Biomarkers for allergen immunotherapy

Abstract

The present invention relates to the field of medical diagnosis. In particular, the present invention relates to methods and kits for determining the therapeutic efficacy of allergen immunotherapy and for measuring or detecting biomarkers in a subject undergoing allergen immunotherapy. For example, the present invention provides a method for determining the efficacy of allergen immunotherapy in a subject, the method comprising the steps of providing a first sample obtained from the subject prior to receiving allergen immunotherapy, providing a second sample obtained from a subject who has received or is receiving allergen immunotherapy, wherein the first and second samples comprise B cells, and determining the level or amount of one or more biomarkers in the B cells of the first and second samples, preferably allergen-specific B cells, wherein the biomarker is selected from the group consisting of IgE, CD29, CD69, IL13Rα, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, SIGLEC10, CD1c, CD23, and IL4Rα, and an increase in the level or amount of one or more biomarkers selected from IgE, CD29, IL13Rα, CD99, FCRL3, FCRL2, SIGLEC10, CD1c, CD23, and IL4Rα in the B cells of the second sample as compared to the first sample indicates the efficacy of allergen immunotherapy in the subject, and / or a decrease in the level or amount of one or more biomarkers selected from CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD1c, CD23, and IL4Rα in the B cells of the second sample as compared to the first sample indicates the efficacy of allergen immunotherapy in the subject.

Description

Technical Field

[0001] The present invention relates to the field of monitoring medical diagnosis, prognosis, and treatment efficacy. In particular, the present invention relates to methods and kits for determining the treatment efficacy of allergen immunotherapy and for measuring or detecting biomarkers in a subject undergoing allergen immunotherapy.

[0002] Cross-reference to prior applications This application claims the priority of Australian Provisional Application No. 2022901401, the entire content of which is incorporated herein by reference.

Background Art

[0003] Allergic diseases are one of the most common chronic immune-mediated disorders, and the clinical severity and range of symptoms can vary extremely widely. As a result, there have been and still are significant challenges in diagnosis, prediction of disease progression / development, and treatment.

[0004] Conventionally, patients have been treated using one of two approaches: (a) non-specific drug therapy through neutralization of effector molecules, e.g., symptom control with antihistamines; and (b) reprogramming of the immune response through specific allergen immunotherapy (AIT).

[0005] AIT through long-term repeated exposure to low doses of allergen has been applied for over a century and is the oldest form of immunotherapy, i.e., redirecting the existing immune response. However, patients typically require treatment for at least several years for AIT to be effective, which can be associated with adverse reactions, so treatment adherence is typically poor.

[0006] Over the past 20 years, the treatment of allergic diseases has changed dramatically with the introduction of biological agents. Specifically, monoclonal antibodies (mAbs) against IgE, type 2 cytokines and their receptors, as well as small molecule inhibitors of signaling pathways, have shown promise in modulating particularly robust responses. However, these therapeutics do not result in a cure, are expensive, are not effective for all patients, and treatment regimens are often very long-term and lifelong.

[0007] Worldwide, pollen (grass, tree or weed) allergy is a major cause of seasonal allergic rhinitis and / or asthma, and house dust mite allergy is a major cause of perennial allergic rhinitis and / or asthma. One specific allergic disease, pollen allergy, is the leading cause of seasonal allergic rhinitis worldwide, and 10-30% of the world's population is sensitized to one or more grass pollen allergens. Allergy to grass pollen is a significant burden on quality of life due to ongoing symptoms of rhinoconjunctivitis throughout the pollen season, anxiety in grassy areas such as public parks, the development of comorbidities such as asthma, and the financial costs of treatment and time off work and school. Furthermore, grass pollen allergy is at the root of thunderstorm asthma, which can overwhelm healthcare facilities as many patients suffer acute asthma exacerbations that can be fatal in extreme cases. For example, during a thunderstorm in Melbourne, Australia in November 2016, over 3,000 people were hospitalized and 10 people died.

[0008] Due to the problem of patients being constrained by potentially costly long-term therapies, there is a great need for objective biomarkers to predict treatment response, disease progression and / or development in patients.

[0009] Just because a prior art is mentioned in the specification does not mean that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art is recognized or suggested as being considered relevant by a person skilled in the art and / or reasonably expected to be combined with other parts of the prior art.

Summary of the Invention

[0010] In one aspect, the present invention is a method for determining the effectiveness of allergen immunotherapy in a subject, comprising: - providing a first sample obtained from the subject before receiving allergen immunotherapy; - providing a second sample obtained from a subject who has received or is receiving allergen immunotherapy, wherein the first and second samples contain B cells; and - determining the level or amount of one or more biomarkers in the B cells of the first and second samples, wherein the biomarker is selected from the group consisting of IgE, CD29, CD69, IL13Rα, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, SIGLEC10, CD1c, CD23, and IL4Rα. Including An increase in the level or amount of one or more biomarkers selected from IgE, CD29, IL13Rα, CD99, FCRL3, FCRL2, SIGLEC10, CD1c, CD23, and IL4Rα in the B cells of the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject, and / or A decrease in the level or amount of one or more biomarkers selected from CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD1c, CD23, and IL4Rα in the B cells of the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject. A method is provided.

[0011] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers selected from IgE, CD29, IL13Rα, CD99, SIGLEC10, CD1c, and CD23 in B cells in the second sample is at least 1.5-fold increased compared to the first sample, which indicates the effectiveness of allergen immunotherapy in the subject.

[0012] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers may be increased by at least 2-fold, which in B cells in the second sample compared to the first sample may include any one or more of IgE, CD29, IL13Rα, CD99, SIGLEC10, and CD1c, indicating the effectiveness of allergen immunotherapy in the subject.

[0013] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers may be increased by at least 2.5-fold, which in B cells in the second sample compared to the first sample may include any one or more of IgE, CD29, IL13Rα, and CD99, indicating the effectiveness of allergen immunotherapy in the subject.

[0014] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers may be increased by at least 3-fold, which in B cells in the second sample compared to the first sample may include any one or more of IgE, CD29, and IL13Rα, indicating the effectiveness of allergen immunotherapy in the subject.

[0015] In one embodiment of this aspect of the present invention, the level or amount of the biomarker may be increased by at least 4-fold, which in B cells in the second sample compared to the first sample may include CD29, indicating the effectiveness of allergen immunotherapy in the subject.

[0016] In one embodiment of this aspect of the present invention, in B cells in the second sample compared to the first sample, the level or amount of one or more biomarkers selected from CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD1c, CD23, and IL4Rα may be reduced to 1 / 1.5 or less, which indicates the effectiveness of allergen immunotherapy in a subject.

[0017] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers may be reduced to 1 / 2 or less, which may include any one or more of CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD23, and IL4Rα in B cells in the second sample compared to the first sample, which indicates the effectiveness of allergen immunotherapy in a subject.

[0018] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers may be reduced to 1 / 2.5 or less, which may include any one or more of CD69, IgD, CXCR4, and FCRL3 in B cells in the second sample compared to the first sample, which indicates the effectiveness of allergen immunotherapy in a subject.

[0019] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers may be reduced to 1 / 3 or less, which may include any one or more of IgD and CXCR4 in B cells in the second sample compared to the first sample, which indicates the effectiveness of allergen immunotherapy in a subject.

[0020] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers may be reduced to 1 / 4 or less, which may include any one or more of IgD and CXCR4 in B cells in the second sample compared to the first sample, which indicates the effectiveness of allergen immunotherapy in a subject.

[0021] In another aspect, the present invention is a method for determining the effectiveness of allergen immunotherapy in a subject, comprising: - providing a first sample obtained from the subject prior to receiving allergen immunotherapy; - providing a second sample obtained from a subject who has received or is receiving allergen immunotherapy, wherein the first and second samples comprise B cells, and - determining the level or amount of one or more biomarkers in the B cells of the first and second samples, wherein the biomarkers are IgG4, CD29, CD23, and IL4Rα. Including, An increase in the level or amount of any one, two, three, or all of IgG4, CD29, CD23, and IL4Rα in the B cells of the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject. Preferably, the B cells are memory B cells (Bmem), more preferably the Bmem are allergen-specific Bmem, and the allergen is the same allergen provided in the allergen immunotherapy. A method is provided.

[0022] In this aspect, the method further comprises determining the level or amount of one or more additional biomarkers. The additional markers may be the biomarkers described in Table 2 and / or Table 3. Preferably, the biomarker is a biomarker that increases by 1.5-fold, 2-fold, 3-fold, or 4-fold or decreases to one-fifth, one-half, one-third, or one-fourth as described in Table 2 and / or Table 3.

[0023] In another aspect, the present invention is a method for determining the effectiveness of allergen immunotherapy in a subject, comprising: - providing a first sample obtained from the subject prior to receiving allergen immunotherapy; - providing a second sample obtained from a subject who has received or is receiving allergen immunotherapy, wherein the first and second samples comprise B cells, and - Determining the proportion or number of IgG + Bmem or IgG4 + allergen - specific Bmem in the first and second samples comprising, An increase in the proportion or number of IgG + Bmem or IgG4 + allergen - specific Bmem in the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in a subject, and provides a method.

[0024] In this aspect, the method further comprises determining the proportion or number of IgG + Bmem or IgG4 + allergen - specific Bmem expressing one or more additional biomarkers. The additional biomarker may be a biomarker described in Table 2 and / or Table 3. Preferably, the biomarker is a biomarker that increases by 1.5 - fold, 2 - fold, 3 - fold or 4 - fold or decreases to 1 / 1.5, 1 / 2, 1 / 3 or 1 / 4 as described in Table 2 and / or Table 3.

[0025] In another aspect, the present invention is a method for determining the effectiveness of allergen immunotherapy in a subject, comprising - providing a first sample obtained from the subject before receiving allergen immunotherapy, - providing a second sample obtained from a subject who has received or is receiving allergen immunotherapy, wherein the first and second samples contain B cells, and - determining the proportion or number of CD29 + allergen - specific Bmem or CD29hi allergen - specific Bmem in the first and second samples comprising, An increase in the proportion or number of CD29 + allergen - specific Bmem or CD29hi allergen - specific Bmem in the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in a subject, and provides a method.

[0026] In this aspect, the method further comprises determining the proportion or number of CD29+ allergen-specific Bmem or CD29hi allergen-specific Bmem that express one or more additional biomarkers. The additional biomarker(s) may be the biomarker(s) described in Table 2 and / or Table 3. Preferably, the biomarker(s) is / are biomarker(s) that increase 1.5-fold, 2-fold, 3-fold or 4-fold or decrease to one and a half, one half, one third or one quarter as described in Table 2 and / or Table 3.

[0027] In another aspect, the present invention is a method for determining the effectiveness of allergen immunotherapy in a subject, comprising: - providing a first sample obtained from the subject prior to receiving allergen immunotherapy; - providing a second sample obtained from a subject who has received or is receiving allergen immunotherapy, wherein the first and second samples comprise B cells, and - determining the proportion or number of CD23+ allergen-specific Bmem or CD23hi allergen-specific Bmem in the first and second samples. Including, An increase in the proportion or number of CD23+ allergen-specific Bmem or CD23hi allergen-specific Bmem in the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject. A method is provided.

[0028] In this aspect, the method further comprises determining the proportion or number of CD23+ allergen-specific Bmem or CD23hi allergen-specific Bmem that express one or more additional biomarkers. The additional biomarker(s) may be the biomarker(s) described in Table 2 and / or Table 3. Preferably, the biomarker(s) is / are biomarker(s) that increase 1.5-fold, 2-fold, 3-fold or 4-fold or decrease to one and a half, one half, one third or one quarter as described in Table 2 and / or Table 3.

[0029] In another aspect, the present invention is a method for determining the effectiveness of allergen immunotherapy in a subject, comprising: - providing a first sample obtained from the subject before receiving allergen immunotherapy; - providing a second sample obtained from a subject who has received or is receiving allergen immunotherapy, wherein the first and second samples comprise B cells; and - determining the proportion or number of IL4Rα+ allergen-specific Bmem or IL4Rαhi allergen-specific Bmem in the first and second samples. The method further comprises: An increase in the proportion or number of IL4Rα+ allergen-specific Bmem or IL4Rαhi allergen-specific Bmem in the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject.

[0030] In this aspect, the method further comprises determining the proportion or number of IL4Rα+ allergen-specific Bmem or IL4Rαhi allergen-specific Bmem that express one or more additional biomarkers. The additional biomarker may be a biomarker described in Table 2 and / or Table 3. Preferably, the biomarker is a biomarker that increases by 1.5-fold, 2-fold, 3-fold or 4-fold or decreases to 1 / 1.5, 1 / 2, 1 / 3 or 1 / 4 as described in Table 2 and / or Table 3.

[0031] In any aspect or embodiment, the allergen-specific B cells, preferably Bmem, are specific to the allergen included in the allergen immunotherapy that the subject has received or is receiving.

[0032] In any aspect or embodiment, the proportion of IgG+Bmem or IgG4+ allergen-specific Bmem is the relative number of IgG+Bmem or IgG4+ allergen-specific Bmem compared to the total number of Bmem or allergen-specific Bmem, or is the number of IgG+Bmem or IgG4+ allergen-specific Bmem cells per microliter of blood.

[0033] In any aspect or embodiment, IgG+ may include all IgG isotypes.

[0034] In any aspect or embodiment, the proportion of CD29+ allergen-specific Bmem or CD29hi allergen-specific Bmem is the relative number of CD29+ allergen-specific Bmem or CD29hi allergen-specific Bmem compared to the total number of allergen-specific Bmem, or is the number of CD29+ allergen-specific Bmem or CD29hi allergen-specific Bmem cells per microliter of blood.

[0035] In any aspect or embodiment, the proportion of CD23+ allergen-specific Bmem or CD23hi allergen-specific Bmem is the relative number of CD23+ allergen-specific Bmem or CD23hi allergen-specific Bmem compared to the total number of allergen-specific Bmem, or is the number of CD23+ allergen-specific Bmem or CD23hi allergen-specific Bmem cells per microliter of blood.

[0036] In any aspect or embodiment, the proportion of IL4Rα+ allergen-specific Bmem or IL4Rαhi allergen-specific Bmem is the relative number of IL4Rα+ allergen-specific Bmem or IL4Rαhi allergen-specific Bmem compared to the total number of allergen-specific Bmem, or is the number of IL4Rα+ allergen-specific Bmem or IL4Rαhi allergen-specific Bmem cells per microliter of blood.

[0037] An increase in the level or amount of one or more biomarkers in B cells in a second sample compared to a first sample may be accompanied by a decrease in the level or amount of one or more biomarkers in the same cells, which indicates the effectiveness of allergen immunotherapy in a subject.

[0038] In some embodiments of the invention, the increase in the level or amount of one or more biomarkers may be a fold change of a different magnitude compared to a decrease in the fold change of the level or amount of one or more biomarkers in B cells in a second sample compared to a first sample, which indicates the effectiveness of allergen immunotherapy in a subject.

[0039] In any aspect of the invention, the second sample may be obtained from a subject at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months after the start of allergen immunotherapy, or after any other period defined herein. In some embodiments of the invention, the second sample, or subsequent samples, may be obtained from a subject between about 1 and 12 months, between about 2 and 12 months, between about 3 and 12 months, between about 4 and 12 months, between about 5 and 12 months, between about 6 and 12 months, between about 1 and 5 years, between about 2 and 5 years, between about 3 and 5 years, between about 4 and 5 years, or up to about 5 years after the start of allergen immunotherapy.

[0040] In any aspect of the present invention, the second sample may be obtained from a subject at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months after the start of allergen immunotherapy. In some embodiments of the present invention, the second sample, or subsequent samples, may be obtained from a subject during 1 - 12 months, 2 - 12 months, 3 - 12 months, 4 - 12 months, 5 - 12 months, 6 - 12 months, 1 - 5 years, 2 - 5 years, 3 - 5 years, 4 - 5 years, or up to 5 years after the start of allergen immunotherapy.

[0041] In any aspect of the present invention, the second sample may be obtained from a subject 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months after the start of allergen immunotherapy.

[0042] In some embodiments of the present invention, the second sample may be obtained from a subject at least after the start phase of allergen immunotherapy. For example, the start phase of allergen immunotherapy may be 4 months, or about 4 months, for sublingual immunotherapy (SLIT) for rye grass pollen (RGP) or house dust mite (HDM) allergy, or 63 days, or about 63 days, for ultra - rush allergen immunotherapy (AIT) for insect venom allergy.

[0043] In any aspect of the present invention, allergen immunotherapy may be administered sublingually (under the tongue), orally, by needle - free injection, by subcutaneous injection (under the skin), or in some cases, by intradermal injection, or by using transcutaneous or intralymphatic administration routes. In a preferred embodiment of this aspect of the present invention, allergen immunotherapy may be administered sublingually.

[0044] In any aspect of the present invention, the B cells may comprise or consist of B memory cells (Bmem). Preferably, the B cells are allergen-specific Bmem. In any embodiment, the Bmem can be identified as having the same or a similar transcriptional profile as clusters 3 and 4 described in the examples. Preferably, cluster 3 mainly IGHM , IGHG3 and IGHG1 are expressed, and cluster 4 is enriched for IGHE , IGHG1 and IGHG4 expression. Cluster 3 preferably has a decreased CXCR4 expression along with an increased expression of ITGB1 and PPP1R18 , and an increased expression of FGR , SIGLEC10 , CD99 , TLE3 as well as a decreased expression of TXNIP , BTG1 and FCER2 compared to Bmem having the same or a similar transcriptional profile as the cells identified as cluster 1 (see examples). Cluster 4 preferably has an increased expression of immune-related genes such as PARM1 , IL13RA1 , ITGB1 , PPP1R18 , SELL , FCER2 etc. and a decreased expression of CXCR4 and CD69 compared to Bmem having the same or a similar transcriptional profile as the cells identified as cluster 1 (see examples). Cluster 4 Bmem can have an increased expression of SELL , FCER2 , IGHE , IL13RA1 , IL4R , PARM1 and ITGB1 , and cluster 3 Bmem IGHM , IGHD , CD1C , PLAC8 , SYK , FGR , FCGR2B , FCRLA , FCRL2 ,FCRL3 and FCRL5 and ITGAX may have increased expression. Cluster 4 Bmem is not shared with clusters 1-3 Bmem ITGB1 and FCER2 and IL13RA1 may have an expression pattern.

[0045] In any aspect of the present invention, the sample may be a body fluid, such as a whole blood sample or serum. Alternatively, the sample may be a tissue sample. The blood sample may be whole blood, buffy coat, peripheral blood mononuclear cells (PBMCs), cord blood, a purified or sorted cell population, or a body fluid. Body fluids include lymph, semen, nasal secretions, bronchial secretions, alveolar fluid, cerebrospinal fluid, endolymph, synovial fluid, intrathoracic fluid, pericardial fluid (pericardial sac fluid), menstrual fluid, or a combination thereof. The tissue sample may be selected from tonsils, lymph nodes, bronchi, nose, or intestine or skin biopsies. The blood sample may be treated, for example, with heparin or ethylenediaminetetraacetic acid (EDTA) to prevent clotting.

[0046] In a preferred embodiment, the sample contains peripheral blood mononuclear cells, which may be enriched or purified for allergen-specific B cells. The enrichment or purification process for allergen-specific B cells may be any suitable cell separation and isolation technique, such as techniques that utilize cell adhesion, density-based centrifugation, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and antibody binding methods that include adapter binding, selective expansion in culture, laser capture microdissection, or erythrocyte rosetting. Most preferably, the enrichment or purification for allergen-specific B cells is performed by FACS.

[0047] In any aspect or embodiment, the biomarker is a protein, nucleic acid, e.g., RNA, or an amplification product. When the biomarker is a nucleic acid or an amplification product, the method includes determining the level or amount of gene or RNA expression. Preferably, the biomarker is one or more nucleic acids comprising a nucleotide sequence derived from a gene or an RNA transcript of the gene, and the gene is IGHE , ITGB1 , CD69 , IL13RA1 , CD99 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , SIGLEC10 , CD1C , FCER2 , and IL4R is one or more of.

[0048] In any aspect or embodiment described herein, when a protein is referred to, the invention may also include, optionally, determining or measuring the presence, level or amount of the corresponding RNA (translated into the protein) or its amplification product.

[0049] In some embodiments of the invention, the method may further include determining the level or amount of one or more biomarkers in B cells in a first and a second sample, and the biomarker is IGHE , PPP1R18 , ITGB1 , PARM1 , c-jun , CD69 , selected from the group consisting of IgE, phostensin and CD29, and an increase in the level or amount of one or more biomarkers selected from IGHE , PPP1R18 , ITGB1 , PARM1 , IgE, phostensin and CD29 in B cells in the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject, and / or, in B cells in the second sample compared to the first sample, c-jun andCD69 A decrease in the level or amount of one or more biomarkers selected from the group consisting of nucleic acids, such as RNA or amplification products, or proteins indicates the effectiveness of allergen immunotherapy in a subject.

[0050] In any aspect of the present invention, the level or amount of one or more biomarkers may be the level or amount of RNA. Preferably, the RNA is either pre-mRNA or mature mRNA, and the change in the level or amount of RNA can be determined using RNA sequencing. Most preferably, the change in the level or amount of RNA can be determined using single-cell transcriptomics.

[0051] In any aspect of the present invention, allergen immunotherapy is for the treatment of sensitization to an allergen. The allergen may be a food-based allergen, an airborne allergen, a drug allergen, a peptide allergen, a goat milk allergen, a plant allergen, an animal allergen, or an arthropod allergen, where the arthropod may be an insect, a myriapod, a spider, or a crustacean (e.g., an insect, a mite, a crustacean).

[0052] When the allergen may be food-based, the allergen may include nuts, sesame, buckwheat, peanuts, milk protein, egg white, etc. Typical food allergens include milk allergens (Bos d 4, 5, and 8), peanut allergens (Ara h 1, 2, 3, 6, 8, and 9), hazelnut (Cor a 9 and 14), cashew nut (Ana o 3), walnut (Jug r 1), Brazil nut (Ber e 1), sesame (Ses i 1), buckwheat (Fag e 3), almond (Pru du 6), peach (Pru p 1 and Pru p 3), shrimp (Pen m 1), and wheat (Tri a 19; omega-5-gliadin, Tri a 20, Tri a 21).

[0053] If the allergen may be airborne, the allergen may include grass pollen, tree pollen, weed pollen, animal dander, house dust mite feces, etc., and fungal allergens.

[0054] Other common airborne allergens include house dust mite feces from Dermatophagoides pteryonyssinus (Der p 1 and 2) or Dermatophagoides farina (Der f 1 and 2); pollen allergens from rye grass (Lol p 1, 5), timothy grass (Phl p 1, 5), American ragweed (Pas n 1), Bermuda grass (Cyn d 1), pigweed (Amb a 1), pellitory species (Par o 1; Par j 1, 2), birch (Bet v 1) and other atmospheric pollens including Olea europaea, Artemisia sp., gramineae, etc.; and animal dander from, for example, cats (Fel d 1) and dogs (Can f 1).

[0055] Other allergens may include venom allergens from honeybees (Api m 1, 3, 10); phospholipases from yellow jacket wasp Vespula maculifrons, common wasp Polistes exclamans and North American hornet Dolichovespula maculata, and venom from jumper ant Myrmecia pilosula.

[0056] Other allergens include, in the case of mold allergy, Alternaria, Aspergillus and Cladosporium species, as well as blood-sucking arthropods such as mosquitoes (Anopheles sp., Aedes sp., Culiseta sp., Culex sp.); flies (Phlebotomus sp., Culicoides sp.), particularly flies including midges, horseflies and Culicoides; ticks (Dermacenter sp., Ornithodoros sp., Otobius sp.); fleas, e.g., fleas belonging to the genus Xenopsylla, the genus Pulex and the genus Ctenocephalides, which may be derived from allergic dermatitis caused by the order Siphonaptera.

[0057] In another aspect of the present invention, there is provided a kit for determining the effectiveness of allergen immunotherapy in a subject, the kit comprising means for detecting changes in the level or amount of transcripts, amplification products and / or proteins, wherein the transcripts, amplification products and / or proteins are IGHE , ITGB1 , CD69 , IL13RA1 , CD99 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , SIGLEC10 , CD1C , FCER2 , IL4R or protein: IgE, CD29, CD69, IL13Rα, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, SIGLEC10, CD1c, CD23, IL4Rα, and optionally PPP1R18 , PARM1 , hostensin, and c-jun selected from the group consisting of any one or more of, and may include a written instruction manual.

[0058] In another aspect of the present invention, a method for measuring or detecting at least one biomarker, comprising the step of obtaining a sample from a subject after allergen immunotherapy, the sample comprising B cells, and IGHE , ITGB1 , CD69 , IL13RA1 , CD99 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , SIGLEC10 , CD1C , FCER2 , IL4R , PPP1R18 , PARM1 and c-jun measuring or detecting in the sample at least one biomarker or a fragment thereof selected from the group consisting of the protein: IgE, CD29, CD69, IL13Rα, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, SIGLEC10, CD1c, CD23, IL4Rα, and phosphostensin or any combination thereof. A method is provided that includes this step.

[0059] In a preferred embodiment, the measurement or detection of at least one biomarker indicates that the subject has responded to the allergen immunotherapy that the subject has received or is receiving. For example, IGHE , ITGB1 , IL13RA1 , CD99 , FCRL3 , FCRL2 , SIGLEC10 , CD1C , FCER2 , IL4R , PPP1R18 , PARM1 if at least one biomarker selected from the group consisting of IgE, CD29, IL13Rα, CD99, FCRL3, FCRL2, SIGLEC10, CD1c, CD23, IL4Rα, and phosphostensin or any combination thereof is not detected or is detected at a lower level in B cells in a sample obtained from the subject prior to allergen immunotherapy, it indicates that the subject has responded to the allergen immunotherapy.

[0060] CD69 、 IGHD 、 CXCR4 、 FCRL3 、 FCRL2 、 FCRL5 、 CD1C 、 FCER2 、 IL4R 、 c-jun 、CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD1c, CD23, IL4Rα, or any combination thereof, when detected or detected at a higher level in a sample obtained from a subject prior to allergen immunotherapy, indicates that the subject has responded to allergen immunotherapy.

[0061] In one embodiment, in B cells in a second sample compared to a first sample, IGHE 、 ITGB1 、 IL13RA1 、 CD99 、 FCRL3 、 FCRL2 、 SIGLEC10 、 CD1C 、 FCER2 、 IL4R 、 PPP1R18 、 PARM1 、IgE, CD29, IL13Rα, CD99, FCRL3, FCRL2, SIGLEC10, CD1c, CD23, IL4Rα and the increase in the level or amount of one or more of the hostensin biomarkers, in the same B cells in the second sample compared to the first sample, CD69 、 IGHD 、 CXCR4 、 FCRL3 、 FCRL2 、 FCRL5 、 CD1C 、 FCER2 、 IL4R 、 c-jun 、CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD1c, CD23 and IL4Rα may be accompanied by a decrease in the level or amount of one or more of them, which indicates that the subject has responded to allergen immunotherapy.

[0062] Preferably, IGHE , ITGB1 , CD99 , SIGLEC10 , FCER2 , IL4R one or more levels of IgE, CD29, CD99, SIGLEC10, CD23, and IL4Rα are higher in a second sample obtained from the subject during or after allergen immunotherapy than in a first sample obtained from the subject before allergen immunotherapy.

[0063] In one embodiment, CD69 one or more levels of and CD69 are lower in a second sample obtained from the subject during or after allergen immunotherapy than in a first sample obtained from the subject before allergen immunotherapy.

[0064] In one embodiment, when compared to the level in a sample obtained from the subject before allergen immunotherapy, IGHE , ITGB1 , CD99 , SIGLEC10 , FCER2 , IL4R one or more increased levels of IgE, CD29, CD99, SIGLEC10, CD23, and IL4Rα may be CD69 associated with a decrease in one or more levels of and CD69.

[0065] In one embodiment, IGHE , ITGB1 , IL13RA1 , CD99 , FCER2 , IL4R one or more levels of IgE, CD29, IL13Rα, CD99, CD23, and IL4Rα may be higher in a second sample obtained from the subject after allergen immunotherapy than in a first sample obtained from the subject before allergen immunotherapy. The increase in level is CD69 , IGHD, CXCR4 , FCRL3 , FCRL2 , FCRL5 , CD1C may be accompanied by a decrease in the level of any one or more of CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, and CD1c.

[0066] In one embodiment, ITGB1 , CD99 , FCRL3 , FCRL2 , SIGLEC10 , CD1C the level of any one or more of CD29, CD99, FCRL3, FCRL2, SIGLEC10, and CD1c may be higher in a second sample obtained from the subject after allergen immunotherapy than in the first sample obtained from the subject before allergen immunotherapy. The increase in level is CD69 , IGHD , CXCR4 , FCRL5 , FCER2 , IL4R may be accompanied by a decrease in any one or more of CD69, IgD, CXCR4, FCRL5, CD23, and IL4Rα.

[0067] In another aspect of the invention, a biomarker panel for determining the effectiveness of allergen immunotherapy in a subject, the following biomarkers: IGHE , ITGB1 , CD69 , IL13RA1 , CD99 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , SIGLEC10 , CD1C , FCER2 , IL4R , PPP1R18 , PARM1 and c-junor a protein: at least one of IgE, CD29, CD69, IL13Rα, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, SIGLEC10, CD1c, CD23, IL4Rα, and hostensin, or any combination thereof, is provided, wherein measurement or detection of at least one biomarker indicates that the subject has responded to allergen immunotherapy.

[0068] In a preferred embodiment of this aspect of the invention, IGHE , ITGB1 , IL13RA1 , CD99 , FCRL3 , FCRL2 , SIGLEC10 , CD1C , FCER2 , IL4R , PPP1R18 , PARM1 , the level of any one or more of IgE, CD29, IL13Rα, CD99, FCRL3, FCRL2, SIGLEC10, CD1c, CD23, IL4Rα, and hostensin is higher compared to its level in a sample obtained from the subject before allergen immunotherapy; and / or CD69 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , CD1C , FCER2 , IL4R , c-jun , a biomarker panel is provided, wherein the level of any one or more of CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD1c, CD23, and IL4Rα is lower compared to its level in a sample obtained from the subject before allergen immunotherapy.

[0069] In another embodiment, a biomarker panel for determining the effectiveness of allergen immunotherapy in a subject, the biomarker panel comprising the following biomarkers: IGHE , ITGB1 , CD69 , CD99 , SIGLEC10 , FCER2 , IL4R、IgE, CD29, CD69, CD99, SIGLEC10, CD23, and IL4Rα, or at least one of any combination thereof, and a panel is provided wherein measurement or detection of at least one biomarker indicates that the subject has responded to allergen immunotherapy.

[0070] More preferably, the biomarker panel, when compared to the level of the same biomarker in a sample obtained from the subject prior to allergen immunotherapy, IGHE , ITGB1 , CD99 , SIGLEC10 , FCER2 , IL4R , may detect an increase in the level of any one or more of IgE, CD29, CD99, SIGLEC10, CD23, and IL4Rα, and this increase may be CD69 accompanied by a decrease in the level of any one or more of

[0071] In another embodiment, a biomarker panel for determining the effectiveness of allergen immunotherapy in a subject, the following biomarkers: IGHE , ITGB1 , CD69 , IL13RA1 , CD99 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , CD1C , FCER2 , IL4R , IgE, CD29, CD69, IL13Rα, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD1c, CD23, and IL4Rα, or at least one of any combination thereof, and a panel is provided wherein measurement or detection of at least one biomarker indicates that the subject has responded to allergen immunotherapy.

[0072] More preferably, the biomarker panel, when compared to the level of the same biomarker in a sample obtained from the subject prior to allergen immunotherapy, IGHE , ITGB1 ,IL13RA1 , CD99 , FCER2 , IL4R , IgE, CD29, IL13Rα, CD99, CD23, and IL4Rα, may detect an increase in the level of any one or more thereof, and this increase is CD69 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , CD1C , CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, and CD1c, may be accompanied by a decrease in the level of any one or more thereof.

[0073] In another embodiment, a biomarker panel for determining the effectiveness of allergen immunotherapy in a subject, the following biomarkers: ITGB1 , CD69 , CD99 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , SIGLEC10 , CD1C , FCER2 , IL4R , CD29, CD69, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, SIGLEC10, CD1c, CD23, and IL4Rα, or any combination thereof, including at least one, and measurement or detection of at least one biomarker indicates that the subject has responded to allergen immunotherapy, a panel is provided.

[0074] More preferably, the biomarker panel, when compared to the level of the same biomarker in a sample obtained from the subject before allergen immunotherapy, ITGB1 , CD99 , FCRL3 , FCRL2 , SIGLEC10 , CD1C , CD29, CD99, FCRL3, FCRL2, SIGLEC10, and CD1c, may detect an increase in the level of any one or more thereof, and this increase is CD69 , IGHD, CXCR4 , FCRL5 , FCER2 , IL4R may be associated with a decrease in the level of any one or more of CD69, IgD, CXCR4, FCRL5, CD23, and IL4Rα.

[0075] In certain embodiments, a panel of biomarkers is used to determine the effectiveness of allergen immunotherapy. Biomarker panels of any size can be used in the practice of the present invention. In certain embodiments, the present invention includes a biomarker panel comprising at least 3, at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or more biomarkers. In one embodiment, the biomarker panel includes means such as polynucleotides, probes, ligands, antibodies, etc. for detecting the presence, increase, or decrease of at least one or more biomarkers.

[0076] In another aspect, the present invention provides a kit, panel, or microarray comprising at least two diagnostic reagents as described herein, wherein each reagent identifies a different biomarker. In one embodiment, the kit includes diagnostic reagents that bind to or individually complex with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more biomarkers.

[0077] In any aspect of the present invention, a biomarker may be its specific peptide fragment or nucleic acid fragment, thereby serving as a substitute for the full-length protein or nucleic acid.

[0078] In any aspect of the present invention, the first and second samples are provided or obtained from the same subject. Alternatively, the first sample may be from a different subject or subjects having the same or related allergies as the subject from whom the second sample is provided or obtained and who has not received allergen immunotherapy.

[0079] In any aspect, instead of providing the first sample and determining the level or amount of one or more biomarkers in the B cells of the first sample, a sample obtained after the initiation of allergen immunotherapy (i.e., the test sample) may be compared to a reference value, or reference range, or reference standard of one or more biomarkers for a subject having the same or related allergies who (a) has not received allergen immunotherapy or (b) has received allergen immunotherapy. In this example, if the level or amount of one or more biomarkers in the B cells of the test sample is the same or similar to the reference value, or reference range, or reference standard of one or more biomarkers for a subject having the same or related allergies who has not received allergen immunotherapy, a determination can be made that the subject is not responding to allergen immunotherapy. In this example, if the level or amount of one or more biomarkers in the B cells of the test sample is different from the reference value, or reference range, or reference standard of one or more biomarkers for a subject having the same or related allergies who has not received allergen immunotherapy, a determination can be made that the subject is responding to allergen immunotherapy.

[0080] In this example, if the level or amount of one or more biomarkers in B cells in a test sample is different from the reference value, or reference range, or reference standard of one or more biomarkers for a subject having the same or related allergies who has received allergen immunotherapy, a determination can be made that the subject is responding to allergen immunotherapy. In this example, if the level or amount of one or more biomarkers in B cells in a test sample is different from the reference value, or reference range, or reference standard of one or more biomarkers for a subject having the same or related allergies who has received allergen immunotherapy, a determination can be made that the subject is not responding to allergen immunotherapy.

[0081] In any embodiment of the present invention, the test sample may be obtained at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months after the start of allergen immunotherapy. In some embodiments of the present invention, the test sample, or subsequent samples, may be obtained from the subject between 6 and 12 months after the start of allergen immunotherapy, or up to 5 years. In some embodiments of the present invention, the test sample may be obtained at least at the start of allergen immunotherapy, for example, 4 months for SLIT for RGP and HDM or 63 days for ultra rush for insect venom allergy (in this example, there may be no sample taken prior to treatment).

[0082] To perform an unmatched sample comparison, typically a normal reference range or reference standard is first defined. Next, the comparison may be made using the median expression level of a particular biomarker if identified using a fluorescence detection method, or may include analysis of the percentage of positive cells for one or more biomarkers.

[0083] In any aspect or embodiment of the invention described herein, the step of determining the level or amount of one or more biomarkers in B cells in a sample (e.g., the first or second sample) may instead be the step of determining the percentage of positive cells for one or more biomarkers in B cells in the sample (e.g., the first or second sample). Further, if an aspect or embodiment of the invention contemplates an increase or, in some cases, a decrease in the level or amount of one or more biomarkers in B cells in a sample (e.g., the second sample being compared to the first sample) compared to another sample, that aspect or embodiment may be carried out by comparing an increase or decrease in the percentage of positive cells having one or more biomarkers. When comparing samples collected before and after allergen immunotherapy, the determining step and the comparing step may include an increase or decrease in the absolute or relative number of positive cells, and this number may be used as a measure indicating the effectiveness of allergen immunotherapy in the subject. For example, in this further aspect of the invention, a method of determining the effectiveness of allergen immunotherapy in a subject, comprising - providing a first sample obtained from the subject before receiving allergen immunotherapy, - providing a second sample obtained from the subject who has received or is receiving allergen immunotherapy, wherein the first and second samples comprise B cells, and - determining the percentage of positive cells having one or more biomarkers in B cells in the first and second samples, wherein the biomarkers are selected from the group consisting of IgE, CD29, CD69, IL13Rα, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, SIGLEC10, CD1c, CD23, and IL4Rα comprising, An increase in the percentage of positive cells having one or more biomarkers selected from IgE, CD29, IL13Rα, CD99, FCRL3, FCRL2, SIGLEC10, CD1c, CD23, and IL4Rα in B cells in a second sample compared to a first sample indicates the effectiveness of allergen immunotherapy in a subject, and / or A method is provided that an decrease in the percentage of positive cells having one or more biomarkers selected from CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD1c, CD23, and IL4Rα in B cells in a second sample compared to a first sample indicates the effectiveness of allergen immunotherapy in a subject.

[0084] In any aspect or embodiment, the B cells are Bmem cells (Bmem), more preferably, IgG4+ Bmem. Typically, the Bmem are allergen-specific Bmem, and the allergen is the same allergen provided in allergen immunotherapy. Thus, the Bmem may be allergen-specific IgG4+ Bmem.

[0085] As used herein, unless the context requires otherwise, the term "comprise" and variations of this term such as "comprising," "comprises," and "comprised" are not intended to exclude further additives, components, integers, or steps.

[0086] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs are given by way of example and will become apparent from the following description with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0087]

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[0088] It is understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or obvious from the text or drawings. All of these different combinations form various alternative aspects of the invention.

[0089] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description given by way of examples and with reference to the accompanying drawings.

[0090] Here, certain embodiments of the invention are referred to in detail. It is understood that the invention is described in conjunction with the embodiments, but the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover any alternatives, modifications, and equivalents, which may be included within the scope of the invention as defined by the claims.

[0091] The present invention provides methods and kits for determining the therapeutic efficacy of allergen immunotherapy and for measuring or detecting biomarkers in a subject undergoing allergen immunotherapy.

[0092] Advantages of aspects or embodiments of the present invention are that the method provides rapid results that can efficiently help understand the efficacy of treatment when a subject is undergoing allergen immunotherapy, by focusing on changes in the level or amount of selection of specific genes and / or proteins. This can determine responsive individuals from non-responsive individuals and allows non-responsive individuals to be treated with different therapies or allergen immunotherapy. Thus, the present invention reduces the time a person is undergoing allergen immunotherapy when it may be mostly or entirely ineffective, thereby reducing the risk of adverse events associated with allergen immunotherapy. Further, the present invention provides increased treatment adherence as it can enable recognition of a treatment response in a subject. There may also be cost savings for patients in jurisdictions where allergen immunotherapy is not government subsidized. In such situations, many patients must make financial decisions regarding which drug therapies they can afford, and thus it is considered advantageous to identify ineffective treatments early.

[0093] General Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition, group of steps or group of compositions shall be construed to include one and more (i.e., one or more) of those steps, compositions, groups of steps or groups of compositions. Accordingly, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise and vice versa. For example, a reference to "a" includes one as well as two or more; a reference to "an" includes one as well as two or more; a reference to "the" includes one as well as two or more, and so on.

[0094] One of ordinary skill in the art will recognize that the present invention is susceptible to variations and modifications other than those specifically described. The present invention is to be understood as including all such variations and modifications. The present invention includes all of the steps, features, compositions and compounds referred to or indicated herein, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0095] One of ordinary skill in the art will recognize many methods and materials similar or equivalent to those described herein, and these methods and materials are considered to be usable in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0096] All patents and publications referred to herein are hereby incorporated by reference in their entirety.

[0097] The present invention should not be limited in scope by the specific embodiments described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions and methods are clearly within the scope of the present invention.

[0098] Unless specifically stated otherwise, any example or embodiment of the invention described in this specification is to be construed as applicable to any other example or embodiment of the invention with the necessary modifications made thereto.

[0099] Unless otherwise specifically defined, all technical and scientific terms used in this specification shall be construed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0100] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures well known to those of ordinary skill in the art. Such techniques are described and explained throughout the literature of sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0101] The term "and / or", e.g., "X and / or Y", is understood to mean "X and Y" or "X or Y", and is to be interpreted as giving clear support to both meanings or either meaning.

[0102] As used herein, the term "derived from" is to be interpreted as indicating that the specified integer can be obtained from a particular source, but not necessarily directly from that source.

[0103] Selected definitions As used herein, the term "allergen" refers to any naturally occurring protein or mixture of proteins or chemical substance / drug that has been reported to induce an allergy, i.e., an IgE-mediated reaction, when an individual is repeatedly exposed to it.

[0104] "Allergy", also referred to herein as "allergy reactivity", is any condition in which there is an unwanted (e.g., type 1 hypersensitivity) immune response (i.e., an allergic response or reaction) to a substance. Such a substance is referred to herein as an allergen. An allergy or allergic condition includes, but is not limited to, allergic asthma, hay fever, hives, eczema, plant allergies, bee sting allergies, pet allergies, latex allergies, mold allergies, cosmetic allergies, food allergies, allergic rhinitis or hay fever, local allergic reactions, anaphylaxis, atopic dermatitis, hypersensitivity reactions and other allergic conditions. An allergic reaction can be the result of an immune response to any allergen. In some embodiments, the allergy is a food allergy. Food allergies include, but are not limited to, milk allergy, egg allergy, nut allergy, fish allergy, shellfish allergy, soybean allergy or wheat allergy.

[0105] As used herein, the term "hypersensitivity" refers to an undesirable reaction resulting from a normal immune response, including allergy and autoimmunity. This excessive reaction of the immune system can be damaging, uncomfortable or even life-threatening. Hypersensitivity reactions require prior sensitization of the host.

[0106] As used herein, the term "allergen sensitization" or "sensitization to an allergen" refers to the production of IgE antibodies following the first exposure to an allergen or antigen that subsequently results in an allergic reaction or allergic reactivity.

[0107] As used herein, the term "immune cell" refers to any cell involved in an immune response. These cells include, but are not limited to, megakaryocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, dendritic cells, natural killer cells, NKT cells, NK-like cells, T cells, B cells and plasma cells.

[0108] The term "recombinant" is understood to mean the product of artificial genetic recombination. Thus, in the context of a recombinant allergen or antigen, this term does not include naturally occurring allergens or antigens. However, if such an allergen or antigen is isolated, it should be considered an isolated allergen or antigen. Similarly, when a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant allergen or antigen. Recombinant proteins also include proteins expressed by artificial recombinant means, for example, if the protein is within the cell, tissue or subject in which it is expressed.

[0109] As used herein, the term "ligand" or "reagent" refers to a molecule that binds to or forms a complex with a biomarker protein, molecular form, or peptide, such as an antibody, antibody mimetic, or equivalent, that binds to or forms a complex with a biomarker, its molecular form, or fragment identified herein with respect to a protein biomarker. In certain embodiments in which biomarker expression is to be evaluated, the ligand can be a nucleotide sequence, such as a polynucleotide or oligonucleotide, primer, or probe.

[0110] As used herein, the terms "antibody," "immunoglobulin," or "Ig" refer to a protein that can specifically bind to one or a few closely related antigens due to the antigen-binding domains contained within the Fv. This term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, deimmunized antibodies, synhumanized antibodies, half antibodies, bispecific antibodies). Antibodies generally contain constant domains, which can be arranged in constant regions or constant fragments or crystallizable fragments (Fc). Exemplary forms of antibodies include a four-chain structure as their basic unit. Full-length antibodies include two covalently linked heavy chains (about 50-70 kD) and two light chains (each about 23 kDa). Light chains generally contain a variable region (if any) and a constant domain, and in mammals, are either κ light chains or λ light chains. Heavy chains generally contain one or two constant domains (s) (if any) linked by a hinge region to additional constant domains (s) by a variable region. Mammalian heavy chains are one of the following types: α, δ, ε, γ, or μ. Each light chain is also covalently bound to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds can vary between different types of antibodies. Each chain has an N-terminal variable region (VH or VL, each about 110 amino acids in length) and one or more constant domains at the C-terminus. The constant domain of the light chain (CL, about 110 amino acids in length) is aligned and disulfide-bonded to the first constant domain of the heavy chain (CH1, 330-440 amino acids in length). The light chain variable region is aligned with the heavy chain variable region. The antibody heavy chain can include two or more additional CH domains (CH2, CH3, etc.) and can include a hinge region between the CH1 and CH2 constant domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.In one example, the antibody is a murine (mouse or rat) antibody or a primate (such as human) antibody. In one example, the antibody heavy chain lacks a C-terminal lysine residue. In one example, the antibody is humanized, semi-humanized, chimeric, CDR-grafted or deimmunized.

[0111] The term "protein" is to be interpreted as including a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently bonded to each other (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently bonded using appropriate chemical or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0112] The term "polypeptide" or "polypeptide chain" is understood from the foregoing paragraphs to mean a series of consecutive amino acids linked by peptide bonds.

[0113] The term "microarray" refers to a regular arrangement of binding / complexing array elements or ligands, such as antibodies, on a substrate.

[0114] As used herein, the term "polynucleotide" (used in the singular or plural) generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for example, polynucleotides as defined herein include, without limitation, single-stranded and double-stranded DNA, DNA containing single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA containing single-stranded and double-stranded regions, which may be single-stranded or more typically double-stranded, or hybrid molecules containing DNA and RNA including single-stranded and double-stranded regions. Further, as used herein, the term "polynucleotide" refers to triple-stranded regions containing RNA or DNA or both RNA and DNA. The term "polynucleotide" specifically includes cDNA. This term includes DNA (including cDNA) and RNA containing one or more modified bases. Generally, the term "polynucleotide" encompasses unmodified polynucleotides in all chemically, enzymatically and / or metabolically modified forms, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.

[0115] As used herein, the term "oligonucleotide" refers to a relatively short polynucleotide of less than 20 bases, including, without limitation, single-stranded deoxyribonucleotides, single-stranded or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNA. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example, using commercially available automated oligonucleotide synthesizers. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques, as well as by the expression of DNA in cells and organisms.

[0116] As used herein, the term "binds" in the context of the interaction of an antibody with an allergen or antigen means that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the allergen or antigen. For example, an antibody recognizes and binds to a specific protein structure rather than to a protein generally. When an antibody binds to epitope "A", in a reaction containing the labeled "A" and the protein, the presence of a molecule containing epitope "A" (or free unlabeled "A") reduces the amount of labeled "A" bound to the antibody.

[0117] As used herein, the term "epitope" (synonymous with "antigenic determinant") is understood to mean the region of a protein (such as an allergen or antigen) to which the antigen-binding domain of an antibody binds.

[0118] As used herein, the term "condition" refers to the breakdown or interference of normal function and should not be limited to any particular condition, including diseases or disorders.

[0119] "Diagnosis" as used herein generally involves a determination as to whether a subject is likely to be affected by a given disease, disorder or dysfunction. One of ordinary skill in the art often makes a diagnosis based on one or more diagnostic indicators, i.e., biomarkers, the presence, absence, or amount of which indicates the presence or absence of a disease, disorder or dysfunction.

[0120] As used herein, the term "prognosis" generally refers to the prediction of the likely course and outcome of a clinical condition or disease. The prognosis of a patient is typically made by evaluating the disease factors or symptoms that indicate a favorable or unfavorable course or outcome of the disease. It is understood that the term "prognosis" does not necessarily refer to the ability to predict the course or outcome of a condition with 100% accuracy. Instead, one of ordinary skill in the art would understand that the term "prognosis" refers to an increased likelihood that a particular course or outcome will occur, i.e., that a patient presenting a given condition is more likely to experience a course or outcome than an individual not presenting the condition.

[0121] As used herein, the term "subject" shall be construed to mean any animal, including a human, e.g., a mammal. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0122] As used herein, the term "reference standard" refers to the source of the reference biomarker level. The "reference standard" is preferably provided by using the same assay technique as that used for measuring the biomarker level of the subject in the reference object or population in order to avoid any errors during standardization. The reference standard may alternatively be a numerical value, a predetermined cut-off point, an average value, an average, a numerical average or a range of numerical averages, a numerical pattern, a ratio, a graphical pattern, a protein mass profile, a protein level profile, a median expression level or a percentage of positive cells from the same biomarker or multiple biomarkers in the reference object or reference population. In a preferred embodiment where the expression of a nucleic acid sequence encoding a biomarker is to be evaluated, the reference standard may be the expression level or expression profile of one or more biomarkers.

[0123] As used herein, the term "percentage of positive cells" is to be construed as meaning the proportion of positive cells having one or more biomarkers when comparing samples collected before and after allergen immunotherapy, and this proportion can be calculated by determining an increase or decrease in the absolute or relative number of positive cells. This measurement can be used in addition to, or as an alternative to, a measurement to determine an increase or decrease in the level or amount of one or more biomarkers to indicate the effectiveness of allergen immunotherapy in a subject.

[0124] Method for determining the effectiveness of allergen immunotherapy In one aspect, the present invention is a method for determining the effectiveness of allergen immunotherapy in a subject, comprising: - providing a first sample obtained from the subject before receiving allergen immunotherapy; - providing a second sample obtained from a subject who has received or is receiving allergen immunotherapy, wherein the first and second samples comprise B cells, and - determining the level or amount of one or more biomarkers in B cells in the first and second samples, or determining the percentage of positive cells having one or more biomarkers in B cells in the first and second samples, wherein the biomarker is selected from the group consisting of IgE, CD29, CD69, IL13Rα, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, SIGLEC10, CD1c, CD23, and IL4Rα; including an increase in the level or amount of one or more biomarkers selected from IgE, CD29, IL13Rα, CD99, FCRL3, FCRL2, SIGLEC10, CD1c, CD23, and IL4Rα, or an increase in the percentage of positive cells having one or more biomarkers, in B cells in the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject, and / or A method is provided that indicates the effectiveness of allergen immunotherapy in a subject, by a decrease in the level or amount of one or more biomarkers selected from CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD1c, CD23, and IL4Rα, or a decrease in the percentage of positive cells having one or more biomarkers, in B cells in a second sample compared to a first sample.

[0125] In any aspect of the present invention, determining the effectiveness of allergen immunotherapy may involve revealing an increase or decrease in the level, amount or proportion of one or more specific biomarkers. The increase or decrease may be a fold change in the expression level of one or more specific biomarkers when comparing samples before and after allergen immunotherapy, or may be represented by the percentage (frequency of cell appearance) of positive cells having the expression of one or more specific biomarkers. The magnitude of the increase or decrease in the level, amount or proportion of one or more specific biomarkers may be any magnitude described herein, including examples and tables, where necessary from the context.

[0126] In one embodiment, one or more biomarkers are one or more nucleic acids comprising a nucleotide sequence derived from a gene or an RNA transcript of a gene, or an amplification product thereof, and the gene is IGHE , ITGB1 , CD69 , IL13RA1 , CD99 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , SIGLEC10 , CD1C , FCER2 and IL4R one or more of.

[0127] In one embodiment of this aspect of the present invention, in B cells in a second sample compared to a first sample, the level or amount of one or more biomarkers selected from IgE, CD29, IL13Rα, CD99, SIGLEC10, CD1c, and CD23 may be increased by at least 1.5-fold, which indicates the effectiveness of allergen immunotherapy in a subject. The one or more biomarkers may be one or more nucleic acids including a nucleotide sequence derived from a gene or an RNA transcript of a gene, or an amplification product thereof, and the gene is IGHE , ITGB1 , IL13RA1 , CD99 , SIGLEC10 , CD1C , and FCER2 one or more of.

[0128] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers may be increased by at least 2-fold, which may include any one or more of IgE, CD29, IL13Rα, CD99, SIGLEC10, and CD1c in B cells in a second sample compared to a first sample, which indicates the effectiveness of allergen immunotherapy in a subject. The one or more biomarkers may be one or more nucleic acids including a nucleotide sequence derived from a gene or an RNA transcript of a gene, or an amplification product thereof, and the gene is IGHE , ITGB1 , IL13RA1 , CD99 , SIGLEC10 and CD1C one or more of.

[0129] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers may be increased by at least 2.5-fold, which may include any one or more of IgE, CD29, IL13Rα, CD99, and IL4Rα in B cells in the second sample compared to the first sample, which indicates the effectiveness of allergen immunotherapy in a subject. The one or more biomarkers may be one or more nucleic acids including a nucleotide sequence derived from a gene or an RNA transcript of a gene, or an amplification product thereof, and the gene is IGHE , ITGB1 , IL13RA1 , and CD99 is one or more of.

[0130] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers may be increased by at least 3-fold, which may include any one or more of IgE, CD29, and IL13Rα in B cells in the second sample compared to the first sample, which indicates the effectiveness of allergen immunotherapy in a subject. The one or more biomarkers may be one or more nucleic acids including a nucleotide sequence derived from a gene or an RNA transcript of a gene, or an amplification product thereof, and the gene is IGHE , ITGB1 , and IL13RA1 is one or more of.

[0131] In one embodiment of this aspect of the present invention, the level or amount of the biomarker may be increased by at least 4-fold, which may include CD29 in B cells in the second sample compared to the first sample, which indicates the effectiveness of allergen immunotherapy in a subject. The biomarker may be a nucleic acid including a nucleotide sequence derived from a gene or an RNA transcript of a gene, or an amplification product thereof, and the gene is ITGB1 .

[0132] In one embodiment of this aspect of the present invention, in B cells in the second sample compared to the first sample, the level or amount of one or more of CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD1c, CD23, and IL4Rα may be reduced to 1 / 1.5 or less, which indicates the effectiveness of allergen immunotherapy in a subject. The one or more biomarkers may be one or more nucleic acids including a nucleotide sequence derived from a gene or an RNA transcript of a gene, or an amplification product thereof, and the gene is CD69 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , CD1C , FCER2 and IL4R one or more of.

[0133] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers may be reduced to 1 / 2 or less, which may include any one or more of CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD23, and IL4Rα in B cells in the second sample compared to the first sample, which indicates the effectiveness of allergen immunotherapy in a subject. The one or more biomarkers may be one or more nucleic acids including a nucleotide sequence derived from a gene or an RNA transcript of a gene, or an amplification product thereof, and the gene is CD69 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , FCER2 and IL4R one or more of.

[0134] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers may be reduced to 1 / 2.5 or less, which may include any one or more of CD69, IgD, CXCR4, and FCRL3 in B cells in the second sample compared to the first sample, and this indicates the effectiveness of allergen immunotherapy in the subject. The one or more biomarkers may also be one or more nucleic acids including a nucleotide sequence derived from a gene or an RNA transcript of a gene, or an amplification product thereof, and the gene is CD69 , IGHD , CXCR4 , and FCRL3 one or more of.

[0135] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers may be reduced to 1 / 3 or less, which may include any one or more of IgD and CXCR4 in B cells in the second sample compared to the first sample, and this indicates the effectiveness of allergen immunotherapy in the subject. The one or more biomarkers may also be one or more nucleic acids including a nucleotide sequence derived from a gene or an RNA transcript of a gene, or an amplification product thereof, and the gene is IGHD and CXCR4 one or more of.

[0136] In one embodiment of this aspect of the present invention, the level or amount of one or more biomarkers may be reduced to 1 / 4 or less, which may include any one or more of IgD and CXCR4 in B cells in the second sample compared to the first sample, and this indicates the effectiveness of allergen immunotherapy in the subject. The one or more biomarkers may also be one or more nucleic acids including a nucleotide sequence derived from a gene or an RNA transcript of a gene, or an amplification product thereof, and the gene is IGHD and CXCR4 one or more of.

[0137] In another aspect, the present invention is a method for measuring or detecting at least one biomarker, comprising: - obtaining a sample from a subject after allergen immunotherapy, the sample comprising B cells, and - in the sample, IGHE 、 ITGB1 、 CD69 、 IL13RA1 、 CD99 、 IGHD 、 CXCR4 、 FCRL3 、 FCRL2 、 FCRL5 、 SIGLEC10 、 CD1C 、 FCER2 、 IL4R 、 PPP1R18 、 PARM1 、 c-jun measuring or detecting at least one biomarker or a fragment thereof selected from the group consisting of IgE, CD29, CD69, IL13Rα, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, SIGLEC10, CD1c, CD23, IL4Rα, and hostecin, or any combination thereof A method is provided that includes:

[0138] In one embodiment, the measurement or detection of at least one biomarker indicates that the subject has responded to the allergen immunotherapy that the subject has received or is receiving.

[0139] In one embodiment, at least one biomarker or a fragment thereof is IGHE 、 ITGB1 、 IL13RA1 、 CD99 、 FCRL3 、 FCRL2 、 SIGLEC10 、 CD1C 、 FCER2 、 IL4R 、 PPP1R18 、 PARM1, selected from the group consisting of IgE, CD29, IL13Rα, CD99, FCRL3, FCRL2, SIGLEC10, CD1c, CD23, IL4Rα, and hostensin, or any combination thereof, at least one biomarker is not detectable or is detected at a lower level in B cells in a sample obtained from a subject prior to allergen immunotherapy.

[0140] In one embodiment, at least one biomarker or a fragment thereof CD69 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , CD1C , FCER2 , IL4R , c-jun , selected from the group consisting of CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD1c, CD23, IL4Rα, or any combination thereof, and at least one biomarker is detectable or is detected at a higher level in a sample obtained from a subject prior to allergen immunotherapy.

[0141] In one embodiment, at least one biomarker or a fragment thereof IGHE , ITGB1 , CD69 , IL13RA1 , CD99 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , SIGLEC10 , CD1C , FCER2 , IL4R , PPP1R18 , PARM1 , c-jun , selected from the group consisting of IgE, CD29, CD69, IL13Rα, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, SIGLEC10, CD1c, CD23, IL4Rα, and hostensin, or any combination thereof.

[0142] In one embodiment, IGHE ,ITGB1 , IL13RA1 , CD99 , FCRL3 , FCRL2 , SIGLEC10 , CD1C , FCER2 , IL4R , PPP1R18 , PARM1 , IgE, CD29, IL13Rα, CD99, FCRL3, FCRL2, SIGLEC10, CD1c, CD23, IL4Rα, and phosphostensin, one or more levels of any of which are higher compared to their levels in a sample obtained from the subject before allergen immunotherapy; and / or CD69 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , CD1C , FCER2 , IL4R , c-jun , one or more levels of CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD1c, CD23, and IL4Rα are lower compared to their levels in a sample obtained from the subject before allergen immunotherapy.

[0143] In one embodiment, IGHE , ITGB1 , CD99 , SIGLEC10 , FCER2 , IL4R , one or more levels of IgE, CD29, CD99, SIGLEC10, CD23, and IL4Rα are higher in a second sample obtained from the subject after allergen immunotherapy compared to their levels in a first sample obtained from the subject before allergen immunotherapy.

[0144] In one embodiment, CD69 , one or more levels of any of and CD69 are lower in a second sample obtained from the subject after allergen immunotherapy compared to their levels in a first sample obtained from the subject before allergen immunotherapy.

[0145] In one embodiment, at least one biomarker or a fragment thereof is IGHE , ITGB1 , CD69 , CD99 , SIGLEC10 , FCER2 , IL4R , selected from the group consisting of IgE, CD29, CD69, CD99, SIGLEC10, CD23, and IL4Rα, or a combination thereof.

[0146] In one embodiment, IGHE , ITGB1 , CD99 , SIGLEC10 , FCER2 , IL4R , one or more levels of any of IgE, CD29, CD99, SIGLEC10, CD23, and IL4Rα are higher compared to their levels in a sample obtained from the subject before allergen immunotherapy; and / or CD69 and one or more levels of any of CD69 are lower compared to their levels in a sample obtained from the subject before allergen immunotherapy.

[0147] In one embodiment, IGHE , ITGB1 , IL13RA1 , CD99 , FCER2 , IL4R , one or more levels of any of IgE, CD29, IL13Rα, CD99, CD23, and IL4Rα are higher in a second sample obtained from the subject after allergen immunotherapy compared to their levels in a first sample obtained from the subject before allergen immunotherapy.

[0148] In one embodiment, CD69 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , CD1CThe level of any one or more of CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, and CD1c is lower in a second sample obtained from a subject after allergen immunotherapy than in a first sample obtained from the subject before allergen immunotherapy.

[0149] In one embodiment, at least one biomarker or a fragment thereof is IGHE , ITGB1 , CD69 , IL13RA1 , CD99 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , CD1C , FCER2 , IL4R selected from the group consisting of IgE, CD29, CD69, IL13Rα, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD1c, CD23, and IL4Rα, or a combination thereof.

[0150] In one embodiment, IGHE , ITGB1 , IL13RA1 , CD99 , FCER2 , IL4R the level of any one or more of IgE, CD29, IL13Rα, CD99, CD23, and IL4Rα is higher compared to its level in a sample obtained from the subject before allergen immunotherapy; and / or CD69 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , CD1C the level of any one or more of CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, and CD1c is lower compared to its level in a sample obtained from the subject before allergen immunotherapy.

[0151] In one embodiment, ITGB1 , CD99 , FCRL3 , FCRL2 ,SIGLEC10 , CD1C The level of any one or more of CD29, CD99, FCRL3, FCRL2, SIGLEC10, and CD1c is higher in a second sample obtained from a subject after allergen immunotherapy than its level in a first sample obtained from the subject before allergen immunotherapy.

[0152] In one embodiment, CD69 , IGHD , CXCR4 , FCRL5 , FCRL2 , IL4R The level of any one or more of CD69, IgD, CXCR4, FCRL5, CD23 and IL4Rα is lower in a second sample obtained from a subject after allergen immunotherapy than its level in a first sample obtained from the subject before allergen immunotherapy.

[0153] In one embodiment, at least one biomarker or fragment thereof is ITGB1 , CD69 , CD99 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , SIGLEC10 , CD1C , FCER2 , IL4R selected from the group consisting of CD29, CD69, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, SIGLEC10, CD1c, CD23, and IL4Rα, or a combination thereof.

[0154] In one embodiment, ITGB1 , CD99 , FCRL3 , FCRL2 , SIGLEC10 , CD1C the level of any one or more of CD29, CD99, FCRL3, FCRL2, SIGLEC10, and CD1c is higher compared to its level in a sample obtained from the subject before allergen immunotherapy; and / or CD69 , IGHD ,CXCR4 , FCRL5 , FCER2 , IL4R , CD69, IgD, CXCR4, FCRL5, CD23, and IL4Rα, the level of any one or more of them is lower compared to its level in the sample obtained from the subject before allergen immunotherapy.

[0155] In any aspect, the biomarker is the biomarker described in Table 2 and / or Table 3. In any aspect, the biomarker is a biomarker that increases by 1.5-fold, 2-fold, 3-fold, or 4-fold or decreases to 1 / 1.5, 1 / 2, 1 / 3, or 1 / 4 as described in Table 2 and / or Table 3.

[0156] Allergy and Sensitization It is understood that the method of the present invention has several important applications, particularly in the field of human medical diagnosis.

[0157] Hypersensitivity reactions due to immunological responses can be classified into four broad classes. In particular, type I hypersensitivity reactions are those immediate allergic reactions mediated by IgE antibodies. In most allergies, such as allergies to food, pollen, and house dust mites, the subject is sensitized to harmless antigens, allergens, by producing IgE antibodies against the antigen, and thus a reaction occurs. Subsequent exposure to the allergen triggers the activation of IgE-binding cells, including mast cells and basophils, in tissues or blood, resulting in a series of responses characteristic of this type of reaction, including degranulation of effector cells, release of histamine, heparin, eosinophil and neutrophil chemotactic factors, leukotrienes, and thromboxanes. The allergic immune response is a response characterized by the production of high levels of IgE antibodies that can be detected in the blood and the production of IgE-specific B cells.

[0158] Conventional tests for allergies include the skin prick test, in which an allergen is injected intradermally or, occasionally, intradermally. An allergic or allergic response causes a rapid formation of wheals and erythema within 30 minutes. Other tests for allergies are known to those skilled in the art and include immunoassay tests such as enzyme-linked immunosorbent assay (ELISA, or EIA) and radioallergosorbent test (RAST). The ELISA test measures the amount of allergen-specific antibodies in the blood, and the RAST test searches for specific allergen-related antibodies to identify allergy triggers.

[0159] The test of the present invention may be used to monitor allergic reactivity or allergen sensitivity in situations where a skin prick test may not be guaranteed, for example, (i) the patient is using a medication known to interfere with the skin prick test, such as an antihistamine, corticosteroid, or certain antidepressants, (ii) the subject cannot tolerate the many injection needle injuries required for a skin test, (iii) the subject has an unstable heart condition, (iv) poorly controlled asthma, severe eczema, dermatitis, psoriasis, or other severe skin conditions and / or (v) there is an excessive or life-threatening allergic reaction during the skin test, for example, there is a history of anaphylaxis.

[0160] The types of allergies include, but are not limited to, food allergies, skin allergies, dust mite or pollen allergies, insect sting allergies, pet allergies, eye allergies, drug allergies, allergic rhinitis, latex allergies, particularly type I IgE-mediated allergic reactions, mold allergies, allergic sinusitis, and cockroach allergies. Food allergies include, but are not limited to, allergies to milk, eggs, peanuts, nuts, soybeans, wheat, fish, and shellfish. Drug allergies include allergies mediated by IgE upon reaction to a substance. The most common drug allergies include penicillin and other related antibiotics, antibiotics containing sulfonamides, anticonvulsants, aspirin, ibuprofen, and other non-steroidal anti-inflammatory drugs (NSAIDs), and chemotherapeutic drugs. The most common skin allergies include eczema (atopic dermatitis), hives (urticaria), and contact dermatitis. The most common form of eye allergy is triggered by outdoor allergens such as pollen from grass, trees, and weeds, indoor allergens such as pet dander, dust mites, and mold, and irritants such as tobacco smoke, fragrances, and diesel exhaust. The most common dust or pollen allergies include dust mites, cockroaches, mold, pollen (e.g., grass, trees, or weeds), pet hair, fur, or feathers. The types of symptoms of allergic reactions include, but are not limited to, excessive mucus production, loss of smell or taste, throat pain and / or cough, fatigue, fever or shivering, facial congestion, headache, toothache, postnasal drip, wheezing, shortness of breath, difficulty breathing, swelling of the throat and mouth, exhalation, vomiting, abdominal distension, diarrhea, stomach pain, cramping abdominal pain, rash, itching (particularly of the nose, eyes, ears, and mouth), redness and watering eyes, swelling around the eyes, hives, swelling of the lips, tongue, or throat, high blood pressure, dizziness and / or fainting, severe asthma episodes (asthma attacks), chronic asthma, and anaphylaxis.

[0161] The subject may be a person with hypersensitivity or allergic to one or more allergens described herein. Allergens include, for example, food-based allergens such as nuts, sesame, buckwheat, peanuts, milk protein, egg white, shrimp, etc. Other allergens of interest include various airborne antigens such as grass pollen, animal dander, house dust mite feces, as well as insect venom and fungal allergens. Typical food allergens include milk allergens (Bos d 4, 5 and 8), peanut allergens (Ara h 1, 2, 3, 6 and 8), hazelnut (Cor a 9 and 14), cashew nut (Ana o 3), walnut (Jug r 1), Brazil nut (Ber e 1), sesame (Ses i 1), buckwheat (Fag e 3), almond (Pru du 6), bovine shrimp (Pen m 1) and wheat (Tri a 19). Common airborne allergens include Dermatophagoides pteryonyssinus (Der p 1 and 2) or Dermatophagoides farina (Der f 1 and 2); ryegrass (Lol p 1, 5), Timothy grass (Phl p 1, 5), American ragweed (Pas n 1), Bermuda grass (Cyn d 1), mugwort (Amb a 1), pellitory species (Par o 1; Par j 1, 2), pollen allergens derived from birch (Bet v 1) and other atmospheric pollens including olive (Olea europaea), Artemisia sp., gramineae, etc.; and, for example, animal dander derived from cats (Fel d 1) and dogs (Can f 1). Other allergens include venom allergens derived from honeybees (Api m 1, 3, 10); phospholipase derived from yellow jacket Vespula maculifrons, common wasp Polistes exclamans and North American hornet Dolichovespula maculata, and venom derived from jumper ant Myrmecia pilosula.Other allergens of interest are mold allergies (especially those from Alternaria, Aspergillus, and Cladosporium species), as well as blood-sucking arthropods such as mosquitoes (Anopheles sp., Aedes sp., Culiseta sp., Culex sp.); flies (Phlebotomus sp., Culicoides sp.), especially flies including midges, black flies, and Culicoides of the order Diptera; ticks (Dermacenter sp., Ornithodoros sp., Otobius sp.); fleas, such as fleas of the order Siphonaptera including the genera Xenopsylla, Pulex, and Ctenocephalides, which are allergens causing allergic dermatitis. The allergen may be secreted by bacteria, such as the bacterium Malassezia (M.) globose, and may be derived from the protein MGL_1304, which is the main allergen for sweat allergy.

[0162] In relation to a subject having rye grass pollen (RGP) allergy, the subject may have moderate to severe seasonal allergic rhinitis (SAR). The subject may or may not have asthma. In one embodiment, the subject may have serum RGP-specific IgE of ≧0.35 kUA / L.

[0163] In relation to a subject having bee venom allergy (BV allergy), the subject may be diagnosed based on a systemic allergic response to a bee sting. In one embodiment, the subject may have serum BV-specific IgE of ≧0.35 kUA / L.

[0164] Reagents and ligands for detecting biomarkers Any aspect of the present invention involves determining the level or amount of one or more biomarkers in B cells. This can be accomplished by measuring or detecting the presence, level, or expression of one or more biomarkers described herein. It can be carried out by a reagent or ligand that binds to or forms a complex with the biomarker in Table 2 or its specific peptide, or a molecular form thereof or a combination of such ligands.

[0165] In one embodiment, such a ligand or reagent preferably binds to a protein biomarker or a specific peptide contained therein, and an antibody that specifically binds to a single biomarker in Table 2 or a specific peptide in that single biomarker is possible. Various forms of antibodies, such as polyclonal, monoclonal, recombinant, chimeric, as well as fragments and components (e.g., CDR, single-chain variable region, etc.) or antibody mimetics or equivalents may be used in place of the antibody. The ligand or reagent itself may be labeled or immobilized.

[0166] Examples of antibodies that bind to the biomarkers in Table 2 are included in Table 1 below.

[0167]

Table 1

[0168] Any combination of labeled or immobilized biomarker reagents or ligands can be assembled into a diagnostic kit or device for the purpose of determining the effectiveness of immunotherapy.

[0169] Thus, the kit or device can include a plurality of reagents or one or more individual reagents. For example, one embodiment of the composition includes a substrate for immobilizing a biomarker or ligand. In another embodiment, the kit also includes any detectable label, immobilization substrate, any substrate for enzyme labeling, as well as other laboratory items.

[0170] Diagnostic reagents, devices, or kit compositions based on the biomarkers of Table 2 described herein or fragments thereof, associated with optionally detectable labels, can be presented in a format of a microfluidic card, chip or chamber, beads or kit adapted for use with assay formats such as sandwich ELISA, multiple protein assay, platform multiplex ELISA such as BioRad Luminex platform, mass spectrometry quantitative assay, or flow cytometry.

[0171] In another embodiment, the reagent ligand is a nucleotide sequence and the diagnostic reagent is a polynucleotide or oligonucleotide sequence that hybridizes to a gene, gene fragment, gene transcript or nucleotide sequence encoding a biomarker of Table 2 or encoding a specific peptide thereof. Such polynucleotides / oligonucleotides can be probes or primers and may themselves be labeled or immobilized. In one embodiment, the polynucleotide or oligonucleotide reagent(s) that hybridize to the ligand are part of a primer-probe set and the kit includes both primers and probes. Each said primer-probe set amplifies different genes, gene fragments or gene expression products encoding different biomarkers of Table 2. For use in compositions, the PCR primers and probes are preferably designed based on intron sequences present in the amplified biomarker gene(s) selected from the gene expression profile. The design of the primer and probe sequences is within the scope of the art after a particular gene target has been selected. The particular methods selected for primer and probe design and the particular primer and probe sequences are not limiting features of these compositions.

[0172] Generally, the optimal PCR primers and probes used in the compositions described herein are typically 17 to 30 bases in length and contain about 20 to 80% G+C bases, such as about 50 to 60% G+C bases. Melting temperatures between 50 and 80 °C, such as about 50 to 70 °C, are typically preferred.

[0173] The method of the present invention includes the detection of biomarkers described herein.

[0174] As used herein, the term "detectable" refers to the generation or change of a signal that is directly or indirectly detectable either by observation or by means of a measuring device. Typically, a detectable response is the generation of a signal where the fluorophore is inherently fluorescent. Alternatively, a detectable response is an optical response that results in a change in the wavelength distribution pattern or intensity of absorbance or fluorescence or light scattering, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters. Other detectable responses include, for example, chemiluminescence, phosphorescence, radiation from radioisotopes, magnetic attraction, and electron density.

[0175] As used herein, the term "label" refers to a chemical moiety or protein that is attached to a reagent or ligand and is directly or indirectly detectable (e.g., by its spectral properties, conformation, or activity) when used in the method.

[0176] The detection label conjugated to the reagent or ligand may be any label that enables separate detection and quantification by flow cytometry. For example, a fluorescent dye. Suitable fluorescent labels are known in the art and include fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridin chlorophyll protein (PerCP), allophycocyanin (APC), Alexa fluor488, Alexa fluor647, Alexa fluor710, Alexa fluor405, cyanine 5 (Cy5), cyanine 5.5 (Cy5.5), Pacific Blue (PacB), Horizon Violet 450 (HV450), Pacific Orange (PacO), Horizon-V500 (HV500), chromo orange, Brilliant Violet 421 (BV421), Brilliant Violet 510 (BV510), Brilliant Violet 605 (BV605), Brilliant Violet 650 (BV650), Brilliant Violet 711 (BV711), Brilliant Violet 785 (BV785), Brilliant Ultra Violet 395 (BUV395), Brilliant Ultra Violet 496 (BUV496), Brilliant Ultra Violet 737 (BUV737), Orange Cytognos (OC)515, quantum dots, and their conjugates (e.g., PE / Cy5, PE / Cy5.5, PE / Cy7, PerCP / Cy5.5, APC / Cy7, APC-H7, APC-Alex750, PE-Texas Red, PE-Dazzle, PE-CF594) conjugated to APC or PerCP, Cytek cFluor (e.g., cFluor V420, V450, V547, B515, B532, BYG575, BYF610, BYG666, BYG690, BYG710, BYG781, R659, R720, R780) or any additional compatible fluorescent dye or fluorescent dye tandem, etc.

[0177] In one example, the antibody is conjugated to (1) Pacific Blue (PacB), Brilliant Violet 421 (BV421) or Horizon V450; (2) Pacific Orange (PacO), Horizon V500 (HV500), BV510, Khrome Orange (KO) or OC515, (3) Horizon BB515, Fluorescein Isothiocyanate (FITC) or Alexa488, (4) Phycoerythrin (PE), (5) Peridin Chlorophyll Protein / Cyanine 5.5 (PerCP-Cy5.5), PerCP or PE-Texas Red, (6) Phycoerythrin / Cyanine 7 (PE-Cy7), (7) Allophycocyanin (APC) or Alexa647, and (8) Allophycocyanin / Hilite 7 (APC-H7), APC-Cy7, Alexa680, APC-A750, APC-C750 or Alexa700.

[0178] In another example, the antibody is conjugated to (1) Brilliant Violet 421, (2) Brilliant Violet 510 (BV510), (3) Brilliant Violet 650 (BV650), (4) Brilliant Violet 786 (BV786), (5) Fluorescein Isothiocyanate (FITC), (6) Peridin Chlorophyll Protein / Cyanine 5.5 (PerCP-Cy5.5), (7) Phycoerythrin (PE), (8) Phycoerythrin / Cyanine 7 (PE-Cy7), (9) Allophycocyanin (APC), and (10) Allophycocyanin / H7 (APC-H7), APC-C750 or APC-Alexa750.

[0179] Suitable labels can be directly or indirectly linked to the reagent or ligand by the use of suitable tags. In a preferred embodiment, the detectable label is linked to streptavidin. Fluorescent dye reagents are useful in panel reactive assays, where a pool of two or more defined reagents or ligands are each conjugated to a different fluorescent dye and added to the sample. Several biomarkers can be assayed at once, allowing multiplexing from a single blood draw. The blood sample is taken from a subject suspected of having an allergy or who has received or is receiving allergen immunotherapy.

[0180] The method of the invention further comprises contacting the sample with one or more, preferably two or more, reagents that allow the identification of one or more biomarkers. Typically, the molecule is bound to a detectable label or is itself a detectable label. For example, the molecule may be a fluorescent dye, antibody, nucleotide probe or enzyme that results in the production of a substrate. Alternatively, the reagent is linked to a tag that facilitates binding to a detectable label. For example, the tag may non-covalently bind to or form a covalent interaction with the detectable label. Suitable tags are known in the art and are described herein.

[0181] For example, the reagent is an antibody that detects the biomarker of interest and the detectable label is a fluorescent dye. Suitable fluorescent dyes are known in the art and are described herein. Several fluorescently conjugated antibodies directed against different phenotypic biomarkers on B cells may be added to the sample to facilitate the detection and discrimination of different cell types. Preferably, the sample is contacted with a panel of fluorescent dye-conjugated antibodies under conditions suitable for binding of the antibody to its respective antigen.

[0182] The sample may be simultaneously contacted with all antibodies, i.e., an antibody cocktail, mixture or composition. However, it may be appropriate to add the antibodies in two or more steps. For example, a two-step incubation can be carried out when both surface membrane and intracellular staining are required. In such cases, surface membrane staining is first carried out, followed by fixation and permeabilization to facilitate cytoplasmic staining. In each case, unlabeled antibodies may be used, but multiple incubation and washing steps may be required. Preferably, in routine diagnostic tests, complex staining is generally not preferred.

[0183] Any suitable phenotypic biomarker can be used to identify the cells of interest. Suitable B cell biomarkers are CD19, CD20, CD79a or CD22, and most preferably, the CD19 antigen.

[0184] To further characterize the immune cells of interest, most preferably B cells, it may be useful to include a panel of fluorescent dye-conjugated antibodies. For example, the antibodies react with biomarkers for the characterization of Bmem or plasma cells, preferably biomarkers selected from the group consisting of CD23, CD27, CD38, CD40, CD80, CD86, CD148, CD180, TACI, CD200, CD73 and CD62L.

[0185] Allergen-specific B cells, such as allergen-specific Bmem, may be identified using labeled allergens (e.g., fluorescently labeled allergens). Fluorescently labeled allergens for the detection of allergen-specific Bmem may provide the highest sensitivity and specificity for detecting the presence of allergen-specific Bmem, or any change in this percentage or number.

[0186] Sample Any biological sample known or suspected to contain B cells, preferably Bmem, is contemplated for use in the present invention.

[0187] As used herein, the term "sample" shall include blood samples, but may also include hematopoietic biological samples such as lymph, leukocyte products, bone marrow, etc., and such derivatives and fractions of such body fluids are also included in this sample. Blood samples are taken, for example, from any site by venipuncture. Blood samples are usually about 1 - 100 ml of whole blood, i.e., 10 5 ~10 7 nucleated blood cells, and as is known in the art, may be treated with anticoagulants such as heparin, EDTA, citrate, citrate dextrose or citrate phosphate dextrose.

[0188] The sample may be a body fluid, such as the blood sample discussed above. Alternatively, the sample may be a tissue sample. The sample may include body fluids and tissue samples.

[0189] The blood sample may be whole blood, buffy coat, peripheral blood mononuclear cells (PBMCs), cord blood, a purified or fractionated cell population or a body fluid. Body fluids include lymph fluid, semen, nasal mucus, bronchial secretions, alveolar fluid, cerebrospinal fluid, endolymph, synovial fluid, intrapleural fluid, pericardial fluid (pericardial sac fluid), menstrual fluid, or a combination thereof.

[0190] The tissue sample may be selected from tonsils, lymph nodes, bronchi, nose or intestine or skin biopsies. Preferably, the tissue sample is processed to form a single cell suspension. The formation of a single cell suspension may be through a mesh filter for tonsils, thymus or lymph nodes. Alternatively, the formation of a single cell suspension may also be by tissue digestion and then the use of a mesh passing filter.

[0191] Specifically, in relation to a biological sample known or suspected to contain B cells, the sample may be blood, bone marrow or lymphoid tissue. The tissue may be selected from tonsils, lymph nodes, bronchi, nasal or intestinal biopsies. Alternatively, the blood sample may be a whole blood sample, buffy coat sample, peripheral blood mononuclear cell (PBMC) sample, cord blood, purified or sorted cell population or body fluid. Body fluids include samples from the group consisting of lymph amniotic fluid, nasal mucus, bronchial secretions, alveolar fluid, endolymph, pericardial fluid (pericardial effusion), ascites, breast milk, or combinations thereof.

[0192] The sample can be taken from any mammal including primates. In particular, humans, mice, and more specifically, mice, horses, cows, sheep, pigs, dogs, cats, etc. Whole blood can be taken from the sample using any acceptable procedure. Using whole blood enables the detection of effector cells such as eosinophils and basophils. Alternatively, the blood sample can be resuspended in a solution that selectively lyses red blood cells, such as ammonium chloride - potassium chloride; ammonium oxalate, etc.

[0193] Detection method The detection of a reagent or ligand linked to a detectable label that binds to a biomarker on B cells can be carried out by flow cytometry or microscopy. These methods are carried out as known in the art. The use of flow cytometry or microscopy can be combined with other cell phenotyping agents.

[0194] When a sample bound to a fluorescent dye selection reagent is used, flow cytometry or microscopy can be used to detect the presence of B cells labeled with the reagent or ligand. Such methods are carried out as known in the art. Biomarker

[0195] For example, a sample is subjected to multi-color flow cytometry and gated for B cells based on forward scatter and side scatter, and typically, subsequently, cell doublets and multiplets are excluded according to conventional criteria, for example, in a forward scatter pulse area versus forward scatter pulse-height bivariate dot plot.

[0196] Monitoring the success of allergy treatment The method of the present invention includes monitoring or determining the success of allergen immunotherapy. For example, monitoring the therapeutic efficacy of anti-IgE therapy or allergen immunotherapy (oral / subcutaneous). Allergen immunotherapy may be allergen-specific or non-allergen-specific (e.g., omalizumab).

[0197] In particular, the present invention relates to the monitoring of the effectiveness of allergen immunotherapy. Allergen immunotherapy (also named desensitization therapy, immunotherapy, desensitization or allergen immunotherapy) includes the immunotherapy of allergic disorders in which patients are vaccinated with increasing doses of allergens for the purpose of inducing immune tolerance. Allergen-specific immunotherapy is the only treatment strategy for treating the underlying cause of allergic disorders. This therapy can reduce the need for drug treatment and the severity of symptoms or completely eliminate hypersensitivity. Allergens can be administered sublingually (under the tongue), by subcutaneous injection (subcutaneously) or in some cases, by intradermal injection.

[0198] The immune system of an individual who has developed an allergy begins to produce IgE by misinterpreting a normally harmless substance as a pathogenic substance. This is called the "primary antibody response." The IgE produced during this response binds to basophils in the bloodstream and to similar types of cells called mast cells in tissues. When a person is re-exposed to that allergen, these basophils and mast cells that were bound to the IgE release histamine, prostaglandins, and leukotrienes, which cause inflammation of the surrounding tissues and result in allergic symptoms. Immunotherapy by repeated exposure to a specific allergen via any of the sublingual, subcutaneous, intradermal, epicutaneous, or intralymphatic routes results in desensitization to the allergen and thus a reduction in the use of overall allergic symptoms and symptom-based treatments. The exact mechanism is not fully understood, but it is recognized that immunotherapy causes a modification of the immune system. This modification results in changes in IgE synthesis and the production of IgE-blocking antibodies, and thus a reduction in the immune system allergic response to a specific allergen due to this production. A shift from a Th2 response to regulatory T cells also occurs. The molecular mechanism of such immunotherapy can be partly interpreted as the induction of allergen-specific IgG, which neutralizes the allergen, instead of the induction of allergen-specific IgE.

[0199] As will be recognized by those skilled in the art, the method of the present invention is highly suitable for monitoring any quantitative changes in allergen-specific B cells in biological samples from patients suffering from allergies and / or undergoing allergen immunotherapy. To that end, IgG-expressing cells (whether they are memory B cells and / or plasma cells) are easily identified and quantified by staining with a reagent or ligand that targets the biomarker of interest, and the reagent or ligand is provided with a detectable label such as a fluorescent dye. Accordingly, a method for monitoring the therapeutic efficacy of allergen immunotherapy is provided. The method includes analyzing a subset of memory B cells in a biological sample isolated from a subject (oral / subcutaneous) undergoing the immunotherapy using the procedures described above herein to detect allergen IgG+ memory B cells.

[0200] A positive response to allergen immunotherapy is - an increase in the level or amount of one or more biomarkers selected from IGHE , ITGB1 , IL13RA1 , CD99 , FCRL3 , FCRL2 , SIGLEC10 , CD1C , FCER2 , IL4R IgE, CD29, IL13Rα, CD99, FCRL3, FCRL2, SIGLEC10, CD1c, CD23, and IL4Rα in B cells in the second sample compared to the first sample - a decrease in the level or amount of one or more biomarkers selected from CD69 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , CD1C , FCER2 , IL4R CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, CD1c, CD23 and IL4Rα in B cells in the second sample compared to the first sample - an increase in the level or amount of one or more biomarkers selected from IGHE , PPP1R18 , ITGB1 , PARM1 IgE, phosphostensin and CD29 in B cells in the second sample compared to the first sample - a decrease in the level or amount of one or more biomarkers selected from c-jun and CD69 in B cells in the second sample compared to the first sample (the first sample is prior to immunotherapy and the second is during or after immunotherapy). Any one or more of these may be present (the first sample is prior to immunotherapy and the second is during or after immunotherapy).

[0201] A positive response to allergen immunotherapy preferably occurs at least 4 months after the start of treatment with allergen immunotherapy, for any period up to 5 years, or for any other period described herein.

[0202] Kit A kit for carrying out the present invention can be provided. For example, the kit may include one or a panel of the reagents or ligands linked to the detectable labels described herein, and optionally, other antibodies that determine the phenotype of the cell of interest. Optionally, the kit of the present invention is packaged together with instructions for use in the methods described herein.

[0203] A still further aspect of the present invention relates to a diagnostic kit comprising reagents for carrying out the methods disclosed herein. In one embodiment, the kit is a diagnostic assay kit for determining the effectiveness of allergen immunotherapy.

[0204] In another aspect of the present invention, a kit for determining the effectiveness of allergen immunotherapy in a subject, comprising means for detecting changes in the levels or amounts of transcripts, amplification products and / or proteins, wherein the transcripts, amplification products and / or proteins are IGHE , ITGB1 , CD69 , IL13RA1 , CD99 , IGHD , CXCR4 , FCRL3 , FCRL2 , FCRL5 , SIGLEC10 , CD1C , FCER2 , IL4R or protein: IgE, CD29, CD69, IL13Rα, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, SIGLEC10, CD1c, CD23, IL4Rα, and optionally, PPP1R18 , PARM1 , hostensin, and c-jun selected from the group consisting of any one or more thereof, and may include written instructions.

[0205] The kit may further comprise any additional reagents, buffers, or devices for use in the method of the present invention. For example, the kit may include reagents for calibrating a flow cytometer, positive controls, negative controls, etc. for creating a standard curve.

Examples

[0206] The following experimental data are as follows: · A patient cohort with RGP allergy (n = 27); 13 patients were treated with SLIT for 4 months, which induced a response predicted with an increase in allergen-specific serum IgE and IgG4 levels; 14 untreated patients and these patients showed a decrease in allergen-specific serum IgE and no change in IgG2 and IgG4 levels. · We describe for the first time the effect of SLIT on allergen-specific memory B cells (Bmem). We identified different Bmem transcriptional profiles and demonstrated using pseudotime analysis that SLIT induces differentiation towards two distinct cell fates with different transcriptional profiles. · SLIT induced differentiation towards a transcriptional profile associated with IGHE and IGHG4-expressing Bmem (pseudotime cluster 4). · The expression of IL13RA1 and IL4R was increased in cluster 4 compared to clusters 1 and 3. · Cluster 4 also expressed higher levels of FCER2 (CD23) compared to clusters 1 and 3. · 14 surface-expressed proteins as candidate markers for successful allergy treatment. · The surface protein expression of IgG4, CD29, and CD23 increased on Lol p 1+Bmem in RGP patients after 4 months of SLIT and did not increase in patients who did not receive SLIT during those 4 months. · The surface expression of IgG4, CD29, and CD23 increased on Api m 1+Bmem in wasp venom allergy patients 63 days after the start of ultra-rush AIT. · The number of CD29+ antigen-specific Bmem at the 4th month of SLIT is a strong predictor of treatment success. · The surface protein expression of IL4Rα was increased on Lol p 1+Bmem in RGP patients after 4 months of SLIT. is described.

[0207] [Example 1] Materials and Methods Study Participants Subjects with grass pollen (RGP) allergy, moderate to severe seasonal allergic rhinitis (SAR), with or without asthma and serum RGP-specific IgE (ImmunoCAP) of ≥ 0.35 kU A / L were recruited from the allergy clinics of The Alfred and Box Hill Hospitals in Melbourne, Victoria, Australia (Alfred Ethics Committee project number 514 / 13). Registration for the study was carried out after a treatment plan decision was made for the upcoming pollen season and after written informed consent.

[0208] All patients were allowed standard treatment with topical symptom relief including antihistamines and topical intranasal corticosteroids. Subjects who received sublingual allergen immunotherapy (SLIT) were treated with a commercially available 5-grass pollen SLIT tablet (Oralair®; Stallergenes, Antony, France) using a daily prescription completed prior to the 2019 Australian grass pollen season for 4 months (April / May - September / October). Treatment with Oralair® included dissolution under the tongue (for at least 2 minutes), followed by swallowing the remainder. The treatment plan included 1 tablet of 100 IR (reactivity index) on day 1; 2 tablets of 100 IR on day 2; 1 tablet of 300 IR per day from day 3 to day 120. Blood samples from all participants were collected prior to treatment initiation (April / May) and after 4 months of treatment (September / October) prior to the start of the Australian grass pollen season.

[0209] Subjects with a history of bee venom (BV) allergy and a systemic allergic response to bee stings and serum BV-specific IgE (ImmunoCAP) of ≥ 0.35 kU A / L were recruited from the Allergy Clinic at Alfred in Melbourne, Victoria, Australia (Alfred Ethics Committee Project Numbers 245 / 16 and 297 / 13). Registration for the study was conducted after the decision to initiate ultra-rush immunotherapy was made and after written informed consent was obtained.

[0210] Participants with neither RGP allergy nor BV allergy were included as controls based on no clinical history of either disease, no detectable specific IgE to RGP, and a negative basophil activation test (BAT) to RGP extract (Monash University Project 2016-0289).

[0211] Exclusion criteria for all subjects were immunodeficiency, AIT within the last 5 years, and treatment with continuous oral corticosteroids and / or β-blockers. The use of symptomatic medications for allergic rhinitis (including antihistamines and topical corticosteroids) was tolerated. The study was conducted in accordance with the principles of the Helsinki Declaration, and written informed consent from each participant was obtained prior to entry.

[0212] Characteristics of participants Fifty RGP-allergic patients (18 - 65 years old; 52% female) with moderate to severe SAR, with or without asthma and serum RGP-specific IgE (ImmunoCAP) of ≥ 0.35 kU A / L were recruited. Twenty-seven participants had samples taken at t = 0, followed by 13 who received SLIT, 14 who received standard treatment only, and 23 who withdrew from further investigation. Seventeen BV-allergic patients with a history of anaphylactic response to bee stings (median age 57 years, range 18 - 69 years; 29% female) and serum BV-specific IgE (ImmunoCAP) of ≥ 0.35 kU A / L were recruited.

[0213] Blood sample collection Heparinized or EDTA blood samples were processed within 24 hours of collection for PBMC isolation and storage. Serum was isolated simultaneously from silica-coated collection tubes. Serum RGP-specific IgE levels were measured by ImmunoCAP at Alfred Pathology Services (Alfred Hospital, Melbourne, Australia) using allergen extracts according to the manufacturer's instructions. Serum IgE specific for Lol p 1 (the major allergen of RGP, ryegrass (Lolium perenne)) was measured by semi-quantitative in-house ELISA as previously described 1、2 by semi-quantitative in-house ELISA.

[0214] Enzyme-linked immunosorbent assay (ELISA) ELISA plates were coated with recombinant monomeric non-biotinylated Lol p 1 (MyBiosource, San Diego, CA, US), blocked with 5% skim milk powder in PBS, and incubated with serially diluted serum samples. For Lol p 1-specific IgG2 and IgG4 ELISAs, the standard curve was prepared using mouse hybridoma-produced Lol p 1-specific antibodies (see next section) as previously described 3It was generated with chimeric Lol p 1-specific IgG2 or IgG4 mAbs prepared from []. In the Lol p 1-specific IgE ELISA, separate wells were incubated with a range of concentrations of purified recombinant human IgE (clone AbD18705; Bio-Rad, Puchheim, Germany) to generate a standard curve for the relative quantification of IgE in serum. Antibodies bound to Lol p 1 were detected using polyclonal rabbit anti-hIgE (Agilent, Santa Clara, CA, US), biotinylated anti-hIgG2 (clone HP6002, Thermo Scientific) or biotinylated anti-hIgG4 (clone HP6025, Sigma Aldrich, St. Louis, MO, US), followed by polyclonal goat anti-rabbit HRP (Promega, Madison, WI, US). The ELISA was developed using TMB (Thermo Fisher Scientific, Waltham, MA, US) before the reaction was stopped with 1 M HCl and absorbance was measured at OD450 nm on a Multiskan microplate spectrophotometer (Thermo Fisher Scientific). Background values were determined using allergen-free wells and subtracted from allergen-specific IgE values, and the results were expressed in arbitrary units (AU).

[0215] Production of recombinant IgG2, IgG4, Lol p 1 and Api m 1 Chimeric IgG2 and IgG4 mAbs specific for Lol p 1 were prepared from previously described mouse hybridoma-produced Lol p 1-specific antibodies 3 from Igh and Igκ generated based on the variable regions. Mouse Igh and Igκ mouse hybridomas using primers specific for the variable regions 4、5PCR with the original mRNA resulted in products cloned into the pWin98 vector containing human IgG2 or the pBAR981 vector containing the human IgG4 constant region. The resulting plasmids were transfected into Expi293F cells (Thermo Fisher Scientific, Scoresby, Australia). Anti-Lol p 1 IgG2 and IgG4 were purified by a protein A column (GE Healthcare Amersham Biosciences, Uppsala, Sweden), and Lol p 1 specificity was confirmed by ELISA (data not shown).

[0216] Recombinant Lol p 1 and Api m 1 were produced in Spodoptera frugiperda 21 (Sf21) insect cells as previously described 6 Briefly, the protein sequences of Api m 1.0101 and Lol p 1.0101 were obtained from the allergen nomenclature website (allergen.org) 7、8 Both constructs were made using an N-terminal leader sequence for Api m 1 for secretion, a 6-His tag for purification, and a BirA tag for biotinylation. The Api m 1 (H67Q) and Lol p 1 (H104V) mutations were introduced to prevent unwanted effects on catalytic activity 9。The Api m 1 and Lol p 1 constructs were codon-optimized for Spodoptera frugiperda and cloned into the pFastBac vector (Thermo Fisher Scientific) prior to their incorporation into the bacmid for baculovirus production. The bacmid was transfected into Sf21 cells, which were subsequently cultured at 27 °C. The supernatant from the infected Sf21 cultures was clarified by centrifugation, and the 6-His-tagged protein was purified through retention on a cobalt column. The supernatant was gravity-fed through a 25-ml column packed with 4 ml of Talon NTA cobalt-agarose beads (Clontech, Mountain View, CA, US). The beads were washed with PBS, and the protein was eluted with PBS, pH 8.5, containing 200 mM imidazole. The eluate was dialyzed against 10 mM TRIS, pH 7.5. The purified recombinant protein was incubated overnight at room temperature with BirA enzyme for target biotinylation (2.5 μg / ml BirA, 12.5 mM ATP, 12.5 mM MgOAc, 62.5 μM D-biotin in 10 mM TRIS containing 62.5 mM bicine-HCl). Subsequent dialysis against PBS was followed by tetramerization at a 4:1 molar ratio of Lol p 1:streptavidin using fluorescent dye-conjugated streptavidin (BV711 and BUV395 conjugates; BD Biosciences, San Jose, CA, US).

[0217] Cell sorting and flow cytometry Isolation of Lol p 1-specific Bmem for single-cell transcriptomics was performed using fluorescence-activated cell sorting. Briefly, 10 million PBMCs were incubated for 15 minutes at room temperature in a total volume of 300 μL with two Lol p 1 allergen tetramers (APC and PE) of a 10-color cocktail and seven antibodies against B cell markers (CD19, CD38, CD27, IgM, IgD, IgG, IgA). Lol p 1-specific memory B cells (Lol p 1+CD19+CD27+CD38dim and Lol p 1+CD19+CD27-CD38dimIgM-IgD-IgG+ or Lol p 1+CD19+CD27-CD38dimIgM-IgD-IgA+) were defined and sorted from four patients before and after SLIT on a 6-laser FACSAria™ Fusion (BD Biosciences, Franklin Lakes, NJ, USA).

[0218] Detailed immunophenotyping of Lol p 1-specific Bmem was performed on 27 pairs of samples from RGP-allergic patients and Api m 1-specific Bmem on 17 pairs of samples from BV-allergic patients. B cell subsets were defined as previously described 10~12 . Briefly, within the CD19+ B cell population, the proportions of IgM+Bmem (CD38dimCD27+IgM+), IgG+Bmem (CD38dimIgD-IgM-IgG+) and IgA+Bmem (CD38dimIgD-IgM-IgA+) were determined. Furthermore, allergen-specific Bmem were quantified and evaluated for Ig isotype and surface marker expression.

[0219] As previously described in detail 32、33 , data were acquired on a 5-laser BD LSRFortessa X-20 (RGP-allergic) and a 5-laser Cytek Aurora with instrument settings, and calibration was performed using standardized EuroFlow SOP. All flow cytometry data were analyzed with FlowJo v10 software (FlowJo LLC, Ashland, OR, USA).

[0220] Single-cell transcriptome profiling FACS-purified Lol p 1+Bmem were labeled with sample tags according to the manufacturer's instructions (Human Single-Cell Multiplexing Kit, BD Biosciences). Single-cell capture, cDNA synthesis, and library preparation were performed using the BD Rhapsody single-cell analysis system (BD Biosciences) for whole transcriptome analysis according to the manufacturer's instructions. Libraries were sequenced in a single run using the Illumina MiSeq platform (Genomics Hub, The Walter and Elisa Hall Institute of Medical Research, Melbourne, Australia). Raw sequences were processed using the SevenBridges platform (SevenBridges, Boston, MA, US) to identify sample tags, cell barcodes, and the number of transcripts per cell. Cells without identifiable sample tags were excluded from further analysis. Single-cell transcriptomics data were analyzed using the SeqGeq v1.6 software package (FlowJo LLC). Read counts were normalized to a total library size of 10,000 reads per cell. Dimension reduction by principal component analysis (PCA) determined 25 principal components derived from the top 200 genes with the most variable gene expression. t-distributed stochastic neighbor embedding (tSNE) analysis was performed with the 25 principal components from PCA. Pseudotime analysis to construct single-cell trajectories was determined using the Monocle v.2 plugin. Data dimensions were reduced by inverse graph embedding using a gene expression filter of 1 and a cell expression filter of 10.

[0221] Statistics Differential gene expression from all pairwise cell transcriptomes before and after SLIT was illustrated by volcano plots, and significance was determined by Bonferroni-corrected Mann-Whitney t-tests. Genes with Q-value < 0.05 and absolute fold change ≥ 1.5 were considered to be significantly and correspondingly different between the subsets analyzed. IGHC Differences in the proportion of transcripts were statistically analyzed by chi-square test.

[0222] Statistical analysis of flow cytometry data was performed using Graphpad Prism (v8.4.1): paired data were analyzed by non-parametric Wilcoxon signed-rank test and unpaired data were analyzed by non-parametric Mann-Whitney U test. In all tests, p < 0.05 was considered significant.

[0223] [Example 2] 4-month SLIT increases serum allergen-specific IgE and IgG4 levels Meta-analyses and clinical trials have confirmed the clinical efficacy of SLIT using a 5-grass mixture. Of the 13 patients in this active study group available after 2 years, 12 (92%) reported clinical usefulness, thereby confirming the efficacy of SLIT in this cohort. There were no significant differences in total, RGP-specific or Lol p 1-specific IgE at recruitment between patients recruited to receive or not receive SLIT (Figures 1B–C). 4-month SLIT increased Lol p 1-specific IgE and IgG4 (Figure 1C). In patients who did not receive SLIT, there was a slight but significant decrease in Lol p 1-specific IgE over the 4 months, while Lol p 1-specific IgG2 and IgG4 levels did not change (Figure 1C).

[0224] [Example 3] Increased expression of IGHE and IGHG4 transcripts in Lol p 1-specific Bmem after SLIT To investigate the immunological effects of SLIT, the inventors performed single-cell transcriptomics on Lol p 1-specific Bmem from four patients before and four months after SLIT. Following electronic gating of CD19+ B cells and CD3+ T cells, Lol p 1-specific Bmem were identified by flow cytometry using double discrimination, i.e., double positivity for two fluorescent Lol p 1 tetramer conjugates (Figure 2A). The Lol p 1 tetramers did not bind to CD3+ T cells (data not shown), and B cells also did not bind to the streptavidin fluorochrome conjugate used to generate these tetramers (data not shown), confirming that the double-positive population constituted bona fide Lol p 1-specific B cells. A total of 189 cells before SLIT and 323 cells after SLIT were subjected to scRNAseq. Outliers for total reads per gene (<150, >15,000) and cells per gene (<30, >650) were excluded, followed by dimensionality reduction by principal component analysis (PCA) based on the 200 most variable genes (Figures 2B–D). This resulted in two main clusters, one enriched in canonical B cell genes MS4A1 (CD20), while the other was enriched in myeloid genes Regarding CD14 . The latter population was considered to consist of monocytes and excluded, leaving a total of 512 B cells for further analysis.

[0225] To examine the Ig gene usage of these Bmem, the inventors IGH assessed the expression of the constant region (Figure 3A), as well as IGK and IGL the expression of the light chain. Each cell expressed one dominant IGH isotype and a single IGK or IGL constant region (Figure 3B). The ratio of IGH and IGHE among all IGHG4 transcripts was significantly increased in Lol p 1-specific Bmem after SLIT (Figure 3C). The IgK or IgLThere was no difference in the expression rate (data not shown). Therefore, after 4 months of SLIT, B cells expressing both IgE and IgG4 as well as IGHE and IGH4 were increased (Figure 1C).

[0226] [Example 4] SLIT promotes the differentiation of Lol p 1-specific Bmem into two putatively temporally distinct transcriptional profiles To further investigate the transcriptional changes of SLIT in Bmem, 15 related differentially expressed genes were identified from Lol p 1-specific Bmem before and after SLIT based on fold change > 1.5 and Q < 0.05 (Table 3). Of these 15 genes, 12 had increased expression levels after SLIT and 3 had decreased. Only 3 genes were considered related biomarkers ( IGHE , ITGB1 , and CD69 ), because either the other genes were not surface-expressed or were the result of donor mixing, and included Ig lambda light chain ( IGLC3 ) and HLA ( HLA-DRB5 ). Most notably, SLIT increased the expression of IGHE (IgE), phosphatase 1F actin cytoskeleton targeting unit PPP1R18 (hostensin), beta1 integrin involved in lymphocyte tissue homing ITGB1 (CD29) and cancer genes associated with IgG+ memory B cells in CD8+ T cell leukemia and transitional memory B cell states PARM1 (Figure 4A). In contrast, SLIT decreased the overall expression of the cancer gene transcription factor JUN (c-jun) and C-type lectin CD69 , and the latter was associated with preventing the release of B cells from lymphoid organs.

[0227] Numerous differentially expressed genes with a <1.5-fold change after SLIT suggest that the SLIT-induced changes in the transcriptional profile of Lol p 1-specific Bmem may have occurred in transcriptionally distinct B cell clusters that cannot be identified by comparing only before and after SLIT. The use of these genes to generate PCA and tSNE analyses did not result in different clustering of Bmem before and after SLIT suggestion. To identify clusters of transcriptional profiles associated with changes induced by SLIT, the inventors performed pseudotime analysis including all differentially expressed genes with significant differences (Q < 0.05) regardless of fold change (n = 272 genes). IGHM and IGHD The B cell clusters expressing were named pseudotime zero by a known class switch from IgM+ B cell precursors. Clustering of all Lol p 1-specific B cells using this pseudotime analysis pathway revealed two different branches of B cell differentiation (Figure 4B). Four different B cell clusters could be identified along this differentiation pathway (Figure 4C). SLIT decreased Bmem in the least differentiated state of cluster 1 (pseudotime = 0 - 2) and drove Bmem towards the most differentiated states in cluster 3 (pseudotime = 5 - 6) and cluster 4 (pseudotime = 6 - 8) (Figure 4C). IGH The heatmap of gene expression demonstrated that the increase in pseudotime was IGHM associated with the transition from to Ig class switch isotypes, particularly in cluster 4 IGHE expression (Figures 4D, 4E). Indeed, Bmem in cluster 4 was IGHE , IGHG1 and IGHG4 enriched for expression, while cells in cluster 3 were IGHM , IGHG3 and IGHG1 predominantly expressed (Figure 4F). SLIT increased the proportion of B cells in clusters 3 and 4, suggesting that SLIT induces Ig class switch and the differentiation of Ag-specific Bmem towards either of these two different transcriptional profiles.

[0228] [Example 5] Gene expression related to the transcription profile induced by SLIT By analyzing the differentially expressed genes in different pseudotime clusters, compared with cluster 1, in cluster 4 IGHE , IGHG4 and IGHG1 were confirmed to increase, while cluster 1 was enriched for IGHM and IGHD . (Figures 5A, B). Compared with cluster 1, cluster 4 also had increased expression of immune-related genes such as PARM1 , IL13RA1 , ITGB1 , PPP1R18 , SELL , FCER2 and decreased expression of CXCR4 and CD69 . (Figure 5B). Similarly, cluster 3 had increased expression of ITGB1 and PPP1R18 along with decreased CXCR4 expression compared with cluster 1. (Figure 5B). Furthermore, cluster 3 showed increased expression of FGR , SIGLEC10 , CD99 , TLE3 and decreased expression of TXNIP , BTG1 and FCER2 . Comparing cluster 3 and 4, cluster 4 had increased expression of SELL , FCER2 , IGHE , IL13RA1 , IL4R , PARM1 and ITGB1 , while cluster 3 had increased expression of IGHM , IGHD , CD1C , PLAC8 , SYK , FGR , FCGR2B , FCRLA , FCRL2 , FCRL3 , FCRL5 and ITGAX . (Figure 5B). In pseudotime cluster 4 compared with clusters 1 - 3ITGB1 , FCER2 and IL13RA1 The unique expression patterns of, and the association of this cluster with IgE and IgG4, which the inventors have already observed to be increased by SLIT, led the inventors to desire to further characterize these cells by visualizing the expression of these genes in space over pseudotime (Figure 5C). The expression of these genes changed as pseudotime progressed while reflecting changes in the Bmem transcriptional profile along the SLIT-induced differentiation pathway.

[0229] To identify potential biomarkers for the initial effects of immunotherapy, three comparisons: pre-SLIT vs. post-SLIT with fold change > 1.5-fold up or down cluster 1 vs. cluster 3 with fold change > 2-fold up or down cluster 1 vs. cluster 4 with fold change > 2-fold up or down were made, and genes encoding surface-expressed proteins that were significantly different with at least one corresponding change among them were selected.

[0230] This analysis yielded 14 transcripts, 2 of which met all three criteria, 6 met two criteria, and the remaining 6 were significantly and correspondingly different in one comparison (Table 2).

[0231] [Example 6] Confirmation of Biomarkers by Flow Cytometry To verify the SLIT-induced changes in gene expression identified by transcriptomics, the inventors evaluated the cell surface expression of Ig isotypes, CD29, CD23, CD213A1, and IL4Rα Lol p 1-specific Bmem by flow cytometry at time points 0 and 4 months from patients who received or did not receive SLIT. In patients who received SLIT, the number of Lol p 1-specific Bmem increased (Figure 6A). The proportion and number of both IgG+ (all IgG isotypes) and more specifically IgG4+ Lol p 1-specific Bmem increased after SLIT, while IgM+ Lol p 1-specific Bmem decreased (Figure 6B). In subjects who did not receive SLIT, no observable changes in the proportion or number of Lol p 1-specific Bmem were seen over a 4-month period (Figure 6A, B). SLIT also increased the proportion and number of CD29hi Lol p 1-specific Bmem and was further associated with increased CD29, CD23, and IL4Rα staining on Lol p 1-specific Bmem (Figures 6C - F and Figure 7). These flow cytometry data are consistent with the inventors' transcriptome data identifying an increased proportion of Lol p 1-specific Bmem expressing FCER2 (CD23), ITGB1 (CD29) and IL4R (IL4Rα) (Figure 5). In contrast, the inventors did not observe any changes in CD213A1 staining on Lol p 1-specific Bmem from patients who received SLIT (Figure 6G). CD29, CD23, and CD213A1 staining on Lol p 1-specific Bmem from patients who did not receive SLIT did not change (Figures 6D - G).

[0232] Since only one subject did not report the benefits of SLIT, the inventors sought to examine whether the number and expression profile of its Lol p 1-specific Bmem were consistent with those of patients who reported the benefits of SLIT (non-responders are indicated by red dots and black lines; Figure 6). This subject showed the greatest decrease in the total, IgG+ and CD29hi Lol p 1-specific Bmem numbers among patients treated with SLIT (Figures 6A, B, D). Furthermore, the MFI of CD29 on Lol p 1-specific Bmem from this non-responder did not change after SLIT (Figure 6E). Although it was only one case, this finding suggests that an increase in CD29hi class-switched Lol p 1-specific Bmem may indicate successful immunotherapy for grass pollen allergy.

[0233] To evaluate the potential of each marker as a predictor of treatment outcome, receiver operating characteristic curves were generated for treated versus untreated patients. To assess the 4-month effect, the first evaluation was the ratio of the markers 4 months after treatment divided by the pretreatment values for total Lol p 1+Bmem, CD29+Lol p 1+Bmem, and the expression levels of CD29 and CD213A1 on Lol p 1+Bmem (Figure 8A). The CD29 expression level on Lol p 1+Bmem was the most predictive with an AUC just below 0.72 (Figure 8A). Subsequently, the inventors evaluated the markers at month 4. These yielded higher AUCs for Lol p 1+Bmem (0.79) and CD29 Lol p 1+Bmem (0.95), while the AUCs for the expression levels of CD29 and CD213A1 did not improve (Figure 8B). Thus, at the 4-month mark of treatment, the CD29+Lol p 1+Bmem number is a very strong indicator of treatment success and is more specific than total Lol p 1+Bmem.

[0234] [Example 7] Evaluation of Biomarkers Following Ultra-Rush AIT for Hymenoptera Allergy To determine whether the changes in allergen-specific Bmem induced by SLIT for RGP allergy are similarly informative for other allergic conditions, these were evaluated on Api m 1-specific Bmem from patients with wasp venom allergy before and 63 days after the start of ultra-rush AIT. Similar to SLIT for RGP, the absolute number of allergen-specific Bmem (Api m 1+) increased on day 63 (Figure 9A). This was mainly the result of IgG-expressing Bmem and included an increase in the IgG4+ subset (Figure 9B). Furthermore, on day 63 of ultra-rush AIT, the absolute number of Api m 1+ Bmem highly expressing CD29 was significantly increased (Figures 9C, D). Additionally, the expression levels of both CD29 and CD23 on Api m 1+ Bmem were significantly increased after AIT, while CD213A1 was unchanged (Figures 9E–G).

[0235] The expression levels of CD29, CD23, and CD213A1 did not change on total B cells after SLIT for RGP allergy or after ultra-rush AIT for wasp venom allergy (Figure 10), strengthening the finding that the effect is antigen-specific.

[0236] Therefore, single-cell transcriptomics of allergen-specific B cells before and after AIT is a sensitive approach for the identification of immunophenotypic changes and can be considered as an initial biomarker for predicting treatment success.

[0237]

Table 2

[0238]

Table 3

[0239]

Table 4

Claims

1. A method for determining the effectiveness of allergen immunotherapy in a subject, - A step of providing a first sample obtained from the subject before receiving allergen immunotherapy. - A step of providing a second sample obtained from a subject who has received or is receiving allergen immunotherapy, wherein the first and second samples contain allergen-specific Bmem, Furthermore - A step of determining the proportion or number of CD29+ allergen-specific Bmem or CD29hi allergen-specific Bmem in the first and second samples, wherein an increase in the proportion or number of CD29+ allergen-specific Bmem or CD29hi allergen-specific Bmem in the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject; or - A step of determining the proportion or number of CD23+ allergen-specific Bmem or CD23hi allergen-specific Bmem in the first and second samples, wherein an increase in the proportion or number of CD23+ allergen-specific Bmem or CD23hi allergen-specific Bmem in the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject; or - A step of determining the proportion or number of IL4Rα+ allergen-specific Bmem or IL4Rαhi allergen-specific Bmem in the first and second samples, wherein an increase in the proportion or number of IL4Rα+ allergen-specific Bmem or IL4Rαhi allergen-specific Bmem in the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject. The method, including the method described above.

2. The method according to Claim 1, wherein the method is: - A step of determining the proportion or number of CD23+ allergen-specific Bmem or CD23hi allergen-specific Bmem in the first and second samples, wherein an increase in the proportion or number of CD23+ and CD29+ allergen-specific Bmem or CD23hi and CD29hi allergen-specific Bmem in the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject; or - A step of determining the proportion or number of IL4Rα+ allergen-specific Bmem or IL4Rαhi allergen-specific Bmem in the first and second samples, wherein an increase in the proportion or number of CD29+ and IL4Rα+ allergen-specific Bmem or CD29hi and IL4Rαhi allergen-specific Bmem in the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject; or - A step of determining the proportion or number of IL4Rα+ allergen-specific Bmem or IL4Rαhi allergen-specific Bmem in the first and second samples, wherein an increase in the proportion or number of CD23+ and IL4Rα+ allergen-specific Bmem or CD23hi and IL4Rαhi allergen-specific Bmem in the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject; or - A step of determining the proportion or number of CD23+, CD29+, and IL4Rα+ allergen-specific Bmem or CD23hi, CD29hi, and IL4Rαhi allergen-specific Bmem in the first and second samples, wherein an increase in the proportion or number of CD23+, CD29+, and IL4Rα+ allergen-specific Bmem, CD23hi, CD29hi, and IL4Rαhi allergen-specific Bmem in the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject. The method, including the method described above.

3. The method according to Claim 1, The level or amount of the CD29 biomarker in the second sample is increased by at least 1.5 times compared to the first sample, and this indicates the effectiveness of allergen immunotherapy in the subject, or The level or amount of the CD29 biomarker in the second sample increased by at least four times compared to the first sample, indicating the effectiveness of allergen immunotherapy in the subjects; and / or The level or amount of the CD23 biomarker in the second sample is at least 1.5 times higher or 1.5 times lower than that of the first sample, indicating the effectiveness of allergen immunotherapy in the subject, or The level or amount of the CD23 biomarker in the second sample increased by at least four times or decreased to less than one-quarter compared to the first sample, indicating the effectiveness of allergen immunotherapy in the subjects; and / or The level or amount of the IL4Rα biomarker in the second sample is at least 1.5 times higher or reduced to 1.5 times or less compared to the first sample, and this indicates the effectiveness of allergen immunotherapy in the subject, or Compared to the first sample, the level or amount of the IL4Rα biomarker in the second sample increased by at least four times or decreased to less than one-quarter, indicating the effectiveness of allergen immunotherapy in the subjects. The aforementioned method.

4. The method according to claim 1, wherein the allergen-specific Bmem is an IgG4+ or IgG4hi allergen-specific Bmem.

5. The method according to claim 1, wherein the method is: - A step of determining the level or amount of one or more biomarkers in the allergen-specific Bmem in the first and second samples, wherein the biomarkers are selected from the group consisting of IgE, CD69, IL13Rα, CD99, IgD, CXCR4, FCRL3, FCRL2, FCRL5, SIGLEC10, and CD1c. It further includes, An increase in the level or amount of one or more biomarkers selected from IgE, IL13Rα, CD99, FCRL3, FCRL2, SIGLEC10, and CD1c in the allergen-specific Bmem of the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject, and / or A decrease in the level or amount of one or more biomarkers selected from CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, and CD1c in the allergen-specific Bmem of the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject. The aforementioned method.

6. The method according to claim 5, Compared to the first sample, in the second sample, In allergen-specific Bmem, the level or amount of one or more biomarkers selected from IgE, IL13Rα, CD99, SIGLEC10, and CD1c is increased by at least 1.5 times, or In allergen-specific Bmem, the level or amount of one or more biomarkers selected from CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, and CD1c is reduced to 1 / 1.5 or less; or The level or amount of one or more biomarkers selected from IgE, CD29, IL13Rα, CD99, SIGLEC10, CD1c, and CD23 has increased by at least 1.5 times, or The level or amount of one or more biomarkers selected from CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, and CD1c has decreased to 1 / 1.5 or less; or The level or amount of one or more biomarkers selected from IgE and IL13Rα has increased by at least three times, and The level or amount of one or more biomarkers selected from CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, and CD1c has decreased to 1 / 1.5 or less; or The level or amount of CD29 has increased by at least four times, and The level or amount of one or more biomarkers selected from CD69, IgD, CXCR4, FCRL3, FCRL2, FCRL5, and CD1c has decreased to 1 / 1.5 or less. This demonstrates the effectiveness of allergen immunotherapy in this group. The aforementioned method.

7. The method according to claim 1, wherein the second sample is obtained from the subject after the initiation of the allergen immunotherapy.

8. The method according to claim 7, wherein the initiation period of the allergen immunotherapy is at least 4 months or at least 63 days.

9. The method according to claim 1, wherein the second sample is obtained from the subject at least four months after the commencement of the allergen immunotherapy treatment.

10. The method according to claim 1, wherein the second sample is obtained from the subject during 6 to 12 months after the start of treatment with the allergen immunotherapy.

11. The method according to claim 1, wherein the second sample is obtained from the subject within five years after the start of treatment with the allergen immunotherapy.

12. The method according to claim 1, wherein the allergen immunotherapy is sublingual or subcutaneous immunotherapy.

13. The method according to claim 1, wherein the sample is a whole blood sample or a serum sample.

14. The method according to claim 13, wherein the sample comprises peripheral blood mononuclear cells.

15. The method according to claim 1, wherein the method is: - A step of determining the level or amount of one or more biomarkers in the allergen-specific Bmem in the first and second samples, wherein the biomarkers are selected from the group consisting of IGHE, PPP1R18, ITGB1, PARM1, c-jun, CD69, FCER2, and IL4R. It further includes, An increase in the level or amount of one or more biomarkers selected from IGHE, PPP1R18, ITGB1, PARM1, c-jun, CD69, FCER2, and IL4R in the allergen-specific Bmem of the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject, and / or A decrease in the level or amount of one or more biomarkers selected from FCER2, IL4R, c-jun, and CD69 in Bmem in the second sample compared to the first sample indicates the effectiveness of allergen immunotherapy in the subject. The aforementioned method.

16. The method according to claim 15, wherein the level or amount of the biomarker is the level or amount of RNA.

17. The method according to claim 16, wherein the RNA is an mRNA precursor or mature mRNA.

18. The method according to Claim 1, The allergen immunotherapy described above is for the treatment of allergen sensitivity, and the allergen is a recombinant allergen; The allergen immunotherapy is for the treatment of sensitivity to an allergen, and the allergen is a synthetic allergen; or The allergen immunotherapy described above is for the treatment of susceptibility to allergens, and the allergen is an environmental allergen. The aforementioned method.

19. The method according to claim 1, wherein the allergen is a grass pollen allergen or a poisonous oat pollen allergen.

20. A method for determining the effectiveness of allergen immunotherapy in a subject, - A step of providing a test sample obtained from a subject who has received or is receiving allergen immunotherapy, wherein the test sample contains an allergen-specific Bmem, - A step of determining the proportion or number of one or more CD29+, CD23+, and IL4Rα+ allergen-specific Bmem or CD29hi, CD23hi, and IL4Rαhi allergen-specific Bmem in the test sample, - A step of comparing one or more proportions or numbers of CD29+, CD23+, and IL4Rα+ allergen-specific Bmem or CD29hi, CD23hi, and IL4Rαhi allergen-specific Bmem in the test sample with the reference value, reference range, or reference standard of CD29+, CD23+, and IL4Rα+ allergen-specific Bmem or CD29hi, CD23hi, and IL4Rαhi allergen-specific Bmem for subjects with the same or related allergies who have not received allergen immunotherapy. Includes, If the proportion or number of one or more CD29+, CD23+, and IL4Rα+ allergen-specific Bmem or CD29hi, CD23hi, and IL4Rαhi allergen-specific Bmem in the test sample is the same as or similar to the reference value, reference range, or reference standard, then it is determined that the subject has not responded to the allergen immunotherapy. or If the proportion or number of CD29+, CD23+, and IL4Rα+ allergen-specific Bmem or CD29hi, CD23hi, and IL4Rαhi allergen-specific Bmem in the test sample differs from the reference value, reference range, or reference standard, it is determined that the subject responded to the allergen immunotherapy. The aforementioned method.

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