NMDA receptor constructs for detecting and isolating nmdar autoantibodies

Abstract

To provide improved NMDAR protein constructs for detection of NMDAR autoantibodies and for treatment of autoimmune diseases associated with NMDAR autoantibodies.SOLUTION: Provided is a soluble N-methyl-D-aspartate receptor (NMDAR) protein construct comprising one or more NMDAR autoantibody epitopes, the construct comprising an extracellular domain (ECD) of the NMDAR subunit GluN1 or a fragment thereof and an ECD of at least one of the NMDAR subunits GluN2A, GluN2B, GluN2C or GluN2D, or fragment thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a soluble N-methyl-D-aspartic acid receptor (NMDAR) protein construct comprising one or more NMDAR autoantibody epitopes, wherein the construct comprises an extracellular domain (ECD) of the NMDAR subunit GluN1 or a fragment thereof, and an ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or a fragment thereof. Further, the present invention relates to an in vitro method for detecting NMDAR autoantibodies in a sample, comprising: a.) preparing a sample suspected of containing NMDAR autoantibodies; b.) preparing the NMDAR protein construct of the present invention as a capture molecule; c.) contacting the sample with the NMDAR protein construct, thereby binding NMDAR autoantibodies from the sample to the NMDAR protein construct; and d.) determining the presence and optionally the amount of the bound NMDAR autoantibodies. In an embodiment, the method of the present invention is applied to the diagnosis, prognosis prediction, disease monitoring, patient stratification and / or treatment monitoring of medical conditions related to autoantibodies against NMDAR, preferably anti-NMDAR encephalitis.

Background Art

[0002] Anti-NMDA receptor encephalitis (NMDAR encephalitis) is the most common form of the increasing number of autoimmune encephalitides (Non-Patent Document 1, Non-Patent Document 2). This disorder mainly affects young women, and patients present with psychiatric and neurological symptoms including seizures and dyskinesia, along with memory loss, hallucinations, and delusions. Current treatment options include glucocorticoids, plasma exchange, along with rituximab and cyclophosphamide. NMDAR encephalitis is caused by the production of autoantibodies that target the extracellular region of the major NMDA receptor subunit GluN1 both in the blood and in the brain. The antibodies alter surface dynamics, induce cross-linking and internalization of the NMDA receptor, and the resulting depletion of the NMDA receptor can account for some of the neurological symptoms observed in patients (Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5). A single recombinant human antibody against GluN1 derived from CSFB cells that induced down-regulation of NMDA receptor function (Non-Patent Document 6) suggests that they are the main pathogenic agents of this disease. This speculation has recently been strengthened by mouse models using active and passive immunization (Non-Patent Document 3, Non-Patent Document 7, Non-Patent Document 8).

[0003] Unlike other forms of antibody-mediated encephalitis and many autoimmune diseases, NMDAR encephalitis does not appear to be associated with specific HLA class II types (Non-Patent Document 9, Non-Patent Document 10). A significant number of female NMDAR encephalitis patients have ovarian teratomas. Interestingly, some of the antibody-secreting cells isolated from the brains of NMDAR encephalitis patients expressed non-mutated / germline antibodies against the NMDA receptor (Non-Patent Document 6, Non-Patent Document 11). The origin of the autoimmune reaction has not been fully elucidated, but it is possible that the majority of the population may have NMDA receptor antibodies, and the presence of NMDA receptor autoantibodies in the serum may constitute a common problem in pregnant women and the elderly with blood-brain barrier disorders. Furthermore, NMDA receptor autoantibodies have been found in patients with neuropsychiatric diseases other than NMDAR encephalitis and are highly likely to contribute to the progression of the disease (Non-Patent Document 4).

[0004] Immunosuppression and plasma exchange or intravenous immunoglobulin are established treatments for NMDAR encephalitis. However, selective removal of the disease-causing antibodies would be preferred as it is expected to show lower side effects. Depleting serum of NMDA receptor antibodies can be applied to NMDAR encephalitis and other disorders associated with NMDA receptor autoantibodies using extracorporeal techniques similar to, for example, plasma exchange. A prerequisite for such an apheresis modality would be the generation of stable proteins from the patient that can bind to NMDA receptor autoantibodies, i.e., soluble antigens against NMDA receptor antibodies.

[0005] The NMDA receptor is assembled from a major GluN1 subunit and regulatory GluN2 / 3 subunits (Non-Patent Document 12). Heterologous expression of GluN1 and its deletion mutants revealed that human IgG isolated from NMDAR encephalitis patients bound to a specific extracellular region called the amino-terminal domain (ATD) of GluN1 (Non-Patent Document 13). Mutations of amino acids in the hinge region of this clamshell-like domain (N368 / 9) suppressed antibody binding, suggesting that the closure of the clamshell or post-translational modification of these amino acids affects or constitutes the epitope recognized by the antibody. Furthermore, the ATD conformation is related to channel opening, and antibodies preferentially bind to the receptor in the activated state (Non-Patent Document 13). The native core NMDA receptor functions as a dimer of GluN1-GluN2 dimers. Within this structure, the ATD of GluN1 directly interacts with the ATD of the GluN2 subunit (Non-Patent Document 14), and their conformations change cooperatively between receptor activation and inhibition (Non-Patent Document 15, Non-Patent Document 16, Non-Patent Document 17). Furthermore, NMDA receptor antibodies can be directed against specific NMDA receptor subtypes defined by the GluN2 subunit in some patients. Antigens that include the GluN2 extracellular domain may therefore be preferred over antigens that contain only GluN1.

[0006] Autoantibodies against NMDA receptors are routinely detected using the Euroimmun cell-based assay (CBA) kit based on a biochip containing acetone-fixed GluN1-expressing heterologous cells, as described in Patent Document 1. However, NMDA receptor autoantibodies often recognize the native conformation of their antigens, and live staining of NMDA receptor-expressing HEK293 cells has been found to be more sensitive than commercial assays using pre-fixed cells for detecting low titers (Non-Patent Document 18). These cell-based assays require visual inspection of results that may be subject to bias. Furthermore, even if routine testing can be automated in such assays, the fact that there are many different antigens on the cell surface in addition to the antigen of interest makes false positive results more likely to occur.

[0007] Therefore, there is an urgent need for a highly sensitive and quantifiable high-throughput method for detecting NMDA receptor autoantibodies. In this direction, a single nanoparticle imaging approach for primary hippocampal neurons can detect NMDA receptor autoantibodies at low titers and is automatable, but is very difficult technically (Non-Patent Document 18). An ELISA that enables comparison of different titers of NMDA receptor autoantibodies based on lysed HEK293 cells expressing the NMDA receptor has been described at an early stage (Non-Patent Document 19). Patent Document 2 discloses a method for detecting antibodies against NR2A (GluN2A) and / or NR2B (GluN2B) in relation to a method for diagnosing stroke in which the amino-terminal fragments of NR2A / NR2B are synthesized and purified, but does not include a combination with the ECD of GluN1. In fact, the disclosed method identifies antibodies specifically directed against GluN2A or GluN2B, has no intention of identifying antibodies directed against GluN1, is unrelated to their association with GluN1, and thus is intended for a completely different use compared to the present invention. Importantly, the method of Patent Document 2 cannot be used to identify antibodies that bind to GluN1 or a part thereof.

[0008] In recent years, cell lines expressing the entire amino-terminal region of GluN1 (amino acids 1 to 561, including the ATD and S1 domains) fused to myc and polyhistidine tags, a tobacco etch virus (TEV) cleavage site, and the transmembrane region of the PDGF receptor have been presented (Non-Patent Document 20). TEV treatment of these cells released the amino-terminal extracellular segment of GluN1. This fragment could be bound to an ELISA plate via an anti-polyhistidine antibody, and monoclonal anti-GluN1 antibodies could be detected. However, the detection sensitivity of this antigen may be limited. In summary, there is no available stable soluble antigen of the NMDA receptor that maintains its native conformation and incorporates GluN1 and GluN2 segments for detecting autoantibodies present, for example, in serum or CSF, for example, in ELISA.

[0009] Therefore, there remains a strong need in the art for the provision of NMDAR protein constructs comprising one or more NMDAR autoantibody epitopes that can be composed of or stabilized by GluN1 and the extracellular domains of GluN2A, GluN2B, GluN2C, and / or GluN2D, or fragments thereof.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0012] In view of the prior art, the technical problem underlying the present invention is to provide an improved NMDAR protein construct for the detection of NMDAR autoantibodies and the treatment of autoimmune diseases associated with NMDAR autoantibodies.

Means for Solving the Problem

[0013] This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

[0014] NMDAR protein construct Accordingly, the present invention relates to a soluble N-methyl-D-aspartic acid receptor (NMDAR) protein construct comprising one or more NMDAR autoantibody epitopes, the construct comprising an extracellular domain (ECD) of the NMDAR subunit GluN1 or a fragment thereof, and an ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or a fragment thereof. In an embodiment, the NMDAR protein construct comprises an extracellular domain (ECD) of the NMDAR subunit GluN1 or a fragment thereof, and an ECD of the GluN2A or a fragment thereof and / or the GluN2B or a fragment thereof of the NMDAR subunit.

[0015] Furthermore, the present invention relates to an N-methyl-D-aspartic acid receptor (NMDAR) protein construct lacking an NMDAR transmembrane domain and containing one or more NMDAR autoantibody epitopes, wherein the construct comprises an extracellular domain (ECD) or a fragment thereof of the NMDAR subunit GluN1 and an ECD or a fragment thereof of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D. In an embodiment, the NMDAR protein construct lacking an NMDAR transmembrane domain comprises an extracellular domain (ECD) or a fragment thereof of the NMDAR subunit GluN1 and an ECD or a fragment thereof of the NMDAR subunit GluN2A and / or an ECD or a fragment thereof of GluN2B.

[0016] Furthermore, the present invention relates to an N-methyl-D-aspartic acid receptor (NMDAR) protein construct containing one or more NMDAR autoantibody epitopes, wherein the construct comprises an extracellular domain (ECD) or a fragment thereof of the NMDAR subunit GluN1, an ECD or a fragment thereof of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, and a dimerization domain. In an embodiment, the NMDAR protein construct comprises an extracellular domain (ECD) or a fragment thereof of the NMDAR subunit GluN1, an ECD or a fragment thereof of the NMDAR subunit GluN2A and / or an ECD or a fragment thereof of GluN2B, and a dimerization domain.

[0017] In addition, the present invention relates to an N-methyl-D-aspartic acid receptor (NMDAR) protein construct comprising one or more NMDAR autoantibody epitopes, wherein the construct comprises an extracellular domain (ECD) of the NMDAR subunit GluN1 or a fragment thereof, and an ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or a fragment thereof, and is present neither intracellularly nor on the cell surface. In an embodiment, the NMDAR protein construct comprises an extracellular domain (ECD) of the NMDAR subunit GluN1 or a fragment thereof, and an ECD of the NMDAR subunit GluN2A or a fragment thereof and / or an ECD of GluN2B or a fragment thereof, and is present neither intracellularly nor on the cell surface.

[0018] The NMDAR protein construct of the present invention can be used for screening NMDA receptor autoantibodies in a patient's serum or CSF.

[0019] The data disclosed herein show that the soluble NMDAR protein construct of the present invention, particularly in embodiments as Fc fusion proteins, can detect antibodies in the sera of patients with anti-NMDA receptor encephalitis. The soluble NMDAR-Fc protein construct (srNR-Fc protein) has several advantages over the state-of-the-art cell- and cell-based assays that use NMDAR expressed on the surface of in vitro cultured cells to detect antibodies. The srNR-Fc protein can be purified and stored. The purified srNR-Fc protein constitutes a clean antigen as compared to heterogeneous cells containing many additional proteins. They enable the generation of very high antigen concentrations that lead to an improvement in sensitivity in antibody detection as compared to cell-based assays (CBA).

[0020] In contrast to known NMDAR constructs of the prior art, the present invention relates to an NMDAR construct comprising at least fragments of two NMDAR subunits that enables the identification of autoantibodies that bind to epitopes formed by residues of two subunits or that are formed or stabilized only by the assembly of two subunits. In contrast, constructs of the prior art that contain only one subunit or a fragment thereof do not bind to and remove such autoantibodies.

[0021] By using the protein constructs of the present invention for the recognition of NMDAR-specific antibodies in a sample, antibodies that bind only to NMDAR and GluN1 can be identified when GluN1 is associated with GluN2 subunits, which leads to the stabilization of the respective epitopes of such antibodies. Since each epitope is stabilized or formed only by the association of GluN1 with the respective GluN2 subunit, such antibodies cannot be identified by a protein or protein construct containing only the GluN1 or GluN2 subunit.

[0022] For example, the NMDAR protein constructs disclosed herein that contain an Fc fragment or other or additional tags may be useful in various diagnostic assays. For example, the diagnostic assay in the form of an ELISA-like assay disclosed in the examples can be used as a companion diagnostic that enables a quantitative high-throughput method for detecting NMDA receptor autoantibodies and detecting autoantibodies in other autoimmune encephalopathies.

[0023] Furthermore, the NMDAR protein constructs can be used to distinguish autoimmune responses to different NMDA receptor compositions that enable the classification of patients.

[0024] As disclosed in the examples provided herein, various srNR-Fc fusion proteins formed by such fusion proteins and NMDAR protein constructs such as dimers produce different signals in response to antibodies / sera from different patients, indicating a patient-specific variant antibody profile. Thus, a set containing different NMDAR protein constructs of the present invention, including different combinations of the ECDs and ATDs of GluN1, GluN2A, GluN2B, GluN2C, and GluN2D, such as the set of srNR-Fc fusions described in the examples, can be used to classify patient-specific antibody profiles, i.e., to distinguish antibodies mainly directed against GluN1 or heteromeric GluN1 / GluN2 structures, and potentially GluN1 / GluN2 structures of a specific composition. The subclassification of NMDAR encephalitis patients may ultimately lead to improved treatment methods.

[0025] Accordingly, the present invention also relates to a set or kit providing two or more NMDAR protein constructs of the present invention, which provide different combinations of GluN1 and one or more of GluN2A - GluN2D, or fragments thereof.

[0026] In an embodiment, the present invention relates to two NMDAR protein constructs of the present invention, one construct comprising the ECD of GluN1 and GluN2A, or a fragment thereof, and the other construct comprising the ECD of GluN1 and Glu2B, or a fragment thereof.

[0027] In an embodiment, the present invention relates to two NMDAR protein constructs of the present invention, one construct comprising the ECD of GluN1 and GluN2A, or a fragment thereof, and the other construct comprising the ECD of GluN1 and Glu2C, or a fragment thereof.

[0028] In an embodiment, the present invention relates to two NMDAR protein constructs of the present invention, one construct comprising the ECDs of GluN1 and GluN2A, or a fragment thereof, and the other construct comprising the ECDs of GluN1 and Glu2D, or a fragment thereof.

[0029] In an embodiment, the present invention relates to two NMDAR protein constructs of the present invention, one construct comprising the ECDs of GluN1 and GluN2C, or a fragment thereof, and the other construct comprising the ECDs of GluN1 and Glu2B, or a fragment thereof.

[0030] In an embodiment, the present invention relates to two NMDAR protein constructs of the present invention, one construct comprising the ECDs of GluN1 and GluN2D, or a fragment thereof, and the other construct comprising the ECDs of GluN1 and Glu2B, or a fragment thereof.

[0031] In an embodiment, the present invention relates to two NMDAR protein constructs of the present invention, one construct comprising the ECDs of GluN1 and GluN2C, or a fragment thereof, and the other construct comprising the ECDs of GluN1 and Glu2D, or a fragment thereof.

[0032] The NMDAR protein constructs of the present invention can comprise four ECDs of GluN subunits that mimic the tetrameric assembly of NMDAR. For example, the constructs of the present invention can comprise two GluN1 ECDs or fragments thereof, and two identical or different ECDs of GluN2A, GluN2B, GluN2C, and / or GluN2D.

[0033] Also, constructs that enable the labeling of B cells expressing NMDA receptor autoantibodies can be used as diagnostic tools and facilitate the isolation of cells and IgG sequence analysis.

[0034] The NMDAR (NMADR) protein constructs of the present invention can be used to detect not only soluble NMDAR autoantibodies but also NMDAR autoantibodies expressed on the surface of B cells present in the sera and CSF of patients. Labeling of B cells expressing NMDA receptor autoantibodies can also be used as a diagnostic tool.

[0035] Furthermore, such constructs can be used to selectively remove antibodies against the NMDA receptor from the sera or CSF of patients.

[0036] Current apheresis protocols deplete serum almost completely of all IgG. The (soluble) NMDAR protein constructs of the present invention can be non-covalently or covalently bound to agarose / sepharose beads or different matrix materials. The resulting matrix can be used to specifically immunodeplete NMDAR autoantibodies from the sera of patients. This approach is efficient and avoids all side effects associated with complete immunodepletion, such as severe infections or impaired wound healing. The immune system is not weakened by this method, in contrast to current treatment options.

[0037] All of the following further preferred embodiments of the invention relate to each NMDAR protein construct described above.

[0038] In an embodiment of the invention, the construct of the invention lacks the NMDAR transmembrane domain.

[0039] Furthermore, the construct can include a dimerization domain and / or a capture domain.

[0040] Importantly, in certain constructs of the invention, the dimerization domain is the capture domain.

[0041] In an embodiment, the dimerization domain includes a leucine zipper and / or a coiled coil domain.

[0042] In certain embodiments of the present invention, the dimerization domain and / or the capture domain comprises or consists of an antibody Fc fragment. In an embodiment, the Fc fragment is a fragment of rabbit IgG Fc.

[0043] The presence of the dimerization and / or capture domain, such as an Fc fragment, can be particularly advantageous since the soluble NMDAR protein construct is stabilized by these domains. In particular, the Fc portion enables long-term storage of constructs with a conserved three-dimensional structure.

[0044] In particular, in the NMDAR protein construct of the present invention, the ECD of GluN1 or a fragment thereof comprises or consists of the amino-terminal domain (ATD) of GluN1 or a fragment thereof.

[0045] In connection with the construct of the present invention, the ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or a fragment thereof, each comprises or consists of the ATD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or a fragment thereof.

[0046] Furthermore, the ECD of GluN2A or a fragment thereof and / or the ECD of GluN2B or a fragment thereof may each comprise or consist of the ATD of GluN2A or a fragment thereof and / or the ATD of GluN2B or a fragment thereof.

[0047] In a particular NMDAR protein construct of the present invention, the ECD of GluN1 and the ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or a fragment thereof, are preferably covalently linked as a fusion protein. Among them, the ECD of GluN1 and the ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or a fragment thereof, may be directly or by a protein linker linked as a fusion protein. In an embodiment, the ECD of GluN1 and the ECD of GluN2A and / or GluN2B, or a fragment thereof, are preferably covalently linked as a fusion protein. Among them, the ECD of GluN1 and the ECD of GluN2A and / or GluN2B, or a fragment thereof, may be directly or by a protein linker linked as a fusion protein.

[0048] In an embodiment, the ECD of GluN1 and the ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or a fragment thereof, are linked by a protein linker comprising one or more repeats of the amino acid sequence GGGGS or consisting of the same.

[0049] In an embodiment, the ECD of GluN1 and the ECD of GluN2A and / or GluN2B, or a fragment thereof, are linked by a protein linker comprising one or more repeats of the amino acid sequence GGGGS or consisting of the same.

[0050] In a particular embodiment of the present invention, the construct comprises one or more protease cleavage sites such as a TEV cleavage site or any other cleavage site recognized by a suitable protease known to those skilled in the art between a part of the construct comprising the ECD of GluN1, the ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or a fragment thereof, and a part of the construct comprising a dimerization domain and / or a capture domain.

[0051] In an embodiment, the construct includes one or more protease cleavage sites, such as a TEV cleavage site or any other cleavage site recognized by a suitable protease known to those skilled in the art, between a portion of the construct that includes the ECD of GluN1, the ECD of GluN2A, and / or the ECD of GluN2B, or a fragment thereof, and a portion of the construct that includes a dimerization domain and / or a capture domain.

[0052] In a preferred embodiment of the NMDAR protein construct of the present invention, the construct is a protein dimer of non-covalently bound monomers, and the construct can be a homodimer or a heterodimer. In certain dimeric constructs of the present invention, the construct can be a heterodimer formed from the ECD of GluN1 or a fragment thereof (as one monomer) and the ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or a fragment thereof (as one monomer). In an embodiment of the dimeric construct, the construct can be a heterodimer formed from the ECD of GluN1 or a fragment thereof (as one monomer) and the ECD of GluN2A or a fragment thereof, and / or the ECD of GluN2A or a fragment thereof. Preferably, the dimer is formed by a dimerization domain composed of each monomer. In a further embodiment, the NMDAR protein construct of the present invention can be a dimer of two monomers, and each monomer contains two ECDs of two NMDAR subunits, such as GluN1 and GluN2A, GluN1 and GluN2B, GluN1 and GluN2C, or GluN1 and GluN2D, or fragments thereof. Thus, it is possible to provide an NMDAR protein construct that mimics a native NMDA receptor (NMDAR, NMDAR receptor), which is a tetramer of four ECDs (provided by two monomers) of NMDAR subunits, or fragments thereof, and thus four subunits, i.e., two GluN1 subunits and two GluN2 subunits. For example, the dimerization of the fusion protein N1-ATD-N2B-ATD-Fc disclosed herein results in the formation of an NMDAR protein construct of the present invention formed by two N1-ATD-N2B-ATD-Fc monomers containing four ATD domains.

[0053] The NMDAR protein constructs of the present invention are advantageous compared to known NMDAR constructs as they allow for the assembly of the ECD or ATD of the GluN1, GluN2A, GluN2B, GluN2C, and / or GluN2D subunits in a given construct. Autoantibodies specific to a particular subunit or combination of subunits can be efficiently bound by the constructs of the present invention, and the potential of this ligand combination is a strong advantage as a series of autoantibodies present in a sample can be evaluated for their differential binding profiles. Furthermore, the ability to combine the ECD or a fragment of the ECD of the GluN2A, GluN2B, GluN2C, and / or GluN2D subunits with a capture domain allows for the flexible use of the constructs, for example, in the context of an ELISA assay, which is advantageous compared to state-of-the-art cell-based ELISA assays.

[0054] Subunit assemblies containing both domains from GluN1 and GluN2, particularly GluN2A, GluN2B, GluN2C, and / or GluN2D, reconstruct the native situation more closely than the presentation of only the GluN1 subunit as in state-of-the-art assays. By combining ligands / subunits, more complete binding, detection, and / or removal of pathogenic autoantibodies can be achieved. For example, as shown in the examples disclosed herein, preferred combinations of subunits in the NMDAR protein constructs of the present invention can include GluN1-ATD and GluN2B-ATD, or GluN1-ECD and GluN2B-ECD, either in a single fusion protein or in a construct containing two proteins that form a heterodimer by assembly via a dimerization domain such as an Fc domain. In the examples, the combinations of fusion protein #1 (N1-ATD-Fc) and fusion protein #6 (N2B-ATD-Fc), fusion protein #8 (N1-ATD-N2B-ATD-Fc) and fusion protein #4 (N1ecd-N2Becd-Fc) were shown to be particularly advantageous for the binding of autoantibodies present in patient samples.

[0055] An in vitro method for detecting NMDAR autoantibodies in a sample Furthermore, the present invention is an in vitro method for detecting NMDAR autoantibodies in a sample, comprising: Preparing a sample suspected of containing NMDAR autoantibodies; Preparing the NMDA protein construct according to any one of the above claims as a capture molecule; Contacting the sample with the NMDAR protein construct, thereby binding NMDAR autoantibodies from the sample to the NMDAR protein construct; Determining the presence, and optionally the amount, of the bound NMDAR autoantibodies; and relates to a method comprising the steps of:

[0056] In an embodiment of the method for detecting NMDAR autoantibodies of the present invention, the NMDAR autoantibodies in the sample are present in solution or on the cell membrane.

[0057] In an embodiment, the method for detecting NMDAR autoantibodies is carried out using a plurality of different NMDAR protein constructs in the sense of the present invention. With respect to the method of the present invention using a plurality of different constructs, the method may additionally comprise the step of determining to which NMDAR protein construct of the plurality of constructs the NMDAR autoantibodies bind, or preferably bind in the largest amount and / or most efficiently. Thus, it may be possible to profile and classify patients providing samples based on the NMDAR autoantibodies determined and their binding characteristics with respect to the NMDAR protein constructs used in the method of the present invention. In this situation, the method of the present invention may be carried out separately for each of the plurality of constructs (parallel determination), or the binding of NMDAR autoantibodies to two or more NMDAR constructs may be determined in a single assay (multiplexing).

[0058] The method of the present invention can include the step of determining the NMDAR autoantibody profile present in the sample.

[0059] In an embodiment of the method of the present invention, the presence and optionally the amount of cells presenting NMDAR autoantibodies on the cell surface present in the above sample may be determined. A major advantage of the method of the present invention is that, together with soluble NMDAR autoantibodies, it is possible to detect NMDAR autoantibodies on the cell surface, particularly on the surface of B cells producing NMDAR autoantibodies.

[0060] In an embodiment, the method of the present invention is applied to the diagnosis, prognosis prediction, disease monitoring, patient stratification and / or treatment monitoring of a medical condition associated with autoantibodies against NMDAR, preferably anti-NMDAR encephalitis, and the sample suspected of containing NMDAR autoantibodies is a sample from a human subject presenting symptoms of having the above medical disorder.

[0061] In an embodiment of the method for diagnosis, prognosis prediction, disease monitoring, patient stratification and / or treatment monitoring of a medical condition associated with autoantibodies against NMDAR, preferably anti-NMDAR encephalitis, the presence of bound NMDAR autoantibodies preferably indicates the presence or the likelihood of a subject having a medical condition associated with autoantibodies against NMDAR, preferably developing anti-NMDAR encephalitis, when the amount of bound NMDAR autoantibodies exceeds an appropriate control such as the amount from a healthy control population.

[0062] The current diagnosis is based on tissue profiling or CBA of the GluN1 subunit of the NMDA receptor. Compared to these, the method of the present invention based on the NMDAR (NMADR) protein construct offers several advantages. For example, the method of the present invention enables the binding or capture of autoantibodies that bind to GluN1 in relation to the GluN2 subunit, and thus also captures autoantibodies that bind to overlapping epitopes or sterically stabilized epitopes. Furthermore, the method of the present invention enables the discrimination of preferential binding of autoantibodies to, for example, GluN1 and GluN2A versus GluN1 and GluN2B versus individual subunits versus GluN1 and GluN2C versus GluN1 and GluN2D, and further combinations of two or more isoforms of the GluN1 and GluN2 subunits.

[0063] A further advantage of the detection method disclosed herein is the fact that it is adaptable to the robustness of the assay and to standardization, such as sFIDA, and even to full automation. Furthermore, the assay can be optimized to function as a single molecule level assay (e.g., SIMOA), thereby achieving quantification of autoantibodies. Moreover, the method of the present invention is more sensitive than state-of-the-art assays for detecting NMDAR autoantibodies due to the reduction of the cell background of mammalian cells, preferably human cells such as HEK cells.

[0064] In the context of the method of the present invention, the NMDAR protein construct can be immobilized on a solid phase before contacting the above sample.

[0065] In an embodiment of the present invention, the soluble NMDAR protein construct of the present invention is provided in an immobilized form. In such an embodiment, the soluble NMDAR protein construct of the present invention may be immobilized on a solid phase after being purified in its soluble form from a suitable expression system. Thus, in the context of such an embodiment of the immobilized NMDAR protein construct of the present invention, "soluble" relates to the previous state of the construct before immobilization.

[0066] Furthermore, the method of the present invention can be implemented as an enzyme-linked immunosorbent assay (ELISA).

[0067] Determination of NMDAR autoantibodies in the context of the method of the present invention involves the following steps: immobilizing NMDAR autoantibodies from a sample by binding them to an NMDAR protein construct immobilized on a solid surface; treating the immobilized NMDAR autoantibodies with a labeled secondary affinity reagent directed against the NMDAR autoantibodies; detecting a signal emitted from the labeled secondary affinity reagent directed against the NMDAR autoantibodies; comparing the signal obtained from the labeled secondary affinity reagent with signals from one or more control samples of pre-determined NMDAR autoantibody concentrations; and can include.

[0068] In an embodiment, the signal is obtained from horseradish peroxidase conjugated to the secondary affinity reagent. In a further embodiment, other labels of the secondary affinity reagent such as fluorescent labels or chemiluminescent labels, and further labels known to those skilled in the art can be used.

[0069] In certain embodiments, the method of the present invention is applied to the treatment guidance of a subject having and / or suspected of developing a medical condition related to NMDAR autoantibodies, and the method includes selecting one or more corresponding NMDAR protein constructs of the present invention for the subsequent treatment of the subject.

[0070] Kit for detecting NMDAR autoantibodies The present invention also relates to a kit for detecting NMDAR autoantibodies in a sample, the kit comprising The NMDAR protein construct of the present invention, and optionally a solid surface for immobilizing the NMDAR protein construct, or the NMDAR protein construct of the present invention immobilized on a solid surface, and a labeled secondary affinity reagent directed to a human NMDAR autoantibody such as a labeled anti-human IgG antibody, and optionally means for detecting a signal emitted from the label, or, the labeled NMDAR protein construct of the present invention, and optionally means for detecting a signal emitted from the label, and optionally, a control sample of a pre-determined NMDAR autoantibody concentration, and comprises.

[0071] The kit of the present invention can be used to detect NMDAR autoantibody-expressing cells, for example by FACS, using the fluorescently labeled construct of the present invention or a fluorescently labeled secondary antibody directed against rabbit Fc. Furthermore, the kit can be used to perform an ELISA for detecting NMDAR autoantibodies present in a sample.

[0072] The present invention also relates to a kit for diagnosing an autoimmune disease associated with an NMDAR autoantibody such as NMDAR encephalitis in a subject by detecting the NMDAR autoantibody, the NMDAR protein construct of the present invention and optionally a solid surface for immobilizing the NMDAR protein construct, or the NMDAR protein construct of the present invention immobilized on a solid surface, and a labeled secondary affinity reagent directed to a human NMDAR autoantibody such as a labeled anti-human IgG antibody, and optionally means for detecting a signal emitted from the label, or, the labeled NMDAR protein construct of the present invention, and optionally, a control sample of a pre-determined NMDAR autoantibody concentration, and relates to a kit comprising.

[0073] A blood treatment device comprising an NMDAR protein construct Furthermore, the present invention relates to a blood treatment device configured to remove NMDAR autoantibodies from the blood or plasma of a person in need of treatment in an extracorporeal blood circuit, the device comprising a matrix having one or more NMDAR protein constructs of the present invention immobilized thereon.

[0074] In an embodiment, the blood treatment device of the present invention is placed within an extracorporeal blood circuit through which a patient's blood passes, and comprises means for transporting blood from the patient's vascular system to the blood treatment device at a defined flow rate and returning the treated blood to the patient.

[0075] A further aspect of the present invention Furthermore, the present invention includes the NMDAR protein constructs disclosed herein for use as a medicament. The present invention also relates to the NMDAR protein constructs disclosed herein for use as a medicament in the treatment of a subject suffering from an autoimmune disease associated with NMDAR autoantibodies, preferably NMDAR encephalitis.

[0076] The present invention also relates to an in vitro method for producing the NMDAR protein constructs of the present invention, the method comprising expressing a nucleic acid sequence encoding the NMDAR protein constructs of the present invention in mammalian cells, preferably human cells, and subsequent isolation of the NMDAR protein constructs. Preferably, the construct is isolated from the cell supernatant after secretion of the protein construct by the cells.

[0077] A major advantage of the present invention is that a soluble NMDAR protein construct can be isolated from the cell culture supernatant of cells modified to express the nucleic acid sequence encoding the NMDAR protein construct of the present invention, particularly as compared to methods that require first isolating the antigen from the cell membrane using either protease cleavage or detergent solubilization.

[0078] Furthermore, the present invention includes the NMDAR protein constructs disclosed herein, which are produced by the disclosed methods for producing the NMDAR protein constructs of the present invention.

[0079] The various embodiments and features of the NMDAR protein constructs disclosed herein also apply to the various embodiments of the methods for detecting NMDAR autoantibodies in a sample, kits for detecting NMDAR autoantibodies, blood treatment devices configured to remove NMDAR autoantibodies from the blood or plasma of a person in need of treatment, and methods for producing the NMDAR protein constructs of the present invention presented herein, and vice versa.

[0080] Detailed Description of the Invention All cited documents, including patent documents and non-patent documents, are hereby incorporated by reference in their entirety and form a part of this specification.

[0081] The present invention relates to a soluble NMDAR protein construct comprising one or more NMDAR autoantibody epitopes, wherein the construct comprises an extracellular domain (ECD) of the NMDAR subunit GluN1 or a fragment thereof, and an ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or a fragment thereof.

[0082] In the context of the present invention, the term "protein construct" may relate to an individual protein or peptide formed by a single amino acid chain. Further, as used herein, the term "protein construct" also includes, for example, two or more proteins, or constructs or complexes of peptides or amino acid chains, covalently linked by, for example, disulfide bridges between individual amino acid chains or other linkers. Further, the term "protein construct" includes protein complexes formed by two or more proteins, or peptides or amino acid chains, by non-covalent interactions such as non-covalent bonds or electrostatic interactions, van der Waals forces, hydrophobic interactions, or others known to those skilled in the art, for example leading to the formation of protein dimers or protein multimers that can be assembled via dimerization or multimerization domains, respectively.

[0083] In an embodiment of the present invention, the protein construct is one or more proteins comprising an extracellular domain (ECD) of the NMDAR subunit GluN1 or a fragment thereof, and an ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or a fragment thereof. Among them, the one or more proteins can be a single protein containing both ECDs or fragments thereof in a single amino acid chain, or the one or more proteins can be, for example, two proteins, each of which contains one or more ECDs or fragments thereof of GluN1 or GluN2 (A, B, C or D), and the two proteins are assembled into a protein complex. In embodiments comprising two (or more) proteins, the two (or more) proteins can be assembled into a complex in which the proteins are covalently linked, for example, by disulfide bridges or other linkers, or in which the proteins assemble by non-covalent bonds / interactions.

[0084] "Peptide", "polypeptide", "polypeptide fragment", "amino acid chain", and "protein" are used interchangeably unless otherwise specified and are used in their ordinary sense, i.e., as a sequence of amino acids. Polypeptides are not limited to a particular length; for example, they can include full-length protein sequences or fragments of full-length proteins, and can include post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc., as well as both other naturally occurring modifications known in the art and other non-naturally occurring modifications.

[0085] "Isolated peptide" or "isolated polypeptide" or "isolated protein construct", etc., as used herein, refers to the in vitro isolation and / or purification of a peptide or polypeptide molecule, or protein construct, from a cellular environment or cell culture supernatant and from association with other components of the cell, i.e., it is not significantly associated with the in vivo substance.

[0086] In the context of the present invention, a dimerization domain is a protein or peptide that can bind to another domain, such as another dimerization domain of another protein or peptide, or any domain that can be integrated. Many examples of dimerization domains are known to those skilled in the art, including the antibody Fc fragment of an antibody, leucine zipper domains, or coiled-coil domains. Dimerization can result in the formation of homo- and heterodimers, meaning the assembly of two identical or two different proteins, respectively. In either case, the dimerization domains may be the same or, in the case of heterodimers, different in the monomers that form the dimer.

[0087] In embodiments of the present invention, the protein construct includes a dimerization domain. Such a construct can be composed of one or more proteins. In the case of the protein construct of the present invention composed of a single protein, it will be apparent to those skilled in the art that the presence of the dimerization domain can lead to the formation of homodimers. Furthermore, it will be immediately apparent to those skilled in the art that a protein construct that includes a dimerization domain and is composed of two or more proteins can include a dimerization domain in each protein. In such embodiments, the dimerization of the two proteins of the protein construct is preferentially mediated by the dimerization domain. In other words, if the protein construct of the present invention is or forms a dimer of two proteins (either a homodimer or a heterodimer), dimerization can be brought about by the dimerization domain contained in each protein. In a preferred embodiment, the dimerization domain is an Fc domain.

[0088] As used herein, the term capture domain refers to a domain or portion of the protein construct of the present invention that can be used to bind the construct of the present invention to a solid phase either by non-covalent interaction or covalent bond. A typical example of such a capture domain is a domain or amino acid sequence that is recognized by a commonly available protein that preferably binds to a capture domain such as an antibody. For example, the Fc fragment of an antibody can serve as a capture domain because there are high-affinity antibodies that specifically bind to these domains. Furthermore, protein tags such as Myc tag, HA tag, HIS tag, etc. can be used as capture domains.

[0089] The possible dimerization domains and capture domains that can be incorporated into the protein constructs of the present invention are diverse, and those skilled in the art can identify suitable variants. Further, in some cases, the dimerization domain can also serve as a capture domain. This is the case, for example, with the antibody Fc fragment, which can form dimers and can be readily bound by commonly available antibodies. Thus, the Fc fragment can function simultaneously as a capture domain and a dimerization domain. Further examples are known to those skilled in the art or can be identified without undue effort.

[0090] Embodiments of the present invention relate to recombinant proteins such as recombinant fusion proteins, which are proteins produced through genetic engineering of fusion genes. This typically involves adding the cDNA sequence of a second protein fragment in-frame with the cDNA of a first protein (fragment) without an intervening stop codon, for example, by ligation or overlap extension PCR. The DNA sequence is then expressed by the cell as a single protein. The protein can be engineered to contain either the complete sequences of both original proteins or only a portion of either. Three or more proteins or fragments can be joined to form complex fusion proteins. There are often so-called linker (or "spacer") peptides between the various parts of the fusion protein, which increases the likelihood that the protein will fold independently and function as expected. Especially when the linker enables protein purification, in protein or peptide fusions, the linker can be engineered with cleavage sites for proteases or chemicals to allow release of two separate proteins. This technique is often used for protein identification and purification by fusing a GST protein, a FLAG peptide, or a hexa-his peptide (6×His tag), which can be isolated using affinity chromatography with nickel or cobalt resin. Dimeric or multimeric chimeric proteins can be produced by genetic engineering by fusing to the original protein a peptide domain that induces dimerization or multimerization of the artificial protein (e.g., streptavidin or leucine zipper).

[0091] Protein linkers assist in the design of fusion proteins by providing an appropriate spacing between domains and support correct protein folding when N- or C-terminal interactions are essential for folding. Generally, protein linkers enable important domain interactions, enhance stability, reduce steric hindrance, and are preferred for use in fusion protein design even when the N- and C-termini can be fused. At least three major types of linkers are flexible, rigid, and (in vivo) cleavable. Flexible linkers can consist of many small glycine residues and confer the ability to curl into dynamic and adaptable shapes. Rigid linkers can be formed by large cyclic proline residues and can be useful when it is necessary to maintain a very specific spacing between domains. (In vivo) cleavable linkers are unique in that they are designed to allow the release of one or more fusion domains under certain reaction conditions such as a specific pH gradient or when in contact with another biomolecule within the cell. The selection and design of appropriate linker sequences are standard procedures known to those skilled in the art. In the case of cleavable linker sequences, those skilled in the art can also select an enzyme or reagent suitable for linker cleavage and design or select the corresponding linker.

[0092] Preferred sequences constituted by the NMDAR constructs of the present invention Table 1 discloses the preferred amino acid sequences constituted by the embodiments of the NMDAR protein constructs of the present invention or the fusion proteins that can be used in the NMDAR protein constructs of the present invention. Table 2 discloses the preferred nucleic acid sequences constituted by the nucleic acid molecules encoding the NMDAR protein constructs of the present invention or the fusion proteins that can be used in the NMDAR protein constructs of the present invention.

[0093] Table 1: Preferred Amino Acid Sequences of the Present Invention

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

[0094] Table 2: Preferred Nucleic Acid Sequences of the Present Invention

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

Table 2-29

Table 2-30

Table 2-31

Table 2-32

Table 2-33

Table 2-34

Table 2-35

Table 2-36

Table 2-37

Table 2-38

Table 2-39

Table 2-40

Table 2-41

Table 2-42

Table 2-43

Table 2-44

Table 2-45

Table 2-46

Table 2-47

Table 2-48

Table 2-49

Table 2-50

Table 2-51

Table 2-52

[0095] The present invention further relates to functionally similar sequences, domains, linkers of each NMDAR protein construct and further elements constituted by such constructs. Protein modifications to the NMDAR protein constructs of the present invention that may occur by substitution of amino acid sequences and nucleic acid sequences encoding such molecules are also included within the scope of the present invention. The substitutions defined herein are modifications made to the amino acid sequence of a protein, whereby one or more amino acids are replaced with the same number of (different) amino acids, producing a protein with an amino acid sequence different from the primary protein. In some embodiments, this modification does not significantly change the function of the protein. Similar to additions, substitutions may be natural or artificial. It is well known in the art that amino acid substitutions can be made without significantly changing the function of a protein. This is particularly true in the case of "conservative" amino acid substitutions, where the modification is a substitution of one amino acid for another with similar properties. Such "conservative" amino acids can be natural or synthetic amino acids that can be substituted without significantly affecting the structure and function of the protein due to size, charge, polarity, and three-dimensional structure. In many cases, many amino acids can be substituted with conservative amino acids without having a detrimental effect on the function of the protein.

[0096] Generally, nonpolar amino acids Gly, Ala, Val, Ile, and Leu; nonpolar aromatic amino acids Phe, Trp, and Tyr; neutral polar amino acids Ser, Thr, Cys, Gln (Gin), Asn, and Met; positively charged amino acids Lys, Arg, and His; and negatively charged amino acids Asp and Glu form a group of conservative amino acids. This list is not exhaustive. For example, Ala, Gly, Ser, and sometimes Cys are well known to be mutually replaceable even though they belong to different groups.

[0097] As described herein, in relation to the present invention, the NMDAR protein constructs of the present invention can be provided at the protein level or in the form of one or more nucleic acids encoding each NMDAR protein construct (which may include two or more proteins).

[0098] The nucleic acid sequences of the present invention include nucleic acid sequences encoding an NMDAR protein construct or individual proteins forming part of the NMDAR protein constructs of the present invention. Protein sequences according to Table 1 and functionally similar sequences represent preferred NMDAR protein constructs of the present invention or parts thereof. Preferred nucleic acid sequences encoding an NMDAR protein construct of the present invention or part thereof are listed in Table 2.

[0099] The NMDAR protein constructs of the present invention can include protein tags that enable easy identification or binding of the NMDAR protein constructs provided by standard techniques, for example, using antibodies directed against the protein tags. Preferred protein tags that can be encoded by the nucleic acid sequences of the present invention are V5 tag, myc tag, HA tag, HIS tag, or antibody Fc fragment. Alternative tags can be used instead of the V5 tag. Such alternatives are well known in the art and can be selected by those skilled in the art.

[0100] In another aspect, the present invention encompasses the NMDAR protein constructs disclosed herein and their use in connection with the methods disclosed herein. In particular, the present invention also relates to nucleic acid molecules encoding the NMDAR protein constructs of the present invention, and in particular to one or more nucleic acid molecules encoding such NMDAR protein constructs or a portion of such constructs, a) one or more nucleic acid molecules comprising a nucleotide sequence encoding an ECD of the NMDAR subunit GluN1 or a fragment thereof, and an ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or a fragment thereof, preferably a dimerization domain and / or a capture domain, b) one or more nucleic acid molecules complementary to the nucleotide sequence described in a), c) one or more nucleic acid molecules that hybridize to the nucleotide sequence described in a) or b) under stringent conditions, d) one or more nucleic acid molecules comprising a nucleotide sequence having sufficient sequence identity that is functionally similar to the nucleotide sequence described in a), b) or c), e) one or more nucleic acid molecules that are degenerate to the nucleotide sequences described in a) - d) as a result of the genetic code, f) one or more nucleic acid molecules described in the nucleotide sequences of a) - e) that are modified by deletions, additions, substitutions, translocations, inversions and / or insertions and are functionally similar to the nucleotide sequences described in a) - e), and is selected from the group comprising.

[0101] Furthermore, the present invention also relates to nucleic acid molecules encoding the NMDAR protein constructs of the present invention, and in particular to one or more nucleic acid molecules encoding such NMDAR protein constructs or a portion of such constructs, g) one or more nucleic acid molecules encoding an ECD of the NMDAR subunit GluN1 or a fragment thereof, and an ECD of the NMDAR subunit GluN2A or a fragment thereof and / or an ECD of GluN2B or a fragment thereof, preferably a dimerization domain and / or a capture domain; h) one or more nucleic acid molecules complementary to the nucleotide sequence described in a); i) one or more nucleic acid molecules that undergo hybridization with the nucleotide sequence described in a) or b) under stringent conditions; j) one or more nucleic acid molecules comprising a nucleotide sequence having sufficient sequence identity functionally similar to the nucleotide sequence described in a), b) or c); k) one or more nucleic acid molecules that are degenerate to the nucleotide sequences described in a) to d) as a result of the genetic code; l) one or more nucleic acid molecules modified by deletion, addition, substitution, translocation, inversion and / or insertion and having functionally similar nucleotide sequences of a) to e) to the nucleotide sequences described in a) to e); selected from the group comprising.

[0102] Thus, the present invention encompasses nucleic acid molecules having at least 60%, preferably 70%, more preferably 80%, particularly preferably 90% sequence identity with the nucleic acid molecule encoding the NMDAR protein construct of the present invention or a part thereof.

[0103] Sequence variants of the nucleic acids and / or proteins described in the claims, which are defined by the provided % sequence identity and maintain the above properties of the present invention, are also included within the scope of the present invention. Although alternative sequences are shown, such variants that maintain essentially the same properties as the provided specific sequences, such as the autoantibody binding properties of each NMDAR protein construct of the present invention, are known as functional analogs or as functionally similar. Sequence identity relates to the percentage of identical nucleotides or amino acids when performing sequence alignment using software such as BLAST.

[0104] One of ordinary skill in the art will understand that as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides have minimal homology or sequence identity to the nucleotide sequences of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Deletions, substitutions, and other changes in sequences that correspond to the described sequence identity are also encompassed by the present invention.

[0105] Description of Autoantigens and Diseases The present invention relates to soluble N-methyl-D-aspartate receptor (NMDAR) protein constructs comprising one or more NMDAR autoantibody epitopes. As used herein, the term "NMDAR autoantibody epitope" relates to an epitope formed by an NMDAR (either by an individual subunit or epitope comprising residues or amino acids of two or more subunits of the NMDAR). Further, the three-dimensional structure of an NMDAR subunit can be stabilized only by the presence of an ECD or fragment of an ECD of another subunit, and certain epitopes can be formed only upon stabilization of such three-dimensional structure. In the context of the present invention, the NMDAR protein constructs and the epitopes formed by the constructs of the present invention can be referred to as autoantigens. The binding itself between an autoantigen and an antibody is an established phenomenon and essentially reflects the physical interaction between any given antibody and its target.

[0106] It is known to those of ordinary skill in the art that various neurological autoimmune conditions in which autoantibodies typically target autoantigens in either the central or peripheral nervous system. However, medical conditions are also known in which autoantibodies are directed against targets present in both the central and peripheral nervous systems. The present invention, therefore, contemplates the use of the NMDAR protein constructs of the present invention in diseases related to autoantibodies that primarily target components of the central nervous system or in which the pathogenic effects of the autoantibodies are caused by autoantibodies targeting autoantigens in the central nervous system.

[0107] As used herein, "central nervous system" or CNS refers to a part of the nervous system consisting of the brain and spinal cord. The CNS is contained within the dorsal body cavity, with the brain housed in the cranial cavity and the spinal cord in the spinal canal. The CNS is divided into white matter and gray matter. This can also be macroscopically observed on brain tissue. White matter consists of axons and oligodendrocytes, and gray matter consists of neurons and unmyelinated fibers. Both tissues contain many glial cells (although white matter contains more glial cells) and are often referred to as the supporting cells of the CNS. From the spinal cord and further towards the spinal cord, the peripheral nervous system in the form of spinal nerves extends. Nerves connect the spinal cord to the skin, joints, muscles, etc., enabling not only the transmission of centrifugal movement but also the transmission of centripetal sensory signals and sensory stimuli. This enables the perception of sensation along with the voluntary and involuntary movements of muscles.

[0108] As used herein, the "peripheral nervous system" (PNS) consists of nerves and ganglia outside the brain and spinal cord. The main function of the PNS is to connect the CNS to the hands, feet, and organs and essentially function as a relay between the brain and spinal cord and the rest of the body. Unlike the CNS, the PNS is not protected by the vertebral column and skull, or the blood-brain barrier.

[0109] New research now shows that autoantibodies can access the CNS (Zong et al Front Immunol. 2017; 8:752), indicating the presence of autoantibody-producing B cells in the CNS. Under normal conditions, immunoglobulins pass through the blood-brain barrier (BBB) at a low rate, and a good example is immunoglobulin G (IgG). The IgG concentration in cerebrospinal fluid (CSF) is approximately 1% of the level in the peripheral circulation. This indicates that when autoantibodies reach the CNS, they can cause diseases as observed in autoimmune encephalitis. In certain situations, the BBB may become more permeable due to stroke, brain trauma, hemorrhage, microangiopathy, or brain tumor, increasing the penetration of antibodies.

[0110] As used herein, the term "autoantibody-mediated mental state" refers to any medical condition that includes the presence of autoantibodies, preferably autoantibodies directed against autoantigens that are primarily targeted in the central nervous system, and in which psychiatric (neuropsychiatric) symptoms are also observed. Many central nervous system disorders, including encephalitis and severe mental disorders, have been demonstrated to be associated with specific neuronal surface autoantibodies (NSAbs). Specific autoantibodies targeting neuronal surface antigens and ion channels have been shown to cause severe mental disorders, i.e., neuropsychiatric symptoms. Many studies have shown the presence of autoantibodies in specific mental states such as schizophrenia and bipolar disorder. Additional disorders are related to neuropsychiatric disorders such as schizophrenia, bipolar disorder, MDD, substance-induced psychosis, Huntington's disease, Alzheimer's disease, and neuropsychiatric systemic lupus erythematosus (Zong et al, Front Immunol. 2017; 8:752).

[0111] In some embodiments, the disease treated or diagnosed using the present invention is an autoimmune encephalopathy or cerebrospinal disorder. "Encephalopathy" typically refers to any disorder or disease of the brain, particularly a chronic degenerative condition. Encephalopathy may refer to permanent (or degenerative) brain damage, or reversible injury. Encephalopathy can be caused by direct injury to the brain, or by diseases remote from the brain. Symptoms often include disability, irritability, agitation, delirium, confusion, drowsiness, coma, stupor, and mental disorders. As used herein, "autoimmune encephalopathy" refers to any brain disease caused by an autoimmune component, including autoimmune encephalitis. As used herein, "autoimmune cerebrospinal disorder" is any disease affecting both the brain and spinal cord due to an autoimmune component.

[0112] Anti-N-methyl-D-aspartic acid (NMDA) receptor encephalitis is a type of encephalitis that frequently occurs in women and is mainly associated with antibodies against the NR1 subunit, but also the NR1 (GluN1) and / or NR2 subunits of the NMDA receptor. Anti-NMDA receptor encephalitis was first described several years ago in a number of large-scale studies that characterized the clinical syndrome in detail (Non-Patent Document 19). Patients with anti-NMDAR encephalitis suffer from severe encephalitis with characteristic clinical multi-stage features that mainly affect children and young women. Anti-NMDAR encephalitis progresses from psychiatric symptoms, memory impairment, and seizures to loss of consciousness, autonomic dysfunction, dyskinesia, and hypoventilation (Dalmau et al. Lancet Neurol. 2011; 10:63-74, Pruess et al. 2010, Neurology. 75(19):1735-9, Pruess et al. 2013, Neurology. 75(19):1735-9). A prominent feature of this disease is the antibody against the NR1 / GluN1 subunit of NMDAR1. This has greatly changed the treatment concept of encephalitis because NMDAR encephalitis was not recognized as a distinct subgroup of encephalitis before 2007. Therefore, NMDAR encephalitis was previously considered encephalitis of unknown cause and had not been adequately treated.

[0113] The N-methyl-D-aspartic acid receptor (also known as the NMDA receptor or NMDAR) is a glutamate receptor and ion channel protein found in nerve cells. The NMDA receptor is one of three types of ionotropic glutamate receptors. The other receptors are the AMPA receptor and the kainate receptor. When glutamate and glycine (or D-serine) bind to the NMDAR, it is activated, and when activated, positively charged ions flow through the cell membrane. The NMDA receptor is very important for controlling synaptic plasticity and memory function. The receptor is usually assembled as a heteromeric complex that interacts with multiple intracellular proteins by three different subunits, NR1, NR2, and NR3. NR1 has eight different isoforms produced by alternative splicing from a single gene. There are four different NR2 subunits (A - D), and the NR3A and NR3B subunits were reported in the latter half of the 20th century. Six separate genes encode NR2 and NR3. According to more recent nomenclature, the subunits are called GluN1, GluN2, and GluN3 instead of NR1, NR2, and NR3, respectively. Alternative variants of the subunits are identified accordingly (e.g., NR2A and NR2B are identified as GluN2A and GluN2B, respectively).

[0114] Each receptor subunit has a modular design. The extracellular domain contains two globular structures, the amino-terminal domain (ATD, also sometimes called the regulatory domain) and the ligand-binding domain. The NR1 subunit binds the co-agonist glycine, and the NR2 subunit binds the neurotransmitter glutamate. The agonist-binding module is linked to a membrane domain consisting of three transmembrane segments and a re-entrant loop that associates with the selectivity filter of the potassium channel. The membrane domain contributes residues to the channel pore and is responsible for the high single-channel conductance, high calcium permeability, and voltage-dependent magnesium block of the receptor. Each subunit has an extensive cytoplasmic domain that contains residues that can be directly modified by a series of protein kinases and protein phosphatases, as well as residues that interact with a number of structural proteins, adapter proteins, and scaffold proteins.

[0115] NMDAR NR1 / GluN1 is a component of the NMDA receptor complex that functions as a calcium-permeable and voltage-dependent magnesium-sensitive heterotetrameric, ligand-gated ion channel. Channel activation requires binding of the neurotransmitter glutamate to the GluN2 subunit, binding of glycine to the GluN1 subunit, and further membrane depolarization to relieve channel inhibition by Mg2+. A number of protein isoforms of the NMDAR NR1 protein are known, for example, but not limited to, those with Gene Bank accession numbers XP_011516885.1, XP_005266130.1, XP_005266129.1, XP_005266128.1, NP_001172020.1, NP_001172019.1, NP_000823.4, NP_015566.1, NP_067544.1. Any one or more of the above sequences or isoforms, or functionally similar derivatives thereof, can be employed in connection with the NMDAR protein constructs of the present invention.

[0116] Regarding GluN2 / NR2, only a single subunit is found in invertebrates, while in vertebrates, four different isoforms of the NR2 subunit are expressed and are designated as NR2A / GluN2A to NR2D / GluN2D (encoded by GRIN2A, GRIN2B, GRIN2C, GRIN2D) according to the nomenclature. They contain binding sites for the neurotransmitter glutamate. Unlike the NR1 subunit, the NR2 subunit is differentially expressed across various cell types and controls the electrophysiological properties of the NMDA receptor. One specific subunit, NR2B, is mainly present within immature neurons and extrasynaptic locations and contains a binding site for the selective inhibitor ifenprodil. NR2B is predominant in the early postnatal brain, whereas the number of NR2A subunits increases and ultimately the NR2A subunit exceeds NR2B. This is called the NR2B-NR2A developmental switch and is worthy of attention due to the various dynamics that each NR2 subunit confers on the receptor. For example, a larger proportion of the NR2B subunit results in NMDA receptors that remain open longer compared to the case of more NR2A.

[0117] NMDAR has various physiological roles and any dysfunctions, either enhancing or reducing activity, and can cause neuropsychiatric disorders such as schizophrenia, bipolar disorder, MDD, substance-induced psychosis, Huntington's disease, Alzheimer's disease, and neuropsychiatric systemic lupus erythematosus (NPSLE). Therefore, NMDAR plays an important role in multiple mental disorders including depression. Furthermore, a subgroup of patients with atypical dementia have anti-NMDAR1 antibodies, and thus are recognized as a medical condition related to autoantibodies against NMDAR, and removal of such NMDAR autoantibodies by non-specific removal of all antibodies has led to clinical improvement in selected cases (Pruess et al. 2010, Neurology. 75(19):1735-9, Doss et al. 2014 Ann Clin Transl Neurol.1 (10):822-32). Additionally, autism can occur in children of mothers suffering from an autoantibody-mediated disorder. Some studies have found a correlation between the presence of circulating maternal autoantibodies in newborns and neurological dysfunction (Fox-Edmiston et al, 2015, CNS Drugs, 29(9):715-724). Specifically, maternal anti-brain autoantibodies that may access the fetal compartment during pregnancy have been identified as one of the risk factors for developing autism spectrum disorder (ASD). The presence of NMDAR-autoantibodies can thus cause autism in the offspring of mothers with the disease, and the present invention is also a potential treatment for such disorders in children and / or a preventive approach to avoid such diseases.

[0118] Thus, any medical condition in which the contribution of NMDAR autoantibodies to the etiology is explained or suggested is eligible as a medical condition related to NMDAR autoantibodies, for example due to the correlation between the occurrence of NMDAR autoantibodies and disease symptoms. Furthermore, the constructs of the present invention can be used to analyze samples from patients suffering from a condition that is explained or suggested as a condition related to NMDAR autoantibodies against the presence of such antibodies, and can then be treated according to the present invention.

[0119] In contrast to anti-NMDAR in autoimmune encephalitis that mainly targets the GluN1 subunit, autoantibodies have been found to target the GluN2 subunit of NMDAR, and these were associated with depression in patients with systemic lupus erythematosus (SLE) (Lapteva et al. Arthritis Rheum (2006) 54(8):2505-14).

[0120] Aspects of in vitro methods for detecting NMDAR autoantibodies Autoantibodies are antibodies (a type of protein) produced by the immune system and directed against one or more of the individual's own proteins. Many autoimmune diseases are associated with and / or caused by such autoantibodies.

[0121] The term "autoimmune disease" refers to any given disease associated with and / or resulting from the presence of autoantibodies. Autoimmune diseases result from an abnormal immune response (autoimmunity) of the body against substances and tissues that are normally present in the body. This can be restricted to a particular organ or can involve specific tissues.

[0122] As used herein, the term "sample" is a biological sample obtained or isolated from a patient or subject. As used herein, "sample" refers to a sample of a body fluid or tissue obtained for the purpose of diagnosing, predicting the prognosis, or evaluating a subject such as a patient. Preferably herein, the sample is a sample of a body fluid such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, pleural effusion, cells, cell extracts, tissue samples, tissue biopsy samples, fecal samples, etc.

[0123] The terms "individual", "subject", or "patient" typically refer to a human, but also refer to other animals including, for example, other primates, rodents, dogs, cats, horses, sheep, pigs, etc. As used herein, "patient" or "subject" can be a vertebrate. In the context of the present invention, the term "subject" includes both humans and animals, particularly mammals, and other organisms.

[0124] As used herein, the terms "diagnosis", "prognosis prediction", and "likelihood assessment" relate to determining the probability that a subject has, or is at risk of having and / or developing, a medical condition associated with NMDAR autoantibodies.

[0125] The terms "diagnosis" and "diagnosing" include the use of NMDAR protein constructs, methods, kits, and further aspects of the invention to determine the presence or likelihood of presence of a medically relevant disorder in an individual. This term also includes devices, methods, and systems for assessing the level of disease activity in an individual. In some embodiments, statistical algorithms are used to diagnose mild, moderate, severe, or fulminant disorders based on criteria developed by Truelove et al., Br. Med. J., 12:1041-1048 (1955). In other embodiments, statistical algorithms are used to diagnose mild-to-moderate, moderate-to-severe, or severe-to-fulminant autoimmune diseases associated with NMDAR autoantibodies.

[0126] The present invention also encompasses the use of a method of disease monitoring, also known as monitoring the progression or regression of an autoimmune disease and treatment monitoring. The term "monitoring" includes the NMDAR constructs disclosed herein for determining an individual's disease state (e.g., the presence or severity of an autoimmune disease), as well as the use of the methods and other aspects of the present invention. In certain examples, the results of a statistical algorithm (e.g., a learning statistical classifier system) are compared to results obtained earlier for the same individual. In some aspects, the kits, constructs, devices, methods, and systems of the present invention can also be used to predict the progression of an autoimmune disease by, for example, determining the likelihood that the autoimmune disease is progressing rapidly or slowly in an individual based on the presence or level of at least one marker (such as one or more NMDAR autoantibodies) in a sample. The present invention can also be used to predict the regression of an autoimmune disease by, for example, determining the likelihood that the autoimmune disease is regressing rapidly or slowly in an individual based on the presence or level of at least one marker in a sample. Additionally, treatment monitoring can be performed, thereby allowing the subject to be monitored for the progression of the disease during the course of any given treatment.

[0127] In aspects of the present invention, the presence or level of NMDAR autoantibodies is determined using an immunoassay or immunohistochemical assay. Non-limiting examples of immunoassays suitable for use in the methods of the present invention include ELISA. Examples of immunohistochemical assays suitable for use in the methods of the present invention include, but are not limited to, immunofluorescence assays such as direct fluorescent antibody assay, IFA assay, anti-complement immunofluorescence assay, and avidin-biotin immunofluorescence assay. Other types of immunohistochemical assays include immunoperoxidase assay.

[0128] In the context of the present invention, the term "affinity reagent" relates to an antibody, peptide, nucleic acid, small molecule, or any other molecule that specifically binds to a target molecule in order to identify, track, capture, or affect its activity. The term "capture" refers to the binding of a target molecule by an affinity reagent.

[0129] The term "secondary affinity reagent" refers to any affinity reagent as defined above, which is used to bind to an antigen that is already bound by another affinity reagent.

[0130] As used herein, the term "antibody" includes a population of immunoglobulin molecules that can be polyclonal or monoclonal and of any isotype, or immunologically active fragments of immunoglobulin molecules. Such immunologically active fragments include the heavy and light chain variable regions that constitute part of an antibody molecule that specifically binds to an antigen. For example, immunologically active fragments of immunoglobulin molecules known in the art as Fab, Fab', or F(ab')2 are included within the meaning of the term "antibody". The term "monoclonal antibody" refers to an antibody made by the same immune cell, which is all clones of a particular parent cell, as opposed to polyclonal antibodies made from several different immune cells. Monoclonal antibodies can have monovalent affinity in that they bind to the same epitope (a part of an antigen recognized by an antibody). There are also engineered bispecific monoclonal antibodies where each "arm" of the antibody is specific for a different epitope. Considering almost all substances, it is possible to produce monoclonal antibodies that specifically bind to that substance, and then the monoclonal antibodies serve to detect or purify that substance.

[0131] In an embodiment, the present invention relates to an in vitro method for detecting NMDAR autoantibodies in a sample. In an embodiment, the method is an immunoassay. Following the addition of a sample solution, the antibodies of the patient contained therein bind to the NMDAR protein construct. For example, antibodies obtained from a patient's serum or feces and bound to the NMDAR protein construct can then be detected using a labeled or labeling reagent and optionally quantified. Thus, according to the present invention, the detection of antibodies in this method can be performed using a labeled reagent by well-known ELISA (enzyme-linked immunosorbent assay) technology. Therefore, the label according to the present invention comprises an enzyme that catalyzes a chemical reaction that can be determined by optical means, in particular using a chromogenic substrate, chemiluminescence or a fluorescent dye. In another preferred embodiment, the autoantibodies are detected by labeling with a weak radioactive substance in a radioimmunoassay (RIA) in which the radioactivity generated is measured.

[0132] As an example of means for detecting a label in the method of the present invention, the presence or level of one or more markers in a sample can be determined using techniques of various immunoassay methods including competitive and non-competitive immunoassay methods (see, for example, Self et al., Curr. Opin. Biotechnol., 7:60-65 (1996)). The term immunoassay method includes, but is not limited to, enzyme immunoassay (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), antigen capture ELISA, sandwich ELISA, IgM antibody capture ELISA (MACELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassay (CEIA); radioimmunoassay (RIA); immunoradiometric assay (IRMA); fluorescence polarization immunoassay (FPIA); and techniques including chemiluminescence assay (CL). Such immunoassay methods can be automated as needed. Immunoassay methods can also be used in combination with laser-induced fluorescence (see, for example, Schmalzing et al., Electrophoresis, 18:2184-2193 (1997), Bao, J. Chromatogr. B. Biomed. Sci., 699:463-480 (1997)). Liposome immunoassay methods such as flow injection liposome immunoassay and liposome immunosensors are also suitable for use in the present invention (see, for example, Rong et al., J. Immunol. Methods, 204:105-133 (1997)). In addition, a nephelometric assay in which the formation of a protein / antibody complex results in an increase in light scattering that is converted to a peak velocity signal as a function of marker concentration is suitable for use in the present invention. The nephelometric assay is commercially available from Beckman Coulter (Brea, California; kit #449430) and can be performed using a Behring Nephelometer Analyzer (Finke et al., J. Clin. Chem. Clin. Biol. Chem., 27:261-276 (1989)).

[0133] The above-described immunoassay is particularly useful for determining the presence or level of one or more NMDAR autoantibodies in a sample (and can also be considered an example of a means for detecting a label).

[0134] In another preferred embodiment of the method according to the invention, the autoantibody is detected in an immunoassay and preferably binds one reactant directly or indirectly to a labeling substance. This allows the method to be flexibly adapted to the possibilities and requirements of different laboratories and their diagnostic testing devices. In an advantageous embodiment, an autoimmune disease-specific antibody is detected in an immunoassay and the antibody is dissolved and present in a liquid phase, preferably a liquid phase diluted in a normal buffer well known to those skilled in the art, or an undiluted body fluid. According to the invention, the detection can also be carried out using a stool sample. Furthermore, the detection method of the present invention can be carried out by binding the construct of the present invention to cells expressing NMDAR autoantibodies on their surface. Detection of cells that bind to the construct of the present invention can occur by direct or indirect labeling of the construct.

[0135] In another preferred embodiment of the present invention, a soluble or solid-phase bound NMDAR protein construct is used to bind an antibody. In a second reaction step, an anti-human immunoglobulin can be employed, preferably selected from the group comprising anti-human IgA, anti-human IgM and / or anti-human IgG antibodies, which anti-human immunoglobulin is a detectable labeled conjugate of two components that can be conjugated with any conventional labeling enzyme, in particular a chromogenic substrate and / or a chemiluminescent substrate, preferably horseradish peroxidase, alkaline phosphatase. The advantage of this embodiment lies in the use of ELISA technology, which is usually available in laboratory facilities, so that the detection according to the present invention can be established in a cost-effective manner. In another preferred embodiment of the present invention, the antibody bound to the NMDAR protein construct of the present invention preferably reacts with an anti-human immunoglobulin selected from the group comprising anti-human IgA, anti-human IgM and / or anti-human IgG antibodies and is detectably bound to fluorescein isothiocyanate (FITC). Similar to the above ELISA, the FITC technique represents a system that is available in many places and thus enables the smooth and low-cost establishment of the detection of the present invention in a conventional laboratory. Those skilled in the art are aware of further standard detection techniques that can be used in connection with the method of the present invention.

[0136] Specific immunological binding of an antibody to a target marker can be detected directly or indirectly via a label. Any given means for detecting these labels can be considered a means for detecting the label according to the method of the present invention. Examples of direct labels include fluorescent tags, luminescent tags, metals, dyes, radionuclides, etc. attached to the antibody. Antibodies labeled with iodine-125 (125I) can be used to determine the level of one or more markers in a sample. Chemiluminescent assays using chemiluminescent antibodies specific for the marker are suitable for highly sensitive non-radioactive detection of marker levels. Antibodies labeled with fluorescent dyes are also suitable for determining the level of one or more markers in a sample. Examples of fluorescent dyes include, but are not limited to, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and Lissamine. Secondary antibodies conjugated to fluorescent dyes are commercially available; for example, goat F(ab')2 anti-human IgG-FITC is available from Tago Immunologicals (Burlingame, CA). Further fluorescent labels are commonly used and known to those skilled in the art.

[0137] Examples of indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, urease, etc. For example, a horseradish peroxidase detection system that produces a soluble product detectable at 450 nm in the presence of hydrogen peroxide can be used with the chromogenic substrate tetramethylbenzidine (TMB). An alkaline phosphatase detection system can be used with a chromogenic substrate such as p-nitrophenyl phosphate, which produces a soluble product easily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG), which produces a soluble product detectable at 410 nm.

[0138] Signals from direct or indirect labels can be analyzed using, for example, a spectrophotometer that detects color from a chromogenic substrate, a radiation measuring instrument such as a gamma counter that detects radiation such as 125I, or a fluorometer that detects fluorescence in the presence of light of a specific wavelength. In the case of detecting enzyme-linked antibodies, quantitative analysis of the amount of the marker level can be performed using a spectrophotometer such as an EMAX microplate reader (Molecular Devices; Menlo Park, California) according to the manufacturer's instructions. If desired, the assays of the present invention can be automated or performed by a robot, and signals from multiple samples can be detected simultaneously.

[0139] A plate reader, also known as a microplate reader or microplate photometer, is a device used to detect biological, chemical, or physical events of samples within a microtiter plate. Plate readers are widely used in the pharmaceutical and biotechnology industries, as well as in research, drug discovery, bioassay validation, quality control, and manufacturing processes in academic institutions. Sample reactions can be assayed, for example, but not limited to, in a 6 - 1536 well format microtiter plate. General detection modes for microplate assays include, for example, but are not limited to, absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization. The camera device related to the present invention is a device suitable for detecting signals of a labeled secondary affinity reagent directed against GP2. The camera device can be provided as being within a plate reader or configured individually. Those skilled in the art are proficient in such devices, which are selected based on the label of the secondary affinity reagent.

[0140] In certain specific embodiments, the present invention provides a method for diagnosing an autoimmune disease or a clinical subtype thereof using the NMDAR protein constructs of the present invention. Various autoimmune disease markers such as biochemical markers, serological markers, genetic markers, or other clinical or ultrasound characteristics are suitable for use and can be combined with a statistical algorithm for classifying samples from an individual as samples of an autoimmune disease. Examples of further markers of autoimmune diseases related to NMDAR autoantibodies suitable for use in the present invention are known to those skilled in the art. Those skilled in the art will know additional markers suitable for use in the statistical algorithms of the present invention.

[0141] In another preferred embodiment of the present invention, the NMDAR protein constructs according to the present application are immobilized on a surface. More specifically, one or more solid-phase bound NMDAR protein constructs disclosed herein bind to organic, inorganic, synthetic and / or mixed polymers, preferably agarose, cellulose, silica gel, polyamide and / or polyvinyl alcohol. Immobilization in the context of the present invention is understood to include various methods and techniques for immobilizing peptides on a specific carrier, for example according to WO 99 / 56126 or WO 02 / 26292. For example, immobilization can help to stabilize the constructs so that their activity is not reduced or adversely affected by biological, chemical or physical exposure, especially during storage or when used in a single batch. Immobilization of the peptides allows for repeated use under technical or clinical routine conditions and further allows samples (preferably blood components) to be reacted with at least one construct according to the present invention in a continuous manner. In particular, this can be achieved by proceeding with the binding of the peptide to other peptides or molecules, or to the carrier, such that the three-dimensional structure of the corresponding molecule, especially the active center mediating the interaction with the autoantibody, is not altered (especially in the case of the active center mediating the interaction with the autoantibody). Advantageously, there is no loss of specificity for the patient's autoantibodies as a result of such immobilization. In the context of the present invention, at least three basic methods can be used for immobilization: (i) Crosslinking: In crosslinking, the peptides are immobilized to each other without adversely affecting their activity. Advantageously, the peptides are no longer soluble as a result of such crosslinking. (ii) Binding to a carrier: Binding to a carrier proceeds, for example, via adsorption, ionic bonding or covalent bonding. Such binding can also occur inside microbial cells, or inside liposomes or other membranes (closed or open structures). Advantageously, the peptides are not adversely affected by such immobilization. For example, multiple or consecutive uses of the carrier-bound peptides are possible, which is advantageous in clinical diagnosis or treatment. (iii) Encapsulation: Encapsulation in the context of the present invention proceeds in particular within semipermeable membranes in the form of gels, fibrils or fibers. Advantageously, the encapsulated peptides are separated from the surrounding sample solution by the semipermeable membrane such that interaction with autoantibodies or fragments thereof is still possible. There are various methods available for immobilization, such as adsorption to an inert or charged inorganic or organic carrier. For example, such a carrier can be a porous gel, aluminum oxide, bentonite, agarose, starch, nylon or polyacrylamide. The immobilization proceeds via physical binding forces and is frequently accompanied by hydrophobic interactions and ionic bonding. Advantageously, such methods are easy to handle and have little effect on the three-dimensional structure of the peptides. Advantageously, the binding can be improved, for example, by using ion exchangers, in particular Sephadex, as a result of the electrostatic binding force between the charged groups of the peptides and the carrier.

[0142] Another method is covalent attachment to a carrier material. Additionally, the carrier may have reactive groups that form isopolar bonds with amino acid side chains. Groups suitable for peptides are carboxy groups, hydroxy groups, and sulfide groups, particularly the terminal amino group of lysine. Aromatic groups offer the possibility of diazo coupling. The surface of microscopic porous glass particles is activated by treatment with silane and can then react with peptides. For example, the hydroxy groups of natural polymers can be activated with bromocyanogen and then coupled with peptides. Advantageously, a large number of peptides can undergo direct covalent attachment to polyacrylamide resins. Encapsulation in a three-dimensional network includes the encapsulation of peptides in ionizable gels or other structures, which is well known to those skilled in the art. More specifically, the pores of the matrix are such that they enable interaction with target molecules while retaining the peptides. In crosslinking, the peptides are converted into polymer aggregates by crosslinking with a bifunctional substance. Such structures are gelatinous and easily deformable and are particularly suitable for use in various reactors. By adding other inert components such as gelatin during crosslinking, advantageous improvements in mechanical and binding properties are possible. In microencapsulation, the reaction volume of the peptide is restricted by a membrane. For example, microencapsulation can be carried out in the form of interfacial polymerization. By immobilization during microencapsulation, the peptides become insoluble and thus reusable. In the context of the present invention, immobilized constructs are in a state where all of their peptides allow for reuse. Restricting the mobility and solubility of peptides by chemical, biological, or physical means advantageously results in lower processing costs, particularly when eliminating autoantibodies from blood components.

[0143] The present invention also relates to a diagnostic kit for determining an autoimmune disease associated with NMDAR autoantibodies, and includes one or more NMDAR protein constructs disclosed herein. The diagnostic kit contains all the necessary reagents specific to the analyte required to perform the diagnostic test. Further, the diagnostic kit may include instructions on how to perform the test using the provided reagents. Optionally, the above diagnostic kit includes instructions regarding combining the contents of the kit and / or providing a formulation for detecting an autoimmune disease associated with NMDAR autoantibodies such as NMDAR encephalitis. For example, the instructions can be in the form of an instruction leaflet or other medium that provides the user with information regarding the type of method in which the substances mentioned are used. Clearly, the information does not necessarily have to be in the form of an instruction leaflet, and for example, the information may be provided via the Internet. For a patient, one advantageous effect of such a kit is that, for example, the patient can determine the actual disease state even while traveling without the doctor having to directly intervene, and adapt the diet and activities accordingly.

[0144] Aspects of the blood treatment device and immobilization of the NMDAR protein construct in the device The present invention also relates to a blood treatment device configured to remove NMDAR autoantibodies from the blood or plasma of a person in need of treatment in an extracorporeal blood circuit, the device comprising a matrix having one or more NMDAR protein constructs of the present invention immobilized thereon.

[0145] Accordingly, as used herein, "matrix" refers to an internal substance through which blood or plasma passes, or a substance within a blood treatment device that provides a surface over which blood or plasma passes. The matrix used in connection with the present invention preferably includes a support to which the NMDAR protein construct is bound. Accordingly, this support serves as a carrier for the NMDAR protein construct, but may also serve other functions.

[0146] As used herein, "support" refers to the portion of the matrix that serves as the "substrate" or "support material" to which the construct according to the invention is attached. Such a support or support material may also be referred to as an "adsorbent substance" or "adsorbent" as used in an "adsorption column" or "column" or "adsorption cartridge". A suitable support according to the invention is uniform over the relevant pH range and temperature, hydrophilic, mechanically and chemically stable, with no or negligible leaching of the NMDAR protein construct during use, and must have good flow characteristics with respect to whole blood and / or plasma, and provide a large surface area for the attachment of the NMDAR protein construct.

[0147] The support can be, for example, a resin, a membrane, or a non-woven material. A "non-woven" material is broadly defined as a sheet, fabric, or web structure joined together by mechanical, thermal, or chemical entanglement (and by perforating a film) without weaving or knitting fibers or filaments. "Resin" refers to an insoluble material that can take the form of a gel or gel beads or microporous beads, or a sponge. Such resins can be natural polymers or biopolymers, synthetic polymers, and inorganic materials. Agarose beads, dextran beads, and cellulose beads are commonly used natural supports. Most synthetic polymers or organic supports are based on derivatives of acrylamide, polystyrene, and polymethacrylate, while porous silica and glass are some of the frequently used inorganic supports.

[0148] According to one embodiment of the present invention, the resin is composed of a polymer selected from the group consisting of alginate, chitosan, chitin, collagen, carrageenan, gelatin, cellulose, starch, pectin, and sepharose; an inorganic material selected from the group consisting of zeolite, ceramics, celite, silica, glass, activated carbon, and carbon; or a synthetic polymer selected from the group consisting of polyethylene (PE), polyoxymethylene (POM), polypropylene (PP), polyvinyl chloride (PVC), polyvinyl acetate (PVA), polyvinylidene chloride (PVDC), polystyrene (PS), polytetrafluoroethylene (PTFE), polyacrylate (PAA), polymethyl methacrylate (PMMA), polyacrylamide, polyglycidyl methacrylate (PGMA), acrylonitrile butadiene styrene (ABS), polyacrylonitrile (PAN), polyester, polycarbonate, polyethylene terephthalate (PET), polyamide, polyaramide, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), polysulfone (PS), polyethersulfone (PES), polyarylethersulfone (PAES(PEAS)), ethylene vinyl acetate (EVA), ethylene vinyl alcohol (EVOH), polyamideimide, polyaryletherketone (PAEK), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polycaprolactone (PCL), polyhydroxyalkanoate, polyetheretherketone (PEEK), polyetherketoneketone (PEKK), polyetherimide (PEI), polyimide, polylactic acid (PLA), polymethylpentene (PMP), poly(p-phenylene ether) (PPE), polyurethane (PU), styrene acrylonitrile (SAN), polybutenoic acid, poly(4-allylbenzoic acid), poly(glycidyl acrylate), polyglycidyl methacrylate (PGMA), acrylonitrile butadiene styrene (ABS), polydivinylbenzene (PDVB), poly(allyl glycidyl ether), poly(vinyl glycidyl ether), poly(vinyl glycidyl urethane), polyallylamine, polyvinylamine, copolymers of the above polymers, and any of these polymers modified by the introduction of functional groups.

[0149] A variety of known methods can be used to immobilize the NMDAR protein construct onto the support and / or matrix according to the present invention. Such immobilization is preferably specific or selective in that it immobilizes the NMDAR protein construct while not immobilizing other proteins and components present in blood or plasma or in vitro samples thereof.

[0150] "Immobilizing" an NMDAR protein construct onto a support providing a matrix that can be used in the device according to the present invention refers to non-covalent or covalent interaction that holds the two molecules together. According to one embodiment of the present invention, this expression refers to a covalent interaction, i.e., a covalently bound NMDAR protein construct. Non-covalent interactions include, but are not limited to, hydrogen bonds, ionic interactions between charged groups, van der Waals interactions, and hydrophobic interactions between non-polar groups. One or more of these interactions can mediate the binding of two molecules to each other. The binding may otherwise be specific or selective, or non-specific.

[0151] According to one embodiment, the NMDAR protein construct comprises an affinity tag for immobilization on a support. The affinity tags can be used for the purification of the protein during production and / or their immobilization on the support of the matrix of the present invention. The affinity tag can be a short polypeptide sequence or an entire protein and can be co-expressed as a fusion partner with the NMDAR protein construct. Different types of affinity tags are well known in the art, and polyhistidine or His6 tags, C-myc tags, and FLAG tags are particularly well described and are options for binding the constructs according to the present invention to a support material. Non-covalent binding of biotin to streptavidin or avidin can also be used to immobilize the NMDAR protein construct to a support. In an embodiment, the binding is mediated by an Fc fragment that forms part of a particular construct of the present invention.

[0152] According to another embodiment of the present invention, the NMDAR protein construct is attached to the support by covalent bonding as further detailed below and / or as described in the prior art. Covalent bonds generally include either non-site-specific covalent bonds of proteins or site-specific covalent bonds of proteins. The support underlying the generation of the matrix needs to provide or facilitate chemical activation and thus enable chemical bonding of the construct. Many binding methods for immobilizing proteins such as NMDAR protein constructs are well known in the art.

[0153] For example, since the activation chemistry must be stable over a wide range of pH, buffer conditions, and temperatures, leaching of the construct can be ignored. The binding method should avoid improper orientation, multi-site binding, or steric hindrance of the construct. The construct density per volume of the matrix can be optimized to facilitate target accessibility and reaction.

[0154] Conjugation can be carried out via common functional groups including amines, alcohols, carboxylic acids, aldehydes and epoxy groups. To directly conjugate to a primary amine on the support surface via an amide bond, carbodiimide compounds can be used to activate the carboxylic acid groups of the protein. The most commonly used carbodiimides are the water-soluble EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) for aqueous crosslinking and the water-insoluble DCC (N',N'-dicyclohexylcarbodiimide) for non-aqueous organic synthesis methods.

[0155] Alternatively, the support can have linkers and / or specific functional groups for binding protein constructs. For example, functionalized resins are commercially available and known to those skilled in the art. A wide range of binding chemistries including primary amines, sulfhydryls, aldehydes, hydroxyls, and carboxylic acids are available in the commercially available supports. Examples of commercially available activated resins are CarboLinkCoupling resin, Profinity™ Epoxy resin, Affi-Gel 10 and 15, Epoxy Activated Sepharose™ 6B, Tosyl Chloride Activated Agarose, and Purolite™ Lifetech™ Methacrylate polymer functionalized with epoxy groups.

[0156] According to one embodiment of the present invention, the support material must be porous, and the pore size ranges from 10 nm to 200 nm. According to another embodiment of the present invention, the support takes the form of beads. According to yet another embodiment of the present invention, the support according to the present invention includes magnetic beads. The magnetic beads are prepared by trapping magnetite within agarose or other polymeric materials, and the NMDAR protein construct according to the present invention is immobilized thereon.

[0157] According to another embodiment of the present invention, the support is a membrane. Membranes as components of the affinity matrix have been used for protein purification because of their simplicity, ease of handling, reduced surface area, and low diffusion limitation compared to gels, resins, and beads. The membrane can take the physical form of hollow fibers or the form of flat membranes. According to one embodiment, the support is a hemodialysis hollow fiber membrane dialysis device, and the filter is a hemodialysis device.

[0158] The hollow fibers or flat membranes used as supports in the devices according to the present invention can be composed of cellulose, cellulose esters (cellulose acetate and cellulose triacetate), poly(methyl methacrylate) (PMMA), polyamide (PA), other nitrogen-containing polymers (polybenzimidazole, polyacrylonitrile (PAN)), polyglycidyl methacrylate (PGMA), polyvinylpyrrolidone (PVP), polysulfone (PS), polyethersulfone (PES), or polyarylether sulfone (PAES). The hollow fiber membranes that can be advantageously utilized to provide the devices according to the present invention preferably have an inner diameter in the range of 100 μm to 500 μm. According to another embodiment of the present invention, specifically when the membrane support is the hemodialysis membrane described above, the hollow fiber membrane can additionally or alternatively be functionalized by the NMDAR protein construct according to the present invention on the inner lumen side of the fibers that can directly interact with the target metabolites in the blood or plasma perfusing the lumen of the hollow fiber membrane. This construct can additionally or alternatively be immobilized on the outer side of the membrane.

[0159] Method for extracorporeal blood treatment The present invention includes a device configured to be disposed in an extracorporeal blood circuit through which a patient's blood passes, comprising means for transporting blood from the patient's vascular system to a blood treatment device at a defined flow rate and then returning the treated blood to the patient, the device being further configured to reduce the level of NMDAR autoantibodies in the blood.

[0160] According to the present invention, the expression "extracorporeal blood purification" preferably refers to a process of removing substances from body fluids by clearance of substances from blood flowing in a bypass circuit outside (extracorporeal) the patient's body. Such substances may include endogenous toxins (i.e., uremic toxins), exogenous poisons (i.e., ethylene glycol or mycotoxins), administered drugs, viruses, bacteria, antibodies, metabolites, and proteins (i.e., IMHA, myasthenia gravis), abnormal cells (i.e., leukemia), and excessive water. Treatment procedures include hemodialysis, including intermittent hemodialysis (HD, HDF, HF) and continuous renal replacement therapy (CRRT); hemoperfusion; plasma exchange, and therapeutic apheresis. Such methods are known to those skilled in the art and the device of the present invention can be incorporated accordingly.

[0161] As used herein, the expression "blood" refers to whole blood containing all components of a living organism's blood, including red blood cells, white blood cells, and platelets suspended in plasma. The expression "plasma" refers to a fluid composed of approximately 92% water, 7% proteins such as albumin, gamma globulin, fibrinogen, complement factors, coagulation factors, etc., and 1% mineral salts, sugars, fats, electrolytes, hormones, and vitamins, which forms a part of whole blood that no longer contains red blood cells, white blood cells, and platelets. In the context of the present invention, the expressions "blood plasma" or "plasma" refer to a specific fraction of plasma as defined above in its standard meaning, such as serum.

[0162] According to one aspect, the blood flow rate in the extracorporeal blood purification circuit is 20 ml / min to 700 ml / min. The typical dialysate flow rate in an extracorporeal circuit including a hemodialysis device for the treatment of renal insufficiency is in the range of 0.5 l / hour to 800 ml / min, in addition to the blood treatment device according to the present invention, or when the hemodialysis device is further configured to bind NMDAR autoantibodies.

[0163] In therapeutic apheresis, whole blood can be processed, or the blood can be separated into its component fractions, for example, by centrifugation or using a cell membrane or filter, and the fraction containing the solute to be removed is specifically processed before being returned to the patient.

[0164] The present invention provides an apheresis treatment in which whole blood or plasma (including the target protein) is removed from the patient's flowing blood and returned to the patient after contact with the device or matrix according to the present invention. The typical blood or plasma flow rates in an extracorporeal circuit perfused with whole blood or plasma by a blood treatment device are in the ranges of 30 ml / min to 200 ml / min and 7 ml / min to 50 ml / min, respectively.

[0165] According to one aspect, the extracorporeal blood circuit according to the present invention is configured to perform hemodialysis. In this case, the device according to the present invention is, for example, a hemodialysis device additionally configured to immobilize / bind the NMDAR autoantibody according to the present invention. This circuit can be operated in different treatment modes including hemodialysis, hemodiafiltration, and hemofiltration modes according to medical needs.

[0166] Unless otherwise specified, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. Also, all publications, patent applications, patents, and other references mentioned in this specification are hereby incorporated by reference in their entirety to form a part of this specification.

[0167] Drawings The present invention will be further described with reference to the accompanying drawings. These are not intended to limit the scope of the present invention, but rather to show preferred embodiments of aspects of the present invention presented for further explanation of the present invention described herein.

Brief Description of the Drawings

[0168]

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Figure 10

DETAILED DESCRIPTION OF THE INVENTION

[0169] Detailed Description of the Drawings Figure 1A. Simplified diagram of an NMDA receptor subunit encompassing the amino-terminal domain (ATD), ligand-binding domain (LBD), plasma membrane (PM) spanning / associated segments (M1-4), and intracellular carboxy-terminal domain (CTD). For details, see Paoletti et al. (2013) Nature Rev Neurosci 14, 383ff (Non-Patent Document 12). Figure 1B. The extracellular domain of the NMDA receptor subunit depicted in A fused to rabbit Fc (rbFc, the black triangle represents a single polypeptide containing CH2 and CH3) yields a soluble antigen. The black lines represent linkers between domains derived from different subunits (see Table 3 for details). Figure 1C. The soluble antigen is expected to form homodimers or heterodimers by rbFc upon (co)expression (showing the selection of possible combinations).

[0170] Figure 2. Cell culture supernatants of HEK293 cells expressing and secreting the designated fusion proteins (Table 3) were captured on a 96-well plate with an anti-rabbit IgG antibody. A single recombinant (rec) human (hu) autoantibody against LGI1 or GluN1 (Non-Patent Document 6) derived from patient CSF cells was applied at 1 μg / ml and detected using a horseradish peroxidase (HRP)-conjugated anti-human IgG antibody, and the captured Fc fusion protein was directly detected using a 0.016 μg / ml HRP-conjugated anti-rabbit (rb) IgG antibody. The ELISA signal is shown as the mean ± SD of two wells in a single experiment after subtracting the signal generated by the anti-human IgG antibody alone. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0171] Figure 3. The cell culture supernatant of HEK293 cells expressing and secreting the designated fusion protein (Table 3) was captured on a 96-well plate with an anti-rabbit Fc antibody. Serum (S3 - S9) from 7 NMDAR encephalitis patients and control serum (control S) were applied at a 1:200 dilution, and human IgG was detected using biotin-conjugated anti-human IgG and HRP-conjugated streptavidin. Panels A - C represent individual experiments. The ELISA signal is shown as the mean ± SD of two wells in a single experiment after subtracting the signal caused by the combination of biotin-conjugated anti-human IgG and HRP-conjugated streptavidin alone. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0172] Figure 4. The cell culture supernatant of HEK293 cells expressing and secreting the designated fusion protein was captured on a 96-well plate with an anti-rabbit Fc antibody. Serum (S10 - S14) from 5 NMDAR encephalitis patients and 3 control sera (S15, S18, S19) were applied at a 1:100 dilution, and human IgG was detected using biotin-conjugated anti-human IgG and HRP-conjugated streptavidin. The ELISA signal is shown as the mean ± SD of two wells in a single experiment after subtracting the signal caused by the combination of biotin-conjugated anti-human IgG and HRP-conjugated streptavidin alone. The NMDAR antibody titers of the sera determined by the Euroimmun cell-based assay are shown below for comparison. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0173] Figure 5. To screen for LGI1 reactivity, the cell culture supernatant of HEK293T cells expressing CSF cell-derived antibody cDNA was applied to LGI1-Fc and NMDA receptor subunit GluN1-ATD-Fc captured on an ELISA plate. Assay examples show the results of controls containing, alongside 13 supernatants, CSF samples (diluted 1:5), human recombinant anti-GluN1, mouse anti-LGI1, and secondary antibodies against human, mouse, and rabbit IgG alone. The signal is shown as the mean ± SD of two wells.

[0174] Figure 6. NR1 (GluN1)-specific human IgG was detected only in whole brain extracts of neonatal mice by ELISA after prenatal NR1 antibody injection, and the concentration increased between P0 and P7. ELISA quantification of NR1-specific human AB in mouse brain extracts revealed an increase in the level of brain-bound IgG from P0 (mean = 10.7 ng) to P7 (mean = 37.4 ng) in the NR1 group.

[0175] Figure 7. Cell culture supernatants of HEK293 cells expressing and secreting the designated fusion proteins (Table 3) were captured on 96-well plates with anti-rabbit IgG antibody. Single recombinant human NMDA receptor autoantibodies (anti-NR-Ab1 and anti-NR-Ab2) or control antibody (mGO53) derived from patient CSF cells were applied at 4.0 μg / ml (anti-NR-Ab1), 3.1 μg / ml (anti-NR-Ab2), and 5.9 μg / ml (mGO53), detected with horseradish peroxidase (HRP)-conjugated anti-human IgG antibody, and the captured Fc fusion proteins were directly detected with 0.016 μg / ml HRP-conjugated anti-rabbit (rb) IgG antibody. ELISA signals are shown as the mean ± SD of two wells in a single experiment after subtraction of the signal generated by anti-human IgG antibody alone. The 490 nm value for anti-rabbit IgG is shown. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0176] Figure 8. The cell culture supernatant of HEK293 cells expressing and secreting the designated fusion protein (Table 3) was captured on a 96-well plate with an anti-rabbit IgG antibody. A single recombinant human NMDA receptor autoantibody (Non-Patent Document 6 or unpublished) or a recombinant human control antibody (Control Ab1) derived from the patient's CSF cells was applied at 1 μg / ml, detected using a horseradish peroxidase (HRP)-conjugated anti-human IgG antibody, and the captured Fc fusion protein was directly detected using a 0.016 μg / ml HRP-conjugated anti-rabbit (rb) IgG antibody. The ELISA signal is shown as the mean ± SD of two wells in a single experiment after subtracting the signal generated by the anti-human IgG antibody alone. PGRN, progranulin. Fc, the constant region of the rabbit IgG1 heavy chain.

[0177] Figure 9. The cell culture supernatant of HEK293 cells expressing and secreting the designated fusion protein (Table 3) was captured on a 96-well plate with an anti-rabbit IgG antibody. A single recombinant human NMDA receptor autoantibody (Non-Patent Document 6 or unpublished) or a control antibody (mGO53) was applied at 0.09 μg / ml (003-102) or 1.8 μg / ml (anti-NR-Ab1, mGO53), detected using a horseradish peroxidase (HRP)-conjugated anti-human IgG antibody, and the captured Fc fusion protein was directly detected using a 0.016 μg / ml HRP-conjugated anti-rabbit (rb) IgG antibody. The ELISA signal is shown as the mean ± SD of two wells in a single experiment after subtracting the signal generated by the anti-human IgG antibody alone. The 490 nm values for 003-102 and anti-rabbit IgG are shown. PGRN, progranulin. Fc, the constant region of the rabbit IgG1 heavy chain.

[0178] The cell culture supernatant of HEK293 cells expressing and secreting the designated fusion protein (Table 3) was captured on a 96-well plate with an anti-rabbit IgG antibody. A single recombinant human NMDA receptor autoantibody (Non-Patent Document 6 or unpublished) derived from the patient's CSF cells was applied at 0.01 μg / ml (003-102), 1 μg / ml (008-218, anti-NR-Ab1) or 10 μg / ml (all others), detected using horseradish peroxidase (HRP)-conjugated anti-human IgG antibody, and the captured Fc fusion protein was directly detected using a 0.016 μg / ml HRP-conjugated anti-rabbit (rb) IgG antibody. The ELISA signal is shown as the mean ± SD of two wells in a single experiment after subtracting the signal generated by the anti-human IgG antibody alone. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0179] Regarding FIGS. 2 to 10, in each figure, the order of the tested constructs in the legend from top to bottom corresponds to the order of the bars from left to right for each condition.

Example

[0180] The present invention will be further described by the following examples. These are not intended to limit the scope of the present invention, but rather to show preferred embodiments of aspects of the present invention presented for further explanation of the present invention described herein.

[0181] Technical Doubts Is it possible to generate recombinant soluble fusion proteins for the labeling, detection, and isolation of NMDA receptor autoantibodies against NMDA receptors with different subunit compositions in patients' sera and CSF?

[0182] Solution The amino-terminal domain (ATD) of the NMDA receptor subunit GluN1 was fused to the constant region of the rabbit IgG1 heavy chain (rbFc), either alone or in combination with the extracellular domain of the NMDA receptor subunits GluN2A or GluN2B, regardless of the presence or absence of additional extracellular domains of GluN1.

[0183] Fc-mediated dimerization of the expressed protein may result in epitopes that closely resemble those of the native NMDA receptor and may confer unprecedented stability to the fusion protein. These soluble recombinant NMDA receptor Fc (srNR-Fc) fusion protein / antigens can detect NMDA receptor autoantibodies against different NMDA receptor subunit compositions present in the sera of NMDAR encephalitis patients and are thus the core subject matter of the present invention.

[0184] The ELISA method used to detect NMDA receptor autoantibodies using the srNR-Fc fusion protein / antigen can be used as a companion diagnostic.

[0185] Detailed Examples Example 1: Exemplary protein constructs of the present invention generated for the experiment. The inventors generated constructs (Figures 1A and 1B, Table 1) encompassing the extracellular portions of the GluN1 and GluN2 subunits, expressed them in HEK293 cells, and isolated the cell culture supernatant containing the Fc fusion protein secreted after 3 days.

[0186] Constructs #1, #2, #3, #5, #6, and #9 (Table 3) are Fc fusion proteins of either the GluN1 or GluN2 domain, and in constructs #4, #7, and #8, both the GluN1 and GluN2B domains are separated by an artificial linker and fused to Fc in a single molecule. The Fc domain is likely to result in dimerization of all the fusion proteins, resulting in GluN1 / GluN2 heterodimers upon co-expression of each of #5, #6, #9, or #3 with #1 or #2, or dimers of GluN1 / GluN2 heterodimers upon expression of constructs #4, #7, and / or #8 (Figure 1C).

[0187] Table 3: Soluble recombinant NMDA receptor rbFc fusion proteins that are composed of and / or represent the NMDAR protein constructs of the present invention.

Table 3-1

Table 3-2

[0188] The inventors established an ELISA to test the ability of the srNR-Fc protein to detect NMDA receptor antibodies in patients' sera. Briefly, the srNR-Fc protein in cell culture supernatants was captured on 96-well plates via anti-rabbit Fc or anti-rabbit IgG antibodies. Serum from NMDAR encephalitis patients or human monoclonal antibodies were applied, and bound antibodies were detected using either biotin-conjugated anti-human IgG and horseradish peroxidase (HRP)-conjugated streptavidin or HRP-conjugated anti-human IgG antibody, along with the HRP substrate ultraTMB.

[0189] Example 2: Soluble NMDA receptor rbFc fusion proteins are recognized by recombinant human GluN1 autoantibodies. Figure 2 shows that all tested srNR-Fc antigens other than the control antigen (progranulin-Fc) were recognized by recombinant human anti-NMDA receptor antibodies. In contrast, there were no antigens recognized by recombinant human anti-LGI1 antibodies. Using an anti-rabbit IgG antibody to detect the rbFc portion of the fusion protein, it was confirmed that the expression and immobilization of all rbFc fusion proteins on the ELISA plates were successful.

[0190] Example 3: The soluble NMDA receptor rbFc fusion protein detects NMDA receptor autoantibodies in the serum of patients. Figure 3 summarizes the results of three ELISA experiments using a set of human sera from patients with NMDAR encephalitis against different srNR-Fc antigens. The data reveals that antigens containing GluN1-ATD-Fc and additional extracellular regions of the GluN1 or GluN2 subunits produced signals similar to those in the sera of most NMDAR encephalitis patients compared to progranulin as a control. Three of the sera reacted strongly with the antigen, while the other four sera reacted only with the selected srNR-Fc antigen, producing lower signals. Diluting the sera 1:200 suggested that the srNR-Fc antigen enables highly sensitive detection of NMDA receptor autoantibodies. The addition of the GluN2 subunit region improved the sensitivity of detection. A subset of NMDAR autoantibodies can recognize GluN1 only in the presence of the assembled GluN2 domain. The antigen that produced the highest signal varied depending on the serum. For example, the antigen containing N1ecd-N2Becd was excellent for detecting antibodies in serum 5, while the antigen containing GluN1-ATD and GluN2B-ATD functioned better in detecting antibodies in serum 7.

[0191] Example 4: Detection of autoantibodies by the soluble NMDA receptor rbFc fusion protein in sera with known anti-NMDA receptor titers. To test how the ELISA based on the srNR-Fc protein differs from the clinical standard assay, the inventors measured sera with known titers from Euroimmun CBA (Figure 4). All sera that were recorded as positive by Euroimmun (S10 - S14) produced a positive signal with at least one of the antigens tested, compared to progranulin and GluN2B-ATD as controls, while sera that were recorded as negative by Euroimmun CBA (S15, S18, S19) did not produce a positive signal. These data indicate that srNR-Fc containing GluN1-ATD specifically detects NMDAR autoantibodies in sera.

[0192] The inventors used two srNR-Fc combinations (N1-ATD-Fc + N2B-ATD-Fc and N1-ATD-N2B-ATD-Fc) expressing the ATDs of GluN1 and GluN2B in this assay. They contain the same amino acids of NMDAR (Table 1), but in one case the antigen is reconstituted from two separate proteins, and in the other construct the ATDs are connected by an artificial linker as a single protein. These two antigens gave equivalent signals with sera S10 - S12, but were very different with sera S13 and S14. The antigen N1-ATD-Fc + N2B-ATD-Fc produced small, equivalent signals in S13 and S14. In contrast, N1-ATD-N2B-ATD-Fc did not detect any NMDAR antibody signal in S13 and detected a strong signal in S14. The molecular composition of N1-ATD-N2B-ATD-Fc may have blocked access to those epitopes of the NMDAR autoantibodies present in S13. This finding emphasizes the need to test several srNR-Fc combinations to detect as many antibodies as possible.

[0193] Example 5: Soluble NMDA receptor rbFc fusion proteins detect subtype-selective recombinant human NMDA receptor autoantibodies. Some autoantibodies were detected by soluble NMDA receptor Fc antigens that include the extracellular domains of two different NMDA receptor subunits, but not by soluble NMDA receptor Fc antigens that contain the extracellular domain of a single NMDA receptor subunit (Figure 7). These NMDA receptor antibodies are not detected in assays based on GluN1-expressing cells.

[0194] Using soluble NMDA receptor Fc antigens, it was determined whether specific subunit combinations are targeted by recombinant human NMDA receptor autoantibodies. NMDA receptor autoantibody 008-218 was detected with equal efficiency by any soluble NMDA receptor Fc antigen that contains the ATDs of GluN1 and GluN2A, or the ATDs of GluN1 and GluN2B. However, anti-NR-Ab1 was detected by a soluble NMDA receptor Fc antigen that contains the ATDs of GluN1 and GluN2B, but not by a soluble NMDA receptor Fc antigen that contains the ATDs of GluN1 and GluN2A (Figure 8). Furthermore, this antibody was not detected by an additional soluble NMDA receptor Fc antigen that contains the ATDs of GluN1 and GluN2C (Figure 9). Thus, the soluble NMDA receptor Fc antigen classifies anti-NR-Ab1 as a GluN1 / GluN2B subtype-selective antibody.

[0195] The soluble recombinant NMDA receptor Fc antigen N1-ATD-N2B-ATD-Fc yielded a higher signal than the assembled antigen N1-ATD-Fc+N2B-ATD-Fc with the GluN1 / GluN2B subtype-selective antibody anti-NR-Ab1 (Figure 8), suggesting that the specific molecular configuration of the Fc fusion protein N1-ATD-N2B-ATD-Fc, in which the ATDs of GluN1 and GluN2B are part of a single protein, provides an advantage in detecting subtype-selective antibodies.

[0196] Example 6: Detection of recombinant human NMDA receptor autoantibodies by three soluble NMDA receptor rbFc fusion proteins. In the tests of three soluble recombinant NMDA receptor Fc antigens, N1-ATD-N2B-ATD-Fc produced the highest signal with several of the human recombinant NMDA receptor autoantibodies investigated, and N1-ATD-Fc + N2B-ATD-Fc produced a signal equal to or greater than the others (Figure 10). These data demonstrate the differential antibody-specific sensitivity of the soluble recombinant NMDA receptor Fc antigens in the sera described in Example 4 and complement the findings thereof.

[0197] Discussion of the Examples The results provided herein serve as a proof of concept. The inventors have concluded that (1) a soluble fusion protein containing the amino-terminal domain of GluN1 and rabbit Fc, heterogeneously expressed and secreted from HEK293 cells, can bind NMDA receptor autoantibodies in the sera of patients, and (2) efficient detection of NMDA receptor autoantibodies by the soluble antigen benefits from the incorporation of the extracellular domain of GluN2. Furthermore, the use of different srNR-Fc antigens may enable the classification of the anti-NMDA receptor immune response in patients.

[0198] Detection of autoreactivity to selected NMDA receptor subtypes may enable differential diagnosis in anti-NMDA receptor encephalitis and other medical conditions associated with antibodies to the NMDA receptor.

[0199] Further examples of experimental uses of the constructs of the present invention ELISA screen for recombinant LGI1 antibodies using an NMDA receptor fusion protein as a control. To generate the mammalian expression constructs used in this experiment, cDNAs corresponding to amino acids 1-558 of human LGI1 (NM_005097.3) and amino acids 1-400 of human GluN1 (NM_007327) were inserted into pFuse-rIgG-Fc1 (in vivo Gen). The resulting plasmids encode the amino-terminal domain (ATD) of hsGluN1 fused to the Fc region (amino acids SKP-PGK) of rabbit IgG linked by either hsLGI1 or the amino acids GSSTMVRS. The chimeric constructs LRR1-EPTP2 and LRR2-EPTP1 encode rabbit Fc fusions of amino acids 1-223 of LGI1 and amino acids 218-545 of LGI2, or amino acids 1-217 of LGI2 and amino acids 224-557 of LGI1, respectively.

[0200] Antibodies that bind to LGI1-Fc and the NMDA receptor subunit GluN1-ATD-Fc were compared by ELISA. A 96-well high-binding microplate (Greiner #655061) coated with donkey anti-rabbit IgG (10 μg / mL, Dianova, #711-005-152) was blocked and incubated with cell culture supernatants of HEK293 cells expressing the Fc fusion protein. Cell culture supernatants containing monoclonal antibodies, CSF samples, or purified antibodies, and horseradish peroxidase (HRP)-conjugated donkey anti-human IgG (1:5000, Dianova, #709-035-149) were applied sequentially. After thorough washing, HRP activity was measured using 1-Step Ultra TMB-ELISA substrate (Thermo Fisher). The presence of immobilized antigen was confirmed by incubation with HRP-conjugated F(ab’)2 donkey anti-rabbit IgG (1:50000, Dianova, #711-036-152). Human recombinant anti-GluN1 antibody 003-102 (Kreye J, Wenke NK, Chayka M, et al. Human cerebrospinal fluid monoclonal N-methyl-D-aspartate receptor autoantibodies are sufficient for encephalitis pathogenesis. Brain 2016; 139:2641-2652 (Non-Patent Document 6)) was used at 10 ng / ml. The results are shown in Fig. 5.

[0201] ELISA quantification of recombinant human NR1 (GluN1) AB in mouse brain extracts using the NMDA receptor fusion protein. The concentration of recombinant human NR1AB #003-102 in brain extracts was determined in 96-well plates coated overnight at 4°C with donkey anti-rabbit IgG (20 μg / mL, Dianova, #711-005-152). After blocking with 2% BSA in PBS / 0.05% Tween-20 (PBS / T) at room temperature, cell culture supernatants of HEK293 cells expressing the amino-terminal domain (amino acids 1–400) of human NR1 (GluN1) fused to rabbit Fc were applied. Mouse brain extracts were diluted 1:25 / 1:100 with 0.4% BSA-PBS / T and added in duplicate. Plates were washed with PBS / T and incubated with horseradish peroxidase (HRP)-conjugated donkey anti-human IgG (1:5000, Dianova, #709-035-149). After washing, HRP activity was measured using 1-Step Ultra TMB-ELISA substrate (Thermo Fisher). The concentration of #003-102 in the extracts was extrapolated from a calibration curve generated using purified #003-102. The results are shown in Fig. 6. References Dalmau, J., Geis, C., and Graus, F. (2017).Autoantibodies to Synaptic Receptors and Neuronal Cell Surface Proteins inAutoimmune Diseases of the Central Nervous System. Physiol Rev 97, 839-887. Dalmau, J., Gleichman, A.J., Hughes, E.G.,Rossi, J.E., Peng, X., Lai, M., Dessain, S.K., Rosenfeld, M.R., Balice-Gordon,R., and Lynch, D.R. (2008). Anti-NMDA-receptor encephalitis: case series andanalysis of the effects of antibodies. Lancet Neurol 7, 1091-1098. Dalmau, J., and Graus, F. (2018).Antibody-Mediated Encephalitis. N Engl J Med 378, 840-851. Gable, M.S., Sheriff, H., Dalmau, J., Tilley,D.H., and Glaser, C.A. (2012). The frequency of autoimmune N-methyl-D-aspartatereceptor encephalitis surpasses that of individual viral etiologies in youngindividuals enrolled in the California Encephalitis Project. 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Malviya, M., Barman, S., Golombeck, K.S., Planaguma, J., Mannara, F., Strutz-Seebohm, N., Wrzos, C., Demir, F., Baksmeier, C., Steckel, J., et al. (2017). NMDAR encephalitis: passive transfer from man to mouse by a recombinant antibody. Ann Clin Transl Neurol 4, 768-783. Mueller, S.H., Farber, A., Pruss, H., Melzer, N., Golombeck, K.S., Kumpfel, T., Thaler, F., Elisak, M., Lewerenz, J., Kaufmann, M., et al. (2018). Genetic predisposition in anti-LGI1 and anti-NMDA receptor encephalitis. Ann Neurol 83, 863-869. Paoletti, P., Bellone, C., and Zhou, Q. (2013). NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. Nat Rev Neurosci 14, 383-400. Sharma, R., Al-Saleem, F.H., Puligedda, R.D., Rattelle, A., Lynch, D.R., and Dessain, S.K. (2018). Membrane-bound and soluble forms of an NMDA receptor extracellular domain retain epitopes targeted in auto-immune encephalitis. BMC Biotechnol 18, 41. 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Explanation of symbols

[0202] Drawing translation Figure 1A NMDA receptor subunits NMDA receptor subunits Figure 1B Soluble recombinant NMDA receptor Fc antigens Soluble recombinant NMDA receptor Fc antigens Figure 1C Homo- and heterodimeric assembly of soluble recombinant NMDA receptor Fc antigens Homo- and heterodimeric assembly of soluble recombinant NMDA receptor Fc antigens Figure 2 rec hsanti-LGI1 rec hs anti-LGI1 rec hsanti-GluN1 rec hs anti-GluN1 anti-rbIgG anti-rbIgG Figure 3A ctrl S control S Figure 3B ctrl S control S Figure 3C ctrl S control S Figure 4 anti-GluN1-Titer: anti-GluN1 titer: neg. negative Figure 5 ctrlSN control SN anti-GluN1 anti-GluN1 no hsAB no hsAB anti-LGI1 anti-LGI1 no mmAB no mmAB anti-rb anti-rb Figure 6 NR1-reactiveIgG [ng / brain] NR1-reactive IgG [ng / brain] CTL control Figure 7 A 450nm or * A 490 nm A 450 nm or * A 490 nm anti-NR-Ab1 anti-NR-Ab1 anti-NR-Ab2 anti-NR-Ab2 anti-rbIgG * anti-rbIgG * Figure 8 anti-NR-Ab1 anti-NR-Ab1 controlAb1 control Ab1 anti-rbIgG anti-rbIgG Figure 9 A 450nm or * A 490 nm A 450 nm or * A 490 nm anti-NR-Ab1 anti-NR-Ab1 anti-rbIgG * anti-rbIgG * Figure 10 anti-NR-Ab1 anti-NR-Ab1 anti-NR-Ab2 anti-NR-Ab2 controlAb1 control Ab1 anti-rbIgG anti-rbIgG

Claims

1. Soluble N-methyl-D-aspartate receptor (NMDAR) protein containing one or more NMDAR autoantibody epitopes A soluble NMDAR protein construct, comprising an extracellular domain (ECD) or a fragment thereof of the NMDAR subunit GluN1 and an ECD or a fragment thereof of at least one of the NMDAR subunits GluN2A, GluN2B, GluN2C, or GluN2D.

2. 2. The NMDAR protein construct of claim 1, wherein the construct lacks the NMDAR transmembrane domain.

3. The NMDAR protein construct of claim 1 or 2, wherein the construct comprises a dimerization domain and / or a capture domain.

4. 4. The NMDAR protein construct of claim 3, wherein the dimerization domain is the capture domain, preferably formed by an antibody Fc fragment.

5. The NMDAR protein construct of any one of claims 1 to 4, wherein the ECD or a fragment thereof of GluN1 comprises or consists of the amino-terminal domain (ATD) or a fragment thereof of GluN1, and / or the ECD or a fragment thereof of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C, or GluN2D comprises or consists of the ATD or a fragment thereof of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C, or GluN2D, respectively.

6. 6. The NMDAR protein construct according to any one of claims 1 to 5, wherein the ECD of GluN1 and the ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or fragments thereof, are covalently linked, preferably as a fusion protein.

7. The NMDAR protein construct according to any one of claims 1 to 6, wherein the construct is a protein dimer of non-covalently linked monomers, and the construct may be a homodimer or a heterodimer.

8. The NMDAR protein construct of claim 7, wherein the construct is a heterodimer formed from (as one monomer) the ECD of GluN1 or a fragment thereof and (as one monomer) the ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D, or a fragment thereof.

9. 1. An in vitro method for detecting NMDAR autoantibodies in a sample, comprising: a. Preparing a sample suspected of containing NMDAR autoantibodies; b. providing an NMDA protein construct according to any one of claims 1 to 8, comprising a capture domain as a capture molecule; c. contacting the sample with the NMDAR protein construct, thereby allowing NMDAR autoantibodies from the sample to bind to the NMDAR protein construct; d. Determining the presence, and optionally the amount, of bound NMDAR autoantibodies; A method comprising:

10. The method of claim 9, wherein the NMDAR autoantibodies in the sample are present in solution or on a cell membrane.

11. The method according to claim 9 or 10, wherein the method is performed with a plurality of different NMDAR protein constructs according to any one of claims 1 to 8, and preferably comprises additionally determining to which NMDAR protein construct of the plurality of constructs the NMDAR autoantibody binds, or preferably binds in the greatest amount and / or most efficiently.

12. 12. The method according to any one of claims 9 to 11, wherein the method is applied to the diagnosis, prognosis, disease monitoring, patient stratification and / or treatment monitoring of a medical condition associated with autoantibodies against NMDAR, preferably anti-NMDAR encephalitis, and wherein the sample suspected of containing NMDAR autoantibodies is a sample from a human subject presenting with symptoms of having said medical disorder.

13. The method according to any one of claims 9 to 12, wherein the method is applied to the therapeutic guidance of a subject having and / or suspected of developing a medical condition associated with NMDAR autoantibodies, the method comprising selecting the corresponding one or more NMDAR protein constructs according to any one of claims 1 to 8 for the subsequent treatment of the subject.

14. A kit for diagnosing an autoimmune disease associated with NMDAR autoantibodies, such as NMDAR encephalitis, in a subject by detecting NMDAR autoantibodies, A NMDAR protein construct according to any one of claims 1 to 8 and optionally the NMDAR protein. A solid surface for immobilizing the construct, or an NMDAR protein construct according to any one of claims 1 to 8 immobilized on a solid surface, and a labeled secondary affinity reagent directed against human NMDAR autoantibodies, such as a labeled anti-human IgG antibody, and a means for detecting a signal emitted by the label, or b. A labeled NMDAR protein construct according to any one of claims 1 to 8; Optionally, c. A control sample with a predetermined NMDAR autoantibody concentration; A kit comprising:

15. 9. A blood treatment device configured for removing NMDAR autoantibodies from the blood or plasma of a person in need of treatment in an extracorporeal blood circuit, the blood treatment device comprising a matrix having one or more NMDAR protein constructs according to any one of claims 1 to 8 immobilized thereon.

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