NMDA Receptor Constructs for Detecting and Isolating NMDAR Autoantibodies

Abstract

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

Description

[Technical field]

[0001] The present invention relates to a soluble N-methyl-D-aspartate receptor (NMDAR) protein construct comprising one or more NMDAR autoantibody epitopes, the construct comprising the extracellular domain (ECD) of the NMDAR subunit GluN1, or a fragment thereof, and the ECD of at least one of the NMDAR subunits GluN2A, GluN2B, GluN2C, or GluN2D, or a fragment thereof.Furthermore, the present invention relates to an in vitro method for detecting NMDAR autoantibodies in a sample, the method comprising:a.) preparing a sample suspected of containing NMDAR autoantibodies;b.) preparing an 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; andd.) determining the presence, and optionally the amount, of bound NMDAR autoantibodies. In an embodiment, the method of the invention is applied to the diagnosis, prognosis, disease monitoring, patient stratification and / or treatment monitoring of a medical condition associated with autoantibodies against NMDAR, preferably anti-NMDAR encephalitis. [Background technology]

[0002] Anti-NMDA receptor encephalitis (NMDAR encephalitis) is the most common form of autoimmune encephalitis that is increasing in number (Non-Patent Document 1, Non-Patent Document 2). The disorder mainly affects young women, and patients present with psychiatric and neurological symptoms including seizures and dyskinesias, along with memory loss, hallucinations, and delusions. Current treatment options include rituximab and cyclophosphamide, along with glucocorticoids and plasma exchange. NMDAR encephalitis is caused by the generation 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 the surface dynamics and induce cross-linking and internalization of NMDA receptors, and the resulting depletion of NMDA receptors may explain some of the neurological symptoms observed in patients (Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5). A single recombinant human antibody against GluN1 derived from CSF B cells induced downregulation of NMDA receptor function (Non-Patent Document 6), suggesting that they are the main pathogenic agent of the disease. This speculation has been recently strengthened by mouse models using active and passive immunization (Non-Patent Document 3, Non-Patent Document 7, Non-Patent Document 8).

[0003] In stark contrast to 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 Document 9, Non-Patent Document 10). A significant number of female NMDAR encephalitis patients have ovarian teratomas. Interestingly, some of the antibody-secreting cells isolated from the brains of NMDAR encephalitis patients expressed non-mutated / germline antibodies against the NMDA receptor (Non-Patent Document 6, Non-Patent Document 11). Although the origin of the autoimmune response has not been fully elucidated, it seems likely that a large proportion of the population may have NMDA receptor antibodies, and the presence of NMDA receptor autoantibodies in serum may constitute a common problem in pregnant women and elderly people with blood-brain barrier disorders. Furthermore, NMDA receptor autoantibodies have been found in patients with neuropsychiatric disorders other than NMDAR encephalitis, likely contributing to disease progression (Non-Patent Document 4).

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

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

[0006] Autoantibodies against NMDA receptors are routinely detected using Euroimmun cell-based assay (CBA) kits based on biochips containing acetone-fixed GluN1-expressing xenogeneic cells as described in US Pat. No. 5,399,663. However, NMDA receptor autoantibodies often recognize the native conformation of their antigens, and live staining of NMDA receptor-expressing HEK293 cells was found to be more sensitive than commercial assays using pre-fixed cells to detect low titers (Non-Patent Document 18). These cell-based assays require visual inspection of the results, which may cause bias. Moreover, even if automated routine testing is possible in such tests, the fact that there are many different antigens on the cell surface, not just the antigen of interest, makes them prone to false positive results.

[0007] Thus, there is an urgent need for a sensitive, quantifiable and high-throughput method for detecting NMDA receptor autoantibodies. In this direction, a single nanoparticle imaging approach on primary hippocampal neurons detects NMDA receptor autoantibodies at low titers and is automatable, but technically very challenging (Non-Patent Document 18). An ELISA was described early on that allows the comparison of different titers of NMDA receptor autoantibodies based on lysed HEK293 cells expressing NMDA receptors (Non-Patent Document 19). Patent Document 2 discloses a method for the detection of antibodies against NR2A (GluN2A) and / or NR2B (GluN2B) in the context of a method for the diagnosis of stroke, in which the amino-terminal fragments of NR2A / NR2B are synthesized and purified, but do not involve combination with the ECD of GluN1. In fact, the disclosed method identifies antibodies specifically directed against GluN2A or GluN2B, but is not intended to identify antibodies directed against GluN1, is independent of their association with GluN1 and is therefore intended for a completely different application compared to the present invention. Importantly, the method of WO 2005 / 023363 cannot be used to identify antibodies that bind to GluN1 or portions thereof.

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

[0009] Thus, there remains a strong need in the art to provide NMDAR protein constructs that contain one or more NMDAR autoantibody epitopes that may be composed of or stabilized by the extracellular domains of GluN1 and GluN2A, GluN2B, GluN2C and / or GluN2D, or fragments thereof. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2012 / 076000 [Patent Document 2] US 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

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Summary of the Invention

Problems to be Solved by the Invention

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

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

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

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

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

[0017] The present invention also relates to an N-methyl-D-aspartate receptor (NMDAR) protein construct comprising one or more NMDAR autoantibody epitopes, the NMDAR protein 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 a fragment thereof, and not present in or on a cell. In an embodiment, the NMDAR protein construct comprises an extracellular domain (ECD) of the NMDAR subunit GluN1 or a fragment thereof, and an ECD of the NMDAR subunits GluN2A or a fragment thereof and / or an ECD of the NMDAR subunits GluN2B or a fragment thereof, and not present in or on a cell.

[0018] The NMDAR protein constructs of the present invention can be used to screen for NMDA receptor autoantibodies in patient serum or CSF.

[0019] The data disclosed herein show that the soluble NMDAR protein constructs of the present invention, especially in their embodiment as Fc fusion proteins, can detect antibodies in the serum of patients with NR encephalitis. Soluble NMDAR-Fc protein constructs (srNR-Fc proteins) have several advantages over state-of-the-art cells and cell-based assays that detect antibodies using NMDAR expressed on the surface of in vitro cultured cells. srNR-Fc proteins can be purified and stored. Purified srNR-Fc proteins constitute clean antigens compared to heterologous cells that contain many additional proteins. They allow the generation of very high antigen concentrations that lead to increased sensitivity in antibody detection compared to cell-based assays (CBA).

[0020] In contrast to the known NMDAR constructs of the state of the art, the present invention relates to NMDAR constructs that comprise at least fragments of two NMDAR subunits, which allows to identify autoantibodies that bind to epitopes formed by the residues of two subunits or that are formed or stabilized only by the assembly of two subunits.In contrast, the state of the art constructs that comprise only one subunit or its fragment do not bind to and remove such autoantibodies.

[0021] By using the protein construct of the invention for the recognition of NMDAR-specific antibodies in a sample, it is possible to identify antibodies that only bind to NMDAR and GluN1 when GluN1 is associated with the GluN2 subunit, which leads to the stabilization of the respective epitopes of such antibodies, which cannot be identified by proteins or protein constructs that contain only GluN1 or GluN2 subunits, since the respective epitopes are stabilized or formed only by the association of GluN1 with the respective GluN2 subunit.

[0022] For example, the NMDAR protein constructs disclosed herein, which comprise Fc fragment or other or additional tags, can be useful in various diagnostic assays.For example, the diagnostic assay in the form of an ELISA-like assay disclosed in the examples can be used as a companion diagnostic to detect NMDA receptor autoantibody and enable quantifiable high-throughput method of detecting autoantibody in other autoimmune encephalopathies.

[0023] Furthermore, NMDAR protein constructs can be used to distinguish autoimmune responses against different NMDA receptor compositions allowing for patient stratification.

[0024] As disclosed in the examples provided herein, NMDAR protein constructs such as various srNR-Fc fusion proteins and dimers formed by such fusion proteins produce different signals in response to antibodies / sera from different patients, and show patient-specific variant 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 antibodies that are primarily directed against GluN1 or heterogeneous GluN1 / GluN2 structures, and potentially against GluN1 / GluN2 structures of specific composition.Subclassification of NMDAR encephalitis patients may ultimately lead to improved treatment methods.

[0025] Thus, the present invention also relates to sets or kits providing two or more NMDAR protein constructs of the invention providing different combinations of one or more of GluN1 and GluN2A-GluN2D, or fragments thereof.

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

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

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

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

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

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

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

[0033] Additionally, constructs that allow for labeling of B cells expressing NMDA receptor autoantibodies can be used as diagnostic tools, facilitating cell isolation and IgG sequence analysis.

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

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

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

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

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

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

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

[0041] In embodiments, the dimerization domain comprises a leucine zipper and / or a coiled-coil domain.

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

[0043] The presence of dimerization and / or capture domains, such as Fc fragments, can be particularly advantageous since these domains stabilize the soluble NMDAR protein constructs, and in particular the Fc portion allows for long-term storage of the construct with a conserved conformation.

[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 the context of a construct of the present invention, the ECD of at least one NMDAR subunit, GluN2A, GluN2B, GluN2C, or GluN2D, or a fragment thereof, comprises or consists of the ATD of at least one NMDAR subunit, GluN2A, GluN2B, GluN2C, or GluN2D, or a fragment thereof, respectively.

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

[0047] In certain NMDAR protein constructs 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 case, 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. In an embodiment, the ECD of GluN1 and the ECD of GluN2A and / or the ECD of GluN2B, or a fragment thereof, are preferably covalently linked as a fusion protein. In this case, the ECD of GluN1 and the ECD of GluN2A and / or the ECD of GluN2B, or a fragment thereof, may be linked directly as a fusion protein or by a protein linker.

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

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

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

[0051] In embodiments, the construct comprises one or more protease cleavage sites, such as a TEV cleavage site or any other cleavage site recognized by a suitable protease known to one of skill in the art, between the portion of the construct comprising the ECD of GluN1, the ECD of GluN2A and / or the ECD of GluN2B, or fragments thereof, and the portion of the construct comprising the dimerization domain and / or the capture domain.

[0052] In a preferred embodiment of the NMDAR protein construct of the present invention, the construct is a protein dimer of non-covalently linked monomers, and the construct can be a homodimer or a heterodimer.In a particular dimeric construct of the present invention, the construct can be a heterodimer formed from the ECD of GluN1 (as one monomer) or a fragment thereof and the ECD of at least one NMDAR subunit of GluN2A, GluN2B, GluN2C or GluN2D (as one monomer) or a fragment thereof.In an embodiment of the dimeric construct, the construct can be a heterodimer formed from the ECD of GluN1 (as one monomer) or a fragment thereof and the ECD of GluN2A (as one monomer) or a fragment thereof and / or the ECD of GluN2A (as one monomer) or a fragment thereof.Preferably, the dimer is formed by a dimerization domain 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, such as GluN1 and GluN2A, GluN1 and GluN2B, GluN1 and GluN2C, or GluN1 and GluN2D, or fragments thereof. Thus, it is possible to provide an NMDAR protein construct comprising four ECDs (provided by two monomers) of an NMDAR subunit, or fragments thereof, thus mimicking the naturally occurring NMDA receptor (NMDAR, NMDAR receptor), which is a tetramer of four subunits, namely two GluN1 subunits and two GluN2 subunits. 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 comprising four ATD domains.

[0053] The NMDAR protein construct of the present invention is advantageous compared to known NMDAR constructs, because it allows the assembly of ECD or ATD of GluN1, GluN2A, GluN2B, GluN2C and / or GluN2D subunits in a given construct.This potential of ligand combination is a strong advantage, because the autoantibody specific for a certain subunit or subunit combination can be effectively bound by the construct of the present invention, and the set of autoantibodies present in a sample can be evaluated for their differential binding profile.In addition, the ECD or ECD fragment of GluN1, GluN2A, GluN2B, GluN2C and / or GluN2D subunits can be combined with a capture domain, which allows the flexible use of the construct, for example, in the context of ELISA assay, which is advantageous compared to the state of the art cell-based ELISA assay.

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

[0055] In vitro methods for detecting NMDAR autoantibodies in samples Furthermore, the present invention provides an in vitro method for detecting NMDAR autoantibodies in a sample, comprising: Providing a sample suspected of containing an NMDAR autoantibody; Providing an NMDA protein construct according to any one of the preceding claims as a capture molecule; contacting the sample with the NMDAR protein construct, thereby allowing NMDAR autoantibodies from the sample to bind to the NMDAR protein construct; determining the presence, and optionally the amount, of bound NMDAR autoantibodies; The present invention relates to a method comprising the steps of:

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

[0057] In an embodiment, the method for detecting NMDAR autoantibodies is carried out using a plurality of different NMDAR protein constructs in the sense of the present invention.For the method of the present invention using a plurality of different constructs, the method may additionally comprise the step of determining to which NMDAR protein construct of said plurality of constructs NMDAR autoantibodies bind, or preferably bind in the greatest amount and / or bind most efficiently.Therefore, it may be possible to profile and classify the patient who provides the sample based on the NMDAR autoantibodies and their binding properties determined for 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 is determined in one assay (multiplexing).

[0058] The method of the invention may comprise the step of determining the NMDAR autoantibody profile present in said sample.

[0059] In an embodiment of the method of the present invention, the presence and optionally the amount of cells presenting NMDAR autoantibody on the cell surface present in the above-mentioned sample can be determined.The great advantage of the method of the present invention is that it can detect NMDAR autoantibody on cell surface, especially on the surface of B cells that produce NMDAR autoantibody, in addition to soluble NMDAR autoantibody.

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

[0061] In embodiments of the methods for diagnosis, prognosis, disease monitoring, patient stratification and / or treatment monitoring of a medical condition associated with autoantibodies to NMDAR, preferably anti-NMDAR encephalitis, the presence of bound NMDAR autoantibodies, preferably an amount of bound NMDAR autoantibodies above a suitable control, such as an amount from a healthy control population, indicates the presence or likelihood of the subject developing a medical condition associated with autoantibodies to NMDAR, preferably anti-NMDAR encephalitis.

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

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

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

[0065] In an embodiment of the present invention, the soluble NMDAR protein construct of the present invention is provided in immobilized form.In such an embodiment, the soluble NMDAR protein construct of the present invention can be purified in its soluble form from suitable expression system and then immobilized on solid phase.Therefore, in the context of such an embodiment of the immobilized NMDAR protein construct of the present invention, "soluble" refers to the previous 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 method of the present invention comprises the following steps: immobilizing NMDAR autoantibodies from the sample by binding to an NMDAR protein construct immobilized on a solid surface; treating the immobilized NMDAR autoantibodies with a labeled secondary affinity reagent directed to the NMDAR autoantibodies; Detecting a signal emitted from the labeled secondary affinity reagent directed to the NMDAR autoantibody; comparing the signal obtained from the labeled secondary affinity reagent with a signal from one or more control samples of predetermined NMDAR autoantibody concentrations; may include.

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

[0069] In certain embodiments, the methods of the invention are applied to the therapeutic guidance of a subject having and / or suspected of developing a medical condition associated with NMDAR autoantibodies, the methods comprising selecting one or more corresponding NMDAR protein constructs of the invention for subsequent treatment of said subject.

[0070] Kit for detecting NMDAR autoantibodies The present invention also relates to a kit for detecting NMDAR autoantibodies in a sample, the kit comprising: An NMDAR protein construct of the present invention and optionally a solid surface for immobilizing said NMDAR protein construct, or an NMDAR protein construct of the present invention immobilized on a solid surface, and a labeled secondary affinity reagent directed against human NMDAR autoantibodies, such as a labeled anti-human IgG antibody, and optionally a means for detecting a signal emitted by the label, or A labeled NMDAR protein construct of the present invention, and optionally a means for detecting a signal emitted from the label; Optionally, a control sample of a predetermined NMDAR autoantibody concentration. 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 the fluorescently labeled secondary antibody directed against rabbit Fc.Furthermore, the kit can be used to carry out ELISA to detect NMDAR autoantibody present in a sample.

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

[0073] Blood Processing Device Containing NMDAR Protein Constructs Furthermore, the present invention relates to a blood treatment device configured for removing NMDAR autoantibodies from the blood or plasma of a person in need of treatment in an extracorporeal blood circuit, the blood treatment device comprising a matrix having one or more NMDAR protein constructs of the present invention immobilized thereon.

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

[0075] Further aspects of the invention Furthermore, the present invention includes the NMDAR protein construct disclosed herein for use as a medicament.The present invention also relates to the NMDAR protein construct disclosed herein for use as a medicament in the treatment of subjects suffering from autoimmune disease associated with NMDAR autoantibody, 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 the 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 the ability to isolate soluble NMDAR protein constructs from cell culture supernatants of cells engineered to express nucleic acid sequences encoding the NMDAR protein constructs of the present invention, particularly as compared to methods that require first isolating the antigen from the cell membrane using either protease cleavage or detergent solubilization.

[0078] Additionally, the present invention includes the NMDAR protein constructs disclosed herein produced by the disclosed methods of producing the NMDAR protein constructs of the present invention.

[0079] The various embodiments and features of the NMDAR protein constructs disclosed herein also apply to 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 in need of treatment, and methods for producing the inventive NMDAR protein constructs presented herein, and vice versa.

[0080] Detailed Description of the Invention All cited documents, both patent and non-patent literature, are hereby incorporated by reference in their entirety.

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

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

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

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

[0085] "Isolated peptide" or "isolated polypeptide" or "isolated protein construct" and the like, as used herein, 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 from association with other components of the cell, i.e., it is not significantly associated with in vivo materials.

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

[0087] In an embodiment of the present invention, the protein construct comprises a dimerization domain. Such a construct may be composed of one or more proteins. For a protein construct of the present invention composed of a single protein, it is clear to a person skilled in the art that the presence of a dimerization domain may lead to the formation of a homodimer. Furthermore, it is readily apparent to a person skilled in the art that a protein construct comprising a dimerization domain and composed of two or more proteins may comprise a dimerization domain in each protein. In such an embodiment, the dimerization of the two proteins of the protein construct is preferentially mediated by the dimerization domain. In other words, when the protein construct of the present invention is or forms a dimer (either a homodimer or a heterodimer) of two proteins, the dimerization may be brought about by a dimerization domain contained in each protein. In a preferred embodiment, the dimerization domain is an Fc domain.

[0088] As used herein, the term capture domain refers to a domain or part of a protein construct of the present invention that can be used to bind the construct to a solid phase, either by non-covalent interactions or by covalent bonds. Typical examples of such capture domains are domains or amino acid sequences that are recognized by commonly available proteins that bind to capture domains, preferably antibodies. For example, the Fc fragment of an antibody can serve as a capture domain, since there are high affinity antibodies that specifically bind to these domains. Furthermore, protein tags such as Myc tags, HA tags, HIS tags, etc. can be used as capture domains.

[0089] There are many different possible dimerization and capture domains that can be incorporated into the protein construct of the present invention, and the skilled person can identify suitable variants. Furthermore, in some cases, it is also possible for the dimerization domain to serve as a capture domain. This is the case, for example, of antibody Fc fragments, which can form dimers and can be easily bound by commonly available antibodies. Thus, the Fc fragment can function simultaneously as a capture domain and a dimerization domain. Further examples are known by the skilled person or can be identified without undue effort.

[0090] An embodiment of the invention relates to recombinant proteins, such as recombinant fusion proteins, which are proteins made through genetic engineering of a fusion gene. This typically involves adding the cDNA sequence of a second protein fragment in frame with the cDNA of the first protein (fragment) without a stop codon in between, for example by ligation or overlap extension PCR. The DNA sequence is then expressed by the cell as a single protein. The protein can be engineered to contain the complete sequence of both original proteins, or only a portion of either. Three or more proteins or fragments can be joined to form complex fusion proteins. There are often so-called linker (or "spacer") peptides between the various parts of the fusion protein, making it more likely that the proteins will fold independently and perform as expected. In protein or peptide fusions the linker may be engineered with a cleavage site for a protease or chemical to allow the release of the two separate proteins, especially if the linker allows for protein purification. This technique is often used to identify and purify proteins by fusing them with GST proteins, FLAG peptides, or hexa-His peptides (6xHis tags), which can then be isolated using affinity chromatography with nickel or cobalt resins. Dimeric or multimeric chimeric proteins can be produced by genetic engineering by fusing peptide domains that induce dimerization or multimerization of the artificial protein to the original protein (e.g., streptavidin or leucine zipper).

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

[0092] Preferred sequences comprised by the NMDAR construct of the present invention Preferred amino acid sequences comprised by the 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 are disclosed in Table 1. Preferred nucleic acid sequences comprised by the nucleic acid molecule 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 are disclosed in Table 2.

[0093] Table 1: Preferred amino acid sequences of the present invention [Table 1-1] [Table 1-2]

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

[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

[0095] The present invention further relates to functionally similar sequences, domains, linkers and further elements constituted by the construct of each NMDAR protein construct. Protein modifications to the NMDAR protein construct of the present invention, which may occur by substitution of amino acid sequences and nucleic acid sequences encoding such molecules, are also included within the scope of the present invention. Substitutions as defined herein are modifications made to the amino acid sequence of a protein, whereby one or more amino acids are replaced with the same number of (different) amino acids, producing a protein that contains an amino acid sequence different from the primary protein. In some embodiments, this modification does not significantly change the function of the protein. As with additions, substitutions can be natural or artificial. It is well known in the art that amino acid substitutions can be made without significantly changing the function of a protein. This is particularly true when the modification concerns "conservative" amino acid substitutions, which are the replacement of one amino acid with another amino acid of similar properties. Such "conservative" amino acids can be natural or synthetic amino acids that, due to 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 by conservative amino acids without adversely affecting the function of the protein.

[0096] In general, the following amino acids are considered to be conservative groups: non-polar amino acids Gly, Ala, Val, Ile, and Leu; non-polar 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, can be substituted for each other, despite belonging to different groups.

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

[0098] The nucleic acid sequence of the present invention includes the nucleic acid sequence that codes for NMDAR protein construct or individual proteins that form part of NMDAR protein construct of the present invention.The protein sequences according to Table 1 and functionally similar sequences represent the preferred NMDAR protein construct of the present invention or parts thereof.The preferred nucleic acid sequences that code for NMDAR protein construct of the present invention or parts thereof are listed in Table 2.

[0099] The NMDAR protein construct of the present invention can comprise a protein tag that allows easy identification or binding of the NMDAR protein construct provided by standard techniques, for example, by using an antibody directed against the protein tag.Preferred protein tags that the nucleic acid sequence of the present invention can code are V5 tag, myc tag, HA tag, HIS tag or antibody Fc fragment.Alternative tags can be used instead of V5 tag.Such alternatives are well known in the art and can be selected by the skilled artisan.

[0100] In another aspect, the present invention encompasses the NMDAR protein constructs disclosed herein and their use in connection with the methods disclosed herein.In particular, the present invention also relates to nucleic acid molecules encoding the NMDAR protein constructs of the present invention, and in particular to one or more nucleic acid molecules encoding such NMDAR protein constructs or parts of such constructs, a) one or more nucleic acid molecules comprising a nucleotide sequence encoding the ECD of the NMDAR subunit GluN1 or a fragment thereof, and the ECD of at least one of the NMDAR subunits GluN2A, GluN2B, GluN2C, or GluN2D, or a fragment thereof, and preferably a dimerization domain and / or a capture domain; b) one or more nucleic acid molecules complementary to the nucleotide sequence of a); c) one or more nucleic acid molecules that undergo hybridization under stringent conditions with the nucleotide sequence according to a) or b); d) one or more nucleic acid molecules comprising a nucleotide sequence having sufficient sequence identity to be functionally similar to the nucleotide sequence of a), b), or c); e) one or more nucleic acid molecules which, as a result of the genetic code, are degenerate to the nucleotide sequences described in a) to d); f) one or more nucleic acid molecules according to the nucleotide sequences of a) to e) which have been modified by deletion, addition, substitution, transition, inversion and / or insertion and which are functionally similar to the nucleotide sequences of a) to e); The compound is selected from the group consisting of:

[0101] Furthermore, the present invention also relates to a nucleic acid molecule encoding an NMDAR protein construct of the present invention, and in particular to one or more nucleic acid molecules encoding such an NMDAR protein construct or a part of such a construct, g) one or more nucleic acid molecules comprising a nucleotide sequence encoding the 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 the NMDAR subunit GluN2B, and preferably a dimerization domain and / or a capture domain; h) one or more nucleic acid molecules complementary to the nucleotide sequence of a); i) one or more nucleic acid molecules which undergo hybridization under stringent conditions with the nucleotide sequence according to a) or b); j) one or more nucleic acid molecules comprising a nucleotide sequence having sufficient sequence identity to be functionally similar to the nucleotide sequence of a), b), or c); k) one or more nucleic acid molecules which, as a result of the genetic code, are degenerate to the nucleotide sequences described in a) to d); l) one or more nucleic acid molecules according to the nucleotide sequences of a) to e) which have been modified by deletion, addition, substitution, transition, inversion and / or insertion and which are functionally similar to the nucleotide sequences described in a) to e); The compound is selected from the group consisting of:

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

[0103] The scope of the present invention also includes sequence variants of the claimed nucleic acids and / or proteins, for example defined by the % sequence identity provided, that maintain the above-mentioned properties of the present invention.Such variants, which show alternative sequences but maintain essentially the same properties as the specific sequences 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 relates to the percentage of identical nucleotides or amino acids when performing sequence alignment 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 code for the polypeptides described herein. Some of these polynucleotides have minimal homology or sequence identity to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Deletions, substitutions and other changes in the sequences that fall within the sequence identity described 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 the ECD or a fragment of the ECD of another subunit, and certain epitopes can only be formed upon stabilization of such conformation. In the context of the present invention, the epitopes formed by the NMDAR protein construct and the construct of the present invention can be referred to as autoantigens. The binding between autoantigens and antibodies is itself a well-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 autoantibody typically targets either central or peripheral nervous system autoantigens.However, medical conditions are also known in which autoantibody is directed against targets present in both central and peripheral nervous systems.The present invention therefore envisages the use of the NMDAR protein construct of the present invention in association with diseases in which autoantibody primarily targets components of the central nervous system, or the pathogenic effects of said autoantibody are caused by autoantibody targeting autoantigens in the central nervous system.

[0107] As used herein, "central nervous system" or CNS refers to the part of the nervous system that consists 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 within the spinal canal. The CNS is divided into white matter and gray matter. This can also be seen macroscopically on brain tissue. White matter consists of axons and oligodendrocytes, while gray matter consists of neurons and unmyelinated fibers. Both tissues contain many glial cells (although white matter contains more glial cells), and are often referred to as supporting cells of the CNS. Extending from and towards the spinal cord is the peripheral nervous system in the form of spinal nerves. Nerves connect the spinal cord to the skin, joints, muscles, etc., and allow for the transmission of efferent motor as well as afferent sensory signals and stimuli. This allows for the perception of sensations, as well as voluntary and involuntary muscle movements.

[0108] As used herein, the "peripheral nervous system" (PNS) consists of the nerves and ganglia outside the brain and spinal cord. The main function of the PNS is to connect the CNS to the limbs and organs, essentially acting 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 spinal column and skull, or the blood-brain barrier.

[0109] Emerging studies now show that autoantibodies can access the CNS (Zong et 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 low rate, a good example being immunoglobulin G (IgG). The concentration of IgG in the cerebrospinal fluid (CSF) is approximately 1% of the level in the peripheral circulation. This indicates that if autoantibodies reach the CNS, they may cause disease, as has been observed in autoimmune encephalitis. In certain circumstances, the BBB may become leaky due to stroke, brain trauma, hemorrhage, microangiopathy, or brain tumors, allowing increased penetration of antibodies.

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

[0111] In some embodiments, the disease treated or diagnosed using the present invention is an autoimmune encephalopathy or encephalomyelopathy. "Encephalopathy" is typically any disorder or disease of the brain, especially a chronic degenerative condition. Encephalopathy may refer to permanent (or degenerative) brain damage, or reversible damage. Encephalopathy may result from direct damage to the brain, or from disease distant 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 with an autoimmune component, including autoimmune encephalitis. As used herein, "autoimmune encephalomyelopathy" is any disease that affects both the brain and spinal cord with an autoimmune component.

[0112] Anti-N-methyl-D-aspartate (NMDA) receptor encephalitis is a type of encephalitis that occurs frequently in women and is associated with antibodies against the NR1 (GluN1) and / or NR2 subunits of the NMDA receptor, mainly the NR1 subunit. Anti-NMDA receptor encephalitis was first described several years ago in several large studies that characterized the clinical syndrome in detail (Non-Patent Document 19). Patients with anti-NMDAR encephalitis suffer from a severe form of encephalitis with characteristic clinical multistage features that mainly 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 (Dalmau et al. Lancet Neurol. 2011; 10:63-74; Pruess et al. 2010, Neurology. 75(19):1735-9; Pruess et al. 2013, Neurology. 75(19):1735-9). The hallmark of this disease is antibodies against the NR1 / GluN1 subunit of NMDAR1. This has significantly changed the therapeutic concept of encephalitis, since NMDAR encephalitis was not recognized as a distinct subgroup of encephalitis before 2007. Thus, NMDAR encephalitis was previously considered an encephalitis of unknown etiology and was undertreated.

[0113] N-methyl-D-aspartate receptors (also known as NMDA receptors or NMDARs) are glutamate receptors and ion channel proteins found in neuronal cells. NMDA receptors are one of three types of ionotropic glutamate receptors. The other receptors are AMPA receptors and kainate receptors. Binding of glutamate and glycine (or D-serine) to NMDARs activates them, allowing positively charged ions to flow across the cell membrane. NMDA receptors are crucial for controlling synaptic plasticity and memory function. Receptors are typically assembled as heterogeneous complexes that interact with multiple intracellular proteins by three distinct subunits, NR1, NR2, and NR3. NR1 has eight different isoforms generated by alternative splicing from a single gene. There are four distinct NR2 subunits (A-D), with NR3A and NR3B subunits reported in the late 20th century. Six separate genes code for NR2 and NR3. According to more recent nomenclature, the subunits are designated GluN1, GluN2 and GluN3 instead of NR1, NR2 and NR3, respectively. Alternative variants of the subunits are identified accordingly (e.g. NR2A and NR2B are identified as GluN2A and GluN2B, respectively).

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

[0115] NMDAR NR1 / GluN1 is a component of the NMDA receptor complex that functions as a heterotetrameric, ligand-gated ion channel with high calcium permeability and voltage-dependent sensitivity to magnesium. Channel activation requires binding of the neurotransmitters glutamate to the GluN2 subunit and glycine to the GluN1 subunit, followed by membrane depolarization to eliminate channel inhibition by Mg2+. Many protein isoforms of the NMDAR NR1 protein are known, including, but not limited to, those in 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 constructs of the present invention.

[0116] With regard to GluN2 / NR2, only a single subunit is found in invertebrates, whereas four different isoforms of the NR2 subunit are expressed in vertebrates, designated by the nomenclature NR2A / GluN2A to NR2D / GluN2D (encoded by GRIN2A, GRIN2B, GRIN2C, and GRIN2D). They contain a binding site 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 present primarily in immature intraneurons and at extrasynaptic locations and contains a binding site for the selective inhibitor ifenprodil. NR2B predominates in the early postnatal brain, whereas the number of NR2A subunits increases, and eventually NR2A subunits outnumber NR2B. This is called the NR2B-NR2A developmental switch, and is notable because of the different dynamics that each NR2 subunit imparts to the receptor. For example, a greater proportion of NR2B subunits results in NMDA receptors that stay open longer compared to more NR2A subunits.

[0117] NMDAR has various physiological roles and any dysfunction, either enhanced or decreased activity, 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). Thus, NMDAR plays an important role in multiple psychiatric disorders, including depression. Furthermore, a subgroup of patients with atypical dementia has anti-NMDAR1 antibodies, thereby qualifying as a medical condition associated with autoantibodies to NMDAR, and removal of such NMDAR autoantibodies by non-specific removal of all antibodies has led to clinical improvement in selected cases (Pruess et al. 2010, Neurology. 75(19):1735-9, Doss et al. 2014 Ann Clin Transl Neurol. 1 (10):822-32). Furthermore, autism can occur in children of mothers suffering from autoantibody-mediated disorders. Some studies have found a correlation between the presence of circulating maternal autoantibodies and neurological dysfunction in newborns (Fox-Edmiston et al, 2015, CNS Drugs, 29(9):715-724). Specifically, maternal anti-brain autoantibodies that may access the fetal compartment during pregnancy have been identified as one of the risk factors for developing autism spectrum disorder (ASD). The presence of NMDAR-autoantibodies may therefore cause autism in the offspring of affected mothers, so the present invention also represents a possible treatment of such disorders in children and / or a preventive approach to avoid such diseases.

[0118] Thus, any medical condition in which the contribution of NMDAR autoantibody to the pathogenesis has been described or suggested qualifies as a medical condition associated with NMDAR autoantibody, for example due to the correlation between NMDAR autoantibody and the occurrence of disease symptoms.Furthermore, the construct of the present invention can be used to analyze samples from patients suffering from a condition described or suggested as a condition associated with NMDAR autoantibody for the presence of such antibodies, and can then be treated according to the present invention.

[0119] In contrast to anti-NMDAR in autoimmune encephalitis, which primarily targets the GluN1 subunit, autoantibodies have been found to target the GluN2 subunit of NMDAR, 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] Embodiments of the in vitro method for detecting NMDAR autoantibodies Autoantibodies are antibodies (a type of protein) produced by the immune system and directed against one or more of an individual's own proteins. Many autoimmune diseases are associated with and / or caused by such autoantibodies.

[0121] The term "autoimmune disease" refers to any given disease associated with and / or caused by the presence of autoantibodies. Autoimmune diseases result from an abnormal immune response of the body against substances and tissues normally present in the body (autoimmunity). This may be restricted 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 fluid or tissue obtained for the purpose of diagnosing, prognosticating, or evaluating a subject of interest, such as a patient. Preferably, the sample herein is a sample of bodily fluid, such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, pleural effusion, cells, cell extracts, tissue samples, tissue biopsy samples, stool samples, etc.

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

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

[0125] The terms "diagnosis" and "diagnosing" include the use of NMDAR protein constructs, methods, kits and further aspects of the invention to determine the presence or absence or possible absence 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 the criteria developed by Truelove et al., Br. Med. J., 12:1041-1048 (1955). In other embodiments, statistical algorithms are used to diagnose mild to moderate, moderate to severe or severe to fulminant autoimmune diseases associated with NMDAR autoantibodies.

[0126] The present invention also encompasses the use of methods of disease monitoring, also known as monitoring the progression or regression of autoimmune disease and therapeutic monitoring. The term "monitoring" includes the use of the NMDAR constructs disclosed herein and the methods and other embodiments of the present invention to determine the disease state 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 to results obtained for the same individual at an earlier time. In some embodiments, the kits, constructs, devices, methods, and systems of the present invention can also be used to predict the progression of an autoimmune disease, for example, by determining the likelihood that an autoimmune disease will progress quickly or slowly in an individual based on the presence or level of at least one marker (such as one or more NMDAR autoantibodies) in a sample. The present invention can also be used to predict the regression of an autoimmune disease, for example, by determining the likelihood that an autoimmune disease will regress quickly or slowly in an individual based on the presence or level of at least one marker in a sample. Therapeutic monitoring can also be performed, thereby monitoring a subject for disease progression over the course of any given treatment.

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

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

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

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

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

[0132] As examples of means for detecting the label in the methods of the present invention, various immunoassay techniques, including competitive and non-competitive immunoassays, can be used to determine the presence or level of one or more markers in a sample (see, e.g., Self et al., Curr. Opin. Biotechnol., 7:60-65 (1996)). The term immunoassay encompasses techniques including, but not limited to, enzyme immunoassays (EIA), such as enzyme-amplified immunoassay technology (EMIT), enzyme-linked immunosorbent assay (ELISA), antigen capture ELISA, sandwich ELISA, IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoretic immunoassay (CEIA); radioimmunoassay (RIA); immunoradiometric assay (IRMA); fluorescence polarization immunoassay (FPIA); and chemiluminescence assay (CL). If desired, such immunoassays can be automated. Immunoassays can also be used in conjunction with laser-induced fluorescence (see, e.g., 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 the present invention (see, e.g., Rongen et al., J. Immunol. Methods, 204:105-133 (1997)). Also suitable for use in the present invention are nephelometric assays, in which the formation of protein / antibody complexes results in an increase in light scattering that is converted into a peak rate signal as a function of marker concentration. Nephelometric assays are commercially available from Beckman Coulter (Brea, Calif.; kit #449430) and can be performed using a Behring Nephelometer Analyzer (Fink et al., J. Clin. Chem. Clin. Biol. Chem., 27:261-276 (1989)).

[0133] The above immunoassays are particularly useful for determining the presence or level of one or more NMDAR autoantibodies in a sample (and can be considered as examples of means for detecting a label).

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

[0135] In another preferred embodiment of the present invention, soluble or solid-phase-bound NMDAR protein constructs are used to bind antibodies. In the second reaction step, anti-human immunoglobulins can be employed, preferably selected from the group including anti-human IgA, anti-human IgM and / or anti-human IgG antibodies, which are two-component detectable label conjugates that can be conjugated with any conventional labeling enzyme, in particular chromogenic and / or chemiluminescent substrates, preferably horseradish peroxidase, alkaline phosphatase. The advantage of this embodiment is the use of ELISA techniques that are usually 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 antibodies bound to the NMDAR protein constructs of the present invention react with anti-human immunoglobulins, preferably selected from the group including anti-human IgA, anti-human IgM and / or anti-human IgG antibodies, and are detectably conjugated to fluorescein isothiocyanate (FITC). As with the above-mentioned ELISA, the FITC technique represents a system that is widely available and therefore allows for a smooth and low-cost establishment of the detection of the present invention in a routine laboratory. The skilled artisan will be aware of further standard detection techniques that can be used in connection with the method of the present invention.

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

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

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

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

[0140] In certain embodiments, the present invention provides a method for diagnosing autoimmune disease or its 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 to classify samples from individuals as samples with autoimmune disease.Additional examples of markers of autoimmune disease associated with NMDAR autoantibodies suitable for use in the present invention are known to those skilled in the art.Those skilled in the art will know additional markers suitable for use in the statistical algorithm of the present invention.

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

[0142] Another method is covalent bonding to the carrier material. Furthermore, the carrier may have reactive groups that form homopolar bonds with amino acid side chains. Suitable groups for peptides are carboxy, hydroxy and sulfide groups, especially 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 silanes and then reacted with peptides. For example, the hydroxy 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 the encapsulation of peptides in ionotropic gels or other structures well known to those skilled in the art. More specifically, the pores of the matrix allow the peptides to interact with target molecules in such a way that they are retained. In crosslinking, peptides are converted into polymer aggregates by crosslinking with bifunctional substances. Such structures are gelatinous and easily deformable, making them particularly suitable for use in various reactors. The addition of other inert components such as gelatin during crosslinking allows advantageous improvement of mechanical and binding properties. In microencapsulation, the reaction volume of the peptides is limited by the membrane. For example, microencapsulation can be carried out in the form of interfacial polymerization. Immobilization during microencapsulation makes the peptides insoluble and therefore reusable. In the sense of the present invention, immobilized constructs are in a state that allows all their peptides to be reused. Restricting the mobility and solubility of peptides by chemical, biological or physical means advantageously leads to lower processing costs, especially when eliminating 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 the necessary analyte-specific reagents required to carry out a diagnostic test. The diagnostic kit may also comprise instructions on how to carry out the test using the provided reagents. The diagnostic kit may optionally comprise instructions on combining the contents of the kit and / or providing 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 medium that provides the user with information on the type of method in which the mentioned substance is to be used. Obviously, the information does not necessarily have to be in the form of an instruction leaflet, for example, the information may be given via the internet. For the patient, one beneficial effect of such a kit is that, for example, the patient can determine the actual disease state even when traveling, without the need for a doctor to deal with him in person, and adapt his diet and activity accordingly.

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

[0145] Therefore, as used herein, "matrix" refers to the internal material through which blood or plasma passes, or the material in blood processing device that provides the surface through which blood or plasma passes.The matrix used in the present invention preferably comprises the support to which NMDAR protein construct is bound.Therefore, this support serves as the carrier of NMDAR protein construct, but may also perform other functions.

[0146] As used herein, "support" refers to the portion of the matrix that serves as a "substrate" or "support material" to which the construct according to the present invention is attached. Such supports or support materials may also be referred to as "adsorbent" or "adsorbents", as used in "adsorption columns" or "columns" or "adsorption cartridges". Suitable supports according to the present invention should be homogeneous, hydrophilic, mechanically and chemically stable over the relevant pH range and temperature, exhibit no or negligible leaching of the NMDAR protein construct during use, have good flow properties for whole blood and / or plasma, and provide a large surface area for attachment of the NMDAR protein construct.

[0147] The support may be, for example, a resin, a membrane, or a nonwoven material. "Nonwoven" materials refer to materials broadly defined as sheets, fabrics, or web structures in which fibers or filaments are not woven or knitted but are bound together by mechanical, thermal, or chemical entanglement (and by perforating films). "Resin" refers to an insoluble material that may take the form of a gel or gel beads or microporous beads, or a sponge. Such resins may be natural or biopolymers, synthetic polymers, and inorganic materials. Agarose beads, dextrose beads, and cellulose beads are commonly used natural supports. Synthetic polymeric 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 invention, the resin is selected from the group consisting of alginate, chitosan, chitin, collagen, carrageenan, gelatin, cellulose, starch, pectin and sepharose; inorganic materials selected from the group consisting of 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 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), styrenic acid (STYRENE), The polymer is composed of a polymer selected from the group consisting of synthetic polymers selected from the group consisting of polyvinyl acrylonitrile (SAN), polybutenoic acid, poly(4-allylbenzoic acid), poly(glycidyl acrylate), polyglycidyl methacrylate (PGMA), acrylonitrile butadiene styrene (ABS), polydivinylbenzene (PDVB), poly(allyl glycidyl ether), poly(vinyl glycidyl ether), poly(vinyl glycidyl urethane), polyallylamine, polyvinylamine, copolymers of the above polymers, and any of these polymers modified by the introduction of functional groups.

[0149] Various known methods can be used to immobilize the NMDAR protein construct to the support and / or matrix according to the present invention.Preferably, such immobilization is specific or selective in that it immobilizes the NMDAR protein construct, whereas other proteins and components present in blood or plasma, or in samples thereof (in vitro), are not significantly immobilized.

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

[0151] According to one embodiment, the NMDAR protein construct comprises an affinity tag for immobilization on a support. The affinity tag can be used for purification of proteins during production and / or for their immobilization on the support of the matrix of the present invention. The affinity tag can be a short polypeptide sequence or an entire protein, which can be co-expressed as a fusion partner with the NMDAR protein construct. Different types of affinity tags are well known in the art, and include polyhistidine or His. 6The tag, C-myc tag and FLAG tag are particularly well described and are the options for binding the construct according to the present invention to support material.Non-covalent binding of biotin to streptavidin or avidin can also be used to immobilize NMDAR protein construct to support.In an embodiment, binding is mediated by the Fc fragment that forms part of certain constructs of the present invention.

[0152] According to another embodiment of the present invention, NMDAR protein construct is covalently attached to support as described below in more detail and / or described in the prior art.Covalent binding generally includes either non-site-specific covalent binding of protein or site-specific covalent binding of protein.The support on which the matrix is ​​generated must provide or facilitate chemical activation, thus allowing chemical binding of construct.Many binding methods for immobilizing proteins such as NMDAR protein constructs are well known in the art.

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

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

[0155] Alternatively, the support may have specific functional groups for binding linkers and / or protein constructs. For example, functionalized resins are commercially available and known to those skilled in the art. A wide range of coupling chemistries are available on the commercially available supports, including primary amines, sulfhydryls, aldehydes, hydroxyls, and carboxylic acids. Examples of commercially available activated resins are CarboLink Coupling resin, Profinity™ epoxy resin, Affi-Gel 10 and 15, epoxy-activated Sepharose™ 6B, trisyl chloride-activated agarose, and Purolite™ Lifetech™ methacrylate polymer functionalized with epoxy groups.

[0156] According to one embodiment of the present invention, the support material must be porous, with pore sizes ranging from 10 nm to 200 nm. According to another embodiment of the present invention, the support is in 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 entrapment of magnetite in agarose or other polymeric material, on which the NMDAR protein construct according to the present invention is immobilized.

[0157] According to another embodiment of the 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 low diffusion limitations compared to gels, resins, and beads. The membrane can take the physical form of a hollow fiber or a flat membrane. According to one embodiment, the support is a hemodialysis hollow fiber membrane dialyzer and the filter is a hemodialysis device.

[0158] The hollow fiber or flat membranes used as supports in the device according to the invention may be composed of cellulose, cellulose esters (cellulose acetate and cellulose triacetate), poly(methyl methacrylate) (PMMA), polyamide (PA), other nitrogen-containing polymers (polybenzimidazole, polyacrylonitrile (PAN)), polyglycidyl methacrylate (PGMA), polyvinylpyrrolidone (PVP), polysulfone (PS), polyethersulfone (PES) or polyarylethersulfone (PAES). The hollow fiber membranes that can be advantageously used to provide the device according to the invention preferably have an internal diameter in the range of 100 μm to 500 μm. According to another embodiment of the invention, in particular when the membrane support is a hemodialysis membrane as described above, the hollow fiber membrane is additionally or alternatively functionalized with an NMDAR protein construct according to the 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 for 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, comprising means for transporting blood from the patient's vascular system to a blood treatment device at a prescribed flow rate, and thereafter returning the treated blood to the patient, the device being further configured to reduce the level of NMDAR autoantibodies in the blood.

[0160] According to the present invention, the expression "extracorporeal blood purification" refers to a process of removing substances from body fluids by clearance of those substances from the blood flowing in a bypass outside the patient's body (extracorporeal). Said substances may include endogenous toxins (i.e. uremic toxins), exogenous toxins (i.e. ethylene glycol or fungal toxins), administered drugs, viruses, bacteria, antibodies, metabolites and proteins (i.e. IMHA, myasthenia gravis), abnormal cells (i.e. leukemia) and excess water. Therapeutic 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 the skilled person and the device of the present invention can be incorporated accordingly.

[0161] As used herein, the expression "blood" refers to whole blood, including all the components of the blood of an organism, including red blood cells, white blood cells and platelets suspended in plasma. The expression "plasma" refers to a fluid composed of approximately 92% water, 7% proteins such as albumin, gamma globulins, fibrinogen, complement factors, clotting factors, and 1% mineral salts, sugars, fats, electrolytes, hormones and vitamins that form part of whole blood that is no longer containing red and white blood cells and platelets. In the context of the present invention, the expressions "blood plasma" or "plasma" refer to a specific fraction of plasma as defined above in its standard 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. Typical dialysate flow rates in an extracorporeal circuit including a hemodialysis machine for the treatment of renal failure are in the range of 0.5 l / h to 800 ml / min, when the hemodialysis machine is further configured to bind NMDAR autoantibodies, or in addition to the blood treatment device according to the invention.

[0163] In therapeutic apheresis, whole blood can be processed, or blood is separated into its component fractions, for example by centrifugation or using plasma membranes or filters, and the fraction containing the solutes that must be removed is specifically treated before being returned to the patient.

[0164] The present invention provides an apheresis process in which whole blood or plasma (containing target proteins) is removed from a patient's flowing blood and returned to the patient after contacting with a device or matrix according to the invention. Typical blood or plasma flow rates in the extracorporeal circuit, in which the blood processing device is perfused with whole blood or plasma, range from 30 ml / min to 200 ml / min, or 7 ml / min to 50 ml / min, respectively.

[0165] According to one embodiment, 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. The circuit can be operated in different treatment modes, including hemodialysis, hemodiafiltration, and hemofiltration modes, depending on medical needs.

[0166] Unless otherwise defined, 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, and all publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0167] drawing The present invention is further illustrated by the accompanying drawings, which are not intended to limit the scope of the invention, but rather depict preferred embodiments of aspects of the invention presented for further illustration of the invention described herein. [Brief description of the drawings]

[0168] [Figure 1-1]1 shows a scheme of a soluble recombinant NMDA receptor Fc (srNR-Fc) antigen / fusion protein. In the context of the present invention, srNR-Fc antigen and srNR-Fc fusion protein are used interchangeably. [Figure 1-2] Same as above [Figure 1-3] Same as above [Diagram 2] FIG. 1 shows that soluble recombinant NMDA receptor Fc fusion proteins are recognized by recombinant human GluN1 autoantibodies. [Figure 3-1] FIG. 1 shows that soluble recombinant NMDA receptor Fc fusion protein detects NMDA receptor autoantibodies in patient sera. [Figure 3-2] Same as above [Figure 3-3] Same as above [Figure 4] FIG. 1 shows autoantibody detection by soluble recombinant NMDA receptor Fc fusion protein in sera with known anti-NMDA receptor titers. [Diagram 5] FIG. 1 shows an ELISA screen for LGI1 antibody using soluble recombinant NMDA receptor Fc fusion protein (fusion protein #1 disclosed herein) as a control. [Figure 6] FIG. 1 shows ELISA quantification of recombinant human NR1(GluN1)AB in mouse brain extracts using soluble recombinant NMDA receptor Fc fusion protein with fusion protein #1 disclosed herein. [Figure 7] FIG. 1 shows that soluble NMDA receptor Fc protein constructs of the invention detect heterodimer-selective recombinant human NMDA receptor autoantibodies. [Figure 8] FIG. 1 shows that soluble NMDA receptor Fc protein constructs of the invention detect subtype-selective recombinant human NMDA receptor autoantibodies. [Figure 9] FIG. 1 shows that soluble NMDA receptor Fc protein constructs of the invention reveal subtype selectivity of recombinant human NMDA receptor autoantibodies. [Figure 10]FIG. 1 shows detection of recombinant human NMDA receptor autoantibodies by three soluble NMDA receptor Fc protein constructs of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0170] Figure 2. Cell culture supernatants of HEK293 cells expressing and secreting the indicated fusion proteins (Table 3) were captured on 96-well plates with anti-rabbit IgG antibodies. Single recombinant (rec) human (hu) autoantibodies against LGI1 or GluN1 (6) derived from patient CSF cells were applied at 1 μg / ml and detected with horseradish peroxidase (HRP)-conjugated anti-human IgG antibodies, and captured Fc fusion proteins were directly detected with 0.016 μg / ml HRP-conjugated anti-rabbit (rb) IgG antibodies. ELISA signals are shown as the mean ± SD of two wells in a single experiment after subtraction of the signal generated by the anti-human IgG antibody alone. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0171] Figure 3. Cell culture supernatants of HEK293 cells expressing and secreting the indicated fusion proteins (Table 3) were captured on 96-well plates by anti-rabbit Fc antibody. Sera from seven NMDAR encephalitis patients (S3-S9) and control serum (Control S) were applied at 1:200 dilution and human IgG was detected using biotin-conjugated anti-human IgG and HRP-conjugated streptavidin. Panels A-C represent separate experiments. ELISA signals are shown as the mean ± SD of two wells in a single experiment after subtraction of the signal caused by the combination of biotin-conjugated anti-human IgG and HRP-conjugated streptavidin alone. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0172] Figure 4. Cell culture supernatants of HEK293 cells expressing and secreting the indicated fusion proteins were captured on 96-well plates by anti-rabbit Fc antibodies. Sera from five NMDAR encephalitis patients (S10–S14) and three control sera (S15, S18, S19) were applied at 1:100 dilution and human IgG was detected using biotin-conjugated anti-human IgG and HRP-conjugated streptavidin. ELISA signals are shown as the mean ± SD of two wells in a single experiment after subtraction of the signal caused by the combination of biotin-conjugated anti-human IgG and HRP-conjugated streptavidin alone. NMDAR antibody titers of sera determined by Euroimmun cell-based assay are shown below for comparison. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0173] Figure 5. To screen for LGI1 reactivity, 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 an ELISA plate. The example assay shows the results of 13 supernatants alongside controls including CSF samples (diluted 1:5), human recombinant anti-GluN1, mouse anti-LGI1, and secondary antibodies against human, mouse, and rabbit IgG alone. Signals are shown as the mean ± SD of duplicate wells.

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

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

[0176] Figure 8. Cell culture supernatants of HEK293 cells expressing and secreting the indicated fusion proteins (Table 3) were captured on 96-well plates with anti-rabbit IgG antibodies. Single recombinant human NMDA receptor autoantibodies (6 or unpublished) or recombinant human control antibodies (control Ab1) derived from patient CSF cells were applied at 1 μg / ml and detected with horseradish peroxidase (HRP)-conjugated anti-human IgG antibodies, and captured Fc fusion proteins were directly detected with 0.016 μg / ml HRP-conjugated anti-rabbit (rb) IgG antibodies. ELISA signals are shown as the mean ± SD of two wells in a single experiment after subtraction of the signal generated by anti-human IgG antibodies alone. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0177] Figure 9. Cell culture supernatants of HEK293 cells expressing and secreting the indicated fusion proteins (Table 3) were captured on 96-well plates with anti-rabbit IgG antibodies. Single recombinant human NMDA receptor autoantibodies (6 or unpublished) derived from patient CSF cells or control antibodies (mGO53) were applied at 0.09 μg / ml (003-102) or 1.8 μg / ml (anti-NR-Ab1, mGO53) and detected with horseradish peroxidase (HRP)-conjugated anti-human IgG antibodies, and captured Fc fusion proteins were directly detected with 0.016 μg / ml HRP-conjugated anti-rabbit (rb) IgG antibodies. ELISA signals are shown as the mean ± SD of two wells in a single experiment after subtraction of the signal generated by anti-human IgG antibody alone. 490 nm values ​​are shown for 003-102 and anti-rabbit IgG. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

[0178] Figure 10. Cell culture supernatants of HEK293 cells expressing and secreting the indicated fusion proteins (Table 3) were captured on 96-well plates with anti-rabbit IgG antibodies. Single recombinant human NMDA receptor autoantibodies (NPL 6 or unpublished) derived from patient CSF cells were applied at 0.01 μg / ml (003-102), 1 μg / ml (008-218, anti-NR-Ab1) or 10 μg / ml (all others) and detected with horseradish peroxidase (HRP)-conjugated anti-human IgG antibodies, and captured Fc fusion proteins were directly detected with 0.016 μg / ml HRP-conjugated anti-rabbit (rb) IgG antibodies. ELISA signals are shown as the mean ± SD of two wells in a single experiment after subtraction of the signal generated by the anti-human IgG antibody alone. PGRN, progranulin. Fc, constant region of rabbit IgG1 heavy chain.

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

[0180] The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention but rather represent preferred embodiments of aspects of the invention presented for further illustration of the invention 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 of different subunit composition in patient serum and CSF?

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

[0183] Fc-mediated dimerization of the expressed proteins may result in epitopes very similar to native NMDA receptors, conferring unprecedented stability to the fusion proteins. These soluble recombinant NMDA receptor Fc (srNR-Fc) fusion proteins / antigens are capable of detecting NMDA receptor autoantibodies against different NMDA receptor subunit compositions present in the serum of NMDAR encephalitis patients, thus forming the core subject of the present invention.

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

[0185] Detailed Example Example 1: Exemplary protein constructs of the invention generated for experiments. We generated constructs encompassing the extracellular portions of the GluN1 and GluN2 subunits (Figures 1A and 1B, Table 1), expressed them in HEK293 cells, and isolated cell culture supernatants containing the secreted Fc fusion proteins 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 GluN1 and GluN2B domains are separated by an artificial linker and fused to Fc in a single molecule. The Fc domain likely leads to dimerization of all fusion proteins, resulting in GluN1 / GluN2 heterodimers upon co-expression of constructs #1 or #2 with #5, #6, #9 or #3, respectively, or dimers of GluN1 / GluN2 heterodimers upon expression of constructs #4, #7 and / or #8 (Figure 1C).

[0187] Table 3: Soluble recombinant NMDA receptor rbFc fusion proteins constituted by and / or representing the NMDAR protein constructs of the present invention. [Table 3-1] [Table 3-2] name Fusion Protein Composition NMDA receptor segments: Amino acids in GenBank entries Number of amino acids in the NMDA receptor part / whole protein hs, human; rb, rabbit; Ecd, extracellular domain; ATD, amino-terminal domain; No, number; Fc, rabbit IgG1 heavy chain constant region.

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

[0189] Example 2: Soluble NMDA receptor rbFc fusion protein is recognized by recombinant human GluN1 autoantibodies. Figure 2 shows that all srNR-Fc antigens tested, except for the control antigen (Progranulin-Fc), were recognized by recombinant human anti-NMDA receptor antibodies. In contrast, no antigens were recognized by recombinant human anti-LGI1 antibodies. Using an anti-rabbit IgG antibody that detects the rbFc portion of the fusion proteins, we confirmed that all rbFc fusion proteins were successfully expressed and immobilized on the ELISA plate.

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

[0191] Example 4: Autoantibody detection with soluble NMDA receptor rbFc fusion protein in sera with known anti-NMDA receptor titers. To test how ELISA based on srNR-Fc protein differs from clinical standard assays, we measured sera with known titers from the Euroimmun CBA (Figure 4). All sera scoring positive in Euroimmun (S10-S14) produced positive signals with at least one of the antigens tested, compared to Progranulin and GluN2B-ATD as controls, whereas sera scoring negative in Euroimmun CBA (S15, S18, S19) did not produce positive signals. These data show that srNR-Fc containing GluN1-ATD specifically detects NMDAR autoantibodies in serum.

[0192] We used two srNR-Fc combinations expressing the ATDs of GluN1 and GluN2B in this assay (N1-ATD-Fc+N2B-ATD-Fc and N1-ATD-N2B-ATD-Fc). They contain the same amino acids of NMDAR (Table 1), but in one case the antigen is reconstituted from two separate proteins, while the other construct contains the ATDs connected by an artificial linker as a single protein. These two antigens gave comparable signals in serum S10-S12, but were significantly different in serum S13 and S14. The antigen N1-ATD-Fc+N2B-ATD-Fc produced small, comparable signals in S13 and S14. In contrast, N1-ATD-N2B-ATD-Fc detected no NMDAR antibody signal at S13 and a strong signal at S14. The molecular architecture of N1-ATD-N2B-ATD-Fc may have hindered the access of NMDAR autoantibodies present in S13 to their epitopes. This finding emphasizes the need to test several srNR-Fc combinations to detect as many antibodies as possible.

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

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

[0195] The soluble recombinant NMDA receptor Fc antigen N1-ATD-N2B-ATD-Fc gave 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), suggesting that the specific molecular organization of the Fc fusion protein N1-ATD-N2B-ATD-Fc suggests that the ATDs of GluN1 and GluN2B are part of a single protein, providing an advantage in detecting subtype-selective antibodies.

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

[0197] Discussion of the Examples The results provided here serve as a proof of concept. We conclude that (1) a soluble fusion protein containing the amino-terminal domain of GluN1 and rabbit Fc, heterogeneously expressed in and secreted from HEK293 cells, is capable of binding NMDA receptor autoantibodies in patient sera, 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 enable classification of anti-NMDA receptor immune responses in patients.

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

[0199] Further examples of experimental uses of the constructs of the invention ELISA screen for recombinant LGI1 antibodies with NMDA receptor fusion protein as a control. To generate the mammalian expression constructs used in this experiment, cDNAs for amino acids 1-558 of human LGI1 (NM_005097.3) and amino acids 1-400 of human GluN1 (NM_007327) were inserted into pFuse-rIgG-Fc1 (invivoGen). The resulting plasmids encode hsLGI1 or the amino terminal domain (ATD) of hsGluN1 fused to the Fc region of rabbit IgG (amino acids SKP-PGK) linked by 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] Antibody binding to LGI1-Fc and the NMDA receptor subunit GluN1-ATD-Fc was compared by ELISA. 96-well high-binding microplates (Greiner #655061) coated with donkey anti-rabbit IgG (10 μg / mL, Dianova, #711-005-152) were blocked and incubated with cell culture supernatants of HEK293 cells expressing Fc fusion proteins. Cell culture supernatants 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 extensive washing, HRP activity was measured using 1-Step Ultra TMB-ELISA substrate (Thermo Fisher). The presence of immobilized antigen was confirmed by incubation with HRP-conjugated F(ab')2 donkey anti-rabbit IgG (1:50000, Dianova, #711-036-152). Human recombinant anti-GluN1 antibody 003-102 (Kreye J, Wenke NK, Chayka M, et al. Human cerebrospinal fluid monoclonal N-methyl-D-aspartate receptor autoantibodies are sufficient for encephalitis pathogenesis. Brain 2016; 139:2641-2652) 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 extracts using NMDA receptor fusion proteins. The concentration of recombinant human NR1 AB #003-102 in brain extracts was determined in 96-well plates coated overnight at 4 °C with donkey anti-rabbit IgG (20 μg / mL, Dianova, #711-005-152). After blocking with 2% BSA in PBS / 0.05% Tween-20 (PBS / T) at room temperature, cell culture supernatants of HEK293 cells expressing the amino-terminal domain (amino acids 1-400) of human NR1 (GluN1) fused to rabbit Fc were applied. Mouse brain extracts were diluted 1:25 / 1:100 in 0.4% BSA-PBS / T and added in duplicate. Plates were washed with PBS / T and incubated with horseradish peroxidase (HRP)-conjugated donkey anti-human IgG (1:5000, Dianova, #709-035-149). After washing, HRP activity was measured using 1-Step Ultra TMB-ELISA substrate (Thermo Fisher). 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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 hs anti-LGI1 rec hs anti-LGI1 rec hs anti-GluN1 rec hs anti-GluN1 Anti-rbIgG Anti-rbIgG Figure 3A ctrl S Control S Figure 3B ctrl S Control S Figure 3C ctrl S Control S Figure 4 Anti-GluN1-Titer: Anti-GluN1 titer: neg. negative Figure 5 ctrl SN Control SN anti-GluN1 no hs AB no hs AB anti-LGI1 no mm AB mm AB no anti-rb Figure 6 NR1-reactive IgG [ng / brain] NR1-reactive IgG [ng / brain] CTL control Figure 7 A 450 nm or * A 490 nm A 450 nm or * A 490 nm Anti-NR-Ab1 Anti-NR-Ab1 Anti-NR-Ab2 Anti-NR-Ab2 Anti-rbIgG * anti-rbIgG * Figure 8 Anti-NR-Ab1 Anti-NR-Ab1 control Ab1 Anti-rbIgG Anti-rbIgG Figure 9 A 450 nm or * A 490 nm A 450 nm or * A 490 nm Anti-NR-Ab1 Anti-NR-Ab1 Anti-rbIgG * anti-rbIgG * Figure 10 Anti-NR-Ab1 Anti-NR-Ab1 Anti-NR-Ab2 Anti-NR-Ab2 control Ab1 Anti-rbIgG Anti-rbIgG

Claims

1. N-methyl-D-aspartate receptor (NMDAR) protein containing one or more NMDAR autoantibody epitopes An NMDAR protein construct lacking the NMDAR transmembrane domain, the construct being a fusion protein comprising the amino-terminal domain (ATD) of GluN1 and the ATD of GluN2B.

2. 2. The NMDAR protein construct of 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 of claim 2, wherein the dimerization domain is the capture domain.

4. An in vitro method for detecting NMDAR autoantibodies in a sample suspected of containing NMDAR autoantibodies, comprising: a. providing an NMDA protein construct according to any one of claims 1 to 3, comprising a capture domain as a capture molecule; b. contacting a sample suspected of containing the NMDAR autoantibody with the NMDAR protein construct, thereby allowing the NMDAR autoantibody from the sample to bind to the NMDAR protein construct; c. Determining the presence of bound NMDAR autoantibodies; and A method comprising:

5. The method of claim 4, wherein the NMDAR autoantibody in the sample is present in a solution or on a cell membrane.

6. The method according to claim 4 or 5, wherein the method is carried out using a plurality of different NMDAR protein constructs according to any one of claims 1 to 3, and further comprises determining to which NMDAR protein construct of the plurality of constructs the NMDAR autoantibody binds.

7. The method according to any one of claims 4 to 6, wherein the method is applied to assess the possibility of a medical condition associated with autoantibodies against NMDAR, and the sample suspected of containing NMDAR autoantibodies is a sample from a human subject presenting with symptoms of having said medical disorder.

8. The method of claim 7, wherein the medical condition associated with autoantibodies to NMDAR is anti-NMDAR encephalitis.

9. A kit for diagnosing an autoimmune disease associated with NMDAR autoantibodies in a subject by detecting NMDAR autoantibodies, comprising: a. an NMDAR protein construct according to any one of claims 1 to 3, or an NMDAR protein construct according to any one of claims 1 to 3 immobilized on a solid surface, and a labeled secondary affinity reagent directed against human NMDAR autoantibodies, and a means for detecting the signal emitted by the label, or b. A labeled NMDAR protein construct according to any one of claims 1 to 3; A kit comprising:

10. The kit according to claim 9 for diagnosing anti-NMDAR encephalitis.

11. 4. A blood treatment device configured for removing NMDAR autoantibodies from the blood or plasma of a person in need of treatment in an extracorporeal blood circuit, the blood treatment device comprising a matrix having one or more NMDAR protein constructs according to any one of claims 1 to 3 immobilized thereon.

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