Use of triiodothyronine autoantibodies as markers of thyroid dysfunction during cancer therapy

Abstract

The present disclosure relates to the field of clinical detection technology, in particular, to the application of triiodothyronine autoantibody as a marker of thyroid dysfunction during cancer treatment. The present disclosure can help clinicians to judge the real thyroid function status of patients and improve the accuracy of diagnosis.

Description

Technical Field

[0001] This disclosure generally relates to the field of clinical testing technology, and more specifically, to the application of triiodothyronine autoantibodies as markers of thyroid dysfunction in the course of cancer treatment. Background Technology

[0002] The thyroid gland is an important organ of the endocrine system. Thyroid dysfunction can affect the normal functioning of the endocrine system, and thyroid hormones play an important regulatory role in cardiovascular and metabolic processes. In recent years, with changes in people's lifestyles and dietary habits, related studies have shown that the incidence of thyroid dysfunction is increasing year by year. When patients experience thyroid dysfunction, it can have adverse effects on their physical condition and quality of life.

[0003] Clinically, discrepancies often arise between thyroid function test results and clinical symptoms during cancer diagnosis and treatment, or irregularities in TSH, free triiodothyronine (FT3), and free thyroxine (FT4) levels. These discrepancies significantly impair physician judgment and consequently negatively impact patient treatment. Eliminating these interfering factors would allow clinicians to more accurately determine a patient's true thyroid function level. Summary of the Invention

[0004] This disclosure covers the following technical solutions:

[0005] The purpose of this disclosure is to provide a detection reagent that specifically binds to triiodothyronine autoantibodies (T3-Ab) for use in the preparation of a kit for diagnosing thyroid dysfunction during cancer treatment.

[0006] Another object of this disclosure is to provide a kit for diagnosing thyroid dysfunction during cancer treatment, which is the kit defined in the application described above.

[0007] The inventors of this disclosure unexpectedly discovered thyroid dysfunction as a side effect during tumor treatment in clinical practice and hypothesized it to be related to T3-Ab. Specifically, in certain preferred formulations, it also showed a strong correlation with thyroid dysfunction during immune checkpoint inhibitor therapy for tumors. Therefore, this disclosure can help clinicians determine a patient's true thyroid function status and improve diagnostic accuracy. Attached Figure Description

[0008] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0009] Figure 1 A linear function fitted to the T3-Ab kit;

[0010] Figure 2 A linear function fitted to the T4-Ab kit;

[0011] Figure 3 Distribution of T3-Ab levels in patients undergoing ICI tumor treatment;

[0012] Figure 4 The curve is for detection using T3-Ab as the biomarker alone;

[0013] Figure 5 Curves for detection using T3-Ab and T4-Ab as joint markers;

[0014] Figure 6 The curves for detection using T3-Ab, FT3, FT4, and TSH as combined markers;

[0015] Figure 7 The curve uses five indicators—T3-Ab, T4-Ab, FT3, FT4, and TSH—as a joint indicator.

[0016] Figure 8 Curves using FT3, FT4, and TSH as joint markers;

[0017] Figure 9 A graph showing the trend of T3-Ab levels during ICI tumor treatment. Detailed Implementation

[0018] Reference will now be made to embodiments of this disclosure in detail, with one or more examples described below. Each example is provided for explanation and not for limitation of this disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to this disclosure without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0019] Unless otherwise stated, all terms used in the disclosure of this disclosure (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Further guidance is provided below for a better understanding of the teachings of this disclosure. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this disclosure.

[0020] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0021] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that, in this disclosure, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0022] The terms “containing,” “including,” and “comprise” as used in this disclosure are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0023] The range of values ​​represented by endpoints in this disclosure includes all values ​​and fractions contained within that range, as well as the endpoints referenced.

[0024] When this document uses the term "about" to refer to a value or parameter, it includes (and describes) an implementation of the value or parameter itself. For example, a description of "about X" includes a description of "X".

[0025] The concentration values ​​mentioned in this disclosure include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may allow fluctuations within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are allowed. For example, 100mM may allow fluctuations within the ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations within ±10% are allowed.

[0026] As used herein, unless otherwise indicated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0027] In this disclosure, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0028] In this disclosure, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0029] In this disclosure, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of implementation methods or embodiments with better effects and should be understood as not constituting a limitation on the scope of protection of this disclosure. In this disclosure, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" parallel solutions. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradictions or mutual constraints, each "optional" statement is independent. In this disclosure, terms such as "further," "even further," and "particularly" are used for descriptive purposes to indicate differences in content, but should not be construed as a limitation on the scope of protection of this disclosure.

[0030] As used in this article, the term "diagnosis" is used in its broadest sense and includes the diagnosis, prognosis, treatment, and monitoring of thyroid dysfunction during cancer treatment. Diagnosis may be accompanied by an assessment of the severity of the thyroid dysfunction.

[0031] As used herein, the term “treatment” and its variations or “improvement” refers to therapeutic treatment in which the aim is to reverse, reduce, improve, suppress, slow, or stop the progression or severity of a disease (e.g., cancer), its associated symptoms, and / or signs. The term “treatment” includes reducing or alleviating at least one adverse effect or symptom of a disease (e.g., cancer).

[0032] In this article, the terms "second" and "third" in "second detection agent" and "third detection agent" are used for descriptive purposes only, to distinguish similar (or related) things or features. They should not be interpreted as indicating or implying relative importance or quantity, nor should they be interpreted as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "second" and "third" serve only as a non-exhaustive enumeration and should be understood as not constituting a closed limitation on quantity.

[0033] As used in this article, “autoantibody” refers to an antibody produced by the immune system of an individual that specifically binds to its “autoantigen,” “autologous antigen,” or “antigenic epitope.”

[0034] As used in this article, the term "specific binding" refers to a higher affinity of a binding molecule for a target molecule compared to its affinity for non-target molecules. A binding molecule that specifically binds to a target molecule essentially does not recognize or bind to non-target molecules; for example, an antibody "specifically binds" and / or "specifically recognizes" another molecule, meaning that this interaction depends on the presence of binding specificity of the molecular structure, such as an antigenic epitope.

[0035] All documents mentioned in this disclosure are cited herein as if they were cited individually. Unless they conflict with the inventive purpose and / or technical solution of this disclosure, all cited documents are cited in their entirety and for all purposes. When citing documents in this disclosure, the definitions of relevant technical features, terms, nouns, phrases, etc., are also cited. When citing documents in this disclosure, examples and preferred embodiments of the relevant technical features may also be incorporated into this disclosure as reference, but only to the extent that they enable the implementation of this disclosure. It should be understood that when the cited content conflicts with the description in this disclosure, this disclosure shall prevail or modifications shall be made adaptively based on the description in this disclosure.

[0036] This disclosure relates to the use of a detection reagent that specifically binds to triiodothyronine autoantibodies in the preparation of a kit for diagnosing thyroid dysfunction during cancer treatment.

[0037] This disclosure is the first to discover that triiodothyronine autoantibodies can serve as a marker of thyroid dysfunction during cancer treatment, hence the above application is proposed.

[0038] In this disclosure, unless otherwise stated, the terms "marker," "biochemical marker," or "thyroid dysfunction marker" are understood by those skilled in the art to refer to "triiodothyronine autoantibody" or a specifically detectable fragment thereof. Their meanings may be used interchangeably depending on the context.

[0039] This biomarker can be used to diagnose thyroid dysfunction during cancer treatment. Such diagnostic tests or methods often provide information that is crucial for the safe and effective use of the corresponding drugs or biologics, helping healthcare professionals determine whether the benefits of a particular cancer treatment product for a patient will outweigh any potential serious side effects or risks.

[0040] The kits provided in this disclosure are for testing biological samples. The terms “biological sample,” “sample,” etc., are used herein in their broadest sense and are generally obtained for use by a subject. The subject is an animal, preferably including at least mammals, such as primates, including humans. Biological samples are typically obtained from a patient. A sample can be any biological tissue or fluid capable of measuring the biomarkers of this disclosure, and typically the sample contains triiodothyronine autoantibodies. Such samples include, but are not limited to, samples that may or may not contain cells; preferred biological samples are blood, serum, or plasma. Such samples also include biopsies (e.g., thyroid biopsies). The term biological sample also includes any material derived from the processing of the biological sample. Derived materials include, but are not limited to, cells (or their progeny) isolated from the sample or proteins extracted from the sample. The processing of a biological sample may involve one or more of the following: filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, etc. For example, the biological sample is mixed with at least one of the following components: a diluent, preservative, stabilizer, buffer, etc., to form a test sample.

[0041] In some implementations, the subjects undergoing cancer treatment have received or are receiving immune checkpoint inhibitor (ICI) therapy. ICIs activate the body's immune system by intervening in immune checkpoints, enhancing the body's anti-tumor capabilities. However, an overactivated immune system may attack non-tumor tissues, resulting in immune-related adverse events (irAEs). While irAEs often indicate effective ICI treatment, uncontrolled damage progression can lead to ICI treatment interruption. Studies show that thyroid immune damage, including thyrotoxicosis and hypothyroidism, is the most frequent among endocrine system AEs associated with ICI treatment, such as anti-PD-1 therapy. However, because the mechanisms by which ICI treatment induces thyroid damage are currently unclear, clinical practice currently only provides hormone replacement therapy rather than etiological treatment for thyroid irAEs. The inventors unexpectedly discovered in clinical practice that thyroid dysfunction side effects during tumor ICI treatment are related to T3-Ab, thus the value of T3-Ab in tumor ICI treatment has profound implications for guiding clinical treatment.

[0042] As used herein, the term "checkpoint inhibitor" refers to an agent that inhibits immune checkpoint proteins or pathways to stimulate or promote the body's antitumor response. Preferred checkpoint inhibitors of this disclosure include inhibitors targeting one or more of the following targets: CTLA-4, PD-1, PD-L1, B7-H3, IDO, KIR, LAG3, TIM-3, TIGIT, VISTA, CD47, GITR, OX40, ADAR1, A2AR, SIRPα, and TLR. Immune checkpoint inhibitors can be commercially available drugs, and are usually antibodies. Therefore, ipilimumab targeting CTLA-4 is preferred; at least one of nivolumab, pembrolizumab, cemiplimab, toripalimab, sintilimab, camrelizumab, tislelizumab, zimberelimab, prolgolimab, and dostarlimab targeting PD-1; at least one of atezolizumab, durvalumab, and avelumab targeting PD-L1; and relatlimab targeting LAG-3.

[0043] In some embodiments, the triiodothyronine autoantibody is selected from IgG and / or IgM antibodies. IgG as referred to in this disclosure includes its various subtypes; for example, for human-derived samples, IgG includes IgG1, IgG2, IgG3, and IgG4. IgM as referred to in this disclosure includes its various subtypes; for example, for human-derived samples, IgM includes IgM1 and IgM2.

[0044] In some embodiments, the cancer arises from any one or more of the following: bone, bone joints, muscles, lungs, trachea, heart, spleen, arteries, veins, blood, capillaries, lymph nodes, lymphatic vessels, lymph fluid, oral cavity, pharynx, esophagus, cardia, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urogenital tract, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testis, vas deferens, penis, eye, ear, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerves, thyroid gland, parathyroid gland, adrenal gland, pituitary gland, pineal gland, islets of Langerhans, thymus, gonads, sublingual gland, and parotid gland. In some embodiments, the cancer is any one or more of the following: lung cancer, stomach cancer, esophageal cancer, liver cancer, colon cancer, gallbladder cancer, and cardia cancer.

[0045] Any method for determining the biomarkers of this disclosure, such as any method that specifically measures triiodothyronine autoantibodies, is applicable. Immunoassays are preferred.

[0046] As an immunoassay, there are no particular limitations, and various enzyme immunoassays, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), indirect immunofluorescence assays (IIF), double monoclonal antibody sandwich immunoassays, polyclonal antibody sandwich immunoassays, immunostaining methods, immunofluorescence methods, Western blotting, biotin-avidin methods, immunoprecipitation methods, colloidal gold agglutination methods, immunochromatography, latex agglutination (LA), immunoturbidimetric assays (TIA), chemiluminescent immunoassays, immunocolloidal gold assays, and immunospot assays can be cited as examples.

[0047] The general characteristics of immunoassays (e.g., ELISA, radioimmunoassay, etc.) are well known to those skilled in the art (see Immunoassay, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996, the contents of which are incorporated herein by reference). Immunoassays for detecting autoantibodies with specific immunological specificity typically require the use of a reagent (antigen) that exhibits a specific immunological reactivity to the relevant autoantibody. Depending on the form of the assay, the antigen may be immobilized on a solid support. The test sample is brought into contact with the antigen, and if an autoantibody with the desired immunological specificity is present in the sample, the autoantibody will react immunologically with the antigen to form an antigen / autoantibody complex, which can then be detected or quantified. Immunoassays for detecting autoantibodies according to this disclosure may be based on standard techniques known in the art. The detection of autoantibodies can be performed in any suitable form, preferably one that allows contact between the sample suspected of containing autoantibodies and the antigen. Examples of the use of antigens to detect autoantibodies are given in the embodiments herein.

[0048] In some preferred embodiments, the immunoassay is based on chemiluminescence.

[0049] In some embodiments, the detection reagent contains triiodothyronine antigen.

[0050] In some embodiments, the triiodothyronine antigen is conjugated to a solid support. Exemplary solid supports include, but are not limited to, filters, membranes, beads (e.g., magnetically or fluorophoretically labeled beads), plates, silicon wafers, glass, metals, plastics, chips, mass spectrometry targets, or matrices. In some embodiments, the solid support is a bead. In some embodiments, the bead is a microsphere. In this disclosure, the term "microsphere" can refer to a sphere, near-sphere, cube, polyhedron, or irregular shape. The diameter of the microsphere is preferably 10 nm to 1 mm, for example, 100 nm, 500 nm, 1 μm, 10 μm, 100 μm, 500 μm; preferably 400 nm to 10 μm.

[0051] In some embodiments, the kit also includes an antibody capable of specifically binding to the Fc fragment of a triiodothyronine autoantibody.

[0052] In some embodiments, the antibody capable of specifically binding to the Fc fragment of a triiodothyronine autoantibody is labeled with a signaling substance.

[0053] Detectable signaling substances known in the art for antibody labeling can be used in this disclosure. Exemplary signaling substances include any one of the following: fluorescent substances, quantum dots, digoxigenin-labeled probes, biotin, radioisotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense materials, chemiluminescent labels, ultrasound contrast agents, photosensitizers, colloidal gold, or enzymes. In some preferred embodiments, the signaling substance is a chemiluminescent reagent. In some preferred embodiments, the signaling substance is at least one of luminol and its derivatives, isoluminol and its derivatives, luciferin, roximate, peroxyoxalate esters, and acridine esters. In some specific embodiments, the signaling substance is ABEI.

[0054] In some embodiments, the kit further comprises one or more of the following: triiodothyronine autoantibody standard, reaction buffer, water, and chemiluminescent substrate solution.

[0055] In some preferred embodiments, the detection reagent further includes a second detection reagent for tetraiodothyronine autoantibodies.

[0056] Compared to T3-Ab detection alone, using T3-Ab and T4-Ab as combined biomarkers to detect thyroid dysfunction during cancer treatment, such as ICI therapy, can significantly improve the sensitivity and specificity of the detection. Exemplary detection results are illustrated in this disclosure. Figure 5 According to the description, in this embodiment, the sensitivity is 86.9% and the specificity is 71.9%.

[0057] In some embodiments, the tetraiodothyronine autoantibody is selected from IgG and / or IgM antibodies.

[0058] In some preferred embodiments, the detection reagent further includes a third detection reagent for quantitatively determining at least one of FT3, FT4, and TSH. That is, for example, the detection reagent detects at least one of triiodothyronine autoantibody, FT3, FT4, and TSH; or the detection reagent detects at least one of triiodothyronine autoantibody, tetraiodothyronine autoantibody, FT3, FT4, and TSH.

[0059] Compared to T3-Ab detection alone, or combined detection of T3-Ab and T4-Ab, or FT3, FT4, and TSH as combined markers, adding more detection markers can further improve the detection effect. Exemplary detection effects are described in this disclosure. Figure 6 , Figure 7 The above description is provided. In these embodiments, the sensitivity was 88.9% and the specificity was 72.1% when using T3-Ab, T4-Ab, FT3, FT4 and TSH as a combined marker; the sensitivity was 94.4% and the specificity was 82.7% when using T3-Ab, T4-Ab, FT3, FT4 and TSH as a combined marker.

[0060] According to another aspect of this disclosure, a kit for diagnosing thyroid dysfunction during cancer treatment is also involved, which is the kit defined in the application described above.

[0061] In this disclosure, the term "kit" may refer to any article (e.g., packaging or container) that includes at least one device and comprises the detection reagents as described in this disclosure. In some embodiments, the kit may further include instructions for use in the methods or steps described in this disclosure, and preferably may also include supplementary reagents and / or components or assemblies.

[0062] The components of the kit can be packaged individually or in combination as needed. The components can be packaged in solution, solid, or test strip form. In some embodiments, at least one component of the kit is a solid, including at least one of lyophilized microspheres, lyophilized cakes, lyophilized powder, and spots dependent on a solid medium. Therefore, components required for autoantibody detection, particularly various enzymes, antibody components, and reaction buffer components, can be provided in lyophilized form.

[0063] According to another aspect of this disclosure, a method for diagnosing thyroid dysfunction during cancer treatment is also provided, comprising measuring the level of triiodothyronine autoantibodies in a patient sample from the course of cancer treatment and comparing the measured values ​​with reference values.

[0064] In the context of this disclosure, the term "reference value" refers to a test result from a control sample. When used to characterize a subject, it refers to a patient who has not experienced thyroid dysfunction during cancer treatment, such as ICI treatment. The term "control sample" refers to one or more samples obtained from a patient who has not experienced thyroid dysfunction during cancer treatment, such as ICI treatment. In some embodiments, patients who have not experienced thyroid dysfunction during cancer treatment, such as ICI treatment, have similar parameters, such as sex, age, and / or body mass index, to subjects who obtained the biological sample to be tested. Exemplary reference values ​​may be determined according to the cut-off values ​​in Embodiment 3 of this disclosure.

[0065] In some implementations, an immunoassay (preferably chemiluminescence) is used to determine the level of triiodothyronine autoantibodies in patient samples from the course of cancer treatment.

[0066] In some typical implementations, the method includes:

[0067] 1) Contact patient samples from the course of cancer treatment with a detection reagent that specifically binds to triiodothyronine autoantibodies, under conditions that allow for the formation of a complex;

[0068] 2) Add a substance with a detectable marker that can specifically recognize the complex formed in step 1);

[0069] 3) Quantify the detectable labeled signal to obtain the amount of triiodothyronine autoantibody.

[0070] As is well known to those skilled in the art, the ideal scenario for diagnosis is one where a single event or process causes multiple diseases, for example, in infectious diseases. In all other cases, a correct diagnosis can be very difficult, especially when the etiology of the disease is not fully understood. As a skilled technician will understand, for a given multifactorial disease, a diagnosis without biochemical markers is 100% specific and 100% sensitive. Determining whether a subject's sample, compared to the said normal control sample, has thyroid dysfunction during cancer treatment can be performed using statistical methods known in the art and confirmed using confidence intervals and / or p-values. In some embodiments, confidence intervals are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99%, and p-values ​​are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001. Furthermore, additional biochemical markers can be used to assess the presence or severity of disease to improve the accuracy of the assessment; in fact, in routine clinical diagnosis, various clinical symptoms and biomarkers are often considered in combination to diagnose, treat, and control underlying diseases. As an example, the method further includes measuring the level of tetraiodothyronine autoantibodies in a patient sample from the course of cancer treatment and comparing the measured value with a reference value. In some embodiments, the method further includes measuring the level of at least one of FT3, FT4, and TSH in a patient sample from the course of cancer treatment and comparing the measured value with a reference value. In some embodiments, the method further includes measuring the level of at least one of tetraiodothyronine autoantibodies, FT3, FT4, and TSH in a patient sample from the course of cancer treatment and comparing the measured value with a reference value. The aforementioned additional biochemical markers can improve the accuracy of this method.

[0071] The embodiments of this disclosure will now be described in detail with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this disclosure. For experimental methods in the following embodiments where specific conditions are not specified, reference should be made to the guidelines given in this disclosure, or to experimental manuals or conventional conditions in the art, or to other experimental methods known in the art, or to the conditions recommended by the manufacturer.

[0072] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0073] Example 1: Preparation of T3-Ab Detector

[0074] I. Preparation of a chemiluminescence indirect detection kit for T3-Ab

[0075] 1. Preparation of T3-Ab calibrators

[0076] (1) Low point calibration (0.4 AU / mL): Using PBS buffer, the T3-Ab stock solution (0.4 mg / mL) was diluted to the required concentration at a ratio of 1:68608.

[0077] (2) High-point calibration (25.6 AU / mL): Using PBS buffer, the T3-Ab stock solution (0.4 mg / mL) was diluted to the required concentration at a ratio of 1:1072.

[0078] 2. Preparation of T3-Ab quality control products

[0079] Quality control (2.2 AU / mL): Using PBS buffer, the T3-Ab stock solution (0.4 mg / mL) was diluted to the required concentration at a ratio of 1:12473.

[0080] 3. Reaction buffer

[0081] Use PBS diluent, formulation: 100 mmol / L phosphate buffer solution containing preservatives.

[0082] 4. T3-coated magnetic spheres

[0083] The antigens that specifically recognize T3-Ab were coated with magnetic beads. The ratio of magnetic beads to antigen was 1 mg: 2 μg, and the concentration of magnetic beads in suspension was 20 mg / mL.

[0084] 5. ABEI-labeled secondary antibodies

[0085] The anti-human secondary antibody hIgG, which identifies T3-Ab, was added in a ratio of 1 mg: 200 μg.

[0086] 6. Chemiluminescent substrate solution

[0087] Sodium hydroxide (NaOH) reacts with hydrogen peroxide (H₂O₂).

[0088] 7. Sample addition process

[0089] The assay was performed using a MAGLUMI X8 chemiluminescence immunoassay analyzer from Shenzhen New Industries Biotechnology Co., Ltd. The instrument loading parameters were as follows: 10 μL sample + 20 μL T3-coated magnetic beads + 150 μL reaction buffer, incubated for 20 min, washed 3 times + 200 μL ABEI-labeled secondary antibody, incubated for 10 min, washed 3 times. Finally, the chemiluminescence substrate solution for the automated immunoassay system was added to initiate the chemiluminescence reaction and generate a light signal. The relative light intensity (RLU) measured by a photomultiplier tube was proportional to the T3-Ab concentration in the sample (or calibrator / control sample).

[0090] II. Performance Validation of T3-Ab Reagent Kit

[0091] 1. Blank limit

[0092] The LOB (Limit of Detection) of the kit prepared in Example 1 is declared to be 0.08 AU / mL. Following YY / T1789.3-2022 "Performance Evaluation Methods for In Vitro Diagnostic Testing Systems Part 3: Limit of Detection and Limit of Quantification", the kit prepared in Example 1 and a MAGLUMI X8 chemiluminescence immunoassay analyzer were used. Three test days were selected, along with two clinical blank samples, and each sample was measured four times repeatedly. The percentage of blank sample test results less than or equal to the declared LOB was calculated. If the percentage was greater than 87%, the declared LOB was considered to be compliant.

[0093] 2. Detection limit

[0094] The LOD (Limit of Detection) of the kit prepared in Example 1 is declared to be 0.12 AU / mL. Following YY / T1789.3-2022 "Performance Evaluation Methods for In Vitro Diagnostic Testing Systems Part 3: Limit of Detection and Limit of Quantification", the kit prepared in Example 1 and a MAGLUMI X8 chemiluminescence immunoassay analyzer were used. Three testing days were selected, two samples were taken at the clinical limit of detection concentration, and four replicate measurements were performed for each sample. The percentage of results in the limit of detection concentration samples that were greater than or equal to the declared LOD was calculated. If the percentage was greater than 87%, the declared LOD was considered to be met.

[0095] Evaluation results:

[0096] Table 1. Results of Blank Limit and Detection Limit

[0097]

[0098] The LOB assessment percentage was 95.83% according to the formula (number of blank samples less than or equal to 0.08 / total number of blank samples) * 100%; the LOD assessment percentage was 100% according to the formula (number of low-concentration samples greater than or equal to 0.08 / total number of low-concentration samples) * 100%, both of which meet the requirements.

[0099] 3. Linear

[0100] The T3-Ab detection kit prepared in this embodiment has a linear interval of [0.16, 10.0]. Following YY / T1789.4-2022 "Performance Rating Method for In Vitro Diagnostic Testing Systems Part 4: Linear Interval and Reportable Interval", different proportion coefficients were selected using high and low value samples. Low-value samples (L) were designated as S1, and high-value samples as S6. The dilution of each concentration sample was calculated based on the dilution ratio. On the same MAGLUMI X8 instrument within one day, calibration and internal quality control tests were performed according to the sample addition process in Example 1. After passing quality control, samples S1-S6 were tested again, with each sample measured four times. Data were recorded and results were statistically analyzed. The data were analyzed using a "linear regression combined with offset method". If r > 0.99, the established linear interval can be preliminarily determined to be acceptable. The specific preparation method is shown in the table below:

[0101] Table 2. Sample preparation methods and theoretical concentrations at each point for linearity testing.

[0102]

[0103] Record the test results for each sample, calculate the mean of each sample test, and use the theoretical concentration of each sample as the x-axis and the test mean of each sample as the y-axis to fit a linear function y = bx + a and a correlation coefficient r, where b is the slope and a is the intercept. Figure 1 As shown, the T3-Ab kit prepared in this method has a linear range of 0.17–9.8 AU / mL and a correlation coefficient r of 0.9963, which meets the linearity requirements.

[0104] III. Preparation of Chemiluminescence Indirect Method T4-Ab Detection Kit

[0105] 1. Preparation of T4-Ab calibrators

[0106] (1) Low point calibration (0.4 AU / mL): Using PBS buffer, the T4-Ab stock solution (0.4 mg / mL) was diluted to the required concentration at a ratio of 1:10496.

[0107] (2) High-point calibration (25.6 AU / mL): Using PBS buffer, the T4-Ab stock solution (0.4 mg / mL) was diluted to the required concentration at a ratio of 1:164.

[0108] 2. Preparation of T4-Ab quality control materials

[0109] Quality control (2.2 AU / mL): Using PBS buffer, the T4-Ab stock solution (0.4 mg / mL) was diluted to the required concentration at a ratio of 1:1908.

[0110] 3. Reaction buffer

[0111] 100 mmol / L phosphate buffer solution, containing preservatives.

[0112] 4. T4-coated magnetic spheres

[0113] The antigens that specifically recognize T4-Ab are coated with magnetic beads. The ratio of magnetic beads to antigen is 1 mg: 5 μg, and the magnetic bead suspension concentration is 20 mg / mL.

[0114] 5. ABEI-labeled secondary antibodies

[0115] The anti-human secondary antibody hIgG, which identifies T4-Ab, was added in a ratio of 1 mg: 200 μg.

[0116] 6. Chemiluminescent substrate solution

[0117] Sodium hydroxide (NaOH) reacts with hydrogen peroxide (H₂O₂).

[0118] 7. Sample addition process

[0119] The assay was performed using a MAGLUMI X8 chemiluminescence immunoassay analyzer from Shenzhen New Industries Biotechnology Co., Ltd. The instrument loading parameters were as follows: 10 μL sample + 20 μL T4-coated magnetic beads + 150 μL reaction buffer, incubated for 20 min, washed 3 times + 200 μL ABEI-labeled secondary antibody, incubated for 10 min, washed 3 times. Finally, the chemiluminescence substrate solution for the automated immunoassay system was added to initiate the chemiluminescence reaction and generate a light signal. The relative light intensity (RLU) measured by a photomultiplier tube was proportional to the T4-Ab concentration in the sample (or calibrator / control sample).

[0120] IV. Performance Validation of T4-Ab Reagent Kit

[0121] 1. Blank limit

[0122] The LOB (Limit of Detection) of the kit prepared in Example 1 is declared to be 0.08 AU / mL. Following YY / T1789.3-2022 "Performance Evaluation Methods for In Vitro Diagnostic Testing Systems Part 3: Limit of Detection and Limit of Quantification", the kit prepared in Example 1 and a MAGLUMI X8 chemiluminescence immunoassay analyzer were used. Three test days were selected, along with two clinical blank samples, and each sample was measured four times repeatedly. The percentage of blank sample test results less than or equal to the declared LOB was calculated. If the percentage was greater than 87%, the declared LOB was considered to be compliant.

[0123] 2. Detection limit

[0124] The LOD (Limit of Detection) of the kit prepared in Example 1 is declared to be 0.12 AU / mL. Following YY / T1789.3-2022 "Performance Evaluation Methods for In Vitro Diagnostic Testing Systems Part 3: Limit of Detection and Limit of Quantification", the kit prepared in Example 1 and a MAGLUMI X8 chemiluminescence immunoassay analyzer were used. Three testing days were selected, two samples were taken at the clinical limit of detection concentration, and four replicate measurements were performed for each sample. The percentage of results in the limit of detection concentration samples that were greater than or equal to the declared LOD was calculated. If the percentage was greater than 87%, the declared LOD was considered to be met.

[0125] Evaluation results:

[0126] Table 3. Results of Blank Limit and Detection Limit

[0127]

[0128] The LOB assessment percentage was 91.67% according to the formula (number of blank samples less than or equal to 0.08 / total number of blank samples) * 100%; the LOD assessment percentage was 100% according to the formula (number of low-concentration samples greater than or equal to 0.08 / total number of low-concentration samples) * 100%, both of which meet the requirements.

[0129] The T4-Ab detection kit prepared in this embodiment has a linear interval of [0.16, 10.00]. Following YY / T1789.4-2022 "Performance Rating Method for In Vitro Diagnostic Testing Systems Part 4: Linear Interval and Reportable Interval", low-value samples L are designated as S1, and high-value samples as S6. Six samples were obtained by diluting according to the dilution ratio, covering the entire linear interval. Specific preparation methods are shown in Table 4. On the same MAGLUMI X8 instrument within one day, calibration and internal quality control tests were performed according to the sample addition process in Example 1 of the instruction manual. After passing quality control, samples S1-S6 were tested again, with each sample measured four times. Data were recorded and results were statistically analyzed. The data were analyzed using a "linear regression combined with offset method." If r > 0.99, it can be preliminarily determined that the established linear interval meets the requirements.

[0130] Table 4. Sample preparation methods and theoretical concentrations at each point for linearity testing.

[0131]

[0132] Record the test results for each sample, calculate the mean of each sample test, and plot the theoretical concentration of each sample on the x-axis and the test mean of each sample on the y-axis to obtain a linear function y = bx + a and a correlation coefficient r, where b is the slope and a is the intercept. The fitted linear function is as follows: Figure 2 As shown, r is 0.9959, r > 0.99, which means that the established linear interval meets the requirements.

[0133] Example 2: Discovery of inducing the body to produce T3-Ab during tumor treatment

[0134] 1. Study population, sample source, and experimental methods

[0135] Study population and sources:

[0136] The study included 62 cancer patients who received chemotherapy and 62 cancer patients who received ICI treatment; all of whom were from the Affiliated Hospital of Jiangsu University.

[0137] Sample type: serum

[0138] Experimental method: Collect 5.0 mL of venous blood into a blood collection tube and let it stand at room temperature. Centrifuge, separate the serum fraction, and store at 2℃~8℃. Use the kit from Example 1 and test the signal on a MAGLUMI X8 instrument.

[0139] Data analysis: Unpaired t-tests were performed on the data using Graphpad 7.0 software. p < 0.05 was considered statistically significant.

[0140] 2. Results Analysis

[0141] Tumor patients receiving chemotherapy alone were used as the control group, and tumor patients receiving immunotherapy (ICI) were used as the treatment group. Serum T3-Ab levels in both the control group (n=62) and the ICI treatment group (n=62) were measured using chemiluminescence immunoassay to assess whether PD-1 blockade immunotherapy affects T3-Ab expression in the body. Figure 3 As shown, compared with the control group, the serum T3-Ab level in tumor patients after receiving PD-1 blockade therapy was significantly higher than that in the control group (p<0.0001). The results indicate that ICI treatment can induce the body to produce T3-Ab, which may be related to the thyroid side effects of irAE.

[0142] Example 3: T3-Ab demonstrates excellent diagnostic performance in diagnosing thyroid adverse reactions associated with ICI treatment.

[0143] 1. Study population and sources:

[0144] The study included 320 cancer patients with thyroid dysfunction after ICI treatment and 100 cancer patients with no thyroid dysfunction after ICI treatment, all from the Affiliated Hospital of Jiangsu University.

[0145] Sample type: serum

[0146] Experimental method: Collect 5.0 mL of venous blood into a blood collection tube and let it stand at room temperature. Centrifuge, separate the serum fraction, and store at 2℃~8℃. Use the kit from Example 1 and test the signal on a MAGLUMI X8 instrument.

[0147] Data Analysis: SPSS software was used to plot ROC curves and perform data analysis.

[0148] 2. Results Analysis

[0149] By statistically analyzing the cut-off values ​​(the cutoff values ​​for positive and negative results) to distinguish whether thyroid dysfunction occurred after ICI treatment, the cut-off values ​​for T3-Ab in the experimental group were 1.315 AU / mL and for T4-Ab were 1.06 AU / mL; while the cut-off values ​​for FT3, FT4, and TSH in the control group were 6.395 pmol / L, 21.85 pmol / L, and 4.21 uIU / mL, respectively.

[0150] ROC curves were also plotted based on the test results, as follows:

[0151] 1) ROC curve of the experimental group:

[0152] ① The curve detected using T3-Ab as the marker alone ( Figure 4 (As shown).

[0153] ② The curve for detection using T3-Ab and T4-Ab as joint markers ( Figure 5 (As shown).

[0154] ③ The curve for joint detection using T3-Ab, FT3, FT4, and TSH as biomarkers ( Figure 6 (As shown).

[0155] ④ A curve using five indicators—T3-Ab, T4-Ab, FT3, FT4, and TSH—as a joint index. Figure 7 (As shown).

[0156] 2) Control group (routine laboratory indicators: FT3, FT4, and TSH) ROC curve:

[0157] Curves using FT3, FT4, and TSH as joint markers ( Figure 8 (As shown).

[0158] Based on the above graph, it can be seen that:

[0159] In the control group, the area under the curve for FT3, FT4 and TSH as combined markers was 0.7204, with a sensitivity of 86.9% and a specificity of 71.9%.

[0160] In the experimental group:

[0161] ① The area under the curve for T3-Ab alone as a marker was 0.6261, with a sensitivity of 85.6% and a specificity of 62.1%;

[0162] ② The area under the curve (AUC) for using T3-Ab and T4-Ab as combined markers was 0.8588, with a sensitivity of 87.1% and a specificity of 74.4%.

[0163] ③ The area under the curve for using T3-Ab, FT3, FT4 and TSH as combined markers was 0.8507, with a sensitivity of 88.9% and a specificity of 72.1%.

[0164] ④ The area under the curve for the combined use of five parameters—T3-Ab, T4-Ab, FT3, FT4, and TSH—was 0.9306, with a sensitivity of 94.4% and a specificity of 82.7%.

[0165] The area under the curve for T3-Ab alone was 0.6261 > 0.5, proving that T3-Ab can effectively diagnose thyroid dysfunction that occurs during ICI treatment.

[0166] Compared with conventional laboratory indicators (FT3, FT4, and TSH), the combined detection of T3-Ab and T4-Ab, the combined detection of T3-Ab and conventional laboratory indicators, and the combined detection of five indicators (T3-Ab, T4-Ab, and conventional laboratory indicators) showed significantly better sensitivity, specificity, and area under the curve than conventional laboratory indicators.

[0167] Example 4: Application of T3-Ab in a kit for auxiliary thyroid function testing during tumor treatment monitoring

[0168] I. Study population and sources:

[0169] Table 3 shows the stages of 7 typical patients before, after the first, second, and third ICI treatments; specific information on the 7 typical patients whose T3 Ab levels were measured is shown in Table 3.

[0170] Sample type: serum

[0171] Experimental method: Collect 5.0 mL of venous blood into a blood collection tube and let it stand at room temperature. Centrifuge, separate the serum fraction, and store at 2℃~8℃. Use the kit from Example 1 and test the signal on a MAGLUMI X8 instrument.

[0172] Data analysis: Graphpad 7.0 analysis software was used to perform statistical analysis on the data.

[0173] II. Results Analysis

[0174] To more comprehensively examine the dynamic changes in serum T3-Ab levels during ICI treatment, we used line graphs to analyze the T3-Ab trajectories of seven typical patients before, after the first, second, and third ICI treatment cycles. The results are shown in Table 5. Figure 8 During ICI treatment, patients showed elevated T3-Ab levels after the first course of treatment, and these levels continued to rise gradually as treatment progressed. This finding is consistent with the patients' thyroid function and clinical presentation. After ICI treatment, patients initially developed hyperthyroidism, which gradually progressed to hypothyroidism, at which point clinicians intervened with levothyroxine. However, during the progression of thyroid dysfunction, other thyroid autoantibodies (TG-Ab, TPO-Ab, TR-Ab) did not show significant changes, and the pathogenesis of irAE (irhysterothyroidism) thyroid side effects remains unclear. Therefore, we hypothesize that ICI-related thyroid dysfunction is closely related to T3-Ab levels. We recommend that clinicians test T3-Ab levels simultaneously with thyroid dysfunction screening to supplement diagnostic results and improve accuracy.

[0175] Table 5. Patient information of typical cases and T3-Ab levels during ICI tumor treatment.

[0176]

[0177] Example 5: T3-Ab demonstrates excellent performance in diagnosing thyroid dysfunction in various immunotherapy-treated cancer patients. Study population, sample source, and experimental methods.

[0178] I. Study population and source: Serum from 420 cancer patients who underwent immunotherapy, including 85 patients with gastric malignant tumors, 93 patients with esophageal malignant tumors, 78 patients with lung malignant tumors, 55 patients with liver malignant tumors, 36 patients with colon malignant tumors, 38 patients with gallbladder malignant tumors, and 35 patients with cardia malignant tumors. All patients were from the Affiliated Hospital of Jiangsu University.

[0179] Sample type: serum

[0180] Experimental Methods: Collect 5.0 mL of venous blood into a blood collection tube and let it stand at room temperature. Centrifuge and separate the serum fraction, store at 2℃~8℃, and use the kit from Example 1, as well as the T3, FT4, and TSH kits, to detect the signals on a MAGLUMI X8 instrument.

[0181] Data analysis: SPSS Statistics 17.0 software was used to analyze the data.

[0182] II. Results Analysis

[0183] To evaluate the superior diagnostic performance of T3-Ab in thyroid dysfunction in cancer patients undergoing immunotherapy, 420 serum samples from patients with different cancer diseases were collected. The T3-Ab levels in these samples were detected using the kit prepared in Example 1. A T3-Ab level > 1.315 AU / mL was defined as a positive thyroid dysfunction. Statistical analysis showed that T3-Ab effectively detected thyroid dysfunction in all cancer patients undergoing immunotherapy. For patients with malignant tumors of the stomach, esophagus, lung, colon, gallbladder, and cardia, the AUC for thyroid dysfunction during immunotherapy was superior to conventional laboratory indicators (FT3, FT4, TSH). The diagnostic performance was best for malignant tumors of the stomach, followed by malignant tumors of the esophagus and colon. The diagnostic performance of T3-Ab for thyroid dysfunction in cancer patients undergoing immunotherapy is shown in the table below.

[0184] Table 6: Diagnostic value of T3-Ab for thyroid dysfunction in different immunotherapy-treated cancer patients

[0185] Illness AUC for T3-Ab diagnosis AUC of routine laboratory indicators Malignant gastric tumors 0.809 0.736 Malignant tumors of the esophagus 0.798 0.765 Malignant lung tumors 0.736 0.701 Malignant liver tumors 0.545 0.628 malignant tumors of the colon 0.711 0.699 Gallbladder malignant tumor 0.606 0.657 Malignant tumor of the cardia 0.634 0.517

[0186] The embodiments described above are merely illustrative of several implementations of this disclosure, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this disclosure, and these all fall within the protection scope of this disclosure. Therefore, the protection scope of this patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Use of a detection agent that specifically binds to triiodothyronine autoantibodies for the preparation of a kit for the diagnosis of thyroid function abnormalities during cancer treatment; said treatment is an immune checkpoint inhibitor treatment; said immune checkpoint inhibitor comprises PD-1; the sample for diagnosis is blood, serum or plasma; said cancer is any one or more of lung cancer, gastric cancer, esophageal cancer, colon cancer and cardia cancer.

2. Use according to claim 1, comprising determining the level of triiodothyronine autoantibodies in a sample from a patient during cancer treatment, and comparing the determined value with a reference value.

3. Use according to claim 2, said determination of the level of triiodothyronine autoantibodies in a sample from a patient during cancer treatment comprising the following steps: 1) contacting a sample from a patient during cancer treatment with a detection agent that specifically binds to triiodothyronine autoantibodies under conditions that allow the formation of a complex; 2) adding a substance with a detectable label that is capable of specifically recognizing the complex formed in step 1); 3) quantifying the signal of the detectable label to obtain the amount of triiodothyronine autoantibodies.

4. Use according to claim 1, said triiodothyronine autoantibodies being selected from the group consisting of IgG and / or IgM type antibodies.

5. Use according to any one of claims 1-4, said kit being suitable for use in an immunoassay.

6. Use according to claim 5, said immunoassay being based on chemiluminescence.

7. Use according to any one of claims 1-4, said detection agent comprising a triiodothyronine antigen.

8. Use according to claim 7, said triiodothyronine antigen being conjugated to a solid support.

9. Use according to any one of claims 1-4, 8, said kit further comprising an antibody capable of specifically binding to the Fc fragment of triiodothyronine autoantibodies.

10. Use according to claim 9, said antibody capable of specifically binding to the Fc fragment of triiodothyronine autoantibodies being labeled with a signal substance.

11. Use according to claim 10, said signal substance being a chemiluminescent reagent.

12. Use according to claim 11, said signal substance being at least one of luminol and its derivatives, isoluminol and its derivatives, lucigenin, lophine base, peroxides of oxalic acid, acridinium esters.

13. Use according to claim 11, said signal substance being ABEI.

14. Use according to any one of claims 1-4, 6, 8, 10-13, said kit further comprising one or more of a triiodothyronine autoantibody standard, a reaction buffer, water, a chemiluminescent substrate solution.

15. Use according to any one of claims 1-4, 6, 8, 10-13, said detection agent further comprising a second detection agent for tetraiodothyronine autoantibodies.

16. Use according to claim 15, said tetraiodothyronine autoantibodies being selected from the group consisting of IgG and / or IgM type antibodies.

17. The use according to any one of claims 1-4, 6, 8, 10-13, 16, wherein the detection agent further comprises a third detection agent for quantitative determination of at least one of FT3, FT4 and TSH.

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