Assays for determining milk allergies

Abstract

Methods of determining the milk phenotype of a subject, methods of determining prior to initiating oral immunotherapy (OIT) in a subject whether the subject will experience sustained unresponsiveness (SU) after completion of the OIT, and methods of determining a milk allergy threshold amount for a subject are presented herein.

Description

[0001] Assays For Determining Milk Allergies

[0002] Field

[0003] The present disclosure is directed, in part, to methods of determining the milk phenotype of a subject, methods of determining prior to initiating oral immunotherapy (OIT) in a subject whether the subject will experience sustained unresponsiveness (S U) after completion of the OIT, and methods of determining a milk allergy threshold amount for a subject.

[0004] Background

[0005] Milk phenotype refers to differentiating milk allergic subjects who are allergic to baked milk and those who are not. This is an important distinction in milk allergic patients for several reasons. First, in day-to-day life, those who are not baked milk allergic can safely consume baked products containing milk (e.g., muffins, bread, etc.) from those who cannot. Second, those who are not baked milk allergic are more likely to outgrow their milk allergy. Accurate phenotyping of milk allergic patients is critical for establishing an allergy management program including dietary and therapeutic options. Currently, phenotyping can only be established through oral food challenges.

[0006] The increasing use of Oral Immunotherapy (OIT) for food allergies, including milk, has led to the clinical question of predicting, before initiating OIT, who will experience sustained unresponsiveness (SU) - that is, who will remain desensitized to milk allergies once OIT is completed.

[0007] To better manage milk allergy, understanding the amount of milk allergy consumed that would cause a reaction is valuable information. Currently, milk allergy management is avoidance.

[0008] Summary

[0009] The present disclosure provides methods of determining the milk phenotype of a subject, the methods comprising determining the level of antibody reactivity in a sample of blood obtained from the subject to one or more milk epitopes chosen from alphas2-10 (INPSKENLC STFCKEVVRNA, SEQ ID NO: 1), betacas-53 (FPPQSVLSLSQSKVLPVPQK, SEQ ID NO: 2), alphas2-13 (STFCKEVVRNANEEEYSIGS, SEQ ID NO: 3), kappacas-41 (TINTIASGEP TSTPTTEAVE, SEQ ID NO: 4), betalac-43 (EVDDEALEKFDKALKALPMH, SEQ ID NO: 5), and betalac-44 (DEALEKFDKALKALPMHIRL, SEQ ID NO: 6); wherein an antibody reactivity above a threshold indicates the subject has a baked milk allergic milk phenotype, and an antibody reactivity’ at or below the threshold indicates the subject does not have a baked milk allergic milk phenotype.

[0010] The present disclosure also provides methods of determining prior to initiating oral immunotherapy (OIT) in a subject whether the subject will experience sustained unresponsiveness (SU) after completion of the OIT, the methods comprising determining the level of antibody reactivity in a sample of blood obtained from the subject to alphas2-10 (INP SKENLCSTFCKEVVRNA, SEQ ID NO: 1) or alphas2-13 (STFCKEVVRNANEEEYSIGS, SEQ ID NO: 3), or a combination thereof; wherein a high antibody reactivity indicates the subject has a low chance of achieving SU, a medium antibody reactivity indicates the subject has a medium chance of achieving SU, and a low antibody reactivity indicates the subject has a high chance of achieving SU.

[0011] The present disclosure also provides methods of determining a milk allergy threshold amount for a subject, the methods comprising determining the level of antibody reactivity in a sample of blood obtained from the subject to a combination of: i) betacas-33 (KVKEAMAP KHKEMPFPKYPV. SEQ ID NO: 7) and betacas-48 (SWMHQPHQPLPPTVMFPPQS, SEQ ID NO: 8); ii) alphasl-35 (KYKVPQLEIVPNSAEERLHS, SEQ ID NO: 9) and alphas2-26 (KHYQKALNEINQFYQKFPQY, SEQ ID NO: 10); or iii) alphal-54 (PSGAWYYVPLGTQY TDAPSF, SEQ ID NO: 11) and alphasl-55 (AWYYVPLGTQYTDAPSFSDI, SEQ ID NO: 12); wherein antibody reactivity that is greater than a threshold of 1.0 indicates that the subject has a low tolerance to milk, and antibody reactivity that is less than a threshold of 1.0 indicates that the subject has a high tolerance to milk.

[0012] Brief Description Of The Drawings

[0013] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several features of the present disclosure.

[0015] Figure 1 shows the alphas2-8,10,13 alignment with P02663 (first 80 amino acids out of 222).

[0016] Figure 2 shows a boxplot of baked milk allergic vs. whole milk (i.e., milk allergic but not baked milk allergic) subjects in the Turkish milk study and their stratification by alphas2-10 (IgE reactivity) and alphas2-13 (IgGE reactivity) (left); and an AUCROC for the same epitope pairing demonstrating an AUC of 95% and optimal sensitivity and specificity of 91% and 94%, respectively (right).

[0017] Figure 3 shows stratification of patients by alphas2-10 (IgE reactivity) alone for the cohorts of Baked Milk Allergic and Whole Milk (milk allergic but not baked milk allergic); another cohort, Outgrown (subjects who outgrew their milk allergy), was included as a reference; the boxplot demonstrates the ability of alphas2-10 to stratify patients by phenotype.

[0018] Figure 4 shows placement of subjects into a low and high milk tolerance group based on the IgE reactivity of two epitopes allows for a tolerance profile for each group to be derived.

[0019] Figure 5 (Panel A, Panel B, and Panel C) shows placement of subjects into a low and high milk tolerance group based on the IgE reactivity of alphas 1-54 and alphas 1-55 at a threshold of 1.0.

[0020] Figure 6 (Panel A, Panel B, and Panel C) shows placement of subjects into a low and high milk tolerance group based on the IgE reactivity of alphasl-35 and s2-26 at a threshold of 1.2.

[0021] Description Of Embodiments

[0022] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0023] The present disclosure provides methods and compositions for determining whether sequential (linear) epitope-specific IgE (ses-IgE) profiling can accurately phenotype baked milk allergic patients vs. baked milk tolerant allergic patients. Currently there is not an accurate tool, except for risky food challenges, to phenotype baked milk allergic patients.

[0024] The present disclosure provides methods of determining the milk phenotype of a subject, the methods comprising determining the level of antibody reactivity in a sample of blood obtained from the subject to one or more milk epitopes chosen from alphas2-10 (INPSKENLC STFCKEVVRNA, SEQ ID NO: 1), betacas-53 (FPPQSVLSLSQSKVLPVPQK, SEQ ID NO: 2), alphas2-13 (STFCKEVVRNANEEEYSIGS, SEQ ID NO: 3), kappacas-41 (TINTIASGEP TSTPTTEAVE, SEQ ID NO: 4), betalac-43 (EVDDEALEKFDKALKALPMH, SEQ ID NO: 5), and betalac-44 (DEALEKFDKALKALPMHIRL, SEQ ID NO: 6). An antibody reactivity above a threshold indicates the subject has a baked milk allergic milk phenotype. An antibody reactivity at or below the threshold indicates the subject does not have a baked milk allergic milk phenotype. The phenotype test described herein differentiates baked milk allergic patients from those who are not baked milk allergic. The sample obtained from the subject can be any sample whereby antibody reactivity to milk peptides can be detected and measures. In some embodiments, the sample is blood. In some embodiments, the sample is serum.

[0025] Determining the level of antibody reactivity' can be carried out by any assay that measures the level of reactivity between an antibody in a subject to a milk peptide. In some embodiments, the assay is a bead-based epitope assay (BBEA).

[0026] The one or more milk epitopes can be chosen from alphas2-10 (INPSKENLCSTFC KEVVRNA, SEQ ID NO: 1), betacas-53 (FPPQSVLSLSQSKVLPVPQK, SEQ ID NO: 2), alphas2-13 (STFCKEVVRNANEEEYSIGS, SEQ ID NO: 3), kappacas-41 (TINTIASGEPTS TPTTEAVE. SEQ ID NO: 4), betalac-43 (EVDDEALEKFDKALKALPMH. SEQ ID NO: 5), and betalac-44 (DEALEKFDKALKALPMHIRL, SEQ ID NO: 6), or any combination thereof. In some embodiments, the level of reactivity' is determined between an antibody and alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1). In some embodiments, the level of reactivity is determined between an antibody and betacas-53 (FPPQSVLSLSQSKVLPVPQK, SEQ ID NO: 2). In some embodiments, the level of reactivity is determined between an antibody and alphas2-13 (STFCKEVVRNANEEEYSIGS, SEQ ID NO: 3). In some embodiments, the level of reactivity is determined between an antibody and kappacas-41 (TINTIASGEPTSTPTTEAVE, SEQ ID NO: 4). In some embodiments, the level of reactivity is determined between an antibody and betalac-43 (EVDDEALEKFDKALKALPMH, SEQ ID NO: 5). In some embodiments, the level of reactivity is determined between an antibody and betalac-44 (DEALEKFDKALKAL PMHIRL, SEQ ID NO: 6).

[0027] The one or more milk epitopes can be chosen from a pair of milk epitopes chosen from: i) alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) and betacas-53 (FPPQSVLSLSQ SKVLPVPQK, SEQ ID NO: 2); u) alphas2-13 (STFCKEVVRNANEEEYSIGS, SEQ ID NO: 3) and kappacas-41 (TINTIASGEPTSTPTTEAVE, SEQ ID NO: 4); in) alphas2-10 (INPSKENLC STFCKEVVRNA, SEQ ID NO: 1) and betalac-43 (EVDDEALEKFDKALKALPMH, SEQ ID NO: 5); iv) alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) and betalac-44 (DE ALEKFDKALKALPMHIRL, SEQ ID NO: 6); v) alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) and betalac-44 (DEALEKFDKALKALPMHIRL, SEQ ID NO: 6); and vi) alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) and alphas2-13 (STFCKEVV RNANEEEYSIGS. SEQ ID NO: 3). In some embodiments, the pair of milk epitopes are alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) and betacas-53 (FPPQSVLSLSQS KVLPVPQK, SEQ ID NO: 2). In some embodiments, the pair of milk epitopes are alphas2-13 (STFCKEVVRNANEEEYSIGS, SEQ ID NO: 3) and kappacas-41 (TINTIASGEPTSTPTT EAVE, SEQ ID NO: 4). In some embodiments, the pair of milk epitopes are alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) and betalac-43 (EVDDEALEKFDKA LKALPMH, SEQ ID NO: 5). In some embodiments, the pair of milk epitopes are alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) and betalac-44 (DEALEKFDKALKALPMH IRL, SEQ ID NO: 6). In some embodiments, the pair of milk epitopes are alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) and betalac-44 (DEALEKFDKALK ALPMHIRL, SEQ ID NO: 6). In some embodiments, the pair of milk epitopes are alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) and alphas2-13 (STFCKEVVRNANEEE YSIGS, SEQ ID NO: 3).

[0028] Any one or more of the milk epitopes described herein can be present on a solid support, such as a bead. The present disclosure also provides compositions comprising any one of more (or pairs thereof) of the milk epitopes present on a solid support, such as a bead.

[0029] An antibody reactivity above a threshold indicates the subject has a baked milk allergic milk phenotype. An antibody reactivity at or below the threshold indicates the subject does not have a baked milk allergic milk phenotype. The threshold can be an antibody reactivity level known to be associated with a subject (or control population) that does not have a baked milk allergic milk phenoty pe (i.e., a control polulation).

[0030] The present disclosure provides methods of predicting sustained unresponsiveness to milk allergy oral immunotherapy using epitope-specific IgE antibody profiling.

[0031] The present disclosure provides methods of determining prior to initiating oral immunotherapy (OIT) in a subject whether the subject will experience sustained unresponsiveness (SU) after completion of the OIT. The methods comprise determining the level of antibody reactivity in a sample of blood obtained from the subject to alphas2-10 (INPSKENL CSTFCKEVVRNA, SEQ ID NO: 1) or alphas2-13 (STFCKEVVRNANEEEYSIGS, SEQ ID NO: 3), or a combination thereof. A high antibody reactivity indicates the subject has a low chance of achieving SU. A medium antibody reactivity indicates the subject has a medium chance of achieving SU. A low antibody reactivity indicates the subject has a high chance of achieving SU.

[0032] The sample obtained from the subject can be any sample whereby antibody reactivity to milk peptides can be detected and measures. In some embodiments, the sample is blood. In some embodiments, the sample is serum.

[0033] Determining the level of antibody reactivity can be carried out by any assay that measures the level of reactivity' between an antibody in a subject to a milk peptide. In some embodiments, the assay is a bead-based epitope assay (BBEA). The milk epitope can be alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) or alphas2-13 (STFCKEVVRNANEEEYSIGS, SEQ ID NO: 3), or a combination thereof. In some embodiments, the level of reactivity is determined between an antibody and alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1). In some embodiments, the level of reactivity' is determined between an antibody and alphas2-13 (STFCKEVVRNANEEEYSIGS, SEQ ID NO: 3). In some embodiments, the level of reactivity is determined between a combination of alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) and alphas2-13 (STFCKEVV RNANEEEYSIGS, SEQ ID NO: 3).

[0034] Any one or more of the milk epitopes described herein can be present on a solid support, such as a bead. The present disclosure also provides compositions comprising any one of more (or pairs thereof) of the milk epitopes present on a solid support, such as a bead.

[0035] A high antibody reactivity, in some embodiments, is greater than 128 Net Median Fluorescent Intensity (NetMFI) and indicates the subject has a low chance of achieving SU. The NetMFI levels are subject to many laboratory conditions (e.g., antibody bead concentrations, dilution factors, reagent lots. etc.). It is the relative amounts that should be consistent. These values are the values used, for example, for this particular embodiment in Luminex Machine’s units. In some embodiments, a low chance of achieving SU is less than 30%, less than 25%, less than 20%, less than 15%. or less than 10% chance of achieving SU. In some embodiments, a low chance of achieving SU is 21%.

[0036] A medium antibody reactivity, in some embodiments, is from 8 to 128 NetMFI and indicates the subject has a medium chance of achieving SU. In some embodiments, a medium chance of achieving SU is from about 31 % to about 69%, or from about 40% to about 60%, or from about 45% to about 55% chance of achieving SU. In some embodiments, a medium chance of achieving SU is 44%.

[0037] A low antibody reactivity, in some embodiments, is less than 8 NetMFI and indicates the subject has a high chance of achieving SU. In some embodiments, a high chance of achieving SU is at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% chance of achieving SU. In some embodiments, a high chance of achieving SU is 80%.

[0038] In some embodiments, when the subject has a low antibody reactivity to alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) or alphas2-13 (STFCKEVVRNANEEE YSIGS. SEQ ID NO: 3), or a combination thereof, OIT can be administered to the subject. In some embodiments, when the subject has a high antibody reactivity to alphas2-10 (INPSKEN LCSTFCKEVVRNA, SEQ ID NO: 1) or alphas2-13 (STFCKEVVRNANEEEYSIGS, SEQ ID NO: 3), or a combination thereof, OIT may not be administered to the subject. The present disclosure provides methods of determining a milk allergy threshold amount for a subject, whereby a subject with milk allergy can be stratified into low and high tolerance groups. The low tolerance subject cannot consumer much milk before a reaction occurs, whereas the high tolerance subject can consume more milk. This allows for better day-to- day diet management than strict avoidance. It also allows for better clinical management, for example, whether to use Milk O1T for those with low tolerance and / or at which amount of milk to begin an OFC.

[0039] The present disclosure provides methods of determining a milk allergy’ threshold amount for a subject. The methods comprise determining the level of antibody reactivity in a sample of blood obtained from the subject to a combination of: i) betacas-33 (KVKEAMAPKH KEMPFPKYPV, SEQ ID NO: 7) and betacas-48 (SWMHQPHQPLPPTVMFPPQS, SEQ ID NO: 8); ii) alphasl-35 (KYKVPQLEIVPNSAEERLHS, SEQ ID NO: 9) and alphas2-26 (KHYQ KALNEINQFYQKFPQY, SEQ ID NO: 10); or iii) alphal-54 (PSGAWYYVPLGTQYTDAPSF, SEQ ID NO: 11) and alphasl-55 (AWYYVPLGTQYTDAPSFSDI, SEQ ID NO: 12). Antibody reactivity that is greater than a particular threshold indicates that the subject has a low tolerance to milk. Antibody reactivity that is less than a particular threshold indicates that the subject has a high tolerance to milk.

[0040] The sample obtained from the subj ect can be any sample whereby antibody reactivity to milk peptides can be detected and measures. In some embodiments, the sample is blood. In some embodiments, the sample is serum.

[0041] Determining the level of antibody reactivity can be carried out by any assay that measures the level of reactivity' between an antibody in a subj ect to a milk peptide. In some embodiments, the assay is a bead-based epitope assay (BBEA).

[0042] In some embodiments, the level of antibody reactivity determined in a sample of blood obtained from the subject can be to a combination of betacas-33 (KVKEAMAPKHKEMPFPK YPV, SEQ ID NO: 7) and betacas-48 (SWMHQPHQPLPPTVMFPPQS, SEQ ID NO: 8). In some embodiments, the level of antibody reactivity determined in a sample of blood obtained from the subject can be to a combination of alphasl-35 (KYKVPQLEIVPNSAEERLHS. SEQ ID NO: 9) and alphas2-26 (KHYQKALNEINQFYQKFPQY, SEQ ID NO: 10). In some embodiments, the level of antibody reactivity7determined in a sample of blood obtained from the subject can be to a combination of alphal-54 (PSGAWYYVPLGTQYTDAPSF, SEQ ID NO: 11) and alphasl-55 (AWYYVPLGTQYTDAPSFSDI. SEQ ID NO: 12).

[0043] Antibody reactivity ratio is mapped to probability of tolerance that is greater than a particular threshold indicates that the subject has a low tolerance to milk. Antibody7reactivity' that is less than a particular threshold indicates that the subject has a high tolerance to milk. The ratio of antibody binding to the “epitope’’ versus antibody binding to the “epitope normalizer” is calculated to produce a ratio score for a subject’s sample. A database of ratio scores with their corresponding tolerated dosing is generated. Thresholds are determined to segregate the sample populations into two or more populations. In some embodiments a single threshold value can be selected to split the database of samples into two distinct groups “low milk tolerance group” and “high milk tolerance group.” In the combination of alphal-54 (PSGAWYYVPLGTQYTDAPSF, SEQ ID NO: 11) and alphasl-55 (AWYYVPLGTQYTDAPSFSDI, SEQ ID NO: 12), a threshold value of 1.0 split the groups into clinically relevant populations. In another embodiment, using a combination of alphasl-35 (KYKVPQLEIVPNSAEERLHS, SEQ ID NO: 9) and alphas2-26 (KHYQKALNEINQFYQKFPQY, SEQ ID NO: 10), a threshold value of 1.2 split the groups into clinically relevant populations.

[0044] In some embodiments, the subject can be stratified as having a low tolerance to milk. Such subjects have about a 33% to 43% chance of tolerating 20 mg of milk, about a 10% to 15% chance of tolerating 100 mg of milk, and about a 0% chance of tolerating 300 mg or greater of milk. In some embodiments, such subjects have about a 38% chance of tolerating 20 mg of milk, about a 13% chance of tolerating 100 mg of milk, and about a 0% chance of tolerating 300 mg or greater of milk.

[0045] In some embodiments, the subject can be stratified as having a high tolerance to milk. Such subjects have about a 66% to 76% chance of tolerating 20 mg of milk, about a 59% to 69% chance of tolerating 100 mg of milk, about a 10% to 18% chance of tolerating 300 mg of milk, and about a 5% to 9% chance of tolerating 1500 mg of milk. In some embodiments, such subjects have about a 71% chance of tolerating 20 mg of milk, about a 64% chance of tolerating 100 mg of milk, about a 14% chance of tolerating 300 mg of milk, and about a 7% chance of tolerating 1500 mg of milk.

[0046] Knowing which tolerance group a subject belongs to, by measuring the IgE reactivity of the pairs of epitopes described herein allows a subject, parent, and / or physician to understand what amount of milk can be safely consumed.

[0047] In order that the subject matter disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the claimed subject matter in any manner. Throughout these examples, molecular cloning reactions, and other standard recombinant DNA techniques, were carried out according to methods described in Mamatis et al., Molecular Cloning - A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989). using commercially available reagents, except where otherwise noted.

[0048] Examples

[0049] Example 1: Phenotyping of Baked vs. Non-Baked Milk Allergic Subjects using Epitope- Specific IgE Antibody Profiling

[0050] A phenotype test is described that differentiates baked milk allergic patients from those who are not baked milk allergic. To achieve this, subjects from a Turkish Milk study where analyzed used the BBEA assay for milk protein allergy epitopes. The subjects of the Turkish milk study included the following two OFC confirmed cohorts: i) baked milk allergic; and ii) cow milk allergic but not baked milk allergic.

[0051] In particular, sixty six (66) ses-IgE antibodies were quantified in blood samples using a bead-based epitope assay spanning milk protein allergens. An optimal pair of ses-IgEs antibodies that phenotype baked milk allergy was determined using regression on 16 baked milk allergic subjects vs. 23 baked milk tolerant subjects. Baked milk allergy status was established by oral food challenge and subjects ranged in age from 0.3 to 12 years of age. Cross validation was performed to establish classification performance.

[0052] After assaying all subjects in these two cohorts, both for IgE and IgG4 reactivity, pairs of milk protein epitopes were assessed in logistic regression models that best separated subjects in the two cohorts. Results of some the of the best pairings of epitopes appear in Table 1 :

[0053] Table 1: Top performing epitope pairs in logistic regression

[0054] Epitope alphas2-10 (reacting to IgE) appears in the majority of top pairings. Furthermore, when alphas2-10 is combined with an epitope (alphas2-13, IgE reactivity) that overlaps with alphas2- 10 strong separation (AUC 85%) is observed indicated that this is a dominant epitope region for the identification of phenotype. Of all pairs of ses-IgE antibodies, a pair binding two epitopes on the alpha S2 casein milk protein allergen were identified with a median cross validated AUC (area under the ROC curve) of 95%. Furthermore, these two epitopes, each of length 20 amino acids, overlap sharing 11 amino acids in common. Cross validation was used to establish performance with a median sensitivity of 88% and median specificity of 96%. Figure 2 shows representative results of alphas2-10 (IgE reactivity) in combination with alphas2-13 (IgE reactivity) by logistic regression giving a fitness score. On the left is a boxplot of baked milk allergic vs. whole milk (i.e. milk allergic but not baked milk allergic) subjects in the Turkish milk study and their stratification by optimal fitness score. On the right is an AUCROC for the same epitope pairing demonstrating an AUC of 95% and optimal sensitivity and specificity of 91% and 94%, respectively.

[0055] Figure 3 shows stratification of patients by alphas2-10 (IgE reactivity) alone for the cohorts of Baked Milk Allergic and WholeMilk (milk allergic but not baked milk allergic). Another cohort, Outgrown (subjects who outgrew their milk allergy) were included as a reference. This boxplot demonstrates the ability of alphas2-10 to stratify patients by phenotype.

[0056] Accordingly, these results indicate that a blood test may be useful as a safe surrogate for food challenges.

[0057] Example 2: Predicting Sustained Unresponsiveness (SU) to Milk Allergy Oral Immunotherapy using Epitope-Specific IgE Antibody Profiling

[0058] It was sought to determine whether sequential (linear) epitope-specific IgE (ses-IgE) profiling can predict if SU can be achieved before OIT is initiated. Specifically, a previously identified ses-IgE antibody that binds to an epitope (named alphas2-10) on the alpha S2 casein milk protein allergen was assessed. This ses-IgE antibody was found to be highly reactive in baked milk allergic patients vs. baked milk tolerant patients.

[0059] To address the question of SU for Milk OIT, the BBEA method using epitopes from milk allerg)’ proteins was applied to 47 subjects who underwent Milk OIT with a follow up assessment after the cessation of OIT. The BBEA analysis was performed on samples collected from the subjects before the initiation of OIT so that the results could potentially predict SU. Both epitope reactivity to IgE and IgG4 was collected in the analysis. The follow up assessment was an OFC to determine if milk allergy desensitization was sustained or not.

[0060] This data was analyzed both for single epitopes and pairs of epitopes using linear regression correlated to the results of the OFC conducted at the follow up assessment. This led to the discover}7of multiple epitopes and epitopes pairs that correlated highly to SU resulting in a blood-based predictor, before treatment, of the probability that OIT would provide benefit.

[0061] In particular, the alphas2-10 ses-IgE antibody was profiled on blood samples, taken prior to initiation of milk OIT, from 47 subjects of ages 7 to 32 years. These subjects underwent milk OIT therapy for a period of 26 months with sustained unresponsiveness assessed by oral food challenge 2 months after cessation of treatment. The epitope reactivity' to IgE was binned into three levels (high, medium, low) and the rate of sustained unresponsiveness of patients in each level determined.

[0062] In a first example, a single epitope, alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) (IgE reactivity) was measured using the BBEA method on blood drawn before treatment. Table 2 shows the predictive power of this epitope at low', medium, and high levels of epitope reactivity to IgE, for predicting SU. Table 2: Percentage of patients achieving SU as determined by levels of alphas2-10 reactivity to IgE

[0063] 93%-94% of all subjects achieved desensitization at the time of completing OIT.

[0064] Specifically, low alphas2-10 reactivity to IgE indicated that there was an 80% chance of achieving SU, and so, an indicator that OIT would very likely provide benefit for the patient. In contrast, a high alphas2-10 reactivity to IgE indicated that there was only a 21% chance of achieving SU, and so, an indicator that OIT would very likely not provide benefit for the patient.

[0065] The same analysis was performed for two overlapping epitopes, alphas2-10 and alphas2-13 (STFCKEVVRNANEEEYSIGS, SEQ ID NO: 3) (IgE reactivity) as in the example above. Table 3 reports the results.

[0066] Table 3: Percentage of patients achieving SU as determined by levels of alphas2-10 and alphas2-13 reactivity7to IgE

[0067] 92%-94% of all subjects achieved desensitization at the time of completing OIT.

[0068] As in the first example, low reactivity with IgE corresponded to high probability (77%) of SU whereas high reactivity to IgE corresponded to low probability (31%) of SU.

[0069] In all three levels of reactivity7at least 93% of patients achieved desensitization by' the end of milk OIT. However, there was a strong association between the alphas2-10 ses-IgE antibody reactivity level and probability of achieving sustained unresponsiveness. Specifically, in the high, medium, and low reactivity levels, the probability of sustained unresponsiveness was 21%, 44% and 80% respectively. Thus, this study provides evidence that IgE reactivity to the alphas2-10 epitope is predictive of the outcome of milk OIT in terms of sustained unresponsiveness. The consequences of this discovery are multifold. First, this is further evidence that the alphas2-10 epitope is informative for both ph eno t ping milk allergy and predictive of milk OIT. suggesting it is central to understanding, managing and treating milk allergy. Second, the measurement of reactivity to this epitope has potential clinical utility’ for determining if a milk allergic patient is likely to benefit, or not benefit, from milk OIT.

[0070] Example 3: Milk Allergy Threshold

[0071] To address the question of milk allergy threshold, the BBEA method using epitopes from milk allergy’ proteins was applied to 38 subjects with milk allergy with highest milk tolerance levels determined by standardize (PRACTALL) OFC. Both epitope reactivity' to IgE and IgG4 was collected in the analysis.

[0072] This data was analyzed for pairs of epitopes using linear regression correlated to the highest milk tolerance levels determined by the OFC. This led to the discovery of epitopes pairs that correlated highly to tolerated levels. From regression models, subjects can then be split into those with high and low scores w hich correlate to high and low tolerated levels of milk.

[0073] Table 4 presents three of the highest performing pairs of epitopes in terms of Pearson and Spearman correlation to OFC-determine milk tolerance levels. The p-values of these correlations are all highly significant.

[0074] Table 4: Correlation of epitope pairs to OFC highest tolerated levels of milk allergen

[0075] Table 4 (cont.)

[0076] Sequences: Betacas-33: KVKEAMAPKHKEMPFPKYPV, SEQ ID NO: 7; Alphasl-35: KYKV PQLEIVPNSAEERLHS, SEQ ID NO: 9; Alphal-54: PSGAWYYVPLGTQYTDAPSF, SEQ ID NO: 11; Betacas-48: SWMHQPHQPLPPTVMFPPQS, SEQ ID NO: 8; Alphas2-26: KHYQKAL NEINQFYQKFPQY, SEQ ID NO: 10; and Alphasl-55: AWYYVPLGTQYTDAPSFSDL SEQ ID NO: 12.

[0077] Using the third model (alphasl-54 and alphasl-55, reactivity to IgE) as an example, at a particular threshold of 1.0, the 38 samples were split into a low and high milk tolerance group (see, Figure 4 and Figure 5). For each of these groups, the tolerance profile for the group was represented by the percentage of patients that can tolerate at least 0 mg, 20 mg, 100 mg, 300 mg, and 1500 mg of milk allergen (Successfully Consumed Doses or SCD). For example, only 38% of subjects in the low tolerance group can tolerate 20 mg of milk whereas 71% of subjects in the high tolerance group can tolerate 20 mg of milk. Similarly, only 13% of subjects in the low tolerance group can tolerate 100 mg of milk whereas 64% of subjects in the high tolerance group can tolerate 100 mg of milk. Similarly, 0% of subjects in the low tolerance group can tolerate 300 mg of milk whereas 14% of subjects in the high tolerance group can tolerate 300 mg of milk. Similarly, 0% of subjects in the low tolerance group can tolerate 1500 mg of milk whereas 7% of subjects in the high tolerance group can tolerate 1500 mg of milk. Using another model (alphasl- 35 and s2-26, reactivity to IgE) as an example, at a particular threshold of 1.2, the samples were split into a low and high milk tolerance group (see, Figure 6).

[0078] Various modifications of the described subject matter, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference (including, but not limited to, journal articles, U.S. and non-U. S. patents, patent application publications, international patent application publications, gene bank accession numbers, and the like) cited in the present application is incorporated herein by reference in its entirety.

Claims

What Is Claimed Is:

1. A method of determining the milk phenotype of a subject, the method comprising determining the level of antibody reactivity in a sample of blood obtained from the subject to one or more milk epitopes chosen from alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1), betacas-53 (FPPQSVLSLSQSKVLPVPQK, SEQ ID NO: 2), alphas2-13 (STFCKEVVRNA NEEEYSIGS, SEQ ID NO: 3). kappacas-41 (T1NT1ASGEPTSTPTTEAVE, SEQ ID NO: 4), betalac-43 (EVDDEALEKFDKALKALPMH, SEQ ID NO: 5), and betalac-44 (DEALEK FDKALKALPMHIRL, SEQ ID NO: 6); wherein an antibody reactivity above a threshold indicates the subject has a baked milk allergic milk phenotype, and an antibody reactivity at or below the threshold indicates the subject does not have a baked milk allergic milk phenotype.

2. The method of claim 1, wherein the antibody reactivity’ is to a pair of milk epitopes chosen from: i) alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) and betacas-53 (FPPQSVLSLSQSKVLPVPQK. SEQ ID NO: 2); ii) alphas2-13 (STFCKEVVRNANEEE YSIGS. SEQ ID NO: 3) and kappacas-41 (TINTIASGEPTSTPTTEAVE. SEQ ID NO: 4); hi) alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) and betalac-43 (EVDDEALEKFD KALKALPMH, SEQ ID NO: 5); iv) alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) and betalac-44 (DEALEKFDKALKALPMHIRL, SEQ ID NO: 6); v) alphas2-10 (INPSKEN LCSTFCKEVVRNA, SEQ ID NO: 1) and betalac-44 (DEALEKFDKALKALPMHIRL, SEQ ID NO: 1); and vi) alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1) and alphas2-13 (STFCKEVVRNANEEEYSIGS, SEQ ID NO: 3).

3. The method of claim 1, wherein the antibody reactivity’ is to alphas2-10 (INPSKE NLC STFCKEVVRNA, SEQ ID NO: 1) and betacas-53 (FPPQSVLSLSQSKVLPVPQK. SEQ ID NO: 2).

4. A method of determining prior to initiating oral immunotherapy (OIT) in a subject whether the subject will experience sustained unresponsiveness (SU) after completion of the OIT, the method comprising determining the level of antibody reactivity in a sample of blood obtained from the subject to alphas2-10 (INPSKENLCSTFCKEVVRNA. SEQ ID NO: 1) or alphas2-13 (STFCKEVVRNANEEEYSIGS, SEQ ID NO: 3). or a combination thereof; wherein a high antibody reactivity indicates the subject has a low chance of achieving SU, a medium antibody reactivity indicates the subject has a medium chance of achieving SU, and a low antibody reactivity indicates the subject has a high chance of achieving SU.

5. The method of claim 4, wherein the level of antibody reactivity is determined for alphas2-10 (INPSKENLCSTFCKEVVRNA, SEQ ID NO: 1).

6. A method of determining a milk allergy’ threshold amount for a subject, the method comprising determining the level of antibody reactivity in a sample of blood obtained from the subject to a combination of: i) betacas-33 (KVKEAMAPKHKEMPFPKYPV, SEQ ID NO: 7) and betacas-48 (SWMHQPHQPLPPTVMFPPQS, SEQ ID NO: 8); ii) alphas 1-35 (KYKVPQLEI VPNSAEERLHS, SEQ ID NO: 9) and alphas2-26 (KHYQKALNEINQFYQKFPQY, SEQ ID NO: 10); or hi) alphal-54 (PSGAWYYVPLGTQYTDAPSF, SEQ ID NO: 11) and alphasl-55 (AWYYVPLGTQYTDAPSFSDI, SEQ ID NO: 12); wherein antibody reactivity that is greater than a threshold of 1.0 indicates that the subject has a low tolerance to milk, and antibody reactivity that is less than a threshold of 1.0 indicates that the subject has a high tolerance to milk.

7. The method of claim 6, wherein a subject that has a low tolerance to milk has about a 33% to 43% chance of tolerating 20 mg of milk, about a 10% to 15% chance of tolerating 100 mg of milk, and about a 0% chance of tolerating 300 mg or greater of milk.

8. The method of claim 7, wherein a subject that has a low tolerance to milk has about a 38% chance of tolerating 20 mg of milk, about a 13% chance of tolerating 100 mg of milk, and about a 0% chance of tolerating 300 mg or greater of milk.

9. The method of claim 6, wherein a subj ect that has a high tolerance to milk has about has about a 66% to 76% chance of tolerating 20 mg of milk, about a 59% to 69% chance of tolerating 100 mg of milk, about a 10% to 18% chance of tolerating 300 mg of milk, and about a 5% to 9% chance of tolerating 1500 mg of milk.

10. The method of claim 9, wherein a subject that has a high tolerance to milk has about a 71% chance of tolerating 20 mg of milk, about a 64% chance of tolerating 100 mg of milk, about a 14% chance of tolerating 300 mg of milk, and about a 7% chance of tolerating 1500 mg of milk.

Images