Early detection of immune checkpoint blockade (ICB)-induced hypophysitis by detecting auto-antibodies against at least one pituitary hormone

Abstract

The invention relates to a method for detecting immune checkpoint blockade (ICB)-induced hypophysitis or the risk thereof in a living being, to a test kit for carrying out the method, and to a method for treating and / or preventing an ICB-induced hypophysitis or the risk thereof in a living being.

Description

Early detection of immuno-checkpoint blockade (ICH)-induced hypophysitis TECHNICAL AREA

[0001] The present invention relates to a method for detecting immuno-checkpoint blockade (ICB)-induced hypophysitis or a risk thereof in a living organism, a test kit for carrying out the method and a method for treating and / or prophylaxis ICB-induced hypophysitis or a risk thereof in a living organism. BACKGROUND

[0002] The introduction of immune checkpoint blockade (ICH) has revolutionized cancer treatment and significantly improved overall survival rates. However, the reactivation of immune cells often leads to immune-related adverse events (irAEs). irAEs vary depending on the type of cancer and reflect common cancer and tissue antigens. Paradoxically, while irAEs can lead to treatment discontinuation, their occurrence can also be associated with tumor regression and improved treatment outcomes.

[0003] Endocrine immune-related adverse events (irAEs) such as diabetes, hypothyroidism, and hypophysitis are caused by permanent tissue damage, necessitating lifelong hormone replacement therapy. ICH-induced hypophysitis is a relatively common irAE observed in melanoma patients. Severe ir-hypophysitis presents with electrolyte imbalances, headache, nausea, and fatigue. Diagnosis involves measuring the levels of pituitary hormones, including adrenocorticotropic hormone (ACTH), thyrotropic hormone (TSH), growth hormone (GH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH).

[0004] From WO 2019 / 195823, a method for predicting and diagnosing immunotherapeutic toxicity is known, which is based on the determination of chemokine and based on cytokine levels, possibly in combination with levels of multiple autoantibodies, in a sample from a patient.

[0005] Biomarkers and mechanistic insights into immune-mediated (ir-) hypophysitis remain insufficient and are currently attributed to a combination of a type II hypersensitivity reaction (mediated by immunoglobulin G) and a type IV hypersensitivity reaction (mediated by T cells). BRIEF SUMMARY OF THE INVENTION

[0006] Against this background, an object of the present invention is to provide biomarkers for ICH-induced hypophysitis. In particular, a method is to be provided by which ICH-induced hypophysitis can be detected early, treated, and possibly even prevented.

[0007] This task is solved by a method for detecting immune checkpoint blockade (ICB)-induced hypophysitis, or a risk thereof, in a living being, comprising the following steps: (1) Providing a biological sample of the organism; (2) Examining the biological sample for the presence of autoantibodies against at least one pituitary hormone; (3) Identifying hypophysitis or a risk thereof if autoantibodies are detected in step (2).

[0008] According to the invention, 'immune checkpoint blockade' (ICB) is understood to mean the targeted inhibition of immune checkpoints in the living organism, such as PD-1, PD-L1 or CTLA-4, by the use of immune checkpoint inhibitors (ICI), e.g. in the context of cancer therapy.

[0009] Hypophysitis is an inflammatory disease of the pituitary gland (hypophysis) caused by autoimmune, infectious, drug-induced (e.g., by ICI), or idiopathic factors. It can lead to pituitary dysfunction, manifesting as hormonal imbalances, including deficiencies in pituitary hormones such as ACTH, TSH, GH, FSH, and LH.

[0010] According to the invention, 'autoantibodies' are defined as immunoglobulins (antibodies) produced by the organism's immune system that recognize the body's own structures, such as specific pituitary hormones or their components, as supposedly foreign and bind to them. The binding of the autoantibodies to the target structures can trigger an immunological reaction and is an indicator of, or a risk for, immunologically induced hypophysitis caused by immune checkpoint blockade (ICH).

[0011] According to the invention, a 'biological sample' is a substance taken from the body of a living being that provides information about its physiological or pathological condition and may potentially contain autoantibodies of the living being, such as blood, serum, plasma, saliva, urine, or other body fluids or tissue samples. In particular, the biological sample is one that may contain antibodies and / or autoantibodies against pituitary hormones.

[0012] According to the invention, a 'pituitary hormone' is a hormone synthesized and secreted by the pituitary gland, including, but not limited to, prolactin (PRL), somatotropin (GH), proopiomelanocortin (POMC) and its derivatives, as well as BPI fold containing family A, member 1 (BPIFA1).

[0013] According to the invention, the 'examination' in step (2) involves applying one or more diagnostic or analytical methods to the biological sample to determine the presence of specific autoantibodies against pituitary hormones. This includes both qualitative and quantitative methods suitable for specifically and selectively detecting autoantibodies. The examination is a key step in diagnosis and risk assessment. A variety of analytical methods are suitable, such as enzyme-linked immunosorbent assay (ELISA). (ELISA), Western blot, immunofluorescence (IF), line immunoassay (LIA), radioimmunoassay (RIA), multiplex immunoassay, immunoprecipitation, flow cytometry, etc.

[0014] According to the invention, the 'living being' is an animal or human being.

[0015] In step (3) of the method according to the invention, 'detection' refers to the detection or diagnosis of immuno-checkpoint blockade (ICB)-induced hypophysitis or an increased risk thereof, based on the detection of specific autoantibodies against pituitary hormones in a biological sample. Detection is performed by analyzing the test results from step (2), in which the presence of autoantibodies against at least one pituitary hormone is determined. The threshold or limit value for the autoantibody indicating hypophysitis or a risk thereof can be determined by standardized procedures or validated test methods known to those skilled in the art, e.g., by receiver operating characteristic (ROC) analysis. Alternatively, the average value orThe average concentration of the autoantibody in a reference sample of a healthy reference organism, such as a healthy human, or the population average is used. Detection is positive if the measured autoantibody levels exceed the defined threshold and negative if they are below this value. When the term "population average" is used here, it can refer to the total population, i.e., all living beings, or preferably human patients, in the local, regional, or national population in which the living being, or preferably the human patient, to be tested lives. The term can also refer to all cancer patients in the local, regional, or national population in which the patient lives, including patients with certain cancer subtypes, particularly melanoma patients.The term can also refer to all healthy patients in the local, regional, or national population in which the person lives. Alternatively, the risk measure can be associated with 1 or 2 standard deviations of the population mean value or with the top third of measured systemic autoantibodies in a population, instead of using a population mean.

[0016] The inventors provide, for the first time, a method for reliably detecting intracranial hepatitis (ICH)-induced hypophysitis. A particular advantage is that detection occurs at a very early stage, i.e., shortly after administration of immune checkpoint inhibitors (ICIs) or induction of immune checkpoint blockade (ICH). The method according to the invention thus represents an "early warning system" for ICH-induced hypophysitis, enabling the early identification and individualized treatment of at-risk patients, as well as the adjustment of ICH if necessary. The method according to the invention can be performed outside the human or animal body, i.e., ex vivo and / or ex vivo.

[0017] Unlike the procedure known from WO 2019 / 195823 (ibid.), the detection of ICH-induced hypophysitis or a risk thereof does not require the determination of the levels of chemokines and / or cytokines in the sample or the organism.

[0018] According to one embodiment of the inventive method, after step (3) the following further step is carried out: (4) Failure to detect hypothyroidism or a risk thereof if no autoantibodies are detected in step (2).

[0019] This further step adds the possibility of explicitly determining a "non-detection" of hypophysitis or a risk thereof if no autoantibodies or autoantibodies below the threshold or limit value are detected. "Non-detection" ensures that even a negative finding is actively interpreted diagnostically, instead of merely stating the absence of positive results. Step (4) according to the invention enables a complete differential diagnosis by systematically integrating both positive and negative results into the diagnostic process. Active "non-detection" is particularly relevant for early detection or risk assessment, as it exempts patients without autoantibodies from further, often burdensome, diagnostic or alternative therapeutic measures, and the intracranial hemorrhage (ICH) can, if necessary, be continued unchanged.This measure increases the reliability of the method and its applicability in clinical practice. Explicitly considering negative findings reduces unnecessary resource expenditure and costs in the medical setting. The possibility of non-... Detection ensures that patients are only monitored more closely if there is an actual risk. The explanations described above for step (3) of the method according to the invention apply accordingly to step (4). "Non-detection" occurs in particular if the threshold or limit value for the autoantibody indicating hypophysitis or a risk thereof is not exceeded.

[0020] According to a further development of the inventive method, the pituitary hormones against which the autoantibodies are directed are selected from the group consisting of: prolactin (PRL), somatotropin (GH), proopiomelanocortin (POMC) and BPI fold containing family A, member 1 (BPIFA1).

[0021] This measure has the advantage that the sample is specifically tested for autoantibodies of particularly high prognostic value. Focusing on a clearly defined group of autoantibodies minimizes the risk of nonspecific or false-positive results. Additionally, the accuracy of the procedure is improved, as only the relevant autoantibodies are examined. Nonspecific autoantibodies directed against other, irrelevant antigens are excluded, which increases the predictive power of the procedure. This also makes the procedure simpler and more cost-effective. Prolactin (PRL) is a peptide hormone synthesized and secreted by the lactotrophic cells of the anterior pituitary gland (adenohypophysis). It is involved in the regulation of various physiological processes, particularly lactation in mammals.Somatotropin, also known as growth hormone (GH), is a peptide hormone produced by the somatotrophic cells of the anterior pituitary gland. It plays a central role in growth regulation and metabolism. Proopiomelanocortin (POMC) is a precursor protein synthesized in the pituitary gland that, through enzymatic cleavage, forms several bioactive peptides, including adrenocorticotropic hormone (ACTH), beta-endorphin, and melanocyte-stimulating hormone (MSH). BPI fold containing family A, member T (BPIFA1) is a protein primarily expressed in the respiratory tract but is also associated with hypophysitis as a potential target autoantigen in immune responses.

[0022] In a further embodiment of the method according to the invention, the biological sample is a blood sample, preferably a blood serum sample.

[0023] This measure narrows the scope of the procedure and makes its execution more precise and practical. The selection of samples reflects the procedure's practical relevance and clinical applicability. Blood and blood serum are established and readily available sample types in medical diagnostics. By focusing on a standardized sample type, diagnostic tests can be better calibrated and validated. Serum is particularly suitable for the detection of autoantibodies because it contains a high concentration of immunoglobulins. This increases diagnostic accuracy and reduces costs, as no special sample preparation procedures (as with tissue samples) are required. Blood serum also offers a more uniform matrix compared to other biological samples, which improves the comparability of test results.

[0024] According to a further embodiment of the inventive method, the biological sample is examined in step (2) by means of a method selected from the group consisting of: Enzyme-Linked Immunosorbent Assay (ELISA), Western Blot, Immunofluorescence (IF), Line Immunoassay (LIA), Radioimmunoassay (RIA), Multiplex Immunoassay, Immunoprecipitation, Flow Cytometry, ImmunoCAP.

[0025] This approach has the advantage of employing methods that have proven particularly suitable for detecting autoantibodies, especially in patient serum. This makes the procedure versatile and applicable in both research laboratories and clinical diagnostic facilities. Users can select the method that best suits their available laboratory equipment or specific requirements (e.g., sensitivity, throughput, time). The integration of modern technologies such as multiplex assays ensures that the procedure remains compatible with current diagnostic advancements. Enzyme-linked immunosorbent assay (ELISA) is a biochemical method that detects specific autoantibodies by binding to an immobilized antigen and subsequent enzymatic color development.'Western blot' is a procedure in which proteins are separated by gel electrophoresis, transferred to a membrane, and targeted by specific autoantibodies. Immunofluorescence (IF) is a method that uses fluorescently labeled antibodies to detect target structures in tissues or cells. Line immunoassay (LIA) is a test format in which antigens are immobilized on strips and autoantibodies are detected through specific binding and enzymatic detection reactions. LIA is useful for the simultaneous detection of multiple autoantibodies. Radioimmunoassay (RIA) is a highly sensitive immunoassay that uses radioactively labeled antigens or antibodies to detect autoantibodies. RIA offers exceptional sensitivity and is suitable for low antibody concentrations. Multiplex immunoassay is an assay that analyzes multiple antigens simultaneously by immobilizing them on different beads or surfaces. It increases diagnostic throughput and reduces sample consumption.Flow cytometry is a method that analyzes cells or particles in suspension by detecting antibody binding using fluorescently labeled probes. It is particularly useful for analyzing cell-bound antigens or antibodies. ImmunoCAP is an automated system for the quantitative measurement of antibodies based on specific binding between antibodies and immobilized antigens. ImmunoCAP enables highly precise, automated diagnostics.

[0026] According to a further embodiment of the method according to the invention, the living being is a human melanoma patient, preferably a malignant melanoma patient.

[0027] This measure focuses the inventive method on patients diagnosed with melanoma, including cutaneous, mucosal, or occult forms, as well as metastatic stages of the disease. In these patients, intracranial hemodialysis (ICH) is increasingly used and leads to therapeutic success in a significant proportion of cases. Therefore, focusing on melanoma patients increases the relevance of the method. Melanoma patients undergoing immunotherapy (ICI) have an increased risk of immune-mediated side effects. The method offers a means of individual risk assessment and monitoring. Particularly in malignant melanoma, the method can help to detect hypophysitis early and avoid serious complications, such as adrenal insufficiency. Insights from the method can help to adapt ICI therapy, e.g. through closer monitoring or targeted prophylaxis in high-risk patients.

[0028] In a further development of the inventive method, ICH was induced in the patient by administering an immune checkpoint inhibitor (ICI).

[0029] This measure aligns the inventive method with a clinically relevant patient group. Since ICI therapy is increasingly used in oncology, the method is future-proof and addresses a growing patient population. Patients can thus be monitored even before the onset of clinical symptoms of hypophysitis, facilitating early detection. The method enables close monitoring to react promptly to potential side effects such as hypophysitis. Through diagnostics, targeted measures such as hormone replacement therapy or adjustment of ICI therapy can be initiated, thereby improving patient safety.

[0030] According to a further development of the inventive method, the ICI is selected from the group consisting of: Ipilimumab, Nivolumab, Pembrolizumab, Cemiplimab, Relatlimab, Sabatolimab, Tiragolumab.

[0031] This measure directs the inventive method to immune checkpoint inhibitors (ICIs) that are particularly frequently used in melanoma patients. The active substances are antibodies that are specifically and selectively directed against immune checkpoint molecules: CTLA-4 (ipilimumab), PD-1 (nivolumab, pembrolizumab, cemiplimab), LAG-3 (relatlimab), TIM-3 (sabatolimab), and TIGIT (tiragolumab). This ensures broad diagnostic applicability of the method. By clearly assigning the diagnosis to ICIs from a known and defined group, the diagnostic target group is refined and focused on clinically relevant cases. The inclusion of newly developed ICIs such as relatlimab and tiragolumab demonstrates that the method is compatible with therapeutic advances.In addition to including established ICIs such as ipilimumab and nivolumab, the procedure also enables diagnosis in patients treated with less frequently used or newly developed ICIs. Physicians. Laboratories can use the procedure specifically in patients treated with the specified ICIs, thus avoiding unnecessary diagnostic testing in other patient groups. The clear naming of the ICIs facilitates the validation and approval of the procedure, as the target group and medical context are precisely defined.

[0032] In a preferred embodiment of the method according to the invention, the detection is an early detection, preferably before the occurrence of clinical symptoms of hypophysitis.

[0033] The first clinical symptoms of immunomodulatory inhibitor (ICI)-induced hypophysitis typically appear approximately 6 weeks to several months after the start of therapy. Early identification of patients at risk for hypophysitis before these timeframes allows for preventive interventions and reduces the likelihood of serious side effects. Early detection enables the timely initiation of therapeutic measures to prevent deterioration, such as starting hormone replacement therapy or adjusting the ICI regimen. Hypophysitis can lead to permanent tissue damage and hormonal dysfunction. Early detection minimizes the risk of such complications.

[0034] Another object of the present invention relates to a test kit for the detection of hypophysitis induced by immune checkpoint blockade (ICH), comprising the following: Recombinant, purified pituitary hormone(s), optionally applied to the surface of a microtiter plate, Secondary antibody, preferably enzyme-labeled, optionally horseradish peroxidase, optionally alkaline peroxidase, optionally wash buffer, preferably phosphate-buffered saline solution with 0.05% Tween-20 (PBS-Tween), optionally substrate solution, preferably tetramethylbenzidine (TMB), Optional serum dilution buffer, preferably PBS and / or Tris buffer, instructions for carrying out the method according to the invention.

[0035] The kit contains all the necessary components for carrying out the inventive method for detecting immunologically induced hypophysitis and enables even non-specialized laboratories to perform the test safely and reliably. The provision of pre-configured reagents and instructions reduces the time required for test preparation and execution. Standardized components (e.g., recombinant pituitary hormones and buffer solutions) ensure consistent test results. The kit can be used in various clinical and research settings, particularly in hospitals, research centers, and diagnostic laboratories. The possibility of expanding the kit with new components, such as additional target antigens or detection methods, ensures its flexibility and adaptability.

[0036] The features, properties, advantages and further developments of the method according to the invention apply accordingly to the kit according to the invention.

[0037] Accordingly, in a further development of the kit according to the invention, the pituitary hormone(s) are selected from the group consisting of: prolactin (PRL), somatotropin (GH), proopiomelanocortin (POMC) and BPI fold containing family A, member 1 (BPIFA1).

[0038] Another object of the invention relates to an autoantibody against at least one pituitary hormone for use in the detection of hypophysitis induced by immune checkpoint blockade (ICH) or of a risk thereof in a living being.

[0039] The features, properties, advantages and further developments of the method according to the invention apply accordingly to the autoantibody(s) according to the invention or their use.

[0040] Another object of the present invention is a method for treating and / or prophylacting immunologically induced hypophysitis or a risk thereof in a living being, comprising the following steps: (1) Providing a biological sample of the organism; (2) Examining the biological sample for the presence of autoantibodies against at least one pituitary hormone; (3) Detection of hypophysitis or risk thereof if autoantibodies are detected in step (2). (4) Treatment and / or prophylaxis of hypophysitis in the living being.

[0041] In this process, the inventive method described at the outset is extended by an additional treatment step. The invention thus also enables active therapy or prophylaxis. Early treatment or prophylaxis can reduce the disease burden and prevent symptoms.

[0042] The features, properties, advantages and further developments of the aforementioned method according to the invention apply accordingly to the treatment method according to the invention.

[0043] In a further development of the treatment method according to the invention, the treatment is selected from the following group: hormone replacement therapy, therapy of inflammation, interruption or discontinuation of ICH, symptomatic therapy.

[0044] Treatment begins after a diagnosis of immunologically induced hypophysitis (ICB) or a risk thereof. A key advantage is the availability of several treatment options, which can be flexibly applied depending on the severity of the condition, the patient's overall health, and the clinical course. Targeted measures to reduce inflammation, regulate hormone levels, and provide symptomatic relief improve the patient's well-being. Hormone replacement therapy can be used to replace missing hormones in cases of hormonal deficiencies. Alternatively, or Additionally, measures can be taken to reduce inflammatory processes in the pituitary gland. Furthermore, the immunological burden can be reduced by discontinuing checkpoint blockade therapy. Symptomatic treatment can address symptoms caused by hypophysitis, such as headaches, visual disturbances, etc.

[0045] Further advantages and features will become apparent from the following description and the accompanying figures. It is understood that the features mentioned above and those to be explained below can be used not only in the combinations specified, but also in other combinations without departing from the scope of the present invention.

[0046] The following are non-restrictive examples with reference to the figures, explained in detail. BRIEF DESCRIPTION OF THE FIGURES Fig. 1 : ELISAs for pituitary autoantibodies in a multicenter discovery cohort. (A) ELISA results from the multicenter confirmatory cohort showing autoantibodies against prolactin (PRL), somatotropin (GH), and proopiomelanocortin (POMC). The absorbance values ​​(Abs 450 nm) for each antigen are shown in a bar chart. Green symbols represent true positive samples with autoantibody levels above the calculated threshold (dashed line), while red symbols indicate false positive samples above the threshold. Data are presented as means ± SD, and the Mann-Whitney U test was used for statistical analysis. These results confirm the presence of autoantibodies in ICH-induced hypophysitis across multiple centers and underscore their potential as predictive biomarkers. (B) Heatmap summarizing the ELISA results of the confirmatory cohort. Of the patients with ICH-induced hypophysitis (n=43), 30 were correctly identified because the absorbance values ​​for at least one of the three proteins were above the threshold. In contrast, only 7 of 19 patients with other immune-mediated toxicities showed elevated values. Sensitivity and specificity were 69.8% and 69.8%, respectively. 63.2% calculated. Grey boxes represent elevated ELISA values, with green representing true positive results and red representing false positive results for the combined biomarker. Fig. 2: New pituitary-associated autoantibodies in ir-hypophysitis. (A) Immunoprecipitation (IP) and mass spectrometry analysis of sera from two patients with ICH-induced hypophysitis identified 274 proteins (red box), four of which were specifically associated with the pituitary gland. These include prolactin (PRL), somatotropin (GH), proopiomelanocortin (POMC), and BPIFA1. In the Venn diagram (left), the 274 proteins identified in the IPs are represented by the blue circle, and their overlap with the 279 proteins elevated in the pituitary gland (data from the Human Protein Atlas) is shown. The four overlapping proteins are highlighted. Enrichment analysis performed with SubcellulaRVis (right) shows that all four identified proteins are extracellular and have the potential to be secreted, which could contribute to their immune recognition in ICH-treated patients. (B) Heatmap summarizing the ELISA results of the discovery cohort. Of the patients with ICH-induced hypophysitis (n=30), 21 were correctly identified because they had above-threshold absorbance values ​​for at least one of the three proteins (PRL, GH, POMC). In contrast, only 6 of 18 patients with other immune-mediated toxicities showed elevated values. The calculated sensitivity and specificity were 70.0% and 66.7%, respectively. Gray boxes are shown in the heatmap. Green represents elevated ELISA values, while green represents true positive detections for the combined biomarker (hypophysitis cases) and red represents false positive detections (other toxicities). Fig. 3: Pituitary-associated autoantibodies in a multicenter confirmatory cohort. (A) ELISA results from the multicenter confirmatory cohort showing autoantibodies against prolactin, somatotropin, and proopiomelanocortin. The absorbance values ​​(Abs 450 nm) for each antigen are shown, with green symbols representing autoantibody levels below the calculated threshold (dashed line) and red symbols representing elevated levels above the threshold. Differences between groups were assessed using statistical analysis (Mann-Whitney U test). These results confirm the presence of autoantibodies in multicenter ICH-induced hypophysitis and underscore their potential as predictive biomarkers. (B) Heatmap summarizing the ELISA results of the confirmatory cohort. Of the patients with ICH-induced hypophysitis (n=43), 30 were correctly identified, with absorbance values ​​above the threshold for at least one of the three proteins. In contrast, only 7 of 19 patients with other immune-mediated toxicities showed elevated values. Sensitivity and specificity were calculated to be 69.8% and 63.2%, respectively. Gray boxes represent elevated ELISA values, while green represents true positives and red represents false positives for the combined biomarker. EXAMPLES 1. Methods Immunoprecipitation for the identification of pituitary autoantigens

[0047] Human pituitary lysates were obtained from four autopsies performed under ethical approval at the Cantonal Hospital of St. Gallen (KSSG) in St. Gallen, Switzerland. Donors were over 18 years of age and had no history of pituitary apoplexy, pressure trauma, immunotherapy, active meningitis, neurodegenerative diseases, or glucocorticoid therapy. Samples were also excluded if death occurred more than 48 hours prior to tissue collection. Upon collection, the pituitary tissue was immediately flash-frozen. Protein lysates were prepared by homogenizing the tissue in lysis buffer (1xPBS with 0.5% NP-40, 1% CHAPS, and complete™, a mini protease inhibitor cocktail). For each immunoprecipitation, 300 pg total protein from the lysate was incubated with 30 pL Protein-G-Agarose-Beads (50% suspension; ROTIGarose-Protein G; Carl Roth) to capture immune complexes.The lysate-bead mixture was incubated overnight at 4 °C with patient serum to enable immunoprecipitation of the target antigens. After thorough washing, the bound proteins were eluted and analyzed by mass spectrometry (gel LC-MS / MS) to identify potential pituitary-specific autoantigens associated with ir-hypophysitis. Protein identification by mass spectrometry (gel-LC-MS / MS).

[0048] A first short SDS-PAGE gel electrophoresis was performed to separate proteins from salts and other low molecular weight molecules, followed by in-gel digestion with trypsin. The subsequently extracted peptides were desalted with C18 StageTips and subjected to LC-MS / MS analysis. LC-MS / MS analyses were performed on an Easy-nLC 1200 UH PLC (Thermo Fisher Scientific) coupled to a QExactive HF Orbitrap mass spectrometer (Thermo Fisher Scientific) or, alternatively, to an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific). Peptides were eluted over a 60-minute segmented gradient, with the 20 most intense peaks selected for fragmentation by higher-energy collision dissociation (HCD). MS data were processed using the MaxQuant Software Suite v.1.6.7.0, and a database search for peptide fragments was performed using the UniProt database and the Andromeda search engine. The identified proteins were compared with the pituitary-specific proteome (The Human Protein Atlas). Enzyme-Linked Immunosorbent Assay (ELISA)

[0049] For coating, 100 pL of 0.05 M carbonate-bicarbonate buffer (pH 9.6) per well of a 96-well flat-bottom plate (MediSorp, Thermo Fisher Scientific) was mixed with 0.1 pg of recombinant prolactin (PRL), somatotropin (GH), or proopiomelanocortin (POMC). The plates were incubated overnight at 4 °C. The following day, the plates were washed once with 225 pL of ELISA wash buffer (PBS + 0.05% Tween-20) and blocked with 150 pL of ELISA blocking buffer (Monster Block; Immunochemistry Technologies / Biomol) for 3 hours at room temperature (RT). After removal of the blocking buffer, 100 pL of diluted patient serum (1:100 in 1x PBS with 0.05% Tween-20 and 1% BSA) were added in duplicates to the wells and then incubated for 1 hour at room temperature. The plates were then washed four times with 225 pL ELISA wash buffer. To detect bound antibodies, 100 pL of a 1:2000 dilution of goat anti-human IgG-HRP (Jackson ImmunoResearch, Cat. No.109-035-003) in ELISA blocking buffer with 0.05% Tween-20 into the wells and the plates were incubated for 1 hour at RT.

[0050] The plates were washed four times with 225 pL of ELI SA wash buffer before 70 pL of TMB substrate (Cell Signaling Technology) was added to each well. The plates were incubated in the dark at room temperature for 20–30 minutes. The reaction was stopped by adding 70 pL of 1 M sulfuric acid, and the absorbance at 450 nm was measured using a TriStar microplate reader (Berthold Technologies). Statistical analysis

[0051] All statistical analyses were performed using GraphPad Prism (10.1.1; GraphPad Software). Absorption data from the ELISAs were analyzed using the Mann-Whitney U test to compare the groups. For all analyses, a p-value > 0.05 was considered non-significant (ns). Statistical significance was reported as follows: p > 0.05 (ns), p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

[0052] To determine optimal thresholds for autoantibody detection, a receiver operating characteristic (ROC) analysis was performed using GraphPad Prism. ROC curves were generated to determine the sensitivity and specificity of ELISA-based biomarkers for immunologically mediated hypophysitis (ICB). Thresholds were determined by maximizing the probability ratio, balancing sensitivity and specificity for optimal diagnostic performance. Sensitivity and specificity values ​​were calculated for both the detection and multicenter confirmatory cohorts, with the thresholds defined by the ROC curve analysis. Index cases Case No. 1

[0053] A 33-year-old man with a history of trunk melanoma (diagnosed in June 2019, BRAF V600E, NRAS wild-type, Breslow thickness unknown due to a fragmented biopsy) was treated with combined checkpoint blockade (ipilimumab and nivolumab) for metastatic disease. Five months after starting therapy, he developed clinical hypophysitis with ACTH levels of 1.1 pmol / L (normal range: 2.0–11 pmol / L), TSH of 0.06 mU / L (0.5–4.4 mU / L), and cortisol of 39 nmol / L (130–630 nmol / L). Case No. 2

[0054] A 53-year-old woman with a history of ulcerated melanoma on her back (diagnosed in December 2017, BRAF wild-type, NRAS Q61 R, Breslow thickness 2.1 mm) was treated with ipilimumab and nivolumab for metastatic disease. Eleven months after starting therapy, she developed clinical hypophysitis with ACTH levels of 1.1 pmol / L (normal range: 2.0–11 pmol / L), TSH levels of 0.09 mU / L (0.5–4.4 mU / L), and cortisol levels of 91 nmol / L (130–630 nmol / L). 2. Results

[0055] To identify potential autoantigens targeted by autoantibodies in ir-hypophysitis, the inventors performed mass spectrometry on immunoprecipitated pituitary lysates incubated with patient sera. Analysis revealed that coprecipitated immunoglobulins bind to four pituitary proteins: prolactin (PRL), somatotropin (GH), proopiomelanocortin (POMC), and BPI fold containing family A, member 1 (BPIFA1), which is present in both patients. To support these findings, the inventors created a discovery cohort of 30 ir-hypophysitis cases, all defined by confirmed ACTH deficiency (Table 1). Anti-CTLA Anti-PD1 2 (6.7%) Tx start until toxicity (months) 4 (3.1 - 5.7) AE to sample (months) 2 (-0.4 - 7.0) Table 1: Patient characteristics of the hypophysitis discovery cohort (n=30); nb: not determined.

[0056] These autoantigen candidates belong to the class of secreted proteins. The researchers focused on the three proteins PRL, GH, and POMC, which have a strong association with the pituitary proteome, while BPIFA1 is predominantly expressed in the olfactory and respiratory epithelium, as well as in the salivary glands. ELISAs using recombinant proteins confirmed the presence of autoantibodies against PRL, GH, and POMC in patient sera (Figure 1A; autoantibody ELISAs against the three hypophysitis antigens in the discovery cohort). Compared to a control group of 18 cases of ir-colitis and ir-hepatitis, ir-hypophysitis patients showed significantly higher absorption values ​​in the PRL and GH assays, with a trend toward increased values ​​for POMC.This suggested that a threshold value in the ELISA results could potentially serve as a biomarker for identifying hypophysitis patients or individuals at risk of developing ir-hypophysitis. A receiver operating characteristic (ROC) analysis was performed. to determine optimal thresholds based on probability ratios (Figure 1B; ROC analysis to determine absorption thresholds as hypophysitis biomarkers). By applying combined thresholds—where elevated autoantibody levels in one of the ELISAs indicated a positive result—the sensitivity in this discovery cohort reached 70% and the specificity 66.7% (Figure 2B; ELISAs of patient sera in the discovery cohort with elevated autoantibodies as biomarkers).

[0057] In a multicenter confirmatory cohort, the inventors validated the presence of autoantibodies against PRL, GH, and POMC in patients with ir-hypophysitis. ELISA results confirmed elevated autoantibody levels in 30 of 43 patients from the validation cohort, based on the thresholds established in the discovery cohort (Figure 3A; autoantibody ELISAs against the three hypophysitis antigens in the confirmatory cohort). All three ELISAs showed significantly higher signals in ir-hypophysitis patients compared to non-melanoma controls. These results demonstrate the reproducibility of autoantibody detection across multiple centers.

[0058] To assess the overall performance of this biomarker combination, a heatmap summarizing the ELISA results of the confirmatory cohort was generated (Figure 3B; ELISAs of patient sera in the confirmatory cohort with elevated autoantibodies as biomarkers). The sensitivity of the combined autoantibody biomarker in identifying hypophysitis cases was 69.8%, with a specificity of 63.2%. Preliminary data suggest that autoantibodies are detectable at study baseline, confirming their potential as predictive markers for hypophysitis in patients receiving ICH treatment. The comparable sensitivity and specificity values ​​observed in both cohorts are promising and reflect balanced biomarker performance with clinically relevant sensitivity and specificity. 3. Conclusion

[0059] Irritable hypophysitis (Ir-hypophysitis) is a common side effect in patients undergoing intracerebral hemorrhage (ICH) and often leads to irreversible tissue damage and long-term complications. Organ dysfunction. In the study underlying this invention, the inventors systematically analyzed the humoral immune response against pituitary antigens in melanoma patients treated with intracranial hepatitis B (ICH) and highlighted potential autoantibody biomarkers associated with this immune-mediated toxicity. The inventors initially identified antibodies directed against pituitary hormones in individual patients in a local cohort and validated them in a multicenter cohort. In two patients for whom samples were available prior to clinical manifestation, the autoantibodies were already detectable at the start of the study, demonstrating a predictive role.

[0060] Since intracranial hypophysitis (IR-hypophysitis) can lead to life-threatening hypocortisolism, early screening for anti-pituitary antibodies using the method according to the invention can enable personalized treatment strategies and timely interventions, particularly in cases where intracerebral hemorrhage (ICH) is not absolutely necessary. The biomarker according to the invention serves as a first-line tool for risk stratification and enables earlier identification of patients at risk for ICH-induced hypophysitis.

Claims

Patent claims 1. Method for detecting immuno-checkpoint blockade (ICH)-induced hypophysitis, or a risk thereof, in a living organism, comprising the following steps: (1) Providing a biological sample of the organism; (2) Examining the biological sample for the presence of autoantibodies against at least one pituitary hormone; (3) Identifying hypophysitis or a risk thereof if autoantibodies are detected in step (2).

2. The method according to claim 1, characterized in that after step (3) the following further step is carried out: (4) Failure to detect hypothyroidism or a risk thereof if no autoantibodies are detected in step (2).

3. Method according to claim 1 or 2, characterized in that the pituitary hormones are selected from the group consisting of: prolactin (PRL), somatotropin (GH), proopiomelanocortin (POMC) and BPI fold containing family A, member 1 (BPIFA1).

4. Method according to one of the preceding claims, characterized in that the biological sample is a blood sample, preferably a blood serum sample.

5. Method according to one of the preceding claims, characterized in that the examination of the biological sample in step (2) is carried out by means of a method selected from the group consisting of: Enzyme-Linked Immunosorbent Assay (ELISA), Western Blot, Immunofluorescence (IF), Line Immunoassay (LIA), Radioimmunoassay (RIA), Multiplex Immunoassay, Immunoprecipitation, Flow Cytometry, ImmunoCAP.

6. Method according to one of the preceding claims, characterized in that the living being is a human melanoma patient, preferably a malignant melanoma patient.

7. Method according to one of the preceding claims, characterized in that the IBC was induced by administration of an immune checkpoint inhibitor (ICI).

8. Method according to claim 8, characterized in that the ICI is selected from the group consisting of: Ipilimumab, Nivolumab, Pembrolizumab, Cemiplimab, Relatlimab, Sabatolimab, Tiragolumab.

9. Method according to one of the preceding claims, characterized in that the detection is an early detection, preferably before the occurrence of clinical symptoms of hypophysitis.

10. Test kit for the detection of immuno-checkpoint blockade (ICH)-induced hypophysitis, comprising the following: Recombinant, purified pituitary hormone(s), optionally applied to the surface of a microtiter plate, Secondary antibodies, preferably enzyme-labeled, optionally horseradish peroxidase, optionally alkaline peroxidase, optionally wash buffer, preferably phosphate-buffered saline with 0.05% Tween-20 (PBS-Tween), optionally substrate solution, preferably tetramethylbenzidine (TMB), optionally serum dilution buffer, preferably PBS and / or Tris buffer, Instructions for carrying out the procedure according to one of claims 1-9.

11. Test kit according to claim 10, characterized in that the pituitary hormone(s) are selected from the group consisting of: prolactin (PRL), somatotropin (GH), proopiomelanocortin (POMC) and BPI fold containing family A, member 1 (BPIFA1).

12. Autoantibodies against at least one pituitary hormone for use in the detection of immuno-checkpoint blockade (ICH)-induced hypophysitis or risk thereof in a living organism.

13. Method for the treatment and / or prophylaxis of immuno-checkpoint blockade (ICH)-induced hypophysitis or risk thereof in a living being, comprising the following steps: (1) Providing a biological sample of the organism; (2) Examining the biological sample for the presence of autoantibodies against at least one pituitary hormone; (3) Detection of hypophysitis or risk thereof if autoantibodies are detected in step (2). (4) Treatment and / or prophylaxis of hypophysitis in the living being.

14. Method according to claim 13, characterized in that the treatment is selected from the following group: hormone replacement therapy, therapy of inflammation, interruption or discontinuation of ICH, symptomatic therapy.

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