Methods for assessing the therapeutic response of rheumatoid arthritis patients to Janus kinase inhibitor therapy.

Abstract

This invention provides a method for evaluating the response of rheumatoid arthritis (RA) patients to Janus kinase (JAK) inhibitor treatment. By detecting specific antibody biomarkers in a patient's biological sample and comparing the data with reference values ​​for treatment response, it is possible to predict whether a patient will respond well to the JAK inhibitor or achieve disease remission. Furthermore, this invention includes a assay kit utilizing target peptide sequences that can bind to specific antibodies and supporting materials for detection, which helps to accurately predict the therapeutic efficacy response to JAK inhibitors.

Description

Methods for measuring the response of patients with rheumatoid arthritis to treatment with Janus kinase inhibitors Technical Field

[0001] The present invention relates to methods for assessing the therapeutic response of patients with rheumatoid arthritis to treatment with a Janus kinase inhibitor. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic arthritis with multiple causes, characterized by persistent synovitis, joint / bone destruction, and decreased quality of life. Treatment options include conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs), biologic disease-modifying anti-rheumatic drugs (bDMARDs), and targeted synthetic disease-modifying anti-rheumatic drugs (tsDMARDs). Janus kinase (JAK) inhibitors achieve therapeutic effects by blocking signaling pathways that activate Janus kinase / signal transducer and activator of transcription (JAK / STAT), a key inflammatory protein in RA. Therefore, they have demonstrated effectiveness in treating RA. Despite the efficacy of biologic disease-modifying antirheumatic drugs and Janus kinase inhibitors in treatment, approximately 15-20% of patients still show adverse reactions to drug treatment. In addition, the heterogeneity of the pathogenesis of rheumatoid arthritis and the pathological conditions associated with additional extra-articular manifestations show the complexity of its clinical management. In addition to affecting the joints, rheumatoid arthritis may also cause different lesions in multiple organs and systems, which makes the management of the disease more challenging. Taken together, these unmet needs pose a major challenge to the precision medicine approach in rheumatoid arthritis. In order to treat rheumatoid arthritis more effectively while reducing the cost burden of using biologics and JAK inhibitors, it is necessary to find biomarkers that can predict the effectiveness of these drugs in patients. This personalized and precise treatment approach will help to manage the disease more accurately while reducing related medical expenses.

[0003] Proteomics studies are being widely used to develop new biomarkers for identifying biomarkers of response to disease-modifying anti-rheumatic drugs (DMARDs). Currently, an unbiased proteomics technique, called phage immunoprecipitation sequencing (PhIP-Seq), employs a synthetic version of the complete human peptidome (T7 peptidome phage display library, T7-Pep). This method uses oligonucleotide library synthesis to encode protein-scale peptide libraries (peptidomes, also known as peptidomides) and display them on phage. Subsequently, immunoprecipitation is performed using a single antibody, followed by high-throughput DNA sequencing analysis. These immune-related peptidomimetics can be simultaneously screened for peptide-associated autoantibodies, providing a powerful approach to identify new biomarkers.

[0004] Summary of the Invention

[0005] The present invention provides a method for assessing the response of rheumatoid arthritis patients to treatment with Janus kinase inhibitors. This method determines a patient's response to treatment by measuring baseline expression levels of specific biomarkers and comparing them with reference expression levels. Based on the determination results, a decision can be made as to whether to administer a Janus kinase inhibitor to the patient. This method can help physicians better tailor treatment plans, providing more effective and precise treatment for patients, thereby improving treatment outcomes and patients' quality of life.

[0006] In the present invention, JAK inhibitors refer to a class of drugs that inhibit the JAK / STAT signaling pathway by targeting one or more enzymes in the Janus kinase (JAK) family. JAK inhibitors interfere with the cytokine signaling related to immune response and inflammation by blocking the activity of kinases such as JAK1, JAK2, JAK3 and / or TYK2, thereby achieving the purpose of treating RA and other related inflammatory diseases. Among them, Tofacitinib is a pan-JAK inhibitor that mainly inhibits JAK1 and JAK3, and also has a certain effect on JAK2 and TYK2. It effectively controls the inflammatory response of RA by reducing the production of proinflammatory cytokines. Baricitinib selectively inhibits JAK1 and JAK2, reduces the signaling of inflammatory factors in RA patients, thereby reducing inflammation and immune response. Upadacitinib and Filgotinib are selective JAK1 inhibitors that specifically target the signaling pathways that JAK1 depends on, and can effectively inhibit the immune response and inflammation associated with RA while reducing the non-target effects on other JAKs. Peficitinib, as a pan-JAK inhibitor, targets all members of the JAK family and comprehensively inhibits multiple signal transduction pathways in RA inflammation. These JAK inhibitors effectively alleviate RA symptoms and control disease progression by interfering with the JAK / STAT signaling pathway to varying degrees.

[0007] The evaluation of the response of RA patients to Janus kinase inhibitors is based on multiple international standards, including the European League Against Rheumatism (EULAR) response criteria, the American College of Rheumatology (ACR) response criteria, the Disease Activity Score (DAS28) 28, the Simplified Disease Activity Index (SDAI) score, and the Clinical Disease Activity Index (CDAI) score. In the present invention, the treatment response and whether the disease has reached a remission state are mainly defined by the EULAR criteria. However, in medical practice, professionals can switch between different standards according to specific circumstances. Therefore, the present invention is not limited to a specific standard, but allows flexible application between different evaluation criteria according to actual clinical needs to ensure the best evaluation of the treatment effect.

[0008] According to one object of the present invention, a method for evaluating the therapeutic response of a rheumatoid arthritis patient to a Janus kinase inhibitor is provided, comprising: (a) providing a biological sample, wherein the biological sample is from a rheumatoid arthritis patient; (b) performing a detection method on the biological sample, wherein the detection method is used to determine the basal expression level of at least one biomarker; wherein the biomarker is freely selected from a group consisting of antibodies, wherein the group consisting of antibodies includes anti-SNRK antibodies, anti-MAGEE1 antibodies, anti-DGKK antibodies, anti-HUWE1 antibodies, anti-GTSF1L antibodies, anti-RIMBP2 antibodies, anti-TACC2 antibodies, anti-PHF14 antibodies, anti-ZNF827 antibodies, anti-FILIP1 antibodies, anti-FRMPD4 antibodies and anti-ZNF329 antibodies; (c) comparing the basal expression level of the biomarker with a reference expression level of the biomarker, wherein when the biomarker is selected from anti-HUWE1 antibodies, anti-DGKK antibodies and SNRK antibodies, the biomarker is wherein, when the baseline expression level of the biomarker is higher than the reference expression level of the biomarker, it is determined that the rheumatoid arthritis patient has a favorable response to treatment with a Janus kinase inhibitor; wherein, when the biomarker is selected from anti-MAGEE1 antibody, anti-ZNF827 antibody, anti-PHF14 antibody, anti-FRMPD4 antibody, anti-RIMBP2 antibody, anti-FILP1 antibody, anti-ZNF329 antibody, anti-GTSF1L antibody and anti-TACC2 antibody, and the baseline expression level of the biomarker is higher than the reference expression level of the biomarker, it is determined that the rheumatoid arthritis patient has a poor response to treatment with a Janus kinase inhibitor; and (d) when the rheumatoid arthritis patient is determined to have a favorable response to treatment with a Janus kinase inhibitor, a therapeutically effective amount of a JAK inhibitor is administered to the patient; when the rheumatoid arthritis patient is determined to have a poor response to treatment with a Janus kinase inhibitor, the patient is not administered treatment with a Janus kinase inhibitor.

[0009] In the method provided by the present invention, the biological sample is a blood sample.

[0010] According to the method provided by the present invention, when the biomarker is selected from anti-HUWE1 antibodies, anti-DGKK antibodies, and SNRK antibodies, if the baseline expression level of the biomarker is higher than the reference expression level of the biomarker, it is determined that the rheumatoid arthritis patient has a good response to treatment with a Janus kinase inhibitor. Therefore, anti-HUWE1 antibodies, anti-DGKK antibodies, and SNRK antibodies are defined as good biomarkers of therapeutic efficacy.

[0011] According to the method provided by the present invention, when the biomarker is selected from anti-MAGEE1 antibodies, anti-ZNF827 antibodies, anti-PHF14 antibodies, anti-FRMPD4 antibodies, anti-RIMBP2 antibodies, anti-FILP1 antibodies, anti-ZNF329 antibodies, anti-GTSF1L antibodies and anti-TACC2 antibodies, and the baseline expression level of the biomarker is higher than the reference expression level of the biomarker, it is determined that the rheumatoid arthritis patient has an adverse reaction to the treatment efficacy of the Janus kinase inhibitor. Therefore, anti-MAGEE1 antibodies, anti-ZNF827 antibodies, anti-PHF14 antibodies, anti-FRMPD4 antibodies, anti-RIMBP2 antibodies, anti-FILP1 antibodies, anti-ZNF329 antibodies, anti-GTSF1L antibodies and anti-TACC2 antibodies are defined as adverse reaction biomarkers of therapeutic efficacy.

[0012] According to the present invention, a method for evaluating the therapeutic response of rheumatoid arthritis patients to Janus kinase inhibitor treatment is provided, wherein the basal expression level is the expression level of the rheumatoid arthritis patient before receiving JAK inhibitor treatment; wherein the reference expression level is determined based on the cutoff point of the biomarker expression level in patients with a good response or an adverse response to treatment.

[0013] The cutoff point was determined through receiver operating characteristic (ROC) curve analysis to ensure an acceptable range of sensitivity and specificity, thereby more accurately assessing the response of rheumatoid arthritis patients to Janus kinase inhibitor treatment. This cutoff point was determined to find a balance in treatment assessment, neither missing patients who responded to treatment nor incorrectly identifying patients who did not respond.

[0014] In one embodiment of the present invention, the test method may be an immunoassay method. In another embodiment, the immunoassay method is selected from the group consisting of enzyme-linked immunosorbent assay, Western blot analysis, immunoprecipitation analysis, radioimmunoassay, and immunochromatographic analysis.

[0015] In one embodiment of the present invention, in the method for evaluating the therapeutic response of rheumatoid arthritis patients to Janus kinase inhibitor treatment, the evaluated Janus kinase inhibitor is selected from JAK1 inhibitors, JAK2 inhibitors, JAK3 inhibitors, TYK2 inhibitors or a combination thereof.

[0016] In another embodiment of the present invention, the Janus kinase inhibitor is selected from a JAK1 inhibitor, a JAK3 inhibitor or a combination thereof.

[0017] In one embodiment of the present invention, in the method for evaluating the therapeutic response of rheumatoid arthritis patients to Janus kinase inhibitor treatment, the evaluated Janus kinase inhibitor is selected from Tofacitinib, Baricitinib, Peficitinib, Upadacitinib and Filgotinib.

[0018] In another embodiment of the present invention, the Janus kinase inhibitor is Tofacitinib, Baricitinib and Upadacitinib.

[0019] In another embodiment of the present invention, the Janus kinase inhibitor is Tofacitinib.

[0020] According to the present invention, a method for evaluating the therapeutic effect of a rheumatoid arthritis patient on a Janus kinase inhibitor is provided, wherein the anti-SNRK antibody can recognize a sequence of continuous amino acids in SEQ ID NO: 1, the anti-MAGEE1 antibody can recognize a sequence of continuous amino acids in SEQ ID NO: 2, the anti-DGKK antibody can recognize a sequence of continuous amino acids in SEQ ID NO: 3, the anti-HUWE1 antibody can recognize a sequence of continuous amino acids in SEQ ID NO: 4, the anti-GTSF1L antibody can recognize a sequence of continuous amino acids in SEQ ID NO: 5, the anti-RIMBP2 antibody can recognize a sequence of continuous amino acids in SEQ ID NO: 6, the anti-TACC2 antibody can recognize a sequence of continuous amino acids in SEQ ID NO: 7, the anti-PHF14 antibody can recognize a sequence of continuous amino acids in SEQ ID NO: 8, the anti-ZNF827 antibody can recognize a sequence of continuous amino acids in SEQ ID NO: 9, the anti-FILIP1 antibody can recognize a sequence of continuous amino acids in SEQ ID NO: 10, the anti-FRMPD4 antibody can recognize a sequence of continuous amino acids in SEQ ID NO: 11. A continuous amino acid sequence in NO:11 and an anti-ZNF329 antibody can recognize a continuous amino acid sequence in SEQ ID NO:12.

[0021] According to the above method, the sequence of consecutive amino acids can be a sequence of 20-50 consecutive amino acids; further, the sequence of consecutive amino acids can be a sequence of 20-30 consecutive amino acids. In other words, the amino acid sequence can be used to bind to the antibody, and the amount of the binding can be used to determine the expression level of the antibody.

[0022] According to one embodiment, the method of the present invention, wherein the biomarker is selected from anti-SNRK antibody or anti-HUWE1 antibody.

[0023] According to one embodiment, the anti-SNRK antibody can recognize a sequence of 20-50 consecutive amino acids in SEQ ID NO: 1.

[0024] According to another embodiment, the anti-HUWE1 antibody can recognize a sequence of 20-50 consecutive amino acids in SEQ ID NO:4.

[0025] According to another embodiment, the anti-SNRK antibody recognizes 20-30 consecutive amino acids located in SEQ ID NO:13.

[0026] According to another embodiment, the anti-HUWE1 antibody can recognize a sequence of 20-30 consecutive amino acids in SEQ ID NO:14.

[0027] According to another embodiment, the biomarker is selected from anti-ZNF827 antibody or anti-RIMBP2 antibody.

[0028] According to one embodiment, the anti-ZNF827 antibody can recognize a sequence of 20-50 consecutive amino acids in SEQ ID NO:9.

[0029] According to another embodiment, the anti-RIMBP2 antibody can recognize a sequence of 20-50 consecutive amino acids in SEQ ID NO:6.

[0030] According to another embodiment, the anti-ZNF827 antibody recognizes 20-30 consecutive amino acids in SEQ ID NO:16.

[0031] According to another embodiment, the anti-RIMBP2 antibody can recognize a sequence of 20-30 consecutive amino acids in SEQ ID NO:15.

[0032] The present invention further provides another method for evaluating the therapeutic response of rheumatoid arthritis patients to Janus kinase inhibitor treatment, comprising: (a) providing a biological sample, wherein the biological sample is from a rheumatoid arthritis patient; (b) performing a detection method on the biological sample, wherein the detection method is used to determine the basal expression level of a biomarker, wherein the biomarker is an anti-SNRK antibody and an anti-HUWE1 antibody, wherein the basal expression level is the expression level of the rheumatoid arthritis patient before receiving the JAK inhibitor treatment; (c) comparing the basal expression level of the biomarker with a first reference expression level of the biomarker and a second reference expression level, wherein the first reference expression level is based on a first cutoff point of the biomarker expression level for patients with good response and poor response after treatment wherein the second reference expression amount is determined based on a second cutoff point of the biomarker expression amount between disease remission and non-disease remission in the patient after treatment; wherein if the baseline expression amount of the biomarker is higher than the first reference expression amount of the biomarker, it is determined that the rheumatoid arthritis patient is likely to have a good response to the Janus kinase inhibitor treatment; wherein if the baseline expression amount of the biomarker is higher than the second reference expression amount of the biomarker, it is determined that the rheumatoid arthritis patient is likely to achieve disease remission after treatment with the Janus kinase inhibitor; and (d) when the rheumatoid arthritis patient is determined to have a good response to the Janus kinase inhibitor treatment or to have achieved disease remission after treatment, a therapeutically effective dose of the JAK inhibitor is administered to the patient.

[0033] According to the methods of the present invention, disease remission refers to a temporary or complete reduction or improvement in disease symptoms or disease states, particularly during the treatment of certain diseases. The present invention provides that the definition of a Janus kinase inhibitor treatment response and disease remission is determined according to the European Union's International Respiratory and Respiratory Response Criteria.

[0034] In the method according to the present invention, the biological sample is a blood sample.

[0035] According to the method of the present invention, the basal expression level is the expression level of the rheumatoid arthritis patient before receiving JAK inhibitor treatment.

[0036] In one embodiment of the present invention, in the method for evaluating the therapeutic response of rheumatoid arthritis patients to Janus kinase inhibitor treatment, the evaluated Janus kinase inhibitor is selected from JAK1 inhibitors, JAK2 inhibitors, JAK3 inhibitors, TYK2 inhibitors or a combination thereof.

[0037] In another embodiment of the present invention, the Janus kinase inhibitor is selected from a JAK1 inhibitor, a JAK2 inhibitor, a JAK3 inhibitor or a combination thereof.

[0038] In another embodiment of the present invention, the Janus kinase inhibitor is selected from a JAK1 inhibitor, a JAK3 inhibitor or a combination thereof.

[0039] In one embodiment of the present invention, the Janus kinase inhibitor is selected from Tofacitinib, Baricitinib, Peficitinib, Upadacitinib and Filgotinib.

[0040] In one embodiment of the present invention, the Janus kinase inhibitor is selected from Tofacitinib, Baricitinib and Upadacitinib.

[0041] In one embodiment, the Janus kinase inhibitor is Tofacitinib.

[0042] In one embodiment of the present invention, the test method may be an immunoassay method. In one embodiment, the immunoassay method is selected from the group consisting of enzyme-linked immunosorbent assay, Western blot analysis, immunoprecipitation analysis, radioimmunoassay, and immunochromatographic analysis.

[0043] According to the method of the present invention, the second reference expression amount for determining disease remission is greater than the first reference amount for determining a good response.

[0044] According to the method of the present invention, the first reference expression level is determined based on the cutoff point of the biomarker expression level in patients with good or poor response to treatment; and the second reference expression level is determined based on the cutoff point of the biomarker expression level in patients with disease remission or no remission after treatment. This cutoff point is based on ROC analysis, and its purpose is to ensure an acceptable range of sensitivity and specificity to more accurately assess the response of rheumatoid arthritis patients to Janus kinase inhibitor treatment.

[0045] According to another embodiment, the anti-HUWE1 antibody can recognize a sequence of 20-50 consecutive amino acids in SEQ ID NO:4.

[0046] In one embodiment, the anti-HUWE1 antibody can recognize a sequence of 20-30 consecutive amino acids in SEQ ID NO:14.

[0047] According to one embodiment, the anti-SNRK antibody can recognize a sequence of 20-50 consecutive amino acids in SEQ ID NO: 1.

[0048] In one embodiment, the anti-SNRK antibody can recognize a sequence of 20-30 consecutive amino acids in SEQ ID NO:13.

[0049] The present invention further provides a reagent kit for evaluating the therapeutic response of rheumatoid arthritis patients to Janus kinase inhibitor treatment, comprising: a target peptide that can recognize a biomarker; a solid support matrix that binds to the target peptide; and a probe-labeled or unlabeled secondary antibody that can bind to the biomarker.

[0050] In one embodiment, the biomarker is anti-SNRK antibody or anti-HUWE1 antibody.

[0051] In the reagent set provided by the present invention, the target peptide that can identify the biomarker is a target peptide that can identify an anti-SNRK antibody or a target peptide that can identify an anti-HUWE1 antibody.

[0052] In one embodiment, the biomarker target peptide capable of identifying anti-SNRK antibodies comprises a continuous sequence of 20 to 50 amino acids in SEQ ID NO: 1.

[0053] In one embodiment, the biomarker target peptide capable of recognizing anti-HUWE1 antibodies comprises a sequence of 20-50 consecutive amino acids in SEQ ID NO: 4.

[0054] In one embodiment, the biomarker target peptide capable of identifying anti-SNRK antibodies comprises a sequence of 20-30 consecutive amino acids in SEQ ID NO: 13.

[0055] In another embodiment, the biomarker target peptide capable of recognizing anti-HUWE1 antibodies comprises a sequence of 20-30 consecutive amino acids in SEQ ID NO: 14.

[0056] In the reagent set provided by the present invention, the Janus kinase inhibitor is selected from JAK1 inhibitors, JAK2 inhibitors, JAK3 inhibitors, TYK2 inhibitors or a combination thereof.

[0057] In another embodiment of the present invention, the Janus kinase inhibitor is selected from a JAK1 inhibitor, a JAK2 inhibitor, a JAK3 inhibitor or a combination thereof.

[0058] In another embodiment of the present invention, the Janus kinase inhibitor is selected from a JAK1 inhibitor, a JAK3 inhibitor or a combination thereof.

[0059] In one embodiment of the present invention, the Janus kinase inhibitor is selected from Tofacitinib, Baricitinib, Peficitinib, Upadacitinib and Filgotinib.

[0060] In one embodiment of the present invention, the Janus kinase inhibitor is selected from Tofacitinib, Baricitinib and Upadacitinib.

[0061] In one embodiment, the Janus kinase inhibitor is Tofacitinib.

[0062] In the reagent set provided by the present invention, the solid support matrix can be selected from the group consisting of ELISA plate, magnetic beads, thin polyvinylidene fluoride membrane (PVDF), resin (agarose beads), polystyene or nitrocellulose membrane.

[0063] In one embodiment, the ELISA plate has a porous structure, which can provide a large surface area to facilitate efficient immobilization of biomolecules such as antibodies or antigens, and is compatible with various detection reagents for highly sensitive immunoassays.

[0064] In one embodiment, the solid support matrix is ​​an ELISA plate.

[0065] According to the reagent set provided by the present invention, the reagent set further includes a first reference expression level and a second reference expression level for comparing the expression levels of biomarkers in patient samples; wherein the first reference expression level is determined based on a first cutoff point of the biomarker expression level for patients with good response and poor response after treatment; wherein the second reference expression level is determined based on a second cutoff point of the biomarker expression level for patients with disease remission and non-disease remission after treatment; wherein the first cutoff point and the second cutoff point are obtained based on ROC analysis, the purpose of which is to ensure an acceptable range of sensitivity and specificity to more accurately assess the response of rheumatoid arthritis patients to Janus kinase inhibitor treatment.

[0066] According to the reagent set provided by the present invention, the reagent set further comprises a sample processing reagent for processing a biological sample, wherein the biological sample is a blood sample.

[0067] According to the reagent set provided by the present invention, the reagent set further comprises at least one standard for establishing a standard curve of biomarker concentration; wherein the standard comprises anti-SNRK antibody and anti-HUWE1 antibody of known concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 shows a heat map of 20 antigens that differentiate RA patients who respond well or poorly to JAK inhibitor therapy using the PhIP-Seq platform.

[0069] Figure 2 shows a receiver operating characteristic (ROC) curve analysis of the 20 individual antigens used to discriminate the therapeutic effect of JAK inhibitor therapy. The X-axis represents the false positive rate (FPR), and the Y-axis represents the true positive rate (TPR).

[0070] Figure 3 shows ROC curve analysis of anti-HUWE1 antibody (A) against peptide SEQ ID NO: 14, anti-SNRK antibody (B) against peptide SEQ ID NO: 13, anti-ZNF827 antibody (C) against peptide SEQ ID NO: 16, and anti-RIMBP2 antibody (D) against peptide SEQ ID NO: 15 for distinguishing good responses from poor responses in patients with rheumatoid arthritis to JAK inhibitor treatment.

[0071] Figure 4 shows ROC curve analysis of anti-HUWE1 antibody (A) against peptide SEQ ID NO: 14, anti-SNRK antibody (B) against peptide SEQ ID NO: 13, anti-ZNF827 antibody (C) against peptide SEQ ID NO: 16, and anti-RIMBP2 antibody (D) against peptide SEQ ID NO: 15, for distinguishing between patients with rheumatoid arthritis who are in remission and those who are not in remission during JAK inhibitor treatment.

[0072] FIG5 shows ROC analysis of anti-HUWE1 antibodies against peptide SEQ ID NO: 14 and anti-SNRK antibodies against peptide SEQ ID NO: 13 for distinguishing good responders from poor responders to JAK inhibitor treatment in rheumatoid arthritis patients.

[0073] FIG6 shows ROC analysis of anti-HUWE1 antibodies against peptide SEQ ID NO: 14 and anti-SNRK antibodies against peptide SEQ ID NO: 13 for differentiating disease remission and non-remission in rheumatoid arthritis patients treated with JAK inhibitors. DETAILED DESCRIPTION

[0074] Various embodiments of the present invention are discussed in more detail below. However, the embodiments may be specific to various applications of the present invention and may be practiced in various specific environments. These embodiments are for illustrative purposes only and do not limit the scope of this disclosure. To evaluate the therapeutic response to treatment with Janus kinase inhibitors, specific test cases will be further illustrated below.

[0075] Example 1

[0076] This study was divided into two phases and included two cohorts. Twelve biologic-naive patients who met the 2010 revised American College of Rheumatology diagnostic criteria for rheumatoid arthritis and received 24 weeks of treatment with a JAK inhibitor (tofacitinib) were consecutively enrolled at the China Medical University Hospital in Taiwan. This study was approved by the hospital's institutional review board (CMUH111-REC3-108), and written consent was obtained from each participant in accordance with the Declaration of Helsinki.

[0077] Assessment of disease activity during treatment response: RA disease activity was assessed using the 28-joints disease activity score (DAS28). Treatment response was assessed at week 24 in two ways: (1) remission if DAS28 < 2.6 after treatment, and (2) good response, moderate response, or poor response according to the European Alliance of Associations for Rheumatology (EULAR) response criteria. According to the definition, a good response is a decrease of more than 1.2 in DAS28 after treatment compared with pre-treatment (ΔDAS28>1.2), and DAS28≤3.2 after treatment; a moderate response is a decrease of more than 1.2 in DAS28 after treatment compared with pre-treatment (ΔDAS28>1.2), and DAS28>3.2 after treatment, or a decrease of 0.6-1.2 in DAS28 after treatment (ΔDAS28 is 0.6-1.2), and DAS28≤5.1 after treatment; an adverse reaction is a decrease of less than ΔDAS28<0.6 after treatment compared with pre-treatment, and DAS28>5.1 after treatment.

[0078] Clinical phenotype:

[0079] Table 1 shows the clinical characteristics of patients with rheumatoid arthritis who received 24 weeks of tofacitinib treatment with respect to EULAR treatment response.

[0080] The data in Table 1 are presented as mean ± standard deviation or median (interquartile range); group differences in numerical variables were compared using the Mann-Whitney U test. *P < 0.05, compared with poor responders, as determined by the Mann-Whitney test. Binary variables were compared using Yates's continuity correction or Fisher's exact test. #p < 0.05, ##p < 0.01, compared with poor responders, as determined by the chi-square test.

[0081] Example 2

[0082] Identify differentially expressed peptides to distinguish between rheumatoid arthritis patients who respond well and poorly to JAK inhibitor treatment.

[0083] Materials and methods:

[0084] (1) Phage immunoprecipitation sequence (PhIP-Seq) analysis: PhIP-Seq is an innovative technology that combines peptide library-based antibody detection with next-generation sequencing. With its sequencing and nucleic acid synthesis capabilities, this technology allows for the efficient identification and characterization of antibody responses. It offers high throughput, excellent sensitivity, and the ability to analyze a large number of peptides simultaneously. PhIP-Seq has significant potential in seroepidemiological studies and can provide valuable insights into antibody responses in different disease contexts.

[0085] (2) Study cohort: Twelve consecutive patients who were naive to biologics and met the 2010 revised diagnostic criteria for rheumatoid arthritis by the American College of Rheumatology were enrolled and received 24 weeks of treatment with the JAK inhibitor, tofacitinib. The institutional review board of the hospital approved this study (CMUH111-REC3-108), and written consent was obtained from each participant in accordance with the Declaration of Helsinki.

[0086] (3) Experimental plan: Serum samples were tested using the PhIP-Seq platform (CDI Laboratories, Inc., Mayaguez, PR, USA). Sample screening was performed on a phage library (approximately 10 11pfu) and 0.2 μL of serum (approximately 2 μg IgG) in 1 mL of PBS (pH 7.4). In addition, a set of 8 independent buffer-only samples without serum (beads only) were included as negative controls. The mixture was rotated overnight at 4°C to allow antibody binding to the phage-displayed peptide target. The next day, 40 μL of a 1:1 protein A / G-coated magnetic bead suspension was added and rotated at 4°C for 4 hours to capture all immunoglobulin G (IgG). The beads were then washed three times with TBS (pH 7.4) containing 0.1% NP-40 using a BRAVO liquid handler (Agilent, Santa Clara, CA, USA). BRAVO then resuspended the beads in 20 μL of Herculase II Fusion Polymerase PCR1 master mix to amplify the library insert. After 20 cycles of polymerase chain reaction (PCR), sample-specific barcoding, and 2 μL of PCR1 product in a subsequent PCR2 reaction, Illumina P5 / P7 adapters were incorporated. After another 20 PCR cycles, PCR2 products were pooled and serialized using an Illumina NextSeq to obtain single-end 50-nucleotide reads.

[0087] (4) Library cloning: An E. coli-cryptoenzyme-optimized library containing approximately 250,000 oligonucleotides encoding 90-amino acid peptides with 45 amino acid overlaps was generated using the Python package pepsyn (https: / / github.com / lasersonlab / pepsyn). The resulting oligonucleotides were first synthesized on a releasable microarray, then amplified by PCR using attached pepsyn adapter sequences, and finally cloned into a modified T7Select 10-3b medium-copy vector. This vector displays the library peptides as a C-terminal fusion of the T7 bacteriophage outer sheath protein 10B and carries a C-terminal FLAG tag. The library peptides were packaged in vitro using the T7Select packaging kit (Millipore) and amplified on plates to obtain an average of 100 plaques / peptide. The quality of the library peptides was assessed by Sanger sequencing of the entire insert of a single plaque, and the clonal distribution was quantified by Illumina sequencing.

[0088] (5) Data evaluation of PhIP-Seq analysis: Differentially expressed peptide signatures were evaluated using the PhIP-Seq platform containing 259,345 peptides. Reads were multiplexed and aligned to the human peptide library using exact matches. Next, the reads in each sample were compared to mock immunoprecipitation (mock-IP) containing only buffer using the R package edgeR, using a negative binomial model. Enrichment values ​​at the selective gene level were obtained by calculating the Z-score (greater than 0) and then hierarchically clustered using Spearman correlation to produce heat maps. The software returned a test statistic and a fold change value for each peptide. For each individual sample with a fold change greater than 5 for the identified enriched peptide (hits), each individual sample was considered "positive". In peptide microarrays, autoantibody responses to antigens on the array were classified as biomarkers if they met all of the following criteria: (I) There was a high penetration fold change (pFC) in the test group. 3 5.0 times), (ii) the frequency with high penetration in the tested group (pFreq 3 50%), and (iii) the frequency % with low penetration in the mixed negative control group (pFreq<20%).

[0089] Results: Based on the fold difference (Z-score) in the primary study data, two distinct expression clusters (top 20 potential biomarkers) were listed to distinguish good and poor responses of RA patients to JAK inhibitors (Figure 1).

[0090] Individual ROC analyses of the top 20 biomarkers clearly showed that some biomarkers (eg, HUWE1, ZNF827, FRMPD4, RIMBP2, and FILIP1) could distinguish good responders from poor responders with high sensitivity and specificity.

[0091] All genes were clustered using a hierarchical analysis (Pearson correlation) and a heat map of gene expression in the mock-IP (mock-IP) profile with a change of more than 5-fold was generated. The color code at the top of the heat map represents the EULAR response to the JAK inhibitor. The color bar represents the calculated Z-score of all individual gene expression (Figure 2).

[0092] Table 2 shows the biomarkers of favorable response to JAK inhibitor therapy. The favorable response biomarkers included anti-DGKK antibodies, anti-SNRK antibodies, and anti-HUWE1 antibodies, with AUCs of 0.743, 0.786, and 0.8, respectively.

[0093] Table 3 shows the biomarkers of adverse reactions to JAK inhibitor treatment. The adverse reaction biomarkers included anti-MAGEE1 antibody, anti-ZNF827 antibody, anti-PHF14 antibody, anti-FRMPD4 antibody, anti-RIMBP2 antibody, anti-FILIP1 antibody, anti-ZNF329 antibody, anti-GTSF1L antibody, and anti-TACC2 antibody, with AUCs of 0.771, 0.9, 0.771, 0.8, 0.8, 0.8, 0.8, 0.8, 0.771, and 0.8, respectively.

[0094] Table 4 shows the gene names and peptide sequences corresponding to anti-SNRK antibody, anti-MAGEE1 antibody, anti-DGKK antibody, anti-HUWE1 antibody, anti-GTSF1L antibody, anti-RIMBP2 antibody, anti-TACC2 antibody, anti-PHF14 antibody, anti-ZNF827 antibody, anti-FILIP1 antibody, anti-FRMPD4 antibody and anti-ZNF329 antibody.

[0095] Example 3

[0096] Validation of candidate peptide fragment biomarkers using indirect enzyme-linked immunosorbent assay (ELISA): Based on the AUC results in Figure 2, anti-ZNF827 antibody, anti-RIMBP-2 antibody, anti-HUWE1 antibody, and anti-SNRK antibody biomarkers were further selected to verify their performance in distinguishing good responses from adverse reactions in patients with rheumatoid arthritis treated with JAK inhibitors (Tofacitinib).

[0097] (1) Materials and methods

[0098] Target peptide sequence:

[0099] SEQ ID NOs: 13, 14, 15, and 16 correspond to target peptide sequences for anti-SNRK antibody, anti-HUWE1 antibody, anti-RIMBP2 antibody, and anti-ZNF827 antibody, respectively. These peptide sequences were synthesized with 95% purity (provided by ThermoFisher Scientific, Massachusetts, USA). Table 5 shows these peptide sequences and the corresponding biomarkers.

[0100] Indirect enzyme-linked immunosorbent assay (ELISA): Target peptides were diluted to 100 nM in carbonate coating buffer and plated onto 96-well microplates overnight at 4°C. After washing with wash buffer (PBS containing 0.02% Triton X100), the plated microplates were blocked with blocking buffer (1 M ethanolamine) for 1 hour at room temperature. All participating plasma or serum samples were diluted (1:500 in 1% BSA) and placed on the blocked microplates for 1 hour at room temperature. Following treatment, the microplates were washed four times and then reacted with HRP (peroxidase conjugate)-conjugated anti-human IgG (1:5000 in PBS containing 1% BSA) for 1 hour. The HRP enzymatic reaction was detected using 3,3'5,5'-tetramethylaniline for 20 minutes and then stopped with 2N sulfuric acid. The absorbance shifted from blue to yellow and was measured at 405 nm.

[0101] (2) Statistical analysis

[0102] All P values ​​were two-tailed. Odds ratios were calculated with the use of Haldane's modified method. Correlation coefficients were obtained using the nonparametric Spearman rank correlation test. A multivariable logistic regression model was developed to evaluate factors predicting treatment response in EULAR. Receiver operating characteristic (ROC) curve analysis was performed using MedCalc v.14 to determine the area under the ROC curve (AUC), sensitivity (SEN), specificity (SPE), and accuracy (ACC).

[0103] (3) Results: ROC analysis was used to evaluate the performance of anti-ZNF827 antibodies, anti-RIMBP2 antibodies, anti-HUWE1 antibodies, and anti-SNRK antibodies in distinguishing good responses from poor responses. As shown in Figure 3, anti-HUWE1 antibodies and anti-SNRK antibodies performed well in predicting EULAR responses to JAK inhibitors (AUCs of 0.768 and 0.814, respectively). At cut-off values ​​of 0.324 and 0.31, anti-HUWE1 antibodies and anti-SNRK antibodies showed the highest predictive ability, with sensitivities (SENs) of 75.0% and 80.0%, specificities (SPEs) of 71.4% and 71.4%, and accuracy (ACCs) of 73.5% and 76.5%, respectively (p < 0.001) (Table 6).

[0104] ROC curves were used to evaluate the performance of anti-ZNF827 antibodies, anti-RIMBP2 antibodies, anti-HUWE1 antibodies, and anti-SNRK antibodies in distinguishing whether remission was achieved. As shown in Figure 4, the optimal cutoff points for anti-HUWE1 antibodies, anti-SNRK antibodies, and anti-RIMBP2 antibodies were 0.355, 0.399, and 0.355, respectively, with AUCs of 0.869, 0.838, and 0.728, respectively. The sensitivities (SENs) were 83.3%, 83.3%, and 75%, respectively, the specificities (SPEs) were 84.8%, 78.8%, and 57.6%, respectively, and the accuracy (ACCs) were 84.4%, 80.0%, and 62.2%, respectively (Table 7).

[0105] Example 4 Anti-HUWE1 Antibody and Anti-SNRK1 Antibody for Evaluating the Therapeutic Response (Good Response and Poor Response) of JAK Inhibitor (Tofacitinib) Treatment

[0106] Table 8 shows the clinical characteristics of patients with rheumatoid arthritis who were evaluated for EULAR good response and poor response after 24 weeks of Tofacitinib treatment. The data are expressed as mean ± standard deviation, number (percentage) or median (interquartile range).

[0107] Figure 5 shows the ROC curves for anti-HUWE1 antibodies and anti-SNRK1 antibodies as good responders after Tofacitinib treatment, which are used to evaluate their diagnostic efficacy. The ROC curve is used to evaluate the performance of anti-HUWE1 antibodies and anti-SNRK antibodies at different sensitivity and specificity thresholds, and the overall efficacy is evaluated based on the area under the curve (AUC). The results showed that the optimal cutoff points for anti-HUWE1 antibodies and anti-SNRK antibodies were 0.362 and 0.381, respectively, the AUCs were 0.74 and 0.823, respectively, the sensitivities (SEN) were 62.5% and 78.1%, the specificities (SPE) were 74.2% and 80.6%, and the accuracy (ACC) were 70.2% and 79.8%, respectively (Table 9).

[0108] Positive Predictive Value (PPV) and Negative Predictive Value (NPV) are important parameters used to evaluate the effectiveness of medical diagnostic tests, providing an assessment of the accuracy of test results relative to the actual disease status. A high NPV shows that the test is very reliable in excluding non-good responders, while a relatively high PPV indicates that it has actual clinical value in predicting good responders. This means that although the proportion of good responders in the overall population is low, the test method can still effectively improve the accuracy of positive results. Overall, this test method has potential for application in different populations, and its clinical efficacy can be further improved through further optimization or combination with other tests.

[0109] The data in Table 8 show the distribution of "good responders" and "non-good responders" in this study. Specifically, good responders comprised 36.8% (39 / 106) of the total sample, while non-good responders comprised 63.2% (67 / 106). Using anti-HUWE1 and anti-SNRK antibodies as predictors of tofacitinib response yielded PPVs of 55.6% and 67.6%, respectively, and NPVs of 79.3% and 87.7%, respectively (Table 8), demonstrating the relative usefulness of this assay in clinical decision-making. Although the proportion of good responders in the overall population is relatively low, this assay significantly increases the probability of correctly identifying good responders among positive results, a significant advantage for clinical decision-making. In other words, this assay provides valuable information in predicting who will benefit from the drug's efficacy, helping physicians more accurately identify patients who should receive treatment.

[0110] Example 5 Anti-HUWE1 Antibodies and Anti-SNRK1 Antibodies for Evaluating the Therapeutic Response (Disease Remission) of JAK Inhibitor (Tofacitinib)

[0111] Table 10 shows the clinical characteristics of patients with rheumatoid arthritis who were evaluated for EULAR disease remission and non-remission after 24 weeks of Tofacitinib treatment. The data are expressed as mean ± standard deviation, number (percentage) or median (interquartile range).

[0112] Figure 6 shows the ROC curves for anti-HUWE1 antibodies and anti-SNRK antibodies as a means of determining whether the disease has achieved remission after Tofacitinib treatment. The ROC curves were used to evaluate the performance of anti-HUWE1 antibodies and anti-SNRK1 antibodies at different sensitivity and specificity thresholds, and the overall efficacy was evaluated using the area under the curve (AUC) as a benchmark. The results showed that the optimal cutoff points for anti-HUWE1 antibodies and anti-SNRK antibodies were 0.472 and 0.452, respectively, the AUCs were 0.682 and 0.728, respectively, the sensitivities (SEN) were 52.9% and 64.7%, the specificities (SPE) were 80.8% and 79.5%, the accuracy (ACC) were 76.6.2% and 70.2%, the positive predictive values ​​(PPV) were 38.1% and 35.1%, and the negative predictive values ​​(NPV) were 87.7% and 93%, respectively (Table 11).

[0113] These results indicate that anti-HUWE1 and anti-SNRK antibodies can be used as indicators to assess the response of rheumatoid arthritis patients to treatment with JAK inhibitors (Tofacitinib). When the expression of anti-HUWE1 or anti-SNRK antibodies reaches the first reference threshold (as shown in Table 9), it can be used to determine whether the patient has responded well to the drug. When the expression of anti-HUWE1 or anti-SNRK antibodies reaches the second reference threshold (as shown in Table 11), it can be further determined whether the patient has achieved disease remission. Therefore, anti-HUWE1 or anti-SNRK antibodies are important biomarkers for the efficacy of JAK inhibitor treatment in patients with rheumatoid arthritis.

[0114] JAK inhibitors currently used to treat rheumatoid arthritis include Tofacitinib, Baricitinib, Peficitinib, Upadacitinib, and Filgotinib. These drugs inhibit the JAK-STAT signaling pathway, reducing immune responses and thereby effectively alleviating symptoms and improving quality of life for patients with rheumatoid arthritis. JAK inhibitors differ in their selectivity for inhibiting different JAK subtypes, resulting in varying efficacy and side effect profiles in clinical practice.

[0115] Tofacitinib is a pan-JAK inhibitor that targets multiple Janus kinase enzymes rather than selectively targeting just one. Specifically, Tofacitinib inhibits JAK1, JAK2, and JAK3, with a strong preference for JAK1 and JAK3. This broad inhibitory effect enables Tofacitinib to effectively reduce the signaling of multiple cytokines involved in the inflammatory process of RA. By blocking these pathways, Tofacitinib can reduce inflammation, alleviate joint damage, and relieve symptoms in RA patients, making it a versatile option for treating the disease.

[0116] Therefore, the present disclosure provides a method for evaluating whether the biomarkers disclosed in the present invention can be used as therapeutic response indicators for JAK inhibitor therapy. This method significantly improves their accuracy, and the content of the present disclosure has potential application value in relevant markets.

[0117] While embodiments of the present disclosure have been broadly described, it is important to note that viable alternative embodiments are possible. Accordingly, the intent and scope of the application appended hereto should not be limited to the depiction of the embodiments provided.

[0118] It will be understood by those skilled in the art that the structure of the present disclosure may be modified and altered while maintaining the scope and essence of the disclosure. Therefore, the present disclosure is intended to include adaptations and alterations to this document as long as they fall within the scope defined by subsequent applications.

Claims

1. A method for evaluating the response of a patient with rheumatoid arthritis to treatment with a Janus kinase inhibitor, comprising: (a) providing a biological sample, wherein the biological sample is from a patient with rheumatoid arthritis; (b) performing a detection method on the biological sample, wherein the detection method is used to determine the basal expression level of the biomarker; wherein the biomarker is an anti-SNRK antibody or an anti-HUWE1 antibody; The basal expression level is the expression level of the rheumatoid arthritis patient before receiving the JAK inhibitor treatment; (c) comparing the basal expression level of the biomarker with a first reference expression level and a second reference expression level of the biomarker; The first reference expression level is determined according to a first cutoff point of the biomarker expression level of patients with good response and poor response after treatment; The second reference expression level is determined according to a second cutoff point of the biomarker expression level between disease remission and non-disease remission of the patient after treatment; When the baseline expression amount of the biomarker is higher than the first reference expression amount of the biomarker, it is determined that the rheumatoid arthritis patient has a possibility of having a good response to the Janus kinase inhibitor treatment; When the baseline expression level of the biomarker is higher than the second reference expression level of the biomarker, it is determined that the rheumatoid arthritis patient is likely to achieve disease remission by the Janus kinase inhibitor treatment; and (d) When the rheumatoid arthritis patient is likely to respond well to Janus kinase inhibitor therapy If the patient is likely to achieve disease remission through treatment, a therapeutically effective dose of the JAK inhibitor is administered to the patient. The method of claim 1 , wherein the biological sample is a blood sample.

3. The method of claim 1, wherein the Janus kinase inhibitor is selected from a JAK1 inhibitor, a JAK2 inhibitor, a JAK3 inhibitor, a TYK2 inhibitor, or a combination thereof.

4. The method of claim 1, wherein the Janus kinase inhibitor is selected from Tofacitinib, Baricitinib, Peficitinib, Upadacitinib and Filgotinib.

5. The method of claim 1, wherein the anti-SNRK antibody can recognize a sequence of 20-50 consecutive amino acids in SEQ ID NO: 1; wherein the anti-HUWE1 antibody can recognize a sequence of 20-50 consecutive amino acids in SEQ ID NO:

4.

6. A reagent kit for evaluating the therapeutic response of patients with rheumatoid arthritis to a Janus kinase inhibitor, the reagent kit comprising: Target peptides that can identify biomarkers; a solid support matrix bound to the target peptide; and a probe-labeled or unlabeled secondary antibody capable of binding to the biomarker. 7 . The reagent set as claimed in claim 6 , wherein the target peptide capable of identifying a biomarker is a target peptide capable of identifying an anti-SNRK antibody or a target peptide capable of identifying an anti-HUWE1 antibody.

8. The reagent set as claimed in claim 7, wherein the biomarker target peptide that can identify anti-SNRK antibody is a continuous 20 to 50 amino acid sequence comprising SEQ ID NO:1; wherein the biomarker target peptide that can identify anti-HUWE1 antibody is a continuous 20-50 amino acid sequence comprising SEQ ID NO:

4. 9 . The reagent set as claimed in claim 6 , wherein the Janus kinase inhibitor is selected from a JAK1 inhibitor, a JAK2 inhibitor, a JAK3 inhibitor, a TYK2 inhibitor or a combination thereof.

10. The reagent set as claimed in claim 6, wherein the Janus kinase inhibitor is selected from Tofacitinib, Baricitinib, Peficitinib, Upadacitinib and Filgotinib.

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