Patent Application
20200191785
The present application claims the benefit to the European applications EP 18213659.8, filed on Dec. 18, 2018, and EP 19171829.5, filed on Apr. 30, 2019, both of which are incorporated by reference in their entirety.
The present application is accompanied by an ASCII text file as a computer readable form containing the sequence listing, titled “Sequence-Listing-as-filed,” created on Nov. 5, 2019, 2019, 3:23:48 PM, with the file size of 43,535 bytes, which is incorporated by reference in its entirety. Applicants hereby state that the information recorded in computer readable form is identical to the written (on paper or compact disc) sequence listing.
The present invention relates to a method for diagnosing AIG, comprising the step of detecting whether autoantibodies against the beta-subunit of the gastric H+/K+-ATPase are present or not in a sample comprising antibodies, with use of a diagnostically useful carrier comprising a solid phase on which an agent for the specific capture of the autoantibody is immobilized, and to a diagnostically useful carrier comprising a solid phase on which an agent for the specific capture of an autoantibody against the beta-subunit of the gastric H+/K+-ATPase is immobilized.
It has been acknowledged that circulating autoantibodies against this H+/K+-ATPase from the parietal cells, also referred to as anti-parietal cell antibodies or APCAs for short, are diagnostic markers for autoimmune gastritis (AIG) and pernicious anaemia. AIG is a chronic disease with a prevalence of approx. 2% in the total population. In patients who suffer from other autoimmune diseases such as type I diabetes, the prevalence is up to 10%.
The H+/K+-ATPase in the parietal cells of the lamina propria mucosae of the gastric mucosa has the function of pumping protons and of thus maintaining an acidic environment in the stomach. It is a heterodimer composed of an alpha-subunit (encoded by the ATP4A gene), which contains the catalytic site and brings about ion transport, and a beta-subunit (encoded by ATP4B), which stabilizes the alpha-subunit and is required for the enzymatic activity.
Damage to the gastric mucosa, referred to as atrophic body gastritis (ABG), occurs in AIG. The degradation of the mucosa leads, then, to a reduced or absent secretion of gastric acid. Said reduced or absent secretion can also be caused by infections due to Helicobacter pylori.
In addition to other possible causes, an autoimmune gastritis can cause a pernicious anaemia (PA), an anaemia involving vitamin B12 deficiency. It is caused by a deficiency of intrinsic factor, a protein which binds and stabilizes the vitamin and is formed by the parietal cells, against which APCAs are directed.
For the correct treatment of an AIG or PA, it is essential to differentiate between a gastritis caused by autoimmunity and a gastritis caused by bacteria or medicaments. Incorrect treatment or lack of treatment can lead to a stomach ulcer or a stomach perforation; moreover, the risk of a stomach cancer increases.
There is therefore a need for diagnostic assays which are suitable for high-throughput use and which can detect diagnostically useful antibodies in readily available sample material from patients with highest possible diagnostic reliability, especially with highest possible sensitivity and/or specificity, particularly in comparison with assays based on the detection of an autoantibody against the alpha-subunit or the heterodimer, comprising alpha-subunit and beta-subunit, of the gastric H+/K+-ATPase.
It should be pointed out that the assay result on its own does not make a diagnosis possible, not least because samples from patients suffering from numerous other diseases such as pancreatitis or diabetes mellitus may have the autoantibody. The assay result can be interpreted by the attending physician together with other symptoms such as vitamin B12 deficiency, hyperchromic anaemia and lymphocyte-infiltration of the gastric mucosa and thus supports the diagnosis without itself providing a sufficient decision-making basis for a definite diagnosis.
What are disclosed in relation to this in the related art are ELISA and immunofluorescence assays which can detect autoantibodies against the heterodimer comprising the ATP4A-subunit and the ATP4B-subunit.
Since 2011, EUROIMMUN Medizinische Labordiagnostika AG has sold an ELISA based on purified native mammalian ATP4 comprising both subunits. In the description of the assay, the specificity is addressed in detail and it is pointed out that said specificity can be optimized via the choice of cut-off value (catalogue No. EA 1361-9601 G).
WO2011/150086 discloses the detection of autoantibodies against the ATP4A-subunit.
Wenzlau et al. disclose the detection of autoantibodies against ATP4A for the diagnosis of autoimmune gastritis in patients who suffer from type I diabetes. A radiobinding assay was used (Wenzlau, J. M., Gardner, T. J., Frisch, L. M., Davidson, H. W. and Hutton, J. C. (2011) Development of a novel autoantibody assay for autoimmune gastritis in TI D individuals, Diabetes Metab Res Rev., 27(8): 887-90).
Rusak et al. disclose that the ATP4A-subunit of the gastric H+/K+-ATPase is the major antigen for APCAs (Rusak, E., Chobot, A., Krzywicka, A. and Wenzlau, J. (2016) Anti-parietal cell antibodies—diagnostic significance, Advances in Medical Sciences, 61: 175-179). They report that better results can be achieved with radioimmunoassays than with ELISA or IFT.
Scharf et al. disclose, in connection with neurological autoimmune diseases, that it was possible to detect autoantibodies against the ATPase heterodimer using an immunofluorescence assay when cells which expressed either ATP4A or ATP4B were used (Scharf, M., Miske, R., Heidenreich, F., Giess, R., Landwehr. P., Blöcker, I., Begemann, N., Denno. Y., Tiede, S., Dahnrich, C., Schlumberger, W., Unger, M., Teegen, B., Stcker, W., Probst, C., Komorowski, L. (2015) Neuronal Na+/K+ ATPase is an autoantibody target in paraneoplastic neurologic syndrome, Neurology, 84: 1-7). In a serum from a patient with neurological symptoms, antibodies against ATP4B, but not against ATP4A, were detected. There is no disclosure of what type of antigens was exactly used and how the method was carried out.
Lahner et al. disclose the detection of autoantibodies against ATP4A and ATP4B using a luminescent immunoprecipitation system (LIPS) from samples from patients who suffered from an atrophic body gastritis. What becomes apparent is that autoantibodies against ATP4A and ATP4B can be detected nearly with the same, high sensitivity and specificity (Lahner, E., Brigatti, C., Marzinotto, I., Carabotti, M., Scalese, G., Davidson, H. W., Wenzlau, J. M., Bosi, E., Piemonti, L., Annibale, B. and Lampasona, V. (2017) Luminescent Immunoprecipitation System (LIPS) for Detection of Autoantibodies Against ATP4A and ATP4B Subunits of Gastric Proton Pump H+/K+-ATPase in Atrophic Body Gastritis Patients, Clinical and Translational Gastroenterology, 8(1):e215).
Burbelo et al. studied the occurrence of antibodies against ATP4B in patients who suffered from type I diabetes (Burbelo, P. D., Lebovitz, E. E., Bren, K. E., Bayat, A., Paviol, S., Wenzlau. J. M., Barriga, K. J., Rewers, M., Harlan, D. M. and ladarola. M. J. (2012) Extrapancreatic Autoantibody Profiles in Type I Diabetes, PLOS ONE, 7(9):e45216).
Using samples from mice, Kontani et al. identified the alpha-subunit as major antigenic protein in AIG (Kontani, K., Taguchi, D., and Takahashi, T. (1992) Involvement of the H+/K+-ATPase alpha subunit as a major antigenic protein in autoimmune gastritis induced by neonatal thymectomy in mice, Clin. Exp. Immunol. 89, 63-67).
Using unpurified beta-subunit in the form of a cell extract, Ma et al. conclude that both the alpha-subunit and the beta-subunit bind to autoantibodies from patients with AIG. A direct comparison of the human alpha- and beta-subunits was not performed (Ma, J. Y., Borch, K. and Mardh, S. (1994) Human Gastric H,K-Adenosine Triphosphatase beta-Subunit is a Major Autoantigen in Atrophic Corpus Gastritis, Scand J. Gastroenterol 29, 790-794).
Both Lahner et al. and Burbelo el al. indicate that only an assay with particularly high sensitivity and specificity such as LIPS or a radiobinding assay is suitable for a reliable diagnosis, in comparison with solid phase-comprising assay systems, by means of which many diagnostically useful epitopes cannot be detected. No reasons are given as to why the ATP4A-subunit was not selected.
The object underlying the invention is achieved by the subject matter of following various embodiments.
In a first aspect, the object underlying the invention is achieved by a method for diagnosing AIG, optionally and/or PA, comprising the step of detecting whether an autoantibody or autoantibodies against the beta-subunit of the gastric H+/K+-ATPase is or are present or not in a sample comprising antibodies, with use of a diagnostically useful carrier comprising a solid phase on which an agent for the specific capture of the autoantibody is immobilized.
In a preferred embodiment, the carrier is selected from the group comprising a microtitre plate, a bead, a blot, preferably western blot, line blot or dot blot, an immunofluorescence assay carrier comprising fixed cells, preferably for a microscopic analysis, a lateral flow device, a cellulose-based polymer, a biochip, a microarray and a chromatography column material.
In a preferred embodiment, the diagnostically useful carrier is an ELISA microtitre plate.
In a preferred embodiment, the sample is selected from the group comprising serum, whole blood, plasma and CSF.
In a preferred embodiment, the method further comprises detecting whether an autoantibody or autoantibodies against intrinsic factor is or are present or not in a sample.
In a preferred embodiment, the autoantibody is a class IgG antibody.
In a second aspect, the object underlying the invention is achieved by a diagnostically useful carrier comprising a solid phase on which an agent for the specific capture of an autoantibody against the beta-subunit of the gastric H+/K+-ATPase is immobilized.
In a third aspect, the object underlying the invention is achieved by a monoclonal antibody or isolated autoantibody which binds specifically against the beta-subunit of the gastric H+/K+-ATPase, particularly preferably in purified form, but preferably not against the alpha-subunit.
In a fourth aspect, the object underlying the invention is achieved by a method for the calibration or for the quality-control of a medical device, comprising the step of contacting the inventive carrier with a liquid comprising the inventive antibody, preferably in known concentration, followed by the step of detecting whether the antibody is present or not in the liquid, particularly preferably with at least semi-quantitative determination of the antibody in the liquid.
In a fifth aspect, the object underlying the invention is achieved by a kit comprising the inventive diagnostically useful carrier, the kit comprising one reagent or more than one reagent, preferably all the reagents, from the group comprising a wash solution, a calibrator solution, an antibody against the beta-subunit of the gastric H+/K+-ATPase, and an agent for the detection of the autoantibody, preferably a secondary antibody, even more preferably a secondary antibody against IgG antibodies.
In a preferred embodiment, the secondary antibody has a detectable label. The detectable label is preferably selected from the group comprising a label which is capable of chemiluminescence, is radioactive or comprises a detectable isotope, an enzymatically active label, a label capable of fluorescence, a compound detectable by NMR spectroscopy and a spin label, preferably a label capable of chemiluminescence, most preferably a label capable of chemiluminescence that generates a chemiluminescent signal upon incubation in a suitable chemiluminescence trigger solution.
In a sixth aspect, the object underlying the invention is achieved by use of an agent for the detection of whether an autoantibody or autoantibodies against the beta-subunit of the gastric H+/K+-ATPase is or are present or not in a sample or of the inventive carrier or of the inventive antibody for the highly specific diagnosis of AIG and/or PA.
In a seventh aspect, the object underlying the invention is achieved by the use of an agent for the detection of whether an autoantibody or autoantibodies against the beta-subunit of the gastric H/K-ATPase is or are present or not in a sample or of a carrier comprising the agent for the production of a kit or a composition for the highly specific diagnosis of AIG and/or PA.
In an eighth aspect, the object underlying the invention is achieved by the use of an autoantibody against the beta-subunit of the gastric H+/K+-ATPase for the diagnosis, preferably the highly specific diagnosis, of AIG. Particularly preferably, the autoantibody of the beta-subunit serves in this connection as sole biomarker, preferably as sole autoimmune biomarker in a test run, even more preferably as sole autoimmune biomarker derived from the gastric H+/K+-ATPase. This can mean that no other autoantibody is detected in parallel in the same sample, particularly no other autoantibody against the gastric H+/K+-ATPase, particularly the alpha-subunit.
The present invention is based on the surprising finding by the inventors that the detection of autoantibodies with optimized diagnostic reliability, especially specificity and sensitivity, can contribute to the diagnosis of AIG when what is detected is whether an autoantibody against the beta-subunit of the gastric H+/K+-ATPase, and not for instance against the alpha-subunit or the heterodimer, is present. This also holds true when a diagnostically useful carrier comprising a solid phase is used, i.e. assay formats which are described as diagnostically unreliable in the related art.
The present invention is further based on the surprising finding by the inventors that the detection of autoantibodies against the extracellular domain of the beta-subunit of the gastric H+/K+-ATPase allows a particularly specific diagnosis. As a result, the number of false-positive diagnoses or findings can be reduced at a high level.
In a preferred embodiment, the assay is carried out using a diagnostically useful carrier comprising a solid phase on which an agent for the specific capture of an autoantibody against the beta-subunit of the gastric H+/K+-ATPase is immobilized. In a more preferred embodiment, the agent is a polypeptide comprising the beta-subunit of the gastric H+/K+-ATPase or a variant thereof. However, molecules derived therefrom or imitating the backbone of the beta-subunit, such as peptidomimetics, peptoids, beta-peptides and peptides comprising unnatural amino acids, are also usable. A person skilled in the art is familiar with such molecules and their design (Pelay Gimeno, M., Glas, A., Koch, O., Grossmann, T. N. (2015) Structure-based design of inhibitors of protein-protein interactions: Mimicking peptide binding epitopes. Angewandte Chemie International Edition. 54(31): 8896-8927). It is equally possible for the beta-subunit to be detected indirectly by, firstly, detecting whether an autoantibody or autoantibodies against the heterodimer comprising alpha-subunit and beta-subunit is or are present or not and, secondly, detecting whether an autoantibody or autoantibodies against the alpha-subunit is or are present or not.
In a preferred embodiment, the term “immobilized”, as used herein, means that the agent for the detection of whether an autoantibody against the beta-subunit of the gastric H+/K+-ATPase is present or not is bound to the solid phase of the carrier, preferably via a covalent bond, electrostatic interaction or via a hydrophobic interaction, the agent remaining exposed to the environment such that it is accessible for autoantibodies in a liquid sample. Various suitable carriers, for example paper, polystyrene, metal, silicone or glass surfaces, microfluidic channels, membranes, beads such as magnetic beads, chromatography column media, biochips, polyacrylamide gels and the like, are described in the literature, for example in Kim, D. and Herr, A. E. (2013), Protein immobilization techniques for microfluidic assays, Biomicrofluidics 7(4), 041501. In this way, the immobilized agent together with the insoluble carrier can be easily separated from an aqueous solution, for example by filtration, centrifugation or decanting. An immobilized agent can be reversibly or irreversibly immobilized. For example, the immobilization is reversible when the agent interacts with the carrier via an ionic interaction, which can be masked by addition of a high concentration of a salt, or when the agent is bound via a cleavable covalent bond such as a disulfide bridge, which can be cleaved by addition of thiol-comprising reagents. In contrast, the immobilization is irreversible when the agent is attached to the carrier via a covalent bond which cannot be cleaved in aqueous solution, for example a bond which is formed by reaction of an epoxy group and an amino group, as is frequently used for binding lysine side chains to affinity columns. The agent, preferably a polypeptide, can be immobilized indirectly, for example by immobilization of an antibody or some other entity having affinity for the agent, followed by the formation of a complex with the effect that the agent-antibody complex is immobilized. Said agent can also be immobilized directly, i.e. for coating the carrier in direct contact. Various options for immobilization are described in the literature, for example in Kim. D. and Herr, A. E. (2013), Protein immobilization techniques for microfluidic assays, Biomicrofluidics 7(4), 041501.
The beta-subunit of the gastric H+/K+-ATPase is preferably the amino acid sequence represented with the UniProtKB sequence entry P51164 or in SEQ ID NO7, more preferably the amino acid sequence represented by SEQ ID NO3, the latter representing the extracellular domain which does not comprise the transmembrane domain, even more preferably the fragment represented in SEQ ID NO5. According to the invention, variants thereof can be used. In this application, all the cited database codes stand for the Uniprot database or other databases, more precisely for the version thereof on the filing date of said application or its earliest priority application. It is particularly preferred that SEQ ID NO3 or a variant thereof is used in the absence of at least one epitope from the transmembrane domain of the beta-subunit having the sequence SEQ ID NO4 and/or at least any epitope of the alpha-subunit, which epitope is reactive towards an autoantibody from the serum of an AIG patient, preferably any epitope of the alpha-subunit.
The alpha-subunit of the gastric H+/K+-ATPase is preferably the amino acid sequence represented by UniProtKB access number P20648 or in SEQ ID NO9.
Intrinsic factor is preferably the amino acid sequence represented by the NCBI GenBank entry NP_005133.2 or in SEQ ID NO10.
In a preferred embodiment, “detecting whether an autoantibody against the beta-subunit of the gastric H+/K+-ATPase is present or not in a sample” is understood to mean that what is detected is whether the sample contains an autoantibody which is directed against the beta-subunit, but not against the alpha-subunit of the same protein. Preferably, an antibody which is directed only against the complex comprising alpha-subunit and beta-subunit is also not detected. In this connection, particular preference is given to generating a first signal indicating that the autoantibody against the beta-subunit has bound and can be distinguished from a signal which arises from a different autoantibody binding to a different autoantigen such as the alpha-subunit or to an autoantigen consisting of amino acids of the alpha- and the beta-subunits. Thus, what can be distinguished is whether an antibody specific for the beta-subunit is binding to the beta-subunit, or whether a different event has taken place, particularly the binding of an autoantibody which binds against the alpha-subunit or the complex composed of alpha-subunit and beta-subunit, but not the beta-subunit alone. In a preferred embodiment, the invention uses, instead of an agent for specific capture, an agent for the specific detection of whether an autoantibody against the beta-subunit of the gastric H+/K+-ATPase is present or not in a sample, preference being given to an agent allowing the detection of whether an autoantibody against the beta-subunit of the gastric H+/K+-ATPase is present or not in a sample. The agent for detection or the agent for specific capture can be a polypeptide or a reagent or a composition for detection or specific capture.
The detection of the autoantibody can be qualitative or quantitative. In a preferred embodiment, the term “qualitative” is understood to mean that what is merely detected is whether the antibody is present or not, but not in what concentration. In particular, there is no establishment of whether the concentration of the antibody exceeds a certain value, which yields further information about the disease in question, for example about the severity of the disease, about the development, about the success of therapy or about certain symptoms. In a preferred embodiment, the term “quantitative” is understood to mean that information going beyond that about being able to detect the antibody is provided. In particular, it is possible to determine an absolute or relative value, or at least an assignment to one of multiple defined concentration ranges.
This can be achieved by the beta-subunit or a variant thereof being used spatially separated from other antigens, preferably autoantigens or epitopes of such autoantigens, which epitopes are reactive towards autoantibodies from samples from AIG patients, especially the alpha-subunit or variants thereof. Alternatively, it is possible to detect whether an autoantibody which binds against the complex comprising alpha-subunit and beta-subunit of the gastric H+/K+-ATPase is present in the sample, and additionally whether an antibody which binds specifically against the alpha-subunit is not present in the sample. If this is namely the case, it can be concluded that an autoantibody which binds specifically against the beta-subunit is present, even though neither the beta-subunit nor a variant thereof is used for the assay. Lastly, there is also the option of immobilizing all the antibodies of the searched class, preferably IgG, that are present in the sample, followed by contacting with the beta-subunit of the gastric H+/K+-ATPase, which either is itself labelled or can be detected via a labelled ligand, preferably a labelled antibody.
For the assessment of whether a patient has an increased risk of suffering from or contracting AIG and/or PA, preference is given to using solely the presence or absence of autoantibodies against the beta-subunit to indicate whether this is the case. By contrast, no attention is paid to the detection of autoantibodies against the alpha-subunit, because it does not indicate such an increased risk.
In a preferred embodiment, the “agent for the specific capture of an autoantibody against the beta-subunit of the gastric H+/K+-ATPase” is understood to mean an agent which can capture an autoantibody against the beta-subunit of the gastric H+/K+-ATPase, but can capture other antibodies, preferably autoantibodies, particularly against the alpha-subunit, less efficiently, most preferably not at all or to a non-detectable extent, with the result that they remain uncaptured in the sample in a detection method according to the invention when contacting the agent or agent-comprising carrier with the sample. Preferably, the agent for the specific capture of an autoantibody against the beta-subunit of the gastric H+/K+-ATPase binds specifically to an autoantibody against the beta-subunit from a sample from an AIG patient. In a preferred embodiment, among all autoantibodies again the H+/K+-ATPase only those binding against the beta subunit, preferably in particular not those binding against the alpha subunit, are used or used as Marker for diagnosing AIG. This autoantibody or these autoantibodies serve as the only autoantibody marker for the diagnosis in line with the present invention. Preferably, epitopes from or derived from the alpha subunit, to which autoantibodies against the alpha subunit, but not autoantibodies against the beta subunit bind, are absent.
In a particularly preferred embodiment, the term “bind specifically”, as used herein, in the case of an antibody means that said antibody binds against the relevant antigen in a binding reaction characterized by a dissociation constant which is 1×10−5 M, more preferably 1×10−7 M, more preferably 1×10−8 M, more preferably 1×10−5 M, more preferably 1×10−10 M, more preferably 1×10−11 M, more preferably 1×10−12 M or a stronger binding reaction. Preferably, the dissociation constant is, in this connection, measured by surface plasmon resonance using a Biacore instrument at 25° C. and in PBS buffer at pH 7 and calculated using the software provided by the manufacturer.
In a preferred embodiment, the term “diagnosis”, as used herein, is understood to mean a procedure which yields information which supports the assessment, or allows it in the first place, of whether a subject is suffering from a disease or has a higher probability of suffering from a disease than an average person or suffered in the past or will suffer in the future, or whether a disease is progressing or how it will develop in the future, or for assessing whether a certain treatment is taking effect. Preferably prior to the diagnosis it is unknown whether or not the subject to be diagnosed suffers from the disease.
Preferably, for the diagnosis or to support the diagnosis, it is sufficient to merely detect whether the antibody is present, it being possible to determine whether detectable concentrations of the antibody are present in the sample. In a preferred embodiment, what is determined is whether the relative concentration of the antibody is higher in a patient to be diagnosed than in an average healthy person. What can be determined is whether the concentration is higher by a factor of 1.1, more preferably 1.2, 1.5, 1, 2, 5, 10, 20, 25, 50, 100, 200, 500, 1000, 10 000 or 100 000, than in a sample from an average healthy person. The higher concentration supports the diagnosis that a patient from whom the sample originates is suffering from AIG or indicates that the patient has an increased probability of suffering from AIG.
The method according to the invention or the use according to the invention is preferably effected in vitro.
A person skilled in the art understands that such a diagnosis generally does not by itself allow a comprehensive diagnosis, but that further aspects must be taken into consideration, for example further parameters to be determined in a sample, medical history, clinical symptoms, anamnesis or the results of imaging methods. Said person further understands that the value of the method according to the invention can consist in it being possible to carry out an indirect diagnosis or differential diagnosis in which the exclusion of a disease indicates that the patient is suffering from a different disease with similar symptoms. In a preferred embodiment, products according to the invention, such as carriers or kits, or methods according to the invention serve for the differentiation between a gastritis caused by autoimmunity and a gastritis caused by bacteria or by medicaments or for the diagnosis of a disease from the group comprising AIG, pernicious anaemia, atrophic body gastritis or vitamin B12 deficiency.
In the event of antibodies being detected, the sample to be investigated is a sample comprising a representative set of antibodies from the patient, who is preferably a mammal, even more preferably a human. Preferably, said sample is selected from the group comprising serum, plasma, serum and CSF.
It is possible to carry out the teaching according to the invention not only by using the wild-type sequences of the specified polypeptides or the reference sequences, as specified here in the form of SEQ ID NOs, collectively referred to henceforth as “full-length polypeptide”, or database codes or in some other way, possibly implicitly, but also by using variants of said sequences.
In a particularly preferred embodiment, the term “variant”, as used herein, means, in this connection, at least one fragment of the full-length polypeptide which is truncated at the C-terminal and/or N-terminal end by one or more than one amino acid compared to the full-length polypeptide. Such a fragment can contain a length of 5, 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150 or 200 successive amino acids from the sequence of the full-length polypeptide, preferably 10 or more.
In a further preferred embodiment, the term “variant”, as used herein, encompasses not only a fragment, but also a polypeptide having amino acid sequences with, in the order of increasing preference, at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity in relation to the full-length polypeptide or in relation to the fragment thereof, with no deletion or non-conservative substitution of an amino acid essential for biological activity, i.e. the ability of an antigen to bind to an antibody specific for the full-length polypeptide antigen. The related art discloses methods for establishing the sequence identity of amino acid sequences, for example in Arthur Lesk (2008). Introduction to bioinformatics, Oxford University Press, 2008, third edition. In a preferred embodiment, the ClustalW software (Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., Higgins, D. G. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948) is used to this end with use of the standard settings.
In addition, a polypeptide used according to the invention or a variant thereof can have chemical modifications, for example isotope label, covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation, etc. In a particularly preferred embodiment, a polypeptide which is used for the detection of an autoantibody against the beta-subunit or used some other way according to the invention and which comprises the beta-subunit or a variant thereof is glycosylated. The glycosylation is preferably accomplished by expression in a cell capable of glycosylation, preferably a mammalian or insect cell, more preferably a mammalian cell.
Variants can also be fusion proteins with amino acids or other known polypeptides or variants thereof, preferably with a purification tag, even more preferably from the group comprising His tag, thioredoxin, maltose-binding protein, streptavidin, glutathione S-transferase or a variant thereof, and be or comprise active parts or domains, preferably with a sequence identity of at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% in relation to the full-length polypeptide. In a preferred embodiment, the term “active parts or domains” is understood to mean a fragment of the full-length polypeptide or a variant which, in both cases, retains at least some of the biological activity of the full-length polypeptide.
It is essential that the variant of the full-length polypeptide has biological activity. In a preferred embodiment, biological activity is the capability of an agent, preferably polypeptide, to capture an autoantibody against the beta-subunit or to bind thereto, preferably bind specifically, which autoantibody occurs in samples from patients who have contracted AIG. The autoantibody from such samples can be obtained as reference for tests. Variants of a secondary antibody serving for the detection of a specifically captured antibody are all molecules which have the same binding specificity as the secondary antibody, preferably the capability of binding against all antibodies of human class IgG. For example, they encompass fragments and derivatives of the secondary antibody.
When producing variants or assessing whether they may exhibit biological activity, a person skilled in the art can orientate himself-herself to the work by da Silva el al. (da Silva, H. D., Gleeson, P. A., Toh, B. H., Van Driel, I. R. and Carbone, F. R. (1999) Identification of a gastritogenic epitope of the H/K ATPase beta-subunit, Immunology 1999, 96, 145-151). They identified SEQ ID NO6 as the peptide comprising the immunodominant epitope. This means that the homologous sequence from the human protein represented in SEQ ID NO5 contains the human epitope. By means of sequence alignments with further mammalian homologues, for example from pig, cattle, primates, goat and others, a person skilled in the art can easily predict reactive variants. By means of mutations and further truncations, a person skilled in the art can generate further variants. Carried out by means of ELISA, as in the examples, said person can confirm that biological activity is preserved.
At the same time, a polypeptide or a variant thereof does not bind, or only binds to a slight extent, against antibodies against the alpha-subunit, preferably the immunodominant epitope thereof, as described in SEQ ID NO8. Homologous epitopes from further homologous mammalian enzymes can be gathered from Nishio et al. and taken into consideration for the design of variants by a person skilled in the art (Nishio, A., Hosono, M., Watanabe, Y., Sakai, M., Okuma, M. and Masuda, T. (1994) Gastroenterology 107, 1408-1414). Preferably, the binding constant of autoantibodies against the beta-subunit, as occur in a sample from a patient, is at least 10, preferably 100, more preferably 1000, more preferably 10 000, more preferably 100 000, even more preferably 1 000 000, even more preferably 10 000 000, times lower—and the strength of the binding reaction accordingly higher—than the binding constant of autoantibodies against the alpha-subunit, as occur in a sample from a patient. The binding constant characterizes, then, the strength of binding of the particular autoantibody against the variant, more precisely a mixture of autoantibodies, as occur in a sample. The binding constant is, in this connection, preferably measured by means of Biacore in PBS buffer at 25° C. For example, the binding constant can be 10−10 M in the case of the binding of autoantibodies against the beta-subunit from a sample, whereas the binding constant of the same autoantibodies against the alpha-subunit from the sample is 10−6 M. The first binding constant is thus 10 000 times lower than the second one. This means that autoantibodies against the beta-subunit bind distinctly more strongly to the polypeptide or the variant than autoantibodies against the alpha-subunit. If the polypeptide or the variant is used according to the invention, only autoantibodies against the beta-subunit are thus bound and detected, but not antibodies against the alpha-subunit.
In a preferred embodiment, the term “autoantibody”, as used herein, is understood to mean an antibody which binds specifically to a structure, preferably an autoantigen, from the organism which produces said antibody. Such an autoantibody has a constant region, as is had by other antibodies of the same class from the same organism. Particularly preferably, the autoantibody is a mammalian autoantibody, even more preferably a human autoantibody, even more preferably a human autoantibody of class IgG. The variable domain is capable of binding specifically against the autoantigen. Preferably, the autoantigen is an epitope derived from or contained in the beta-subunit, preferably SEQ ID NO5 or a variant thereof.
In a preferred embodiment, the agent for capture is an isolated and/or purified polypeptide, which is particularly preferably recombinant. In this connection, an “isolated” polypeptide can be understood to mean that the polypeptide has been removed from its natural environment or the environment in which it had been expressed. In a preferred embodiment, a “purified” polypeptide is understood to mean a polypeptide which has been enriched with respect to the concentration and/or purity in its natural environment by using chromatographic methods, preferably selected from the group comprising affinity chromatography, gel-filtration chromatography and ion-exchange chromatography. In a particularly preferred embodiment, a polypeptide is purified when it has a purity of at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 990/or 99.5%, which can be estimated visually or, preferably, by measurement of band intensities on an SDS-PAGE gel after Coomassie staining.
A polypeptide can be provided in the form of a tissue or a cell, preferably a recombinant tissue or a recombinant cell. The cell is preferably a eukaryotic cell, more preferably an insect, plant or mammalian cell, even more preferably a mammalian cell such as a human cell, most preferably an HEK293 cell.
A polypeptide as agent can be provided in folded form or in unfolded form and is preferably folded. Preferably, folding is measured by means of CD spectroscopy in a buffer which allows the measurement and in which the protein can assume its three-dimensional folding (see, for example, Banaszak, L. J. (2008), Foundations of Structural Biology, Academics Press, or Teng, Q. (2013), Structural Biology: Practical Applications, Springer), for example in 10 mM sodium phosphate buffer at pH 7. Preferably, the protein assumes folding recognized by an antibody to be detected.
In a preferred embodiment, an antibody is, after capture, detected by a method from the group comprising the measurement of immunodiffusion, immunoelectrophoresis, light scattering, agglutination, radioactivity, enzymatic activity, (electro)chemiluminescence and immunofluorescence. In the case of a detection by radioactivity, enzymatic activity, (electro)chemiluminescence and immunofluorescence, it is possible to use a detectable label. Said label is preferably attached to a secondary antibody which binds to the antibody to be detected. Detection methods are, for example, described in Zane, H. D. (2001), Immunology—Theoretical & Practical Concepts in Laboratory Medicine, W. B. Saunders Company, particularly in chapter 14.
The invention provides a kit comprising the carrier according to the invention, the kit comprising one reagent or more than one reagent, preferably all the reagents, from the group comprising a wash solution, at least one calibrator solution, an antibody against the beta-subunit of the gastric H+/K+-ATPase, and an agent for the detection of the autoantibody, preferably a secondary antibody, even more preferably a secondary antibody against IgG antibodies, or the beta-subunit of the gastric H+/K+-ATPase or a variant thereof, preferably having a detectable label, a dilution buffer for the dilution of the sample, a positive control, a chemiluminescence trigger solution and a substrate capable of chemiluminescence. Instead of the chemiluminescence trigger solution and the substrate capable of chemiluminescence, a chromogenic solution e.g. for an ELISA can also be contained.
The autoantibody detection can be quantitative or semi-quantitative, preferably semi-quantitative. In a preferred embodiment, it is understood that “semi-quantitative” means that what is determined is whether the measurement result lies above a certain threshold value, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 RU/ml. The value in RU/ml is, in this connection, preferably measured by means of ELISA (EA 1361-9601 G) from EUROIMMUN Medizinische Labordiagnostika AG.
A calibrator solution, as used in the method according to the invention, is understood to mean a solution comprising a defined amount of an antibody which binds against the same antigen as the autoantibody to be detected and preferably belongs to the same IgG class as the antibody to be detected, the antigen preferably being the beta-subunit. The calibrator may comprise an amount of the antibody which is known in absolute or relative terms. For example, one calibrator solution may comprise ten times as much antibody as another one. The antibody present in a defined amount can be an autoantibody from a sample or a recombinant monoclonal antibody. Preferably, more than two, preferably 3, 4, 5 or 6, calibrator solutions having to different concentrations of the antibody are placed into the kit or used according to the invention, in particular investigated in parallel to a sample, with the result that a calibration curve for the semi-quantitative or quantitative determination of the autoantibody against the beta-subunit can be plotted. In a preferred embodiment, the calibrator comprises an autoantibody or antibody against the beta subunit of the gastric H+/K+-ATPase, preferably less or no detectable amounts of or no autoantibody or antibody against the alpha subunit.
According to the invention, a monoclonal antibody or isolated autoantibody which binds specifically against the beta-subunit of the gastric H+/K+-ATPase is used particularly preferably in recombinant, isolated and/or purified form, most preferably against SEQ ID NO5. Such an antibody can serve as positive control for methods according to the invention and also as reagent for the immobilization of the beta-subunit or as reagent for competitive assay formats, for which it can optionally have a detectable label.
In a preferred embodiment, the captured autoantibody is detected by means of chemiluminescence. For this purpose, the autoantibody can be contacted with a ligand which is capable of chemiluminescence and which binds to the autoantibody. A ligand capable of chemiluminescence can be a secondary antibody which binds to the constant domain of the autoantibody or can be the beta-subunit of the gastric H+/K+-ATPase or a variant thereof which binds to the variable domain. The ligand capable of chemiluminescence can be an enzyme capable of chemiluminescence, for example luciferase, peroxidase, alkaline phosphatase and β-galactosidase, which can convert a substrate capable of chemiluminescence without being consumed at the same time (Kricka, L. J. (2003). Clinical applications of chemiluminescence. Analytica chimica act, 500(1): 279-286). The ligand capable of chemiluminescence can, however, also comprise an organic compound capable of chemiluminescence and without enzymatic activity, which compound emits a chemiluminescent signal in the presence of a chemiluminescence trigger solution, for example an acridinium ester (Weeks, I., Beheshti, I., McCapra, F., Campbell, A. K., Woodhead, J. S., Acridinium esters as high specific activity labels in immunoassay. Clin Chem 29: 1474-1479 (1983)) or luminol or a derivative thereof capable of chemiluminescence. This compound is consumed in the reaction.
In the case of an acridinium ester, the trigger solution used is a mixture of hydrogen peroxide and hydroxide, for example in the form of sodium hydroxide. The acridinium ester compound is, then, oxidized under alkaline conditions, and this generates light-emitting acridone. The signal is released in the form of a light flash (flash luminescence) which typically lasts 1-5 s. The short duration of the emission requires that the reaction be initiated and measured directly at the detector.
Preferably, chemiluminescence as detection method is used with a carrier which is a bead.
In a further preferred embodiment, the captured autoantibody is detected by means of ELISA. For this purpose, the agent for specific capture is immobilized in at least one well, preferably multiple wells, preferably at least 2, 4, 6, 8, 10, 12, 16, 20 or 24 wells, of an ELISA microtitre plate. A sample to be analysed is added to at least one well, and 2, 3, 4, 5 or more calibrator solutions are added to other wells. The captured antibody is then preferably detected via a secondary antibody having an enzymatically active label.
According to the invention, use is made of an agent for the specific capture of an autoantibody against the beta-subunit of the gastric H+/K+-ATPase for the calibration or for the quality-control of a medical device. In a preferred embodiment, “calibration” is understood to mean that calibrator solutions are used to obtain measurement values which confirm that the instrument is providing reliable measurement values within a required concentration range or is allowing a reliable differentiation between the presence or absence of the autoantibody in a sample. In a further preferred embodiment, “control” can mean that what is verified is that the instrument is working reliably in the sense that a sample comprising the autoantibody is actually measured as positive and a sample without the autoantibody is actually measured as negative. In a further embodiment, “control” means that what is checked is whether a batch of the agent or an instrument under given conditions has the capability, preferably still sufficient capacity, to bind the autoantibody. The medical device can be a diagnostic product such as the inventive carrier. Alternatively, it can be a therapeutic device, for example for apheresis, which likewise comprises an agent for the specific capture or detection of the autoantibody.
In a preferred embodiment, the invention provides for use of a reagent or agent or polypeptide for the detection of an autoantibody against the beta-subunit or use of a nucleic acid encoding the beta-subunit or a variant thereof or a nucleic acid which hybridizes to said nucleic acid under stringent conditions or a vector or a cell comprising the nucleic acid or the vector for the production of a kit for the diagnosis of a disease. In a preferred embodiment, a cell comprising the vector, and preferably not comprising a vector encoding the alpha-subunit or an epitope or a variant thereof, is provided and cultured under conditions allowing the expression of the nucleic acid, followed by isolation and/or purification of the expression product, followed by use of the expression product as agent for the specific capture of an antibody in a method or product according to the present invention.
In a preferred embodiment, the present invention provides an apparatus for the analysis of a sample from a patient for the detection of an autoantibody against the beta-subunit, the detection indicating an increased probability of a disease, preferably AIG and/or PAG, the apparatus comprising:
The present patent application lists novel polypeptides and/or nucleic acids. They have the following sequences:
The invention is elucidated below on the basis of exemplary embodiments with reference to the figures. The embodiments described are merely exemplary in every respect and not to be understood as limiting, and various combinations of the cited to features are encompassed by the scope of the invention.
Sensitivity was investigated by using sera from patients for whom the diagnosis of AIG was made. On the other hand, specificity was investigated by using sera from clinically normal blood donors for whom it can be assumed that they do not suffer from AIG.
The human cDNA described by Genbank access number BC 167780 and encoding the ATP4A-subunit was ligated into the vector pTriEx (yielding SEQ ID NO11).
The human cDNA described by Genbank access number BC029059 and encoding the ATP4B-subunit was likewise ligated into the vector pTriEx (yielding SEQ ID NO12).
Production of Substrates for Indirect Immunofluorescence HEK293 cells cultured on glass slides in D-MEM comprising an addition of 10% (v/v) foetal calf serum were transfected with the plasmid DNA encoding ATP4A or ATP4B, individually and in combination, and additionally, for the production of a negative control, with the empty pTriEx vector by means of 25 kDa linear polyethyleneimine and fixed after 48 hours. For the fixation, the glass slides with the adherent cells were first washed with PBS and then fixed alternately with 1.8% formalin solution in PBS for 5 minutes and in acetone at room temperature for 10 minutes. Finally, a wash and blocking step was carried out in PBS comprising an addition of 1% (w/v) BSA (PBS (1% w/v BSA)) at room temperature for 5 minutes.
All the incubation steps were carried out at room temperature.
Immunofluorescence Analysis
The serum or plasma samples to be analysed were incubated on the substrate in a 1:10 dilution in PBS (1% w/v BSA) for 30 minutes. Thereafter, the substrates were washed in PBS (0.2% v/v Tween 20) for 5 minutes. Bound human immunoglobulin of class IgG was detected using fluorescein-labelled anti-human IgG in a 30-minute incubation step. Excess conjugate was removed with PBS (0.2% v/v Tween 20) for 5 minutes before evaluation on an immunofluorescence microscope. Immunofluorescence was evaluated semi-quantitatively by indication of the intensity of the specific fluorescent signal in intensities of 0 (no fluorescence), trace (borderline signal), 1 (faintly positive), 2, 3 and 4 (highly positive).
Purification of the Extracellular Domain of ATP4B (SEQ ID NO2) Expressed Using Vector pTriEx-1 in HEK293 Cells:
HEK293 cells cultured in D-MEM comprising an addition of 10% (v/v) foetal calf serum were transfected with the plasmid DNA encoding spGh*-H6-ATP4B(ec) by means of 25 kDa linear polyethyleneimine. On day 5 after transfection, the cell culture supernatant was removed, clarified by centrifugation, and used for the purification of the recombinant protein by means of immobilized metal-chelate affinity chromatography (IMAC) using standard protocols. The purified protein stained with Ponceau S is shown in
ELISA Analysis:
The experiments were carried out as described in Dahnrich et al. (2013) Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy, Clinica Chimica Acta 421, 213-218, with the exception of the extracellular domain of ATP4B (SEQ ID NO2) being used as the antigen.
In brief, microtitre plates (Nunc, Germany) were coated overnight with 0.7 μg/ml antigen in PBS at 4° C. and washed three times with wash buffer (0.05% (w/v) Tween 20 in PBS). Non-specific binding sites were blocked by means of a one-hour incubation with 0.1% casein (w/v) in PBS.
Samples of human serum were diluted 1:100 in sample buffer (0.05% (w/v) Tween 20, 1% (w/v) casein in PBS) and incubated for 30 minutes, followed by three wash steps with wash buffer. In addition to two positive samples from clinically characterized patients, seven further samples from patients were investigated.
Bound antibodies were detected by means of a 30-minute incubation with anti-human IgG HRP conjugate (EUROIMMUN, Germany) which was diluted 1:1000 in sample buffer. Thereafter, washing was carried out as described above, followed by addition of 3,3′,5,5′-tetramethylbenzidine (EUROIMMUN Medizinische Labordiagnostika AG) and a 15-minute incubation. All the incubation steps were carried out at room temperature.
Optical density (OD) was measured at 450 nm using an automatic spectrophotometer (Spectra Mini, Tecan, Crailsheim, Germany). The cut-off was ascertained by ROC curve analysis (maximum sum of sensitivity plus specificity).
For comparison, the assay was also carried out using the conventional anti-PCA ELISA (IgG) (EUROIMMUN Medizinische Labordiagnostika AG, order number EA 1361 G).
Results:
The experiments were carried out as described in Example 1, with the exception of use of sera from clinically normal blood donors.
Result:
The results show that, with the exception of the sample from the first-mentioned patient, it is also possible to detect the autoantibody against the gastric H+/K+-ATPase in the samples from patients suffering from AIG when only the extracellular domain of the beta-subunit of the gastric H+/K+-ATPase is used as antigen.
Thus, there is consequently practically no loss of sensitivity compared to the conventional ELISA.
Using the alpha-subunit of the gastric H+/K+-ATPase, practically all the samples appear, by contrast, as false-negative.
The results of Example 2 show that it is possible to achieve a considerable specificity gain of 6% compared to the conventional assay.
It should be emphasized that the conventional ELISA already exhibits a considerable specificity of 93%. Achieving a further increase in specificity to this high level is technically challenging in diagnostics and a considerable contribution over the related art.
Specificity and diagnostic reliability as a whole are increased when the method according to the invention is used.
Unless otherwise described, the experiment was carried out using the same reagents as described in Example 1.
In the present ChLIA, antigen-coated magnetic beads are used as solid phase for the specific detection of autoantibodies of immunoglobulin class IgG against ATP4B. The magnetic beads were produced using tosyl-activated M-280 Dynabeads (Invitrogen, catalogue number 14203, 14204) in accordance with the instructions from the manufacturer.
The assay is carried out in an automated manner in a random access analyser (RA Analyzer 10, product no. YG 0710-0101, EUROIMMUN Medizinische Labordiagnostika AG). In the first analysis step, coated magnetic beads are incubated with diluted patient samples in sample buffer (catalogue number LL9012, EUROIMMUN Medizinische Labordiagnostika AG). If the samples are anti-ATP4B-positive, specific antibodies bind to the antigen under these conditions.
In a second analysis step, acridinium ester-labelled anti-human IgG (catalogue number LK0711-G, EUROIMMUN Medizinische Labordiagnostika AG) conjugate is added, which binds to the specific antibodies.
Thereafter, the reaction mixture is admixed with trigger solutions (catalogue numbers LL0713-1 (A) and LL0713-2 (B), EUROIMMUN), which induce a chemiluminescence reaction.
The resultant light signal is outputted in relative light units (RLU). The intensity of the light signal is proportional to the quantity of the bound antibodies.
The results are shown in Table 3. A check with 20 sera from blood donors showed a specificity of 100%; with the conventional assay, it was only 95%. Data relating to sensitivity are shown in Table 4 and
| Number | Date | Country | Kind |
|---|---|---|---|
| 18213659.8 | Dec 2018 | EP | regional |
| 19171829.5 | Apr 2019 | EP | regional |
