 1020, wherein the subject is IgA deficient. [The present invention 1034] The method of claim 1020, wherein the subject is adhering to a gluten-free diet. [The present invention 1035] The method of the present invention 1020, wherein the synthetic polypeptides of the array are configured to have at least 80% sensitivity and 90% specificity for detecting a healing state in a subject suffering from celiac disease and adhering to a gluten-free diet. [The present invention 1036] A method for identifying a synthetic polypeptide for detecting a curative state in a subject suffering from celiac disease, comprising one or more of the following steps: estimating antibody binding strength for an array of synthetic tTG-DGP neoepitopes, the antibody binding strength values ​​being associated with corresponding peptide sequences; removing background noise using background normalization modeling performed by an expectation maximization algorithm; applying vector machine modeling to the training set of peptides to construct a hyperplane and maximize the difference in the training data between the two classes (celiac and non-celiac), thereby determining a set of disease-associated peptide sequences of the tTG-DGP complex; determining the sensitivity and specificity of each peptide identified as a disease-associated peptide sequence of the tTG-DGP complex; and Identifying a further set of immunogenic epitopes of the tTG-DGP complex based on the sensitivity, specificity and predictability of celiac disease associated with the peptides of the set. [The present invention 1037] 1036 , An array for detecting a curative state in a subject suffering from celiac disease, said array comprising a synthetic polypeptide, said synthetic polypeptide being identified by the method of the present invention. [The present invention 1038] 1. An array for detecting a cured state in a subject suffering from celiac disease, the array comprising one or more sequences selected from the group consisting of SEQ ID NOs: 1 to 172. [The present invention 1039] An array of features attached to a surface at positionally defined locations, said features comprising at least one synthetic polypeptide identified by the method of the present invention 1036. [The present invention 1040] A method for identifying an autoimmune disorder in a subject, comprising: Contacting a sample derived from the subject with any one of the arrays 1001 to 1019 and 1037 to 1039 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 disease. [The present invention 1041] The method of claim 1040, wherein said autoimmune disorder is celiac disease. [The present invention 1042] The method of the present invention 1040, comprising a detection sensitivity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% for said autoimmune disorder. [The present invention 1043] The method of the present invention 1040, comprising a detection specificity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% for said autoimmune disorder. [The present invention 1044] A substantially purified peptide and / or recombinant peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 172, or any one or more biologically active fragments or variants thereof. [The present invention 1045] 1. 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. [The present invention 1046] A method for determining the degree of celiac disorder or celiac-related disorder in a patient, the method comprising measuring the reactivity of a serum sample from the patient that has been contacted with a preparation comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 172, or any one or more biologically active fragments or variants thereof. [The present invention 1047] 1. A biomarker for celiac disease comprising a polypeptide epitope of a celiac antibody, wherein said polypeptide epitope is selected from the group consisting of SEQ ID NOs: 1-172, or any one or more biologically active fragments or variants thereof. [The present invention 1048] An agent comprising one or more peptides of the invention 1047. [Brief description of the drawings]

[0029] This application will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the subject matter, there are shown in the drawings exemplary embodiments of the subject matter; however, the subject matter disclosed herein is not limited to the specific methods, devices, and systems disclosed. Additionally, the drawings are not necessarily drawn to scale; sizes and relative dimensions of components may be exaggerated to facilitate understanding. Like numbers refer to like elements throughout. [Figure 1] 1 is a flow chart for biomarker selection, training set analysis, and validation set analysis, according to an embodiment. [Figure 2A] 1 shows a proposed scheme for on-array peptide synthesis according to an embodiment (SEQ ID NOs: 176-187, respectively, in order of appearance). [Figure 2B] FIG. 2B illustrates the deamidation of a 12-mer GP according to an embodiment. FIG. 2B discloses SEQ ID NOs: 184, 188, 185, 189, 190, 185, and 191, in order of appearance, respectively. [Figure 3A] 1 illustrates the preparation of a wafer substrate, according to an embodiment. [Figure 3B] 1 illustrates pillars of a substrate, according to an embodiment. [Figure 3C] 1 illustrates exemplary AFM measured roughness and calculated density of a substrate according to an embodiment. [Figure 4] 1 illustrates peptide array synthesis, according to an embodiment. [Diagram 5] Examples of combined epitopes of the tTG-DGP complex according to an embodiment are shown (SEQ ID NOs: 192 to 194, respectively, in the order listed). [Figure 6] Figure 6A shows immunoreactivity against tTG peptide, according to an embodiment, and Figure 6B shows immunoreactivity against neoepitopes of the tTG-DGP complex, according to an embodiment. [Figure 7] Figure 7A shows immune reactivity against epitopes of the tTG-DGP complex in untreated, treated but not cured, and treated cured CeD patients, as well as healthy control patients, according to an embodiment. Figure 7B shows principal component analysis of immune reactivity against neoepitopes of the tTG-DGP complex, according to an embodiment. [Figure 8] 1 shows a comparison of antibody binding levels of tTG-immunoglobulin A complexes with antibody binding levels of tTG-DGP complexes in treated but not cured CeD patients according to an embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Those skilled in the art will readily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be used without departing from the principles of the invention described herein.

[0031] Detailed Description definition Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0032] As used herein, the term "wafer" refers to a thin slice of semiconductor material, such as silicon or germanium crystals, commonly used in the manufacture of integrated circuits. Wafers can come in a variety of sizes, with dimensions along one direction ranging from, for example, 25.4 mm (1 inch) to 300 mm (11.8 inches), and thicknesses ranging from, for example, 275 μm to 775 μm.

[0033] As used herein, the term "photoresist" or "resist" or "photoactivatable material" refers to a photosensitive material that changes its solubility in solution when exposed to ultraviolet or deep ultraviolet radiation. Photoresists can be organic or inorganic compounds and are 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 receive light become soluble in the 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 receive light become insoluble in the photoresist developer. The portions of the photoresist that are not exposed to light are dissolved by the photoresist developer.

[0034] As used herein, the term "photomask" or "reticle" or "mask" refers to an opaque plate with transparent patterns or holes that allow light to pass through. In a typical exposure process, the pattern on the photomask is transferred to a photoresist.

[0035] The term "coupling molecule" or "monomer molecule" as used herein includes any natural or synthetic amino acid whose amino group is protected with a fluorenylmethoxycarbonyl or 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. Further examples are described below.

[0036] As used herein, the term "coupling" or "coupling process" or "coupling step" refers to a process of forming a bond between two or more molecules, such as linking molecules or coupling molecules. The bond can be a covalent bond, such as a peptide bond. A peptide bond can be a chemical bond formed between two molecules when a carboxyl group of one coupling molecule reacts with an amino group of the other coupling molecule, releasing a molecule of water (H2O). This is a dehydration synthesis reaction (also known as a condensation reaction), and typically occurs between amino acids. The resulting CO-NH bond is referred to as a peptide bond, and the resulting molecule is an amide.

[0037] As used herein, the terms "biomolecule," "polypeptide," "peptide," or "protein" are used interchangeably to describe a chain or polymer of amino acids linked together by bonds. Thus, as used herein, the term "peptide" includes dipeptides, tripeptides, oligopeptides, and polypeptides. The term "peptide" is not limited to any particular number of amino acids. In some embodiments, a peptide comprises 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, can be a "native" or "wild-type" molecule, meaning that it occurs naturally in nature; or it can be a "mutant," "variant," "derivative," or "modification," meaning that it is created, modified, derived, or in some way different or altered from a native molecule or from another molecule, such as a mutant.

[0038] 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 the peptide from the support and extends therefrom, thereby increasing the distance between the substrate surface and the growing peptide. Generally, this reduces steric hindrance with the substrate for reactions involving the peptide (including unimolecular folding reactions and multimolecular binding reactions), thereby improving the performance of assays for measuring one or more embodiments of peptide function.

[0039] 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 is a water-based developer that contains tetramethylammonium hydroxide. The developer is used to define the initial pattern using a commercially available photoresist. Example 1 below describes the use of the developer.

[0040] As used herein, the term "protecting group" refers to a group that is introduced into a molecule by chemical modification of a functional group for the purpose of obtaining chemoselectivity in a subsequent chemical reaction. Chemoselectivity refers to directing a chemical reaction along a desired path compared to an alternative to obtain a preselected product. For example, the use of tboc as a protecting group allows for selective removal of the protecting group using a photomask and a photoacid generator, thus providing chemoselectivity for peptide synthesis where the photomask directs the occurrence of a predetermined peptide coupling reaction at a defined location.

[0041] 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, ordered locations, thereby forming a microscopic array.

[0042] The term "microarray system" as used herein refers to a system that is typically composed of biomolecular probes arranged in a predetermined format on a solid surface such as a glass, plastic, or silicon chip, as well as equipment required for handling the samples (automated robotic equipment), equipment required for reading reporter molecules (scanners), and equipment required for analyzing the data (bioinformatics tools).

[0043] As used herein, the terms "patterned area" or "pattern" or "location" refer to the areas on a substrate where different features are grown. These patterns can be defined using a photomask.

[0044] The term "derivatization" as used herein refers to the process of chemically modifying a surface to make it suitable for the synthesis of biomolecules. In general, derivatization includes the following steps: rendering the substrate hydrophilic, adding aminosilane groups, and attaching linker molecules.

[0045] 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 commonly capped with an acetic anhydride molecule to prevent further formation of peptide bonds.

[0046] 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.

[0047] As used herein, the term "dye molecule" generally refers to a dye, which is a colored substance that can be bound to a substrate and is useful for detecting binding between features on an array and a molecule of interest.

[0048] As used herein, the terms "immunological binding" and "immunological binding properties" refer to non-covalent interactions of the type that occur between an immunoglobulin molecule (or a variant thereof, such as, for example, an scFv) and its antigen for which the immunoglobulin is specific.

[0049] The term "biological sample" as used herein refers to a sample derived from a biological tissue or biological fluid that can be assayed for an analyte(s) 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, or cells derived therefrom. Biological samples may also include sections of tissue, such as frozen sections taken for histological purposes. Samples are typically obtained from human patients, but the assays can be used to detect the analyte(s) 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 diluted with an appropriate buffer, if necessary, or concentrated, if necessary.

[0050] 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.

[0051] The term "subject" includes, inter alia, an individual, a patient, a target, a host, or a recipient, regardless of whether the subject is a human or a non-human animal, including mammalian species, and even avian species.Thus, the term "subject" includes humans, non-human primates (e.g., gorillas, marmosets, African green monkeys), livestock animals (e.g., sheep, cows, pigs, horses, donkeys, goats), experimental 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.

[0052] The term "antigen" as used herein refers to a molecule that induces an immune response by a subject's immune system, e.g., 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 with the release of various cytokines in response to the antigen). Antigens can be exogenous, endogenous, or self-antigens. Exogenous antigens are those that enter the body from the outside by inhalation, ingestion, or injection. Endogenous antigens are those that are produced in previously normal cells as a result of normal cellular metabolism or due to viral or intracellular bacterial infection. Self-antigens are normal proteins or protein complexes present in the host's body, but that are capable of stimulating an immune response.

[0053] As used herein, the term "epitope" or "immunoreactive region" refers to a unique feature on the molecular surface of an antigen that can bind to a component of the adaptive immune system, such as an antibody or a T-cell receptor. An antigenic molecule can present several surface features that can act as an interaction point with a specific antibody. Any such molecular unique feature can constitute an epitope. Thus, an antigen has the ability to bind to several unique antibodies, each of which is specific for a particular epitope.

[0054] As used herein, the term "antibody" or "immunoglobulin molecule" refers to molecules that are naturally secreted by a particular type of cell of the immune system: B cells. There are five different naturally occurring isotypes of antibodies: IgA, IgM, IgG, IgD, and IgE.

[0055] 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).

[0056] 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.

[0057] 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 damage to the subject's own tissues. Celiac disorder, lupus erythematosus, and rheumatoid arthritis are examples of autoimmune disorders. Autoimmune disorders can be induced by environmental factors.

[0058] The term "percent identity" or "percent 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 visual evaluation. Depending on the application, the percent "identity" can 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.

[0059] For sequence comparison, typically one sequence is used as a reference sequence to compare with test sequences. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the sequence identity of the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0060] Optimal alignment of sequences for comparison can be accomplished, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), 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 (see generally Ausubel et al., supra).

[0061] One example of an algorithm that is suitable for determining percent 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. Percent identity scores can be calculated using the default settings of this program, available at the National Center for Biotechnology Information website as of the priority date of this application.

[0062] As used herein, the term "biologically active fragment" or variants thereof refers to a polypeptide capable of inducing substantially the same or greater T cell response in a gluten-sensitive subject as compared to the polypeptide from which it is derived (e.g., GP or tTG). In another embodiment, the biologically active fragment is capable of inducing at least 50%, more preferably at least 75%, of a T cell response in a gluten-sensitive subject as compared to the peptide from which it is derived. In certain embodiments, the biologically active fragment is 14, 13, 12, 11, 10, 9, 8 amino acids in length, and not less than 7 amino acids in length. Deletions and / or additions at either end of any peptide are specifically contemplated. Examples of biologically active fragments disclosed herein include SEQ ID NOs: 1-127.

[0063] As used herein, the term "celiac disease," also known as "CeD," refers to a chronic inflammatory disorder of the small intestine. The disease encompasses a range of symptoms, 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 osteoporosis and a substantially increased risk of developing intestinal malignancies (lymphomas and carcinomas).

[0064] The term "gluten-sensitive" refers to any one or more of the symptoms of celiac disease or a condition in which a subject exposed to gluten or its peptide fragments exhibits an inappropriate T cell response. In subjects without gluten sensitivity, ingestion of gluten elicits little or no T cell response. In contrast, subjects with gluten sensitivity exhibit inappropriate CD4 T cell responses to peptides derived from gluten following ingestion. + A T cell-mediated immune response is observed.

[0065] The terms "immune tolerance", "immunological tolerance", "tolerance" or "desensitize" are defined herein as suppressing the immunological reactivity of a subject to gluten, thereby rendering a sensitized or sensitive subject less sensitive, desensitized or non-responsive to gluten. Immune tolerance can occur, for example, upon exposure to a mucosal surface of a tolerogenic antigen fragment of gluten as defined herein. Mucosal administration of antigen, both at high and low doses, can result in immune tolerance, suppressing 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 is believed to occur through inactivation or clonal deletion of Th1 and Th2 cells. In contrast, tolerance to low doses of antigen results in bystander immune suppression mediated by stimulation of Treg cells to produce inhibitory cytokines such as interleukin-4 (IL-4), interleukin-10 (IL-10) and TGFβ.

[0066] As used herein, the term "inducing immune tolerance" refers to causing, producing, or causing immune tolerance to gluten in a subject sensitive to gluten.

[0067] The term "hypersensitivity" is defined herein as an abnormal physiological sensitivity to gluten.

[0068] The term "anergy" refers to a state of reversible unresponsiveness or hyporesponsiveness of T cells (or B cells) to antigens.

[0069] As used herein, "Treg" refers to a subclass of T cells whose primary role is to terminate T cell-mediated immunity during immune responses and to suppress autoreactive T cells that have escaped negative selection in the thymus. As used herein, the term "Treg response" refers to a T cell subclass that is characterized by a T cell phenotype characterized by expression of the forkhead family transcription factor FOXP3 (forkhead box p3) and / or the MHC class II-associated protein LAG-3, and / or 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 is also characterized by the differentiation and proliferation of 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 + The expression of FOXP3 may be determined by analysis. This is conveniently achieved by flow cytometry. In addition, the level of FOXP3 mRNA may be determined in mononuclear cells from peripheral blood or spleen by quantitative reverse transcriptase polymerase chain reaction (PCR) to quantify Treg cells. In addition, the induction of Treg response in vivo may be evaluated by measuring Treg-associated cytokines produced by mononuclear lymphocytes from peripheral blood or lymph nodes. Treg cells generally show higher levels of expression of anti-inflammatory cytokines such as IL-10 and TGFβ, and the presence of these mediators may be determined by methods known in the art, such as flow cytometry, immunohistochemical staining, or ELISA.

[0070] The term "T cell stimulatory peptide" or "stimulatory peptide" refers to a peptide or epitope that is capable of activating T cells.

[0071] The terms "activate" or "activating" or "activation" in relation to T cells refer to the binding of a costimulatory molecule by a T cell, accompanied by MHC molecules on one cell presenting epitopes to an appropriate T cell receptor on a second (T) cell, thereby eliciting a "T cell response."

[0072] As used herein, the term "toxic peptide" refers to a peptide that stimulates T cell activation in a subject.

[0073] As used herein, the term "expansion" refers to the proliferation and amplification of a T cell population following T cell activation.

[0074] The term "immunodominant" refers to the subunit (epitope) of a peptide that is most easily recognized by the immune system and thus most influences the specificity of an induced immune response, such as a T cell response. "Immunodominant" may be used interchangeably with "dominant" herein.

[0075] As used herein, the term "modulating a T cell response" refers to modulating or regulating the T cell response in a subject sensitive to gluten, thereby suppressing or decreasing the T cell response to gluten.

[0076] As used herein, the term "modifying cytokine secretion" refers to altering or changing to some extent the secretion of cytokines by a gluten-sensitive subject, thereby suppressing or reducing gluten sensitivity in the subject. This term encompasses both increasing the secretion of a particular cytokine or combination of cytokines, and suppressing the secretion of a particular cytokine or combination of cytokines.

[0077] As used herein, the term "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 are at least about 3 amino acids in length, and can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids in length.

[0078] The term "polyepitope" refers to the presence of two or more epitopes (peptides) linked together in a single polypeptide chain.

[0079] As used herein, the terms "antigen" and "immunogen," as well as variations thereof, are generally used interchangeably and refer to epitope-containing structures recognized by the immune system.

[0080] 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. "Gluten peptides" refers to peptides derived from or within one or more gluten proteins.

[0081] As used herein, the term "gliadin," also known as "GP," refers specifically to the hydroalcohol soluble fraction of gluten derived from wheat, including, but not limited to, gluten derived from Triticum aestivum.

[0082] The term "glutenin" refers to the aqueous alcohol insoluble fraction of gluten, particularly, but not limited to, from wheat, e.g., Triticum aestivum.

[0083] As used herein, "hordein" or "barley hordein" refers to gluten derived from barley, Hordeum vulgare.

[0084] As used herein, "secalin" or "rye secalin" refers to gluten derived from rye, Secale cerale.

[0085] The term "avedin" or "oat avedin" as used herein 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 that are made up of proteins that play a key role in activating the body's immune system to respond to foreign organisms. In humans and other animals, HLA is also called the "major histocompatibility complex" (MHC).

[0086] "Tissue transglutaminase", also known as "tTG" as used herein, is an important factor in celiac disease, as it promotes gluten-specific T cell responses. tTG 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 interactions lead to proinflammatory CD4 T cell responses. Thus, the term "deamidation" refers to the conversion of glutamine to glutamic acid or asparagine to aspartic acid. As used herein, the term deamidation refers specifically to the conversion of glutamine to glutamic acid in gluten, a process that enhances the propensity of gluten peptides to activate T cells.

[0087] The term "agent" as used herein 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., one composition containing a first and second peptide as defined herein and another composition containing a third peptide). If in different compositions, they are preferably in close proximity, such as in a kit. Thus, the method of the invention contemplates providing (e.g., administering to a subject) the individual component peptides and / or polynucleotides of the agent of the invention in a single composition (vaccine) or in different compositions, sequentially or in combination.

[0088] Before describing the disclosed embodiments in more detail, it is to be understood that the disclosure is, of course, not limited to particular embodiments described. The scope of the disclosure will be limited only by the appended claims, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0089] Where numerical ranges are provided, the disclosure includes each value within that range, to the tenth of the unit of the lower limit unless expressly stated otherwise, and every value between the upper and lower limits of that range, and every other value recited in that range or values ​​therein. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also included in the disclosure, with any end specifically excluded in the range stated therein. When the stated range includes one or both ends, ranges excluding either or both of those ends are also included in the disclosure.

[0090] Certain ranges may be presented herein with the term "about" before the numerical values. As used herein, the term "about" is used to provide literal support for the exact number to which the term is attached, as well as for a number that is close to, or before or after the number to which the term is attached. In determining whether a number is close to, or before or after a specifically recited number, the number that is close to, or before or after, may be a number that provides a substantial equivalent to the specifically recited number in the context in which it is presented.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these disclosed embodiments belong. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the disclosed embodiments, representative exemplary methods and materials are described herein. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0092] All publications and patents cited in this specification are incorporated by reference as if each individual publication or patent was specifically and individually incorporated by reference as if it were disclosing and describing the methods and / or materials in connection with which the publication is cited.

[0093] 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.

[0094] peptide The present disclosure relates to 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 CeD. In some embodiments, the polyepitope-containing peptides are antigens that modulate T cell responses in subjects with sensitivity to gluten or with CeD. Examples of these polyepitope-containing peptides and celiac active peptides are shown in Table 1.

[0095] Table 1. tTG-DGP complex peptide sequences with immunogenic specificity for CeD TIFF2025011239000001.tif131128TIFF2025011239000002.tif24275TIFF2025011239 000003.tif24275TIFF2025011239000004.tif24275TIFF2025011239000005.tif166128

[0096] Disclosed herein are methods for identifying novel polyepitope-containing peptides and uses of the novel polyepitope-containing peptides. The novel polyepitope-containing peptides can include epitope sequences of proteins that stimulate antibody production in subjects with autoimmune disease and epitope sequences of bioactive polypeptides that generate an immune response in subjects with autoimmune disease. The proteins that stimulate antibody production in subjects with autoimmune disease can include autoantigens. For example, the proteins can include tTG. In an embodiment in which the autoimmune disease is CeD, the bioactive polypeptides that generate an immune response can include GPs, such as alpha gliadin, beta gliadin, gamma gliadin, or omega gliadin, or another wheat-related protein or peptide. In a further embodiment, the bioactive polypeptides that generate an immune response can include DGPs. Thus, in certain embodiments, the novel polyepitope-containing peptides can include epitopes of the tTG-DGP complex. Uses of arrays or formulations containing the novel polyepitope-containing peptides disclosed herein can include research applications, therapeutic purposes, medical diagnostics, and / or stratification of one or more patients or subjects.

[0097] The novel polyepitope-containing peptides and / or components thereof may also include biologically active variants. Biologically active variants include peptides that differ from a given peptide by one or more amino acids, which are known in the art as homologues. For example, variants may include one or more amino acid substitutions in any of one or more peptides. As used herein, the term "substituted" or "substitution" includes substitution, exchange, addition, insertion, omission, and / or deletion of amino acid residue(s) (so that variants may also be fragments). In particular, this refers to peptides with conservative substitutions that do not preclude or significantly impede use in the methods of the invention. Preferably, a biologically active variant is capable of generating a substantially equivalent or greater T cell response in a subject sensitive to gluten compared to the peptide from which it is derived. In another embodiment, a biologically active variant is capable of generating at least 50%, more preferably at least 75%, of a T cell response in a subject sensitive to gluten compared to the peptide from which it is derived.

[0098] 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.

[0099] In one embodiment, fewer than five, more preferably fewer than four, more preferably fewer than three, more preferably fewer 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.

[0100] In another embodiment, the percentage identity between a particular sequence (variant) and a 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, e.g., at least about 96%, 97%, 98%, 99% or more. Percentage identity can be determined using readily available software packages, such as BLAST (www.ncbi.nlm.nih.gov / ) and GAP. Natural 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 specifically contemplated.

[0101] Substitutions may be conservative amino acid substitutions, where the substituted amino acid has similar structural or chemical properties as the corresponding amino acid in the reference sequence, or may be non-conservative amino acid substitutions, so long as the desired activity is maintained.

[0102] Examples of conservative amino acid substitutions include replacing an aliphatic or hydrophobic amino acid, such as alanine, valine, leucine, and isoleucine, with another; replacing a hydroxyl-containing amino acid, such as serine and threonine, with another; replacing an acidic residue, such as glutamic acid or aspartic acid, with another; replacing an amide-containing residue, such as asparagine and glutamine, with another; replacing an aromatic residue, such as phenylalanine and tyrosine, with another; replacing a basic residue, such as lysine, arginine, and histidine, with another; and replacing small amino acids, such as alanine, serine, threonine, methionine, and glycine with another.

[0103] Peptide variants can be generated by mutagenesis or other chemical methods. Alanine scanning is a useful technique for identifying important amino acids. In this technique, amino acid residues are replaced with Ala and the effect on the activity of the peptide is determined. For example, cysteine ​​residues can be substituted to minimize dimer formation through disulfide bonds. In this method, each of the amino acid residues of a peptide is analyzed to determine the important regions of the peptide. Means for preparing such peptides are well understood in the art.

[0104] In addition to naturally occurring amino acids, non-naturally occurring or modified amino acids are 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.

[0105] As used herein, the phrases "protecting group" and "blocking group" refer to modifications to peptides that protect the peptide 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, teyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups, such as benzyloxycarbonyl (Cbz); aliphatic urethane protecting groups, such as t-butoxycarbonyl (Boc) or 9-fluorenylmethoxy-carbonyl (FMOC); pyroglutamic acid, and amidation. Numerous other modifications which result in increased potency, sustained activity, ease of purification, and / or increased half-life are known to those of skill in the art.

[0106] In one embodiment, one or more glutamic acid residues of one or more peptides may be generated by tTG activity on the peptide, hi another embodiment, this reaction occurs in vivo after administration.

[0107] The peptide may include one or more modifications, which may be natural post-translational modifications or artificial modifications. The modifications may provide, for example, chemical moieties such as amino, acetyl, acyl, carboxy, hydroxy, or halogen (e.g., fluorine) groups, or carbohydrate groups (typically, for example, by replacing hydrogen in a C-H bond). Typically, the modifications are present at the N-terminus or C-terminus. Furthermore, one or more peptides may be PEGylated, where PEG (polyethyleneoxy group) increases longevity in the bloodstream. One or more peptides may also be combined with other proteins or specific binding agents as fusion proteins or chimeric proteins to target specific parts of target cells.

[0108] Peptide variants may be obtained where the peptide has been chemically modified at the level of the amino acid side chains, amino acid chirality, and / or the peptide backbone.

[0109] Certain peptides described herein may exist in particular geometric or stereoisomeric forms. The present invention contemplates that all such forms are within the scope of the present invention, including cis-(Z) and trans-(E) isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof. Additional asymmetric carbon atoms may be present in substituents, such as alkyl groups. All such isomers, as well as mixtures thereof, are contemplated as being within the scope of the present invention.

[0110] In another example, to prevent cleavage by peptidases, any of one or more of the peptides may contain non-cleavable peptide bonds in place of particularly sensitive peptide bonds to obtain a more stable peptide. Such non-cleavable peptide bonds may include beta amino acids.

[0111] In certain embodiments, any of one or more of the peptides may include a functional group, for example in place of a fragmentable peptide bond, which facilitates inhibition of serine-, cysteine-, or aspartic acid-type proteases, as appropriate. For example, the present invention includes peptidyl diketones or 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 have been provided on other peptide molecules, and general routes for their synthesis are known.

[0112] 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 maintains a particular activity of interest (e.g., inducing tolerance). The rationale behind the use of peptidomimetics is that the peptide backbone of a protein exists primarily to orient the amino acid side chains in a manner that facilitates molecular interactions, such as between T cells and MHC peptides, antibodies and antigens, enzymes and substrates or scaffold proteins, etc. Peptidomimetics are designed to allow molecular interactions similar to those of natural molecules. Mimetics include olefins, phosphonates, aza-amino acid analogs, etc. Those skilled in the art will readily understand methods for designing peptide mimetics and can utilize them to design mimetics of peptides as defined herein.

[0113] Peptides may 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 may also be performed to identify regions of the peptide that exhibit specific structural motifs. Manipulation, translation, prediction of secondary structure, hydrophilicity and hydrophobicity profiles, prediction and plotting of open reading frames, and determination of sequence homology may be accomplished using computer software programs available in the art. Other methods of structural analysis may 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).

[0114] The peptide, fragment, or variant may be in the form of a salt, preferably in the form of a pharma- ceutically acceptable salt. The "pharma-ceutically acceptable salt form" includes conventional non-toxic salts of peptides, or quaternary ammonium salts, such as those derived from non-toxic organic or inorganic acids. Conventional non-toxic salts include, for example, salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfonic acid, phosphoric acid, nitric acid, and the like; 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, isothioic acid, and the like.

[0115] The peptides can be provided in the agent or vaccine as separate peptides or linked, for example, in a polyepitope structure. In one embodiment, the peptides can be presented in a single polypeptide chain (polyepitope string), i.e., in a linear or cyclic arrangement. In another embodiment, the peptides can be presented in a multiple antigen presentation system, especially based on a dendrimer backbone such as polylysine. The polylysine backbone provides a non-linear branched arrangement of epitopes. This system has advantages over polyepitope strings, such as the ability to induce a complete T cell response, since the peptides do not interfere with each other or are less likely to be cleaved into cryptic epitopes.

[0116] Conjugates One or more peptides can be conjugated with compound by using standard method.The examples of compound that peptide can be conjugated include but are not limited to radioisotope, fluorescent label, chemiluminescent compound, enzyme label, free radical, avidin-biotin label, bacteriophage label, compound that extends the half-life of peptide in subject, adjuvant, MHC molecule or its fragment.

[0117] The compound may facilitate detection and / or isolation of the conjugated peptide or may increase its immunogenicity.

[0118] As used herein, "conjugated" means coupled via a covalent or non-covalent bond. Although covalent bonds are preferred, the compound may be linked to the peptide via non-covalent complexation, for example, via hydrogen bonds or electrostatic interactions, hydrophobic interactions, etc.

[0119] Common 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 There is Eu.

[0120] Common fluorescent labels include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine.

[0121] Common chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters. Common bioluminescent compounds include luciferin, luciferase, and aequorin.

[0122] Common enzyme labels include alkaline phosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, glucose oxidase, and peroxidase.

[0123] In some embodiments, a non-specific linker is used between the compound and the peptide to be conjugated. Such linkers are not involved in the activity of the peptide. Rather, the linker can function as a spacer between the peptide and the functional moiety. The use of the linker is the immobilization of the peptide for purposes such as purification or detection support. Alternatively, the linker can allow the attachment of the compound to the peptide, which allows the specific delivery of the peptide to a specific target, for example, a cell or tissue, in space or time. When used as a vaccine, one or more peptides can be coupled with a linker that acts as a spacer between the peptide and the immunogenic carrier, or can be coupled with a linker that allows improved binding between the peptide and the immunogenic carrier, preventing the formation of cryptic epitopes.

[0124] In certain embodiments, one or more 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), using any of several conjugation chemistries known in the art, to enhance their immunogenicity. 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 act as a spacer between the peptide and the immunogenic carrier.

[0125] When used as a diagnostic agent, one or more peptides are preferably conjugated to an immunogenic carrier that has not been used before for vaccination, which prevents the diagnostic agent from reacting with antibodies formed against the carrier fraction of the vaccine when monitoring for successful vaccination.

[0126] In some embodiments, the compound is an MHC class II molecule or a peptide-binding fragment thereof. The MHC class II molecule may be purified from a biological sample. Alternatively, the MHC class II molecule may be recombinantly produced. The peptide-binding fragment of the MHC class II molecule may be obtained, for example, by enzymatic cleavage of the purified molecule or recombinant intact molecule. Alternatively, the peptide-binding fragment may be recombinantly produced. In a preferred embodiment, the compound is a recombinant two-domain MHC class II molecule.

[0127] 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, 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 associated with a peptide as defined herein through a covalent or non-covalent interaction. In certain 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 covalently linked to an MHC molecule in a directed manner (i.e., rather than randomly attached), it is generally linked to the carboxy terminus of the molecule so as to minimize interference with the peptide antigen linked to the amino terminus.

[0128] 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 the 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 CeD. 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 drugs (e.g., doxorubicin, daunorubicin, methotrexate, cytotoxins, and antisense RNA), antibodies against cytotoxic T cell surface molecules, lipases, and radioisotopes that emit "hard" radiation, e.g., beta rays.

[0129] antigen presenting cells The agents and / or peptides defined herein may be delivered, for example, by APCs carrying the first, second and third peptides, one or more biologically active fragments or variants thereof, and / or polynucleotides encoding one or more of them.

[0130] Preferably, the APC is selected from the group consisting of dendritic cells, macrophages, B lymphocytes, and hepatic sinusoidal endothelial cells that express MHC class II molecules that have a common MHC phenotype with the subject. For example, the APC is selected from the group consisting of HLA-DQ2 (e.g., HLA DQA1 * 05 and HLA DQB1 * 02), and / or HLA DQ8. APCs used for this purpose may be isolated from the subject to which they are administered after loading, or may be obtained from an allotype-matched subject.

[0131] "Loading" an APC means incubating or transfecting the APC with a peptide, one or more biologically active fragments or variants thereof, or a polynucleotide encoding one or more of them. Loading an APC can be accomplished using conventional nucleic acid transfection methods, such as lipid-mediated transfection, electroporation, and calcium phosphate transfection.

[0132] Peptide Production The peptides can be prepared in any suitable manner, for example, they can be produced recombinantly and / or synthetically.

[0133] Peptides may be synthesized by standard chemical techniques, such as synthesis by automated procedures using commercially available peptide synthesizers. Typically, peptide analogs are prepared by solid-phase peptide synthesis, which may involve coupling each protected amino acid residue to a resin, preferably a 4-methylbenzhydrylamine resin support, by activation with dicyclohexylcarbodiimide to give peptides with C-terminal amides. Alternatively, chloromethyl resin (Merrifield resin) may be used to give peptides with a free carboxylic acid at the C-terminus. After attachment of the last residue, the protected peptide-resin is treated with hydrogen fluoride to cleave the peptide from the resin and deprotect the side chain functional groups. The crude product may be further purified by gel filtration, high pressure liquid chromatography (HPLC), partition chromatography, or ion exchange chromatography.

[0134] Optionally, and as outlined above, various groups may be introduced into the agent peptide during synthesis or during expression that allow for linkage to other molecules or surfaces, for example, cysteine ​​to create thioesters, histidine to link to metal ion complexes, carboxyl groups to form amides or esters, amino groups to form amides, etc.

[0135] 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; whereas the "coupled" and "ligated" systems begin with a DNA template, which is transcribed into RNA and subsequently translated.

[0136] Alternatively, the peptides may be produced by transfecting host cells with an expression vector containing a polynucleotide(s) encoding one or more peptides.

[0137] For recombinant production, a recombinant construct containing a sequence encoding one or more 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, bombardment or infection.

[0138] One or more 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 growing 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 retained for further purification of the peptide or its variants.

[0139] 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, baculovirus, herpes virus, and retrovirus. Polynucleotides can be introduced into the expression vector by conventional procedures known in the art.

[0140] The polynucleotide encoding one or more peptides may 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, E. coli lac or trp, phage lambda PL promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or viruses. The expression vector may also include a ribosome binding site for translation initiation and a transcription terminator.

[0141] An expression vector can also include an origin of replication and a selection marker, such as the ampicillin resistance gene of E. coli, that permits selection of transformed cells, i.e., cells that express the heterologous polynucleotide. A nucleic acid molecule encoding one or more peptides can be incorporated into the vector in frame with translation initiation and termination sequences.

[0142] One or more peptides can be recovered and purified from recombinant cell cultures (i.e., from the cells or from the culture medium) by well-known methods, such as 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.

[0143] It is preferred to use recombinant techniques to produce glycosylated peptides. It is preferred to use mammalian cells, such as COS-7 cells and Hep-G2 cells, in recombinant techniques to produce glycosylated peptides.

[0144] Peptides can also be prepared by cleavage of longer peptides, particularly from food extracts.

[0145] Pharmaceutically acceptable peptide salts can be synthesized from peptides containing a basic or acidic moiety by conventional chemical methods. In general, 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.

[0146] Methods for identifying peptide sequences Disclosed herein are novel synthetic polyepitope-containing peptide sequences generated by novel discovery and generation methods. Disclosed herein are novel epitope sequences generated by novel epitope discovery and generation methods. In one embodiment, the method of generating novel synthetic peptide sequences involves the discovery of novel epitope sequences in polypeptides that can bind to antibodies or elicit an immune response in subjects with autoimmune disease and that stimulate antibody production in subjects with autoimmune disease. Once epitope sequences are discovered, they are recombined with other epitope sequences discovered or with random sequences to generate novel synthetic polypeptide sequences that have better 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 in a peptide array configured to contact the sample.

[0147] In some embodiments, as illustrated in FIG. 1, a method for identifying novel epitopes comprises the steps of: 1) generating a first plurality of overlapping polypeptide fragments, each of which comprises a portion of a native active protein or polypeptide; 2) determining the specificity and sensitivity of autoimmune disorder-correlated antibodies for each of the polypeptide fragments by contacting an array comprising the polypeptide fragments with a sample from a subject having an autoimmune disorder; 3) selecting a polypeptide fragment that exceeds a predetermined threshold for binding sensitivity and / or specificity or has a maximum sensitivity and / or specificity value for the set of polypeptide fragments; 4) selecting a polypeptide fragment that exceeds a predetermined threshold for binding sensitivity and / or specificity, or has a maximum sensitivity and / or specificity value for the set of polypeptide fragments; From the polypeptide fragments identified in step 3, identifying the occurrence of epitope sequences within the polypeptide fragments; 5) generating a second plurality of synthetic polypeptides, each comprising at least two epitope sequences from step 4, and optionally comprising at least one random polypeptide sequence; 6) determining the specificity and sensitivity for each of the synthetic polypeptides generated in step 5 by contacting an array comprising the synthetic polypeptide fragments with a sample from a subject having an immune disorder; and 7) selecting the synthetic polypeptides from step 6 that exceed the specificity and sensitivity thresholds for use as biomarkers for autoimmune disorders. Optionally, steps 5-7 can be repeated to further increase the sensitivity and / or specificity of the synthetic polypeptides for binding antibodies associated with autoimmune disorders. This method generates a plurality of novel synthetic polypeptides useful for diagnosing and treating autoimmune disorders (e.g., CeD).

[0148] In one embodiment, the autoimmune disorder is CeD. In one embodiment, the proteins that generate the novel synthetic polypeptide are GP and tTG. In one embodiment, GP is α-gliadin, β-gliadin, γ-gliadin, or ω-gliadin.

[0149] Identification of antigen epitopes As disclosed herein, methods are provided for identifying epitopes of proteins such as GP and tTG, for use in the generation of novel synthetic polypeptide sequences for use in the diagnosis and treatment of autoimmune diseases. In one embodiment, a full-length polypeptide sequence is divided into overlapping polypeptide fragments of a discrete length. In one embodiment, each polypeptide fragment is 6-15 amino acids long. In one embodiment, each polypeptide fragment is 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long. In a preferred embodiment, each polypeptide fragment is 12 amino acids long. The amount of overlap between polypeptide fragments of a full-length polypeptide can be determined by a polypeptide fragment step size, which refers to the distance between the respective N-terminal or C-terminal amino acids of each polypeptide fragment as determined by the full-length polypeptide. An illustration of an embodiment in which the step size is 2 amino acids is shown in FIG. 2A, where the length of the polypeptide fragments is 12 amino acids. As a result, the overlap between adjacent polypeptide fragments is 10 amino acids. This overlap allows more accurate determination of the active epitope sequence in the 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 a preferred embodiment, the step size is 2 amino acids. A step size of 1 amino acid can also be used to improve accuracy, although it requires the generation of more fragment polypeptides.

[0150] Based on the above scheme for generating polypeptide fragments, the fragment polypeptides are synthesized in an array for screening for samples with antibodies that correlate with autoimmune disorders. Binding of the antibodies 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 believed to be 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. The 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 a length of 3 amino acids.

[0151] In some embodiments, for example, where the polypeptide comprises a biologically active polypeptide such as a GP, epitope pairs are identified in the polypeptide fragment that exceed a threshold of binding specificity and / or sensitivity to autoimmune positive samples. These epitope pairs can then be used to generate novel biologically active sequences, as described below.

[0152] Generation of novel biologically active sequences The epitopes identified from the native bioactive polypeptides described above are used to generate and synthesize novel synthetic bioactive polypeptide sequences in an array for further screening. In one embodiment, each novel synthetic bioactive polypeptide comprises at least one epitope identified by the methods disclosed herein. In another embodiment, each novel synthetic bioactive polypeptide comprises at least two epitopes identified by the methods disclosed herein. In some embodiments, each novel synthetic bioactive polypeptide comprises two, three, four, or five epitopes identified by the methods described herein. In some embodiments, each novel synthetic bioactive polypeptide comprises 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 includes two of the three amino acid epitope sequences identified by the methods disclosed herein and at least one randomly generated polypeptide sequence, generating a novel synthetic bioactive polypeptide sequence of 12 amino acids. In an embodiment, the novel synthetic bioactive polypeptide sequence is selected from SEQ ID NOs: 1-172. In an embodiment, a plurality of novel synthetic bioactive polypeptide sequences are synthesized on an array for contact with samples to determine the sensitivity and specificity of each novel synthetic bioactive polypeptide sequence for detecting samples having an autoimmune disorder. In an embodiment, a novel synthetic bioactive polypeptide having high sensitivity and / or specificity for detecting an autoimmune disorder is selected for further modification of the random polypeptide sequence around the epitope 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.

[0153] In some embodiments, a polypeptide array is generated having a plurality of synthetic bioactive polypeptide sequences provided herein. In some embodiments, 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 generated by the methods disclosed herein. In some embodiments, 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: 1-172. In some embodiments, the polypeptide array exhibits greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sensitivity for detection of an autoimmune disorder in subjects suspected of having an autoimmune disorder. In certain embodiments, the polypeptide array exhibits greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% detection specificity for an autoimmune disorder in a subject suspected of having an autoimmune disorder.

[0154] Peptide sequences and methods of use array Methods of using the substrates, formulations, and / or arrays are also disclosed herein. Uses of the arrays disclosed herein include research applications, therapeutic purposes, medical diagnostics, and / or stratification of one or more patients.

[0155] Any array described herein can be used as a research tool or for research applications. In some embodiments, the array can be used for high-throughput screening assays. For example, enzyme substrates (i.e., peptides on the peptide array described herein) can be tested for the presence or absence of enzyme substrate(s) on the array by, for example, detecting and identifying at least one change in the features of the array.

[0156] Arrays can also be used in screening assays for ligand binding to determine substrate specificity, or to identify peptides that inhibit or activate proteins. Labeling techniques, protease assays, and binding assays useful in carrying out these methods are generally well known to those of skill in the art.

[0157] In some embodiments, arrays can be used to present a known protein sequence as a sequence of overlapping peptides. 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.

[0158] In some embodiments, the antigen presentation of the array includes at least one region, i.e., a region that spans the entire antigen sequence of a known protein, via epitope sliding; and the immunoreactive regions of the antigen are determined by contacting one or more clinical samples on the array, or on a plurality of different arrays, reducing the set of peptide sequences required to present the known protein antigen, and the array is used in a method.

[0159] In some embodiments, the sample is applied to an array with a plurality of random peptides. The random peptides can be screened and BLAST searched to determine homologous domains with, for example, 90% or more identity to a given antigen sequence. In some aspects, the entire antigen sequence can then be synthesized and used to identify potential markers and / or causes of the disease of interest.

[0160] In some embodiments, the array is used in 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.

[0161] 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 disease. Biomarkers can be expressed, absent, or at different levels in an individual depending on the disease state, stage of the disease, 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.

[0162] The array can also be used for therapeutic purposes, e.g., for the identification of one or more bioactive agents. A method for identifying a bioactive agent can include applying a plurality of test compounds to the array and identifying at least one test compound as a bioactive agent. The test compound can be a small molecule, an aptamer, an oligonucleotide, a chemical, a natural extract, a peptide, a protein, a fragment of an antibody, an antibody-like molecule, or an antibody. The bioactive agent can be a therapeutic agent or a modulator of a therapeutic target. The therapeutic target can include phosphatases, proteases, ligases, signaling molecules, transcription factors, protein transporters, protein sorters, cell surface receptors, secreted factors, and cytoskeletal proteins.

[0163] In another embodiment, the array can be used to identify drug candidates for therapeutic use. For example, when 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 target antibodies in diseases.

[0164] In some embodiments, the array is also provided for use in medical diagnostics. The array can be used to determine the response to administration of a drug or vaccine. For example, an individual's response to a vaccine can be determined by detecting the individual's antibody levels using an array with peptides that represent epitopes recognized by antibodies generated by an induced immune response. Another diagnostic application is to test an individual for the presence of a biomarker, by taking a sample from the subject and testing the sample for the presence of one or more biomarkers.

[0165] 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 therapeutic treatment. Arrays can be used to identify known biomarkers to determine appropriate treatment groups. For example, samples from subjects with a disease state can be applied to the array. Binding to the array can indicate the presence of biomarkers for that disease state. Previous studies can show that biomarkers are associated with positive outcomes after treatment, whereas the absence of biomarkers is associated with negative or neutral outcomes after treatment. Because a patient has biomarkers, medical professionals can stratify the patient into groups that will receive treatment.

[0166] 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 as 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 bodily fluid, such as amniotic fluid, aqueous humor, vitreous fluid, bile, serum, breast milk, cerebrospinal fluid, earwax, chyle, endolymph, perilymph, feces, female vaginal fluid, gastric acid, gastric juice, lymph, mucus, peritoneal fluid, pleural fluid, pus, saliva, sebum, semen, sweat, synovial fluid, tears, vaginal secretions, vomit, or urine.

[0167] In some embodiments, a method of identifying a vaccine candidate can include obtaining an array as disclosed herein in contact with a sample from a subject previously administered a 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 that comprise subsequences derived from a source protein having a known sequence.

[0168] Also disclosed herein is a method for fabricating an array. In some embodiments, the arrays disclosed herein can be synthesized in situ on a surface, for example, on a substrate disclosed herein. In some cases, photolithography is used to create the array. For example, a mask can be used to control the irradiation or exposure of a linker molecule with a protecting group to a specific location on a prepared surface. At the exposed location, the protecting group is removed, and one or more reactive moieties are newly exposed 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 positionally defined locations on a surface (see, e.g., U.S. Pat. No. 5,143,854 to Pirrung et al., and U.S. Patent Application Publication Nos. 2007 / 0154946, filed Dec. 29, 2005, 2007 / 0122841, filed Nov. 30, 2005, 2007 / 0122842, filed Mar. 30, 2006, 2008 / 0108149, filed Oct. 23, 2006, and 2010 / 0093554, filed Jun. 2, 2008, each of which is incorporated by reference herein). Other preferred methods and compositions useful for synthesizing features on arrays, including the fusion peptides described herein, are disclosed in PCT Publication No. WO2016 / 040703, "Peptide Microarrays and Novel Biomarkers For Celiac Disease," published March 17, 2016, the entire contents of which are incorporated by reference herein.

[0169] Vaccines and Administration The present invention also provides vaccines comprising synthetic polypeptide-containing peptides, 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. Some embodiments of vaccines and vaccine administration can be found in PCT Publication No. WO2016 / 040703, published March 17, 2016, entitled "Peptide Microarrays and Novel Biomarkers For Celiac Diseases," the entire contents of which are incorporated herein by reference. EXAMPLES

[0170] The following examples illustrate methods for identifying biomarkers for celiac disease. The biomarkers include a set of peptides from known antigens in celiac disease, including, but not limited to, alpha, beta, gamma, and omega gliadin, their deamidated modifications, and tTG. The method includes 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 sequences of the known celiac antigens either two amino acids or three amino acids at a time. FIG. 2A illustrates the identification of 12-mer sequences based on shifting only two amino acids along the alpha / beta gliadin sequence. The identification of the tTG 12-mer sequence can be done in a similar manner as for the gliadin peptides, but is not explicitly described herein. FIG. 2B illustrates the deamination of one or two glutamines at a time in the 12-mer GP to enlarge the DGP portion of the peptide library. The peptide library was then synthesized on a microarray, as described in more detail below. Coupling yields during peptide synthesis on the array were continuously monitored for peptide yield, purity, and sequence fidelity using fluorescence, mass spectrometry, and monoclonal antibody-bound substrate assays.To identify biomarkers based on B-cell epitopes of tTG and DGP, peptide microarrays containing 2.1 million different peptides derived from peptide libraries of tTG and DGP, including triplicate repeats of each peptide, were synthesized, extracted, and placed on a plate containing 96 pillars.

[0171] Example 1: Wafer Substrate Preparation Premium 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. The wafers were deposited with 1000 Å thermal oxide by dry oxidation at 1000° C. for 2 hours in a furnace under pure oxygen atmosphere. The wafers were spin coated with commercial photoresist P5107 at 2000 rpm for 40 seconds using a Sokudo RF3S Coat / Develop Track. The wafers were exposed at 248 nm using a reverse zero layer mask using 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). For the oxide etch, the wafers were subjected to a wet oxide etch using buffered hydrofluoric acid prepared by mixing 5 parts 40 wt% ammonium fluoride (Sigma) with 1 part 49 wt% hydrofluoric acid (Sigma) for 1 minute. The wafers were then stripped with Nanostrip (CyanTek) for 24 hours and finally rinsed with DI water and ultrasonically cleaned in DI water for 10 minutes. This process, illustrated in Figure 3A, resulted in a substrate having a portion of features that included thermal oxide and measured 1000 Å in height, and a portion of non-features that included silicon.

[0172] A DI 5000 AFM system was used to measure the roughness of the substrate and calculate the density. Figure 3B shows the pillars illustrated in Figure 3A and their dimensions formed after the process described above. Figure 3C illustrates the root mean square (RMS) roughness of the substrate. The density of the substrate was calculated to be about 100-150 pM.

[0173] Example 2: Wafer surface derivatization The wafer was thoroughly washed with DI water for 5 min and spin-coated with a solution of 1.25% (v / v) 3-aminopropyltriethoxysilane [APTES] (Sigma Aldrich) in N-methyl-pyrrolidone [NMP] (BDH) and then left at room temperature for 15 min. The wafer was cured at 120°C for 60 min under N2 atmosphere. The wafer was then spin-coated with a coupling solution of 2 wt% Fmoc-Gly-OH (Anaspec), 2 wt% HOBt (Anaspec), and 2 wt% N,N'-diisopropylcarbodiimide [DIC] (Sigma Aldrich) in NMP and baked at 60°C for 5 min. This allowed for coupling of Fmoc-glycine with the free amines present in the APTES. The wafer was then rinsed with NMP and capped with 50% (v / v) acetic anhydride mixed with 50% NMP to cap any remaining free amines that were not coupled. 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 NMP containing 5% (v / v) piperidine (Sigma Aldrich) and baking at 80°C for 300 seconds. The linker Fmoc-(PEG)4-COOH (Anaspec) was then coupled to the wafer surface with a coupling solution containing 2% by weight of linker, 2% by weight of HOBt (Anaspec), and 2% by weight of N,N'-diisopropylcarbodiimide [DIC] in NMP and baking at 90°C for 120 seconds. The wafer was then rinsed with NMP and capped with 50% (v / v) acetic anhydride mixed with 50% NMP to cap any residual uncoupled free amines. To complete the surface derivatization process, the wafer was stripped with acetone and IPA.

[0174] Example 3: Peptide array synthesis The steps taken to synthesize peptides on an array are illustrated in FIG. 4 and have been previously described in detail.

[0175] 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. Then, 2 wt% Fmoc-amino acid (Anaspec) was added to the solution, and 2 wt% HOBt (Anaspec) was added. Finally, 1 wt% tetrazolethione was added to the solution. The solution was then filtered using a 0.05 μm filtration device.

[0176] Carbodiimide formation mechanism Photoactivated carbodiimide coupling was carried out as follows. TIFF2025011239000006.tif30131

[0177] Tetrazolethione was used, which upon exposure to 248 nm triggered a ring-opening mechanism and released a carbodiimide to activate the carboxylic acid group of the amino acid coupled to the wafer. 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 acids to form stable esters for efficient coupling.

[0178] Amino acid coupling A base resist solution containing 1 wt% polymer and 3 wt% piperidine in NMP was spin coated onto the wafer at 3000 rpm for 30 seconds and soft baked on a hot plate at 65°C for 1 minute. The wafer was then baked at 80°C for 300 seconds. The Fmoc protection was removed from all features, leaving unprotected amine groups. An input amino acid activation solution ... input amino acid was exposed to a dose of 120 mJ / cm. 2 The wafer was exposed using a reticle exposing the desired features that need to be coupled with 1000 nm of ... This entire process was repeated for each individual coupling layer of amino acids that were designed to be coupled to complete the synthesis of the peptide chains attached to the array surface.

[0179] Side chain protection removal After completing the peptide synthesis, residual side chain protection present on any coupled amino acids was removed to achieve peptide biological activity. The side chain protection removal solution was prepared by mixing 95 wt% trifluoroacetic acid [TFA] (Sigma Aldrich) and 5 wt% DI water. The wafer was reacted with the side chain protection removal solution for 90 min. This step was followed by successive washing of the wafer with TFA (5 min), IPA (5 min), NMP (5 min), neutralization with NMP containing 5 wt% DIEA (Alfa Aesar) (5 min), and then successive washing of the wafer with NMP (5 min) and IPA (5 min).

[0180] Example 4: Generation of a novel synthetic CeD biomarker from a combination of tTG-DGP complexes for the diagnosis of CeD To identify novel biomarkers of CeD, serum samples were obtained from an exploratory cohort of 90 patients with biopsy-proven CeD and 79 healthy control patients. 3 The clinical characteristics of the exploratory cohort are shown in Table 2A below.

[0181] Table 2A. Clinical characteristics of the exploratory cohort TIFF2025011239000007.tif43128

[0182] Serum samples were also obtained from a validation cohort of 82 patients diagnosed with CeD and 217 control patients. The clinical characteristics of the validation cohort are shown in Table 2B below. The validation cohort was used to validate the diagnostic utility of the biomarkers discovered in the exploratory cohort. The 82 patients diagnosed with CeD in the validation cohort included 4 patients with IgA deficiency.

[0183] Table 2B. Clinical characteristics of the validation cohort TIFF2025011239000008.tif43128

[0184] For solid-phase peptide synthesis, silicon-based wafers (300 mm diameter) with 100 nm height and thermal oxide coated feature and non-feature portions were created using photolithography and inductively coupled plasma deep etching techniques. The surface of the prepared silicon-based wafer contained a monolayer of aminosilanes providing peptide attachment sites, and peptide synthesis was carried out using fluorenylmethoxycarbonyl (Fmoc) chemistry. After removal of the Fmoc protection, the unprotected amines were coupled with the desired incoming Fmoc amino acids using a specific reticle that activated only the desired sites where the incoming amino acid should be coupled. This process was repeated for each individual layer of amino acids to generate the desired peptide sequence on each feature portion.

[0185] A set of approximately 66,000 12-mer peptides with sequences that are lateral shifted by two amino acids from the α, β, γ, and Ω fractions of gliadin were synthesized on silicon-based wafers. In addition, in these synthetic GPs, each glutamic acid was exchanged for a glutamine position to mimic the deamidation of GP (DGP). Using a peptide microarray immunoassay, we assessed the predominant 3-mer GP sequence with high antibody binding strength relative to the native peptide, DGP, and CeD. 3 Overlapping 12-mer peptides and tTG of various lengths were also synthesized following a similar scheme as for GP. In addition, novel combination sequences, which are combinations of the major 3-mer GP sequence and tTG subsequences, were synthesized on silicon-based wafers. For example, the novel combination sequence In TIFF2025011239000009.tif6170 (SEQ ID NO: 173), YGDGVS (SEQ ID NO: 174) is derived from tTG (positions 245-250), and TIFF2025011239000010.tif6170 is the main 3mer GP sequence. The method for selecting new combination tTG-DGP sequences is shown in FIG.

[0186] FIG. 5 shows examples of combined epitopes of tTG-DGP complexes according to embodiments. Specifically, FIG. 5 shows examples of three different ways of combining tTG and GP segments. YGDGVS (SEQ ID NO: 174) is at position 245-250 of the tTG peptide, and PEQ and PEP are two important 3-mer amino acids of GP. Top row 1, YGDGVS (SEQ ID NO: 174) is followed by PEQ and PEP. Middle row, YGDGVS (SEQ ID NO: 174) is between PEQ and PEP. Bottom row, PEQ and PEQP (SEQ ID NO: 175) are followed by YGDGVS (SEQ ID NO: 174). E indicates glutamic acid; tTG, tTG; Q, glutamine; Y, tyrosine; D, aspartic acid; G, glycine; V, valine; P, proline; F, phenylalanine; S, serine.

[0187] The fluorescent peptide microarray platform was used to estimate the antibody binding intensity of each novel synthetic tTG-DGP neoepitope. Using a region of interest stitching program JAVA, image files obtained from scanning the peptide microarray chip were converted into individual antibody binding intensity values ​​calculated using the median foreground intensity, and then a binary log transformation was applied to stabilize the variance. Each antibody binding intensity value is associated with the corresponding peptide sequence.

[0188] A random forest model was used to remove low-confidence peptide sequences of the tTG-DGP complex. 16 A random forest classifier was trained to detect portions of peptide sequences whose values ​​were not found within the 95% linear regression confidence band of a single linear regression analysis of the multiple assays (performed using the rapmad [Robust Analysis of Peptide MicroArray Data] R-package). 17Additionally, background normalization modeling was also applied, which was performed using the expectation-maximization algorithm (implemented using the R-package) to place blank spots, but the sequences were not synthesized.

[0189] After eliminating background noise and low-confidence peptide sequences, support vector machine modeling was performed. 18 was applied to a training set of approximately 55,000 samples to construct a hyperplane and maximize the difference in training data between two classes (CeD and non-CeD) (performed using a Python package) with the aim of identifying disease-associated peptide sequences of tTG-DGP complexes. Based on the results of the support vector machine training, the identified disease-associated peptide sequences were then tested on unknown samples to calculate the prediction accuracy, sensitivity, and specificity. Furthermore, receiver operating characteristic (ROC) curve analysis was performed to determine the sensitivity and specificity of each peptide. The threshold of the ROC curve for each peptide was determined by selecting the value with the highest sensitivity and specificity. Furthermore, principal component analysis, hierarchical cluster analysis using heatmap, and random forest multivariate analysis were performed using R- or Python packages. 19

[0190] Example 5: Correlation between immunoreactivity of novel synthetic CeD biomarkers and CeD severity The synthesized tTG peptide fragments were tested with serum samples from the discovery cohort to determine immunoreactivity to the tTG fragments. Figures 6A-6B show heat maps showing immunoreactivity to tTG and tTG-DGP complexes according to an embodiment. Specifically, Figure 6A shows immunoreactivity to tTG peptide according to an embodiment. No significant difference in immunoreactivity was observed between serum samples from CeD patients and control patients.

[0191] Synthetic 12-mer neoepitopes derived from tTG and native peptides or the primary 3-mer motif of DGP were also tested in serum samples from the discovery cohort to identify immunogenic epitopes, defined as any sequence with an area under the ROC curve value of >0.7. A total of 172 immunogenic epitopes of the tTG-DGP complex were identified. The sequences for each of the 172 immunogenic epitopes of the identified tTG-DGP complex are shown in Table 1 above. As previously mentioned, each epitope in Table 1 demonstrated high sensitivity and specificity in diagnosing CeD in healthy control patients, with an area under the ROC curve of >0.7.

[0192] 6B shows the immunoreactivity against the neoepitope of the tTG-DGP complex according to the embodiment. As shown in FIG. 6B, the antibody binding intensity of the neoepitope of the tTG-DGP complex was significantly increased in serum samples from CeD patients, whereas the immunoreactivity was minimal or nearly zero in controls.

[0193] In an exploratory cohort, the identified set of neoepitopes derived from tTG-DGP complexes showed very high sensitivity (99%) and specificity (100%) in diagnosing CeD. To validate the discriminatory power of this set of tTG-DGP complexes, serum samples from a validation cohort of 82 patients with CeD and 217 control patients were analyzed in a blinded study. This set of tTG-DGP complexes distinguished CeD cases from controls with high accuracy, achieving a sensitivity of 99% and a specificity of 100%. Notably, high sensitivity and specificity were observed when using these neoepitopes to distinguish CeD cases from controls, compared with current serological tests for CeD, such as tTG-IgA and DGP-IgA (especially the tTG-IgA and DGP-IgA ELISA tests). Table 3 below compares the sensitivity, specificity, overall accuracy, positive predictive value (PPV), and negative predictive value (NPV) of the neoepitope of the tTG-DGP complex with current serological tests for CeD, including the tTG-IgA and DGP-IgA ELISA tests, in diagnosing CeD. Overall, the neoepitope of the tTG-DGP complex showed equal or better diagnostic accuracy for identifying CeD than clinically available serological tests.

[0194] Table 3. Predictive value of tTG-DGP complex for current serodiagnostic tests for CeD diagnosis TIFF2025011239000011.tif57168

[0195] To compare the immunoreactivity to epitopes of DGP, tTG, and tTG-DGP complex, we tested serum samples from selected disease controls without CeD and villous atrophy, consisting of 10 patients with autoimmune enteropathy, 6 patients with CVID with enteropathy, and 11 patients with drug-induced enteropathy. We found that the immunoreactivity to neoepitopes of tTG-DGP complex in these disease controls was significantly lower than that in patients with CeD and similar to that in other control patients. In addition, 4 patients with complete IgA deficiency were included in the validation set of CeD patients. All of these patients were negative for tTG-IgA, but the immunoreactivity to neoepitopes of tTG-DGP complex, especially IgG immunoreactivity, was increased in these IgA-deficient patients. Furthermore, the patients with intestinal villous atrophy but not CeD did not show any immunoreactivity to neoepitopes of tTG-DGP complex.

[0196] Example 6: Evaluation of novel synthetic CeD biomarkers to determine healing status in CeD patients adhering to a GFD To evaluate the identified biomarkers for identifying the mucosal healing status of patients treated for CeD, serum samples were collected from mucosa of treated and cured CeD patients (n=85), treated but not cured CeD patients (n=81), untreated CeD patients (n=82), disease control patients (n=27), and healthy control patients (n=217). Mucosal healing status was defined as persistent villous atrophy under adherence to the GFD or histological recovery (without villous atrophy). Patients with uncured CeD and refractory CeD were not included in this study. Mucosal healing status in the small intestine was classified based on pathological reports; treated CeD patients with partial or total villous atrophy were classified into the treated but not cured CeD group. Disease control patients were defined as villous atrophy without CeD. The 27 disease control patients consisted of 10 patients with autoimmune enteropathy, 6 patients with common variable immunodeficiency-associated enteropathy, and 11 patients with drug-induced enteropathy.

[0197] Table 4 shows the characteristics of patients treated with CeD according to the healing status of the mucosa. Patients with treated and healed CeD mucosa were, on average, younger than patients with treated but not healed CeD mucosa, but the genders were similar (73% and 72% of patients, respectively, were female). Patients with treated but not healed CeD mucosa adhered longer to the GFD than treated and healed CeD patients, which was not statistically significant (P = .16). Seven percent of patients with treated and healed CeD mucosa were positive for tTG-IgA, whereas 27% of patients with treated but not healed CeD mucosa were positive for tTG-IgA, and about three-quarters of patients with treated but not healed CeD mucosa were negative. In addition, 48% of patients with treated but not healed CeD mucosa were positive for DGP-IgA, whereas 9% of patients with treated but healed CeD mucosa were positive for DGP-IgA.

[0198] Table 4. Characteristics of cured and non-cured patients treated for CeD TIFF2025011239000012.tif83160

[0199] 7A-7B show the immune reactivity to the epitopes of the tTG-DGP complex based on the antibody binding intensity according to an embodiment. Specifically, FIG. 7A-7B show the immune reactivity to the neo-epitopes of the tTG-DGP complex in the treated CeD patients according to the cured state. FIG. 7A shows the immune reactivity to the epitopes of the tTG-DGP complex in the untreated CeD, the treated but not cured CeD, and the treated and cured CeD patients, as well as the healthy control patients, according to an embodiment. As shown in FIG. 7A, the untreated CeD and the treated but not cured CeD patients show a greater antibody binding intensity compared to the healthy control patients, the treated and cured CeD patients, and the disease control patients with villous atrophy due to autoimmune enteropathy, common variable immunodeficiency-associated enteropathy, or drug-induced enteropathy.

[0200] FIG. 7B shows a principal component analysis of immunoreactivity to neoepitopes of the tTG-DGP complex according to an embodiment. Specifically, FIG. 7B shows the correlation between the level of immunoreactivity to the tTG-DGP complex and the CeD phenotype. Treated and cured CeD patients and healthy control patients are displayed together in the PCA plot.

[0201] Overall, as shown in Figure 7A, immunoreactivity to neoepitopes of the DGP-tTG complex was stronger in patients with untreated but unhealed CeD mucosa than in patients with treated and healed CeD mucosa and in control patients. The mean antibody binding intensities of neoepitopes derived from the tTG-DGP complex were significantly different among the five groups (P<.001). The immunoreactivity decreased stepwise according to the intestinal mucosal injury status, with the highest mean (SD) reactivity in patients with untreated but unhealed CeD mucosa (32.5 [16.4]), followed by patients with treated but unhealed CeD mucosa (15.1 [7.5]), treated and healed CeD mucosa (5.5 [3.4]), control patients (1.3 [0.5]), and disease controls (1.3 [0.4]). Furthermore, in the principal component analysis shown in Figure 7B , patients with treated and healed CeD mucosa and control patients formed clusters, whereas patients with treated but unhealed CeD mucosa and patients with untreated CeD mucosa were similarly distributed.

[0202] FIG. 8 shows a comparison of antibody binding levels of tTG-immunoglobulin A complexes and tTG-DGP complexes in treated but not cured CeD patients according to an embodiment. More specifically, FIG. 8 shows that the neo-epitopes of the tTG-DGP complexes can diagnose treated but not cured CeD mucosa compared to the tTG-IgA enzyme-linked immunosorbent assay (ELISA) test. Although about 75% of treated but not cured CeD patients were negative for tTG-IgA, most of these patients showed increased immune reactivity to the neo-epitopes of the tTG-DGP complexes. Table 5 below compares the sensitivity, specificity, overall accuracy, positive predictive value (PPV), and negative predictive value (NPV) of the neo-epitopes of the tTG-DGP complexes in identifying the cured status of treated but not cured CeD patients with current serological tests for CeD, such as the tTG-IgA and DGP-IgA ELISA tests. Compared with the tTG-IgA ELISA test (especially the tTG-IgA and DGP-IgA ELISA tests), the neoepitope of the tTG-DGP complex showed high sensitivity (84%) and specificity (95%) for identifying the healing status in patients with treated but unhealed CeD mucosa, with a positive predictive value of 0.94 and a negative predictive value of 0.86.

[0203] Table 5. Predictive value of tTG-DGP complexes versus current serodiagnostic tests for identifying treated CeD cured status TIFF2025011239000013.tif54167

[0204] Biomarker discovery via the highly efficient mass production platform of ultra-high density peptide microarrays presented herein provides an efficient method for mapping antigens and determining novel epitopes through combination with immune enhancing sequences. Using a peptide microarray based on 2.1 million 9mer-15mer peptides, each overlapping by 3 or 6 amino acids, providing extremely high density coverage of immunogenic proteins maximizing the ability to identify informative peptides, we have demonstrated the efficacy and utility of this technology to identify novel epitopes that are 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 test panels for autoimmune diseases, including celiac disease. Furthermore, by designing peptide microarrays with lateral shifts of one amino acid, we were able to evaluate the contribution of individual amino acids of the antigen to antibody binding and achieve higher mapping resolution for the target antigen.

[0205] All previous methods for in situ synthesis of microarrays were based on photolithography 5、22-24is based on an individually addressable deprotection step followed by coupling of monomers to the selectively deprotected sites. The method described herein includes a generalized deprotection followed by selective activation, which provides two advantages: 1) very high fidelity peptide synthesis and 2) a significant reduction in the time required for each step. This allows for a very large number of steps, up to 400, in the synthesis of peptide microarrays with very little yield loss. In some embodiments, the combination of high fidelity and short reaction times allows for much higher yields and high volume chip production capacity. Other advantages include cost reduction 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 very large density of the microarrays allows for large scale biomarker validation as well as molecular diversity required for biomarker discovery. The method reduces the chip size to 0.5×0.5 mm, which fits into any diagnostic well plate format, such as 96-well, 384-well, 1396-well, etc. 2 The size of the ELISA kit can be reduced to as small as 1 mm, making it ideal for mass production of routine diagnostic tests. This allows even smaller samples to be used for routine diagnostic tests.

[0206] The method disclosed herein is a non-invasive, widely available, low-cost, and versatile method that is ideal for routine medical testing purposes and represents a powerful new tool for biomarker discovery, by using the disclosed peptide microarrays.

[0207] 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.

[0208] All references, issued patents, and patent applications cited throughout the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0209] Finally, it should be noted that the language used herein has been chosen primarily for ease of reading and instructional purposes, and not for the purposes of precisely delineating or limiting the subject matter of the present invention. Accordingly, the disclosure of the present invention is intended to be illustrative, and not limiting, of the scope of the invention.

[0210] References TIFF2025011239000014.tif228159TIFF2025011239000015.tif238158TIFF2025011239000016.tif162158

[0211] Sequence information SEQUENCE LISTING <110> VIBRANT HOLDINGS, LLC MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH <120> TTG-DGP BIOMARKERS FOR MONITORING CELIAC DISEASE <150> US 62 / 742,863 <151> 2018-10-08 <160> 194 <170> PatentIn version 3.5 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 1 Phe Glu Asp Gly Ile Leu Glu Gln Pro Pro Glu Gln 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 2 Pro Phe Pro Gln Lys Thr Val Glu Ile Pro Glu Gln 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 3 Phe Pro Leu Arg Asp Ala Pro Glu Gln Gln Pro Glu 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 4 Phe Pro Gln Gln Pro Phe Trp Leu Thr 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 Phe Asp Val Phe Ala His Pro Phe Pro Phe Pro Gln 1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 6 Ala Trp Cys Pro Ala Asp Phe Pro Glu Glu Gln Pro 1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 7 Phe Pro Glu Pro Ala Pro Ser Gln 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 Ala Glu Val Ser Leu Gln Glu Gln Pro Pro Glu Gln 1 5 10 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 9 Glu Met Ile Trp Asn Phe Pro Phe Pro Glu Gln Pro 1 5 10 <210> 10 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 10 Glu Gln Pro Pro Glu Gln Ala Glu Val Ser Leu Gln 1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 11 Phe Pro Glu Gln Pro Glu Tyr Gly Asp Gly Val Ser 1 5 10 <210> 12 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 12 Pro Phe Pro Pro Glu Gln Ala Leu Leu Val Glu Pro 1 5 10 <210> 13 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 13 His Asp Gln Asn Ser Asn Gln Pro Phe Gln Pro Glu 1 5 10 <210> 14 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 14 Pro Phe Pro Ser Val Asp Ile Leu Arg 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 Glu Gln Pro Leu Thr Gln Gln Gly Phe Glu Gln Pro 1 5 10 <210> 16 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 16 Phe Pro Glu Phe Pro Glu Val Val Asn Phe Glu Ser 1 5 10 <210> 17 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 17 Gln Pro Phe Gln Pro Glu Tyr Asn Ser Ala His Asp 1 5 10 <210> 18 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 18 Asp Leu Cys Arg Glu Lys Pro Glu Gln Glu Gln Pro 1 5 10 <210> 19 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 19 Glu Lys Leu Val Val Arg Pro Glu Gln Gln Pro Glu 1 5 10 <210> 20 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 20 Phe Pro Gln Pro Gly Tyr Glu Gly Trp Glu Gln Pro 1 5 10 <210> 21 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 21 Gln Pro Glu Gln Pro Glu Tyr Gln Gly Ser Ser Phe 1 5 10 <210> 22 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 22 Pro Phe Pro Asn Arg Ser Leu Ile Val Gln Pro Phe 1 5 10 <210> 23 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 23 Asp Cys Thr Leu Ser Leu Pro Glu Gln Gln Pro Glu 1 5 10 <210> 24 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 24 Pro Phe Pro Ser Val Asp Ser Leu Thr Phe Pro Glu 1 5 10 <210> 25 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 25 Asp Ala Val Glu Glu Gly Gln Pro Glu Pro Glu Gln 1 5 10 <210> 26 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 26 Ala Ser Thr Gly Tyr Gln Gln Pro Glu Pro Phe Pro 1 5 10 <210> 27 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 27 Phe Glu Gly Arg Asn Tyr Phe Pro Glu Phe Pro Gln 1 5 10 <210> 28 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 28 Glu Gln Pro Leu Gln Asn Pro Leu Pro Gln Pro Phe 1 5 10 <210> 29 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 29 Gly Trp Gln Ala Leu Asp Phe Pro Gln Pro Phe Pro 1 5 10 <210> 30 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 30 Pro Glu Gln Arg Lys Leu Val Ala Glu Phe Pro Glu 1 5 10 <210> 31 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 31 Gln Pro Glu Pro Val Pro Val Arg Ala Phe Pro Gln 1 5 10 <210> 32 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 32 Pro Phe Pro Gln Pro Phe Val Phe Ala Glu Val Asn 1 5 10 <210> 33 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 33 Gln Pro Phe Leu Ala Glu Arg Asp Leu Phe Pro Glu 1 5 10 <210> 34 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 34 Pro Glu Gln Pro Glu Gln Val Asp Gln Gln Asp Cys 1 5 10 <210> 35 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 35 Glu Gln Pro Ser Gly Met Val Asn Cys Glu Gln Pro 1 5 10 <210> 36 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 36 Phe Pro Glu Leu Cys Ala Arg Thr Val Pro Phe Pro 1 5 10 <210> 37 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 37 Pro Phe Pro Leu Leu Phe Asn Ala Trp Pro Phe Pro 1 5 10 <210> 38 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 38 His Leu Asn Lys Leu Ala Pro Glu Gln Gln Pro Glu 1 5 10 <210> 39 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 39 Glu Gln Pro Asn Ala Pro Ile Gly Leu Pro Phe Pro 1 5 10 <210> 40 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 40 Phe Pro Glu Arg Glu Ala Phe Thr Arg Glu Gln Pro 1 5 10 <210> 41 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 41 Phe Pro Gln Pro Phe Pro Ala Ala Val Ala Cys Thr 1 5 10 <210> 42 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 42 Gln Pro Phe Pro Glu Gln Tyr Cys Cys Gly Pro Val 1 5 10 <210> 43 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 43 Glu Gln Pro Gln Ser Met Asn Met Gly Pro Phe Pro 1 5 10 <210> 44 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 44 Cys Arg Leu Leu Leu Cys Pro Glu Gln Pro Glu Gln 1 5 10 <210> 45 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 45 Ile Pro Thr Arg Val Val Phe Pro Glu Glu Gln Pro 1 5 10 <210> 46 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 46 Gln Pro Phe Leu His Met Gly Leu His Gln Pro Glu 1 5 10 <210> 47 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 47 Pro Phe Pro Leu Ser Leu Glu Ala Ser Gln Pro Glu 1 5 10 <210> 48 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 48 Phe Pro Gln Asn Gly Arg Asp His His Gln Pro Phe 1 5 10 <210> 49 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 49 Gln Pro Glu Asn Asn Thr Ala Glu Glu Phe Pro Glu 1 5 10 <210> 50 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 50 Pro Phe Pro Leu Asp Pro Thr Pro Gln 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 Ala His Ile Thr Asn Asn Glu Gln Pro Glu Gln Pro 1 5 10 <210> 52 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 52 Phe Pro Gln Lys Val Arg Met Asp Leu Gln Pro Phe 1 5 10 <210> 53 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 53 Phe Pro Glu Met Gly Ser Asp Phe Asp Gln Pro Phe 1 5 10 <210> 54 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 54 Pro Glu Gln Lys Ser Val Gly Arg Asp Gln Pro Glu 1 5 10 <210> 55 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 55 Ile Lys Val Arg Ala Leu Pro Phe Pro Pro Glu Gln 1 5 10 <210> 56 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 56 Phe Pro Glu Asn Phe His Cys Trp Val Pro Glu Gln 1 5 10 <210> 57 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 57 Gly Arg Val Val Ser Gly Phe Pro Gln Gln Pro Phe 1 5 10 <210> 58 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 58 Gln Pro Glu Pro Phe Pro Ala Ser Thr Gly Tyr Gln 1 5 10 <210> 59 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 59 Ala Ala Val Ala Cys Thr Phe Pro Gln Pro Phe Pro 1 5 10 <210> 60 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 60 Pro Phe Pro Pro Glu Gln Trp Met Thr Arg Pro Asp 1 5 10 <210> 61 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 61 Pro Glu Gln Glu Gln Pro Trp Val Glu Ser Trp Met 1 5 10 <210> 62 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 62 Gln Pro Glu Pro Val Tyr Val Gly Arg Phe Pro Glu 1 5 10 <210> 63 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 63 Pro Glu Gln Asn Tyr Glu Ala Ser Val Gln Pro Phe 1 5 10 <210> 64 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 64 Glu Gln Pro Gln Pro Phe Val Val Asp Trp Ile Gln 1 5 10 <210> 65 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 65 Gln Pro Glu Gln Pro Glu Tyr Pro Glu Gly Ser Ser 1 5 10 <210> 66 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 66 Pro Phe Pro Pro Lys Gln Lys Arg Lys Gln Pro Phe 1 5 10 <210> 67 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 67 Gln Pro Phe Asn Phe Gly Gln Phe Glu Glu Gln Pro 1 5 10 <210> 68 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 68 Gln Pro Glu Gln Pro Phe Val Asn Ala Asp Val Val 1 5 10 <210> 69 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 69 Ala Leu Leu Val Glu Pro Pro Phe Pro Pro Glu Gln 1 5 10 <210> 70 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 70 Glu Gly Asp Leu Ser Thr Gln Pro Phe Gln Pro Phe 1 5 10 <210> 71 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 71 Pro Glu Gln Asn Cys Asn Asp Asp Gln Gln Pro Phe 1 5 10 <210> 72 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 72 Pro Phe Pro Thr Arg Ala Asn His Leu Pro Glu Gln 1 5 10 <210> 73 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 73 Asp Gln Gly Val Leu Leu Pro Glu Gln Gln Pro Glu 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 Cys Gly Thr Phe Pro Gln Gln Pro Phe 1 5 10 <210> 75 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 75 Phe Pro Gln Leu Val Leu Glu Arg Cys Gln Pro Phe 1 5 10 <210> 76 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 76 Gln Pro Phe Glu Gln Pro Val Val Thr Asn Tyr Asn 1 5 10 <210> 77 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 77 Gly Leu Tyr Arg Leu Ser Gln Pro Phe Glu Gln Pro 1 5 10 <210> 78 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 78 Ala Asp Ala Val Tyr Leu Pro Glu Gln Gln Pro Phe 1 5 10 <210> 79 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 79 Phe Pro Gln Ser Glu Gly Thr Tyr Cys Gln Pro Glu 1 5 10 <210> 80 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 80 Phe Pro Gln Ser Asn Leu Leu Ile Glu Pro Glu Gln 1 5 10 <210> 81 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 81 Glu Asn Pro Glu Ile Lys Phe Pro Gln Pro Phe Pro 1 5 10 <210> 82 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 82 Gln Pro Phe Gln Glu Tyr Val Leu Thr Phe Pro Gln 1 5 10 <210> 83 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 83 Gln Pro Phe Ser Trp Ile Gly Ser Val Phe Pro Gln 1 5 10 <210> 84 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 84 Glu Asp Ile Thr His Thr Glu Gln Pro Gln Pro Phe 1 5 10 <210> 85 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 85 Cys Gln Arg Val Lys Tyr Gln Pro Glu Pro Glu Gln 1 5 10 <210> 86 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 86 Glu Ile Pro Asp Pro Val Phe Pro Gln Gln Pro Glu 1 5 10 <210> 87 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 87 Glu Gly Ala Gly Leu Thr Gln Pro Glu Pro Glu Gln 1 5 10 <210> 88 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 88 Gln Pro Glu Ser Phe Val Leu Gly His Pro Glu Gln 1 5 10 <210> 89 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 89 Pro Glu Gln Lys Asn His Gly Cys Gln Glu Gln Pro 1 5 10 <210> 90 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 90 Pro Phe Pro Pro Gln Glu Lys Ser Glu Glu Gln Pro 1 5 10 <210> 91 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 91 Gln Pro Phe Pro Val Glu Ala Gly Glu Phe Pro Glu 1 5 10 <210> 92 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 92 Glu Gln Pro Met Ala Glu Glu Leu Val Phe Pro Glu 1 5 10 <210> 93 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 93 Ile Lys Ile Arg Ile Leu Pro Phe Pro Pro Glu Gln 1 5 10 <210> 94 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 94 Ile Leu Asp Ile Cys Leu Pro Phe Pro Phe Pro Gln 1 5 10 <210> 95 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 95 Phe Pro Glu Leu Thr Leu His Phe Glu Phe Pro Glu 1 5 10 <210> 96 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 96 Asp Leu Tyr Leu Glu Asn Gln Pro Phe Pro Glu Gln 1 5 10 <210> 97 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 97 His Thr Tyr Lys Tyr Pro Pro Phe Pro Phe Pro Gln 1 5 10 <210> 98 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 98 Glu Gln Pro Phe Pro Glu Val Ile Ile Gly Pro Ala 1 5 10 <210> 99 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 99 Asp Gly Ser Val His Lys Phe Pro Glu Pro Phe Pro 1 5 10 <210> 100 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 100 Phe Pro Gln Leu Glu Gly Cys Thr Phe Phe Pro Glu 1 5 10 <210> 101 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 101 Gln Pro Glu Arg Cys Asp Leu Glu Leu Gln Pro Phe 1 5 10 <210> 102 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 102 Gln Pro Glu Thr Lys Ala Arg Phe Pro Gln Pro Glu 1 5 10 <210> 103 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 103 Phe Pro Gln Arg Asn Glu Phe Gly Glu Phe Pro Glu 1 5 10 <210> 104 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 104 Cys Trp Val Phe Ala Ala Phe Pro Gln Gln Pro Glu 1 5 10 <210> 105 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 105 Phe Pro Glu Leu Ala Glu Lys Glu Glu Gln Pro Glu 1 5 10 <210> 106 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 106 Gln Pro Phe Pro Phe Pro Trp Asp Asn Asn Tyr Gly 1 5 10 <210> 107 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 107 Phe Pro Gln Arg Arg Ser Ser Pro Val Phe Pro Glu 1 5 10 <210> 108 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 108 Glu Ser Asn Leu Ile Lys Pro Glu Gln Gln Pro Phe 1 5 10 <210> 109 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 109 Asp Leu Leu Pro Leu His Glu Gln Pro Phe Pro Glu 1 5 10 <210> 110 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 110 Asp Cys Leu Thr Glu Ser Gln Pro Phe Pro Glu Gln 1 5 10 <210> 111 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 111 Gly His Phe Ile Leu Leu Pro Glu Gln Gln Pro Glu 1 5 10 <210> 112 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 112 Phe Ser Glu Lys Ser Val Phe Pro Glu Gln Pro Glu 1 5 10 <210> 113 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 113 Gln Pro Glu Glu Gln Pro Thr Val Ser Tyr Asn Gly 1 5 10 <210> 114 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 114 Gly Glu Glu Val Lys Val Pro Glu Gln Pro Glu Gln 1 5 10 <210> 115 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 115 Glu Pro Val Ile Asn Ser Gln Pro Glu Pro Glu Gln 1 5 10 <210> 116 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 116 Glu Glu Glu Arg Gln Glu Glu Gln 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 His His Thr Ala Asp Leu Gln Pro Glu Gln Pro Glu 1 5 10 <210> 118 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 118 Gly Thr Lys Tyr Leu Leu Pro Phe Pro Phe Pro Glu 1 5 10 <210> 119 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 119 Glu Gln Pro Thr Phe Thr Val Glu Gly Pro Phe Pro 1 5 10 <210> 120 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 120 Gly Glu Ile Gln Gly Asp Gln Pro Glu Gln Pro Phe 1 5 10 <210> 121 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 121 Pro Phe Pro Leu Pro Val Ala Leu Glu Phe Pro Glu 1 5 10 <210> 122 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 122 Cys Ile Leu Tyr Glu Lys Glu Gln Pro Phe Pro Glu 1 5 10 <210> 123 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 123 Gln Pro Phe Pro Lys Phe Leu Lys Asn Gln Pro Glu 1 5 10 <210> 124 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 124 Phe Pro Gln Leu Thr Phe Ser Val Val Pro Glu Gln 1 5 10 <210> 125 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 125 Pro Phe Pro Glu Gln Pro Val Val Thr Gly Pro Ala 1 5 10 <210> 126 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 126 Glu Lys Tyr Arg Asp Cys Phe Pro Glu Pro Glu Gln 1 5 10 <210> 127 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 127 Pro Phe Pro Thr Ala Thr Val Val Asp Gln Pro Glu 1 5 10 <210> 128 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 128 Pro Phe Pro Leu Asp Val Asn Pro Lys Gln Pro Phe 1 5 10 <210> 129 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 129 Phe Pro Gln Gln Gly Ser Ala Lys Phe Gln Pro Glu 1 5 10 <210> 130 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 130 Pro Phe Pro Arg Asp Glu Arg Glu Asp Gln Pro Phe 1 5 10 <210> 131 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 131 Glu Gln Pro Glu Gln Pro Val Arg Arg Gly Gln Pro 1 5 10 <210> 132 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 132 Pro Phe Pro Ser Val Pro Leu Cys Ile Gln Pro Glu 1 5 10 <210> 133 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 133 Ile Leu Gly Glu Pro Lys Gln Pro Phe Gln Pro Glu 1 5 10 <210> 134 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 134 Phe Pro Gln Pro Phe Pro Val Ser Pro Met Ser Trp 1 5 10 <210> 135 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 135 Gln Pro Glu Leu His Lys Leu Val Val Gln Pro Glu 1 5 10 <210> 136 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 136 Gly Phe Ile Tyr Gln Gly Phe Pro Gln Pro Phe Pro 1 5 10 <210> 137 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 137 Glu Gln Pro Glu Gln Pro Ala His Ile Thr Asn Asn 1 5 10 <210> 138 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 138 Glu Glu Tyr Val Cys Arg Phe Pro Glu Pro Phe Pro 1 5 10 <210> 139 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 139 Pro Glu Gln Pro Asp Leu Gln Pro Gly Gln Pro Glu 1 5 10 <210> 140 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 140 Gln Pro Phe Phe Pro Gln Thr Thr Pro Ala Asn Ala 1 5 10 <210> 141 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 141 Glu Gln Pro Leu Thr Glu Glu Gln Lys Gln Pro Glu 1 5 10 <210> 142 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 142 Glu Ser Asp Lys Leu Lys Gln Pro Phe Pro Glu Gln 1 5 10 <210> 143 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 143 Glu Gln Pro Asn Gly Ile Leu Gly Pro Glu Gln Pro 1 5 10 <210> 144 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 144 Pro Phe Pro Gln Glu Ala Gly Thr Lys Phe Pro Gln 1 5 10 <210> 145 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 145 Glu Glu Thr Gly Met Ala Pro Phe Pro Glu Gln Pro 1 5 10 <210> 146 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 146 Pro Phe Pro Met Ala Met Arg Ile Arg Gln Pro Phe 1 5 10 <210> 147 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 147 Gln Pro Glu Leu Leu Gly Arg Trp Asp Gln Pro Glu 1 5 10 <210> 148 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 148 Asp Ala Pro Phe Val Phe Gln Pro Phe Gln Pro Phe 1 5 10 <210> 149 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 149 Ile Glu Tyr Phe Arg Asn Glu Gln Pro Phe Pro Glu 1 5 10 <210> 150 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 150 Gln Pro Glu Ser Thr Lys Tyr Asp Ala Gln Pro Phe 1 5 10 <210> 151 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 151 Cys Leu Ile Leu Leu Asp Gln Pro Glu Pro Phe Pro 1 5 10 <210> 152 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 152 Phe Pro Glu Arg Cys Leu Gly Ile Pro Glu Gln Pro 1 5 10 <210> 153 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 153 Glu Gln Pro Asn Ile Pro Trp Asn Phe Pro Phe Pro 1 5 10 <210> 154 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 154 Gly Asp Lys Ser Glu Met Pro Glu Gln Phe Pro Glu 1 5 10 <210> 155 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 155 Pro Phe Pro Phe Pro Gln Tyr Leu Asp Ser Glu Glu 1 5 10 <210> 156 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 156 Phe Pro Glu Asn Ser Tyr Leu Leu Ala Pro Glu Gln 1 5 10 <210> 157 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 157 Glu Gln Pro Arg Ala Ile Lys Glu Gly Gln Pro Phe 1 5 10 <210> 158 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 158 Cys Thr Val Leu Arg Cys Phe Pro Gln Glu Gln Pro 1 5 10 <210> 159 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 159 Pro Glu Gln Lys Tyr Gly Gln Cys Trp Phe Pro Gln 1 5 10 <210> 160 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 160 Glu Gly Asp Trp Thr Ala Pro Glu Gln Glu Gln Pro 1 5 10 <210> 161 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 161 Pro Glu Gln Gln Pro Phe Ala Asp Ala Val Tyr Leu 1 5 10 <210> 162 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 162 Pro Phe Pro Leu Lys Ala Val Lys Gly Glu Gln Pro 1 5 10 <210> 163 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 163 Glu Gln Pro Ser Ser Glu Glu Arg Glu Pro Phe Pro 1 5 10 <210> 164 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 164 Glu Gln Pro Arg Asp Cys Ser Arg Arg Pro Glu Gln 1 5 10 <210> 165 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 165 Glu Gln Pro Asn Val Ile Ile Gly Pro Phe Pro Glu 1 5 10 <210> 166 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 166 Pro Glu Gln Leu Leu Asn Leu Asn Leu Pro Glu Gln 1 5 10 <210> 167 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 167 Glu Gln Pro Ser Leu Gln Leu Thr Thr Phe Pro Glu 1 5 10 <210> 168 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 168 Pro Glu Gln Asn Leu Glu Pro Phe Ser Gln Pro Phe 1 5 10 <210> 169 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 169 His Lys Ser Ile Asn Arg Phe Pro Glu Glu Gln Pro 1 5 10 <210> 170 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 170 Glu Gln Pro Leu Arg Arg Trp Lys Asn Pro Glu Gln 1 5 10 <210> 171 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 171 Pro Phe Pro Lys Asn Ala Gly Arg Asp Glu Gln Pro 1 5 10 <210> 172 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 172 Glu Leu Glu Thr Asn Gly Pro Phe Pro Gln Pro Phe 1 5 10 <210> 173 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 173 Tyr Gly Asp Gly Val Ser Gln Pro Glu Gln Pro Phe 1 5 10 <210> 174 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 174 Tyr Gly Asp Gly Val Ser 1 5 <210> 175 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 175 Pro Glu Gln Pro 1 <210> 176 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 176 Met Lys Thr Phe Leu Ile Leu Val Leu Leu Ala Thr 1 5 10 <210> 177 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 177 Thr Phe Leu Ile Leu Val Leu Leu Ala Thr Ile Val 1 5 10 <210> 178 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 178 Leu Ile Leu Val Leu Leu Ala Thr Ile Val Ala Thr 1 5 10 <210> 179 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 179 Leu Val Leu Leu Ala Thr Ile Val Ala Thr Ala Thr 1 5 10 <210> 180 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 180 Leu Leu Ala Thr Ile Val Ala Thr Ala Thr Thr Ala 1 5 10 <210> 181 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 181 Ala Thr Ile Val Ala Thr Ala Thr Thr Ala Val Arg 1 5 10 <210> 182 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 182 Ile Val Ala Thr Ala Thr Thr Ala Val Arg Phe Pro 1 5 10 <210> 183 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 183 Ala Thr Ala Thr Thr Ala Val Arg Phe Pro Val Pro 1 5 10 <210> 184 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 184 Ala Thr Thr Ala Val Arg Phe Pro Val Pro Gln Leu 1 5 10 <210> 185 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 185 Thr Ala Val Arg Phe Pro Val Pro Gln Leu Gln Pro 1 5 10 <210> 186 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 186 Val Arg Phe Pro Val Pro Gln Leu Gln Pro Gln Asn 1 5 10 <210> 187 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 187 Cys Thr Ile Ala Pro Phe Gly Ile Phe Gly Thr Asn 1 5 10 <210> 188 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 188 Ala Thr Thr Ala Val Arg Phe Pro Val Pro Glu Leu 1 5 10 <210> 189 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 189 Thr Ala Val Arg Phe Pro Val Pro Glu Leu Gln Pro 1 5 10 <210> 190 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 190 Thr Ala Val Arg Phe Pro Val Pro Gln Leu Glu Pro 1 5 10 <210> 191 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 191 Thr Ala Val Arg Phe Pro Val Pro Glu Leu Glu Pro 1 5 10 <210> 192 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 192 Tyr Gly Asp Gly Val Ser Pro Glu Gln Pro Phe Pro 1 5 10 <210> 193 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 193 Pro Glu Gln Tyr Gly Asp Gly Val Ser Pro Glu Pro 1 5 10 <210> 194 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 194 Pro Glu Gln Pro Glu Pro Tyr Gly Asp Gly Val Ser 1 5 10

Claims

**Claim 1** A method for detecting a state of remission in a subject suffering from celiac disease, comprising: obtaining a sample from the subject, wherein the sample partially contains the target antibody; contacting an array of linear synthetic polypeptides with the sample, wherein each linear synthetic polypeptide contains at least two epitope sequences derived from an alpha-gliadin, beta-gliadin, gamma-gliadin, or omega-gliadin polypeptide, or any combination thereof, and at least one epitope sequence derived from tissue transglutaminase; identifying an antibody binding strength value for each of the linear synthetic polypeptides in the array; and determining the remission state of the subject based on the identified antibody binding strength values for each of the linear synthetic polypeptides in the array. **Claim 2** The method according to claim 1, wherein at least one of the at least two epitope sequences derived from the alpha-gliadin, beta-gliadin, gamma-gliadin, or omega-gliadin polypeptide contains a deamidated polypeptide sequence. **Claim 3** The method according to claim 1, wherein the target antibody is an IgA or IgG antibody. **Claim 4** The method according to claim 1, wherein the at least two epitope sequences derived from the alpha-gliadin, beta-gliadin, gamma-gliadin, or omega-gliadin polypeptide are discontinuous in the gliadin polypeptide. **Claim 5** The method according to claim 1, wherein each of the at least two epitope sequences derived from the alpha-gliadin, beta-gliadin, gamma-gliadin, or omega-gliadin polypeptide is 3 amino acids in length. **Claim 6** The method according to claim 1, wherein each linear synthetic polypeptide further contains at least one randomly generated polypeptide sequence. **Claim 7** The method according to claim 1, wherein the linear synthetic polypeptide is 12 amino acids in length. **Claim 8** The method according to claim 1, wherein the linear synthetic polypeptides of the array are configured to have at least 90% sensitivity and 90% specificity for the detection of celiac disease after contacting the microarray with the sample. The method according to claim 1, wherein the array comprises a fluorescence array. Identifying the antibody binding strength value for each of the linear synthetic polypeptides in the array, imaging the array after contacting the array with the test sample; identifying the fluorescence emission value for each of the linear synthetic polypeptides; and identifying the antibody binding strength value for each of the linear synthetic polypeptides based on the identified fluorescence emission values The method according to claim 1, comprising: The method according to claim 1, wherein the linear synthetic polypeptide in the array comprises one or more of the sequences selected from the group consisting of SEQ ID NOs: 1 to 172. The method according to claim 1, wherein the subject has IgA deficiency. The method according to claim 1, wherein the subject adheres to a gluten-free diet. The method according to claim 1, wherein the linear synthetic polypeptide of the array is configured to have at least 80% sensitivity and 90% specificity for detecting a cured state in a subject suffering from celiac disease and adhering to a gluten-free diet. A method for determining the degree of celiac disorder or celiac-related disorder in a patient, the method comprising measuring the reactivity of a serum sample of the patient contacted with a preparation comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 172 or any one or more biologically active fragments or variants thereof.

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