Immunoglobulin detection and related therapies

Abstract

The present invention relates to improved methods for the detection of immunoglobulin M (IgM), immunoglobulin A (IgA), and immunoglobulin E (IgE) antibodies, and to new therapies for pathogenic antibody- and antibody complex-mediated diseases and conditions.

Description

Technical Field

[0001] Field of Invention The present invention relates to an improved method for detecting immunoglobulin M (IgM), immunoglobulin A (IgA) and immunoglobulin E (IgE) antibodies, as well as new treatments for diseases and conditions mediated by pathogenic antibodies and antibody complexes.

Background Art

[0002] Background of the Invention Autoimmune diseases and other conditions mediated by pathogenic endogenous antibodies pose complex therapeutic challenges. For many patients suffering from renal failure, liver failure or heart failure, the last resort is often organ transplantation, but human leukocyte antigen (HLA) sensitization is a major barrier to organ transplantation. Highly sensitized patients have high levels of anti-HLA antibodies, which target the transplanted organ and are likely to severely damage the organ. The abundance of such antibodies directly affects the likelihood of finding a compatible donor organ. For example, many highly sensitized patients with renal failure continue to have a debilitated condition due to long-term dialysis, accompanied by high costs, a decline in quality of life, and an increased mortality rate. Immunosuppressive agents can suppress early graft loss due to acute rejection, but are less effective in preventing graft loss due to chronic rejection and increase the risk of serious complications such as life-threatening infections and cancer.

[0003] Pathogenic endogenous antibodies and immune complexes can also cause a wide range of autoimmune diseases and conditions, whereby patients have antibodies that recognize self-antigens, and these antibodies mediate inflammation and tissue damage.

[0004] The diagnosis and treatment of autoimmune diseases and other endogenous pathogenic conditions require highly sensitive assays to detect endogenous pathogenic antibodies and therapies to mitigate their harmful effects. However, antibodies are complex molecules, and current immunoassays for their detection can be unreliable and insufficiently sensitive. Current methods for detecting IgM antibodies, such as anti-HLA IgM antibodies, are repurposed from methods for detecting IgG antibodies (Paantjens, et al., Pulm. Med., 2011). When interference issues are suspected in methods for detecting IgG, it is common to use reducing agents such as dithiothreitol (DTT) to destroy IgM and prevent IgM from blocking IgG (Paantjens, et al., Pulm. Med., 2011). There is a demand for improved assays for detecting antibodies, particularly for detecting IgM antibodies and further classes of antibodies other than IgG. There is also a need for new methods for treating patients with pathogenic antibodies.

[0005] Immurefidase (IdeS), an immunoglobulin G-degrading cysteine ​​protease, is an IgG endopeptidase currently under development as a rapid desensitization therapy in kidney transplantation. Immurefidase exhibits high specificity and cleaves all subclasses of human IgG. Furthermore, it has been suggested that immurefidase also cleaves pathological anti-HLA IgM (Zhang et al. 2019, Am J Transplant. 2019; 19 (suppl 3)). [Overview of the Initiative]

[0006] Summary of the Invention The present invention provides an improved method for detecting IgM antibodies, IgA antibodies, or IgE antibodies in a sample, comprising contacting the sample with IgG cysteine ​​protease under conditions that enable the generation of IgG cysteine ​​protease activity, and contacting the sample with an agent suitable for detecting IgM antibodies, IgA antibodies, or IgE antibodies.

[0007] In a preferred embodiment, the method of the present invention is for detecting IgM antibodies. The inventors have shown in examples that IgM antibodies can form immunocomplexes with IgG, which interfere with the detection of antigen-specific IgM antibodies. Since these complexes can be cleaved by IgG cysteine ​​proteases, the use of IgG cysteine ​​proteases would provide an improvement to any method for detecting IgM antibodies.

[0008] In certain embodiments, the method of the present invention is for detecting IgA antibodies. IgA antibodies are known to form immune complexes with IgG in a similar manner to IgM antibodies. Since such complexes may cause interference with any method for detecting antigen-specific IgA antibodies, the use of IgG cysteine ​​protease would provide an improvement to any method for detecting IgA antibodies, as demonstrated for IgM antibodies in the examples.

[0009] In certain embodiments, the method of the present invention is for detecting IgE antibodies. IgE antibodies are known to form immune complexes with IgG in a similar manner to IgM antibodies. Since such complexes may cause interference with any method for detecting antigen-specific IgE antibodies, the use of IgG cysteine ​​protease would provide an improvement to any method for detecting IgE antibodies, as demonstrated for IgM antibodies in the examples.

[0010] In a preferred embodiment, the polypeptides include IgG cysteine ​​proteases, IdeS or IdeZ polypeptides, such as polypeptides having a sequence that is at least 80% identical to SEQ ID NOs: 2, 4, or 5, or polypeptides having a sequence that is at least 85%, 90%, 95%, or 99% identical. These polypeptides have established specificity for IgG, as confirmed in the examples, and are therefore effective in eliminating IgG interference without affecting IgM, IgA, or IgE levels.

[0011] In certain embodiments, the agent suitable for detecting IgM antibodies, IgA antibodies, or IgE antibodies is an anti-IgM antibody, an anti-IgA antibody, or an anti-IgE antibody. Such antibodies are readily available. The antibodies may be immobilized on a solid substrate such as beads or plates to assist in sample handling and improve throughput. In certain embodiments, the agent suitable for detecting IgM antibodies, IgA antibodies, or IgE antibodies is labeled to aid detection.

[0012] The method of the present invention is particularly useful for detecting IgM antibodies, IgA antibodies, or IgE antibodies against specific antigens. Examples demonstrate that IgM-IgG immune complexes cleaved by IgG cysteine ​​protease can provide false-positive results in assays for target-specific antibodies. Therefore, in certain embodiments, a sample is contacted with the antigen of interest under conditions that allow for the isolation of antibodies that specifically bind to the antigen of interest. The antigen of interest may be, for example, human leukocyte antigen (HLA), erythrocyte antigen, or drug antigen.

[0013] The method of the present invention is particularly useful for analyzing patient samples, such as serum samples from patients diagnosed with a condition requiring organ transplantation or from patients diagnosed with a disease selected from Table A.

[0014] The present invention also provides a kit for carrying out the method of the present invention, comprising IgG cysteine ​​protease and an agent for detecting IgM antibodies, IgA antibodies, or IgE antibodies, and optionally comprising an antigen of interest such as human leukocyte antigen (HLA), erythrocyte antigen, or drug antigen.

[0015] The present invention also provides a method for treating a disease or condition that is entirely or partially mediated by a pathogenic IgM antibody, a pathogenic IgA antibody, or a pathogenic IgE antibody, the method comprising administering an IgG cysteine ​​protease. The inventors have demonstrated in examples that an IgM antibody can form an immune complex with an IgG antibody that can be cleaved by an IgG cysteine ​​protease. IgA and IgE antibodies will likely form complexes similar to those with IgG antibodies. Since these immune complexes are expected to mediate disease processes, IgG cysteine ​​proteases would be useful in treating diseases or conditions mediated by pathogenic IgM antibodies, pathogenic IgA antibodies, or pathogenic IgE antibodies. The present invention also provides an IgG cysteine ​​protease for use in a method for treating a disease or condition that is entirely or partially mediated by a pathogenic IgM antibody, a pathogenic IgA antibody, or a pathogenic IgE antibody.

[0016] The present invention also provides a method for treating in a subject a disease or condition that is entirely or partially mediated by pathogenic IgG antibodies, wherein the subject is determined to be anti-IgG IgM antibodies, anti-IgG IgA antibodies, or anti-IgG IgE antibodies, and the method comprises administering IgG cysteine ​​protease. The inventors have demonstrated in examples that IgM antibodies can form immune complexes with IgG antibodies that can be cleaved by IgG cysteine ​​protease. IgA and IgE antibodies will likely form complexes similar to those with IgG antibodies. Since these immune complexes are expected to amplify the effects of pathogenic IgG antibodies, IgG cysteine ​​protease would be particularly effective in treating patients with anti-IgG IgM antibodies or anti-IgG IgA antibodies. In a further preferred embodiment, the present invention provides IgG cysteine ​​protease for use in a method for treating in a subject a disease or condition that is entirely or partially mediated by pathogenic IgG antibodies, wherein the subject is determined to be anti-IgG IgM antibodies, anti-IgG IgA antibodies, or anti-IgG IgE antibodies.

[0017] The present invention also provides a method for treating diseases or conditions that are mediated, whole or partially, by a complex of an IgG antibody with an IgM, IgA, or IgE antibody, the method comprising administering an IgG cysteine ​​protease. The inventors have demonstrated in examples that an IgM antibody can form an immune complex with an IgG antibody that can be cleaved by an IgG cysteine ​​protease. IgA and IgE antibodies will likely form a complex similar to that of an IgG antibody. Since these immune complexes may mediate disease processes, an IgG cysteine ​​protease would be effective in treating diseases mediated by a complex of an IgG antibody with an IgM, IgA, or IgE antibody. In a further preferred embodiment, the present invention provides an IgG cysteine ​​protease for use in a method for treating diseases or conditions that are mediated, whole or partially, by a complex of an IgG antibody with an IgM, IgA, or IgE antibody. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 - Schematic diagram showing IgM and its disulfide bond. [Figure 2] Figure 2 - No cleavage of human IgM was observed after incubation with immunofidase. [Figure 3] Figure 3 shows that we successfully purified IgG from serum using the CaptureSelect IgG-CH1 Affinity Matrix. [Figure 4] Figure 4 - Immurefidase activity was observed in HLA I + II antibodies (IgG, HI). MFI cutoff > 3000 [Figure 5] Figure 5 - HLA I + II antibody (IgG, EDTA) vs. HLA I + II antibody (IgM, EDTA) [Figure 6] Figure 6 - Comparison of effects observed in neat serum and anti-HLA IgM antibodies after IgG removal. [Figure 7] Figure 7 - Detection limits of assays - Anti-human IgM (PE) [Figure 8]In sensitized patients, Imurifidase does not digest human IgM [Figure 9] Figure 9 - A) Relative intensities (MFI) of SAB-HLA class I and II specific IgM in serum samples 24 hours after administration compared to pre-administration serum samples of sensitized ESRD patients. Each point represents SAB-HLA beads in patient serum. The cutoff for positive beads is 500 in the ether of the sample, and only positive beads are included in the analysis. B) Beads that increased in the 24-hour sample. C) Beads that decreased in the 24-hour sample. For reference, IgG SAB-HLA of the pre-administration serum is also included in the graph. Individual HLA beads are connected by lines. Individual patients are labeled with different point and line types. [Figure 10] Figure 10 - Digestion of purified human IgM and IgG was analyzed by SDS-PAGE gel (4 - 20%). Purified human IgM (A) and IgG (B) were incubated with a wide range of concentrations of Imurifidase at 37°C for 2 hours. The high molecular weight IgM sample (A) was reduced with DTT and separated by SDS-PAGE gel. The IgG sample (B) was heated for 3 minutes and separated by SDS-PAGE gel without reduction. [Figure 11] Figure 11 - Serum samples from 4 ESRD patients were incubated with a high concentration of Imurifidase. Digestion of human IgM was analyzed by SDS-PAGE gel (A) and Western blot (B). [Figure 12] Figure 12 - SDS-PAGE evaluation of serum samples before and after IgG removal. A) includes sample forms of patients 02-922, 02-923, 02-925 and 02-926. B) includes sample forms of patients 02-927, 02-928 and 02-929. [Figure 13]Figure 13 - IgG-free serum samples from sensitized ESRD patients analyzed for SAB-HLA class I and II (IgM) before and 24 hours after immunofidase administration. In the figure, N is the number of single antigen beads that reached the threshold of 1000 MFI in the pre-administration serum of that particular patient (from the analysis for IgM in neat serum [Figure 4]), and only positive beads are included in the analysis. A) Patient 02-922. B) Patient 02-923. C) Patient 02-925. D) Patient 02-926. E) Patient 02-927. F) Patient 02-928. G) Patient 02-929. [Figure 14] Figure 14-1 compares serum samples from one sensitized ESRD patient (02-927) before and 24 hours after immunofidase administration, using in vitro incubation with 6 and 30 μg / mL immunofidase for 1 hour. Serum samples were analyzed for SAB-HLA classes I and II (IgG and IgM). [Figure 15] Figure 15 - The mechanism we propose is that IgG-DSA-complexed IgM binds to SAB-HLA beads. The IgM detection antibody recognizes the IgM in the complex, resulting in a false IgM signal. After immunofidase treatment, these IgG-IgM complexes are cleaved, and a much lower but true IgM signal will be recorded in the SAB HLA assay. [Figure 16] Figure 16 - Serum samples from sensitized ESRD patients analyzed for SAB-HLA class I and II specific IgG, before and 24 hours after immunofidase administration. For clarity, only single antigen beads reaching the threshold of 3000 MFI in the pre-administration serum were included in the analysis. N indicates the number of positive beads for patients 02-922(A), 02-923(B), 02-925(C), 02-926(D), 02-927(E), 02-928(F), and 02-929(G). [Figure 17] Figure 17 - Beads that change slightly before and after IgM administration. Lines and shapes are as shown in the legend.

[0019] A brief explanation of arrays Sequence ID 1 is the complete sequence of IdeS, including the N-terminal methionine and signal sequence. It is also available as NCBI reference sequence ID WP_010922160.1. Sequence ID 2 is the mature sequence of IdeS lacking the N-terminal methionine and signal sequence. It is also available as Genbank acceptance number ADF13949.1. Sequence ID 3 is the complete sequence of IdeS, including the N-terminal methionine and signal sequence. It is also available as NCBI reference sequence ID WP_014622780.1. Sequence ID 4 is the mature IdeZ sequence, lacking the N-terminal methionine and signal sequence. Sequence ID 5 is the sequence for a hybrid IdeS / Z. The N-terminus is based on IdeZ lacking an N-terminal methionine and signal sequence. Sequence IDs 6 to 25 are exemplary protease sequences for use in the method of the present invention. Sequence ID 26 is the sequence of the IdeS polypeptide. It includes the sequence of Sequence ID 2 (internal standard pCART124) with additional N-terminal methionine and histidine tags. Sequence ID 27 is the sequence of the IdeS polypeptide. It includes the sequence of Sequence ID 4 (internal standard pCART144) with additional N-terminal methionine and histidine tags. Sequence ID 28 is the sequence of the IdeS / Z polypeptide. It includes the sequence of Sequence ID 5 (internal standard pCART145) with additional N-terminal methionine and histidine tags. Sequence ID 29 is the continuous sequence PLTPEQFRYNN, which corresponds to positions 63-73 of sequence ID 3. Sequence ID 30 is the continuous sequence PPANFTQG, corresponding to positions 58-65 of sequence ID 1. Sequence ID 31 is the sequential sequence DDYQRNATEAYAKEVPHQIT, which corresponds to positions 35-54 of Sequence ID 3. Sequence ID 32 is the continuous sequence DSFSANQEIRYSEVTPYHVT, which corresponds to positions 30-49 of sequence ID 1. Sequence IDs 33 to 55 are nucleotide sequences that encode the proteases defined above. Sequence IDs 56 to 69 are exemplary protease sequences for use in the method of the present invention. Sequence ID 70 is a continuous sequence NQTN corresponding to positions 336-339 of sequence ID 1. Sequence ID 71 is the continuous sequence DSFSANQEIR YSEVTPYHVT, which corresponds to positions 30-49 of sequence ID 1. Sequence IDs 72 to 86 are nucleotide sequences encoding polypeptides disclosed herein. Sequence ID 87 is the sequence SFSANQEIRY SEVTPYHVT, which corresponds to positions 31-49 of sequence ID 1. Sequence ID 88 is the sequence DYQRNATEAY AKEVPHQIT, which corresponds to positions 36-54 of the IdeZ polypeptide NCBI reference sequence ID WP_014622780.1. Sequence ID 89 is the sequence DDYQRNATEA YAKEVPHQIT, which may be present at the N-terminus of the polypeptide of the present invention.

[0020] Detailed description of the invention Methods for detecting antibodies The inventors have demonstrated in examples that IgM antibodies can form immune complexes with IgG antibodies. Such immune complexes can bind to both the target antigen and the agent for detecting IgM when no IgM antibody is present to react with the target antigen. Therefore, such immune complexes may produce false-positive results in assays for detecting IgM antibodies, particularly antigen-specific IgM antibodies. The examples also demonstrate that these problematic complexes can be cleaved with IgG cysteine ​​protease. IgA and IgE antibodies are known to form complexes with IgG in a similar manner to IgM. Therefore, the present invention provides an improved method for detecting IgM, IgA, and IgE antibodies, comprising contacting the sample with IgG cysteine ​​protease under conditions that allow for the generation of IgG cysteine ​​protease activity.

[0021] The interference discovered by the inventors is unusual in that it does not block the signal, but rather results in an increase of an inappropriate signal. The solution developed by the inventors using IgG cysteine ​​protease is particularly effective because it does not affect any of the IgM, IgA, or IgE antibodies to be detected. Other techniques to address the interference identified by the inventors, such as removal with anti-IgG antibodies or removal by column, would likely result in a decrease in IgM, IgA, and / or IgE levels.

[0022] The present invention provides a method for detecting IgM antibodies, IgA antibodies, or IgE antibodies in a sample, comprising: contacting the sample with IgG cysteine ​​protease under conditions that enable the generation of IgG cysteine ​​protease activity; and contacting the sample with an agent suitable for detecting IgM antibodies, IgA antibodies, or IgE antibodies.

[0023] Various immunoassays are available for detecting IgM, IgA, and IgE antibodies, and these can be adapted to include a step of contacting the sample with IgG cysteine ​​protease.

[0024] In certain embodiments, the method of the present invention is used in a solid-phase assay such as ELISA, where the agent for detecting the antibody is immobilized in the wells of the plate. Contact between the sample and IgG cysteine ​​protease may be performed in the same well.

[0025] In certain embodiments, the method of the present invention is used in a solid-phase assay such as a multiplex bead assay, where an agent for detecting antibodies is immobilized on beads. Different agents for detecting antibodies and / or antigens for different purposes may be immobilized on different beads. In certain embodiments, the method of the present invention is used in a single-antigen bead assay. In certain embodiments, the method of the present invention uses the Luminex® platform.

[0026] In certain embodiments, the method of the present invention is used in solid-phase assays such as ELISA or CAP FEIA (ImmunoCAP) tests, where the target antigen of interest is immobilized on a well or a solid substrate such as cellulose. The sample is then brought into contact with the immobilized antigen, into contact with IgG cysteine ​​protease, and into contact with an agent for detecting the antibody class of interest.

[0027] In certain embodiments, the method of the present invention is used in a surface plasmon resonance assay, in which an agent for detecting the antibody is immobilized on the surface of a sensor chip.

[0028] In certain embodiments, the method of the present invention is used in a nephelometry assay, in which contact between an agent suitable for detecting antibodies and a sample is performed in solution.

[0029] In certain embodiments, the method of the present invention is used in a biolayer interference assay, in which an agent for detecting antibodies is immobilized on a biosensor chip.

[0030] In a preferred embodiment, a suitable agent for detecting IgM antibodies, IgA antibodies, or IgE antibodies is an anti-IgM antibody, an anti-IgA antibody, or an anti-IgE antibody. Such antibodies are well known and widely available. In a particular embodiment, a suitable agent for detecting IgM, IgA, or IgE antibodies is labeled. Labeling can be used to aid detection.

[0031] In a preferred embodiment, the method of the present invention is for detecting IgM, IgA, or IgE antibodies specific to a target of interest. As described above, various immunoassays for detecting such antibodies are known and can be adapted to include a step of contacting the sample with IgG cysteine ​​protease. Generally, target-specific antibodies can be detected by contacting the sample with the antigen of interest under conditions that allow for the isolation of antibodies that specifically bind to the antigen of interest. In a particular embodiment, the antigen of interest is immobilized on a solid substrate or solid-phase matrix, such as beads or plates. The antigen may be coated onto the solid substrate. In a particular embodiment, the beads or plates are polystyrene. In a particular embodiment, the beads are or paramagnetic microspheres. In a particular embodiment, the beads are labeled. Multiple beads may be labeled differently to allow for the distinction of different beads and the detection of multiple different antibodies in a single assay. In a particular embodiment, the method of the present invention includes sorting the beads using flow cytometry.

[0032] Determining an appropriate cutoff value for any immunoassay utilizing the cleavage step by IgG cysteine ​​protease is within the ordinary capability of those skilled in the art. In many cases, a cutoff >1000 MFI is used, and >3000 MFI was used in the examples.

[0033] In certain embodiments, the methods of the present invention utilize multiple agents for detecting multiple target antigens and / or IgM, IgA, or IgE antibodies. Such diverse assays enable the detection of antibodies against a large number of different antigens in a single assay, and such assays would benefit from the improvements provided by the use of IgG cysteine ​​proteases. For example, in certain embodiments, the method involves contacting a sample with multiple HLA class I and / or HLA class II antigens, such as at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigens. In exemplary assays, beads may be coated with two alleles of HLA-A, -B, and -C at each locus, or HLA-DR, -DQ, and -DP, thereby maintaining expression rates similar to those observed on the cell surface. Alternatively, the beads may be coated with multiple copies of a single antigen.

[0034] Accordingly, the present invention provides a method for detecting IgM, IgA, or IgE antibodies specific to a target antigen in a sample, comprising: contacting the sample with IgG cysteine ​​protease under conditions that allow for the generation of IgG cysteine ​​protease activity; contacting the sample with the target antigen, optionally immobilized on a solid substrate such as a plate or beads; and contacting the sample with an agent suitable for detecting IgM, IgA, or IgE antibodies. Such a method would generally also include a standard washing step. The step of contacting the sample with IgG cysteine ​​protease may be performed before or after contacting the sample with the target antigen, but would generally be performed before contacting the sample with an agent suitable for detecting IgM, IgA, or IgE antibodies.

[0035] In certain embodiments, the method of the present invention is carried out in ELISA format. In such an assay, the wells of the assay plate will typically be coated with an antibody target. Next, an IgG protease is added to the wells, followed by the addition of a sample to be tested for IgM, IgA, or IgE antibodies specific to the target coating the well. The protease and the sample are allowed to interact under conditions suitable for IgG cysteine ​​protease activity. After a suitable interval, the assay plate is washed, and a detection antibody that specifically binds to IgM, IgA, or IgE antibodies is added under conditions suitable for binding to the target-specific antibody. The detection antibody binds to any intact target-specific antibody bound to the target in each well. After washing, the amount of detection antibody present in a well is proportional to the amount of target-specific antibody bound to that well. The detection antibody can be directly or indirectly labeled or conjugated to other reporter systems (such as enzymes) to determine the amount of detection antibody remaining in each well.

[0036] Any suitable sample containing or suspected to contain IgM, IgA, or IgE antibodies can be used in the method of the present invention. In a preferred embodiment, the sample is a sample obtained from a patient, such as a serum sample.

[0037] Detection of target-specific antibodies In a preferred embodiment, the method of the present invention detects the presence of IgM antibodies, IgA antibodies, or IgE antibodies specific to a particular target or set of targets. As shown above, various immunoassays for detecting such antibodies are known and can be adapted to include a step of contacting the sample with IgG cysteine ​​protease. Generally, target-specific antibodies can be detected by contacting the sample with the antigen of interest under conditions that allow for the isolation of antibodies that specifically bind to the antigen of interest.

[0038] In a preferred embodiment, the antigen of interest is a human antigen. Endogenous antibodies against such human antigens are often pathogenic; for example, autoantigens can cause autoimmune diseases, and donor-specific antigens can cause transplant rejection. In a preferred embodiment, the antigen of interest is an antigen associated with autoimmune diseases, such as human leukocyte antigens (HLA), erythrocyte antigens, or antigens selected from Table A below. Detection of such antibodies is difficult and requires particular accuracy, because clinicians often need to accurately and subtly evaluate a patient's antibody signature when diagnosing autoimmune diseases or determining whether a patient can receive a transplant from a particular donor. Therefore, the improvements achieved by the method of the present invention are particularly useful for detecting IgM antibodies, IgA antibodies, or IgE antibodies specific to these HLA, erythrocyte antigens, or antigens associated with autoimmune diseases.

[0039] In a more preferred embodiment, the method of the present invention is for use in detecting anti-drug antibodies. In such embodiments, the antigen of interest is a drug, such as an antibody or peptide drug, and IgM, IgA, or IgE antibodies against that drug are detected. Since such anti-drug antibodies may reduce the efficacy of the drug, their detection is useful in ensuring that treatment can be adjusted as required to maintain efficacy. Because high precision is required for the proper adjustment of treatment, the method of the present invention is particularly useful for detecting anti-drug antibodies.

[0040] Methods for treating diseases or conditions mediated by pathogenic antibodies and immune complexes. In a preferred embodiment, the present invention provides a method for treating a disease or condition that is mediated whole or partially by a pathogenic IgM antibody, a pathogenic IgA antibody, or a pathogenic IgE antibody, the method comprising administering an IgG cysteine ​​protease. The inventors have demonstrated in examples that an IgM antibody can form an immune complex with an IgG antibody that can be cleaved by an IgG cysteine ​​protease. IgA and IgE antibodies will likely form a complex similar to that of the IgG antibody. Since these immune complexes are expected to mediate disease processes, IgG cysteine ​​proteases would be useful in treating diseases or conditions mediated by pathogenic IgM antibodies, pathogenic IgA antibodies, or pathogenic IgE antibodies.

[0041] The present invention also provides IgG cysteine ​​protease for use in methods for treating diseases or conditions that are entirely or partially mediated by pathogenic IgM antibodies, pathogenic IgA antibodies, or pathogenic IgE antibodies. The present invention also provides the use of IgG cysteine ​​protease in the manufacture of pharmaceuticals for use in methods for treating diseases or conditions that are entirely or partially mediated by pathogenic IgM antibodies, pathogenic IgA antibodies, or pathogenic IgE antibodies.

[0042] In certain aspects, diseases or conditions that are entirely or partially mediated by pathogenic IgM antibodies, pathogenic IgA antibodies, or pathogenic IgE antibodies are diseases selected from Table A.

[0043] In certain embodiments, patients having a disease or condition that is entirely or partially mediated by pathogenic IgM antibodies, pathogenic IgA antibodies, or pathogenic IgE antibodies, to be treated according to the present invention, do not exhibit significant levels of anti-autoIgG antibodies. In certain embodiments, patients having a disease or condition that is entirely or partially mediated by pathogenic IgM antibodies, pathogenic IgA antibodies, or pathogenic IgE antibodies, to be treated according to the present invention, exhibit levels of anti-autoIgG antibodies or other IgG antibodies that are below pathogenic or within the normal range. The immune complexes identified by the inventors in the examples may mediate the disease in the absence of anti-autoIgG antibodies, and therefore IgG cysteine ​​proteases would be useful for treating such diseases or conditions in the absence of significant levels of anti-autoIgG antibodies.

[0044] In a further preferred embodiment, the present invention provides a method for treating a disease or condition that is entirely or partially mediated by a pathogenic IgG antibody, wherein the subject is determined to exhibit anti-IgG IgM antibody, anti-IgG IgA antibody, or anti-IgG IgE antibody, and the method comprises administering IgG cysteine ​​protease. The inventors have demonstrated in examples that IgM antibodies can form immune complexes with IgG antibodies that can be cleaved by IgG cysteine ​​protease. IgA and IgE antibodies will likely also form complexes similar to those with IgG antibodies. Since these immune complexes are expected to amplify the effects of pathogenic IgG antibodies, IgG cysteine ​​protease would be particularly effective in treating patients with anti-IgG IgM antibody, anti-IgG IgA antibody, or anti-IgG IgE antibody.

[0045] The present invention also provides an IgG cysteine ​​protease for use in a method of treating a subject with a disease or condition that is entirely or partially mediated by a pathogenic IgG antibody, wherein the subject is determined to be an anti-IgG IgM antibody, an anti-IgG IgA antibody, or an anti-IgG IgE antibody. The present invention also provides the use of an IgG cysteine ​​protease in the manufacture of a pharmaceutical for use in a method of treating a subject with a disease or condition that is entirely or partially mediated by a pathogenic IgG antibody, wherein the subject is determined to be an anti-IgG IgM antibody, an anti-IgG IgA antibody, or an anti-IgG IgE antibody.

[0046] In a further preferred embodiment, the present invention provides a method for treating diseases or conditions that are mediated whole or partially by complexes of IgG antibodies with IgM, IgA, or IgE antibodies, the method comprising administering IgG cysteine ​​protease. The inventors have confirmed in examples that IgM antibodies and IgG antibodies can form complexes that can be cleaved by IgG cysteine ​​protease. IgA antibodies and IgE antibodies are expected to form similar complexes. Such immune complexes are pathogenic and may cause diseases, for example, in the kidneys or blood vessels. Therefore, in such a preferred embodiment, the present invention provides a method for treating vasculitis, granulomatosis with polyangiitis (Wegener's granulomatosis), IgA nephropathy, or systemic lupus erythematosus, comprising administering IgG cysteine ​​protease. IgA nephropathy is caused by the accumulation of IgA antibodies in the kidneys, and this accumulation may be partially caused by IgA-IgG complexes similar to those confirmed in the examples. As demonstrated in the examples, cleaving such complexes with IgG cysteine ​​proteases may reduce or prevent the accumulation of IgA in the kidneys, thereby preventing, treating, or alleviating the symptoms of the disease. Vasculitis, granulomatous vasculitis with polyangiitis, and systemic lupus erythematosus may be caused by autoantibodies and immune complexes that can be cleaved by IgG cysteine ​​proteases according to the present invention. IgG-IgE complexes may cause or exacerbate acute allergic reactions or chronic inflammatory allergic diseases; therefore, in certain embodiments, the present invention provides IgG cysteine ​​proteases for use in methods of treating or preventing acute allergic reactions or chronic inflammatory allergic diseases. In certain such embodiments, the subject being treated may not exhibit or may not exhibit significant levels of anti-autoIgG antibodies or other pathogenic IgG antibodies. Instead, the complexes of IgG antibodies with IgM, IgA, or IgE antibodies are pathogenic in themselves.

[0047] The present invention also provides IgG cysteine ​​protease for use in methods of treating diseases or conditions that are mediated whole or partially by a complex of an IgG antibody and an IgM, IgA, or IgE antibody. The present invention also provides the use of IgG cysteine ​​protease in the manufacture of pharmaceuticals for use in methods of treating diseases or conditions that are mediated whole or partially by a complex of an IgG antibody and an IgM, IgA, or IgE antibody.

[0048] The pathogenic IgG, IgM, IgA, or IgE antibodies that form cleavable and therapeutic immune complexes according to the present invention may typically be specific to antigens targeted in autoimmune diseases or other conditions that are entirely or partially mediated by the antibody. Table A lists such diseases and associated antigens. The proteases of the present invention can be used to treat any of these diseases or conditions. The proteases are particularly effective in treating or preventing autoimmune diseases that are entirely or partially mediated by pathogenic IgG antibodies. Furthermore, the proteases are particularly effective in treating such diseases in patients who have anti-IgG IgM antibodies, anti-IgG IgA antibodies, or anti-IgG IgE antibodies. The proteases are also particularly effective in treating or preventing autoimmune diseases that are entirely or partially mediated by pathogenic IgM antibodies, IgA antibodies, or IgE antibodies.

[0049] [Table 1-1]

[0050] [Table 1-2]

[0051] [Table 1-3]

[0052] The IgG cysteine ​​protease according to the present invention may also be used for therapeutic or prophylactic purposes. In therapeutic use, the protease is administered to a subject already suffering from a disorder or condition in an amount sufficient to cure, alleviate, or partially cessate one or more of the condition or its symptoms. Such therapeutic treatment may result in a reduction in the severity of disease symptoms or an increase in the frequency or duration of asymptomatic periods. An amount sufficient to achieve this is defined as a “therapeutic effective dose.” In prophylactic use, the protease is administered to a subject who has not yet shown symptoms of a disorder or condition in an amount sufficient to prevent or delay the onset of symptoms. Such an amount is defined as a “preventive effective dose.” The subject may have been identified by any appropriate means as being at risk of developing the disease or condition.

[0053] In the method of the present invention, the protease may be co-administered with an immunosuppressant. In the method of the present invention, the protease is preferably administered by intravenous infusion, but may be administered by any suitable route, such as intradermal, subcutaneous, transdermal, intramuscular, intra-arterial, intraperitoneal, intra-articular, intraosseous, or other suitable route of administration. The amount of protease administered may be between 0.01 mg / kgBW and 2 mg / kgBW, between 0.05 and 1.5 mg / kgBW, between 0.1 mg / kgBW and 1 mg / kgBW, preferably between 0.15 mg / kg and 0.7 mg / kgBW, most preferably between 0.2 mg / kg and 0.3 mg / kgBW, and particularly between 0.25 mg / kgBW. The protease may be administered to the same subject multiple times, provided that the amount of anti-drug antibodies (ADAs) in the serum of the subject capable of binding to the protease does not exceed a threshold determined by the clinician. The amount of ADA in the serum of a target that can bind to the protease may be determined by any appropriate method, such as the drug-specific CAP FEIA (ImmunoCAP) test or the titer assay.

[0054] Organ transplantation The therapeutic method of the present invention may be particularly useful in the context of organ transplantation. The organ may be selected from the kidney, liver, heart, pancreas, lung, or small intestine. The subject to be treated may preferably be sensitized or highly sensitized. "Sensitized" means that the subject expresses antibodies against human major histocompatibility (MHC) antigens (also called human leukocyte antigens (HLA)). The subject may have IgM, IgA, or IgE anti-HLA antibodies, or the subject may have IgG anti-HLA antibodies and anti-IgG IgM, IgA, or IgE antibodies. Anti-HLA antibodies originate from allogeneically sensitized B cells and are usually present in patients who have been previously sensitized due to blood transfusion, previous transplantation, or pregnancy (Jordan et al., 2003).

[0055] Whether a candidate transplant recipient is sensitized can be determined by any preferred method. For example, a panel-reactive antibody (PRA) test may be used to determine whether a recipient is sensitized. A PRA score > 30% is typically understood to mean that the patient is at “high immunological risk” or “sensitized.” Alternatively, a cross-matching test may be performed, in which a blood sample from a candidate transplant donor is mixed with a sample from the intended recipient. A positive cross-match means that the recipient has antibodies that react to the donor sample, indicating that the recipient is sensitized and should not be transplanted. Cross-matching tests are typically performed as a final check immediately before transplantation.

[0056] IgG cysteine ​​protease The inventors have demonstrated that using IgG cysteine ​​protease in a method for detecting IgM, IgA, or IgE antibodies leads to improved specificity and sensitivity because the protease can cleave immune complexes that may interfere with the detection of IgM, IgA, and IgE antibodies. The IgG cysteine ​​protease used in the present invention is specific to IgG and does not have significant cleavage activity against the antibody to be detected. Therefore, in certain embodiments, the protease cleaves IgG but not IgM. In certain embodiments, the protease cleaves IgG but not IgA. In certain embodiments, the protease cleaves IgG but not IgE. In certain embodiments, the protease cleaves IgG but not IgM, IgA, or IgE.

[0057] In a preferred embodiment, the protease used in the method of the present invention is immunofidase (IdeS) (an immunoglobulin G-degrading enzyme of S. pyogenes). IdeS is an extracellular cysteine ​​protease produced by the human pathogen S. pyogenes. IdeS was originally isolated from serotype M1 group A streptococcus, but the ideS gene has now been identified from all group A streptococci tested. IdeS has very high substrate specificity, and its sole substrate has been identified as IgG. IdeS catalyzes single proteolytic cleavage in the lower hinge region of the heavy chains of all subclasses of human IgG. IdeS also catalyzes equivalent cleavage of the heavy chains of several subclasses of IgG from various animals. IdeS efficiently cleaves IgG into Fc and F(ab')2 fragments in a two-step mechanism. In the first step, one (primary) heavy chain of IgG is cleaved, producing a single cleaved IgG (scIgG) molecule with a non-covalently bonded Fc molecule. The scIgG molecule is essentially an intermediate product, retaining the remaining (secondary) heavy chain of the original IgG molecule. In the second step of the mechanism, this primary double chain is cleaved by IdeS, releasing an F(ab')2 fragment and a homodimer Fc fragment. These are the products commonly observed under physiological conditions. Under reducing conditions, the F(ab')2 fragment may dissociate into two Fab fragments, and the homodimer Fc may dissociate into its constituent monomers. Sequence ID 1 is the complete sequence of IdeS, including the N-terminal methionine and signal sequence. It is also available as NCBI reference sequence number WP_010922160.1. Sequence ID 2 is the mature sequence of IdeS, lacking the N-terminal methionine and signal sequence. It is also available as Genbank acceptance number ADF13949.1.

[0058] In an alternative embodiment, the protease for use in the method of the present invention is IdeZ, an IgG cysteine ​​protease produced by Streptococcus equi ssp. Zooepidemicus, a bacterium mainly found in horses. Sequence ID 3 is the complete sequence of IdeZ, including the N-terminal methionine and signal sequence. It can also be used as NCB reference sequence number WP_014622780.1. Sequence ID 4 is the mature sequence of IdeZ, lacking the N-terminal methionine and signal sequence.

[0059] In an alternative embodiment, the protease for use in the method of the present invention is a hybrid IdeS / Z, such as the sequence of SEQ ID NO: 5. The N-terminus is based on IdeZ lacking an N-terminal methionine and a signal sequence.

[0060] In a preferred embodiment, the protease for use in the present invention may include or be derived from SEQ ID NOs: 2, 4, or 5. The protease for use in the present invention may include an additional methionine (M) residue at the N-terminus and / or a tag at the C-terminus to facilitate expression and isolation therefrom in a standard bacterial expression system. Suitable tags include histidine tags, which may be directly bound to the C-terminus of the polypeptide or indirectly bound by any suitable linker sequence, such as 3, 4, or 5 glycine residues. The histidine tag typically consists of 6 histidine residues, but can be longer, typically up to 7, 8, 9, 10, or 20 amino acids, or shorter, for example, 5, 4, 3, 2, or 1 amino acid.

[0061] In a further preferred embodiment, the protease for use in the present invention may substantially consist of, or be composed of, any one of the sequences of SEQ ID NOs: 6 to 25. These sequences represent IdeS and IdeZ polypeptides with increased protease activity and / or decreased immunogenicity. Each of SEQ ID NOs: 6 to 25 may optionally contain an additional methionine at the N-terminus and / or a histidine tag at the C-terminus. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C-terminus by a linker of 3x glycine residues or 5x glycine residues.

[0062] In a further preferred embodiment, the protease for use in the present invention may substantially consist of, or be composed of, any one of the sequences of SEQ ID NOs. 56 to 69. These sequences represent an IdeS polypeptide with increased protease activity and / or decreased immunogenicity. Each of SEQ ID NOs. 56 to 69 may optionally contain an additional methionine at the N-terminus and / or a histidine tag at the C-terminus. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C-terminus by a linker of 3x glycine or 5x glycine residues.

[0063] In a further preferred embodiment, the protease for use in the present invention substantially consists of, or may consist of, any one of the sequences of SEQ ID NOs: 6 to 25, which may have up to three (e.g., 1, 2, or 3) amino acid substitutions. Each of SEQ ID NOs: 6 to 25 and its variants may optionally contain an additional methionine tag at the N-terminus and / or a histidine tag at the C-terminus.

[0064] In a further preferred embodiment, the protease for use in the present invention substantially consists of, or may consist of, any one of the sequences of SEQ ID NOs. 56 to 69, which may have up to three (e.g., 1, 2, or 3) amino acid substitutions. Each of SEQ ID NOs. 56 to 69 and its variants may optionally contain an additional methionine tag at the N-terminus and / or a histidine tag at the C-terminus.

[0065] The polypeptides of the present invention typically have a length of at least 100, 150, 200, 250, 260, 270, 280, 290, 300, or 310 amino acids. The polypeptides of the present invention typically have a length of 400, 350, 340, 330, 320, or 315 amino acids or less. It is understood that any of the lower limits listed above can be combined with any of the upper limits listed above to provide a range for the length of the polypeptides of the present invention. For example, the polypeptide may have a length of 100 to 400 amino acids, or a length of 250 to 350 amino acids. The polypeptide is preferably 290 to 320 amino acids in length, and most preferably 300 to 315 amino acids in length.

[0066] The primary structure (amino acid sequence) of the protease of the present invention is based on the primary structure of IdeS, IdeZ, or IdeS / Z, specifically on the amino acid sequence of SEQ ID NO: 2, 4, or 5, respectively. The sequence of the protease of the present invention may include variants of the amino acid sequence of SEQ ID NO: 2, 4, or 5 that are at least 80% identical to the amino acid sequence of SEQ ID NO: 2, 4, or 5. The variant sequence may be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 2, 4, or 5. The variant may be identical to the sequence of SEQ ID NO: 2, 4, or 5, except that it includes one or more specific modifications identified in WO2016 / 128558 or WO2016 / 128559. The identity of sequence SEQ ID NO: 2, 4, or 5 can be measured over a region of at least 50, at least 100, at least 200, at least 300 or more consecutive amino acids in the sequence shown in SEQ ID NO: 2, 4, or 5, or more preferably over the entire length of SEQ ID NO: 4 or 5.

[0067] The proteases for use in the present invention may be IdeS, IdeZ, or IdeS / Z polypeptides containing variants of the amino acid sequence of SEQ ID NOs. 2, 4, or 5, which are modified by adding, deleting, or substituting amino acids into the sequence of SEQ ID NOs. 2, 4, or 5. Such modifications are preferably conservative amino acid substitutions. Conservative substitutions involve substituting an amino acid with another amino acid having a similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acid they substitute for. Alternatively, a conservative substitution may involve introducing another aromatic or aliphatic amino acid in place of an existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art.

[0068] IgG cysteine ​​protease activity may be evaluated by any suitable method, for example, by incubating a polypeptide with an IgG-containing sample and determining the presence of an IgG cleavage product. A preferred method is described in publication WO2016 / 128559. A preferred assay is an ELISA-based assay, such as the one described in WO2016 / 128559. In such an assay, the wells of the assay plate are typically coated with an antibody target, such as bovine serum albumin (BSA). Next, a sample of the polypeptide to be tested is added to the well, followed by a sample of a target-specific antibody, which in this example is an antibody specific to BSA. The polypeptide and antibody are allowed to interact under conditions suitable for IgG cysteine ​​protease activity. After a suitable time, the assay plate is washed, and a detection antibody that specifically binds to the target-specific antibody is added under conditions suitable for binding to the target-specific antibody. The detection antibody binds to any intact target-specific antibody bound to a target in each well. After washing, the amount of detection antibody present in a well is proportional to the amount of target-specific antibody bound to that well. The detection antibody can be directly or indirectly labeled or conjugated to other reporter systems (such as enzymes), allowing the amount of detection antibody remaining in each well to be measured. The higher the potency of the test polypeptide in the well, the less intact target-specific antibody remains, and therefore the less detection antibody there is. Typically, at least one well on a given assay plate contains IdeS instead of the polypeptide being tested, allowing a direct comparison of the potency of the test polypeptide to that of IdeS. IdeZ or IdeS / Z may also be included for comparison.

[0069] Other assays can determine the potency of a test polypeptide by directly visualizing and / or quantifying IgG fragments resulting from cleavage of IgG by the test polypeptide. This type of assay is also described in WO2016 / 128559. Such assays typically involve incubating a sample of IgG with different concentrations of the test polypeptide (or one or more of IdeS, IdeZ, and IdeS / Z as controls) in a titration system. The products resulting from incubation at each concentration are then separated using gel electrophoresis, e.g., SDS-PAGE. The total IgG and fragments resulting from IgG cleavage are then identified by size and quantified by the intensity of staining with an appropriate dye. A larger amount of cleavage fragments indicates greater potency of the test polypeptide at a given concentration. The polypeptide of the present invention will typically produce detectable amounts of cleavage fragments at lower concentrations (lower points in the titration system) than IdeZ and / or IdeS. This type of assay can also determine the amount of different fragments resulting from each cleavage event, thus enabling the identification of test polypeptides that are more effective at cleaving the first or second heavy chain of the IgG molecule. The polypeptides of the present invention may be more effective at cleaving the first chain of the IgG molecule than the second chain, particularly when IgG is of the IgG2 isotype. The polypeptides of the present invention may be more effective at cleaving IgG1 than IgG2.

[0070] Polypeptide production Polypeptides such as those disclosed herein may be produced by any suitable means. For example, polypeptides may be synthesized directly using standard techniques known in the art, such as Fmoc solid-phase chemistry, Boc solid-phase chemistry, or solution-phase peptide synthesis. Alternatively, polypeptides may be produced by transforming cells, typically bacterial cells, with nucleic acid molecules or vectors encoding the polypeptide. The production of polypeptides by expression in bacterial host cells is described and illustrated in WO2016 / 128559.

[0071] Polypeptide-containing compositions and formulations The present invention also provides compositions comprising proteases for use in the therapeutic methods of the present invention. For example, the present invention provides compositions comprising one or more polypeptides of the present invention and at least one pharmaceutically acceptable carrier or diluent. The carrier(s) must be “acceptable” in the sense that they are compatible with the other components of the composition and do not dramatically alter the subject to which the composition is administered. Typically, the carrier and the final composition are sterile and pyrogen-free.

[0072] The formulation of suitable compositions can be carried out using standard pharmaceutical chemistry and methodologies, all of which are readily available to those skilled in the art. For example, a formulation can be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances such as wetting agents or emulsifiers, pH buffers, and reducing agents may be present in the excipients or vehicles. Suitable reducing agents include cysteine, thioglycerol, thioreducin, and glutathione. Excipients, vehicles, and auxiliary substances are generally pharmaceuticals that do not induce an immune response in the individual receiving the composition, and they can be administered without excessive toxicity. pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, thioglycerol, and ethanol. Pharmacologically acceptable salts, such as mineral salts like hydrochloride, hydrobromide, phosphate, and sulfate; and salts of organic acids such as acetate, propionate, malonate, and benzoate may also be included. A complete discussion of pharmaceutically acceptable excipients, vehicles, and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0073] Such compositions may be prepared, packaged, or sold in forms suitable for bolus or continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dose forms, such as ampoules or multi-dose containers containing preservatives. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and embeddable sustained-release or biodegradable formulations. Such compositions may further contain, but are not limited to, one or more additional components, such as suspending agents, stabilizers, or dispersants. In one embodiment of a parenteral administration composition, the active ingredient is provided in a dry form (e.g., powder or granules) for reconstitution in a suitable vehicle (e.g., sterile pyrogen-free water), and the reconstituted composition is subsequently administered parenterally. Compositions may be prepared, packaged, or sold in the form of sterile injectable aqueous or oily suspensions or solutions. These suspensions or solutions can be formulated according to known art and may contain, in addition to the active ingredient, additional components such as dispersants, wetting agents, or suspending agents as described herein. Such sterile injectable formulations may be prepared, for example, using water or a non-toxic, parenterally acceptable diluent or solvent such as 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixatives such as synthetic mono or diglycerides.

[0074] Other useful and parentally-administrable compositions include those containing the active ingredient in microcrystalline form, liposomal formulations, or as components of biodegradable polymer systems. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymer materials or hydrophobic materials such as emulsions, ion exchange resins, poorly soluble polymers, or poorly soluble salts. Compositions may be suitable for administration by any suitable route, such as intradermal, subcutaneous, transdermal, intramuscular, intra-arterial, intraperitoneal, intra-articular, intraperiosteal, or other suitable routes of administration. Preferred compositions are suitable for administration by intravenous infusion.

[0075] kit The present invention also provides a kit for carrying out the method of the present invention. The kit of the present invention comprises an IgG cysteine ​​protease as defined and discussed above, and an agent for detecting IgM antibodies, IgA antibodies, or IgE antibodies as defined and discussed above. In certain embodiments, the kit also includes an antigen of interest, such as a human leukocyte antigen (HLA), an erythrocyte antigen, or a drug antigen. As described above, the antigen of interest may be immobilized on a solid substrate, such as a plate or beads, by coating or other means.

[0076] General matters It should be understood that the different applications of the disclosed products and methods may be adapted to the specific needs of the art. It should also be understood that the terms used herein are solely for the purpose of describing specific aspects of the invention and are not intended to limit them.

[0077] Furthermore, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context specifically indicates otherwise. Thus, for example, a reference to "polypeptide" includes "polypeptides (plural)."

[0078] Unless otherwise prohibited, the steps of the methods disclosed herein may be performed in any suitable order, and the order in which the steps are described should not be considered limiting.

[0079] In this specification, the term "polypeptide" is used in its broadest sense to refer to compounds of two or more subunits of amino acids, amino acid analogs, or other peptide mimetic compounds. Thus, the term "polypeptide" includes short peptide sequences, as well as longer polypeptides and proteins. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acids, including both D and L optical isomers, as well as amino acid analogs and peptide mimetic compounds.

[0080] The terms "patient" and "subject" are used interchangeably and typically refer to humans. References to IgG typically refer to human IgG unless otherwise specified.

[0081] Amino acid identity can be calculated using any suitable algorithm. For example, the PILEUP and BLAST algorithms can be used (typically in their default settings) to calculate homology or to line up sequences (identify identical or corresponding sequences, etc.), as described, e.g., Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that, when aligned with words of identical length in a database sequence, either match or satisfy a certain positive-valued threshold score T. T is called the neighbor word score threshold (Altschul et al, above). These first neighbor word hits serve as seeds to initiate a search for HSPs containing them. The word hit is extended in both directions along each sequence as long as the cumulative alignment score can increase. The extension of the word hit in each direction stops when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of residue alignments with a negative score of 1 or more; or when it reaches the end of either sequence. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment.The BLAST program uses the following initial settings: word length (W) 11, BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919), alignment (B) 50, expected value (E) 10, M=5, N=4, and comparison of both strands.

[0082] The BLAST algorithm performs statistical analysis of the similarity between two sequences (e.g., see Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two polynucleotide or amino acid sequences occurs by chance. A sequence is considered similar to another sequence if, for example, the smallest sum probability in a comparison between the first and second sequences is less than approximately 1, preferably less than approximately 0.1, more preferably less than approximately 0.01, and most preferably less than approximately 0.001. Alternatively, the UWGCG package provides the BESTFIT program (e.g., used in its initial settings) which can be used to calculate identity (Devereux et al (1984) Nucleic Acids Research 12:387-395).

[0083] All publications, patents, and patent applications cited herein, whether above or below, are incorporated herein by reference in their entirety. [Examples]

[0084] Example 1 Unless otherwise noted, the methods used are standard biochemical and molecular biology techniques. Examples of appropriate methodological textbooks include Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley and Sons, Inc.

[0085] Introduction Immurefidase (IdeS), an immunoglobulin G-degrading cysteine ​​protease, is an IgG endopeptidase currently under development as a rapid desensitization therapy in kidney transplantation. Immurefidase is highly specific and cleaves all subclasses of human IgG. Due to its specific protease activity, it is known to effectively inhibit IgG Fc-mediated effector functions such as antibody-dependent cell phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cell-mediated cytotoxicity (CDC).

[0086] The purpose of this study is to further evaluate whether immunofidases affect IgM antibodies (Abs), particularly in sensitized end-stage renal disease (ESRD) patients, and to identify new applications for immunofidases.

[0087] Materials and methods Serum samples from sensitized end-stage renal disease (ESRD) patients Serum samples from sensitized end-stage renal failure patients enrolled in a Phase II clinical trial (13-HMedIdeS-02;NCT02224820) and administered immunofidase at 0.12 or 0.25 mg / kg were investigated. The study was conducted before immunofidase administration and 24 hours after administration.

[0088] Treatment with imulifidase Immurefidase (P16-0041701) was used for the in vitro procedure.

[0089] SDS-PAGE and Immunoblot Human IgG and IgM were separated under non-reducing conditions using 4-20% Mini-PROTEAN TGX Stain-Free gel (Bio-Rad) in Tris-glycine-SDS buffer. This was done using immunofidase-cleared human IgM (1 mg / mL) (#16-16-090713-M, Athens Research & Technology) or serum samples from sensitized ESRD patients. The gel was transferred to a 0.45 μm nitrocellulose membrane, blocked with 5% skim milk (NFM), and incubated with PE-labeled donkey anti-human IgM (#IGM-PEC1, One Lambda) in PBS-Tween. The membrane was washed and analyzed using a PE-appropriate blot setting. Signals were acquired using a ChemiDoc MP system (Bio-Rad).

[0090] IgG purification using CaptureSelect Affinity Matrix For the purification of serum samples, use CaptureSelect. TM IgG-CH1 Affinity Matrix (#194320005, ThermoFisher Scientific) was used. CaptureSelect TM The Affinity Matrix purifies recombinant human Fab fragments and IgG from complex raw materials in a single step. This Affinity Matrix recognizes all four subclasses of IgG (IgG1, IgG2, IgG3, and IgG4), regardless of light chain type (copper / lambda). Serum samples were analyzed by SDS-PAGE for IgG evaluation and purification.

[0091] Single-antigen bead HLA assay for Class I and Class II (One Lambda / ThermoFisher) Serum samples from sensitized ESRD patients treated with immunofidase at 0.12 or 0.25 mg / kg were investigated. Test samples were examined before immunofidase administration and 24 hours after administration. All serum samples were pre-treated with EDTA to overcome the prozone effect. Serum was tested for both HLA class I and class II anti-HLA antibodies using commercially available Single antigen Beads (LABScreen Single antigen, #LS1A04, #LS2A01, One Lambda) on the Luminex platform. Serum was first incubated with LABScreen beads for 30 minutes and washed three times with wash buffer. Phycoerythriltin (PE)-labeled goat anti-human IgG or PE-labeled donkey anti-human IgM was added, incubated for 30 minutes, and washed twice. Fluorescence emission of PE from each bead was detected using a LABScan 100 analyzer. HLA specificity was determined by comparing the reaction patterns of the test serum with lot-specific worksheets defining the antigen sequences. The results were interpreted using HLA Fusion software (One Lambda) and displayed as mean fluorescence intensity (MFI).

[0092] Rheumatoid factor (RF) Serum samples from sensitized ESRD patients were screened for rheumatoid factor (IgM-RF) (Labmedicin Skane, Clinical Immunology and Transfusion Medicine, Lund, Sweden).

[0093] Immunoglobulin (Ig) class and IgG subclass The immunoglobulin classes and IgG subclasses were measured before and after purification using CaptureSelect Matrix in serum samples from two healthy volunteers (Labmedicin Skane, Clinical Immunology and Transfusion Medicine, Lund, Sweden).

[0094] result No cleavage of purified human IgM was observed after incubation with immunofidase. The test samples were separated on an SDS-PAGE gel, and the cleavage ability of immunofidases to purified human IgM was evaluated. Before heating, DTT was added to digest IgM and break the disulfide bonds (see Figure 1). Even after incubation with high concentrations (maximum 200 μg / mL) of immunofidase at 37°C for 2 hours, no digestion of purified human IgM was observed (see Figure 2).

[0095] We successfully purified IgG from serum using CaptureSelect Affinity Matrix. We successfully purified serum samples using CaptureSelect IgG-CH1 Affinity Matrix, and compared to untreated serum samples, we were unable to detect intact IgG bands on the SDS-PAGE gel (see Figure 3).

[0096] Treatment with immunofidase has a significant impact on the level of circulating IgG-specific anti-HLA antibodies. The ability of donor-specific antibodies (DSAs) to cleave IgG by immunofidase has been previously reported to allow patients who have cross-matched positively with deceased donors to receive transplants without requiring several weeks of pre-transplant treatment (Jordan et al., 2017; Lonze et al., 2018; Lorant et al., 2018).

[0097] Serum samples from sensitized ESRD patients were examined before and 24 hours after immunofidase administration. This study also successfully detected cleavage of anti-HLA IgG antibodies by immunofidase. A significant decrease in the levels of both class I and class II circulating anti-HLA IgG antibodies was observed in all seven patients (see Figure 4).

[0098] Immurefidase therapy does not affect the circulating levels of IgM-specific anti-HLA antibodies. The ability of donor-specific antibodies (DSAs) to possess IgM specificity by cleaving IgM with immunofidase was presented at the ATC Meeting 2019 (X. Zhang, S. Jordan, 2019. Anti-HLA IgM Antibodies Are Reduced in Highly-HLA Sensitized Patients Transplanted after Imlifidase (IdeS) Treatment).

[0099] Serum samples from sensitized ESRD patients treated with immunofidase were examined before and 24 hours after immunofidase administration. A decrease in circulating anti-HLA IgM antibodies after immunofidase treatment was detected in 2 out of 7 patients (subjects 02-927 and 02-929) (see Figure 5).

[0100] However, when serum samples were purified using CaptureSelect Matrix (IgG removal), there was no effect on the level of circulating anti-HLA IgM antibodies after immunofidase treatment (see Figure 6).

[0101] No cleavage of human IgM by immunofidase was detected. Purified human IgM was titrated (1250 to 4.8 ng / well) to confirm the detection limit of the assay. DTT-reduced human IgM was isolated on an unstained SDS-PAGE gel. The reduced heavy chain was located at 75 kDa and was visible down to 39 ng / well at the settings used. After transferring to a nitrocellulose membrane, protein rearrangement was checked using the unstained gel setting for the nitrocellulose membrane. The membrane was blocked and developed with PE-labeled donkey anti-human IgM (One Lambda). The 75 kDa heavy chain was visible down to 19 ng / well. Some bands at the highest IgM concentrations were also visible at approximately 200 kDa and 50 kDa (see Figure 7).

[0102] Serum samples from four sensitized ESRD patients (02-925, 02-927, 02-928, and 02-929) were treated in vitro with immunofidase and PBS, respectively. Serum samples and purified IgM were treated with DTT, separated on SDS-PAGE gel, transferred to a nitrocellulose membrane, blocked with PE-labeled donkey anti-human IgM (One Lambda), and developed. The 75 kDa heavy chain band was clearly stained, and no difference was observed between samples treated with immunofidase and those treated with immunofidase (see Figure 8).

[0103] Measurement of rheumatoid factor (RF) All serum samples examined for this report were diagnosed as rheumatoid factor (IgM-RF) negative.

[0104] Measurement of purified immunoglobin (Ig) class and IgG subclass using CaptureSelect Matrix To determine whether purification using CaptureSelect IgG-CH1 Affinity Matrix affects the Ig composition in human serum, serum samples from two healthy volunteers were measured for immunoglobulin classes and IgG subclasses before and after purification using CaptureSelect Matrix. The results are shown in Table B.

[0105] [Table 2]

[0106] conclusion In some patients, anti-HLA IgM signaling decreased after treatment with IgG cysteine ​​protease. However, this decrease in HLA-IgM signaling was not observed in serum from which IgG had been removed. The initially high anti-HLA IgM signaling is likely a result of IgG-complexed IgM on the surface of LABScreen HLA beads. Thus, while human IgM is not cleaved by IgG cysteine ​​protease, IgG-complexed IgM may interfere with the way IgM is detected, potentially generating a false signal. The decrease in anti-HLA IgM signaling observed in certain patients after treatment with IgG cysteine ​​protease suggests that IgG cysteine ​​protease can cleave the problematic complex.

[0107] Zhang et al.'s abstract (2019, Am J Transplant. 19 (suppl 3)) claims that IgG cysteine ​​protease immunofidase can reduce pathological anti-HLA IgM, but in this case, the immunofidase would not be useful for detecting any antigen-specific IgM and would not be useful for treating diseases mediated by IgG and IgM complexes. Surprisingly, this study demonstrates that IgG cysteine ​​protease can reduce antigen complexes consisting of IgG and IgM by cleaving IgG. Furthermore, the data highlight that in assays for detecting IgM antibodies, a method to remove IgG is necessary to ensure a true IgM signal, and the use of IgG-degrading cysteine ​​protease is particularly suitable for this purpose. Similarly, IgG cysteine ​​protease may be useful in therapies aimed at reducing antibody complexes consisting of IgG and other Ig isotypes, such as IgM, IgA, or IgE.

[0108] Example 2 Introduction The purpose of this study was to further evaluate the specificity of immunofidases by investigating whether they have any effect on human anti-HLA IgM in sensitized ESRD patients, or whether immunofidases may have any indirect effect on anti-HLA IgM SAB signaling in assays routinely used in clinical practice. The data and samples obtained in Example 1 were analyzed in more detail, and further experiments were conducted.

[0109] Materials and methods Serum samples from sensitized end-stage renal disease (ESRD) patients ESRD patients undergoing dialysis who were on the waiting list for kidney transplants at the Department of Surgery and Transplant Surgery, Uppsala University Hospital in Sweden, were eligible for the clinical trial (13-HMedIdeS-02; NCT02224820) if, in at least two separate single-antigen bead analyses, one or more antibodies showed antibody reactivity to ≥2 identified anti-HLA antibodies with a mean fluorescence intensity (MFI) greater than 3000. Eight patients enrolled received immunofidase intravenously once or twice daily (0.12 mg / kg body weight × 2 [n=3]; 0.25 mg / kg × 1 [n=3], or 0.25 mg / kg × 2 [n=2]). This clinical trial was conducted in accordance with the ethical principles derived from the Declaration of Helsinki. All ethical and regulatory approvals were obtained before any patient was exposed to any procedure related to the trial. Following IRB approval for the use of clinical samples for scientific research (EudraCT Number: 2013-005417-13; Diary Number: 2014 / 131, approved EPN 2014-04-16), all samples were remarked and anonymized.

[0110] In vitro treatment with imurifidase For in vitro processing, Idefirix® (Hansa Biopharma AB, Sweden) was used. Imurefidase, a 35 kDa monomeric active substance, is a recombinant protein expressed in E. coli. In reported in vitro studies, imurifidase was diluted to a final concentration of 0.002–200 μg / mL in PBS alone or PBS (BSA 0.05%), and the samples were incubated at 37°C for 1 or 2 hours.

[0111] SDS-PAGE and Immunoblot Immurefidase-treated purified human IgM (1 mg / mL) (#16-16-090713-M, Athens Research & Technology) or human IgG (1 mg / mL) (IVIg, Gamunex®) and IgM from serum samples of sensitized ESRD patients were separated on 4-20% Mini-PROTEAN TGX Stain-Free gels (#456-8093, Bio-Rad) in Tris-Glycine-SDS buffer (#161-0732, Bio-Rad) after DTT (#D9163, Sigma) treatment (IgM only). The gels were transferred to a 0.45 μm nitrocellulose membrane (#LC2001, Novex), blocked with 5% skim milk powder (OXOID), and PE-labeled donkey anti-human IgM (#IGM-30 PEC1, One) in PBS-Tween. The membrane was incubated with Lambda. The membrane was washed and analyzed using a blot setting suitable for phycoerythrin (PE). The signal was acquired using the ChemiDoc MP system (Bio-Rad).

[0112] CaptureSelect TM IgG purification using Affinity Matrix For the purification of serum samples, use CaptureSelect. TM AffinityMatrix (#194320005, ThermoFisher Scientific) was used. CaptureSelect TMAffinityMatrix purifies recombinant human Fab fragments and IgG from complex raw materials in a single step. This Affinity Matrix can recognize all four subclasses of IgG (IgG1, IgG2, IgG3, and IgG4), regardless of the light chain type (copper / lambda). Serum samples were analyzed by SDS-PAGE for IgG evaluation and purification.

[0113] Single-antigen bead HLA assay (One Lambda) of Class I and II anti-HLA antibodies Serum samples from sensitized ESRD patients treated with immunofidase at 0.12 or 0.25 mg / kg were investigated. Test samples were examined before immunofidase administration and 24 hours after administration. All serum samples were pre-treated with ethylenediaminetetraacetic acid (Ultrapure 0.5 M EDTA, pH 8.0, REF 15575-038, Invitrogen, Grand Island, NY, USA) in a final solution of 5 mM to overcome the prozone effect. Serum was tested for both HLA class I and class II anti-HLA antibodies using Single antigen Beads (LABScreen Single antigen, #LS1A04, lot 010, #LS2A01 lot 012, One Lambda, Canoga Park, CA) on a Luminex platform. Serum was first incubated with LABScreen beads for 30 minutes, and then the beads were washed three times with wash buffer. Phycoerythrin (PE)-labeled goat anti-human IgG (#LS-AB2, One Lambda, Canoga Park, CA) or PE-labeled donkey anti-human IgM (#IGM-PEC1, One Lambda, Canoga Park, CA) was added, incubated for 30 minutes, and washed twice. PE fluorescence emission from each bead was detected using a LABScan 200 analyzer. HLA specificity was determined by comparing the reaction patterns of the test serum with lot-specific worksheets defining the antigen sequences. Results were interpreted using HLA Fusion software (version 4.3, One Lambda) and displayed as the mean fluorescence intensity (MFI) of the raw data.

[0114] Data analysis of single-antigen bead HLA assays The mean fluorescence intensity (MFI) values ​​for the specificity of each antibody were determined using a baseline formula within the HLA Fusion 4.3 software (OneLambda). A baseline MFI threshold of 3,000 was used to assign positive reactions to all reporter antibodies (IgG-PE and IgM-PE). Baseline raw MFI data from each test serum were transferred to a Microsoft Office Excel spreadsheet for analysis and comparison between different test conditions.

[0115] Rheumatoid factor (RF) Serum samples from ESRD-sensitized patients were diagnosed for rheumatoid factor (IgM-RF) levels (Labmedicin Skane, Clinical Immunology and Transfusion Medicine, Lund, Sweden).

[0116] Immunoglobin (Ig) class and IgG subclass analysis Immunoglobulin classes and IgG subclasses were measured in serum samples from two healthy volunteers before and after purification using CaptureSelect™ Matrix (Labmedicin Skane, Clinical Immunology and Transfusion Medicine, Lund, Sweden).

[0117] result Immurefidase cleaves circulating anti-HLA IgG antibodies. Serum samples from sensitized ESRD patients (N=7) were examined before and 24 hours after immunofidase administration. The ability of immunofidases to cleave IgG, such as anti-HLA IgG antibodies, was confirmed, as established in previous studies (Jordan et al., 2017; Lonze et al., 2018; Lorant et al., 2018; Jordan et al., 2020; Schinstock, 2020) (Figure 16). A clear decrease in circulating anti-HLA IgG levels was observed in all patients tested (Figure 16).

[0118] Immurefidase treatment affects the assay signal in anti-HLA IgM SAB assays using neat serum. The samples were also analyzed for SAB-HLA IgM (Figure 10). A decrease in anti-HLA IgM signaling was observed in 2 out of 7 patients before immunofidase administration and 24 hours after administration (Figures 10E and 10G; patients 02-927 and 02-929). Antigen MFI values ​​in patients 02-927 and 02-929 decreased by an average of approximately 26-fold (32 antigens) and 15-fold (7 antigens), respectively. What these antigens had in common was that the pre-administration intensity corresponding to the beads in the IgG SAB-HLA assay was very high for the same antigen (minimum 11000 MFI).

[0119] In patients 02-923 and 02-925 (Figures 10B and 10C), there were beads with increased levels in the SAB-HLA IgM assay 24 hours after administration. In patient 02-923, increases were observed in beads containing HLA-A and HLA-B antigens (13 antigens), increasing to an average of five times the pre-administration value. In patient 02-925, increases were observed in beads containing HLA-DQ antigens (10 antigens), increasing to an average of two times the pre-administration value. The pre-administration values ​​of the same antigens in IgG SAB-HLA assay beads were extremely high (minimum 22000 MFI), within the range of expected bead saturation (generally occurring at >20000 MFI) (McCaughan et al., 2019). Therefore, the presence of high levels of IgG may adversely affect the number of available binding sites for low-affinity IgM in the assay. When SAB-HLA beads were present at low levels in the IgG assay, there was generally little difference in IgM assays between samples before administration and 24 hours later (Figure 17).

[0120] Purified human IgM is not affected by immunofidase treatment. We tested whether immunofidase has the ability to cleave purified human IgM using SDS-PAGE. Even at high concentrations up to 200 μg / mL, no cleavage of purified human IgM by immunofidase was observed (Figure 10A). On the other hand, human IgG treated in the same way was already cleaved into scIgG at 0.2 μg / mL, and completely cleaved at 2 μg / mL (Figure 10B).

[0121] IgM in human serum treated with immunofidase is not cleaved. The cleavage of human IgM in serum samples from four sensitized ESRD patients (02-925, 02-927, 02-928, and 02-929) was investigated after in vitro treatment with high-concentration immunofidase. Serum samples (and purified IgM) after immunofidase treatment were reduced, separated on SDS-PAGE (Figure 11A), and subjected to Western blotting using PE-labeled anti-human IgM (Figure 11B). The intact 75kDa heavy chain band of intact human IgM was clearly stained, and no difference in intensity was observed between those treated with immunofidase and those not, clearly demonstrating that serum IgM is not cleaved by immunofidase in vitro.

[0122] CaptureSelect TM Purification of IgG from serum using Affinity Matrix All serum samples obtained from the 7 patients were processed using CaptureSelect. TM We successfully removed IgG using AffinityMatrix. Compared to untreated serum samples, IgG could not be detected on the gel (Figure 4). All serum samples examined for this report were diagnosed as rheumatoid factor (IgM-RF) negative by the clinical laboratory methods used (Analysportalen Skane, Lund Sweden).

[0123] To confirm that the CaptureSelect Matrix primarily removes IgG and not other immunoglobulins, serum samples were measured for IgG, IgA, IgM, and IgG subclasses. Due to the required serum volume for this analysis, healthy volunteers were used instead of patient samples to demonstrate the principle in combination with the gel (Figure 12). All subclasses of IgG were removed to below the detection limit. Other immunoglobulin classes were reduced by approximately 50% from their initial levels by this procedure.

[0124] Immurefidase treatment does not affect anti-HLA IgM antibodies in serum after IgG removal. The pre- and post-administration effects observed in Figure 1 were completely eliminated in the IgG-removed sample (Figure 13). The anti-HLA IgM signal was generally lower (~80%) in the IgG-removed sample compared to the pre-removal sample (comparison of Figure 9 and Figure 13), likely due to dilution by washing in the removal protocol. In patient 02-925, for some HLA-DQ antigens, a similar pattern of increase in the 24-hour sample compared to pre-administration was observed both before removal (Figure 9B) and after removal (Figure 13), although the increase was reduced to 46% instead of 115%. In patient 02-927 (Figure 13), 10 beads remained that were higher (on average 3 times) in the pre-IgG removal administration compared to the 24-hour sample. These were the beads that decreased the most before removal (Figure 9C), and the mean MFIs for these beads were 15186 and 1023 before and after IgG removal, respectively. The MFI for the same beads in the 24-hour sample was the same before and after IgG removal. The same pattern was observed with 02-929, but with fewer beads. The continued reduction in these beads may be due to the fact that these IgM antibodies have an affinity for IgG rather than HLA, and are therefore co-removed along with IgG. In vitro treatment of serum samples with immunofidase before the SAB-HLA IgG / IgM test yields results equivalent to IgG removal (Figure 14).

[0125] Consideration The data presented in Example 2 confirm the conclusions drawn in Example 1. This study evaluated the specificity of immunofidase activity with respect to IgM. We can confidently conclude that immunofidase does not possess detectable activity against IgM, even in vitro in a PBS buffer system under ideal conditions.

[0126] The data presented herein demonstrate that the decrease in IgM signaling after immunofidase treatment in the SAB-HLA IgM assay is likely due to artifacts that are resolved by the immunofidase treatment.

[0127] The mechanism we propose involves IgG-DSA-complexed IgM binding to SAB-HLA beads (Figure 15). The IgM detection antibody recognizes the IgM in the complex, resulting in an artifactual IgM signal. After immunofidase treatment, these IgG-IgM complexes are cleaved, and a much lower but true IgM signal will be recorded in the SAB HLA assay. Therefore, these data demonstrate the usefulness of immunofidase treatment in detecting IgM antibodies.

[0128] This model is supported by several of our observations.

[0129] Firstly, under ideal conditions, even with very high concentrations of immunofidase, there is no detectable enzymatic activity in human IgM.

[0130] Secondly, the appearance of artifacts in some patients can clearly be linked to the presence of IgM and particularly high IgG DSA levels. This combination appears to be a necessary condition for the development of a false-positive IgM signal, and this signal disappeared when pre-administration patient serum samples were treated with immunofidase in vitro before performing the test.

[0131] Thirdly, false-positive signals in pre-administration samples disappear after IgG removal in all observed cases. Furthermore, this reduction in false-positive IgM signals is primarily observed in antigen beads where particularly high levels of IgG DSA signaling are present in the SAB-IgG assay.

[0132] It is interesting that the presence of RF was not detected in the patient even using standard clinical testing methods.

[0133] Our results demonstrate that immunofidase enzymatic activity can efficiently dissolve IgM-IgG complexes. This may have clinical implications in patients where IgG-IgM complex formation contributes to the exacerbation of autoimmune disease symptoms.

[0134] Interestingly, in contrast to the false-positive signals mentioned earlier, some patients also showed an increase in IgM signaling after immunofidase treatment, particularly when antigen beads exhibiting high IgG signaling. This is explained by the reduction in steric hindrance of DSA IgG, which allows DSA IgM to bind and generate a signal after serum Ig levels have decreased following immunofidase treatment.

[0135] Therefore, it appears that high IgG DSA concentrations carry a risk of both false-positive and false-negative IgM DSA signals in SAB-HLA IgM testing. In the worst-case scenario, this could jeopardize physicians' ability to make correct clinical decisions or lead to decisions based on inaccurate data.

[0136] While IgM assays are not currently widely used in clinical decision-making, the presence of IgG IgM aggregates in IgG SAB HLA may similarly mislead IgG DSA signaling, potentially leading to unnecessarily long waiting times for these patients.

[0137] Some HLA centers employ methods such as EDTA addition, thermal inactivation, DTT (Dithiothreitol) addition, and serial dilution to overcome the prozone effect problem, and these methods can improve the predictive accuracy of SAB-HLA assays. Our results show that in vitro treatment of test samples with immunofidase or IgG removal before performing SAB-HLA testing can improve the signal on some beads, reduce the risk of false negatives, and simultaneously reduce the risk of false positive IgM signals. Therefore, in vitro treatment of samples with immunofidase before testing may be a novel method to improve the accuracy and reliability of IgM SAB-HLA assays. In conclusion, the effect of immunofidase on the IgG / IgM complex that we have confirmed is expected to be useful in both clinical and diagnostic applications.

Claims

1. A method for detecting IgM antibodies in a sample containing an immune complex of IgM antibodies and IgG antibodies, Includes the following: method: a. Contacting the sample with IgG cysteine ​​protease under conditions that enable the generation of IgG cysteine ​​protease activity; and b. Contact the sample with an agent suitable for detecting IgM antibodies.

2. The method according to claim 1, wherein the IgG cysteine ​​protease is an IdeS or IdeZ polypeptide.

3. The method according to claim 1 or 2, wherein the IgG cysteine ​​protease is a polypeptide having a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to sequence number 2 or 4, or the IgG cysteine ​​protease comprises or consists of any one sequence of sequence numbers 6 to 25 and 56 to 69.

4. The method of claim 3, wherein the sequence comprises an additional methionine at the N-terminus and / or a histidine tag at the C-terminus.

5. The method according to any one of claims 1 to 4, wherein the agent suitable for detecting the IgM antibody is an anti-IgM antibody.

6. The method of claim 5, wherein the agent is fixed to a solid substrate.

7. The method according to any one of claims 1 to 6, further comprising contacting the sample with a target antigen under conditions that enable the isolation of an antibody that specifically binds to the target antigen.

8. The method of claim 7, wherein the target antigen is a human leukocyte antigen (HLA), an erythrocyte antigen, or a drug antigen.

9. The method according to claim 7 or 8, wherein the target antigen is immobilized on a solid substrate.

10. The method of claim 6 or 9, wherein the solid substrate is a bead or a plate.

11. The method according to any one of claims 1 to 10, wherein the sample is a sample obtained from a patient.

12. The method of claim 11, wherein the sample is a serum sample.

13. The method of claim 11 or 12, wherein the sample is obtained from a patient diagnosed with a condition requiring organ transplantation, or from a patient diagnosed with a disease selected from Table A.

14. The method according to any one of claims 1 to 13, wherein the method is an ELISA, a single antigen bead assay, a surface plasmon resonance assay, a nephelometry assay, or a biolayer interference assay.

15. A kit for carrying out the method of any one of claims 1 to 14, comprising an agent for detecting IgG cysteine ​​protease and IgM antibody.

16. The kit according to claim 15, further comprising the target antigen.

17. The kit according to claim 16, wherein the target antigen is a human leukocyte antigen (HLA), an erythrocyte antigen, or a drug antigen.

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