NMDA receptor construct 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, 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. 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 embodiments, 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 autoimmune encephalitis, which is increasing in number (Non-Patent Literature 1, Non-Patent Literature 2). This disorder primarily 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 rituximab and cyclophosphamide, along with glucocorticoids and plasmapheresis. NMDAR encephalitis is caused by the production of autoantibodies that target the extracellular domain of the major NMDA receptor subunit GluN1, both in the blood and in the brain. The antibodies alter surface dynamics and induce crosslinking and internalization of the NMDA receptor, and the resulting NMDA receptor depletion can explain some of the neurological symptoms observed in patients (Non-Patent Literature 3, Non-Patent Literature 4, Non-Patent Literature 5). A single recombinant human antibody against GluN1 derived from CSFB cells in which downregulation of NMDA receptor function was induced (Non-Patent Literature 6) suggests that they are the main pathogens of this disease. This hypothesis has recently been strengthened by mouse models using active and passive immunity (Non-Patent Literature 3, Non-Patent Literature 7, Non-Patent Literature 8).

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

[0004] Immunosuppression and plasmapheresis or intravenous immunoglobulin therapy are established treatments for NMDAR encephalitis. However, selective removal of disease-causing antibodies would be preferable, as it is expected to have fewer side effects. Serum depletion from NMDA receptor antibodies can be applied to NMDAR encephalitis and other disorders associated with NMDA receptor autoantibodies, for example, using in vitro techniques similar to plasmapheresis. A prerequisite for such apheresis would be the production of NMDA receptor autoantibodies from the patient, i.e., stable proteins capable of binding soluble antigens to NMDA receptor antibodies.

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

[0006] Autoantibodies against the NMDA receptor are routinely detected using Euroimmun cell-based assay (CBA) kits based on biochips containing acetone-fixed GluN1-expressing xenoplasmic cells, as described in Patent Document 1. However, NMDA receptor autoantibodies often recognize the native three-dimensional structure of their antigens, and live staining of NMDA receptor-expressing HEK293 cells has been found to be more sensitive than commercially available assays using pre-fixed cells for detecting low titers (Non-Patent Document 18). These cell-based assays require visual inspection of results that may introduce bias. Furthermore, even if the usual automated testing is possible in such tests, false positive results are likely to occur due to the fact that there are many different antigens on the cell surface, not just the antigen of interest.

[0007] Therefore, there is an urgent need for a highly sensitive, quantifiable, high-throughput method for detecting NMDA receptor autoantibodies. In this regard, a single nanoparticle imaging approach to primary hippocampal neurons can detect NMDA receptor autoantibodies at low titers and is automatable, but is technically very difficult (Non-Patent Literature 18). An ELISA that allows comparison of NMDA receptor autoantibodies at different titers based on lysed HEK293 cells expressing the NMDA receptor has been described at an early stage (Non-Patent Literature 19). Patent Literature 2 discloses a method for detecting antibodies against NR2A (GluN2A) and / or NR2B (GluN2B) in relation to a stroke diagnostic method in which the amino-terminal fragments of NR2A / NR2B are synthesized and purified, but does not involve combination with GluN1 ECD. In fact, the disclosed method identifies antibodies specifically directed to GluN2A or GluN2B, but is not intended to identify antibodies directed to GluN1, and is unrelated to their association with GluN1, and is therefore intended for a completely different application compared to the present invention. Importantly, the method described in Patent Document 2 cannot be used to identify antibodies that bind to GluN1 or a portion thereof.

[0008] In recent years, cell lines expressing the entire amino-terminal region of GluN1 fused to myc tags and polyhistidine tags (including amino acids 1-561, ATD and S1 domains), the tobacco ecchi disease virus (TEV) cleavage site, and the transmembrane region of the PDGF receptor have been presented (Non-Patent Literature 20). TEV treatment of these cells released the amino-terminal extracellular segment of GluN1. This fragment could be conjugated to an ELISA plate via an anti-polyhistidine antibody, allowing for the detection of monoclonal anti-GluN1 antibodies. However, the detection sensitivity of this antigen may be limited. In summary, there are no available stable soluble antigens of the NMDA receptor that maintain their native conformation and incorporate GluN1 and GluN2 segments for detection, for example, in ELISA, such as in serum or CSF.

[0009] Therefore, there remains a strong need in the art for providing an NMDAR protein construct comprising GluN1 and one or more NMDAR autoantibody epitopes that may be composed of, or stabilized by, GluN2A, GluN2B, GluN2C, and / or GluN2D extracellular domains or fragments thereof. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] International Publication No. 2012 / 076000 [Patent Document 2] U.S. Patent Application Publication No. 2003 / 096331 [Non-patent literature]

[0011] [Non-Patent Document 1] Dalmau et al., 2017 [Non-Patent Document 2] Dalmau and Graus, 2018 [Non-Patent Document 3] Hughes et al., 2010 [Ref. 4] Jezequel et al., 2017a [Ref. 5] Ladepeche et al., 2018 [Ref. 6] Kreye et al., 2016 [Ref. 7] Jones et al., 2018 [Ref. 8] Malviya et al., 2017 [Ref. 9] Kim et al., 2017 [Ref. 10] Mueller et al., 2018 [Ref. 11] Wenke et al., 2019 [Ref. 12] Paoletti et al., 2013 [Ref. 13] Gleichman et al., 2012 [Ref. 14] Lee and Gouaux, 2011 [Ref. 15] Lee et al., 2014 [Ref. 16] Tajima et al., 2016 [Ref. 17] Zhu et al., 2016 [Ref. 18] Jezequel et al., 2017b [Ref. 19] Dalmau et al., 2008 [Ref. 20] Sharma et al., 2018 [Summary of the Invention] [Problems to be Solved by the Invention]

[0012] In light of prior art, the underlying technical problem of 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 clause. Preferred embodiments of the present invention are provided by the dependent clause.

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

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

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

[0017] Furthermore, the present invention relates to an N-methyl-D-aspartate receptor (NMDAR) protein construct comprising one or more NMDAR autoantibody epitopes, wherein the construct comprises the extracellular domain (ECD) or a fragment thereof of the NMDAR subunit GluN1 and the ECD or a fragment thereof of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C, or GluN2D, and is not present inside or on a cell. In embodiments, the NMDAR protein construct comprises the extracellular domain (ECD) or a fragment thereof of the NMDAR subunit GluN1 and the ECD or a fragment thereof of the NMDAR subunit GluN2A and / or the ECD or a fragment thereof of GluN2B, and is not present inside or on a cell.

[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 demonstrate that the soluble NMDAR protein construct of the present invention, particularly in its embodiment as an Fc fusion protein, can detect antibodies in the serum of NR encephalitis patients. The soluble NMDAR-Fc protein construct (srNR-Fc protein) has several advantages over state-of-the-art cell and cell-based assays that detect antibodies using NMDAR expressed on the surface of in vitro cultured cells. The srNR-Fc protein can be purified and stored. Purified srNR-Fc proteins constitute a clean antigen compared to heterogeneous cells containing many additional proteins. They enable the generation of very high antigen concentrations, leading to improved sensitivity in antibody detection compared to cell-based assays (CBAs).

[0020] In contrast to NMDAR constructs of known status, the present invention relates to an NMDAR construct comprising at least fragments of two NMDAR subunits, which enables the identification of autoantibodies that bind to an epitope formed by residues of two subunits, or that are formed or stabilized only by an assembly of two subunits. In contrast, a state-of-the-art construct comprising only one subunit or its fragment does not bind to and remove such autoantibodies.

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

[0022] For example, NMDAR protein constructs disclosed herein, including Fc fragments or other or additional tags, may be useful in a variety of diagnostic assays. For instance, the ELISA-like assay-based diagnostic assay disclosed in the Examples may be used as a companion diagnostic to detect NMDA receptor autoantibodies and enable a quantifiable, high-throughput method for detecting autoantibodies in other autoimmune encephalopathy.

[0023] Furthermore, NMDAR protein constructs can be used to differentiate autoimmune responses to different NMDA receptor compositions, enabling patient classification.

[0024] As disclosed in the examples provided herein, various NMDAR protein constructs, such as srNR-Fc fusion proteins and dimers formed by such fusion proteins, produce different signals in response to antibodies / serums from different patients and exhibit patient-specific mutant antibody profiles. Therefore, a set of different NMDAR protein constructs of the present invention, including different combinations of ECD and ATD 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., distinguish between GluN1 or hetero-GluN1 / GluN2 structures and antibodies that are potentially primarily directed towards GluN1 / GluN2 structures of a specific composition. Subclassification of NMDAR encephalitis patients could 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 that provide one or more of GluN1 and GluN2A-GluN2D, or different combinations thereof.

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

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

[0028] In embodiments, the present invention relates to two NMDAR protein constructs, wherein one construct comprises the ECD of GluN1 and GluN2A, or a fragment thereof, and the other construct comprises the ECD of GluN1 and Glu2D, or a fragment thereof.

[0029] In embodiments, the present invention relates to two NMDAR protein constructs, wherein one construct comprises the ECD of GluN1 and GluN2C, or a fragment thereof, and the other construct comprises the ECD of GluN1 and Glu2B, or a fragment thereof.

[0030] In embodiments, the present invention relates to two NMDAR protein constructs, wherein one construct comprises the ECD of GluN1 and GluN2D, or a fragment thereof, and the other construct comprises the ECD of GluN1 and Glu2B, or a fragment thereof.

[0031] In embodiments, the present invention relates to two NMDAR protein constructs, wherein one construct comprises the ECD of GluN1 and GluN2C, or a fragment thereof, and the other construct comprises the ECD of GluN1 and Glu2D, or a fragment thereof.

[0032] The NMDAR protein construct of the present invention may include four ECDs of the GluN subunit that mimic the tetrameric assembly of an NMDAR. For example, the construct of the present invention may include two GluN1ECDs or fragments thereof and two equal or different ECDs of GluN2A, GluN2B, GluN2C and / or GluN2D.

[0033] Furthermore, the construct, which enables the labeling of B cells expressing NMDA receptor autoantibodies, can be used as a diagnostic tool and facilitates cell isolation and IgG sequence analysis.

[0034] The NMDAR (NMADR) protein construct 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 patient's serum and CSF. Labeling of B cells expressing NMDA receptor autoantibodies can also be used as a diagnostic tool.

[0035] Furthermore, such a construct can be used to selectively remove antibodies against the NMDA receptor from a patient's serum or CSF.

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

[0037] The following further preferred embodiments of the present invention relate to each of the NMDAR protein constructs described above.

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

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

[0040] Importantly, in the specific construct of the present invention, the dimerizing domain is a capture domain.

[0041] In the 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 capture domain comprises or consists of an antibody Fc fragment. In the embodiments, the Fc fragment is a rabbit IgGFc fragment.

[0043] The presence of dimerization and / or capture domains, such as Fc fragments, can be particularly advantageous because these domains stabilize the soluble NMDAR protein construct. 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 relation to 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 contain or consist of the ATD of GluN2A or a fragment thereof and / or the ATD of GluN2B or a fragment thereof, respectively.

[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. In this embodiment, 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 linked directly as a fusion protein or by a protein linker.

[0048] In this embodiment, the ECD of GluN1 and the ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C, or GluN2D, or fragments thereof, are linked by a protein linker that includes or consists of one or more repeats of the amino acid sequence GGGGS.

[0049] In this embodiment, the ECD of GluN1 and the ECD of GluN2A and / or GluN2B, or fragments thereof, are linked by a protein linker that includes or consists of one or more repeats of the amino acid sequence GGGGS.

[0050] In a particular embodiment of the present invention, 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 including the ECD of GluN1, the ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C, or GluN2D, or a fragment thereof, and a portion of the construct including a dimerization domain and / or a capture domain.

[0051] In the 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 containing the ECD of GluN1, the ECD of GluN2A and / or the ECD of GluN2B, or fragments thereof, and a portion of the construct containing the dimerization domain and / or capture domain.

[0052] In preferred embodiments of the NMDAR protein construct of the present invention, the construct is a protein dimer of non-covalent monomers, and the construct may be a homodimer or a heterodimer. In certain dimer constructs of the present invention, the construct may be a heterodimer formed from the ECD or fragment thereof of GluN1 (as one monomer) and the ECD or fragment thereof of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C, or GluN2D (as one monomer). In embodiments of the dimer construct, the construct may be a heterodimer formed from the ECD or fragment thereof of GluN1 (as one monomer) and the ECD or fragment thereof of GluN2A (as one monomer), and / or the ECD or fragment thereof of GluN2A. Preferably, the dimer is formed by dimerization domains composed of each monomer. In further embodiments, the NMDAR protein construct of the present invention may be a dimer of two monomers, each monomer comprising two ECDs of two NMDAR subunits, e.g., 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 includes four ECDs of an NMDAR subunit (provided by two monomers) or fragments thereof, and thus a tetramer of four subunits, namely two GluN1 subunits and two GluN2 subunits, which mimics the naturally occurring NMDA receptor (NMDAR, NMDAR receptor). For example, dimerization of the fusion protein N1-ATD-N2B-ATD-Fc disclosed herein results in the formation of the NMDAR protein construct of the present invention, formed by two N1-ATD-N2B-ATD-Fc monomers containing four ATD domains.

[0053] The NMDAR protein construct of the present invention is advantageous compared to known NMDAR constructs because it allows for the assembly of ECDs or ATDs of the GluN1, GluN2A, GluN2B, GluN2C, and / or GluN2D subunits in a given construct. The potential of this ligand combination is a strong advantage because autoantibodies specific to certain subunits or combinations of subunits can be efficiently bound by the construct of the present invention, allowing for the evaluation of a range of autoantibodies present in a sample based on their differential binding profiles. Furthermore, the ability to combine ECDs or ECD fragments of the GluN1, GluN2A, GluN2B, GluN2C, and / or GluN2D subunits with capture domains allows for flexible use of the construct, for example, in ELISA assays, 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, more closely reconstruct the natural context 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 construct of the present invention may include GluN1-ATD and GluN2B-ATD, or GluN1-ECD and GluN2B-ECD, in either a single fusion protein or a construct containing two proteins that form a heterodimer by assembly via a dimerizing 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), and fusion protein #8 (N1-ATD-N2B-ATD-Fc) and fusion protein #4 (N1ecd-N2Becd-Fc) were shown to be particularly advantageous for binding to autoantibodies present in patient samples.

[0055] In vitro method for detecting NMDAR autoantibodies in a sample Furthermore, the present invention relates to an in vitro method for detecting NMDAR autoantibodies in a sample, Prepare a sample suspected of containing NMDAR autoantibodies, The NMDA protein construct described in any one of the above claims is prepared as a capture molecule, The above sample is brought into contact with the above NMDAR protein construct, thereby binding the NMDAR autoantibody from the above sample to the above NMDAR protein construct. The presence of bound NMDAR autoantibodies, and, if necessary, the determination of their quantity, This includes methods.

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

[0057] In the embodiments, 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 include a step of determining which of the above plurality of NMDAR protein constructs to which the NMDAR autoantibodies bind, preferably in the maximum amount and / or most efficiently. Thus, it may be possible to profile and classify patients providing samples based on the NMDAR autoantibodies and their binding characteristics determined 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 (multiplexed).

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

[0059] In embodiments of the method of the present invention, the presence and, optionally, the amount of cells present on the cell surface of NMDAR autoantibodies in the sample may be determined. A major advantage of the method of the present invention is that, in addition to soluble NMDAR autoantibodies, NMDAR autoantibodies can be detected on the cell surface, particularly on the surface of B cells that produce NMDAR autoantibodies.

[0060] In the embodiments, 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 NMDARs, preferably anti-NMDAR encephalitis, wherein the sample suspected of containing NMDAR autoantibodies is a sample from a human subject exhibiting symptoms of the above-mentioned medical disorder.

[0061] In embodiments of methods for diagnosing, predicting the prognosis of, monitoring the disease, stratifying patients, and / or monitoring treatment of a medical condition associated with autoantibodies against NMDARs, preferably anti-NMDAR encephalitis, the presence of bound NMDAR autoantibodies, preferably exceeding the amount of a suitable control such as the amount from a healthy control population, indicates the presence or possibility of a subject developing a medical condition associated with autoantibodies against NMDARs, preferably anti-NMDAR encephalitis.

[0062] Current diagnostics are based on tissue profiling or CBA expressing the GluN1 subunit of the NMDA receptor. Compared to these, the present invention's method, based on the NMDAR (NMADR) protein construct, offers several advantages. For example, the present invention's method allows for the binding or capture of autoantibodies that bind to GluN1 in relation to the GluN2 subunit, and therefore also captures autoantibodies that bind to overlapping or sterically stabilized epitopes. Furthermore, the present invention's method allows for the distinction of preferred binding of autoantibodies to further combinations, for example, GluN1 and GluN2A vs. GluN1 and GluN2B vs. individual subunits vs. GluN1 and GluN2C vs. GluN1 and GluN2D, and two or more isoforms of the GluN1 and GluN2 subunits.

[0063] Further advantages of the detection method disclosed herein are the robustness of the assay and its adaptability to standardization such as sFIDA, and even to full automation. Furthermore, the assay can be optimized to function as a single-molecule 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 cellular background of human cells, preferably mammalian cells such as HEK cells.

[0064] In the present invention, the NMDAR protein construct can be immobilized on a solid phase before contact with the sample.

[0065] In embodiments of the present invention, the soluble NMDAR protein construct of the present invention is provided in an immobilized form. In such embodiments, the soluble NMDAR protein construct of the present invention may be purified in its soluble form from a suitable expression system and then immobilized on a solid phase. Therefore, in the context of such embodiments of the immobilized NMDAR protein construct of the present invention, "soluble" refers to the prior state of the construct before immobilization.

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

[0067] The determination of NMDAR autoantibodies in the context of the present invention involves the following steps: A process for immobilizing NMDAR autoantibodies from a sample by binding to an NMDAR protein construct immobilized on a solid surface, A step of treating the immobilized NMDAR autoantibody with a labeled secondary affinity reagent directed towards the NMDAR autoantibody, A step of detecting a signal emitted from the above-mentioned labeled secondary affinity reagent directed towards an NMDAR autoantibody, The process involves comparing the signal obtained from the labeled secondary affinity reagent with the signals from one or more control samples with predetermined NMDAR autoantibody concentrations. It can include...

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

[0069] In a particular embodiment, the method of the present invention is applied to the treatment guidance of a subject having and / or suspected of having a medical condition related to an NMDAR autoantibody, the method comprising selecting one or more corresponding NMDAR protein constructs of the present invention for subsequent treatment of the subject.

[0070] NMDAR autoantibody detection kit The present invention also relates to a kit for detecting NMDAR autoantibodies in a sample, the kit comprising: The present invention relates to an NMDAR protein construct, a solid surface for immobilizing the NMDAR protein construct, or the NMDAR protein construct immobilized on a solid surface, a labeled secondary affinity reagent directed at a human NMDAR autoantibody such as a labeled anti-human IgG antibody, and a means for detecting a signal emitted from the label, or The present invention comprises a labeled NMDAR protein construct, and a means for detecting a signal optionally emitted from the label, Optionally, a control sample with a predetermined NMDAR autoantibody concentration, It is equipped with.

[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 ELISA to detect NMDAR autoantibodies present in a sample.

[0072] The present invention also provides a kit for diagnosing autoimmune diseases related to NMDAR autoantibodies, such as NMDAR encephalitis, in a subject by detecting NMDAR autoantibodies. The present invention relates to an NMDAR protein construct, optionally a solid surface for immobilizing the NMDAR protein construct, or the NMDAR protein construct immobilized on a solid surface, a labeled secondary affinity reagent directed towards a human NMDAR autoantibody such as a labeled anti-human IgG antibody, and optionally means for detecting a signal emitted from the label. The labeled NMDAR protein construct of the present invention, Optionally, a control sample with a predetermined NMDAR autoantibody concentration, Regarding a kit that includes the following features.

[0073] Blood processing device containing NMDAR protein construct Furthermore, the present invention relates to a blood processing apparatus configured to remove NMDAR autoantibodies from the blood or plasma of a person requiring processing in an extracorporeal blood circuit, wherein the apparatus comprises a matrix having one or more NMDAR protein constructs of the present invention immobilized on the matrix.

[0074] In the embodiment, the blood processing device of the present invention is placed in an extracorporeal blood circuit through which the patient's blood passes, and includes means for transporting blood from the patient's vascular system to the blood processing device at a predetermined flow rate, and returning the processed blood to the patient.

[0075] Further aspects of the present invention Furthermore, the present invention includes an NMDAR protein construct disclosed herein for use as a pharmaceutical. The present invention also relates to an NMDAR protein construct disclosed herein for use as a pharmaceutical in the treatment of subjects suffering from autoimmune diseases associated with NMDAR autoantibodies, preferably NMDAR encephalitis.

[0076] The present invention also relates to an in vitro method for producing the NMDAR protein construct of the present invention, comprising expressing a nucleic acid sequence encoding the NMDAR protein construct of the present invention in mammalian cells, preferably human cells, and subsequently isolating the NMDAR protein construct. 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, compared to methods that require the initial isolation of antigens from the cell membrane using either protease cleavage or surfactant solubilization, soluble NMDAR protein constructs can be isolated from the cell culture supernatant of cells modified to express the NMDAR protein constructs of the present invention.

[0078] Furthermore, the present invention includes the NMDAR protein construct disclosed herein, which is produced by the disclosed method for producing the NMDAR protein construct of the present invention.

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

[0080] Detailed description of the invention All cited documents, both patent and non-patent documents, by reference, constitute an entire 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 the extracellular domain (ECD) or a fragment thereof of the NMDAR subunit GluN1 and the ECD or a fragment thereof of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C, or GluN2D.

[0082] In relation to the present invention, the term "protein construct" may refer to individual proteins or peptides formed by a single amino acid chain. Furthermore, as used herein, the term "protein construct" also includes constructs or complexes of two or more proteins, peptides, or amino acid chains covalently linked by, for example, disulfide bridges or other linkers between individual amino acid chains. Moreover, the term "protein construct" includes protein complexes formed by two or more proteins, 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, which lead to the formation of protein dimers or protein polymers that can be assembled, for example, via dimerization or polymerization domains.

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

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

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

[0086] In relation to the present invention, a dimerizing domain is any domain contained in or that can be integrated into a protein or peptide that can bind to another domain, such as another dimerizing domain of another protein or peptide. Many examples of dimerizing domains, including antibody Fc fragments of antibodies, leucine zipper domains, or coiled-coil domains, are known to those skilled in the art. Dimerization can result in the formation of homodimers and heterodimers, which mean the assembly of two identical or two different proteins, respectively. In either case, the dimerizing domains may be identical, or, in the case of heterodimers, they may be different in the monomers that form the dimer.

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

[0088] As used herein, the term "capture domain" refers to a domain or portion of a protein construct of the present invention that can be used to bind the construct to a solid phase by either non-covalent interactions or covalent bonds. Typical examples of such capture domains are domains or amino acid sequences recognized by commonly available proteins that bind to the capture domain, such as antibodies. 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 tags, HA tags, and HIS tags can be used as capture domains.

[0089] The possible dimerization and capture domains that can be incorporated into the protein construct of the present invention are diverse, and those skilled in the art can identify appropriate variants. Furthermore, in some cases, the dimerization domain can also function as a capture domain. This is the case, for example, with antibody Fc fragments that can form dimers and can be readily bound by commonly available antibodies. Thus, Fc fragments can function simultaneously as both capture and dimerization domains. 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, for example, ligation or overlap extension PCR, adding the cDNA sequence of a second protein fragment to a frame having the cDNA of a first protein (fragment) without containing a stop codon in between. The DNA sequence is then expressed by cells as a single protein. The protein can be manipulated to contain the complete sequences of both original proteins, or only parts of either. Three or more proteins or fragments can be linked together to form complex fusion proteins. Between the various parts of the fusion protein, there are often so-called linker (or "spacer") peptides, which increase the likelihood that the proteins will fold independently and function as expected. In protein or peptide fusion, the linker may be manipulated by a cleavage site for a protease or chemical, allowing the release of two separate proteins, especially when the linker enables protein purification. This technique is often used for protein identification and purification by fusing GST proteins, FLAG peptides, or hexahis peptides (6×His tag), which can then be isolated using affinity chromatography with nickel or cobalt resins. Dimeric or polymeric chimeric proteins can be produced by genetic engineering by fusing a peptide domain that induces dimerization or polymerization of the artificial protein to the original protein (e.g., streptavidin or leucine zipper).

[0091] Protein linkers assist in the design of fusion proteins by providing appropriate spacing between domains and support correct protein folding when N-terminal or C-terminal interactions are essential for folding. Generally, protein linkers are preferred for use in fusion protein design even when the N-terminus and C-terminus can be fused, as they enable critical domain interactions, enhance stability, reduce steric hindrance, and allow for N-terminal and C-terminal fusion. There are at least three main types of linkers: flexible, rigid, and (in vivo) cleavable. Flexible linkers can consist of many small glycine residues, giving them the ability to curl into dynamic and adaptable shapes. Rigid linkers can be formed from large cyclic proline residues and may be useful when it is necessary to maintain highly specific spacing between domains. (In vivo) cleavage 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 in the cell. The selection and design of appropriate linker sequences are standard procedures known to those skilled in the art. In the case of cleavage linker sequences, those skilled in the art can also select enzymes or reagents suitable for linker cleavage and design or select the corresponding linkers.

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

[0093] Table 1: Preferred amino acid sequence 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, and elements further comprised of such constructs of each NMDAR protein construct. Protein modifications to the NMDAR protein constructs of the present invention, which may occur by substitutions of amino acid sequences and nucleic acid sequences encoding such molecules, are also within the scope of the present invention. Substitutions as defined herein are modifications made to the amino acid sequence of a protein, thereby replacing one or more amino acids with the same number of (different) amino acids, producing a protein with an amino acid sequence different from that of the primary protein. In some embodiments, this modification does not significantly alter the function of the protein. As with additions, substitutions may be natural or artificial. It is well known in the art that amino acid substitutions can be made without significantly altering the function of a protein. This is especially true when the modification relates to “conservative” amino acid substitutions, where one amino acid is substituted for another amino acid with similar properties. Such “conservative” amino acids may be natural or synthetic amino acids that, due to their size, charge, polarity, and conformation, can be substituted without significantly affecting the structure and function of the protein. In many cases, many amino acids can be substituted with conservative amino acids without adversely affecting the function of the protein.

[0096] Generally, the following amino acids are considered conserved: 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. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser, and sometimes Cys are substitutable for each other despite belonging to different groups.

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

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

[0099] The NMDAR protein construct of the present invention may include a protein tag that enables easy identification or binding of the NMDAR protein construct to a standard technique, for example, using an antibody directed against the protein tag. Preferred protein tags that the nucleic acid sequence of the present invention can encode are V5 tags, myc tags, HA tags, HIS tags, or antibody Fc fragments. Alternative tags can be used instead of V5 tags. 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 use thereof in relation to the NMDAR protein constructs disclosed herein and 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 parts thereof. a) One or more nucleic acid molecules comprising a nucleotide sequence encoding an ECD or fragment thereof of an NMDAR subunit GluN1, an ECD or fragment thereof of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C, or GluN2D, and 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 undergo hybridization with 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 to be functionally similar to the nucleotide sequence described in a), b), or c), e) One or more nucleic acid molecules that, as a result of the genetic code, are degenerated into the nucleotide sequences described in a) to d), f) One or more nucleic acid molecules described in a) to e) that are modified by deletion, addition, substitution, rearrangement, inversion and / or insertion and are functionally similar to the nucleotide sequences described in a) to e), It is selected from the group that includes it.

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

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

[0103] For example, sequence variants of nucleic acids and / or proteins described in the claims, defined by the provided % sequence identity and maintaining the above-mentioned properties of the present invention, are also included in the scope of the present invention. Such variants, which show alternative sequences but maintain essentially the same properties as the specific sequence provided, for example, the autoantibody binding properties of each NMDAR protein construct of the present invention, are known as functional analogs or functionally similar. Sequence identity refers to the percentage of identical nucleotides or amino acids when sequence alignment is performed using software such as BLAST.

[0104] Those skilled 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 frequency are specifically intended by the present invention. Deletions, substitutions, and other alterations 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 a soluble N-methyl-D-aspartate receptor (NMDAR) protein construct comprising one or more NMDAR autoantibody epitopes. As used herein, the term “NMDAR autoantibody epitope” refers to an epitope formed by NMDAR (either by individual subunits or epitopes comprising residues or amino acids of two or more subunits of NMDAR). Furthermore, the conformation of an NMDAR subunit can only be stabilized by the presence of an ECD or fragment of another subunit, and certain epitopes can only be formed upon such conformational stabilization. In connection with the present invention, the NMDAR protein construct and the epitopes formed by the construct of the present invention may be referred to as autoantigens. The binding between an autoantigen and an antibody is an established phenomenon and essentially reflects the physical interaction between any given antibody and its target.

[0106] Those skilled in the art are aware of various neurological autoimmune conditions in which autoantibodies typically target autoantigens in either the central or peripheral nervous system. However, medical conditions in which autoantibodies are directed against targets present in both the central and peripheral nervous systems are also known. The present invention therefore envisions the use of the NMDAR protein construct of the present invention in relation to diseases in which autoantibodies primarily target components of the central nervous system, or in which the pathogenic effects of such autoantibodies are caused by autoantibodies targeting autoantigens in the central nervous system.

[0107] As used herein, the “central nervous system” or CNS refers to the 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 housed in the spinal canal. The CNS is divided into white matter and gray matter, which can also be seen macroscopically in brain tissue. White matter consists of axons and oligodendrocytes, while gray matter consists of neurons and unmyelinated fibers. Both tissues contain many glial cells (white matter contains more glial cells), which are often called the supporting cells of the CNS. The peripheral nervous system, in the form of spinal nerves, extends from and toward the spinal cord. Nerves connect the spinal cord to the skin, joints, muscles, etc., enabling the transmission of not only efferent movement but also afferent sensory signals and stimuli. This enables the perception of sensation, along with voluntary and involuntary muscle movements.

[0108] As used herein, the “peripheral nervous system” (PNS) consists of nerves and ganglia outside the brain and spinal cord. The primary function of the PNS is to connect the CNS to the limbs and organs, and to essentially act 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 by the blood-brain barrier.

[0109] New research now shows that autoantibodies can access the CNS (Zonget al Front Immunol. 2017; 8:752) and that autoantibody-producing B cells are present in the CNS. Under normal conditions, immunoglobulins cross the blood-brain barrier (BBB) ​​at a slow rate, a good example being immunoglobulin G (IgG). The concentration of IgG in cerebrospinal fluid (CSF) is approximately 1% of the level in peripheral circulation. This suggests that when autoantibodies reach the CNS, they can cause disease, as observed in autoimmune encephalitis. In certain circumstances, the BBB may become more vulnerable due to stroke, traumatic brain injury, hemorrhage, microangiopathy, or brain tumors, potentially increasing antibody penetration.

[0110] As used herein, the term “autoantibody-mediated mental state” refers to any medical condition involving the presence of autoantibodies, preferably autoantibodies directed primarily to autoantigens targeted in the central nervous system, where 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 nerve surface autoantibodies (NSAbs). Specific autoantibodies targeting nerve surface antigens and ion channels have been shown to cause severe mental disorders, i.e., neuropsychiatric symptoms. Numerous studies have shown the presence of autoantibodies in certain mental conditions, such as schizophrenia and bipolar disorder. Additional disorders include neuropsychiatric disorders such as schizophrenia, bipolar disorder, MDD, substance-induced psychosis, Huntington's disease, Alzheimer's disease, and neuropsychiatric systemic lupus erythematosus (Zonget al, Front Immunol. 2017; 8:752).

[0111] In some embodiments, the diseases treated or diagnosed using the present invention are autoimmune encephalopathy or cerebrospinal cord disorders. "Encephalopathy" is typically any disorder or disease of the brain, particularly a chronic degenerative state. Encephalopathy may refer to permanent (or degenerative) brain injury or reversible injury. Encephalopathy may result from direct injury to the brain or from a disease detached from the brain. Symptoms often include disability, irritability, agitation, delirium, confusion, somnolence, stupor, coma, 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 cord disorder" refers to any disease affecting both the brain and spinal cord caused by an autoimmune component.

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

[0113] The N-methyl-D-aspartate 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 ionizing glutamate receptors; the other two are the AMPA receptor and the kainate receptor. Glutamate and glycine (or D-serine) bind to the NMDAR, activating it, and when activated, positively charged ions flow across the cell membrane. The NMDA receptor is crucial for regulating synaptic plasticity and memory function. The receptor is typically assembled as a heterogeneous complex interacting with multiple intracellular proteins via three distinct subunits: NR1, NR2, and NR3. NR1 has eight distinct isoforms, generated by alternative splicing from a single gene. There are four distinct NR2 subunits (A-D), and NR3A and NR3B subunits were reported in the late 20th century. Six separate genes encode NR2 and NR3. According to more recent nomenclature, the subunits are called GluN1, GluN2, and GluN3, respectively, instead of NR1, NR2, and NR3. Subunit variants are identified accordingly (for example, NR2A and NR2B are identified as GluN2A and GluN2B, respectively).

[0114] Each receptor subunit has a modular design. The extracellular domain contains two spherical structures: an amino-terminal domain (ATD, sometimes called the regulatory domain) and a 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 reminiscent of a potassium channel selectivity filter. The membrane domain contributes residues to the channel pore, responsible for the receptor's high unit conductance, high calcium permeability, and voltage-dependent magnesium blocking. Each subunit has a broad cytoplasmic domain containing residues that can be directly modified by a range of protein kinases and protein phosphatases, as well as residues that interact with numerous structural proteins, adapter proteins, and scaffold proteins.

[0115] NMDARNR1 / GluN1 is a component of the NMDA receptor complex, functioning as a ligand-gated ion channel that is a heterotetramer with high calcium permeability and voltage-dependent sensitivity to magnesium. Channel activation requires the binding of the neurotransmitter glutamate to the GluN2 subunit, glycine to the GluN1 subunit, and further membrane depolarization to eliminate Mg2+-mediated channel inhibition. Many protein isoforms of the NMDARNR1 protein are known, including, but are 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, and NP_067544.1. Any one or more of the above sequences or isoforms, or functionally similar derivatives thereof, may be employed in connection with the NMDAR protein construct 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, nominally designated as NR2A / GluN2A to NR2D / GluN2D (encoded by GRIN2A, GRIN2B, GRIN2C, and GRIN2D). 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 particular subunit, NR2B, is primarily found in immature neurons, both intraneuronally and extrasynaptically, and contains a binding site for the selective inhibitor ifenprodil. NR2B is dominant in the early postnatal brain, while the number of NR2A subunits increases, eventually surpassing NR2B. This is called the NR2B-NR2A developmental switch and is noteworthy for the various dynamics each NR2 subunit exerts on the receptor. For example, a higher proportion of NR2B subunits results in NMDA receptors remaining open longer compared to cases with more NR2A subunits.

[0117] NMDARs have various physiological roles, either enhancing or reducing activity, and any dysfunction, 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, NMDARs play an important role in several mental disorders, including depression. Furthermore, a subgroup of patients with atypical dementia have anti-NMDAR1 antibodies, thereby being recognized as a medical condition associated with autoantibodies against NMDARs, and the removal of such NMDAR autoantibodies by nonspecific removal of all antibodies has resulted in clinical improvement in selected cases (Pruesset al. 2010, Neurology. 75(19):1735-9, Doss et al. 2014 Ann Clin Transl Neurol.1 (10):822-32). In addition, autism may occur in children of mothers with autoantibody-mediated disorders. Several studies have found a correlation between the presence of circulating maternal autoantibodies in newborns and neurological dysfunction (Fox-Edmistonet 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). Since the presence of NMDAR-autoantibodies can therefore cause autism in the offspring of mothers with the disorder, the present invention also represents a potential treatment of such disorder and / or a preventive approach to avoid such disorder in children.

[0118] Thus, any medical condition in which the contribution of NMDAR autoantibodies to the etiology is explained or suggested is eligible as an NMDAR autoantibody-related medical condition, for example, due to the correlation between the NMDAR autoantibodies and the onset of disease symptoms. Furthermore, the constructs of the present invention can be used to analyze samples from patients suffering from conditions explained or suggested as NMDAR autoantibody-related conditions in the presence of such antibodies, and subsequently treated with the present invention.

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

[0120] A method for detecting NMDAR autoantibodies in vitro. Autoantibodies are antibodies (a type of protein) produced by the immune system that are directed against one or more proteins of the individual itself. 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 arise from an abnormal immune response (autoimmunity) of the body to substances and tissues normally present in the body. This may be limited to certain organs or involve certain tissues.

[0122] As used herein, the term "sample" refers to a biological sample obtained or isolated from a patient or subject. As used herein, "sample" refers to a sample of bodily fluids or tissues obtained, for example, for the purpose of diagnosis, prognosis prediction, or evaluation of a subject, such as a patient. Preferably in this specification, a sample is a sample of bodily fluids such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, pleural fluid, cells, cell extracts, tissue samples, tissue biopsy samples, or stool samples.

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

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

[0125] The terms “diagnosis” and “diagnosing” include the use of NMDAR protein constructs, methods, kits and further embodiments of the present invention to determine the presence or likelihood of a medically relevant disorder in an individual. The terms also include 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 autoimmune diseases associated with NMDAR autoantibodies ranging from mild to moderate, moderate to severe, or severe to fulminant.

[0126] The present invention also encompasses the use of disease monitoring methods, also known as monitoring the progression or regression of autoimmune diseases and treatment monitoring. The term “monitoring” includes the use of the NMDAR constructs disclosed herein, as well as the methods and other embodiments of the present invention, for determining the disease status of an individual (e.g., the presence or severity of an autoimmune disease). In certain examples, the results of a statistical algorithm (e.g., a learning statistical classifier system) are compared with results obtained earlier for the same individual. In some embodiments, the kits, constructs, apparatus, methods, and systems of the present invention can also be used to predict the progression of an autoimmune disease by determining, for example, the likelihood that the autoimmune disease will progress rapidly or slowly in an individual based on the presence or level of at least one marker (e.g., one or more NMDAR autoantibodies) in a sample. The present invention can also be used to predict the regression of an autoimmune disease by determining, for example, the likelihood that the autoimmune disease will progress rapidly or slowly in an individual based on the presence or level of at least one marker in a sample. Treatment monitoring can also be performed to monitor the progression of the disease in a subject during any given course of treatment.

[0127] In aspects of the present invention, the presence or level of NMDAR autoantibodies is determined by an immunoassay or immunohistochemical assay. A non-limiting example of an immunoassay suitable for use in the method of the present invention is ELISA. Examples of immunohistochemical assays suitable for use in the method of the present invention include, but are not limited to, direct immunofluorescence assays, IFA assays, anti-complement immunofluorescence assays, and avidin-biotin immunofluorescence assays. Other types of immunohistochemical assays include immunoperoxidase assays.

[0128] In relation to the present invention, the term "affinity reagent" refers to antibodies, peptides, nucleic acids, small molecules, or any other molecules that specifically bind to a target molecule in order to identify, track, capture, or influence 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 has already been bound by another affinity reagent.

[0130] As used herein, the term “antibody” includes a population of immunoglobulin molecules, which may be polyclonal or monoclonal and of any isotype, or immunologically active fragments of immunoglobulin molecules. Such immunologically active fragments include 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 in the meaning of the term antibody. The term “monoclonal antibody” refers to an antibody produced by the same immune cell, which is a clone of a specific parent cell, as opposed to a polyclonal antibody produced from several different immune cells. Monoclonal antibodies can have monovalent affinity in that they bind to the same epitope (part of the antigen recognized by the antibody). There also exist engineered bispecific monoclonal antibodies, in which each “arm” of the antibody is specific to a different epitope. For almost any substance, it is possible to produce a monoclonal antibody that specifically binds to that substance, and then the monoclonal antibody functions to detect or purify that substance.

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

[0132] As an example of means for detecting labels in the method of the present invention, the presence or level of one or more markers in a sample can be determined using various immunoassay techniques, including competitive and non-competitive immunoassays (see, for example, Selfet al., Curr. Opin. Biotechnol., 7:60-65 (1996)). The term immunoassay encompasses, but is not limited to, techniques including enzyme-mediated immunoassays (EIA), such as enzyme amplification immunoassay (EMIT), enzyme-linked immunosorbent assay (ELISA), antigen capture ELISA, sandwich ELISA, IgM antibody capture ELISA (MACELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoretic immunoassay (CEIA); radioimmunoassay (RIA); immunoradioassay (IRMA); fluorescence-polarized immunoassay (FPIA); and chemiluminescence assay (CL). Such immunoassays can be automated as needed. Immunoassays 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 immunoassays such as flow injection liposome immunoassays and liposome immunosensors are also suitable for use in this invention (see, for example, Rongenet al., J. Immunol. Methods, 204:105-133 (1997)). Furthermore, turbidimetric assays in which the formation of protein / antibody complexes results in increased light scattering, which is converted into a peak velocity signal as a function of marker concentration, are also suitable for use in this invention. The turbidimetric assay is commercially available from BeckmanCoulter (Blair, California; kit #449430) and can be performed using a Behring Nephelometer Analyzer (Finket al., J. Clin. Chem. Clin. Biol. Chem., 27:261-276 (1989)).

[0133] The immunoassay method described above 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 labels).

[0134] In another preferred embodiment of the method according to the present invention, autoantibodies are detected by immunoassay, preferably by directly or indirectly conjugating one reactant to a labeling substance. This allows the method to be flexibly adapted to the capabilities and requirements of different laboratories and their diagnostic equipment. In one advantageous embodiment, autoimmune disease-specific antibodies are detected by immunoassay, and the antibodies are dissolved in a liquid phase, preferably a liquid phase diluted in a common buffer well known to those skilled in the art, or in an undiluted body fluid. According to the present invention, detection can also be carried out using a stool sample. Furthermore, the detection method of the present invention can be carried out by conjugating the construct of the present invention to cells expressing NMDAR autoantibodies on their surface. Detection of cells conjugated 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 conjugate an antibody. In the second reaction step, an anti-human immunoglobulin selected from the group including anti-human IgA, anti-human IgM, and / or anti-human IgG antibodies may be employed, the anti-human immunoglobulin being a detectable labeled conjugate of two components that can be conjugated with any conventional labeling enzyme, particularly 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 typically available in laboratory facilities, so that detection according to the present invention can be established in a cost-effective manner. In another preferred embodiment of the present invention, the antibody conjugated to the NMDAR protein construct of the present invention reacts with an anti-human immunoglobulin selected from the group including anti-human IgA, anti-human IgM, and / or anti-human IgG antibodies, and is detectably conjugated to fluorescein isothiocyanate (FITC). Similar to the ELISA described above, the FITC technology represents a system available in many locations and therefore enables the smooth and low-cost establishment of the detection of the present invention in a typical laboratory setting. Those skilled in the art will recognize further standard detection techniques that can be used in connection with the method of the present invention.

[0136] The specific immunological binding of an antibody to a target marker can be detected directly or indirectly via labeling. Any given means for detecting these labels can be considered a means for detecting the label according to the method of the present invention. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, etc., attached to antibodies. Antibodies labeled with iodine-125 (125I) can be used to determine the level of one or more markers in a sample. Chemiluminescent assays using marker-specific chemiluminescent antibodies 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, β-phycoerythrin, R-phycoerythrin, rhodamine, Texas Red, and lysamine. Secondary antibodies conjugated to fluorescent dyes are commercially available; for example, goat F(ab')2 anti-human IgG-FITC is available from TagoImmunologicals (Burlingame, California). Further fluorescent labeling is commonly used and known to those skilled in the art.

[0137] Examples of indirect labeling include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, and urease. For example, a horseradish peroxidase detection system can be used that produces a soluble product detectable at 450 nm in the presence of hydrogen peroxide using the chromogenic substrate tetramethylbenzidine (TMB). An alkaline phosphatase detection system can be used with the chromogenic substrate 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 labeling can be analyzed using, for example, a spectrophotometer that detects color from a chromogenic substrate, a radiation detector such as a gamma counter that detects 125I, or a fluorometer that detects fluorescence in the presence of light of a specific wavelength. In the case of enzyme-bound antibody detection, quantitative analysis of marker-level amounts can be performed using a spectrophotometer such as the EMAX microplate reader (Molecular Devices; Menlo Park, California) according to the manufacturer's instructions. If desired, the assay of the present invention can be automated or performed by a robot to simultaneously detect signals from multiple samples.

[0139] A plate reader, also known as a microplate reader or microplate photometer, is an instrument used to detect biological, chemical, or physical events in a sample 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 in, for example, 6- to 1536-well format microtiter plates, but are not limited to these. Common detection modes for microplate assays include, for example, absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization. The camera device relating to the present invention is suitable for detecting the signal of a labeled secondary affinity reagent directed toward GP2. The camera device may be provided within the plate reader or as a separately configured device. Those skilled in the art are familiar with such devices, which are selected based on the labeling of the secondary affinity reagent.

[0140] In certain embodiments, the present invention provides a method for diagnosing autoimmune diseases or their clinical subtypes using the NMDAR protein construct 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 statistical algorithms for classifying samples from individuals as autoimmune disease samples. 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 of additional markers suitable for use in the statistical algorithm of the present invention.

[0141] In another preferred embodiment of the present invention, the NMDAR protein construct according to this application is immobilized on a surface. More specifically, one or more solid-phase NMDAR protein constructs disclosed herein are bound to organic, inorganic, synthetic and / or mixed polymers, preferably agarose, cellulose, silica gel, polyamide and / or polyvinyl alcohol. In the sense of the present invention, immobilization is understood to include various methods and techniques for immobilizing peptides on a particular carrier, for example, according to International Publication No. 99 / 56126 or International Publication No. 02 / 26292. For example, immobilization can help stabilize the construct so that its activity is not reduced or adversely affected by biological, chemical or physical exposure, particularly during storage or single-batch use. Peptide immobilization allows for repeated use under conventional technical or clinical conditions and further enables the reaction of a sample (preferably a blood component) with at least one construct according to the present invention in a continuous manner. In particular, this can be achieved by using various immobilization techniques to ensure that the three-dimensional structure of the corresponding molecule, especially the peptide, is not altered (particularly at the active site that mediates interaction with autoantibodies) while the binding of the peptide to another peptide or molecule, or to a carrier, proceeds. Advantageously, no loss of specificity to the patient's autoantibodies results from such immobilization. In the sense of the present invention, at least three basic methods can be used for immobilization: (i) Crosslinking: In crosslinking, peptides are immobilized on each other without adversely affecting their activity. Advantageously, the peptides are no longer soluble as a result of such crosslinking. (ii) Binding to the carrier: Binding to the carrier proceeds, for example, via adsorption, ionic bonding, or covalent bonding. Such binding can also occur within microbial cells, liposomes, or other membranes (closed or open structures). Advantageously, the peptide is not adversely affected by such immobilization. For example, the carrier-bound peptide can be used multiple times or consecutively, which is advantageous in clinical diagnosis or treatment. (iii) Encapsulation: In the sense of the present invention, encapsulation proceeds in particular within a semipermeable membrane in the form of a gel, fibril, or fiber. Advantageously, the encapsulated peptide is separated from the surrounding sample solution by the semipermeable membrane so that interaction with the autoantibody or its fragments is still possible. Various methods can be used for immobilization, such as adsorption onto an inert or charged inorganic or organic carrier. For example, such carriers may be porous gels, aluminum oxide, bentonite, agarose, starch, nylon, or polyacrylamide. Immobilization proceeds via physical bonding forces, frequently involving hydrophobic interactions and ionic bonding. Advantageously, such methods are easy to handle and have little effect on the three-dimensional structure of the peptide. Advantageously, binding can be improved as a result of electrostatic bonding forces between the charged groups of the peptide and the carrier, for example, by using an ion exchanger, particularly Sephadex.

[0142] Another method is covalent bonding to a support material. Furthermore, the support may have reactive groups that form isopolar bonds with amino acid side chains. Suitable groups for peptides are carboxyl groups, hydroxyl 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 can be activated by treatment with silane and then react with peptides. For example, the hydroxyl groups of natural polymers can be activated with bromocyanogen and then coupled with peptides. Advantageously, many peptides can undergo direct covalent bonding with polyacrylamide resins. Encapsulation in three-dimensional networks includes encapsulation of peptides in ionizing gels or other structures, which are well known to those skilled in the art. More specifically, the pores of the matrix allow interaction with target molecules in a manner that holds the peptides. In crosslinking, peptides are converted into polymer aggregates by crosslinking with a bifunctional material. Such structures are gelatinous and readily 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 limited by the membrane. For example, microencapsulation can be carried out in the form of interfacial polymerization. Immobilization during microencapsulation makes the peptide insoluble, thus making it reusable. In the sense of the present invention, an immobilized construct is in a state where all of its peptides are reusable. Limiting the mobility and solubility of peptides by chemical, biological, or physical means advantageously results in lower processing costs, particularly when removing autoantibodies from blood components.

[0143] The present invention also relates to a diagnostic kit for determining autoimmune diseases associated with NMDAR autoantibodies, comprising one or more NMDAR protein constructs disclosed herein. The diagnostic kit comprises all necessary analytes-specific reagents required to perform the diagnostic test. The diagnostic kit may also include instructions on how to perform the test using the provided reagents. The diagnostic kit may optionally include instructions on how to combine the contents of the kit and / or provide a formulation for detecting autoimmune diseases associated with NMDAR autoantibodies, such as NMDAR encephalitis. For example, the instructions may be in the form of an instruction leaflet or other media providing the user with information on the type of method by which the substances mentioned are used. Obviously, 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. One advantageous effect of such a kit for a patient is that, for example, the actual disease state can be determined and diet and activity adapted accordingly, even if the patient is traveling, without the need for a doctor to directly intervene.

[0144] Embodiments of a blood processing device and immobilization of NMDAR protein constructs in the device. The present invention also relates to a blood processing apparatus configured to remove NMDAR autoantibodies from the blood or plasma of a person requiring processing in an extracorporeal blood circuit, wherein the apparatus comprises a matrix having one or more NMDAR protein constructs of the present invention immobilized on the matrix.

[0145] Therefore, as used herein, “matrix” refers to an internal substance through which blood or plasma passes, or a substance within a blood processing apparatus 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 an NMDAR protein construct is bound. Thus, this support serves as a carrier for the NMDAR protein construct, but may also perform other functions.

[0146] As used herein, “support” refers to the portion of the matrix that acts as a “substrate” or “support material” to which the construct according to the present invention is bound. Such a support or support material may also be referred to as “adsorbent” or “adsorbent material” as used in an “adsorption column” or “column” or “adsorption cartridge.” A suitable support according to the present invention must be 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, have good flow properties to whole blood and / or plasma, and provide a large surface area for the attachment of the NMDAR protein construct.

[0147] The support material may be, for example, a resin, a film, or a nonwoven material. “Nonwoven” material refers to a material broadly defined as a sheet, cloth, or web structure formed by mechanically, thermally, or chemically entangled fibers or filaments (and by perforating the film) without weaving or knitting them together. “Resin” refers to an insoluble material that can take the form of a gel, gel beads, microporous beads, or sponge. Such resins may be natural polymers or biopolymers, synthetic polymers, and inorganic materials. Agarose beads, dextrose beads, and cellulose beads are commonly used natural supports. Synthetic polymer or organic supports are mostly based on derivatives of acrylamide, polystyrene, and polymethacrylate, while porous silica and glass are some frequently used inorganic supports.

[0148] According to one embodiment of the present invention, the resin is an inorganic material selected from the group consisting of alginates, chitosan, chitin, collagen, carrageenan, gelatin, cellulose, starch, pectin and Sepharose; zeolites, ceramics, Celite, silica, glass, activated carbon and charcoal; or 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, polyaramid, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polysulfone (PS), polyethersulfone (PES), polyarylethersulfone (PAES (PEAS)), ethylene vinyl acetate (EVA), ethylene vinyl Nyl alcohol (EVOH), polyamide-imide, polyaryl ether ketone (PAEK), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polycaprolactone (PCL), polyhydroxyalkanoate, polyether ether ketone (PEEK), polyether ketone ketone (PEKK), polyetherimide (PEI), polyimide, polylactic acid (PLA), polymethylpentene (PMP), poly(p-phenylene ether) (PPE), polyurethane (PU), styrene acrylonitrile (SAN) The polymer is composed of a polymer selected from the group consisting of 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 synthetic polymers selected from the group consisting of any of these polymers modified by the introduction of functional groups.

[0149] Various known methods can be used to immobilize NMDAR protein constructs onto the support and / or matrix according to the present invention. Such immobilization is preferably specific or selective in that it immobilizes NMDAR protein constructs rather than immobilizing other proteins and components present in blood or plasma, or in vitro.

[0150] "Immobilizing" an NMDAR protein construct onto a support that provides a matrix usable in the apparatus according to the present invention refers to a non-covalent or covalent interaction that holds two molecules together. According to one embodiment of the present invention, this expression refers to a covalent interaction, i.e., a covalently bonded 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 nonpolar groups. One or more of these interactions can mediate the binding of two molecules to each other. The binding may otherwise be specific, selective, or nonspecific.

[0151] According to one embodiment, the NMDAR protein construct includes an affinity tag for immobilization on a support. The affinity tag can be used for the purification of proteins during production and / or their immobilization onto a support of the matrix of the present invention. The affinity tag may 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, with polyhistidine or His6 tags, C-myc tags and FLAG tags being particularly well described and offering options for binding the construct according to the present invention to a support material. Non-covalent binding of biotin to streptavidin or avidin may also be used to immobilize the NMDAR protein construct to a support. In embodiments, 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 covalently attached to a support as described below in further detail and / or in the prior art. The covalent bond generally includes either non-site-specific covalent bonding of the protein or site-specific covalent bonding of the protein. The support underlying the formation of the matrix needs to provide or facilitate chemical activation, thus enabling the 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, buffering conditions, and temperatures, construct leaching can be ignored. The bonding method should avoid improper orientation, multi-site bonding, or steric hindrance of the construct. The construct density per unit volume of the matrix can be optimized to facilitate target accessibility and reaction.

[0154] Covalent bonding can be achieved via common functional groups, including amines, alcohols, carboxylic acids, aldehydes, and epoxy groups. Carbodiimide compounds can be used to activate carboxylic acid groups of proteins for direct conjugation to primary amines on the supporting surface via amide bonds. The most commonly used carbodiimides are water-soluble EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) for aqueous crosslinking and water-insoluble DCC (N',N'-dicyclohexylcarbodiimide) for non-aqueous organic synthesis.

[0155] Alternatively, the support may have specific functional groups for linking linkers and / or protein constructs. For example, functionalized resins are commercially available and known to those skilled in the art. A wide range of bonding chemistry, including primary amines, sulfhydryls, aldehydes, hydroxyls, and carboxylic acids, is available in the above-mentioned commercially available supports. Examples of commercially available activated resins include CarboLinkCoupling resins, Profinity® epoxy resins, Affi-Gel 10 and 15, epoxy-activated Sepharose® 6B, trisilloride-activated agarose, and epoxy-functionalized Purolite® Lifetech® methacrylate polymers.

[0156] According to one embodiment of the present invention, the support material must be porous, with a pore size in the range of 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, the support according to the present invention comprises magnetic beads. The magnetic beads are prepared by trapping magnetite in agarose or other polymer material, to which the NMDAR protein construct according to the present invention is immobilized.

[0157] According to another embodiment of the present invention, the support is a membrane. Membranes, as components of affinity matrices, have been used in protein purification due to their simplicity, ease of handling, reduced surface area, and lower diffusion restriction compared to gels, resins, and beads. The membrane can take the physical form of hollow fibers or the form of a plate membrane. According to one embodiment, the support is a hemodialysis hollow fiber membrane dialysis apparatus, and the filter is a hemodialysis apparatus.

[0158] The hollow fiber or plate membrane used as a support in the apparatus according to the present invention may consist 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 polyarylethersulfone (PAES). The hollow fiber membrane that can be advantageously utilized to provide the apparatus according to the present invention preferably has 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 is additionally or alternatively functionalized by an NMDAR protein construct according to the present invention on the luminal side of the fiber, which can directly interact with target metabolites in the blood or plasma perfusing the lumen of the hollow fiber membrane. This construct may additionally or alternatively be immobilized on the outside of the membrane.

[0159] Methods of extracorporeal blood processing The present invention includes a device configured to be placed in an extracorporeal blood circuit through which a patient's blood passes, and comprising means for transporting blood from the patient's vascular system to a blood processing device at a specified flow rate, and then returning the processed blood to the patient, the device 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 the process of removing substances from body fluids by clearance of substances from the blood flowing in an external (outside the body) bypass circuit of the patient. These substances may include endogenous toxins (i.e., uremic toxins), exogenous toxins (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 excess fluid. 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 apparatus of the present invention can be incorporated accordingly.

[0161] As used herein, the term “blood” refers to whole blood, including all components of an organism’s blood, such as red blood cells, white blood cells, and platelets suspended in plasma. The term “plasma” refers to a fluid consisting of approximately 92% water, 7% proteins such as albumin, gamma globulin, fibrinogen, complement factors, and coagulation factors, and 1% mineral salts, sugars, fats, electrolytes, hormones, and vitamins, which form a portion of whole blood that no longer contains red blood cells, white blood cells, and platelets. In connection with the present invention, the term “bloodplasma” or “plasma” refers to a specific fraction of plasma as defined above in its standard sense, such as serum.

[0162] According to one embodiment, the blood flow rate in the extracorporeal blood purification circuit is 20 ml / min to 700 ml / min. The typical flow rate of dialysate in an extracorporeal circuit, including a hemodialysis machine for the treatment of renal failure, is in the range of 0.5 l / hour to 800 ml / min, in addition to the blood processing device according to the present invention, or when the hemodialysis machine 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 plasma 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 process in which whole blood or plasma (including the target protein) is removed from the patient's flowing blood, contacted with the device or matrix according to the present invention, and then returned to the patient. The typical blood or plasma flow rates in an extracorporeal circuit in which the blood treatment device is perfused with whole blood or plasma are in the ranges of 30 ml / min to 200 ml / min or 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 herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, all publications, patent applications, patents, and other references mentioned herein 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] [Figure 1-1]This figure shows a scheme for the soluble recombinant NMDA receptor Fc (srNR-Fc) antigen / fusion protein. In relation to the present invention, the terms srNR-Fc antigen and srNR-Fc fusion protein are used interchangeably. [Figure 1-2] Same as above [Figure 1-3] Same as above [Figure 2] This figure shows that the soluble recombinant NMDA receptor Fc fusion protein is recognized by recombinant human GluN1 autoantibody. [Figure 3-1] This figure shows that soluble recombinant NMDA receptor Fc fusion protein can detect NMDA receptor autoantibodies in a patient's serum. [Figure 3-2] Same as above [Figure 3-3] Same as above [Figure 4] This figure shows the detection of autoantibodies by soluble recombinant NMDA receptor Fc fusion protein in serum with known anti-NMDA receptor titers. [Figure 5] This figure shows an ELISA screen for LGI1 antibody using a soluble recombinant NMDA receptor Fc fusion protein (fusion protein #1 disclosed herein) as a control. [Figure 6] This figure shows the ELISA quantification of recombinant human NR1(GluN1)AB in mouse brain extract using soluble recombinant NMDA receptor Fc fusion protein #1 disclosed herein. [Figure 7] The present invention provides a soluble NMDA receptor Fc protein construct for the detection of heterodimer-selective recombinant human NMDA receptor autoantibodies. [Figure 8] The present invention demonstrates that the soluble NMDA receptor Fc protein construct can detect subtype-selective recombinant human NMDA receptor autoantibodies. [Figure 9] This figure shows that the soluble NMDA receptor Fc protein construct of the present invention clarifies the subtype selectivity of recombinant human NMDA receptor autoantibodies. [Figure 10]This figure shows the detection of recombinant human NMDA receptor autoantibodies using three soluble NMDA receptor Fc protein constructs of the present invention. [Modes for carrying out the invention]

[0169] Detailed description of the drawing Figure 1A. A simplified diagram of the NMDA receptor subunit, including 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 Literature 12). Figure 1B. The extracellular domain of the NMDA receptor subunit depicted in A, fused to rabbit Fc (rbFc; black triangles represent a single polypeptide containing CH2 and CH3), generates a soluble antigen. 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 during (co)expression (a selection of possible combinations is shown).

[0170] Figure 2. Cell culture supernatant (Table 3) of HEK293 cells expressing and secreting the specified fusion protein was captured on a 96-well plate with anti-rabbit IgG antibody. A single recombinant (rec) human (hu) autoantibody against LGI1 or GluN1 (Non-Patent Literature 6) derived from patient CSF cells was applied at 1 μg / ml and detected using horseradish peroxidase (HRP)-conjugated anti-human IgG antibody. The captured Fc fusion protein was directly detected using 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 anti-human IgG antibody alone. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

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

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

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

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

[0175] Figure 7. Cell culture supernatant (Table 3) of HEK293 cells expressing and secreting the specified fusion protein was captured on a 96-well plate with anti-rabbit IgG antibody. Single recombinant human NMDA receptor autoantibodies (anti-NR-Ab1 and anti-NR-Ab2) or a 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), and detected using horseradish peroxidase (HRP)-conjugated anti-human IgG antibody. The captured Fc fusion protein was directly detected using 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 anti-human IgG antibody alone. 490 nm values ​​are shown for anti-rabbit IgG. PGRN, progranulin. Fc is the constant region of the rabbit IgG single 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 recombinant human control antibody (Control Ab1) derived from the patient's CSF cells was applied at 1 μg / ml, 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.

[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 control antibody (mGO53) was applied at 0.09 μg / ml (003-102) or 1.8 μg / ml (anti-NR-Ab1, mGO53), 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. The 490 nm values for 003-102 and anti-rabbit IgG are shown. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0178] Figure 10. Cell culture supernatant (Table 3) of HEK293 cells expressing and secreting the specified fusion protein was captured on a 96-well plate with anti-rabbit IgG antibody. A single recombinant human NMDA receptor autoantibody derived from patient CSF cells (Non-Patent Literature 6 or unpublished) was applied at 0.01 μg / ml (003-102), 1 μg / ml (008-218, anti-NR-Ab1), or 10 μg / ml (all others) and detected using horseradish peroxidase (HRP)-conjugated anti-human IgG antibody. The captured Fc fusion protein was directly detected using 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 anti-human IgG antibody alone. PGRN, progranulin. Fc, constant region of rabbit IgG monoheavy chain.

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

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

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

[0182] solution The amino-terminal domain (ATD) of the NMDA receptor subunit GluN1 was fused to the constant region (rbFc) of the rabbit IgG1 heavy chain, either alone or in combination with the extracellular domain of the NMDA receptor subunit GluN2A or GluN2B, with or without the additional extracellular domain of GluN1.

[0183] Fc-mediated dimerization of the expressed protein may result in an epitope very similar to that of the native NMDA receptor, potentially giving the fusion protein unprecedented stability. These soluble recombinant NMDA receptor Fc(srNR-Fc) fusion proteins / antigens can detect NMDA receptor autoantibodies against different NMDA receptor subunit compositions present in the serum of NMDAR encephalitis patients, and thus form the core subject of this invention.

[0184] ELISA, a method used to detect NMDA receptor autoantibodies using srNR-Fc fusion proteins / antigens, can be used as a companion diagnostic.

[0185] Detailed Examples Example 1: An exemplary protein construct of the present invention generated for an experiment. The inventors generated constructs (Figures 1A and 1B, Table 1) containing the extracellular portions of the GluN1 and GluN2 subunits, expressed them in HEK293 cells, and isolated the cell culture supernatant containing the secreted Fc fusion protein 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, while in constructs #4, #7, and #8, both the GluN1 and GluN2B domains are separated by an artificial linker and fused to the Fc as a single molecule. The Fc domain is likely to result in dimerization of all fusion proteins, resulting in a GluN1 / GluN2 heterodimer when co-expressed with #5, #6, #9, or #3 of construct #1 or #2, respectively, or a dimer of the GluN1 / GluN2 heterodimer when expressed with constructs #4, #7, and / or #8 (Figure 1C).

[0187] Table 3: Soluble recombinant NMDA receptor rbFc fusion proteins composed of and / or representing the NMDAR protein construct of the present invention. [Table 3-1] [Table 3-2] name Fusion protein composition NMDA receptor portion: Amino acids in GenBank Entry Number of amino acids in a portion / total protein of the NMDA receptor hs, human. rb, rabbit. Ecd, extracellular domain. ATD, amino-terminal domain. No, number. Fc, constant region of rabbit IgG single chain.

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

[0189] Example 2: The soluble NMDA receptor rbFc fusion protein is recognized by recombinant human GluN1 autoantibody. Figure 2 shows that all srNR-Fc antigens tested, except for the control antigen (progranulin-Fc), were recognized by recombinant human anti-NMDA receptor antibody. In contrast, no antigens were recognized by recombinant human anti-LGI1 antibody. Using an anti-rabbit IgG antibody to detect the rbFc portion of the fusion protein, we confirmed the successful expression and immobilization of all rbFc fusion proteins on ELISA plates.

[0190] Example 3: Soluble NMDA receptor rbFc fusion protein detects NMDA receptor autoantibodies in the patient's serum. Figure 3 summarizes the results of three ELISA experiments using sets of human serum from NMDAR encephalitis patients against different srNR-Fc antigens. The data reveal that antigens including GluN1-ATD-Fc, as well as additional extracellular regions of GluN1 or GluN2 subunits, evoked signals similar to those in most NMDAR encephalitis patient serums, compared to progranulin as a control. Three of the serums reacted strongly with the antigen, while the other four reacted only with selected srNR-Fc antigens, emitting weaker signals. A 1:200 dilution of the serum suggested that srNR-Fc antigens enabled highly sensitive detection of NMDA receptor autoantibodies. The addition of the GluN2 subunit region improved detection sensitivity. A subset of NMDAR autoantibodies can recognize GluN1 only in the presence of the assembled GluN2 domain. The antigen that evoked the best signal varied across serums. For example, antigens containing N1ecd-N2Becd performed well in detecting antibodies in serum 5, while antigens containing GluN1-ATD and GluN2B-ATD performed better in detecting antibodies in serum 7.

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

[0192] In this assay, the inventors used two srNR-Fc combinations expressing GluN1 and GluN2B ATD (N1-ATD-Fc+N2B-ATD-Fc and N1-ATD-N2B-ATD-Fc). These combinations contain the same amino acids of the NMDAR (Table 1), but in one case the antigen is reconstituted from two separate proteins, while the other construct contains ATD linked by an artificial linker as a single protein. These two antigens gave similar signals in serum S10-S12, but differed significantly in serum S13 and S14. The antigen N1-ATD-Fc+N2B-ATD-Fc produced small, similar signals in S13 and S14. In contrast, N1-ATD-N2B-ATD-Fc did not detect any NMDAR antibody signal in S13, but detected a strong signal in S14. The molecular structure of N1-ATD-N2B-ATD-Fc may have prevented NMDAR autoantibodies present in S13 from accessing their epitopes. This finding highlights the need to test several srNR-Fc combinations to detect as many antibodies as possible.

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

[0194] Using soluble NMDA receptor Fc antigens, we determined whether specific subunit combinations were targeted by recombinant human NMDA receptor autoantibodies. NMDA receptor autoantibodies 008-218 were detected with comparable efficiency by either soluble NMDA receptor Fc antigens containing GluN1 and GluN2A ATDs or GluN1 and GluN2B ATDs. However, anti-NR-Ab1 was detected by soluble NMDA receptor Fc antigens containing GluN1 and GluN2B ATDs, but not by soluble NMDA receptor Fc antigens containing GluN1 and GluN2A ATDs (Figure 8). Furthermore, this antibody was not detected by additional soluble NMDA receptor Fc antigens containing GluN1 and GluN2C ATDs (Figure 9). Therefore, soluble NMDA receptor Fc antigens classify anti-NR-Ab1 as a GluN1 / GluN2B subtype-selective antibody.

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

[0196] Example 6: Detection of recombinant human NMDA receptor autoantibodies using three soluble NMDA receptor rbFc fusion proteins. In testing of three soluble recombinant NMDA receptor Fc antigens, N1-ATD-N2B-ATD-Fc produced the highest signal among several investigated human recombinant NMDA receptor autoantibodies, while N1-ATD-Fc + N2B-ATD-Fc produced a signal equal to or better than the others (Figure 10). These data support the findings by demonstrating differential antibody-specific sensitivity of soluble recombinant NMDA receptor Fc antigens in the serum described in Example 4.

[0197] Consideration of the Examples The results presented herein serve as proof of concept. The inventors conclude that (1) a soluble fusion protein containing the amino-terminal domain of GluN1 and rabbit Fc, heterogeneously expressed and secreted in HEK293 cells, can bind NMDA receptor autoantibodies in patient serum, and (2) efficient detection of NMDA receptor autoantibodies by soluble antigens benefits from the incorporation of the extracellular domain of GluN2. Furthermore, the use of different srNR-Fc antigens may allow for the classification of the patient's anti-NMDA receptor immune response.

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

[0199] Further examples of experimental applications of the construct of the present invention ELISA screening against recombinant LGI1 antibody using NMDA receptor fusion protein as a control. To generate the mammalian expression construct used in this experiment, cDNA for amino acids 1-558 of human LGI1 (NM_005097.3) and amino acids 1-400 of human GluN1 (NM_007327) was inserted into pFuse-rIgG-Fc1 (invivoGen). The resulting plasmid encodes the amino-terminal domain (ATD) of hsGluN1 fused to the Fc region (amino acids SKP-PGK) of rabbit IgG linked by hsLGI1 or 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 binding 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 supernatant of HEK293 cells expressing the Fc fusion protein. Cell culture supernatant containing monoclonal antibodies, CSF samples, or purified antibodies were sequentially applied with horseradish peroxidase (HRP)-conjugated donkey anti-human IgG (1:5000, Dianova, #709-035-149). After thorough washing, HRP activity was measured using 1-Step Ultra TMB-ELISA substrate (Thermo Fisher). The presence of immobilized antigens was confirmed by incubation with HRP-coupled 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 aresufficient for encephalitis pathogenesis. Brain 2016; 139:2641-2652 (Non-patent Literature 6)) was used at 10 ng / ml. The results are shown in Figure 5.

[0201] ELISA quantification of recombinant human NR1(GluN1)AB in mouse brain extract using 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 supernatant of HEK293 cells expressing the amino-terminal domain (amino acids 1-400) of human NR1 (GluN1) fused to rabbit Fc was applied. Mouse brain extracts were diluted 1:25 / 1:100 with 0.4% BSA-PBS / T and added in duplicate. The 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 a 1-Step Ultra TMB-ELISA substrate (Thermo Fisher). The concentration of #003-102 in the extract was estimated from a calibration curve prepared using purified #003-102. The results are shown in Figure 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, AJ, Hughes, EG, Rossi, JE, Peng, X., Lai, M., Dessain, SK, Rosenfeld, MR, Balice-Gordon, R., and Lynch, DR (2008). Anti-NMDA-receptor encephalitis: case series and analysis 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. Clin Infect Dis54, 899-904. 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[0202] Drawing translation Figure 1A NMDA receptor subunits Figure 1B Soluble recombinant NMDA receptor Fc antigens Figure 1C 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 Figure 3A Ctrl S (Synchronized S) Figure 3B Ctrl S (Synchronized S) Figure 3C Ctrl S (Synchronized S) Figure 4 anti-GluN1 titer: neg. negative Figure 5 ctrlSN (Control SN) anti-GluN1 no hsAB hs AB none anti-LGI1 no mmAB mm AB not included anti-rb Figure 6 NR1-reactiveIgG [ng / brain] CTL control Figure 7 A 450nm or * A 490 nm A 450 nm or * A 490 nm anti-NR-Ab1 anti-NR-Ab2 anti-rbIgG * anti-rbIgG * Figure 8 anti-NR-Ab1 controlAb1 (control Ab1) anti-rbIgG Figure 9 A 450nm or * A 490 nm A 450 nm or * A 490 nm anti-NR-Ab1 anti-rbIgG * anti-rbIgG * Figure 10 anti-NR-Ab1 anti-NR-Ab2 controlAb1 (control Ab1) anti-rbIgG

Claims

1. N-methyl-D-aspartate receptor (NMDAR) protein containing one or more NMDAR autoantibody epitopes A protein construct, wherein the construct is a fusion protein comprising the amino-terminal domain (ATD) of GluN1 and the ATD of an NMDAR subunit GluN2A or GluN2C, and lacking an NMDAR transmembrane domain.

2. The NMDAR protein construct according to claim 1, wherein the construct comprises a dimerization domain and / or a capture domain for binding the construct to a solid phase.

3. The NMDAR protein construct according to claim 2, wherein the dimerization domain is the capture domain.

4. The NMDAR protein construct according to any one of claims 1 to 3, wherein the construct comprises the amino-terminal domain (ATD) of GluN1 and the ATD of the NMDAR subunit GluN2A.

5. An in vitro method for detecting NMDAR autoantibodies in a sample, a. Prepare an NMDA protein construct according to any one of claims 1 to 4, which includes a capture domain as a capture molecule, b. Contacting a sample suspected of containing an NMDAR autoantibody with the NMDAR protein construct, thereby binding the NMDAR autoantibody from the sample to the NMDAR protein construct, c. To determine the presence of bound NMDAR autoantibodies, Methods that include...

6. The method according to claim 5, wherein the NMDAR autoantibody in the sample is present in solution or on the cell membrane.

7. The method according to claim 5 or 6, wherein the method is carried out using a plurality of different NMDAR protein constructs as described in any one of claims 1 to 4, and further comprises determining which of the plurality of constructs the NMDAR protein construct to which the NMDAR autoantibody binds.

8. The method according to any one of claims 5 to 7, wherein the method is applied to assess the possibility of a medical condition associated with an autoantibody against NMDAR, and the sample suspected of containing an NMDAR autoantibody is a sample from a human subject exhibiting symptoms of having the medical disorder.

9. The method according to claim 8, wherein the medical condition associated with the autoantibody against the NMDAR is anti-NMDAR encephalitis.

10. A kit for diagnosing autoimmune diseases associated with NMDAR autoantibodies in a subject by detecting NMDAR autoantibodies, a. An NMDAR protein construct according to any one of claims 1 to 4, or an NMDAR protein construct according to any one of claims 1 to 4 immobilized on a solid surface, a labeled secondary affinity reagent directed towards a human NMDAR autoantibody, and means for detecting a signal emitted from the label, or b. A labeled NMDAR protein construct according to any one of claims 1 to 4, A kit that includes the following:

11. The kit according to claim 10 for diagnosing NMDAR encephalitis.

12. A blood processing device configured to remove NMDAR autoantibodies from the blood or plasma of a person requiring processing in an extracorporeal blood circuit, wherein the device comprises a matrix having one or more NMDAR protein constructs according to any one of claims 1 to 4 immobilized on the matrix.

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